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Adoptively transferred tumor-specific T cells expanded ex vivo using HSV amplicons encoding
4-1BBL persist in the host and demonstrate anti-tumor activity in vivo
Kyung H. Yi1,4
, Hovav Nechushtan5, William J. Bowers
2,3, Dien G. Pham
5, Yu Zhang
5,
Khaled A. Tolba
5
, Eckhard R. Podack
4
, Howard J. Federoff
1,2,3
, and Joseph D. Rosenblatt
4,5,6,7
Departments of Microbiology and Immunology1, Neurology2, and the Center for Aging and
Developmental Biology3, University of Rochester School of Medicine, Rochester, NY 14620,
Department of Microbiology and Immunology4, University of Miami School of Medicine,
Miami, FL 33136, and University of Miami Sylvester Comprehensive Cancer Center5, Miami,
FL 33136.
Address correspondence to: Joseph D. Rosenblatt, University of Miami SylvesterComprehensive Cancer Center, Division of Hematology/Oncology, 1475 NW 12th
Avenue (D8-4), Suite 3300, Miami, FL 33136. Phone: 305-243-4860; Fax: 305-243-9161; email:
Running title: 4-1BBL in adoptive immunotherapy
Key words: Costimulation, T lymphocytes, adoptive immunotherapy, HSV amplicons, 4-1BBL
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Footnotes
6 This work was supported by National Institutes of Health Grant RO1 CA87978 and RO1
CA74273, the Leukemia and Lymphoma Society of America, and the Rochester Nathan Shock
Center.
7 Address correspondence and reprint requests to Dr. Joseph Rosenblatt, University of Miami
Sylvester Comprehensive Cancer Center, Division of Hematology/Oncology, 1475 NW 12th
Avenue (D8-4), Suite 3300, Miami, FL 33136. Email address: [email protected]
8Abbreviations used in this paper: 4-1BBL, 4-1BB ligand; LLC, Lewis lung carcinoma; HSV,
herpes simplex virus I; LacZ, β -galactosidase; MOI, multiplicity of infection.
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Abstract
4-1BB is a T cell costimulatory receptor which binds its ligand 4-1BBL, resulting in
prolonged T cell survival. We studied the anti-tumor effects of adoptively transferred tumor-
specific T cells expanded ex vivo using tumors transduced with herpes simplex virus (HSV)
amplicons expressing 4-1BBL as a direct source of antigen and costimulation. We constructed
HSV amplicons encoding either 4-1BBL (HSV.4-1BBL) or B7.1 (HSV.B7.1) costimulatory
ligands. Lewis lung carcinoma cells expressing ovalbumin (LLC/OVA) were transduced with
HSV.4-1BBL, HSV.B7.1, or control HSV and used to stimulate GFP+ OVA-specific CD8+ T
cells (OT-1/GFP). Naïve or ex vivo-stimulated OT-1/GFP were then adoptively transferred into
LLC/OVA tumor-bearing mice. Mice receiving OT-1/GFP stimulated with HSV.4-1BBL had
significantly decreased tumor volumes compared to untreated mice (P<0.0001) or mice into
which naïve OT-1/GFP were transferred (P=0.0003). In contrast to HSV.4-1BBL-expanded OT-
1/GFP, transfer of HSV.B7.1-stimulated OT-1/GFP did not protect mice from tumor. Higher
percentages of OT-1/GFP cells were seen in the peripheral blood for the HSV.4-1BBL-
stimulated OT-1/GFP group compared to other experimental groups. Splenocytes from mice that
received HSV.4-1BBL-stimulated OT-1/GFP exhibited increased cytolytic activity against
LLC/OVA and higher percentages of OT-1/GFP cells, which expressed CD44 and the memory
marker Ly-6C, compared to controls. Increased tumor-free survival was observed 45 days
following adoptive transfer of HSV.4-1BBL-stimulated OT-1 compared to transfer of naïve OT-
1 into tumor-bearing mice. Tumor-specific T cells stimulated ex vivo using tumor transduced
with HSV.4-1BBL expand in vivo following adoptive transfer, resulting in tumor eradication and
the generation of tumor-specific T cells of memory phenotype.
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Introduction
Adoptive transfer of autologous tumor-specific T cells is a promising procedure for
cancer immunotherapy. T cells isolated from tumor-infiltrating lymphocytes, tumor-draining
lymph nodes, or peripheral blood contain tumor-specific cells, which can be expanded ex vivo
and transferred back into the host to treat established disease. Several types of cancers including
metastatic melanoma (1), renal cell carcinoma (2), and glioma (3), show encouraging results
when treated with ex vivo-expanded cytotoxic T lymphocytes. Clinical trials have indicated that
immune ablation is an effective preconditioning procedure that can increase T cell responses
after adoptive transfer (1). Additional strategies are needed to improve generation of more potent
tumor-reactive T cells for adoptive transfer. Areas of development include optimization of in
vitro T cell expansion and culture, characterization of potent T cells for transfer, and preservation
of function and survival of transferred cytotoxic T cells. Under favorable circumstances,
adoptive transfer has the potential to induce long-lasting effects via the establishment of
immunologic memory.
