Studies of the Immune System of HOX11 Transgenic Mice for Lymphoma Immunotherapy
Suzana Rosic-Kablar, M.D.
A thesis subrnitted in conformation with the requiremenb for the degree of Master of Science
Graduate department of Laboratory Medicine and Pathobiology University of Toronto
@ Copyright by Suuna Rosic-Kablar, 1999
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Studies of the Immune System of HOX11 Transgenic Mice for Lymphorna
lrnmunotherapy
A thesis submitted in conformation with the requirernents for the degree of Master of Science
Graduate department of Laboratory Medicine and Pathobiology University of Toronto
1999
Suzana Rosic-Kablar, M.D.
Abstract
We assessed the applicability of dendntic (DC) cell based
immunotherapeutic protocols for the treatment of B cell lymphoma, using the
HOXI1 transgenic mouse model. These mice develop large B cell lyrnphomas in
the spleen. Towards this goal, we analyzed dendritic and T cells from HOX1 1
mice. Results indicate that HOX11 DCs express normal levels of ceil surface
molecules and have normal ability to present foreign antigens, while T cells show
normal proliferative response to mitogenic stimulation, foreign MHC and OVA
protein. In addition, HOXI1 derived peptides or lymphoma cell lysates are
sufficiently immunogenic to induce specific immune responses in naïve mice.
However, HOXl1 derived T cells do not show induction of immune responses.
Our results suggest that lymphoma development in HOXll rnice is associated
with the toleration of lymphoma antigen specific T cells. making HOXll
transgenic mice a good model ta develop DC-based immunotherapeutic
protocols for the treatment of nonfollicular lymphomas.
ACKNOWLEDGMENTS
I would Iike to thank my supervison, Drs. lan Dube and Margaret Hough for
being patient, supportive and encouraging throughout my studies.
Dr. Dubé believed in me and gave me the opportunity to be a member of his
research team. He provided a great environment in which I could leam and I
considered myself lucky to be a part of it.
I had the opportunity to know Dr. Hough as a supewisor and a friend.
Margaret has continually challenged me throughout this project with intriguing
questions and has provided me with valuable advice and direction in both work
and life.
My appreciation also goes to Dr. Serge Jothy who provided me with important
feedback at my cornmittee meetings and always reminded me how wonderful
science is, even when baniers are faœd.
Thanks to my committee members Drs. Charles Catzavelos, Corrinne Lobé
and David Spaner for providing adviœ and guidance du ring my graduate studies.
My gratitude also goes to al1 rnembers of the Dubé research team who were
always ready to provide moral and technical support. In this respect I would
especially like to thank Dr. Carolyn Lutzko, Kin Chan and Renita Yap.
Thanks to Drs. Marciano Reis and Megan Lim for sharing their knowledge in
hernopoiesis and for the hours spent in reviewing the histopathology slides.
Thanks to the members of Sunnybrook Animal Facility for technical support
and assistance with animal husbandry.
A special thanks goes to Dr. Barbara Guinn who introduced me to many of the
challenging techniques that I used in my project and turned our relationship into a
great friendship.
My special gratitude goes to my mother for her love and support. She has
always encouraged hard work and stood behind any of my decisions.
Finally, but most importantly, I would like to thank rny husband, Nijaz and my
son, Adi who believed in me and gave me the support and tirne I needed to
complete this degree. Thanks is such a small word considering how much you
deserve.
4
Table of Contents
CHAPTER 1 1
LNTRODUCTION
1.1 HEIMATOPOIETTC CELL OEUGM, GROWTH AND DIFFERENTIATIoN
1.2 MALIGNANT HEMATOPOIESIS
1.3 NEOPLA~MS OF m L~LMUNE SYSTEM
1.4 NON-HODGKM L'YWPHOMA
1.4. I Developmenz and Classtjkation
1-42 Incidence and Etiology
1.4 3 Trearment and Prognosii of NhZ
1.5 TUMOR IMMUNOLOGY
I . 5 I Tumor anrigens
1-52 me Immune System 's Rote in Tumorigeneiris
15.2.1 B Cells and Antibody-Dependent Killing
1 -5.2.2 T Ce11 Mediated Cytotoxicity
1.5.3 Mechankm by Which Tumor Ce Ils Escape an Immune Response
1.6 ~ ~ U N O T H E R A P Y FOR NON-HODGKM LYMPKOMA (NHL)
1.6.1 Eady Approaches of Lymphoma Immun0 fherapy
1.6.2 Crarenr Concepts of Lymphoma Zinmunotherapy
1.7 DENDRITIC CELLS AS PROFESSIONAL ANTIGEN P R E S ~ G CELLS
1.7.1 *gin and Generation of Cuitwed De&-tic Cells
1.7.2. Rule of Denilritic Celk in T cell Activation
1.8 D m m c CELLS AND OR hm-mrw
1.8.1 Denchitic Cell Vaccination in Murine ModeIs: Preclinical Sludies
1.8.2 Dendriric Cell Vaccination for Human Cancer: Clinical Trrals
1.8.2.1 Non-Hodgkin Lymphoma
1.8.2.2 Malignant Melanoma B.
1.8.2.3 Multiple *elorna
'1.8.2.4 Ofher Cancers
1.8.3 F u m e Prospects for DC Based Cancer Vaccines
1 -9 HOX 1 1 TRANSGENK MICE
1.9.1 Homeobox Genes in Hematopoiesis and Hematologic Malignancies
1.9.2 Production of HOX1 1 Trmgenic Mice
1.9.3 Lymphoma Developrnenr In HOXl 2 Mce
1.10 SWY RATIONALE
1.1 1 HYPoTHESIS
1.12 OBJEC~VES
CHAPTER 2 48
~ ~ T E R U L S AND METHODS
2.1 ANIMALS
2.2 REAGENTS USED FOR TKE ~ O L A T I O N AND C U L T U ~ G OF DCS AND T CELLS
2.2.1 Growth Factors
2.2.2 Collagenase D
2.2.3 Bovine Serum Albumine (BSA)
2.2.4 Preparation of Steriked Nylon Wool Columrts
2.3 bfATERIALS USED FOR MIXED LYMPHOCYE W ~ O N W R ) AND CYTOTOXIC T
LYMPHOCYTE (Cil) ASSAY
2.3.1 Cell Lines Used ai^ Targets in CTL Assqv
2.3.2 Production of h vitro Transcribed (m RNA
2.3.3 Preparation of Tumor Lysate
2.4 GENERATION OF DC CULTURES
2-41 Dendritic Cell I.solationfiom Spleen
2.4.1.1 Density gradient centrifugation using Bovine Senim Albumin @SA)
2.4.1.2 Separation using MACS CD1 l c MicroBeads
2.4.2 Dendritic Cell Isolation fiom Bone Marrow
2.5 DENDRlIK CELL SURFACE PHENoTYPE CHARACIFRIZA~ON
2 .3 1 Flow Cytomeiric Anabsis of Spleen and Bone M m o w Dendritic CelLs
2.6 T CELL ISOLATION AND ENRICHMENT
2.6.2 Isolation of T celkfi.orn Peripheral Blood
2.6.2 Isolation of T Cells fiom Spleen and Thymus
2.6.2.1 Separation with MACS System
2.6.2.2 Isolation with Nylon WooI Columns
2.7 OPT[MIZATION OF MIXED LYMPHOCYTE REACTION (MLR)
2.7.1 Optirniration of T Cell Prolijeraiive Respowes
2.8 FUNCTIONAL STUDiES USMG HOXI1 DWED DCS
3 8.1 Presenfarion of Foreip A2HC by HOXl l Derived DCs
2-82 Presenfarion of O VA Protein by HUXl l Derived DCs
2.9 FUNCTIONAL STUDIES USMG HOX11 DERIVED T CELLS
2.9.1 T Cell Prolferution Assay
2.9.2 T Cell Aesponses tu Foreign MHC Anrigens
2.9.3 T Cefl Responses ro O VA Prorein
2.10 ~WUREMENT OF CYTOTOXIC T LYMPHOCYTE (CTL) ACTIV~TY
2.1 O. 1 In Vitro CTL
3 10.2 In Vivo CTL
2.1 1 ASSESSMENT OF I M M U N O G E N E I C ~ OF HOXI 1 DENVED Amx~ms 2.1 1. 1 Induction of CTL Response ro in Vitro Tramcribed (rm HOXI 1 RNA
2.11.2 Induction of CTL Response to HOXI1 Tumor Lysutes
RESULTS
3.1 LDENTTFICATION OF HOXl 1 TP.SGEN1C MCE
3.2 O P ~ Z A T I O N OF MIXED LYMPHOCYTE REAC~ON (MLR)
3.3 CHARA~RIZATION OF HOX11 TRANSGENIC M m DERIVED D E N D ~ C CELLS
3.3.1 ESTABLISHMENT OF DENDRITIC CEU CULWRES
3.3.2 Phenotype Characterïzation
3- 3.3 HOXI i Derived DCs: Presenraiion of Foreign MHC
3.3.4 HOXI i Derived DCs: Presentaiion of OVA Prolein
3.4 CHARACTERIZATION OF HOXI I TRANSGEMC MICE DEWED T CELLS
3 - 4 1 Prolijeration Assay (PNA Stimulation)
3.42 T Cell Prol~erative Responses to Foreign MHC
3.43 T Ceil Proliferaiïve Responses to OVA Protein
3 -5 MEASUREMENT OF CWOTOXIC T LYMPHOCYTE (Cm) A c w m
3.5.1 In Viîro CTL Assay
3.22 in vivo CTL Assay
3 -6 AsSESSMENT OF IMMtMoGENEICITY OF HOX 1 1
3.6.1 T CeZi Prof$erative Responses upon the Stimulaion with IVT RNA
3.6.2 Inducrion of Primary CTL Respome zo XVTNOX'l RNA in Nafie Mice 114
3.6.3 Inducfion of CTL Response u p n St i~~dat ion with Timor Lysares 120
CHAPTER 4 124
0
D~scussxo~ AND FUTURE EXPWUMENTS
vii
List of Figures
Figure 1 : PCR analysis of HOX11 transgenic and nontransgenic mice .............. ... ... 74
Figure 2: Optimization of MLR ........................................................................ ïï
Figure 3: Flow cytometric analysis of HOXl1 and nontransgenic splenic DCs ........... 81
Figure 4: Flow cytometric analysis of HOXI 1 and nontransgenic bone marrow
Derived DCs .................................................................................. 82
Figure 5: Presentation of foreign MHC by HOXI1 derived DCs ............................. 86
Figure 6: HOXI1 derived DCs presenting OVA protein ........................................ 88
Figure 7: HOXi 1 derived T cell proliferation assay (PHA) ..................................... 91
Figure 8: HOX11 derived T cell proliferative responses to foreign MHC ................... 94
Figure 9: HOX11 derived T cell proliferative responses to DOTAP-OVA pulsed
DCs ............................................................................................. 96
Figure 10: ln vitro CTL assay using OVA pulsed DCs ........................................... 99
Figure 11 : In vivo CTL assay using OVA protein pulsed DCs ............................... 102
Figure 12: MLR stimulated with IVT HOXI 1 RNA pulsed DCs used for immunization . . ....................................................................................... ln VIVO. 105
........... Figure 1 3: CTL assay after immunization with IVT HOX11 RNA pulsed DCs 108
............ Figure 14: In vitro CTL assay using DCs pulsed with lymphoma cell lysates 113
List of Illustrations
...... Illustration 1 : Tissue sections from HOX11 transgenic mice showing lymphorna -42
Illustration 2: HOX11 derived DCs in culture .................................................... -81
Illustration 3: In vitro CTL assay using HOX11 transgenic mice dedved DCs pulsed
....................................................................... with OVA protein 102
Illustration 4: In vitro restimulation of splenocytes isolated from HOX11 transgenic.
nontransgenic and C57BU6 after irnmunization ............................... 116
List of Tables
Table 1 : Nurnber of DCs obtained from spleens of HOX11 transgenic mice ............... 80
Table 2: Nurnber of HOXl 1 bone manow derived DCs ......................................... 81
Table 3: Flow cytometric analysis of HOXl1 and nontransgenic spleen derived DCs.83
Table 4: Flow cytometric analysis of HOX11 and nontransgenic bone marrow
denved DCs .................................................................................... -87
Table 5: In vitro CTL assay using OVA protein pulsed DCs .................................. 105
Table 6: In vivo CTL using DC pulsed with OVA ................................................. 109
..... .............. Table 7: In vivo CTL assay using DCs pulsed with IVT HOX11 RNA ... 115
Table 8: In vjtm CTL assay using DCs pulsed with lymphoma cell lysates .............. .121
Chapter 1
ln traduction
1.1 Hematopoietic Cell Origin, Growth and Differentiation
Hematopoiesis is a process through which blood cells grow, divide and
differentiatel. All ceIl types are derived from a pool of hematopoietic stem œlls
(HSCs), which reside in the bone manow and have the unique ability, under
the appropriate conditions, to give rise to al1 of the different mature blood cells.
As the cells differentiate, their capacity for replication and self-renewal
declines. Thus, the less differentiated cells in a given pathway are rare but
replicate actively whereas the mature cells are more numerous but generally
are mitotically inert. Some daughter cells remain as HSCs (self-renewing cells),
while othen commit to any one of several alternative differentiation pathways
(myelo-erythroid progenitors and lymphoid progenitor cells) that lead to the
production of three general classes of cells: (1) erythrocytes, responsible for
oxygen transportation; and (2) myeloid and (3) lyrnphoid cells, both critical to
host defense.
The mature cells of the myeloid lineage include neutrophils, monocytes,
mast cells, eosinophils, basophils and megakaryocytes. Mature cells of the
lymphocyte lineage include B lymphocytes, T lymphocytes and natural killer
(NU) cells. B cells express immunoglobulin (lg) receptors, which recognize
soluble antigens whereas T cells express T cell receptor FCR) molecules that
recognize antigenic peptides in the context of major histocompatibility
molecules on the surface of antigen presenting cells. Consequer!tly, B and T
lymphocytes. with other blood cells. play important roles in host immune
responses and in the elimination of foreign pathogens.
1.2 Malignant Hernatopoiesis
Vast numbers of mature blood cells are produced daily in the
hematopoietic system. Hematopoiesis is precisely controlled at several levels:
(1) the maintenance of a lifelang pool of HSCs; (2) regulation of cornmhent,
proliferation and differentiation of cells at al1 stages of each hematopoietic
pathway; and (3) modulation of the activities of each pathway in response to
physiological demands. Normal hematopoietic cell activities are coordinated so
that the rate of loss due to natural death of mature differentiated cells closely
approxirnates the rate of appearance of new cells from the less mature
proliferating cell pool*. The transformation of a normal hematopoietic progenitor
ce11 to one with malignant potential can result from a variety of causes. It may
be induced by chemical. physical and viral carcinogens or may occur
spontaneously by random mutation or gene rearrangements. For example, the
mechanisms by which chemical carcinogens induce neoplastic transformation
predominantly reflect the mutagenic adivity of the carcinogens? Physical
carcinogens, such as radioactivity or ultraviolet radiation. often induce damage
to cellular DNA resulting in a variety of cancers of epithelial cell origin'. Viral
oncogenesis is of particular interest because several human DNA viruses have
been found to contain potential onwgenes that have been associated with the
development of malignancies. These include links between Epstein-Ban virus
(EBV) and Burkitt's lymphoma and some forms of Hodgkin's disease, and
human papillomavirus and skin cancer. In addition to DNA viruses, a class of
human retroviruses. the human T cell Ieukemia viruses (HTLV) are responsible
for the development of a subset of T cell leukemias. Another important
oncogenic retrovirus is human immunodeficiency virus (HIV). which is
responsible for the development of acquired irnmunodeficiency syndrome
(AIDS), a disease that among other symptoms. makes its vidims susceptible to
nume rous cancers inciuding non-Hodgkin's lymp homa (NHL) and Kaposi's
sarcoma (KS).
Advances in molecular biology have provided the tools to better
understand the essential role of many cellular oncogenes in normal growth and
development, and in the transformation of normal cells into malignant cells.
Proteins expressed by advated oncogenes are involved in the process of
transformation. Oncogene activation may occur by a variety of mechanisms
including gene amplification, mutation. and chrornosomal translocation. Gene
translocations are frequently observed in the malignant cells of patients with
leukemia and lymphoma. The most commonly obseived translocation in 6 cell
tumon places the c-MYC oncogene next to the immunoglobulin gene locisI6.
Aberrant MYC expression can lead to the development of malignant tumors,
presumably by interfering with the normal control of cell g r m . These
translocations are seen in over 90% of endemic and sporadic Burkitt's
lymphomas. T lymphocyte derived tumors frequently contain translocations
involving the T cell receptor gene loci. In pathologic conditions resuiting from
oncogene activation, an individual cell may acquire the potential to produce
daughter cells that proliferate independently of extemal growth signals and this
rnay represent the basis of a malignant proœss? Such transformed cells rnay
be less sensitive to normal cell death pathways and may also fait to respond to
the regulatory signals responsible for normal growth and tissue repair. The
additional accumulation of mutations within the expanding monoclonal
populations often results in genotypic and phenotypic heterogeneity. Genomic
abnormalities may also eventually result in invasive growth through normal
tissue boundaries and metastasis to distant organs. following entry into blood
and lymph channels.
The immunologic responses of the host to the growth of malignant cells
are based on the host's immune system and the way in which the host defense
is triggered to promote tumor eradication. Immune rejection of malignant cells
is often difficult to accomplish because tumor cells have many similarities to
normal cells, despite often exhibiting an abnormal ability to proliferate, an ability
to spread throughout the host, and capacities to interfere with normal organ
functions.
1.3 Neoplasms of the lmmune System
Neoplasms of the hematopoietic system are cancers that directly affect
the host immune defense. They are similar to other neoplasms in many
respects, but considering the crucial role of the host immune system, some
notable differenœs occur. First. sinœ neoplastic lymphocytes may circulate in
the penpheral blood and lymphatic systems. like their non-neoplastic
counterparts, many lymp hoid neoplasms are widely disseminated at diagnosis.
A second feature of neoplasms of the immune system is that they often retain
some functional characteristics of normal cells. For example. neoplastic T cells
may secrete cytokines and neoplastic plasma cells usually secrete
immunoglobulins. but generally the functional propertïes of such transformecl
cells do not occur in response to physiological stimuli. These cellular functions
become autonomous. often contributing to further tumor development and
clonal expansion.
Neoplasms of the hematopoietic system are classified into leukemias
and lymphomas. The leukemias are hernatologic neoplasms in which rnalignant
cells are present in the bone marrow and the blood, whereas lymphomas are
localized proliferations of lymphoid cells forrning a solid tissue mas. However,
many hematologic neoplasms show both patterns, to varying degrees, making
distinctions between leukemia and lymphoma sometimes rather difficult in
clinical practice. Neoplastic cell classification and the establishment of a correct
diagnosis is important for the selection of the appropriate therapy.
1.4 Non-Hodgkin Lymphoma
7.4.1 Development and Classfication
Lymphomas are divided into Hodgkin's disease and non-Hodgkin
lymphoma. NHLs make up about 5% of al1 cancers and 85% of al1 lymphomas.
In 1832. Thomas Hodgkin first described the neoplastic transformations of cells
that reside predominantly within lymphoid tissues. The f o m he described is
now called Hodgkin's disease. Despite their diversity, al1 of the other malignant
lymphomas are refened to as non-Hodgkin's lymphomas (NHLs). Primary sites
of NHL growth are the organs of the immune system such as lymph nodes,
spleen. tonsils and thymus. The stomach, small intestine and skin may also be
affected. The development of lyrnphomas reflects in large part the relatively
unrestrained growth of lyrnphoid cells that locally invade and disrupt normal
tissue as well as metastasize and growth in distant organs. Lymphoid
neoplasrns can be divided into 6 or T cell in origin. Furthemore. since normal
lymphocytes go through several discrete stages of development, lymphoid
malignances may arise from cells arrested at a particular stage of
differentiation? Consequently a cell population that has undergone malignant
proliferation often shows a combination of surface markers that are known to
arise during discrete stage of T or 6 lymphopoiesis1o.
Appropriate classification is of significant importance because of the
multiple subgroups that are associated with distinct clinical outcornes. A variety
of systems have been used to classify NHL subtypes according to their
morp holog ical appearance, p henotype and clinical behavior of the disease. The
Rappaport classification is based on assessrnent of the overall pattern of lymph
node architecture as well as the cytological classification of the neoplastic cell
subdividing NHL into diffuse and follicular lyrnphomasll. In follicular
lymphomas the neoplastic cells appear to recapitulate normal lymphoid
germinal centers while in difise types normal cortical and paracortical lymph
node architecture is largely affeded. The International Working Formulation,
developed in 1982. defined 10 major subtypes of lyrnphoma, categorized as
low, intemediate or high grade. More recently, in 1994, the International
Lyrnphoma Study Group developed the Revised European-American
Lyrnphoma (REAL) classificationl2. It describes several lymphoma types with
distinct clinical and morphological features and defines the most important
immunophenotypic, cytogenetic, and molecular features of each.