4-1BB (CD137, ILA, TNFRSF9) is a 30-kDa type I transmembrane glycoprotein
belonging to the TNF receptor superfamily (4, 5). Expression of the transmembrane form of 4-
1BB has been observed in a broad range of both myeloid and lymphoid cells, including CD4+
and CD8+ T cells, intraepithelial lymphocytes, natural killer cells, monocytes, and dendritic cells
(5-7). 4-1BB is usually induced on cell surfaces following activation, but dendritic cells express
4-1BB constitutively (7). This is in contrast to CD28, which is expressed on the naïve T cell. The
temporal expression of 4-1BB suggested that CD28 might relay an initial costimulatory signal
followed by 4-1BB signaling to further shape the T cell response. Ligation of 4-1BB induces
cytokine secretion, especially IFN- γ, and significant cell proliferation and survival of T cells in
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vitro and in vivo (5, 8-13). In vivo studies show 4-1BB costimulation plays a crucial role in the
generation of effector and memory cytotoxic T lymphocytes (CTLs) by increasing the overall
number of CTL produced (9, 14).
Manipulation of 4-1BB responses has been suggested for purposes of tumor
immunotherapy. Administration of agonistic anti-4-1BB monoclonal antibody (mAb) greatly
enhanced tumor-specific CTL responses in mice and eradicated established tumor even in tumor
models considered to be poorly immunogenic (15). In addition, antibodies against 4-1BB
remarkably improved the anti-tumor effects seen with adoptive transfer of CD8+ T cells specific
for tumor antigens (16-18). 4-1BB-mediated anti-tumor effects have been ascribed to the
prevention of programmed cell death in T cells, thereby promoting accumulation of anti-tumor
effector populations (16, 19).
Previous experiments have demonstrated effectiveness of 4-1BBL8 gene transduction for
anti-tumor immunity (20-23). Melero et al. first reported using a retroviral vector to stably
transduce 4-1BBL into P815 mastocytoma and AG104A sarcoma cell lines (22). Mice inoculated
with P815 tumor cells expressing 4-1BBL developed a strong CTL response and long-term
immunity against wild-type tumor. In subsequent trials using B cell lymphoma (20) and
squamous cell carcinoma (23) cell lines, gene transfer of 4-1BBL by retrovirus also induced CTL
and reduced tumor growth. Other studies using replication-defective adenovirus for gene
delivery showed enhanced survival and systemic protection against hepatic metastases induced
by colon carcinoma with a combination of 4-1BBL and IL-12 gene transfer (21).
We used herpes simplex virus I (HSV) amplicons as a vehicle for gene transfer of 4-
1BBL. HSV amplicons were used to transduce costimulatory molecules into tumor cells because
of their broad cellular tropism, large transgene capacity, and very high levels of gene expression.
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Because of the ability of HSV amplicons to readily transduce primary tumor cells, we reasoned
that HSV amplicons encoding 4-1BBL could be used to facilitate direct antigen presentation by
tumor cells and to expand tumor-specific effector populations for adoptive transfer. We have
noted activation of several toll-like receptors (TLRs) including TLR2 and TLR9 by
administration of HSV amplicons in macrophage cells lines as well as human chronic
lymphocytic leukemia (CLL) (Tolba K et al. unpublished data). HSV transduction can lead to
cytokine induction and NKG2DL expression of transduced tumor cells as well as activation of
anti-tumor CD8+ T cells (unpublished data). By triggering the innate immune response, HSV
amplicons may serve to facilitate a more specific adaptive response. We also hypothesized that
4-1BBL-expanded CD8+ T cells would show desirable effector properties, including in vivo
expansion and therapeutic efficacy in an immune replete host, as well as potentially confer a
memory response.
We used HSV.4-1BBL amplicons to transduce tumor for purposes of activating and
expanding tumor-specific CD8+ OT-1 cells in vitro, and studied the behavior of adoptively
transferred ex vivo expanded cells in LLC/OVA tumor-bearing mice. Our studies demonstrate
that 4-1BBL expressed from HSV amplicons (HSV.4-1BBL) has the potential to induce
significant expansion of cytolytic T cells in vitro and in vivo and that the adoptive transfer of
expanded T cells may result in reduction of tumor growth in vivo as well persistence of
CD44hi
Ly-6Chi
tumor-specific memory T cells.
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Materials and Methods
Animals and cells
C57BL/6 mice were obtained from Jackson Laboratories. OT-1 TCR transgenic mice, a
gift from Dr. M. Bevan, were bred to wild-type C57BL/6 mice to generate mice heterozygous for
the OT-1 TCR transgene. OT-1 mice express a transgenic TCR that is specific for OVA (257-
264) (SIINFEKL) peptide bound to H-2Kb. GFP+ mice were obtained by permission of the
producers (24); they were bred with OT-1 mice to generate OT-1/GFP T cells expressing green
fluorescent protein. Mice were maintained in pathogen-free animal facilities at the University of
Miami, and all procedures were performed in agreement with the Institutional Animal Care and
Use Committee, according to the National Institutes of Health guidelines for animal use.
Lewis lung carcinoma (LLC) stably transfected with ovalbumin (LLC/OVA) in pAC-
Neo-ova was (25) and grown in IMDM plus 10% FBS, penicillin (50 units/ml), streptomycin (50
µg/ml), 20 µM 2-ME (I-10), and 1 mg/ml Geneticin.
Antibodies
Directly conjugated monoclonal antibodies, including PE-conjugated anti-mouse CD3ε, Cy-
Chrome- (PE-Cy5-) or PE-conjugated anti-mouse CD8a, FITC-conjugated anti-mouse CD4,
FITC-conjugated anti-mouse V2α, and PE-conjugated anti-mouse Vβ5.1,5.2 (BD PharMingen,
San Diego, CA), were used to stain for T cells. Prior to staining, spleen cells were treated with
purified anti-mouse CD16/CD32 (Fc-γIII/II receptor, BD PharMingen) to block Fc-mediated
binding. Antibodies used to assess activation and/or differentiation states of T cells include: anti-
CD44-PE (eBiocscience), anti-human Granzyme B-PE (Caltag, Burlingame, CA), anti-mouse
CD25-biotin (7D4) (BD PharMingen) or anti-mouse-Ly-6C-biotin (BD PharMingen) followed
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by streptavidin-PE (Sigma, St Louis, MO), and purified anti-CD107a (1D4B) (BD PharMingen)
followed by anti-rat IgG (H+L)-PE (Caltag). Tumor transduction was verified by staining for
human B7.1 with anti-human CD80-FITC (BD PharMingen) and for murine 4-1BBL with anti-
4-1BBL (BD PharMingen), anti-rat IgG (H+L)-biotin (Caltag), and streptavidin-PE (Sigma).
Purification of CD8+
OT-1 cells
Spleen cells from OT-1 TCR transgenic mice were purified by positive column selection using
MACS anti-CD8a (Ly-2) MicroBeads (Miltenyi Biotec, Auburn, CA). Splenocytes were
resuspended in 90 µl of buffer (PBS containing 0.5% BSA) and 10 µl of Ly-2 microbeads per
107 cells. After 15 min incubation at 4°C, cells were washed and used for magnetic separation on
LS columns. Purified cells were >95% CD8+ as judged by FACS analysis using antibodies (BD
PharMingen) to CD8, variable (V) region α (Vα2), and Vβ (Vβ5.1,5.2) chains.
HSV-amplicon-vector construction and helper virus-free packaging
The cDNA of murine 4-1BBL with a KpnI restriction enzyme site 5’ and NheI site 3’ was
amplified by RT-PCR from RNA of C57BL/6 spleen stimulated with LPS (15 µg/ml) for 24
hours using the 5’ primer: 5’-GGTACCGCCATGGACCAGCACACACTTG-3’ and the 3’
primer: 5’-GCTAGCTTCCCATGGGTTGTCGGGTTTCAC-3’ based on its published
nucleotide sequence (26). The cDNA was inserted into pCR-Script Amp SK(+) (Stratagene, La
Jolla, CA) (415.pCRScript) and again amplified by PCR, but with BamHI at its 5’ and the stop
codon plus EcoRI at its 3’ using the 5’ primer: 5’-
TCGGATCCGTAATGGACCAGCACACACTTG-3’ and the 3’ primer: 5’-
GAGAATTCTCATTCCCATGGGTTGTCGGGTTTCAC-3’. The complete murine 4-1BBL
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cDNA was then cloned into the polylinker region of the HSV-1 amplicon vector pHSVPrPUC
(27) using the BamHI and EcoRI restriction enzyme sites and sequence verified. The cDNA of
human B7.1 was cloned into HSVPrPUC as previously described (28).
Packaging of virus vectors into helper-virus-free replication-defective HSV-1 viral
amplicons was performed as described previously (29, 30). Purified and concentrated viral
pellets were resuspended in PBS and stored at -80oC until use. Vector titers were determined as
described previously by using expression and transduction titering methods (31). Amplicons
containing the gene for Escherichia coli β -galactosidase (LacZ) were prepared using the same
vector system described above as a control for HSV transduction.
OT-1/GFP in vitro expansion for in vivo administration
LLC/OVA tumor cells were treated with 2.5mM EDTA, washed in I-10, and resuspended at 106
cells/100 µl in polypropylene tubes. LLC/OVA cells were transduced with either helper virus-
free HSV.4-1BBL (MOI=1) or HSV.B7.1 (MOI=1) and incubated at 37oC for 1 h before being
transferred to a six-well plate. One day later, transduced LLC/OVA were resuspended at 5 x 106
cells/ml, treated with 0.4 mg/ml mitomycin C for 20 min at 37oC in PBS, and washed three times
in RPMI 1640 plus 10% FBS, penicillin (50 units/ml), streptomycin (50 units/ml), and 50 µM 2-
ME (R-10). Freshly isolated OT-1/GFP cells were then plated with mitomycin C-treated
LLC/OVA at an E:T ratio of 3:2 for three days in 24-well plates. Each well was plated with 2.4 x
106
OT-1/GFP cells plus 1.6 x 106
tumor cells in 2 ml, for a total of 4 x 106
cells/2ml.
Supplemented R-10 media (0.5 ml) was added to each well after 2 days, and cells were harvested
for in vivo adoptive transfer on the third day of co-culture.
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Intracellular staining and flow cytometry
Cells were washed, blocked for Fc γIII/II receptors using anti-CD16/32 (BD PharMingen), and
stained on the surface with fluorochrome-conjugated anti-CD4 and anti-CD8a antibodies (BD
PharMingen) in PBS at 4oC for 20 min. Cells were then washed with PBS and fixed using
Cytofix/Cytoperm buffer (BD PharMingen) for 20 min at 4oC. 0.1% saponin/1%FBS in PBS was
used to wash, stain with fluorochrome-conjugated antibodies, and wash again. Cells were
analyzed using an LSR flow cytometer (BD Biosciences, San Jose, CA) and CellQuest software
(BD Biosciences).