Currently, lymp homas are graded as aggressive (intemediate and hig h
grade) and indolent tumors (low-grade lymphomas). Aggressive lymphomas
are generally large cell lymphomas with rapid cell division and rapid
progression of d isease. Indolent lymphomas are small cell lymphomas and,
although patients may be asymptomatic for prolonged periods, these diseases
usually progress to an acute phase-
1.4.2 Incidence and Etiology
The number of new cases of NHL in Canada in 1999 is estimated to be
5,800. Based on the American Cancer Society statistics, NHLs are the second
leading cause of cancer deaths in patients aged 15 to 34 and the third leading
cause of cancer death in patients aged 35 to 54. Although childhood cancer is
rare, NHL is the third most common pediatnc cancer and accounts for 6
percent of childhood cancers. An increase in the incidence of NHL between
1973 and 1987 was larger than the increase during that period for any other
major cancer except rnelanoma and lung cancer? Eight to 27 percent of the
increase was associated with the development of NHL in association with
AIDS. For children with AIDS. the rate of developing NHL is about ten times
higher than that of adults with AIDS.
The etiology of most types of NHLs is unknown. However, several
factors suggest possible causal relationships. In addition to chernical and viral
etiologies, NHL has been obsewed as a late complication of prior
chemotherapy and radiation therapy for unrelated disorders. Patients with
Hodgkin's disease who were treated with conventional therapy exhibit an
increased risk of developing secondary large-cell lymphornas'4. The incidence
of NHL is increased nearly 100-fold for patients undergoing organ
transplantation necessitating chronic imrnunosuppression, and it is greatest
during the first year posttransplantl? Diseases of inherited and acquired
irnmunodeficiency as well as autoimmune diseases are associated with an
increased incidence of lymphoma, indicating that immune deregulation may
significantly contribute to the pathogenesis of lymphoma16.
7.4.3 Treatmenf and Prognosk of NHL
The treatrnent of lymphoma is detenined according to the grade or
claçsificatic?n of the disease at the time of diagnossq? Non-Hodgkin
lymphomas often demand multidisciplinary treatment regimes to achieve
optimal cure rates's. Aggressive lymphomas are rapidly growing tumors thus
are generally treated using aggressive chemotherapyl? This results in a high
percentage of durable, long-temi disease-free survival and with approximately
45 percent of patients being cured using a variety of chemotherapeutic
regimens. Unforhinately, a significant nurnber of patients with aggressive
lymphoma either fail to respond or eventually have disease relapse. Prognosis
then is poor and almost al1 patients in this category can be expected to die of
progressive lymphoma.
lndolent lymphomas are slow growing tumors and patients diagnosed
with this type of cancer can have median survivals of 7.5 to 9 years. For
patients with early stage disease. especially those under 40 years of age, local
radiation is the standard treatrnent? A standard treatment for advanced
indolent lymphomas has not yet k e n determined. Chemotherapy with one
drug or combinations of dnigs is effective in treating recurrences. Although
generally regarded as an indolent disease, slow growing tumors are
progressive and ultimately fatal. lndolent lymphomas that progress into more
aggressive types are generally more difficult to treat with standard therapies
than lymphomas that are aggressive at diagnosis.
Relapse following initial therapy confers a poor outlook with overall
survival of less than I O percent? The principal curative approach involves
aggressive chemotherapy in combination with radiation therapy. Autologous
bone marrow transplantation has also been considered as a form of primary
treatment. Such treatrnent requires further validation before it is acœpted as
standard practice21. In addition, allogeneic bone manow transplantation has
been performed on a small number of patients with resistant disease.
Unfortunately, this procedure is restricted due to the need for a matched donor
and hig h treatrnent related mortalip.
1.5 Tumor lmmunology
The discussion of tumor immunology will be categorized as follows: (1)
antigenic properties of transformed cells, (2) consequences of the growth of
malignant cells, (3) host immune responses to tumor cells, (4) tumor
immunosurveillance and (5) modulation of the immune system to recognize
and promote tumor eradication.
7 - 5 7 Tumor antigens
Malignant cells may express tumor antigens that can be recagnized by T
cells or antibodies. These fall into two major categories, which are tumor
specific antigens and turnor associated antigens. Tumor specific antigens
(TSA) are found only on tumor cells and therefore represent ideal targets for an
irnmunologic attack. Although the existence of TSA has been refened through
studies in animal models, their identification in human tumors has been ditficult
and thus the existence of these antigens remains a debated issue. In one of
the early studies, Prehn et al. induœd sarcomas in inbred strains of mice. by
painting mouse skin with the chernical carcinogen. methylcholanthren*.
Arising tumon were transplanted inducing growth and eventually killing the
new host, demonstrating the existence of antigens expressed exclusively by
tumor cells. In contrast, the injection of tumor cells into the original host
resulted in a specific immunologic rejection of the tumor, indicating the
presence of activated tumor antigen specific T cells. Altematively, irndiated
tumor cells used to immunize secondary syngeneic mice induced immune
responses that protected hosts from additional tumor transplants. One potential
example of a TSA found in human malignancy may be the specific lg idiotype
expressed by 6 cell lymphoma (see below pg. 22)
Tumor associated antigens (TAA) are found on both tumor cells and
normal cells. and rnost TAA identified to date represent differentiation antigens.
An example of the diversity of TAAs is seen in the phenotype of human
melanoma24. Melanome cells express highly immunogenic surface proteins
that can stimulate immune responses ri, vivo. Several of the melanoma
antigens isolated to date are also expressed within normal tissues. MAGE-1 is
expressed in testis, whereas tyrosinase, MART-1 and gplOO are present in
rnelanocytes and retniel pigment epithelium. When cancers from different sites
are studied, the expression of tyrosinase, MART-1 and gp100 appear lirnited to
melanoma, in contrast to MAGE-1. -2, -3 that are found in a variety of tumors24.
The best-characterized human TAAs are the on cofetal antigens 25.26.
These antigens are expressed during embryogenesis but are absent or very
difFicult to detect in normal adult tissue. The prototype antigen is
carcinoernbryonic antigen (CEA), which is greatly increased on human colon
cancers, breast cancers and non-small-cell lung can~ers2~. Assays for senim
CEA are used to monitor the spread of carcinoma or its recurrence after
primary treatment28-30. Moreover, human studies have suggested that antî-
turnor T cell immunity can be elicited to tumors expressing CE@.
Other types of TAAs are lymphoid-specific or differentiation antigens that
are expressed in normal cells reflecting distinct stages of differentiation. These
lymphoma-associated antigens may be aberrantly expressed on some tumor
cells. For example, malignant lymphomas ansing during a specific stage of 6
cell development may be diagnosed as derived from the 6 cell lineage by the
detection of surface markers characteristic for nomal pre-B cells. such as
CD10. Tumors arising from more mature B cells are characterired by the
presence of surface immunoglobulin3~.
1.5.2 The Immune System's Role In Tumongenesis
The concept of host immune surveillance, in which the immune system
provides the framework for recognition and eradication of developing
immunogenic tumor cells was first proposed by F. M. Bumet32. The immune
surveillance hypothesis has since stimulated rnany experimental studies al1
aimed at detennining whether the immune system has a general role in
controlling tumor growth. Under this model. al1 of the effector components of
the immune system potentially contribute to the eradication of tumor cells.
However. specific immune responses are classified into two types: humoral
and cellular irnmunity, rnediated by B and T cells. respecüvely.
1 - 5.2. ? B Cells and Antibody-Dependent Killriig
The development of monoclonal antibody technology by Kohler and
Milstein in 1975 resulted in the generation of reagents pemitting precise
characterization of ceIl surface antigens associated with B cell
differentiation33.34. The initial stages of B cell differentiation are independent of
antigens, but subsequent difFerentiation requires antigens and proœeds
optimally in the presence of T cellderived factors. Mature 6 cells eventually
arise that synthesize and express immunoglobulins on their surface. After
interacting with antigens and T cells, B cells clones differentiate into plasma
cells that produce multiple copies of a single antibody with the ability to bind a
particular antigen35-36.
There are two major mechanisms by which antibodies may mediate
tumor cell lysis. The first involves complernent-dependent IgG and IgM
antibodies37. These antibodies bind to antigenic sites on target cells and
promote attachment of complement components that create pores in the
target's cell membrane, resulting eventually in cell death due to loss of osmotic
and structural integrity. An alternative mechanism is antibodydependent
cellular cytotoxicity (ADCC) in which antibodies, usually of the IgG class, act as
bridges between tumor targets and effector cells that express Fc receptors,
particularly natural killer (NK) cells, macrophages and granulocytes. ADCC is a
more efficient lytic mechanism than cornplement-mediated cytotoxicity,
requiring fewer antibody molecules per cell for a cytotoxic response37.
Antibodies that bind to human TAA have been found in sera from
patients with various cancers, such as melanorna and renal ceIl cancer?
Approximately 10 percent of patients with melanorna have antibodies that bind
to gp75, which is a melanosorne-rnembraneassociated protein found in both
melanomas and normal rnelanocytes38. Antibodies may also be fomied against
antigens induced by EBV. herpes sirnplex virus 2 (HSV-2) and papillomavirus.
In addition to their role as antibody producing cells. B cells also play a
role in the immune system as antigen presenting cells (APC) in a secondary
immune response. 6 cells can take up extracellular antigens via lg molecules
expressed on the cell surface. Sinœ B cells express both MHC class I and
class II molecules, these antigens can be processed and presented for the
activation of CD4 or CD8 T cells. However, they do not express costirnulatory
molecules required for the activation of naïve T cells, thus B cells can not
function as APC in a prirnary immune response.
7.5.2.2 T Cell Mediated Cytotoxicity
T lymphocyte progenitors arise in bone marrow, migrate to the thymus
where they proliferate and differentiate into mature CD4 and CD8 thymocytes.
Part of the maturation process involves selection of thymocytes expressing
functional T cell receptor ( K R ) that can interact with either MHC class I or
class II molecule but do not recognize self peptides. After selection, mature
thymocytes expressing productive TCR and either CD4 or CD8 CO-receptor
migrate from the thymus and circulate in the penphery as mature T cells. Over
90 percent of thymocytes generated in the thymus are eliminated during the
maturation process. Mature T cells mediate cellular immune responses with the
specifïcity of interaction against different antigens being mediated by the T cell
receptor. In contrast to B cells, which can recognize antigens in the fluid phase,
T cells recognize antigenic peptides cornplexed to major histocornpatibility
complex (MHC) class I or class II molecules presented on the surface of
antigen presenting cellsJ?
T cells play important roles in host defenses against tumor and foreign
antigens (see illustration on pg.16). There are two T cell subsets: CD4 helper T
cells mediate their effect by the secretion of cytokines to acüvate other effector
cells, while CD8 cytotoxic T cells also secrete cytokines but primarily mediate
their effect by direct lysis of tumor cells4Oe41. Extracellular derived peptide
antigens are presented to CD4 T cells by antigen presenting cells in the context
of peptide-MHC dass II complexes. CD4 T cells differentiate into (wo types of
effector cells. TH1 cells produœ INF-y and 11-2 cytokines, which contribute to
the activation of macrophages and induction of IgG antibodies. TH2 c e k
secrete 11-4, 11-5 and IL-IO, which initiate humoral immune responses by
activating naïve antigen-specific B cells to produce IgM antibodies. CD8 T cells
recognize peptides derived from proteins that are processed within the
cytoplasm of the cells and are presented by MHC class I molecules. These T
cells differentiate into cytotoxic T cells that kill foreign antigen presenting cells.
The precise mechanism of T cell mediated cytotoxicity is not known, but
cytotoxii
TNF recepto
Fas band
r
:as
virus-infected cell bacteria
macrophage
antigen-specific B cell --
O " 1 I F - i l F I L I l I L ( IL-. 1 Gmnzyrnes TNF-P TNF-a TN F-P IL-5 GM-CSF Fas ligand TNF-a CD40 ligand (IL-2) 11-1 0 CD40 ligand TGF-B
Fas ligand
CD4 and CD8 T cells in host defense: The three main types of effector T cells pro- duce distinct sets of molecules
Source: Imrnunobiology, Janeway CA and Travers P;Garland Publishing Inc, 1997
perforins, lym p hotoxins, direct membrane interactions and induction of
apoptosis by Fas-Fas ligand pathway have been proposed4*+.
The role of CTLs in rejecüng tumor cells has been demonstrated in
numerous studies. For example, in the case of melanoma patients, antitumor
CTLs have been shown to recognize and lyse cells expressing the MEGA-1
TAA presented by HLA-Al'? Furthemore. in vitro studies have shown that
human sarcomas, renal cell cancers, breast cancers, ovarian cancers, head
and neck cancers as well as HTLV-I induced leukemias are lysed by T cells
that recognize TAA in the context of MHC antigens.
Other cell types, such as natural killer (NK) cells and macrophages, also
play significant roles in tumor immunology. NU cells represent a firçt Iine of host
defense against the proliferation of transforrned cells at both primary and
metastatic sites. They provide an efiector rnechanism recruited by T cells to
supplement specific antitumor responses. These cells are large granular
lymphocytes which have been shown by in vitro studies to possess the ability
to lyse certain tumor cell lines in vit#. Cytolysis by NK cells is mediated by
the release of cytotoxic factors and the use of perforins that puncture holes in
the target cells membraned? Like NK cells, macrophages are also important in
tumor immunology not only as potential effector cells to mediate tumor lysis but
also as antigen presenting cells to initiate antitumor immune responses. The
rnechanisms by which macrophages recognize tumor cells and mediate their
lysis are not defined, but activated macrophages bind to and lyse transfomied
cells in marked preference to normal cells. Intercellular transfer of lysosomal
products, release of neutral proteinases, TNFu and 11-2 al1 rnay contnbute to
macrophage mediated cytotoxicity, depending on the macrophage-activating
factors (MAK) responsible for activation48.
1.5.3 Mechanisms by Which lurnor Celk Escape an Immune Response
Many potential mechanisms that rnay allow tumor cells to escape
immune rejection by the host have been identified49eso. One of the hypotheses
is the lack of antigen expression by tumor cells, because antigens rnay not
appear at the cell surface or rnay not be adequately processed or presented in
the context of appropriate MHC determinants. Although most cells within a
tumor express potentially immunogenic antigens, a subpopuIation of tumor
cells rnay lose expression of these antigensS1. This loss rnay result in a
reduction or elimination of tumor antigens that can be recognized by the
immune system resulting in the outgrowth of a variant subpopulation of tumor
cells that become the dominant population.
For T cells to recognize an antigen, it must be degraded into small
peptides that are transported to the endoplasmic reticulum and presented by
MHC class I molecules on the cell surface or via endosomes and presented by
class II molecules. Studies have shown that some tumor cells have defective
antigen processing rnachinery, with the result that class I MHC molecubs are
not loaded with peptides and transported to the cell surface5? In addition, MHC
expression rnay be down regulated and in such circumstances even potentially
highly immunogenic tumor antigens may not be presented to the immune
s ysterns?
In addition to the first signal received via the TCR through its interaction
with MHC-peptide cornplex. T cells require a second signal. which is provided
by costimulatory molecules 87-1 and 87-2 (CD80 and CD86)54. These
molecules interact with CD28 molecules expressed on T cells inducing its full
activity. TCR ligation by peptides in the absence of the second signal results in
T cell anergy or a state of non-responsiveness. Since most tumor cells do not
express costimulatory molecules required for the activation of naïve T cells, it
has been hypothesized that tumor antigen specific T cells are rendered non-
responsive upon encountering TAA expressed on malignant ceIls5557.
There are many other mechanisms by which tumor cells may non-
specifically interfere with the induction of immunity in the host. Some tumors
can produce a vaMy of soluble factors such as transforrning growth factor4
(TGF-B) that can affect immune effectors. TGF-B has antiproliferative effeds
on a wide variety of cell types, including T and B lymphocytes. It also
suppresses the production of rnost lymphokines and reduces the cellular
expression of MHC class II mole cul es^. In addition to TGF-B. 11-10 may
prevent the difFerentiation of monocytes into dendntic cells or induce
suppression of MHC class II expression and consequent loss of the accessory
functions of macrophages and dendritic cells5? In fact, both TGF-13 and 11-10
are expressed by lymphoma c ~ I I s ~ O - ~ ~ .
1.6 lmmunotherapy for Non-Hodgkin Lymphorna (NHL)
The majority of NHL patients are not cured with conventional therapy,
although signifiant advances have been made in the treatrnent of NHL62. Only
20-50 percent of patients with intermediate- and high-grade lyrnphoma
experience prolonged diseasefree survival and nearly al1 patients with low-
grade NHL relapse63. Based on the overall treatment results, rising incidence
rates that contribute to a slight increase in mortality rate and the reality that
chemotherapy cannot be further increased due to unacceptable toxicity , other
strategies for treatrnent of NHLs, including immunotherapy, have been
increasingly investigated. Cancer immunotherapy is a mode of cancer
treatment that stimulates the natural host immune system to destroy tumor
cells thereby mediating cancer regression, in contrast with conventional
therapies, which act by directly attacking, or killing tumor cells. A better
understanding of the factors involved in the induction of an immune response
and the biology of tumor progression have created new interest in
immunotherapy for the treatment of many malignancies including lymphoma. In
addition, technological advances that permit the isolation of lymphocyte
subpopulations, identification and purification of tumor antigens, growth of
selected antigen specific T cells, and amplification of immune responses with
cytokines have created new approaches for the development of
immunotherapeutic treatment of cancer.
1.6.1 Early Approeches of Lymphoma lmmunotherapy
Early cancer immunotherapeutic approaches used recombinant
cytokines and immune celis, in particular lAK cells and TIL, to increase
immune responses. lAK cells are a phenotypically heterogeneous population
of cells with the major effector cells being NK cells. Tumor-infiltrating
lymphocytes (TILs) can be isolated directly from tumors and expanded in vitm
with 11-2. Murine studies showed that treatment of mice with high doses of 11-2
alone or in conjunction with LAK cells could mediate the regression of
established liver and lung metastasis, using a variety of tumor rnodels. These
studies fostered the development of human clinical protocols. In clinical trials
based on this approach, Ki vitro treatment of peripheral blood cells with 11-2
induces large numben of LAK cells but did not significantly increase specific
killing of autologous lymphorna cells when used as a vaccine in vivo64. ln
clinical trials involving TlLs, TlLs from more than 50 patients with rnalignant
melanoma were expanded in culture with IL-2. These in vitro expanded cells
appeared to have specific cytotoxic reactivity against autologous tumor cells
with tumor regression being reported in about thirty-eight percent of patients65.
In addition to LAK and TIL, treatment with monoclonal antibodies (mAbs)
directed against turnor surface antigens provided a rational therapeutic
approach for some types of cancers. For example, antibodies specific for CD20
rnolecule, which is expressed on B cells have been shown to produce clinical
responses when administered to B ceIl lymphorna patients who have previously
failed chemotherapya6. Conjugation of cytotoxic drugs, toxins, or radioisotopes
to the antibody are other approaches used to deliver a lethal hit directly to
tumor cells without requiring the participation of host effector ce l ls~ . Studies
utilizing radiolabeled forrns of anti-CD20 have demonstrated potentially curative
effects and have also been tested for clinical uses? Evidence fmm other
cancers such as colon or breast cancer have also shown reduction in mortality
and support a rote of monoclonal antibodies in cancer treatment.
7.6.2 Current Concepts of Lymphoma lmmunotherapy
Current strategies for irnmunotherapy of 6 cell NHL are focused on
approaches to induce effective antilymphoma specific T ceIl mediated immune
responses. Two main strategies in B cell lymphoma immunotherapy are being
explored. First, since most B cell lymphomas are characterized by the clonal
expansion of a single B cell with a unique rearrangernent of both the heavy and
light immunoglobulin genes, their specific B cell receptors (idiotype, Id) are not
only clonal markers but also tumor specific antigens to which an immune
response might be directed68. Vaccination with either Id bound to keyhole
limpet hemacyanin (KLH) alone or in combination with GM-CSF has shown
efficacy against established tumors in murine models and to some extent. in
humans69-70. However, there are several disadvantages to this approach. For
example, it has been demonstrated that clonal expansion in low grade NHL
could potentially lead to loss of recognition by the effector cells introduced by
Id-vaccines if the antigen components are altered. In addition, production of Id-
vaccines is highly labor intensive and cosUy, and thus would not be practical as
a general treatrnent for NHL6gS70.