Blood collection and preparation for flow cytometry
Sodium heparin from 10 ml Vacutainer blood collection tubes (Becton Dickinson, Franklin
Lakes, NJ) was resuspended in PBS (5 ml) and aliquoted into microfuge tubes (100 µl/tube).
Mice were gently warmed with heat lamp, and blood (approximately 5 drops) was collected from
the tail into microfuge tubes. Blood was transferred into flow tubes containing 3 ml of ACK
lysing buffer. Samples were kept at room temperature for 5 min and centrifuged at 1400 rpm.
Treatment with ACK lysing buffer was conducted twice more, and the cells were resuspended in
PBS for antibody staining.
Detection of GFP+ cells in frozen tissue sections
Spleens were frozen in O.C.T. compound (Sakura Finetek, Torrance, CA) with dry ice and stored
at -80oC until sectioning. Tissues were cut 6 µm thick using an electronic cryotome (Shandon,
Pittsburgh, PA) and adhered onto Superfrost plus glass slides (VWR, West Chester, PA). Slides
were kept cold to prevent diffusion of GFP and exposed in a closed-lid container to vapor from a
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napkin soaked with 37% formaldehyde (Sigma) at -20oC for 24 h, as first described by Jockusch
et al. (32). Tissues were then outlined with ImmEdge pen (Vector Laboratories, Burlingame,
CA), washed with PBS, and counterstained with 1 µg/ml Hoechst 33342 (Sigma) for 15 min at
37oC. After washing with PBS, slides were mounted with Prolong Gold antifade reagent
(Molecular Probes/Invitrogen, Eugene, OR). Sections were viewed using a Leica DMIRB
Inverted Microscope (Bannockburn, IL) and captured with MetaMorph Imaging System
(Molecular Devices Corporation, Downingtown, PA).
Tumor measurements and statistics
Tumors were measured at two different diameters (D1 and D2) using a caliper. Tumor volume
was calculated with the following formula: Tumor volume = 4/3π((D1+D2)/4)3.
For calculations of statistical significance, commercial software (StatView; SAS Institute, Cary
NC) was used. Regression analysis was used for computing P values.
Cytotoxic T lymphocyte (CTL) assay
Splenocytes were harvested and passed through a nylon mesh to make a single-cell suspension.
Following red blood cell lysis with ACK buffer, splenocytes were enumerated and incubated
with mitomycin C-treated LLC/OVA at a ratio of 10:1 for 5-6 days with recombinant murine IL-
2 (10-20 U/ml) in R-10 media. LLC/OVA and LLC targets (2 x 10 6 each) were labeled with 300
µl
51
Cr (150 µl/10
6
cells) for 5 h and washed in R-10 media. Splenocytes were collected,
counted, spun down to add fresh R-10 media, and plated in 96-well round-bottom plates
according to the indicated E:T (effector:target) ratios in 100 µl. Each sample dilution was plated
in triplicate. Targets were then washed three times to remove excess 51Cr and plated at 5 x 104
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cells/100 µl/well. Plates were briefly spun at 1400 rpm to increase contact between effectors and
targets and incubated at 37oC for 8 h. After plates were spun at 1400 rpm for 5 min, supernatant
(100 µl) was collected from each well and added to 2ml of Ready Safe Liquid scintillation
cocktail for aqueous samples (Beckman Coulter, Fullerton, CA). Samples were counted on a LS
6500 multi-purpose scintillation counter (Beckman Coulter). Percent lysis = (Sample counts –
Spontaneous counts)/(Maximum counts) * 100. Maximum counts were assessed from target lysis
with Triton X-100.
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Results
Tumors transduced by HSV.4-1BBL express murine 4-1BBL and can induce proliferation of
CD8+ OT-1 T cells.
Murine 4-1BBL cDNA was cloned into the herpes simplex virus I (HSV) amplicon
vector pHSVPrPUC and packaged into replication defective HSV-1 viral particles, or amplicons
(HSV.4-1BBL) using a helper virus-free methodology (Fig. 1A) (29, 30). The helper virus-free
system employs a bacterial artificial chromosome (BAC) that encodes the entire HSV genome
minus its cognate cleavage/packaging signals. This method leads to the production of virions that
contain only amplicon genomes, without the propagation of cytotoxic helper virus particles (Fig.
1A). Human B7.1 was also packaged into HSV amplicons (HSV.B7.1) as previously described
(28) and used in the following experiments to compare 4-1BB costimulation with stimulation
through the CD28 receptor. Both mouse and human B7.1 can stimulate T cell CD28 receptors of
either species (24).
Once the HSV.4-1BBL and HSV.B7.1 plasmids were each packaged into amplicons, we
tested the ability of the amplicons to transduce Lewis lung carcinoma stably expressing
ovalbumin (LLC/OVA) and the parental LLC tumor cell line. As a control for effects of HSV
amplicon transduction on endogenous costimulatory ligand expression, HSV.LacZ, which
encodes bacterial β-galatosidase, was used. Both LLC and LLC/OVA cells transduced with
HSV.B7.1 or HSV.4-1BBL showed high levels of expression of B7.1 or 4-1BBL, respectively,
by day 2 as shown by flow cytometric analysis (Fig. 1B). Several other murine tumor cell lines,
including T cell lymphoma EL4, B cell lymphoma A20, melanoma B16-F10, and mammary
adenocarcinoma EMT6, were also readily transduced by both amplicons (data not shown).
HSV.LacZ did not induce either B7.1 or 4-1BBL on transduced LLC/OVA (Fig.1B). In addition,
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HSV.B7.1 did not induce 4-1BBL and vice versa (data not shown). Therefore, HSV amplicons
can be used to efficiently express B7.1 and 4-1BBL on tumor cells.
In order to more accurately follow the effects of 4-1BBL on T cells that are specific for
tumor, we utilized the adoptive transfer of CD8+ OT-1 T cells, which express a transgenic TCR
specific for OVA257-264 (SIINFEKL) peptide bound to H-2Kb, in our in vivo mouse experiments.
Adoptive transfer of T cells from TCR transgenic mice into normal recipients provided a means
of monitoring antigen-specific T cells during a response (33, 34). We used OT-1/GFP cells that
were derived from OT-1 transgenic mice backcrossed into GFP+ mice to facilitate monitoring.
Following adoptive transfer into normal C57BL/6J mice, OT-1/GFP cells were easily detectable
by GFP+ fluorescence.
Functionality of B7.1 or 4-1BBL on transduced LLC/OVA tumor cells was subsequently
assessed by using the tumor cells to induce proliferation of CD8+ OT-1 T cells (Fig. 1C).
LLC/OVA cells, which are recognized by OT-1, were transduced with HSV.B7.1, HSV.4-1BBL,
or HSV.LacZ. One day post-transduction LLC/OVA cells were treated with mitomycin C to
prevent proliferation and used to stimulate CD8+ OT-1 cells for 3 or 5 days. At day 3, robust
proliferation was seen for OT-1 cells stimulated with tumor transduced with either HSV.B7.1 or
HSV.4-1BBL (7-fold and 6-fold, respectively, compared to non-transduced tumor) (Fig. 1C).
LLC/OVA cells transduced with HSV.LacZ did not augment proliferation of OT-1 in vitro.
OT-1 stimulated with HSV.4-1BBL-transduced LLC/OVA continued to proliferate
vigorously at day 5 (4-fold greater than with non-transduced tumor), although OT-1 stimulated
with HSV.B7.1-transduced LLC/OVA proliferated at levels similar to non-transduced tumor
(Fig. 1C). This suggested that initial proliferation could be prolonged in the presence of 4-1BB
costimulation relative to that seen with costimulation through CD28. OT-1 cells which were
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cultured with parental LLC tumor with or without costimulatory ligands did not induce
proliferation, signifying an absolute requirement for signal one in T cell activation (data not
shown).
OT-1 cells costimulated ex vivo with 4-1BBL display an activated effector phenotype.
The phenotype of expanded OT-1/GFP cells was characterized after three days of co-
incubation with tumor cells (Fig. 1D). CD44 is expressed on activated T cells and functions in
lymphocyte homing and adhesion. CD25 (IL-2Rα) is a component of the high-affinity IL-2
receptor and is also upregulated on activated T cells. Granzyme B is a serine protease stored in
the granules of cytotoxic T cells along with perforin (35). CD107a (LAMP-1) is a widely
expressed intracellular antigen that appears on the T cell surface following activation-induced
degranulation and has been used to enumerate activated antigen-specific CD8+ cytotoxic T cells
(36). OT-1/GFP stimulated ex vivo with either HSV.4-1BBL- or HSV.B7.1-transduced
LLC/OVA expressed high levels of CD44, intracellular granzyme B, and CD107a and modestly
increased levels of CD25, indicating that they were activated and capable of cytotoxic activity
(Fig. 1D). HSV.LacZ-stimulated OT-1/GFP also expressed CD44 and CD107a, but at lower
levels than were observed for those stimulated with HSV.4-1BBL or HSV.B7.1. Naïve OT-
1/GFP cells were used as a negative control for activation and did not express any of the
aforementioned activation markers. Ly-6C is a marker for previously activated T cells and
memory CD8+ T cells (37). Expression of Ly-6C was the highest in HSV.4-1BBL-stimulated
OT-1/GFP, compared to HSV.B7.1- and HSV.LacZ-stimulated OT-1/GFP and naïve OT-1/GFP
(Fig. 1D).
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T cells costimulated with 4-1BBL ex vivo readily expand in vivo in response to tumor.