A second strategy for B cell lymphoma immunotherapy is based on
knowledge that normal 6 cells do not express costirnulatory molecules and
therefore can not activate naïve T cells. However, 8 cells can function as APC
for the activation of experienced T cells. Consequently malignant B cells could
potentially function as APC, although clinically significant immune responses in
patients with B cell lymphoma are not readily detected. A second
immunotherapeutic strategy for the treatment of B cell NHL has attempted ta
enhance the ability of B lymphoma cells to function as APC by transfecting
malig nant cell Iines with 87 wstimulatory molecules required for the activation
of primary T cell responses71. In addition to transfection of primary B cell
lymphoma cells, it is possible to upregulate the expression of 67 molecules.
One approach for the induction of 87-1 and 8-2 molecules in normal and
malignant B cells is by cross-linking of CD40 molecules necessary for B cell
proliferation and activation72. This approach has the advantage that co-
stimulation and adhesion as well as antigen presentation is enhanced. CD40-
activated lymphoma celfs can be used to induce T cell response either as a
vaccine or for in vitro stimulation of T cells. These in vitro activated T cells are
capable of mediating effedor responses against the CWO-activated follicular
lymphoma. Once primed, these T cells should rewgnize the wild-type tumor
celI~73~74.
Several animal tumor model systems support the relevance of exploring
87 CO-stimulatory pathway in the generation of tumor specific immune
responses. In some experimental models an effective antitumor immune
response was initiated in animals irnmunized with tumor cells genetically
altered to express 87 molecules~=75. When 87 is transfected into a highly
malignant melanoma ceIl Iine and then implanted into an immunocompetent
mouse, tumon initially grew but then completely regressed over three to four
weeks. Further, these animals were protected against a subseqwnt
rechallenge with the parental cell line. However, expression of these molecules
has not proven to be sufficient in al1 tumor models76. In clinical trials, a breast
cancer cell line was transduced with B7 to vaccinate patients in combination
with GM-CSF as an adjuvantT
In addition to the above described approaches, transfection of various
cytokine genes such as GM-CSF or 11-2 that assist in host immune cell
recruitment and activation, into tumor cells has also been assessed with the
idea that providing cytokines to cytotoxic T cells would bypass the need for
CD4 helper T cells78.79.
1.7 Denditic Cells as Professional Antigen Presenting Cells
Studies carried out more than 20 year ago led to the identification of
dendritic cells (DC). Since DCs have been shown to be particularly effective
and potent APCs, their use in cancer immunotherapy protocols is emerging as
a viable alternative to radiation or chemotherapy. This new strategy of
enhancing the recognition of tumor cells by the immune system is based on the
presentation of tumor antigens by dendritic cells80. Theoretically, autologous
dendritic cells presenting tumor antigens may be used as vaccines to trigger an
antitumor specific T cell response.
As professional antigen presenting celk (APC), dendntic cells (DC) are
critical for the initiation of ceIl-mediated immune responses. DCs express high
levels of MHC class I and class II molecuies. and therefore have the
exceptional ability to present class I and class II restricted peptides and
activate either CD8 or CD4 T cellsatla. They also express the additional
accessory molecules required for T cell activation, including CD40,
costirnulatory molecules B7- 1 /CD80 and B7-2/CD86, intercellular ad hesion
molecules I CAM4 /CDS, ICAM-3/CD50, Iyrnp hocyte function-associated
proteins LFA-IdCD1 1 a, LFA03/CD58 and Mac-VCDl 1 b, CD1 1 c and HSA
molecules56~83. DCs are specialized to take up, process and present antigen.
and have the capacity to stimulate naïve T cells as part of a primary immune
responseV
1.7.1 Ongin and Generation of Cultured Dendntic Cells
DCs were first recognized by Steinman and Cohn in 1973. They were
characterized on the basis of their morphology. low density and migratory
capacity. DCs circulate from pen'pheral tissues to lymphoid organs, where they
corne into contact with mature T cefls85. Mernbers of the DC family include
interstitial DCs which are found in mainly al1 tissues but are primarily
concentrated along epithelial and body cavity surface, the Langerhan's cefk
(LCs) of the skin, and the interdigitating cells of the thymusm.
DCs differentiate from bone marrow derived CD34+ granulocyte-
macrophage precursor ce l l~8~. Immature DC precursors exit the bone manow
and circulate via the bloodstream to reach peripheral tissues. Immature DCs
are highly efficient at antigen uptake and proœssing but do not present these
processed peptides at the cell surface. Thus, at this early stage of
development, they are unable to activate T ~ells88~". Mature DCs migrate
through lymphatic vessels to T cell areas of lymph nodes, where they forrn
clusters with T cells, to maximize encounters with rare antigen reactive
clonesg0. DCs maturation results in the loss of their ability to uptake antigens,
but their capacity to present antigens and stimulate T cells is greatly increased.
Maturation also coincides with the upregulated expression of adhesion and CO-
stimulatory molecules. Ligation of CD40 on the surface of activated DCs by
CD40 ligand expressed by T cells induces further elevated expression of 87-1
and B7-2 molecules. In addition, DCs initiate the secretion of high levels of IL-
12, which supports the development of Tnl CD4 T cells and the maturation of
CTLs91. DCs also enhance the proliferation and maturation of B cells either
directly or by stimulating CD4 T cells to produce cytokinesgz.
In addition to DCs derived from CD34' myeloid precursors, lymphoid
derived DC subpopulations have also been identifiedg? Lymphoid DC
precursors have little or no capacity to fonn myeloid cells, indicating that they
are of lymphoid lineage rather than a myeloid-related lineage. The
developrnent of thymic DCs and thymocytes is Iinked via a common precursor
at an early stage of thymocyte developmentw. Thymic DCs induce the death of
the developing, serreacting T cells, rather than presenting foreign antigens.
These DCs, as well as a subgroup of DCs in spleen and lymph nodes, express
some markers normally expressed on lymphoid cells.
DC cultures rnay be established using BM. peripheral blood and spleen
cells or early thymic precursor ceIl. DCs may be expanded in vitro by
supplementing spleen or bone marrow derived cell cultures with GM-CSF and
IL4, although lymphoid-related DCs do not require GM-CSFS3.95w% The
development of DCs in culture is rapid, with expanded populations reaching
DCs purity of more than 85%. Cultured DCs are similar to freshly isolated DCs
in their expression of surface antigens. their ability to stimulate allogeneic T
cells in vitro and to proœsses and present antigens.
7.7.2. Role of Dendntic Cells in T ce// Activation
Antigens that are captured by antigen presenting cells (APC) such as
dendritic cells (DC), become enclosed within membrane-lined vesicles,
undergo a series of alterations, which includes cleavage of antigens into
peptides by denaturation and proteolityc digestiong7(see illistration on pg.28)
The resulting peptides then associate with major histocompatibility complex
(MHC) proteins and are transported to the APC cell surface. There are two
different classes of MHC proteins, each of which is recognized by one of the
two major subpopulations of T lymphocytes. Class I MHC molecules are
expressed by
for Ag "Or (TCR) / & ~
l MHC
C
Activation of the naive T cells in the proces of immune response: Interaction between APC and T cells
Adapted from Medical Immunology, Stites DP, Ten Al and Parslow TG, 1997
virtually al1 somatic cell types and are used to present antigen to CD8 T cells.
Thus, almost any cell can present antigen to cytotoxic T cells and serve as the
target of a cytotoxic response. Class II MHC molecules are expressed by DCs,
macrophages and B cells and are necessary for antigen presentation to CD4-
helper T cellsg*. Since helper cell activation is important for immune responses,
class Il-bearing APCs play a crucial role in mediating such responses. T cells
express a number of adhesion molecules, such as leukocyte fundional
antigen-1 (LFA-1) and CD2 that bind ligands on the surface of DCs. and
stabilize T cells and DCs interactions.
Despite their complexity, the signals delivered by TCRs are not sufficient
to fully activate T cells. T cell activation requires a second signal delivered by
costimulatory molecules8J. CD28 is a costimulatory molecule expressed on al1
CD4 T cells and a number of CD8 T cells. CD28 binds the cell surface
molecule B7 expressed on DC, and together fully activate T cells to proliferate,
differentiate and perfonn their effector functionsg? Activation of helper T cells
leads to the production of cytokines that promote cellular and humoral immune
responses, whereas activation of tumor antigen specific T cells results in the
lysis of the tumor antigen-beanng ~ e l l s ~ ~ ~ 0 2 .
1.8 Dendritic Cells and Antitumor lmmunity
The rote of DCs in T cell activation and the demonstrated ability to
manipulate DCs to present tumor antigens has generated great interest in the
use of DCs as cancer vaccines and to their recent evaluation in pilot clinical
trials. One of the most promising areas of DCs research has been the
development of OC-based strategies for enhancing immune responses against
tumors.
7.8.7 Dendritic Cell Vaccination in Murine Models: Preclinical Studies
The induction of anti-tumor immune responses by the injection of
antigen-loaded DCs has been studied extensively in animals. Antigen-loading
of DCs can be achieved through pulsing of DCs in vitro with protein or peptide,
tumor cell lysates, tumor-derived RNA ennched for tumor specific mRNA, or
transfection of DCs with cDNA encoding tumor antigensf03-107.
Protein loading of DCs is accomplished by CO-culturing of DC and protein
preparations in the presence of a lipid carrier such as DOTAPW The protein is
taken into the DC, processed intracellulary, and class I and class II restncted
peptide fragments presented at the DC surface, for the activation of CD4 and
CD8 T cells. DCs rnay also be pulsed with MHC class 1 or class II restricted
peptides to enhance either CD8 or CD4 T cell responseslo? Numerous studies
have clearly established the effectiveness of tumor antigen or peptide pulsed
DCs to induce potent antitumor immune responses and to provide protective
tumor immunity from a subsequent lethal tumor challenge104~108-110.
The approach of using tumor lysate-pulsed DCs offers the potential
advantage of increasing T cell immune response to tumor-associated antigens
that may not be obtained by pulsing DCs with single tumor peptideslo? Pulsing
DCs with tumor cell lysates avoids the problem of defining TAAs and facilitates
the presentation of a large anay of antigens derived from a heterogeneous
population of tumor cells thus reducing the risk of %scape mutationsn. In a
recent study, Fields et al. demonstrated that immunization of healthy mice
subcutaneously (SC. ) with tumor cell lysate-pulsed DCs provided strong
protection against a subsequent challenge with a lethal dose of the viable
parental tumor cells and rnediated reduction in the number of experimentally
established pulmonary metastasesW
The amount and purity of tumor tissue used for the isolation of proteins,
peptides or turnor-lysates is often insufficient for vaccination. Recently
investigators have focused on discovering more effective methods of delivering
tumor antigens to DCs, not requiring knowledge of the relevant tumor peptide
sequences. A potential advantage of using RNA rather then proteins or
peptides as a source of tumor antigens is that sufficient amounts of antigen can
be generated from very small amounts of tumor tissue using PCR amplification
techniques. Boczkowski et al. demonstrated that rnunne DCs could be
efficientiy transfected with RNA encoding the chicken ovalbumin (OVA) antigen
and used as a potent APC for the induction of T cells mediated responses'o3.
In addition, the ability to transfect human DCs with CEA RNA and the induction
of CTL response with these DCs in vitro has been also rep0rtedlll1~~2. cDNAs
cloned in bacterial plasmids can also be used for the production of RNA. by in
vitro transcription, allowing the applicability of RNA-based vaccines to patients
with very small tumors. Another promising strategy for DC loading wlh tumor
antigens is direct transfection with cDNA libraries derived from tumor cellsfl3.
Although DCs pulsed with cDNA have been shown to be effective in reversing
tumor progression. the generation of cDNA expression libraries is labor-
intensive and therefore pulsing DCs with mRNA will likely prove to be more
advantageousl14. In addlion to the above strategies. the fusion of tumor celfs
to DCs has also proven to be an efficient method for presenting tumor antigens
and inducing an anti tumor immune respon~e115*'~6.
Thus. various shidies h mouse tumor rnodels suggest that DCs pulsed
with tumor antigens in vitm and administrated to murine rnodels may initiate an
effective antitumor immune response. Such immunity protects animais against
inoculation with tumor cells and often causes suppression of tumor cell growth
and even regression of established tumor~105~117*118.
7.8.2 Dendntic Ce// Vaccination for Human Cancer Clinical Trials
Based on the results of animal studies, several investigators have
speculated that the administration of manipulated DCs may also be effective in
generating cellular immunity to tumors in humansllg. The development of
methods for obtaining large numbers of human DCs has made testing this
hypothesis feasible. Clinical trials of OC vaccination are based on two general
methods including purification of immature DCs precunon from peripheral
blood, and in vitm differentiation of DCs from peripheral blood monocytes or
CD34+ hematopoietic progenitor cells120- The potential of isolated human DCs
to prime tumor antigen specific T cells in vitm has been reported repeatedfy.
7.8.2.1 Non-Hodgkin Lymphoma
Hsu et al. first reported the intravenous administration of autologous DCs
pulsed rii vitro with idiotypic proîeins into patients for the treatrnent of NHL121.
In that study, peripheral bloodderived DCs from four patients with low-grade
lymphoma were pulsed with KLH or tumorderived idiotype protein and infused
intravenously. Each patient received several DC infusions with a final given five
to six months later. The treatment approach resutted in cellular protiferative
responses specific to the patient's own idiotypic protein, as welf as humoral and
cellular responses to the control KLH protein. In addition, clinical responses
were also found- Complete remission of pericardial and periaortic masses were
seen in one patient, who remained in remission for 42 months. In the case of
another patient, polymerase chain reaction (PCR) analysis of blood and bone
marrow for tumor specific DNA became negative and complete remission was
found for more than 36 months after vaccination. The two other patients
exhibited stabilization of disease. Wih respect to safety of idiotype-pulsed DC
vaccinations in patients with NHL, no delay or long-terni toxicity was identified
in this study.
A similar approach being evaluated in clinical studies. demonstrated that
vaccination with DCs pulsed with idiotype protein directly coupled to KLH may
improve immunogenicity and tumor protection in a murine lymphorna model
when compared with vaccination using idiotype-pulsed DCsl*.
1.8.2.2 Malignant Melanoma
The characterizaüon of melanoma antigens recognized by T cells lead to
the initiation of a variety of melanoma vaccine trials, using antigen pulsed APC.
One of the first studies, done by Mukherji and colleagues, involved the
treatment of patients with advanced melanoma with GM-CSF expanded and
MAGE-1 MHC class 1-restricted peptide pulsed monocytes. Following
intradermal injection, induction of melanoma-reactive CTL responses was
detected in patients with advanced melanoma. although no significant
therapeutic responses were seen123.
In a similar study by Nestle and colleagues, DCs were pulsed with either
a cocktail of HLA class I restricted peptides including gp100, MART-1,
tyrosinase, MAGE-1 and MAGE-3 peptides or with tumor lysates, if patient's
HLA haplotype was not HLA class I specific and was inappropriate for peptide
loadingl24. To induce CD4 T cell response and to improve immunogenicity KLH
was included during antigen pulsing. Patients received 6-10 intralymphatic
injection, with only minor toxicity noted, which was limited at the injection site.
Regression of tumors was seen in five of the sixteen patients, including two
patients that received only tumor lysate pulsed DCs, applicable to cancers
lacking defined tumor antigensl*?
1.8.2.3 Mulftiple Myeloma
Multiple myeloma is a late B cell disorder involving malignant
transformations of a mature, antibody secreting B dl. The lg idiotype
expressed by the transfonned cells (M protein) represents a tumor specific
antigen, which may be used as a target antigen by host T cells. Preclinical
studies have dernonstrated that host T cells could recognize the idiotypic
deteminants of myeloma proteins (M protein)W Additional studies assessed
the therapeutic benefits of injecting rnyeloma patients with penpheral blood
derived DCs pulsed with M protein after the patients had received high dose
chernotherapy, total body irradiation and autologous penpheral blood stem cell
transplantation. DCs injection was followed by five subcutaneous booster
injections of idiotype coupled to KLH. ldiotype specific T cell proliferation and
cytolytic responses were induced but it was observed only in a group of
patients who achieved complete remission prior to vaccination. ln a similar
study carried out by Wen et al, results demonstrated the induction of anti-
idiotypic T cell response but no significant clinical responses, respectively'27.
1.8.2.4 Other Cancers
Prostate cancer is another type of malignancy that may be amenable to
immunotherapy, since severai prostate tissue associated antigens are now
being characterized. These include prostatic alkaline phosphatase (PAP),
prostate specific membrane antigen (PSMA) and prostate-specific antigen
(PSA). Valone et al. used PAP protein to pulse penpheral blood derived DCs.
These cells were injected into twelve patients, which induœd T cell proliferative
responses to PAP protein128. However. when monocyte derived DCs were
pulsed with PSMA and then administrated to eighty-two patients. only two of
the patients mounted T cell responses against PSMA peptides129JJo.
DC based immunotherapy has also been assessed for the treatrnent of
renal carcinoma. In one study monocyte derived DCs were pulsed with KLH
and autologous tumor cell l ysa te~~3~ . Only two patients completed the
treatment with one exhibiting reduction of tumor rnass and proliferative
responses of peripheral blood monocytes against tumor ceIl lysates-
Although vanous studies in murine model systerns and human clinical
trials have demonstrated the in vivo activity of antigen-pulsed DCs to function
as vaccines against tumor challenges, the animal model systems used are
artificial. In rnost experiments, genes encading foreign proteins have been
introduced into tumor cells to serve as model tumors antigens, making them
highly irnmunogenic. In contrast, most spontaneously arising human cancers
are nonimmunogenic. Moreover, in most transplantable tumor models, animals
with healthy immune systems are useds? However. rnalignancies usually
develop spontaneously as a result of the expression of a specific genetic
abrnormality. Clona1 expression and the accumulation of genetic abnormalities
leads to the generation of a heterogenous population of tumor cells, some of
which have the ability to escape host immune rejection.
7.8.3 Future Prospects for DC Based Cancer Vaccines
Vaccination of cancer patients with antigen pulsed DCs illustrates the
powerful antigen-presenting capacity of DCs for induction of an antitumor-
specific immune response. The general safety of DC vaccination in patients
and therapeutic potential of this approach have been amply demonstrated.
Several variables should be considered in the future studies:
1) Since both peripheral blood and monocyte derived DCs have shown
eficiency but have never been compared the optimal source of DCs for
clinical use must be determined;
2) It remains to be determined whether DCs pulsed with autologous tumor cell
antigens induce the maximum antitumor response or whether more effort
should be directed at the identification of tumor specific antigens that would
serve as a general antigen to induce an anti tumor response in al1 patients
with a particular malignancy;
3) The sufficient amount of DCs and the most efficient methods for antigen
loading of DCs that will permit the presentation of large array of tumor
antigens, and
4)- The optimal route and frequency of administration that is sufficient for
induction of tumor specific immune responses.
Immune responses may be further enhanced by the development of
techniques that may improve antigen presenting properties of tumor cells and
to inactivate the immunosuppressive factors produced by tumor cells-
1.9 HOX11 Transgenic Mice
1.9.1 Homeobox Genes in Hematopoiesis and Hematologic Malignancies
Horneobox genes are a family of genes containing the conserved
domain of 61-amino acid132. Homeobox genes are classifed into class I HOX
genes and divergent homeobox genes. Thirty-eight known class I mammalian
HOX genes are arranged in four clusters designated HOX A, BI C and Dl while
more than 200 divergent horneobox genes are located outside the HOX loci
often without forming clusters133*134.
The products of homeodomain containing genes, homeoproteins,
function as transcription factors and play an important role in forming the body
structure during fetal developmentr3? In addition. many of these genes are
expressed in hematopoietic cells and in leukemia cel ls136~~~~. Genetic alteration
and aberrant expression of homeobox genes have been reported in human
leukemias. In early studies hurnan leukemia cell lines were used to determine
the expression pattern of HOX genes138. Subsequent studies used normal
peripheral blood and bone marrow cells to determine which HOX genes are
expressed during normal hematopoiesisl3? Results from these studies showed
general preference of the HOX genes from each cluster in their expression
pattern. The genes of HOX A, 6 and C clusters are predominantly expressed in
rnyelornonocytic, erythroid and lymphoid lineage, respectively140. However,
HOX genes of each cluster play a role in not only a single lineage but also
hernatopoietic cells. For example. Care et al. showed that HOX 6 cluster genes
are expressed in PHA activated T cells, while Petrini et al. demonstrated the
expression of several HOX B locus genes in lymphoid cell lines with a natural
killer phenotype141-142.
As with class I HOX genes. divergent homeobox genes are also
expressed in normal and malignant hematopoietic cells143. One of the genes
that belong to this family is the HOX11 gene. HOXI 1 was originally isolated
from the breakpoint of the nonrandom t(l O;l4)(qZ4;q 1 1) chromosome
translocation, which is found in the malignant cells of 5% of patients with T cell
acute lymphoblastic leukemias (T-ALL)lun14? Another group reported that
thirty-three percent of pediatnc T-ALL patients showed overexpression of the
HOXll gene in their leukemic blast cells. although normal T cells did not
express HOXI1 as detennined by RT-PCRW Sirnilar HOX11 rearrangements
have been also seen in some cases of B Iineage malignancy.
HOX11 is nomally expressed during murine ernbryonic development in
the surface ectodem, certain brachial arches, as well as within cranial nerves
innervating these structures147-149. Human Iiver and CD34' bone marrow cells
show HOXlI expression, but it does not appear to be expressed in normal T
cells, although one report suggests that mature T cells may express HOX11150-
152. Expression of HOX11 is also found in the splanchnic mesoderm. which
ultimately gives rise to the spleen. It is interesting to note that in HOX11
deficient mica, spleen formation is inliated during early embryogenesis, which
is followed by atrophy of the organ due to apoptosis. These miœ are norrnal at
birth but show no spleen thus indicating that HOX11 may contribute to the
survival of the primordial cells, which develop into the spleen147.
1.9.2 Production of HOXl1 Ti-ansgenic Mice
Transgenic technology offers possibillies for generating precise animal
models for human genetic disease. Rapid generation tirne and the ease of
maintaining inbred strains make mouse models useful mammalian genetic
systems. One of the beneficial applications of this technology has been the
study of the behavior of overexpressed or ectopically expressed oncogenes.
While transgenic animals bear the introduced oncogenes in every tissue.
expression of that gene may either be widespread or directed to a particular
cell Iineage, depending upon the regulatory sequences chosen.
Much work has been done in Our laboratory to generate transgenic mice
for characterizing the oncogenic potential of HOX11. We have developed a
HOXll transgenic mouse modal for human lymphoma that may be more
consistent with spontaneous lymphoma growth found in patients. The
oncogenic potential of HOX11 was assessed by placing the human HOX11
cDNA under the transcriptional control of munne immunoglobulin heavy chain
(IgH) prornoter and enhancer sequences with expression directed in B
lymphocytes. The transgene (IgHp -HOXI 1 ) was injected into pronuclei of a
fertilized ovum and three lines, C2, C5 and D l 1 were obtained. Each line
contained approximately 2 4 copies of the transgene integrated in head-to-tail
orientation. Histopathologic analyses showed that low nurnbers of HOX11
transgenic mice develop lymphoid hyperplasia dun'ng their first year of life and
85 percent of mice developed lymphorna in the second year of life 1%.
7.9.3 Lymphoma Development in HOXl1 Mice
Detailed analyses confimed that HOXl 1 transgenic mice developed
mature B cell lymphomas153. Assessment of the effects of HOXl l transgene
expression on lymphopoiesis indicated that low level expression of HOX11 in
B-cells resulted in no significant effects on normal lymphopoiesis. However. al1
HOXl1 transgenic rnice died during their second year of life with the average
age of death being 14 to 15 month. The lymphomas were predominantly
detected in spleen with progression to an aggressive phenotype characterized
by metastasis to the lymph nodes. thymus. liver, lungs. kidney and pancreas.
The B cell origin of the lymphomas was confirmed by analyzing thymuses
infiltrated with lymphoma cells. Since the lymphoma cells expressed both IgM
and IgD. it is most likely they are mature B cells. Furthemore. the injection of
SClD mice with spleen cells obtained from HOXI 1 transgenic mice confimed
the malignant phenotype of these cells- This study provided the first direct
evidence that expression of HOX11 leads to rnalignant transformation- The
long latency period suggests that other genetic abnonnalities in conjunction
with ectopic expression of HOX11 in lymphoma cells are required for the
development of lymphomas. The course of lyrnphomagenesis in tiûX11
transgenic mice is similar to a subtype of human, indolent nonfollicular
lymphoma temed splenic marginal zone lymphoma.
Illustration 1. Tissue sections from HOXlt transgenic mice stained with
hematoxylin and eosin showing lyrnphoma.
(A) HOX11 transgenic spleen (B) Lymphoma cells x 200 in spleen (C)
Thymus (D) Lung (E) Liver (F) Kidney
For example, the lymphocytes in HOX11 hyperplastic spleens are small and
show maturation to plasma cells. Progression to lymphoma is associated with
enlarged marginal zones, early marginal zone lymphorna and the appearance
of large lymphoma cells. Therefore. HOX11 transgenic mice provide a clinically
relevant model in which to assess novel approaches for the treatrnent of
indolent nonfollicular 8 cell lymphoma-
1 -1 0 Study Rationale
Making up about 5% of al1 cancers and 85% of al1 lymphomas. non-
Hodgkin lymphoma is a significant cause of morbidity and mortality'w. It is
primarily seen in older individuals and dMded into B and T celk lymphoma by
origin. 8 cell tumors are most common and have better prognosis than T cell
tumors. Lyrnphomas may be classified into indolent and aggressive tumors.
Conventional treatments, such as radiation or chemotherapy are most effective
against aggressive lymphoma. In cases where patients are not cured. they
generally have a short life span. Patients with tumors that grow slowly may
have a much better prognosis. However, some indolent lymphomas may
transform into aggressive types, which are generally more difficult to treat with
standard therapies.
In addition to killing tumor cells, conventional treatments cause damage
in normal cells and produce side effeds, and often do not result in cures.
Based on these data, several different approaches to improve cure rate and
survival are being investigated. One of the most promising is cancer
immunotherapy involving a new strategy based on enhancing the recognition of
turnor cells by the host immune system through the presentation of tumor
antigens by dendritic cellss7Jos.1ss1s7- Animal expenments as well as clinical
experience indicates that the immune system can recognize and kill tumor
cells. However the presence of tumor antigens by themselves is often
inadequate to stimulate the host immune system to control tumor growth. The
cumulative cunent knowledge suggests an approach to cancer
immunotherapy. in which autologous DCs from tumor-bearing hosts are
expanded ex vivo, pulsed with potential tumor antigens and reinfused into
patients to induce tumor specific T cells including CTLs. Data discussed above
suggest that DC may be used as a physiological adjuvant in vivo to activate
antigen-specific T cells and wnsequently induce T cell dependent humoral
responsesll2.114.158.159.
Mode1 systems cunently used to assess DC based therapeutic
modalities often involve treating miœ after subcutaneous or intravenous
injection of tumor cells into healthy mi~eio3~105. Although tnese experiments
show very promising results including prevention of tumor developrnent and
reduction of metastasis to distant organs, results of human clinical trials are not
conclusive. One possible reason is that immunosurveillance of tumor cells by
the cellular immune systern is often ineffective in vivo, which is reflected in the
outgrowth of tumors that are apparently unable to evoke an immune
response". One explanation for this is that the tumor cells have downregulated
expression of the cell surface molecules such as costirnulatory molecules or
MHC. In addition. it is also possible that T cells with receptors specific for tumor
antigens have been rendered anergic.
Our laboratory recently developed a transgenic mouse model for 6
lineage lymphomas. Sinœ lymphoma developrnent in these mica is
spontaneous with a long latency period, we believe that this transgenic model
is a unique system in which to assess the therapeutic potential of dendritic
cells, Before these studies c m be initiated, however, it is essential to show that
HOXI1 transgenic mice have a competent immune system capable of initiating
an immune response against foreign antigens. Furthemore it is important to
determine whether the process of lymphomagenesis results in the generation
of TAA to which an immune response can be induced.
1 -1 1 Hypothesis
HOXl 1 transgenic mice spontaneously develop lymphomas, which
ultirnately evade the host immune system and therefore may be used as a
model system to develop dendritic cell based cancer vaccines. We hypothesize
that young HOXI 1 mice have functionally competent dendritic and T cells and
that HOX11 lymphomas express TAA that can be recognized by naïve T cells.
We also hypothesize that lymphomagenesis in HOXll transgenic mice is
associated with the development of a comprornised immune system andior the
development of the host T cell tolerance to lymphoma associated antigens.
1 -1 2 Objectives
The objectives of the experiments undertaken in this thesis were to:
assess the functionality of DCs from HOXll transgenic mice including
number, phenotype and capacity for antigen presentation,
asssss the proliferative response of T cells from HOX11 transgenic mice to
mitogenic stimulators and foreign proteins presented by DCs,
assess the cytolytic activity of antigen specifïc cytotoxic T cells from HOXl1
transgenic rnice, and
assess the immunogenicity of HOX11 denved peptides or HOX11
lymphomas using either in vitm transcribed HOXI 1 RNA or HOXl1
lymphoma cell lysates as a source of tumor antigens
In future studies, tumor antigen pulsed DCs may be used as cell
vaccines to abrogate or prevent lymphoma development in this unique murine
mode1 system. Eventually, a better understanding of DC-based immunotherapy
may lead to human clinical trials.
Chapter 2
Materials and Methods
2.1 Animals
HOXll transgenic mice were originally produced in Our laboratory and
we have ample access to them in our breeding colony153. Transgenic mice
were identified by amplification of HOX11 DNA (20 ng) using the Polymerase
Chain Reaction (PCR), by using the forward primer, 5'-
AACCGCAGATACACAAAGGA-3' and the reverse primer, 5'-
TGGGCCAGGCTCTTCTGGAA-3'. Samples were first incubated at 95°C for 5
min and then subjected to 35 cycles of PCR amplification. PCR condition were
as followed: denaturation at 94°C for 1 min, prÏmer annealing at 62°C for 1 min,
extension at 72°C for 2 min and a final incubation at 72°C for 10 min- The
product was detected in 2.5% agarose gels. In addition, CDI, C57BU6 (H-2b)
and C3H (H-2*) mice were purchased from Charles River (Quebec, Canada)
and bred and maintained in microisolators under pathogen-free conditions in
the animal facility of the Sunnybrook Hospital Research Institute. All animal
manipulations were in accordance with Sunnybrook Health Science Center
Animal Care Committee guidelines.
2.2 Reagents Used for the Isolation and Culturing of DCs and T cells
2.2.1 Growth Factors
The interleukin-4 (114) producing cell Iine was cultured in RPMl 1640
(GIBCO BRL) supplemented with 10% heat inactivated FCS (Gibco), 2 mM L-
glutamine (Gibco), 100 Ufml penicillin (Gibco) and 100 mg/ml streptomycin
(Gibco)W Cultures were left without feeding untir viability dropped below 80%
(as determined by trypan blue staining), at which tirne. cells were harvested.
centrifuged for 10 min at 200xg (Beckman) and supernatant filter sterilized and
stored at -20°C.
Granulocyte macrophage-colony stirnulator factor (GM-CSF) was
produced from COS-7 cells transduced with the rnurine GM-CSF cDNA (a gift
of Dr. Nicholas Gough, Victoria, Australia). COS-7 cells were cultured in 100
mm plates in DMEM (Sigma) supplemented with t O % FCS. Before
transfection, cells were washed with TS and then TD. TD contained 140 rnM
NaCI, 5 mM KCI, 25 mM Tris and 0.5 rnM NaH2P04, pH 7.5. TS contained TD
supplemented with 1 mM MgClz and 1 mM CaCI2.
Transfection method
10pg of GM-CSF cDNA was diluted in 2 ml of TS and mked with 2 ml of TS
containing 1 mg/ml DEAE-Dextran, making a final volume of 4 ml of DNA
solution. The DNA solution was added ta the cells and incubated at 37°C. After
50 min, the DNA solution was replaced wRh new TS containing 20% glycerol,
for 2 min. TS was removed and cells washed with TS and then with DMEM
containing 5% FCS. Cells were placed in fresh DMEM containing 5% FCS and
200 pM Cloroquine and incubated at 37OC for 2 hours. at which time, fresh
DMEM (S%FCS) was added and cells incubated for 48 hours at 37OC, 5%
C02. After incubation, the culture supernatant was filter sterilized and stored at
-20°C.
2.2.2 Collagenase D
Collagenase D was purchased from Boehringer Mannheim and since the
activity is fisted in a specific units called Wunsch units and the protocol required
Mandl units, the entire Collagenase D via1 was dissolved to 4000 Mandl Ulml in
HBSS to a final volume determined by the following equation:
mllvial = (x Wunsch Ufml) x (750 Mandl UMlunsch U) x 1000 mghial / 4000
Mandllml
x = specific activity listed in the package insert
1 Wunsch unit = 750 Mandl units
Calcium containing HBSS is essential to preserve Collagenase D activity. since
it is a Ca" dependent enzyme.
2.2.3 Bovine Serum Albumin (BSA)
DCs from the spleen were isolated by BSA density gradient centrifugation. To
prepare the 35% BSA solution, 186 ml PBS. 29ml of 1 N NaOH. and 65ml
water was combined in a 1-liter beaker. 106 g BSA (Cohn fraction V, Integren)
was placed ont0 the surface of the solution without stirring and refrigerated
ovemight to allow the albumin to dissolve. After 24 hours. a small sample was
removed and the refractive index measured. The correct density of BSA was
1 -080 g/mI which conesponded to a refractive index between 1 -384 and 1 -385
at 25°C. The solution was filter sterilized (Nalgene. 500 ml) and stored for 3
months at 4°C-
2.2.4 P reparation of Sterilized Nylon Wool Columns
Nylon Wool (Fisher Scientific) was placed in a Cliter beaker and
saturated with an excess volume of 1 % HCI, The contents were boiled for 5 to
10 min to remove contaminants and then washed at least 10x with water.
Nylon wool was completely dried at 25°C and wool combed to remove knots.
Columns were made by placing 0.5-0.8 g or 1.0-1.5 g of nylon wool inside 5
and 10 ml disposable syringes (Becton Dickinson). respectively. Columns were
autoclaved at 1 1 O°C for 15 min at slow exhaust (no dry cycle) and stored for
months in a dry place, at 25OC (room temperature). A total of -10' cells were
added to 5 ml columns and 2-3 xlo8 cells were added to 10 ml columns..
2.3 Materials Used for Mixed Lymphocyte Reaction (MLR) and Cytotoxic T
Lymphocyte (CTL) Assay
a OVA protein (grade VI. Sigma Chemical Co., 1 g) was dissolved in RPMl
1640 to a final concentration of 200mglml and stored at -20°C.
a Phytohemagglutinin (PHA) (Sigma Chemical Co., 1 mg/ml) was dissolved in
RPMA 1640 and stored at -20°C.
[ 3 ~ ] thymidine (Amersham) was received as a stock of 1mCi. 1 ml of th#
stock was dissolved in 19 ml of RPMl 1640 and stored at 4°C. The working
concentration was 20uCi per well (200 pl).
2.3.1 Cell Lines Used as Targets in CTL Assay
The EL4 ceii Iine (American Type Culture Coilection, Rockville, MD, TI&
39) was established from a T cell thymoma derived from a C57BU6 (H-zb)
rnouse'61. The EG7-OVA cell line (ATCC. CRL-2113) was derived by
transfection of EL4 cells with a plasmid carrying chicken ovalbumin (OVA) and
the neomycin (G418) resistanœ genel62. E.G7-OVA cells contain a single copy
of the inserted plasmid and translate and secrete OVA protein. Cell lines were
maintained in DMEM medium (EL-4) and RPMl 1640 medium (E.G7-OVA)
supplemented with 10% heat inactivated FCS, 2 mM L-glutamine, 100 Ulml
penicillin and 100 rnglml streptomycin. In addition, EG7-OVA culture media
was adjusted to contain 1.5 gA sodium bicarbonate. 4.5 glL glucose. 1 O mM
HEPES. 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol and 0.4 mgh l
G418.
In addition to the EL4 and E.G7-OVA cell lines described above, the K3P
cell line was also used as a target in CTL assays. The K3P was cell line was
derived from cells obtained from a T-ALL patient with a t( l O;l4) chromosome
translocation and established according to published methodst63. Cytogenetic
analysis indicated that the cell line had lost both the normal and derivative
chromosomes 10 but retained the derivative chromosome 14 bearing the short
and proximal long amis of 14 and the translocated chromosome 10 material
distal to the breakpoint in 10q24. The cell Iine expressed the 2.lkb HOXll
transcript and was used to isolate the HOXI1 cDNA1W. K3P cells were
maintained in complete RPMI-1640 medium supplemented with 10% heat
inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml
streptomycin.
2.3.2 Production of In vitro Tiiinscribed (IVT) RNA
The pGEMHOXl1 vector was generated by cloning nucleotides 165-2058
of the human HOXll cDNA into the Smal site of the multiple cloning site of
pGEM7Zf (Promega. Madison, WI) thereby placing the HOX11 cONA
downstrearn of the T7 prornoter. The vector was Iinearized with BamHl or
Hindlll to minimize plasmid sequences from the in vitro transcribed mRNA. In
vitro transcription of HOXl1 was carried out at 37OC for one hour using the
Promega RiboprobeO in vitro Transcription System according to the
manufacturer's instructions. Template DNA was digested with RNase free
DNase 1 and RNA was recovered by triasoVchlorofon extraction followed by
centrifugation and the pellet was washed with 70% ethanol. The pellet was air-
dried and resuspended in sterile water. RNA was quantitated by measunng the
optical density (OD) at 260-280 nm and stored at -80°C.
2.3.3 Preparation of Tumor Lysate
Tumor biopsies from HOXll mice that had developed lymphoma were
removed under the sterile conditions using forceps and scissors and placed in
PBS. Nonmalignant tissue was removed and tumor cells disperseci to create
single cell suspensions. Cells were transferred into 2 ml cryopreservation tubes
(Falcon) and lysed using the following procedure. First, tubes were placed in
iiquid nitrogen for 5 min and then lef€ ai 25°C to thaw completely. This freezel
thaw procedure was repeated 6x. After the last thaw cycle, cells were
centrifuged at 14Oxg for 10 min to remove large particles, and supernatant
passed through a sterile MiIlex@-HV 0.22 prn filter (Millipcre) and aliquots
stored at -80%.
2.4 Generation of DC Cultures
The main objective of this initial expen'ment was to detemine the most
efficient technique for the isolation of DCs from spleen and bone marrow of
HOXI 1 mice. Further, we tested culture conditions under which we would
routinely obtain sufficient number of DCs to be used in different assays (see
below).
2.4.1 Dendn'tic Cell Isolation from Spleen
2.4.1.1 Density gradient cenHfugation using Bovine Semm Albumin (BSA)
Collagenase digested splenocyte suspensions were centrifuged in dense
bovine serum albumin to obtain a fraction with a low buoyant density enriched
for DCs.
Spleens were removed and plaœd in 60 mm tissue culture dishes
containing 5 ml of Collagenase D solution. Each spleen was injected with 100
pl of Collagenase D using a 22-G needle attached to a 5 ml syringe. Forceps
were used to tease the spleen into srnall fragments. Dishes were placed in a
37°C incubator for 45-60 minutes to release DCs. At the end of the incubation
period, the fragments were placed into a 70 pn nybn cell strainer (Falcon),
pressed through the strainer using a plunger from 5 ml syringe, and celi
suspensions collected into 50ml polypropylene tubes (Falcon). Tubes were
centrifuged in a refrigerated centrifuge (Beckman) held at 4OC for 10 min at
280x9. After centrifugation. supematants were removed and pellets
resuspended in 35% BSA (1 mwspleen). Cell suspensions were transferred to
the 6 ml polypropylene tubes (Falcon), overlaid with 1.5 ml of 4OC RPMl 1640
to fomi a sharp interface, and low density cells obtained after a 15 min
centrifugation (Beckman, L8-70 Ultracentrifuge) (on a 35% BSA gradient) at
9500xg and 4°C. The centrifuge was set up for slow acceleration and with the
break turned off. Using a pipette, low density cells were carefully collected from
the interface region, taking the full volume of RPMl 1640 and the top 1 ml of
BSA. Cells were resuspended in wld (4°C) RPMl 1640 and centrifuged for 10
min (280xg, 4OC), after which supematants were removed. and cells
resuspended in 5-10 ml of DCs medium (RPMI 1640 medium supplemented
with 10% FCS (Gibco). 50 mM 2-mercaptoethanol (Sigma), 10 mM HEPES
(Sigma). 2 mM glutamine (Gibco). 100 Ulml penicillin (Gibco), 50 pglml
streptomycin (Gibco), and 20 nglml GM-CSF). Cell viability was detemined by
trypan blue exclusion and 4 ml of the DC suspension (- 1 4 x lo5 cells/ml) was
placed in 60 mm tissue culture dishes. After 90 minutes, medium containing the
non-adherent cells was removed and adherent cells cultured ovemight. All
tissue culture procedures were done under sterile conditions in a level 2
containment facility.
2.4.1.2 Separation using MA CS CD f fc MicroSeads
For MACS systern separation. colloidal super-paramagnetic MicroBeads
conjugated to monoclonal hamster anti-mouse CDl lc antibodies were
purchased from Milteny Biotech. The CD1 1c antibody clone N418 is specïfic for
the leukocyte integrin expressed primarily but not exclusively on mouse DCs.