HSV.4-1BBL amplicons were used as a means for activating and expanding tumor-
specific cytotoxic CD8+ OT-1/GFP T cells in vitro. We then studied the extent of expansion of
adoptively transferred T cells in vivo, as well as the effects of adoptive transfer of the ex vivo-
expanded T cells on tumor growth. Ex vivo-stimulated OT-1/GFP were adoptively transferred
into LLC/OVA tumor-bearing mice, and anti-tumor response was assayed. A schematic of the
experimental procedures is given in Figure 2. Mice were bled at several time points following
adoptive transfer of OT-1/GFP in order to detect expansion of OT-1/GFP in the peripheral blood
(Fig. 3A). Six days following adoptive transfer, the number of OT-1/GFP was significantly
greater in the LLC/OVA tumor-bearing group given OT-1/GFP stimulated with 4-1BBL
compared to tumor-bearing groups given naïve or B7.1-stimulated OT-1/GFP. Non-specific
expansion of stimulated OT-1/GFP was determined by transferring OT-1/GFP into non-tumor-
bearing mice. Greater numbers of OT-1/GFP were seen in the peripheral blood following transfer
of 4-1BBL-stimulated OT-1/GFP into non-tumor-bearing mice than naïve or B7.1-stimulated
OT-1/GFP, indicating that expansion of 4-1BBL-stimulated OT-1/GFP had occurred in vivo in
the absence of viable tumor. The number of 4-1BBL-stimulated OT-1/GFP were significantly
greater in the tumor-bearing mice compared to non-tumor-bearing mice, suggesting that 4-
1BBL-stimulated OT-1/GFP cells were able to expand in vivo in a tumor-specific manner (Fig.
3A).
The total numbers and percentages of tumor-specific OT-1/GFP cells in relation to the
CD8+ population were determined in the spleen of LLC/OVA tumor-bearing mice 17 days post-
adoptive transfer. The percentage of GFP+ cells in the spleen was the greatest in the tumor-
bearing group given OT-1/GFP stimulated with HSV.4-1BBL-transduced tumor (19.8% of
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CD8+ cells) vs. HSV.B7.1-stimulated tumor (0.1%) or no tumor (1.1%) (Fig. 3B). Substantial
numbers of OT-1/GFP were also detected in the spleen of non-tumor-bearing mice 17 days after
transfer of 4-1BBL-stimulated OT-1/GFP (7% of CD8+ cells), suggesting that tumor-specific 4-
1BBL-stimulated T cells can also persist in vivo in the absence of antigen (Fig. 3B).
When the total number of OT-1/GFP in the spleen was determined 17 days following
adoptive transfer, there were greater overall numbers of OT-1/GFP present in the spleen when
stimulated with 4-1BBL compared to B7.1 or to no stimulation, as seen in the peripheral blood
(Fig. 3C). Spleens from mice in each of the groups were sectioned to observe the presence and
localization of GFP+ cells 32 days following adoptive transfer of OT-1/GFP. The spleens from
mice given HSV.4-1BBL-stimulated OT-1/GFP showed the greatest degree of infiltration with
GFP+ cells (Fig. 3D). These results indicate that tumor-specific T cells stimulated ex vivo with
HSV.4-1BBL-transduced tumor can expand and persist in vivo.
CD8+ T cells expanded in vitro with 4-1BBL decrease tumor growth and increase survival.
We tested for growth of LLC/OVA tumor following adoptive transfer of HSV amplicon-
stimulated T cells or controls. LLC/OVA tumor-bearing mice treated with HSV.4-1BBL-
activated OT-1/GFP had statistically significant lower tumor burden than untreated mice (P <
0.0001) or mice treated with an identical number of HSV.B7.1-stimulated OT-1/GFP (P <
0.0001) or naïve OT-1/GFP (P = 0.0003) (Fig 4A). Naïve OT-1/GFP treatment did not reduce
tumor burden significantly when compared to the untreated group (P = 0.06). Administration of
HSV.B7.1-activated OT-1/GFP did not have major inhibitory effects on tumor size when
compared to no treatment or to naïve OT-1/GFP transfer (P > 0.05) (Fig. 4A).
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OT-1, either naïve or stimulated with HSV.4-1BBL-tansduced tumor, was administered
into LLC/OVA tumor bearing mice in order to determine effects on overall survival (Fig. 4B).
The group treated with naïve OT-1 had significantly better survival than those untreated
(p=0.001, two-sided log-rank test). Four (80%) of 5 mice treated with 4-1BBL-stimulated OT-1
survived 45 days compared with 2 (20%) of 10 mice given naïve OT-1, and is significant
(p=0.04, one-sided log-rank test). Those mice that survived to day 45 were completely tumor-
free.
4-1BBL-stimulated CD8+ OT-1 cells retain CTL activity and possess a memory phenotype.
We measured CTL activity in mice that had received adoptively transferred OT-1/GFP
cells. Splenocytes were harvested and cultured with LLC/OVA and recombinant IL-2 for six
days. Effectors from the HSV.4-1BBL-activated OT-1/GFP group had substantially higher levels
of CTL activity against LLC/OVA when compared to splenocytes from the HSV.B7.1-
stimulated OT-1/GFP, naïve OT-1/GFP, and untreated groups (Fig. 5). When restimulated in
vitro for 6 days, splenocytes from the HSV.B7.1 group showed lower CTL activity (14% lysis at
E:T = 50:1) against LLC/OVA compared to the HSV.4-1BBL group (41% lysis at E:T = 50:1)
and was similar to that of the naïve OT-1/GFP group (17% lysis at E:T = 50:1) (Fig. 5). These
results indicate that HSV.4-1BBL can be used for in vitro expansion of tumor-specific T cells for
the purpose of adoptively transferring them into tumor-bearing host so as to augment anti-tumor
responses. They also suggest that T cells expanded in this fashion may have more favorable
effector characteristics than those obtained through HSV.B7.1-mediated stimulation.