Using this technique DCs can be efficiently enriched.
lsolated spleens (4-6) were placed in a 60 mm tissue culture dishes with
Collagenase D solution (5ml) and the procedure for the spleen preparation
followed as described above. After incubation, cell suspensions were prepared
and cells washed in 15 ml of buffer followed by centrifugation at 200x0 for 10
min (4°C). The bmer solution consisted of phosphate buffered saline (PBS) pH
7.2, supplemented with 0.5% BSA. Cell pellets were resuspended in 400 pl of
buffer per 1 o8 total cells. 100 pl of CD1 1c MicroBeads per 10' cells were added
and cells were incubated at 6°-120C. After 15 min cells were washed by adding
4-8 ml of buffer and centrifuged at 200x9 for 10 min. Cell pellets were
resuspended in 500 pl of buffer per 108 cells and passed through a separation
column (column type MS+/RS+ for up to 2x10' total cells), which was held in
the magnetic field of the MACS separator. The magnetically labeled CD1 lc'
DCs were retained in the column while the unlabeled CD116 cells passed
through. Columns were washed with 500 pl of buffer and removed from the
magnetic field. Retained CD1 1c' DCs were eluted as the positively selected
cell fraction using 1 ml of OC medium and cultured under the same conditions
as BSA isolated DCs (above). To obtain a highly pure population of DCs for
some experiments, the magnetic selection step was repeated.
2.4.2 Dendn'tic Ce11 Isolation from Bone Manow
After removing al1 muscle tissues from the femun using forceps and
scissors, the bones were plaœd into 35 mm culture dish with 4°C RPMl 1640
medium. Both ends of the bones were cut with scissors, and the marrow was
flushed out using 3 ml of RPMl 1640 and a 5 ml syringe with a 25-gauge
needle. Red blood cells were lysed using 3 ml of lysis bMer (0.165 M
ammonium chlonde and Tris, pH 7.4) at 25OC for 10 min. Bone marrow cells
were washed in RPMl 1640 supplemented with 2% FCS followed by
centrifugation for 10 min at 200x9. Cells were plaœd in 60 mm tissue culture
dishes in RPMl supplemented with 10% FCS, 50 mM 2-mercaptoethanol, 10
mM HEPES, 2 mM glutamine. 100 Ufml penicillin, 50 pglml streptomycin, 20
nglml GM-CSF and 20 nglml 114. The cell concentration was - 1-5 x 10'
cellslml. The cultures were incubated at 37*C, 5% CO2 and fed every 2 days by
removing 75% of the medium and returnhg a similar volume of fresh, warm
(37°C) DC medium. On day 7, cells were harvested by gentle pipetting. Further
purification of DCs was done using CD1 1 c MicroBeads (described above).
2.5 Dendritic Cell Surface Phenotype Charactenzation
We used flow cytometric analysis to determine the phenotype of HOXI 1
spleen and bone mamw denved DCs, since the expression of specific
molecules on the surface of DCs is essential for their function as APCs-
2.5.1 Flow Cytometric Analysis of Spleen and Bone Manow Dendritic Cells
Two groups of six mice (HOX11 x C57BU6. F4) were used to detennine the
phenotype of spleen derived DCs. One group consisted of three HOXI1
transgenic and three non-transgenic mice (three months old) while another
included nine month old mice. To evaluate bone marrow derived DCs, HOXI 1
(HOX11 x C57BU6, F4) and non-transgenic mice were used. Each group
consisted of three mice (three months old). Spleen derived DCs were isolated
by BSA density gradient centrifugation, while bone marrow derived DCs were
isolated using CDl lc MicrBeads (MACS system). Both techniques were
described above. DCs (-1o6) were stained with primary antibodies in HANK'S
medium supplemented with 2% FCS (HF) on ice for 30 min. The cells were
subsequently washed twice with HF and resuspended in HF containing 1 pl/rnl
propidium iodide. Antibodies used in this study were: Fluoresœin
isothiocyanate (FITC)-labeled monoclonal antibodies to CD1 lc , CD1 1 b (Mac-
1). Ml169 (HSA), CD80 (87-1). CD86 (87-2). CD40. CD3 and 8220, al1
purchased from PharMingen (Mississauga, Canada); FITC-labeled H ~ K ~ (MHC
class 1) and I A ~ (MHC class II) purchased from Cedarlane (Homby, Canada).
FITC-labeled polyclonal anti-rat IgG (PharMingen) was used as negative
controls. Flow cytometric analysis was performed using a Coulter Elite
(Beckman Coulter) flow cytorneter equipped with EXP02 software (Applied
Cytometry System).
2.6 T Ceii Isolation and Enrichment
The aim of this experiment was to determine the best method for the
isolation of sufficient numbers of an enriched T cell population from HOX11 and
control mice. These T cells were used for the optimization of the mixed
lymphocyte reactions (described below) followed by dfierent functional studies
to characterize HOXl 1 derived T cells.
2.6.1 Isolation of T cells f m Peripheral Blood
Blood was obtained by cardiac puncture using a 19-gauge needle and 5 ml
syringe filled with RPMl 1640 with 100 U/ml heparin (-2 ml/mouse). Blood was
transferred into 14 ml polypropylene tubes (Corning). diluted 1:2 in medium and
carefully layered onto the density gradient, FicolCPaque, using a pipette to
produce a clean interface between the tw layers (the proportion of blood ta
Ficoll was 1:3). Tubes were centrifuged at 400x9 at 20°C. After 25 min. the
white opaque mononuclear fraction from the interface between the diluent
(medium) and Ficoll-Paque was removed using a disposable pipette and the
cells washed at least two times with 5-10 ml of medium to remove Ficoll-Paque
(300x9, for 10 min).
2.6.2 Isolation of T Cells from Spleen and Thymus
2.6.2.7 Separa tion with MA CS System
T cells were isolated from spleens using either the MACS separation
system or nylon wool columns. For MACS separation. a single cell suspension
was prepared from spleens (described above) or thymuses (same procedure
as with spleen cells) and red cells lysed with 0.165 M NHdCI-Tris pH 7.4. The
protocol for the MicroBeads separation was similar to the method for the DCs
isolation from spleen, previously described, with the only difference being the
type of MicroBeads used for T cell isolation. CD90 (Thy 1.2) MicroBeads were
used since CD90 is expressed on thymocytes. peripheral T cells and some
interepithelial T cells. Thymus or spleen cell suspensions depleted of red cells
were washed. and labeled with CD90 MicroBeads (1 Op1 of beads/l o7 total cells)
and passed through separation columns. The rnagnetically retained CD90' T
cells were eluted as a positively selected cell fraction. Eluted cells were -90%
CD3+ as deterrnined by flow cytometric analysis.
2.6.2.2 Isolation with Nylon Wool Columns
T cell enrichment by nylon wool is based on the adherent properties of B
cells and accessory cells to nylon wool at 37°C in the presence of FCS. In
contrast T cells do not adhere but pass through the columns. Sterilization of the
nylon wool and assembly of the columns was described above. Columns were
washed with warm (37OC) RPMl 1640 supplemented with 5% FCS, sealed with
parafilm and incubated at 37°C. 5% CO2 (with 1 ml of medium remaining in the
columns). After 1 hour, columns were rinsed with 5 ml of warm medium and 1
ml of the spleen cell suspension (prepared as described above) was added to
columns and incubated at 37°C for 45 min. After incubation, columns were
washed with 15 to 20 ml of warm medium, T cells collected into 50 ml tubes
(Falcon) and concentrated by centrifugation (200x9, 10 min).
2.7 Optimization of Mixed Lymphocyte Reaction (MLR)
The MLR assay was used to measure T cell proliferation ability after
stimulation. Suspensions of responder T cells were cultured with stimulator
cells and based on the activating stimuli, an allogeneic or syngeneic MLR was
established. The activating stimuli in an allogeneic MLR were the mismatched
MHC class I and class II antigens processed and presented by allogeneic
stimulator cells (DCs). while in syngeneic MLR OVA protein pulsed stimulator
cells (DCs) were used to present foreign antigen and induœ proliferation of
responder cells (T cells). Parameters affecting the magnitude of T cell
proliferative responses include cell concentration, type of medium, type and
concentration of activation agent, type of responding T cells and culture time.
We optimized experimental conditions with respect to these variables.
2.7.1 Optimization of T CeIl Pmliferative Responses
To optimize MLR. a dose-response assay was performed using different
sources and nurnbers of responder T cells. T cells were isolated from spleen,
peripheral blood and thymus using the previously described techniques.
T h e replicate wells were set up containing each of the following:
T cell proliferation was induced by phytohemagglutinin (PHA, Sigma) at a
concentration of 3 4 pgfml. Cells were plated at different concentration (as
presented in the table above), cultured in 96-well U-bottom plates (Corning)
and I~H] thymidine (1 pCilwell, Amersham) added on days 3. 4. and 5 to
determine the time of the maximum T celI proliferative response. Cells were
harvested 1848 hou= later, using a Titertek-cell harvester. I~H] thymidine
incorporation was measured by liquid scintillator(Wallac-1205 Betaplate).
Spleen
Peripheral blood
Thymus
2.8 Çunctional Studies Using HOX11 Derived DCs
2.8.1 Presentation of Foreign MHC by HOX7 7 Derived DCs
The prirnary aim in this experiment was to test the ability of HOX11
spleen derived DCs to process and present their own MHC class I and class II
0.5
1 .O
1 .O
3.0
2.0
5.0
8.0 4.0
5.0
6.0
molecules and to induce proliferation of allogeneic T cell isolated from C3H
mice (H-23.
Four month old HOX11 transgenic (bred in the CD4 genetic
background), C3H (H-2*) and C578U6 (H-2b) female mice were used in this
experiment. 1 x Io4 irradiated (>ZOO0 rad) spleen derived DCs from HOX11
transgenic mice were cultureci for 5 days in 96-well plates with 1 x 10' splenic T
cefls isolated from C3H and C57BU6 mice. Thirty six hours before assay
completion, i 3 ~ ] thymidine (1 pCi1well) was added to each well. At completion,
plates were harvested and 1 3 ~ ] thymidine uptake measured in a liquid
scintillation counter. Responses were reported as mean cpm from triplicate
samples. As a positive control, C3H DCs were plated with C57BU6 T cells,
while as a negative control, HOXI1 derived DCs were mixed with autologous
HOX11 derived T cells.
2.8.2 Presentation of OVA Protein by HOXI 1 Derived DCs
Samples from three month old HOX11 transgenic and normal female mice
were used in this assay. OVA protein (grade VI-Sigma) was dissolved in OC
culture medium at 40, 60, 100 and 120 pg/ml to detemine the best
concentration of OVA protein required for maximum proliferation of autologous
T cells. 1 x 105 spleen DCslml were plated in 6-well dishes and incubated
(37OC, 5% COz) in 1 ml of culture medium containing OVA protein and 2%
FBS. After 30 min, 4 ml of OC medium supplemented with 6% FBS was added
and cells were incubated overnight. After 18 hours, DCs were washed,
irradiated at > 2000 rad and added (1 x lo4 per well) to naïve autologous T
cells (1 x l o5 per well) in 96-well plates. After 5 days. PH] thymidine was
added, and cells harvested 36 hr later and thymidine uptake measured in a
liquid scintillation counter. Proliferative responses were reported as mean cpm
frorn triplicate samples. As a negative control. T cells were either cultured alone
or with unpulsed DCs.
2.9 Functional Studies Using HOXI 1 Derived T Cells
2.9.1 T Cell Proliferation Assay
Mice ranging in ages from 3.5 to 18 month of age were used. MicroBead
purified T cells from spleen were plated in 96-well plates at concentrations of 3
x 1 o5 per well. PHA was added at a concentration of 5 pgiml. The final volume
was 200 pl per well. Cells were cultured for 3 days at which tirne VH] thymidine
was added and plates incubated at 37OC in 5% COz for 38 hr. Cells were then
harvested and ['HI thymidine incorporation measured. Data are presented as
the difference in cpm between stimulated and non-stimulated T cells.
2.9.2 T Ce11 Responses to Foreign MHC Antigens
Four rnonth old HOX11 transgenic, C57BU6 and C3H female mice were
used in this experiment. T cells were isolated from the spleens of HOXll
transgenic mice using the MACS systern and plated in 96-well plates (1 x
105/well) with C3H irradiated spleen DCs (1 x 104 per well). On day 5, PH]
thymidine was added and cells were harvested. Thymidine uptake was
measured using a liquid scintillation counter (Wallac-1205 Betaplate).
Proliferative responses were reported as mean cpm from triplicate samples. As
a positive control. C57BU6 cells were cuitured with C3H DCs. As a negative
control, C3H T cells and HOX11 derived T cells were mixed with autologous
C3H DCs and HOXl 1 transgenic DCs, respectively-
2.9.3 T Ce11 Responses to O VA Pmtein
Three month old HOX11 transgenic, normal female mice were used in
this experiment. The protocol used was similar to that previously described to
assess the ability of HOXll derived DCs to present OVA protein. The only
modification was that OVA protein was incubated with N-(1-(2,3-
dioleoyloxy)propyl]-N, N. N-trimethylammonium methylsulfate (DOTAP)
(Boehringer Mannheim), which is a lipid component that increase antigen
uptake by DCs. 1 x 1 o4 DCs were pulsed overnight with DOTAP-OVA protein
(D0TAP:OVA was 100pg:40pg and 100pg:lOOpg). After a 18 hour incubation,
cells were irradiated and added to naïve HOXll transgenic mice derived T
cells in 96-well plates at the concentration of 1 x 104 cells/well DCs and 1 x lo5
cells/well T cells. After 5 days in culture, [ 3 ~ ] thymidine was added and uptake
measured. As negative controk, T cells were elher cultured alone or with
unpulsed DCs.
2.1 0 Measurement of Cytotoxic T Lymphocyte (CTL) Activity
This method was used to examine ability of HOX11 transgenic mouse
derived DCs to present antigen and induce primary CTL responses as well as
to test the capacity of HOX11 derived CTL to lyse different target cells. The
CTL killing assay is frequently used to measure the activity of CTL. generated
b y precursor T lymphocytes following stimulation by specific antigens
presented by DCs. The most sensitive method to assay CTL adivity is JAM
test @ust Another Method). This assay is based on the evidence that target
cells that are killed by CTL effector cells undergo apoptosis, which results in
the degradation of their ONA into srnall fragments. Target cells are labeled with
[ 3 ~ ] thymidine. washed and then mixed with cytotoxic effector cells. If target
cells are killed their DNA fragments will be washed through a filter, whereas
target cells, which are not killed uptake the PH] thymidine that will be captured
by the filter and measured. The percent lysis can be calculated by comparing
the amount of PH] thymidine bound to the filters in the presence and absence
of effector cells.
CTL activitv determination
%Spontaneous lysis = [(T-S)m x 100
T - total counts wells
S - spontaneous release wells
This value is not used to calculate % cytotoxicity, but is important because high
levels of spontaneous lysis can seriously distort the expenmental results.
Specific cytotoxicity for each expenmental point was measured by the formula:
% Cytotoxicity = ((S-€11 x 100
S - spontaneous release
E - the average values of the replicates from expenments
T and S value were measured, for each CTL assay, using the following
procedure:
100p1 of target cells were added to control wells containing 100p1 of
medium (to measure spontaneous target cell lysis during assay) as well as on
a separate plate to measure total counts. All plates were centrifuged for 2 min
at 400xg at room temperature. A separate plate containing cells used to
measure total munts was harvested immediately while other plates were
incubated for 5 hrs at 37°C. After incubation, plates were harvested and
radioactivity counted on al1 filters (from test plate and total count plate) by a
liquid scintillation counter. Percentage of cytotoxicity was measured as the total
counts as well as spontaneous release for each experiment.
2.1 O. 7 In Vitro CTL
This assay was used to determine the ability of OVA protein pulsed
HOXl1 derived DCs to induce CTL responses in vitm.
Six month old HOXI1 (HOXI1 x C57BU6, F5). non-transgenic and
C57BU6 female mice were used in this assay. Bone marrow derived DCs (1 x
I o 5 cells/ml) were pulsed with OVA protein in the presence of DOTAP as
descri bed a bave. After ovemight incubation, cells were washed , irradiated with
>2000 rads and added to naïve responder syngeneic T cells (1 x 106 cellshi)
in 24-well plate at responderlstimulator ratio of 20:l. The cultured medium was
complete RPMl 1640 supplemented with 1mM sodium pyruvate and l x
nonessential amino acids. After 6 days, the T cells were hawested, adjusted to
1 x 10~1rnl and restimulated with freshly pulsed DCs. Twelve to fourteen days
after the first stimulation, T cells were harvested and tested for cytolytic activity
with either OVA protein pulsed autologous BM DCs, EG7-OVA, EL4 or
unpulsed DCs serving as target cells. Based on preliminary studies to optimize
the time for the best PH] thymidine uptake, target cells were labeled with [=HI
thymidine for 5-12 hours and washed and centrifuged for two times for 7 min at
1200 rpm. In the blockinglinhibition experiment using anti-MHC class I mAb,
OVA protein pulsed target cells were incubated at 37OC for 30 min with anti-H-
2~~ mAb before use in cytotoxicity assays. The final rnAb concentration was
50pg/ml. Cytotoxic T cells and target cells were plated in 96-well plate dishes at
effectorltarget ratio of 50:l. 20:1 and 10:l using RPMl 1640 medium with 10%
FBS. In addition, plates to measure spontaneous target lysis and total counts
were prepared as described above. All plates were centrifuged. further
incu bated, harvested and radioactivity counted as described above. Each
assay was perforrned in triplicates with spontaneous release < 15% (see
a bove).
2.1 O. 2 ln Vivo C TL
Based on the results of the in vitro study, we next determined whether
HOXl1 derived DCs were able to induce primary CTL immune responses to
OVA protein in vivo.
Three month old HOX11 transgenic (HOXI 1 x C57BU6-F5). non-
transgenic littermates and C57BU6 female mice were used in this assay. Bone
marrow cells were placed in culture medium supplemented with GM-CSF and
11-4 to expand DCs, while DCs isolated from spleens using CD1 l c Microbeads
were pulsed with OVA protein in presenœ of DOTAP as descnbed above. After
18 hours, pulsed cells were irradiated with >2000 rads, washed three times in
PBS and injected intraperitonealy (i-p.) into recipient mice at a concentration of
5 x 105 cellslml in 2004 PBS. Seven days post immunization mice received a
second round of immunization using expanded, CD? 1c Microbeads purified BM
derived DCs that were freshly pulsed with OVA protein in the presence of
DOTAP and irradiated. Seven to eight days after the second injection with OVA
puised DCs, spleen cells from primed mice were removed and red cells lysed.
Splenocytes (2 x I o 6 cells/ml) were cultured with either 1 x Io5 celldml OVA
protein pulsed DCs irradiated at 2000 rads or E.G7-OVA cells inadiated at
20,000 rads, as a stimulators. The same culture medium describeâ for in vitro
CTL was used and plates were incubated for 5 days at 37OC and 5% CO. In
addition, bone marrow cells isolated from prirned mice were cultured in GM-
CSF and I L 4 to expand DCs and serve as autologous target cells. Effector T
cells were harvested on day 5 and assayed for antigen specific CTL activity.
Targets used to asses CTL adivity were autologous DCs pulsed with OVA
protein, E.G7-OVA, EL4 and unpulsed autologous DCs. Target cells were
labeled with ('HI thymidine for 5-12 hous and washed two times and
centrifuged for 7 min at 1200 rpm. The killing assay was set up at
effectodtarget ratios of 50:l to 5 3 in 200~1 of RPMl 1640 with 10% FBS in 96-
well plates. In addition, plates to measure spontaneous target lysis and total
counts were prepared as described above. All plates were centrifuged, further
incu bated , harvested and radioactivity coun ted as described above. Each
assay was perfomed in triplicate with spontaneous release being c 20%.
2.1 1 Assessrnent of Immunogeneicity of HOXl1 Derived Antigens
2.1 7.1 Induction of CTL Response fo In Vitro lmnscribed (IV? HOXl1 RNA
Pulsing of DCs with IVT HOXI 7 RNA
Two to three month old HOX11 transgenic (HOX11 x C57BU6-F5),
normal and C57BU6 female mice were used in this assay. CD1 lc' DCs were
isolated from spleens by MACS systern. Negatively labeled cells were washed
and served as a source of 1 cells, which were purified by nylon wool columns.
Bone marrow cells were cultured in medium supplemented with GM-CSF and
11-4 to expand BM-DCs (to be useâ later as APC in a second immunization).
DOTAP (in 250-500 pl RPMI-1640) was incubated with IVT RNA (2-5 pgl250-
500 pl RPM 1-1 640) (described above) at room temperature for 20 minutes after
which the complex was added to the 5-10 x 105 cells/ml spleen derived DCs
and incubated at 37OC for 4 hours. In addition. sorne DCs were pulsed with
DOTAP-OVA protein (as descnbed above) and used as controls. DCs were
washed with PBS, centrifuged at 200x0 for 1 O min. irradiated at ,2000 rads
and used as stimulators in an MLR as well as to prime mice in an in vivo CTL
experiment as descnbed below.
MLR Assay Using DCs Pulsed with HOXl1 I M RNA
The main objective of this experiment was to determine whether DCs
pulsed with IVT HOXll RNA could activate T cells isolated from HOX11
transgenic and naïve C57BU6 mice. DCs pulsed with IVT HOXll RNA were
used as stimulator cells, while nylon wool purified T cells were used as
responder cells. MLR assay was set up in 96-well plates at the
responder/stimulator ratio 1O:l. On day 5. thymidine was added and cells
were harvested. Thymidine uptake was measured using a liquid scintillation
counter. Proliferative responses were reported as mean cpm from triplicate
sarnples. PHA and unpulsed DCs were used as controls.
In Vivo Induction of CTL Response to IVT HOX11 RNA
Our primary aim was to determine whether HOXll derived peptides.
presented by DCs could be used to induce antigen specific CTL response in
vivo in HOXl1 transgenic and naïve mice.
DCs were pulsed with IVT HOXll RNA. inadiated, washed in PBS
(three times) and 3 x 10' cells/ml in 2004 PBS were injected intraperitonealy
@p.) into recipient mice. Seven days post immunization mice received a
second round of immunization using expanded, CDllc* irradiated bone
manow DCs that were freshly pulsed with IM HOXI 1 RNA or OVA protein.
Seven to eight days after the previous injection, spleen cells from primed mice
were removed, red cells lysed and in v i t . CTL assays were set up. For in vifro
CTL, splenocytes (2 x 1 o6 cells/ml) were cultured with inadiated IVT HOXII
RNA pulsed DCs (1 x 10' cellslml), which sewed as a stimulator'57. The same
culture medium described for in vitro CTL was used and plates were incubated
for 5 days. In addition. bone manow cells from primed mice were cultured in
media supplemented with GM-CSF and 11-4 to expand DCs, which served as
autologous target cells. Effector T cells were harvested on day 5 and assayed
for anti-HOX11 peptide specific CTL activity. Targets used to asses CTL
activity were autologous DCs pulsed with HOX11 IVT RNA, OVA protein, K3P.
EG7-OVA and EL4 cells. All target cells were labeled with [ 3 ~ ] thymidine for 5-
12 hours and washed two times and centrifuged for 7 min at 1200 rpm. Killing
assays were set up at effectwnarget ratios of 50:l to 12.5:1 in 200pl of RPMl
1640 with 10% FBS in 96-well plates. In addition, plates to measure
spontaneous target lysis and total counts were prepared as described above.
All plates were œntrifuged . further incu bated. harvested and radioactivity
counted as described above. Each assay was perfonned in tripkate with
spontaneous release being < 20%.
2.1 1.2 Induction of CTL Resporise to HOX11 Tumor Lysates
Pulshg of DCs with Tumor Ce11 Lysates and In Vitro CTL Assay
HOXll transgenic and C57BU6 bone marrow cells were cultured in
medium supplemented with GM-CSF and 114 to expand DCs and expanded
DCs purÎfied using CD1 1c Microbeads. Spleens were removed, red cells iysed
and spleen cells cryopreserved as a source of T cells. Bone mamw derived
DCs were incubated with freezelthawed tumor lysates ovemight in DCs
cultured medium. After 18 hours DCs were harvested, irradiated with 2000 rads
washed and used as stimulators in CTL assays. T cells to serve as responder
cells were isolated from spleen ceils suspensions using nylon wool columns.
CTL assays were set up in 24-well plates (Corning), in RPMl supplemented
medium (described above), at responder/stimulator ratio 20:l. The same day
the CTL assay was set up, additional T cells were isolated from another
C57BU6 mouse and used as additional responder cells. After five days in
culture, T cells were harvested, adjusted to 1 x 1 o6 cellslml and then stimuiated
with DCs pulsed with tumor cell lysates. After a total of 12 days in culture, T
cells were harvested and tested for cytolytic ability using different target cells,
including DCs pulsed with tumor lysates, DCs pulsed with IVT HOXll RNA or
unpulsed DCs. All target cells were labeled with thymidine for 5 hou= and
washed two times for 7 min at 1200 rpm. Killing assays were set up at effector
to target ratio of 80:l to 1O:l in 200~1 of RPMl 1640 with 10% FBS in 96-well
plates. In addition. plates were prepared to measure both spontaneous target
cell lysis and total counts as described above. All plates were centrifuged.
further incubated, harvested and radioactivity counted as described above.
Each assay was perforrned in triplicate with spontaneous release c 20%.
Chapter 3
RESULTS
3.1 Identification of HOX11 Transgenic Mice
PCR analysis was used to distinguish between control (non-transgenic)
and HOX11 transgenic mice as described in the Materials and Methods. As
shown in Figure 1, the primers amplify both endogenous murine (mHOX11)
and HOXI 1 transgenic (HOXI 1 Tg) fragment.
3.2 Optimization of Mixed Lymphocyte Reaction (MLR)
The MLR is one of the most widely used methods to evaluate overall T
cell proliferative responses as well as the ability of DCs to present antigens.
However, to utilize this technique several parameters must be deterrnined
including the source and concentration of T cells, the concentration of
activation agent, and cuiture times. In the following study, T celk were isolated
from spleens, peripheral blood and thymuses from three C57BU6 mice as
described in the Methods. To deterrnine the optimum cell number required for
maximum proliferation of T cells induced by the mitogenic stimulator, PHA,
different combinations of cell concentrations were plated and T cell proliferative
responses assayed by TH] thymidine incorporation. The data presented in Fig.
2 indicated that T cell proliferation was dependent on the number of T cells
plated. The best proliferation of spleen derived T cells was obtained using 2 x
10' cells while 3 x 10"as optimal for PB derived T cells.
Figure 1. PCR analysis used to dîstinguish HOXII transgenic (HOX11
Tg) and non-transgenic mice. Amplification of both endogenous and
HOXI 1 transgenic fragment was observed. Lane 1 contained a negative
control, and lines 2-1 5 contained amplified DNA from transgenic and non-
transgenic miœ followed by CD-1 non-transgenic DNA (lane 16), positive
transgenic DNA (lane 17). and a plasmid containing the enüre HOXI1
cDNA (lane 18).
Figure 2. Optimization of MLR. Spleen, peripheral blood (PB) and thymus denved
T cells from C57BU6 mice were stimulated with PHA and T cell proliferation
measured by PH] thymidine uptake, after 3 days in culture for spleen and 5 days
for peripheral blood and thymic T cells.
Dose-response for Spleen T cells
OTcell alone PHA
O
Dose-response for PB T cells
Dose-reaponse for Thymus T cells
When these concentrations of cells were used, 10-1 2 fold higher proliferation
was observed, when compared wÏth unstimulated T cells. Although high
concentrations of thymocytes were used (8 x 10' cellolml), the maximum
proliferation was only 3 foM above unstimulated T cells. One possible
explanation for this result couM be the low frequency of responding T cells in
the thymus. The results also suggested that the highest proliferation was
obtained on day 3 (for spleen T celts) or day 5 (for PB-T cells) after PHA
stimulation (Fig. 2).
3.3 Characterization of HOXI1 Transgenic Mice Derived Dendritic Cells
3.3.1 Establishment of Dendn'ic Ce11 Cultures
Spleen derived DCs were purified by either BSA density gradient
centrifugation or MACS CD1 1c magnetic separation and DC cell nurnben
expanded in culture supplemented with GM-CSF. Initially, DCs adhered to the
tissue culture dishes but became nonadherent after ovemight culture.
Nonadherent cells were then transferred to new T25 flasks and fed with GM-
CSF supplemented medium every 3-4 days. After 7-10 days the surface of the
fiasks generally became covered with tightly adherent strornal-like cells and
loosely detached clusters of DCs were apparent. The clusters expanded rapidly
in number and sire. After 10 days, high numbers of spontaneously released
cells were observed. These cells were subcultured and upon transfer most of
the cells adhered within 1-3 days, fonning new clusters (Illustration 2). The final
yield of DCs isolated from HOX11 transgenic mice ranged from 2 x 1 O' to 5 x
10' DCS per spleen (Table 1).
1 #DC 1 HOXll transgenic 1
Table.? Number of DCs obtained from spleen of HOXI 1 transgenic rnice
(spleen) #1
Bone marrow derived DCs were cultured in the presence of GM-CSF and 11-4.
mice 2x105
Since GM-CSF also induces proliferation of macrophages, 11-4 was added to
cultures to suppress the proliferation of macrophages. Cells were washed to
remove nonadherent granulocytes without dislodging clusters of developing
DCs that were loosely attached to the marrow stroma (Picture 2). Optimal
yields (5 x I o5 DCs per mouse) and purity of DCs were obtained when the
aggregates were harvested on day 7 followed by CD1 1 c magnetic separation
(Table 2). If the clusters were allowed to overgrow without subculturing or
without GM-CSF, the aggregates dissociated and did not refom, indicating that
these factors are critical for normal DC development and proliferation.
#DC (mouse-
Table.2 Number of HOXl 1 bone marrow derived DCs
HOX11 transgenic ' mice
BM) #I #2
4 x 1 0 5 3 x 105
Illustration 2. Dendritic cells isolated frorn HOXll transgenic mice. A and
B represent spleen DCs 18 hr after isolation and culture in medium
supplemented with GM-CSF, while C represents bone mamw derived
DC-cluster after 6 days in the culture supplemented with GM-CSF and IL-
4.
3.3.2 Phenotype Characteniation
DCs isolated from the spleens of non-transgenic and HOX11 transgenic
mice and cultured ovemight were analyzed for the expression of cell surface
antigens associated with DC function by fl ow cytometric analysis (Fig.3 and 4).
The nonadherent cells analyzed were 80.90% DCs. Staining of cells with MHC
class I and class II antibodies indicated that both control and HOX11 derived
DCs express high levels (measured in percentage) of MHC molecubs.
Furthemore, both control and HOXI 1 DCs expressed moderate levels of the
macrophage/granulocyte marker CD1 1 b (Mac-1). normally expressed at levels
ranging from low to high on difterent DC subpopulations. They also expressed
moderate levels of costimulatory molecules CD80 (87-1). CD86 (87-2), HSA
and CD40. The cells did not express or expressed low levels of the T ceII
specific antigen, CD3 and B lineage specific marker 8220. (Fig. 3)
Table 3. Flow cytometric analysis of HOX11 and non-transgenic spleen derived DCs. Results are presented in percentage of cells that stained with specific antibody
Cells isolated from the bone marrow of HOX11 transgenic and
nontransgenic littermate mice and cultured in medium supplemented with GM-
CSF and 11-4 were also phenotypically characterized by flow cytometry
analysis. After 6 days, floating and easily detached cels from aggregates were
harvested, purified using the MACS system and stained with antibodies specific
for surface antigens reflecting DCs ability to function as APC.
Figures 3 and 4 Flow cytometric analysis of HOX11 transgenic and non-
transgenic DCs isolated from either spleen (Fig.3) or bone marrow
(FigA). Solid histogram represents unstained cells. while open and
shaded histograms represent HOXI1 transgenic and non-transgenic
derived DCs , respectively.
HOX11 Spleen Derived Dendritic Cells
MHC I MHC II
HSA Mac-1 CD3 8220
Figure 3. Surface Phenotype Characterization
HOX11 Bone Marrow Derived Dendritic Cells
MHC I MHC II HSA
Figure 4. Surface Phenotype Characterization
HOX11 transgenic and control DCs stained strongly with rnAb to MHC class II
and HSA. Moderate levels of MHC class 1, CD80, CD86 and CD40 staining of
al1 DCs sam ples was noted. Staining with monocyte/macrop hage specific
antigen mAb, CDllb, was weak or undetectable. In addition, cultures
contained only rare B cells (8220) (Fig. 4). To further confirm these finding, a
larger group of mice should be used for isolation of DCs from bone marrow.
Table. 4 Flow cytometric analysis of HOX11 and non-transgenic bone marrow dedved DCs. Results are presented in percentage of cells that stained with specific antibody.
Oh cells
In summary, these studies indicated that we were able to isolate and
expand normal numbers of DCs from the spleen and bone marrow of HOX11
transgenic mice. DCs responded normally to stimulation by growth factors such
as GM-CSF. In addition, HOX11 derived DCs expressed normal levels of MHC
class I and class II molecules required for antigen presentation, costirnulatory
molecules such as B7-1, 87-2 and HSA required for the activation of naïve T
cells and the CD40 activation antigen.
3.3.3 HOXl 1 Derived DCs: Presentation of FoMgn MHC
The capacity of HOX11 derived DCs to prime naïve alfogeneic T cells in
vitro was assessed by measuring T cell proliferative responses to specific
foreign MHC presented by DCs. For this experirnent, HOX11 derived DCs were
used as stimulators (presenting their own MHC as an antigen), white C3H and
MHC I [ MHC II CDl lc , HOXI1 44% , Non-Tg ) 39%
15% 67% 56%
87-1 32%
12% 1 35%
87-2 9% 6%
CD40 31% 30%
HSA 92% 86%
Mac-1 8220 1% 5% 2% 3%
C57BU6 derived T cells were used as allogeneic responder cells. As shown in
Fig. 5 stimulation of naïve C3H and C57BU6 derived T cells with HOX11
derived DCs (bred in the CD-1 genetic background) resulted in an 8-10 fold
increase in proliferation relative to the unstimulated control T cens. As a
positive control, C3H derived DCs (H-2*) were used to stimulate C57BU6 (H-
2 9 derived T cells. This stimulation resulted in a 9-12 fold increase in
proliferation relative to unstimulated T celk thereby suggesting that HOXll
derived DCs were similar to C3H derived DCs in their ability to present self-
peptides and activate allogeneic T cells. Negative controls in this experiment
included MLR in which HOX11 or C3H derived DCs were used to stimulate
autologous T cells. Under these conditions, no T cell proliferation was detected.
Similar results were obtained in three additional experiments (Fig . 5).
3.3.4 HOXI1 Derived DCs: Presentation of OVA Protein
Prior to initiating these experiments. we detemined that the optimum
conditions required to induce maximum proliferation of T cells were obtained
when 1 x I o5 DCsfml were pulsed with 60 pg/ml of OVA protein. To detennine
whether HOXI 1 derived DCs were able to present a foreign protein and induœ
a specific T cell proliferative response, OVA pulsed DCs were prepared and
used as stimulators in a MLR. Autologous T cells were isolated from two
HOXl1 transgenic and non-transgenic rniœ used as responder cells. The
results of the expenments showed that HOXI1 derived splenic DCs pulsed with
Figure.5 HOXll miœ (bred in the CD-1 genetic background) derived DCs
presenting their own MHC antigens. Spleen derived DCs were used as
stimulator cells and cultured with C3H (H-2k) and C57BU6 (H-2b) derived T
cells that served as responder cells. As a positive control, C3H derived DCs
were cultured with C57BU6 derived T cells, while as a negative control HOX11
C3H derived DCs were cultured with autologous T cells. Proliferation was
measured by the incorporation of PH] thymidine and reported as mean counts
per min with standard deviation. The same experiment was repeated three
times with similar results.
PRESENTATION OF FOREIGN MHC BY HOX11 DERIVED DCs
Fig u re.6 HOXI 1 transgenic mice denved DCs presenting OVA protein. Spleen
derived DCs from HOXI 1 transgenic and non-transgenic (control) mice were
pulsed with OVA, irradiated and used as stimulator cells in MLR. T cells were
isolated from spleen and served as responder cells. Cultures were incubated for
5 days and T cells proliferative responses to OVA protein measured by
incorporation of [ 3 ~ ] thymidine. The same experiment was repeated three times
and error bars present the standard deviations among the experiments.
CONTROL AND HOX11 DERIVEO DCs PRESENTING OVA PROTEIN FOR THE ACTIVATION OF AUTOLOGOUS T CELLS
OVA protein induced 5-6 fold higher proliferation of T cells, relative to unpulsed
control DCs. In addition, no difference in the stimulatory responses between
HOX11 transgenic (5-6 fold) and non-transgenic mice derived DCs (6-7 fold)
was noted. This experiment was repeated three times with similar results. (Fig.
6)
3.4 Characterization of HOX1I Transgenic Mice Derived T Cells
3.4.1 Proliferation Assay (PHA Stimulation)
These experiments were initiated to compare the ability of normal and
HOXl l derived T cells to proliferate in response to mitogenic stimulation. A
number of agents can activate T cells resulting in cytokine production, cytokine
receptor expression and T cell proliferation. The mitogen used in these
experiments was phytohemagglutinin (PHA) which activates T cells by cross-
linking the TCR. TCR-negative cells will not respond to PHA. Ta test the ability
of HOXI 1 derived T cells to proliferate upon stimulation with PHA, T cells were
isolated from spleens from a total of 5 HOX11 transgenic and 5 non-transgenic
mice ranging in age from 3 to 18 months. T cell proliferation was assessed
using a standard PH] thymidine incorporation assay.
Data shown in Fig. 7 demonstrated that PHA stimulation induced an 80-
100 fold increase in proliferation of stimulated HOXI 1 derived T cells as
compared to unstimulated T cells. Furthemore, the proliferative ability of T
cells isolated from HOXI 1 transgenic mice was similar when compared to age
and sex matched non-transgenic mice (75-90 fold) (Fig. 7).
Figure 7. HOXI 1 transgenic mice denved T cells proliferative responses upon
the stimulation with PHA. Spleen derived T cells isolated from HOXI1 transgenic
and non-transgenic (control) mice were stimulated with PHA for 3 days and T cell
proliferation measu red by PH J thymidine incorporation. The same experiment
was repeated five times using miœ ranging in aga from 3 to 18 rnonth. Enor ban
present the standard deviations among the experiments.
HOXl1 DERIVED T CELL PROLIFERATION ASSAY
( P W
3.4.2 T Ceii Proliferative Responses to Foreign MHC
To assess the ability of HOX11 derived T cells to proliferate after
stimulation by foreign MHC antigens. T cells were isolated from spleens of
HOX11 transgenic and non-transgenic female mice and used as a responder
cell population in an allogeneic MLR. Each group consisted of three mice. C3H
derived, irradiated DCs (H-zk) were used as stimulator cells presenting foreign
MHC antigen. As shown in Fig. 8, HOXl l derived T celfs proriferated in
responds to allogeneic MHC antigens, presented by C3H derived DCs. Under
these conditions, a 12-15 fold increase in proliferation of T cells was detected
when compared with unstimulated T cells. As a positive control, C3H derived
DCs (H-2k) were used to stimulate C57BU6 derived T cells (H-2b). This
stimulation resulted in 1 3-1 7 fold increase in proliferation as compared with
unstimulated T cells and suggested that HOX11 derived T cells did not difFer in
their proliferative responses when compared with C57BU6 derived T cells. In
additional control experiments neither HOXl l transgenic nor C3H derived T
cells proliferated upon stimulation with autologous DCs (Fig. 8).
3.4.3 T Ce11 Proliferative Responses to OVA Protein
To address the question of whether HOXl1 transgenic mice derived T
cells were functionally capable of responding to specific foreign antigens. we
used DCs pulsed with OVA protein ta stimulate autologous T cells. The
proliferative responses of T cells isolated from two HOX11 transgenic and non-
transgenic mice were compared in MLR. AS shown in figure 9, HOX11 derived
Figure 8 HOX11 mice (bred in the CD-1 genetic background) derived T cells
responding to foreign MHC antigen. Spleen derived T cells were used as
responder cells. C3H (H-2k) derived DCs were used as stimulator cells
presenting their own MHC antigens. As a positive control, C3H derived DCs
were cultured with C57BU6 (H-2b) derived T cells, while as a negative control
HOX11 and C3H derived DCs were cultured with autologous T cells.
Proliferation was measured by the incorporation of [)HI thymidine and reported
as mean counts per min with standard deviation. The same experiment was
repeated three timas with similar results.
HOX11 DERIVED T CELL PROLIFERATIVE RESPONSES TO
FOREIGN MHC
Figure 9 Proliferative responses of HOXI 1 transgenic mice derived T cells
to OVA protein in MLR. T cells were isolated from HOX11 transgenic and
non-transgenic (non-Tg) mice and cultured with autologous DCs pulsed
with OVA protein in the presence of DOTAP using different D0TAP:OVA
ratio. After 5 days proliferation of T cells was measured by the
incorporation of [ 3 ~ ] thymidine. The same expenment was repeated three
times with similar results. Error bars present the standard deviations
among the mice in each group.