The phenotype of OT-1/GFP cells in each of the groups was characterized in freshly
isolated splenocytes 17 days post-adoptive transfer of OT-1/GFP (Fig. 6). GFP+ cells from both
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tumor-bearing and non-tumor-bearing mice which had received HSV.4-1BBL-stimulated OT-
1/GFP or the control OT-1/GFP indicated were studied for levels of CD44 and Ly-6C, a CD8+
memory cell marker (Fig. 6) (37). Significantly higher levels of CD44+ OT-1/GFP were seen in
mice which had received HSV.4-1BBL-stimulated OT-1/GFP (12.7% in tumor-bearing mice,
3.8% in non-tumor-bearing mice) than were seen for either HSV.B7.1-stimulated (0.1% in
tumor-bearing and non-tumor-bearing mice) or naïve (0.4% in tumor-bearing mice, 0.1% in non-
tumor-bearing mice) OT-1/GFP transfer (Fig. 6A). Similarly, HSV.4-1BBL-stimulated OT-
1/GFP also demonstrated higher levels of Ly-6C expression in comparison to the other groups,
suggesting differentiation into memory cells was more pronounced in the HSV.4-1BBL-treated
group (Fig. 6B). HSV.B7.1-stimulated OT-1/GFP cells had considerably lower levels of Ly-6C
expression compared to HSV.4-1BBL-stimulated OT-1/GFP, although costimulation had
occurred in vitro (Fig. 6B). This indicates that upon adoptive transfer of 4-1BBL-stimulated T
cells, the T cells are maintained at high levels and exhibit phenotypic attributes of memory cells.
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Discussion
The overall purpose of these studies was to understand the potential utility of the TNFR
family member 4-1BB as a means of expanding and activating tumor-specific CD8+ T cells for
adoptive immunotherapy. We were particularly interested in the purported ability of 4-1BB to
sustain nascent responses and prevent activation-induced cell death in T cells (38). This effect on
T cell survival is dependent on TRAF 2 signaling and NF-κB activation (39, 40), which in turn
induces Bcl-xL and Bfl-1, two pro-survival members of the Bcl-2 family. We reasoned that cells
expanded using 4-1BB costimulation might have favorable effector characteristics and persist in
vivo potentially leading to tumor eradication as well as improved survival.
We investigated effects of 4-1BB engagement on anti-tumor immunity by using HSV
amplicons to express high levels of 4-1BBL on tumor cells. HSV.4-1BBL was able to efficiently
transduce a variety of tumor cell lines, including LLC/OVA. LLC/OVA transduced with HSV.4-
1BBL were used to stimulate CD8+ TCR-transgenic OT-1 T cells. OT-1 cells responded in vitro
by proliferating and expressing activation markers, namely CD44, CD25, Ly-6C, CD107a, and
intracellular granzyme B, indicating priming of cytolytic activity and differentiation into
cytolytic effectors.
HSV.4-1BBL amplicons were used to transduce LLC/OVA tumors in vitro for purposes
of ex-vivo expansion of tumor-specific OT-1/GFP T cells. Adoptive transfer of OT-1/GFP T cells
that had been expanded ex vivo using HSV.4-1BBL-transduced LLC/OVA appeared to confer
greater protection against LLC/OVA growth compared to naïve OT-1/GFP, or OT-1/GFP cells
incubated with HSV.B7.1-transduced tumor. Compared to splenocytes from the other groups,
splenocytes from mice treated with HSV.4-1BBL-expanded OT-1/GFP showed higher
percentages of tumor-specific OT-1/GFP+ cells, exhibited greater CTL activity, and expressed
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higher levels of CD44 and the CD8+ memory marker Ly-6C. Therefore, T cell costimulation
with 4-1BBL expressed on tumor cells may be useful in facilitating expansion of tumor-specific
T cells in vivo as well as ex vivo.
We utilized the adoptive transfer of TCR-transgenic CD8+ OT-1/GFP T cells in order to
more accurately follow the effects of 4-1BBL on T cells that were specific for defined tumor-
related antigens in our in vivo mouse experiments. This overcame a major difficulty in isolating,
monitoring, and characterizing effective CD8+ CTL responses since antigen-specific CD8+ T
cells are present in very low numbers in the animal (33, 34). Special consideration needs to
taken, however, in order to apply information gained from this model to the human setting.
Instead of expanding a population of naïve TCR-transgenic T cells all of which are specific for
tumor as in our experiments, in humans T cell expansion starts from a heterogeneous population
in which relatively rare T cells can recognize tumor. Current cell culture techniques require
several months to produce sufficient numbers of cells from single clones (41, 42).