HOX11 DERNED T CELL PROLIFERATIVE RESPONSES TO DOTAP-
OVA PULSED DCs (pglml)
10 - 9 - 8 - 7 - 6 - 5 - 4 - 3 -
I HOX11 T cells O Non-Tg T cells
2 - 1 - O - m I
DCs presenting OVA protein induœd 10 to 14 fold increase in proliferation of T
cells when compared to unstimulated T cells. MLRs were set up with cells
isolated from HOXl1 and non-transgenic mice. Furthemore, T cells isolated
from non-transgenic mice showed a 8-13 fold increase in proliferation upon
stimulation by OVA presenting DCs as compared to unstimulated T cells.
These results indicated that HOXI 1 derived T cells showed normal levels of
proliferation upon activation by foreign antigens. We also analyzed whether
HOXl 1 det-ived DCs could uptake OVA protein more efficiently in the presence
of lipid (DOTAP). When these data were compared to those from Fig. 6 the
results suggested that the lipid component was not essential to deliver protein
into DCs. This experiment was repeated three times with similar results, using
total of six mice in each group. (Fig. 9)
3.5 Measurement of Cytotoxic T Lymphocyte (CTL) Activity
3.5.7 ln Vitro CTL Assay
These studies were initiated to compare the ability of HOX transgenic and
non-transgenic (control) derived DCs to present OVA protein and generate
OVA-specific CTL responses in vitro. OVA protein is taken up by DCs and
degraded into the peptides. These are further processed and presented as a
class I or class II restricted peptides by MHC molecules for the activation of
CD8 and CD4 T cells. In our assays, no additional cytokines were added and
thus the CTL assay measured not only the ability of antCOVA specific CD8 T
cells, but also indirectly assayed the ability of OVA specific CD4 T cells to
!Ilustration 3. In vitro CTL essay using HOXll transgenic mice derÏved
DCs pulsed with OVA protein. Picture 4A represents target cells (DCs
pulsed with OVA protein), 48 represents effector cells (cytotoxic T cells),
while picture 4C represents mixed effectorharget cell in attempt to induce
specific killing of target cells.
Figure 10 Induction of CTL response in vitro using DCs pulsed with OVA
protein in the presence of DOTAP. Spleen derived DCs from HOXll
transgenic, non-transgenic and C57BU6 mice were pulsed with OVA protein
for 18 hr, irradiated and used as stimulator cells. T cells were isolated from
spleen of 3 transgenic and non-transgenic mice and used as responder cells.
CTL assay was done after two rounds of stimulation and percentage of specific
lysis measured using different target cells (EG 7-OVA. DCs pulsed with OVA
protein and irrelevant EL4 cells) The same experiment was repeated three
tirnes with similar results. Error bars present standard deviation among the
experiments.
IN WTRO CTL ASSAY USlNG OVA PULSE0 DCs
5 0 : l 20:1 1 O : l u n p u l s e d D C s
D C - O V A
50:1 20:1 1 O : u n p u l s e d ant i -H- D C s 2 K b
,*rH 0 x 1 1
N o r m a t
+ C 5 7 B L / 6
Target E.G.7-OVA I OC pulsed with OVA I EL4
T cells #1 + Table. 5 In vitro CTL assay using OVA protein pulsed DCs. Table
present ability of cytotoxic T cells to lyse specific target cells. Data in
percentage are presented for each mouse with average percentage for each
group of mice including HOX11, non-transgenic and C57BU6 rnice.
produce cytokines required by cytatoxic T cells. T cells obtained from three
HOX11 transgenic and three non-transgenic mice were activateci in vitro with
OVA protein pulsed DCs and the resulting T cells evaluated for antigen specific
target cells lysates using the JAM test. Data presented in figure 10 showed that
both control and HOXI 1 transgenic cytotoxic T cells were similar in their ability
to lyse OVA protein pulsed autologous DC targets or E-G7-OVA target cells. In
contrast, there was no or littte response when CTL were rnixed with EL4 or
unpulsed autologous DC targets. Of importance, the anti-OVA cytolytic activity
of the effector T cells was blocked by the addition of a n t i - ~ - 2 ~ ~ mAb, indicating
that the CTL activity detected in these assays was MHC class I restricted (Fig.
1 O).
3.5.2 ln vivo CTL Assay
To test the in vivo ability of HOX11 transgenic DCs presenting foreign
peptides to induce CTL responses, HOXI 1 transgenic, non-transgenic and
C57BU6 mice (two mice in each group) were immunized on two separate
occasions with DCs pulsed with OVA protein. Mice were immunized by
intraperitoneal (1.P) injection of 5 x 10' irradiated OVA pulsed DCslml. with
seven day interval between injections. Sevan days after the second
immunization, mice were sacrificed and isolated splenocytes restimulated in
vitro with either OVA pulsed DCs or E.G7-OVA cells. Cytotoxic activity of
splenocytes was tested using autologous OVA pulsed DCs, E.07-OVA, EL4
Figure 11A and 11 B. Priming of OVA-specific CTLs in mice immunized with DCs
pulsed with OVA protein. Spleen and bone rnarrow DCs were isolated from
HOX11 transgenic. C578U6 and non-transgenic mice, pulsed with OVA protein,
irradiated and injected I.P. into the mice on two separate occasions. Seven days
after the second injections, splenocytes were restimulated in vitro either with DCs
pulsed with OVA or with EG7-OVA tumor cells for 5 days. Cytotoxicity was
measured using a JAM test and presented as percentage. This experiment was
done once using two mice in each group. Error bars represent standard deviation
within the each group.
IN VlVO CTL ASSAY USlNG OVA PROTEIN PULSED DCs
(CTL RESTIMULATED IN WTRO WlTH OVA PULSED DCsL
Target OC pulseci with OVA
+C57BM
+ Normal
,+ HOX11
Target EL4 œlls
Figure 11A.
IN VIVO CTL ASSAY USING OVA PROTEIN PULSED DCs
(CTL RESTIMULATED IN VITRO WlTH E.G7-OVA)
Target E.G7-OVA cells
2 5 3 12.5:1
Target: EL4 cells
Figure 11 B.
Non-Tg T L T
1 DCs pulsed with OVA
r r C
C L
- - -
-
-
-
Table. 6a In vivo CTL using DC pulsed with OVA (restimulated in vitm with DC-OVA)
1 Target
Table. 6b In vivo CTL using DC pulsd with OVA (mtimulatmî in vttn> with E.G.7-OVA)
and autologous unpulsed DCs as targets. As shown in figure 11, HOXl1
derived DCs pulsed with OVA protein were able to induce a strong and specific
in vivo CTL response as rneasured by the percentage of lysed target cells (DC-
OVA pulsed and E-G7-OVA cells). A small percentage of target cells were
lysed in control samples. Since OVA protein is an H-2b restricted protein.
C57BU6 derived cells were used as positive controls in this assay. A
cornparison of the stimulation potential of HOX11 transgenic DCs with C57BU6
and non-transgenic mice derived DCs revealed sirnilar percentages of
cytotoxicity thus demonstrating similar functional ability of DCs and CTL among
different groups of mice (Fig.11).
3.6 Assessrnent of Immunogeneicity of HOXi 1
The ability of HOX11 protein derived peptides and HOXI1 tumor cell
lysates to induce an immune response in naïve mice was assessed using MRL
and CTL assays. To assess the immunogenicity of HOXl1 derived peptides
human HOX11 cDNA was used for the production of in vitro transcnbed (IVT)
HOXlf RNA, while tumor cells, isolated from a seventeen month old HOX11
mouse, served as source of the tumor lysates. It was demonstrated by
histopathofogical analysis that this mouse had splenic lymphoma with
dissemination of lymphoma cells to the lungs, kidney. liver and thymus.
3.6.1 T Cell Proliferative Responses upon the Stimulation with /VT RNA
The ability of DCs pulsed with IV1 HOX11 RNA to stimulate C57BiJ6,
non-transgenic and HOXI 1 transgenic T cells was assessed in MLR assays.
Results using T cells from naïve. C57BU6 and non-transgenic miœ
demonstrated a 4 to 7 fold increase of T cell proliferation after stimulation with
DCs pulsed with IVT HOX11 RNA as compared to unstimulated T cells or T cell
stirnulated with unpuised DCs (Fig. 12)- This indicated that DCs were efficîently
pulsed with IVT HOXll RNA using previously described technique and
strongly suggested that the T cell proliferation was the result of the presence of
HOXl 1 peptide. It also suggested that HOXI1 derived peptides are suffciently
immunogenic to activate T cells in naTve mice. However. HOXl l derived T
ceils showed less than a 1 fold increase in proliferation as compared to 4-7 fold
increase in proliferation of C57BU6 or non-transgenic T cells. These results
suggested that T cells from HOXll transgenic mice were nonresponsive to
HOXI 1 protein, which is constitutively expressed by B cells in HOX11
transgenic mice.
Figure 12 T cell proliferative responses upon the stimulation with HOXl1 RNA
as tested by MLR. DCs isolated from spleen of HOXI 1 transgenic, C57BU6 and
non-transgenic rnice were pulsed with IVT HOX11 RNA, irradiated and cultured
with spleen derived T cells. After 5 days proliferative responses of T cells were
measured by the [ 3 ~ ] thymidine incorporation. The experiment was done once.
MLR STIMULATED WlTH IVT HOX11 .. RNA PULSED DCs USED FOR
IMMUNIZATION IN VIVO
U T cells alone DC-HOX1 1 RNA
I D C unpulsed
T cell
3.6.2 Induction of Primary CTL Response to IVT HOXI 1 RNA in Naïve Mice
Based on the findings from the in vitro experiments, we used an
experirnental system in which primary CTL responses to HOX11 derived
peptide were induœd and evaluated in vivo- Spleen and bone marrow DCs
were isolated from HOX11 transgenic. non-transgenic and C57BU6 mice and
pulsed with HOX11 RNA synthesized in vitro (IVT HOX11 RNA) from a plasmid
encoding the human HOXI1 cDNA. DCs pulsed with OVA protein served as a
positive control. Three groups of mice (five HOX11. six non-transgenic and six
C57BU6 mice) were immunized on two separate occasions with IVT HOXl l
RNA or OVA protein pulsed DCs to stimulate an in vivo HOX11 or OVA specific
CTL response. CTL activity was assayed using difTerent targets such as DCs
pulsed with IVT HOX11 RNA or K3P cells. As shown in figure 13, IVT HOX11
RNA pulsed DCs were capable of ïnducing HOX11 specific responses in naïve
C57BU6 and non-transgenic mice, although HOX11 transgenic DCs pulsed
with IVT HOX11 RNA did not induce a notable CTL responses. Our data
suggest that T cells from HOXll transgenic mice have either been deleted
during thymic selection or have been rendered non-responsive to HOX11-
derived peptides as a result of constitutive expression of the human HOXl l
transgene in B lymphocytes. (Fig. 13).
Table 7 In vivo CTL assay using OC8 pulsed with IVf HOX11 RNA. Cytotoxic T celi
ability of HOXl1 transgenic, C57BU6 and non-transgenic mice to lyse specific target
cells is measured in percentage and presented for each mouse used in this assay with
average for HOX11, C57BU6 and non-transgenic group of mice.
Target
E:T
C57BU6 T cells #1
#2
I #3
#4
Average % lysis
Non-Tg T cells #I
#2
#3
#4
Average % lysis
HOXIi T celIs #1
#2
#3
Average % lysis
DC pulsed with IVT -
K3P
12.5:1
13%
14Oh
18Oh
13Oh
15% '
13%
15%
19%
24Oh
18%
6Oh
0%
0%
2%
HOX11 RNA EL4
50:1
27%
38%
24%
29%
' 2511
14Oh
25%
21%
15%
30%
40%
27%
38%
33%
35%
4%
9%
9%
9%
19%
19%
18%
20°h
25%
21%
10%
1%
0%
4 O h
Illustration 4. Restimulation of splenocytes isolated from HOX11 transgenic,
non-transgenic and C57BU6 mice after immunization with DCs pulsed with IVT
HOX11 RNA. Splenocytes were restimulated in vifm with pulsed DCs.
Development of clusters presenting expanded anti-HOX11 specific T cells were
observed in cultures containing cells derived from C57BU6 (B) and non-
transgenic (C) mouse, but in a culture containing T cells from HOX11 mouse no
clusters were noted (A). Picture D present T cell clusters expanded from
splenocytes from HOXl 1 transgenic mouse immunized with DCs pulsed with
OVA and restimulated in vitro.
Figure. 13 in vivo induction of HOXll RNA-specific CTL in HOXlI transgenic
and naïve (C57BU6 and non-transgenic) mice immunized with DCs pulsed with
IVT HOXl l RNA. Spleen and bone marrow derived DCs were pulsed with IVT
HOXI 1 RNA, irradiated and injected i.p. into the mice on two separate occasions
(with the interval of 7 days). Seven days after the second injection. splenocytes
were restimulated in vitro with IVT HOXl l RNA pulsed DCs and pementage of
lysis measured by JAM test. DC pulsed with IVR HOXl1 RNA. E.G.7-OVA and
EL4 cells were used as target cells. This experiment was done once with total of
four C57BU6, four non-transgenic and three HOXl1 transgenic mica. Error bars
present standard deviations within the each group.
CTL ASSAY AFTER IMMUNIZATION WlTH IVT HOX11 RNA PULSED
DCS
DCHOXll RNA
.r
40% - -. - 30% - 0 .- X 0 20% - O CI Target: EL4
B
e h -
Wh - .% 40% O
*-
:: Ph- u O C
rnh-
looh
OO/o 1 1 1
501 B.1 1251
E
3.6.3 Induction of CTL Response upon Stimulation witb Tumor Lysates
This experiment was designed to compare the ability of T cells isolated
from HOXll transgenic mice with lymphoma and naïve C57BU6 mice to
generate CTL responses in vitro after stimulation with lymphoma antigens.
Since the expansion of DCs from bone rnarrow cells requires 6 days. it was
necessary to cryopreserve spleen cells as a source of T cells. To ensure that
CTL responses were fymphoma antigen specific response and not an artifact
induced by the freezing procedure, fresh T cells were isolated from the spleen
of a syngeneic C57BU6 mouse on the day the CTL assay was set up. In so
doing, we were able to compare fresh and frozen T cell responses. As shown
in figurel4, presentation of tumor antigens by C57BU6 derived DCs resulted in
the induction of CTL activity as measured by percentage of lyses of dïfFerent
target cells (DCs pulsed with either IVT HOXl1 RNA or tumor cells lysats).
However, a lymphoma antigen specific response was seen only in assays
using C57BU6 derived T cells but not when HOX11 transgenic rnouse derived
T cells were used. These results lead us to several conclusions. First, they
suggested that HOXl 1 transgenic mouse derived lymphoma ceIl lysates were
a good source of tumor associated antigens, which when presented by DCs.
were sufficiently immunogenic to induce T cell response in naïve mice. Second,
the low percentage of killing found in HOXll derived T cells support findings
from the in vivo experiments using IVT HOXI 1 RNA suggesting that HOX11
transgenic T cells have been rendered non-responsive to HOXI 1 derived
peptides. Furthemore, since the T cells used in above experiment were
isolated from the teminally il1 mouse in which the lymphoma developed, the
results provide preliminary data suggesting that the process of
lymphomagenesis in HOX11 transgenic mice is associated with the induction of
a nonresponsive state of T cells specific for HOX11 antigens.
Target
E:T
HOX11 frozen T
cells C57BU6 frozen T
cells C57BU6 fresh T cells
Table 8 In vitro CTL assay using DCs pulsed with lymphoma cell lysates. Ability
of cytotoxic T cells from teminally il1 HOXt l transgenic mouse and control C57BU6
mice to lyse specific target cells is presented (in percentage).
DC pulsed with IVT HOXl i RNA
20:L
2%
10%
13%
DC pulsed with Lymphoma Cell
Lysates 80:l 40:l
EL4
9%
28%
25%
20:l
5%
12%
15%
8O:L
16%
30%
33%
80:l
5%
7%
1
5%
15%
17%
40:l
10%
24%
21%
40: 1
3%
2%
/
Figure. 14 Induction of C I L response N, vitro by DCs pulsed with lymphoma ceIl
lysates. Bone mancw derived DCs from tenninally il1 HOXl1 mouse and control
C57BU6 mice were pulsed with lymphoma cell lysates and used as stimulator
cells in CTL assay. T cells were isolated from spleens and used as autologous
responder cells. CTL assay was done after two rounds of stimulation using DC
pulsed with tumor cell lysates, DCs pulsed with IVT HOXll RNA and unpuised
DCs as target cells.
IN VITRO CTL ASSAY USlNG DCs PULSED WlTH LYMPHOMA CELL LYSATES
Target- DC-HOXI 1 RNA
w I rn 80: 1 40:1 20: 1
Target- DC-Tum-Lys.
-C- HOX11 T cells
+CS7BU6 T cells
+C57BU6 f'fesh T cells
80:1 40:1
Target- unpulsed DCs
Chapter 4
Discussion and Future Experiments
4.1 Discussion
Non-Hodgkin's lymphomas are a heterogeneous group of disorders
characterized by malignant proliferation of subpopulations of B or T lymphoid
cells. The nurnber of new cases of NHLs has increased in Western countries
since 1960. More then 85% of NHL patients have advanced disease at the time
of initial presentation. Aggressive chemotherapy is the primary form of therapy
for h ig h and intemediate-grade NHLs. Since lymphomas disseminate widely
by hematogenous routes, radiation therapy is usually considered an adjunct to
chernotherapy rather than a primary treatrnent. Relapse rates post bone
marrow transplantation are also high making the overall prognosis for patients
with NHL poor, despite improvements achieved by cornbined radiation and
chernotherapy.
Given the increasing evidence that chemotherapy, radiotherapy, and
stem cell transplantation approaches do not cure the majority of patients with 6
cell lymphoma, new investigative treatrnents are being consideredl? Several
studies have shown that the host defense against tumors is reliant on the
immune systern, supporting a potential for cancer immunotherapylm-1. Most
cancer irnmunotherapeutic approaches are based on modification of the host
immune system to promote tumor eradication. Further, since most tumors are
poorly immunogenic and escape host immune detection much research has
been focused on the identification of tumor specific antigens and the
modification of tumor cells to make them more immunogenic and more
susceptible to endogenous effector mechanisrns. Studies using animal models
and pilot human clinical trials are underway to determine whether potential
tumor specific antigens can indeed be recognized by CTLs and, consequently,
induce tumor eradication.
Areas of immunotherapy specifically directed at the treatrnent of 6 cell
lymphoma, which are currently being evaluated in clinical trials, incfude: (1)
vaccination with idiotype derived peptide epitopes presented on professional
APC, (2) vaccination with CD40 activated lymphoma cells and (3) adoptive
transfer of lymphoma specific T cells 166.167.
In addition to these strategies for cancer immunotherapy, DC-based
immunotherapy is rapidly emerging as a potentially viable alternative.
Increased interest in DCs has been based on the hypothesis that only
optimized antigen presentation by APCs will induce T cell activation and
suffîcient anti tumor immune responses in vivo to eradicate tumor cells. The
pivota1 role of DCs in the induction of cellular immune responses has focused
attention on DCs as a potentially powerful tool for enhancing anti-tumor
immunity. Results obtained in animal models. using mouse transplantable
tumor cells have demonstrated the effectiveness of tumor peptide pulsed DCs
in stimulating both naïve CD4 and CD8 T cells in vitro and in vivo 102,168.
Furthemore, immunization of mice with DCs pulsed with tumor lysates are able
to induce protective imrnunity against subsequent lethaf challenges with live
tumor cells '05. In animais model studies using transplantable 6 cell lyrnphoma
a single immunization with DCs pulsed with tumor specfic idiotype protein was
shown to induce regression of tumors as well as protecting mice against future
inoculations with lethal doses of tumor cells169.
Results from human clinical trials in which patients received several D G
idiotype infusions dernonstrated clinical responses. However the therapeutic
benefits were less dramatic than would have been predicted based on the
results from murine studies 121 36%
One possible reason for the poorer than expected results obtained in
pilot clinical trials of DCs based immunotherapy is the discrepant pattern of the
animal lymphoma models currently used to assess DC based therapeutic
modalities, compared to human lymphoma. The best responses in animal
studies were in fact obtained when non-tumor bearing animals were immunized
with tumor antigen presenting DCs, pnor to tumor ~halIenge103*~05. When
immunization occurred after the establishment of the tumor, outcomes were
significantly poorer. Therefore, these murine transplantable tumor model
systems are not reflective of the process of tumor progression that occurs in
patients with NHLs in which tumor cells arise spontaneously, acquire
mutations, escape immune detection and invade distant sites. Thus model
systems are required in which tumors arise spontaneously and in which tumor
progression is no separated from the host immune systern and the local tumor
environment.
A long terni goal of our laboratory research is to assess the applicability -
of DC based immunotherapeutic protocols for the treatment of non-follicular B
cell lymphoma. To achieve our goals we used a HOX11 transgenic rnouse
model. which was produced in our laboratory by introdwing a transgenic
vector, consisting of the human HOX11 cDNA under the transcriptional control
of the murine IgH promoter and enhancer, thus driving the expression of the
HOX11 transgene during the B lineage development. HOX1 1 transgenic rnice
were healthy at birth and analysis of cells isolated from bone rnanow, thymus,
spleen and lymph nodes revealed normal B and T lymphopoiesis. However,
eig hty-five percent of mice spontaneously developed lymphoma during their
second year of He. Lymphomas were of a mature B cell phenotype with the
spleen being the main organ of tumor development. The lymphomas developed
in HOXll transgenic mice were low grade, indolent lymphomas with slow
progression to a hig hly malignant phenotype. Moreover, the lymphomas were
clinically and morphologically similar to human splenic marginal zone
lymphoma (SMZL) (as determined by Dr. Megan Lim, Sunnybrook 8 Women'
College Health Science Centre). Patients with SMZL are initially asymptomatic,
but at the tirne of clinical presentation, the disease has usually disseminated,
which is associated with a very poor prognosis even after aggressive
chemotherapy. Therefore, HOX1 1 transgenic mice provide a clinically relevant
model for a subtypa of human indolent lymphoma, which will be a powerful
systern to assess novel approaches for the treatment of NHL.
Given the complexity of currently available animal models, the main
advantage of this model is the predictable spontaneous development of low to
intermediategrade mature 6 cell lymphomas with a premalignant phase k ing
detected as earîy as four weeks of age. This animal model may serve as a
unique pre-clinical model to study immunotherapeutic approaches to lymphoma
therapy.
Normal APC and T cell functioris are essential for induction of proper
antitumor responses in vivo. It was therefore necessary to carefully
characterize the functional properties of the immune system of HOXll
transgenic miœ before proceeding with studies designed to asses DC based
irnmunotherapeutic protocols for lymphoma therapy. Several reasons lead us
to select this strategy. First, it haî been well documented that although IgH
promoter and enhancer sequences prirnarily direct expression throughout B
cell developrnent, leaky expression in T lymphocytes and myeloid cells is
frequently seen. Therefore it was critical to assess whether HOX11 transgenic
T cells and DCs were functionally cornpetent. Second, the HOXI1 gene is
nonnally not expressed in T and B cells and the ectopic expression of this
transcription factor could activate expression of different cytokines or adhesion
molecules that could perturb the ability of HOXI1 transgenic mice to induce an
immune response. For example, abundant expression of 11-10, a potent
immunosuppressive cytokine, may prevent DC accumulation and antitumor
immunity, as demonstrated in some animal models170. Based on this
knowledge. we analyzed the functional properties of HOX11 derived DCs and T
cells.
Characterization of HOXI 1 Derived DCs
DCs were isolated from spleen and bone rnarrow and ouf analysis
demonstrated that HOXI1 transgenic mice had nonal numbers of DCs. These
DCs could be expanded in culture suppleniented with GM-CSF and 11-4. Under
these conditions Î t was possible to reliably obtain large numben of HOXl l
DCs, further indicating that HOXll DCs showed a nomal proliferative
response to cytokines.
Phenotype Characterization
It is well established that proper antigen presentation and T cell
activation are dependent on the expression of specific cell surface antigens by
DCs. FACS analysis was therefore used to characterize the phenotype of
HOXl 1 transgenic mouse derived DCs171-173. Splenic DCs isolated from
HOX11 transgenic rnice of different ages showed a typical DCs surface antigen
phenotype when compared to DCs isolated from non-transgenic mice. These
cells were mainly mature DCs as demonstrated by the presence of suiface
markers specifically expressed at that stage (CDllc, CD80, CD86, CD40,
MHC class I and class II). Phenotypic analysis of bone mamw derived DCs
revealed several DCs subpopulations in both control and HOXI 1 transgenic
mice. These included subpopulations of DCs expressing high levels of MHC
class II and HSA molecules, moderate levels of MHC class 1, CD80 and CD86
molecules. and low levels of CD1 l c and Mac-1. These expression profiles are
typical of an immature stage of DC development
These results are important with respect to both spleen and bone marrow
derived DCs. since MHC class I and class II molecules are necessary for the
antigen presentation and consequently activation of CD8 and CD4 T cells,
respectively. Furthemore. CD80 and CD86 are CO-stimulatory molecules also
required for T cell activation. while CD40 is required for DC activation and
maturation. Thus, the results document that the profile of antigens expressed
by HOXll mice DCs is consistent with a normal OC phenotype. Some
exampies of differences in levels of expression were seen among HOX11
transgenic mice, but this was also observed among normal mice, and therefore
probably represents normal variability of expression of cell surface antigens
detected on DCs isolated from different mice.
Presentation of MHC in Allogeneic MLR
The main objective of the next experiment was to evaluate the ability of
DCs to present antigens using allogeneic MLR. HOX11 derived DCs
(stimulator) were cultured with either C3H or C57BU6 derived T cells
(responder). The proliferation ability of responder cells were wmpared with
those obtained from cultures containing C3H derived DCs (stimulator. H-2k)
and C57BU6 T cells (responder. H-2b). When HOXll derived DCs were
compared with C3H denved DCs with respect to their ability to stimulate
C57BU6 T cells, similar results were observed. Since this assay requires OC to
be able to present their own MHC proteins and present them at the ce11 surface
for the activation of allogeneic T cells, our resuîts indicate that HOX11 derived
DCs are functionally competent in their ability to process and present antigens.
Presentation of OVA Pmtein in MLR and CTL Assays
Both in vitro and in vivo expenmental systems were next used to assess
the ability of HOX11 derived DCs to present foreign antigens. Recently, several
studies showed that DCs can present antigenic epitopes denved from
exogenous antigens onto MHC class I molecules and induce an antigen
specific CTL response when pulsed with soluble proteins such as OVA
proteinla*.
The efficiency of HOXl1 denved DCs to present foreign antigens was
first tested in syngeneic MLR assays. in which OVA protein was presented by
HOXl 1 transgenic mice dedved DCs to autologous T cells. The results
demonstrated the ability of HOXll derived DCs to present OVA protein by
inducing 4-6 fold higher proliferation of stimulated T cells when compared to
non-stimulated T cells. Also, when these results were compared with those
obtained with DCs derived from non-transgenic mice, no differences were
found. Since protein loading of DCs requires uptake, intracellular processing
and presentation by both MHC class I and class II molecules. HOXI1
transgenic derived DCs demonstrated a nomal ability to present both class I
and class II restricted antigens and subsequently activate CD4 and CD8 T
cells.
In vitro CTL
To assess the induction of MHC class I restricted CTL responses by
HOX11 derîved DCs, transgenic and control DCs were pulsed with OVA protein
and used to stimulate autologous T cells using in vito CTL assaysW
Expanded OVA specific T cells were able to lyse OVA antigen expressing
targets. whereas M e or no lysis were detected if T cells were expanded with
unpulsed DCs or when an irrelevant tumor cell line was used as a target cells.
Furthemore, anti-OVA cytolytic activity of these effedor T cells was blocked by
the addition of an t i -~ -2~ rnAb and blocking the presentation of OVA by DCs,
indicating that these CTLs were indeed MHC class I restricted. Therefore, our
results demonstrated that HOXI 1 derived DCs can be ef'ficiently pulsed with
OVA protein and used as antigen presenting cells to activate the cytolytic
activity of antigen specific CTL cells.
ln vivo CTL
In vivo experiments were undertaken to examine the capacity of HOX11
transgenic derived DCs ta present OVA protein and induce antigen specific,
primary immune responses. It is well demonstrated that bone rnarrow derived
DCs can present exogenous OVA protein and induce high level of CTL
responses in vivo and protective antitumor immunityl74. Moreover, the
identification of MHC class I (H-2b) restricted be presented by MHC class I
molecules and activated cytotoxic T cells10g. epitope that is recognized by CD8
CTL in C57BU6 mice indicated that OVA antigens can For the current study,
HOX11 transgenic, non-transgenic and C57BU6 mice were immunized on two
separate occasions with syngeneic spleen and bone marrow derived DCs
pulsed with OVA protein. Similar levels of CTL adivity were observed among
HOXI 1, C57BU6 and non-transgenic mice. These results are in accordance
with our in vitro results and confimi that HOXi1 derived DCs are indeed
efficient antigen presenting cells. capable of inducing OVA specific CTL
responses in HOXl1 transgenic rnice.
Characterization of HOX11 Derived T Celis
Proliferative Response to PHA
For our studies it was neœssary to assess the functional properties of T
cells isolated from HOX11 transgenic mice, despite the fact that it was
previously reported that thymocyte development was unperturbed in HOXI1
mice with phenotypically normal T cell subpopulations in the thymus and
periphery. In wntrast to cytotoxicity, proliferation is not a specific effector
function of T lymphocytes, although it has been widely used to assess the
overall immunocompetence of an animal. One of the methods to study T cell
proliferative responses involves the stimulation of T cells with mitogenic
stimulators. T cells isolated from HOXI 1 transgenic and non-transgenic mice
were compared with respect to their proliferation responses to PHA. Our results
demonstrated no difference in the proliferative ability between non-transgenic
and HOXI 1 transgenic derived T cells.
Proliferative Response to Foreign MHC
To evafuate more specifically T cell proliferative responses, allogeneic
MLRs were used. in which T cell proliferation was assessed upon the
stimulation with allogeneic DCs. HOX11 transgenic and C57BU6 (H-2b) derived
T cells were cultured with C3H (H-2k) derived DCs with similar levels of
proliferation between the two groups of mice being observed. Ttius, this result
further supports the conclusion that HOXI 1 transgenic T cells are functionally
competent.
ProMerative Responses to OVA Protein in MLR and CTL Assays
T cell proliferative responses were also evaluated using DCs pulsed with
OVA protein as a stimulator. This is a more specific assay than allogeneic MLR
because foreign protein presented by both classes of MHC molecules on DCs
must be recognized by autologous T cells for activation of CD4, which secrete
cytokines required for the activation CD8 T cells. HOXI 1 transgenic and non-
traiisgenic derived T cells were used as responder cells. which were stimulated
with OVA pulsed autologous DCs. The results demonstrated comparable
proliferative responses between HOX11 transgenic and control rnice. Thus
HOXl1 transgenic derived T cells are normal with respect to their proliferative
responses upon stimulation with foreign antigens.
In vitro CTL
CTL activity of HOX11 transgenic derived T cells was further detennined
by in vitro and in vivo assays. Since cytolyüc activity may be induced by a large
number of antigens, we used OVA protein rather than a single OVA peptide
epitope to pulse autologous DCs and stimulate production of OVA specific CTL
responses in vitro. HOXI 1 transgenic and non-transgenic derived T cells were
used to expand sufficient number of CTLs by Wo rounds of stimulation with
pulsed DCs. All mice used in these assays had been backcrossed for more
then 4 generation ont0 C57BU6 background. since OVA protein is specific for
that strain of mice. The results indicated that primed cytotoxic T cells from
HOX11 transgenic mice had a similar ability to lyse OVA expressing target cells
as was found with normal and C57BU6 derived cytotoxic T cells. In contrast,
there was little or no response when irrelevant target cells were used.
Combined, these data support the conclusions drawn from allogeneic MLR,
that T cells derived from HOX11 transgenic mice are functionally competent to
induce an immune response against foreign antigens.
In vivo CTL
For in vivo assessrnent of the generation of anti-OVA CTL activity,
HOXl 1 transgenic derived T cell HOXI 1, non-transgenic and C57BU6 mice
were immunized on two separate occasions with spleen and bone marrow
derived DCs pulsed with OVA protein- These immunizations resulted in
activation of OVA specific T cells in each mouse group, which were shown in in
vitro assays to be capable to lysing different target cells expressing OVA. The
generation of OVA specific CTL activity requires the activation, in vivo, of both
CD4 and CD8 T cells by MHC class I and class II restricted peptides. These
observations reflect normal functions of HOXl 1 derived T cells.
Combined, these data indicate that HOXli DCs and T cells from healthy
mice are phenotypically and functionally normal in their ability to process.
present and respond to foreign antigens. Furthermore, the expression of the
HOX11 transgene and lymphoma development do not compromise these cells,
supporting our hypothesis that HOX11 mice will provide a very powerful model
s ystem in wh ich to assess DCs based immunotherapeutic protocols.
Assessrnent of Immunogenicity of HOX11
Other critical questions addressed in this study are: (1) whether HOXj1
protein derived peptides are sufficiently immunogenic to induce HOXf 1 specific
immune response in naïve mice and (2) whether HOX11 iymphoma
progression in HOXl l transgenic mice is associated with the induction of
Iymphoma antigen-specific T cell tolerance.
Induction of Immune Responses in Naïve Mice
To address the first question, C57BU6 and non-transgenic mice were
injected on two separate occasions with bone manow or spleen denved DCs
pulsed with IVT HOX11 RNA. These RNA pulsed DCs were capable of
stirnulating primary CTL responses in vivo, inducing killing of target cells
expressing HOXl l (K3P) and autologous DCs pulsed wlh IVT HOX11 RNA.
This result indicates that HOXI 1 derived peptides are sufficiently immunogenic
to induce specific T cell responses. In addition to IV1 HOX11 RNA. tumor
lysates (from terminally il1 HOXI1 transgenic mouse) were used to stimulated
C57BU6 derived DCs. These tumor lysates presenting DCs induced a CTL
immune response as detemined by in vitro CTL assays. Data suggested that
CTL specific proliferative responses were induced and these cells were
capable of lysing autologous DCs pulsed with tumor Iysates. The main finding
of this expenment was that tumor cell developing in HOXI1 transgenic mice
can serve as effective immunogens for the presentation of MHC class I and
class II restricted antigens. which may function for the activation of both CD4
and CD8 T cells. However. this was shown only in vitm using a small group of
C57BU6 mice, and additional in vivo expenments must be done to draw a firm
conclusion.
Induction of immune Responses in HOX1 i Transgenic Mice
To address the second question, similar experiments to those described
above were used to evaluate the induction of antigen specific CTL responses in
HOX11 transgenic mice upon the stimulation with IVT HOXll RNA and tumor
lysates pulsed DCs. For in vivo studies. HOXll transgenic mice were
immunized with IVT HOXI I RNA pulsed DCs on two separate occasions and
results indicated that HOXll derived T cells were not activated by DCs
presenting HOXl l protein derived antigens. with no induction of antigen-
specific CTL being observed. These findings were predictable given that the
HOX11 protein is constitutively expressed by B cells in transgenic mice and
would thus represent a 'self" protein. Since previous experiments
demonstrated the immunogenic~ of the HOXll protein, one possible
explanation for this result is that anti-HOX11 specific T cells were either deleted
during thymocyte development of rendered non-responsive.
To address the question of whether lymphoma progression in HOXl l
transgenic mice is associated with toleration of lymphoma antigen specific T
from a terminally il1 HOXl1 transgenic mouse with lymphoma were stimulated
in vitro with autologous tumor lysates pulsed DCs. There was no expansion of
specific CTL in culture and antigen specific target cells lysis was not detected.
Although this expenment neeâs to be expanded to include a large? number of
HOXll transgenic mice with lymphoma with both in vitro and in vivo assays
used, this result is of significance because of its accordance with our in vitro
results. lt indicates again that lymphomagenesis in HOX11 miœ is associated
with the toleration of anti-lymphorna antigen-specific T cells. HOX11 mice thus
represent a clinically relevant madel in which to assess novel therapeutic
approaches for the treatrnent of indolent nonfollicular lymphoma.
Studies proposed by this master's thesis advanced our understanding
regarding the relationship between expression of HOX11 transgene and
inductior: of antitumor immune responses in HOX11 transgenic miœ. Also,
these studies helped us reveal that HOX11 protein derived peptides and
lymphoma antigens are sufficiently immunogenic to induce immune responses.
Finally, these studies have provided preliminary data with respect to a possible
mechanisrns by which lymphomas develop in HOXl1 transgenic mice leading
likely to non-responsive state in anti-tumor specific HOX11 derived T cells.
4.2 Future Studies
Numerous studies have shown that preventative imrnunization before the
inoculation of tumor cells into healthy rnice can be highly effective at inducing
tumor specific immune responses, resulting in rejection of tumor cells.
However, vaccination of a tumor-bearing host in the late stage of tumor
developrnent usually fails to induce specific immunity. These findings lead to
the conclusion that specfic mechanisms are used by malignant cells, which are
responsible for a lack of efficient immune response. One possible mechanism
of tumor evasion is the development of antigen-specific T cell anergy, which
may occurs as an early event during tumar progression leading to a deficient
antigen-specîfic T cell responsesl?? Toleranœ to tumor antigens may result
from a decreased clonal frequency (peripheral deletion) or the induction of
proliferative unresponsiveness (clonal anergy). In addition, self-antigens may
be the most relevant and abundant antigens expressed by malignant cells and
the host's immune system must be tolerant of these antigens. The same
rnechanisms that mediate toleranœ to self-antigens may represent a potential
barrier for the full development of effective immune responses against antigens
expressed by tumors. In the current study, antigen-specific T cell toleranœ to
HOXI 1 derived peptides was observed since HOXl1 denved T cells were not
activated by autologous HOXI 1 RNA. In addition, DCs pulsed with lymphoma
cell lysates also failed to induce immune response. However, to better evaluate
the potential immunogenicity of tumor lysates isolated from HOX11 transgenic
mice with lymphoma, more studies need to be done.
(1) Future experiments will include injection of HOXl1 denved DCs pulsed with
lymphoma cell lysate into Young, healthy HOXI1 transgenic recipients. If
HOXI 1 lymphomagenesis is associated with the generation of lymphorna
associated antigens, recipient mice will mount Ipphoma-antigen specific
CTL responses. These studies will suggest the existence of different
antigens. in addlion to HOX11, presented on lyrnphoma cells and that the
lack of an immune response in sick rnice is due to the toleranœ of
lymphoma antigen specific T cells.
(2) There is evidenœ to suggest that CTLA4 (negative regulator of T cell
function that binds to the 67 molecule) activation result in the down
regulation of an immune response. Furthemore, the blockade of C T W
can lower the fhreshold for T ceH activation. Moreover, in vivo blockade of
CTLA4 may induce the regression of established tumors. We will design
future studies using CTLA4 blocking antibodies in an attempt to reverse
the non-responsive state of lymphoma antigen specific T cells in HOX11
transgenic rnice. HOX11 transgenic mice will be injected with CTLA-4
blocking antibodies and then immunized with tumor lysate or IVT HOX11
RNA pulsed DCs, while some mice will receive only pulsed DCs. We
anticipate that these manipulations will also increased levels of anti
lymphoma immune responses in HOXI 1 transgenic mice receiving CTLA-4
and antigen pulsed DCs.
(3) Different experiments will be initiated to detenine whether the anergic
state in HOX11 transgenic mice with lymphoma can be reversad by
culturing tumor antigen presenting DCs and T cells with various cytokine
combination (11-2, 11-12 or INF-y). We will also detemine whether the
injection of CD40 activation antibody facilitates the in vivo presentation of
lymphoma associated antigens and the activation of specific T cells
These experiments should provide important data, which wRI assist in the
developrnent of immunotherapeutic protocols for the treatrnent of NHL.
4.3 Summary
We have demonstrated that DCs isolated from HOX11 transgenic mice
show a typical DCs surface antigen phenotype when cornpared to DCs isolated
from normal mice. In addition. we used two stringent experimental systems to
assess the antigen presenting ability of HOXII derived DCs: MLR and CTL
assays. The data obtained suggest that HOX11 derived DCs are able to
present both foreign MHC antigens and OVA-protein. Consistent with this are
the in vivo findings that HOXI 1 mice injected with OVA pulsed DCs are able to
activate both MHC class I and class II restricted OVA specific T cells. HOXl1
derived T cells show normal overall proliferative responses (PHA) and normal
proliferative responses to foreign MHC. These cells are also able to proliferate
upon stimulation with autologous OVA protein pulsed DCs.
We also demonstrated that HOX11 peptides or lymphoma cell lysates
are sufficiently immunogenic to induce specific immune responses in naïve
mice, since irnmunization with pulsed DCs induced proliferation of CTLs able to
lyse target cells. However, HOX11 derived T cells were not activated in vivo by
DCs pulsed with IVT HOX11 RNA. Moreover, DCs pulsed with lymphoma cell
lysates failed to activate autologous T cells isolated from the HOX11 mouse in
which the lymphoma developed. These data suggest that lymphoma
developrnent in HOX11 mice is associated with the tolerization of lymphorna
antigen specific T cells. Overall, these studies pave the way to use HOXll
transgenic mice modal to develop DC based immunotherapeutic protocols for
the treatrnent of spontaneously aflsing indolent nonfollicular lymphomas.
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