Other groups have examined the possibility of utilizing 4-1BB costimulation for the
generation of tumor-reactive T cells for adoptive immunotherapy and have determined that
addition of an agonistic anti-4-1BB antibody to in vitro culture enhances expansion and survival
of T cells. Murine fibrosarcoma MCA 205 tumor-draining lymph node (TDLN) cells were
expanded in vitro for 5 days with IL-2 and antibodies to CD3, CD28, and 4-1BB and found to
produce greater numbers of T cells in vitro than those without anti-4-1BB, due to fewer apoptotic
and necrotic cells (43). Anti-CD3/anti-CD28/anti-4-1BB-costimulated TDLN also produced
more of the type 1 cytokine IFN- γ and granulocyte macrophage colony-stimulating factor (GM-
CSF) and less of the type 2 cytokine IL-10 compared to those stimulated without anti-4-1BB
(43). When anti-CD3/anti-CD28/anti-4-1BB-expanded TDLN cells were adoptively transferred
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into MCA 205 tumor-bearing mice, significantly fewer metastatic lesions in the lungs were seen,
and survival was prolonged compared to those mice treated with TDLN cells stimulated without
anti-4-1BB (43). IFN- γ secretion was shown to be important in the 4-1BB-costimulated-TDLN
effects using neutralizing antibody (43). IL-10 neutralization, on the other hand, resulted in
significantly enhanced tumor regression (43). It would, therefore, be interesting to further study
effects of HSV.4-1BBL-transduced tumor on IFN- γ-/- T cells in order to assess the importance of
IFN- γ in generating adept effector cells in our system.
Strome et al. also observed that the combined use of anti-CD3/anti-CD28/anti-4-1BB in
activating T cells for adoptive immunotherapy resulted in the generation of T cells that were
more effective than those activated by anti-CD3 alone or anti-CD3/anti-CD28 in mediating anti-
tumor reactivity (17). Intravenous adoptive transfer of dual CD28/4-1BB-costimulated T cells
into mice bearing disseminated micrometastasis of a poorly immunogenic A9P melanoma
resulted in a 60% cure rate.
Unlike the previous experiments, we used ligands to CD28 and 4-1BB and employed
them separately to differentiate the effects of CD28 and 4-1BB costimulation. When other
groups used anti-CD3 plus agonistic anti-4-1BB antibody, without anti-CD28 antibody, in
culture to expand a polyclonal T cell population for adoptive transfer, they were not successful in
generating tumor-reactive T cells (17, 18). Kim et al. observed that TDLN cells activated in vitro
with anti-CD3/anti-4-1BB were less potent than anti-CD3-activated cells in mediating tumor
regression in vivo (18). In their study, the proliferation of TDLN cells in IL-2 following anti-
CD3/anti-4-1BB activation was significantly enhanced compared to anti-CD3 alone. However,
the increased proliferation resulted in the expansion of non-tumor-reactive T cells. Based on our
experiments, we can potentially overcome the difficulty of generating non-tumor-reactive clones
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by using HSV.4-1BBL amplicons to transduce tumor cells for specific stimulation of tumor-
reactive T cells.
It is notable that Kim et al. added anti-4-1BB into culture in soluble form, taking into
consideration crosslinking due to APCs (18), and Strome et al. used plate-fixed anti-4-1BB
antibodies in their in vit ro T cell expansion assays (17); however, both groups did not generate
potent in vivo effectors through 4-1BB costimulation using solely anti-CD3 and anti-4-1BB
antibodies. Some suggest that the lack of CD28 costimulation during in vitro culture in their
studies diminished activation of tumor-primed reactive T cells. In contrast, our studies have
shown that triggering the 4-1BB pathway using 4-1BBL, instead of anti-4-1BB antibody,
independent of CD28 costimulation, can generate potent tumor-reactive T cells that are efficient
in tumor eradication and can persist in vivo.
Maus et al. have studied ex vivo expansion of human polyclonal and MHC tetramer-
sorted antigen-specific cytotoxic T cells using K562 erythromyeloid cell lines stably transfected
to express 4-1BBL and the Fc γ receptor CD32 to bind anti-CD3 and anti-CD28 antibodies on the
surface (12). They demonstrated that these artificial APCs activate and rapidly expand
polyclonal or sorted antigen-specific CD8+ T cells with substantial increases in expansion and
recovery of T cells. In addition, apoptosis of cultured CD8+ T cells was decreased using 4-
1BBL-expressing anti-CD3/anti-CD28-coated K562 cells versus anti-CD3/anti-CD28-coated
K562. Although CD8+ T cells initially stimulated with anti-CD3/CD28-coated beads proliferate
and produce IL-2, they can become unresponsive to re-stimulation in vitro (44). Maus et al.
determined that the addition of 4-1BB costimulation facilitated the proliferation of T cells
following re-stimulation with anti-CD3 and anti-CD28. Real-time quantitative RT-PCR showed
that the addition of 4-1BB costimulation increased mRNA expression levels of IL-2 and Bcl-xL,
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two genes involved in T-cell survival and proliferation, respectively, in polyclonal CD8+ T cell
cultures. Therefore, 4-1BBL in combination with CD28 costimulation was found to expand and
increase survival in cytotoxic T cells ex vivo. Our studies show that 4-1BBL ligand stimulation
alone, without prior CD28 costimulation, is also able to expand tumor-specific T cells, which
were observed to elicit anti-tumor responses in vivo. In contrast to the work by Maus et al. in
which 4-1BBL is stably transduced to a “generic” artificial APC, an advantage to using the
highly efficient HSV amplicon system to express 4-1BBL is that it provides a theoretical means
by which to expand tumor-specific effector populations using autologous tumor from patients
without first pre-sorting tumor-reactive T cells. Using this system, T cells targeting unidentified
tumor antigens are able to be expanded specifically.
Another advantage to using HSV amplicons is their capacity to impart a strong innate
stimulus to transduced cells, including macrophage cell lines and human chronic lymphocytic
leukemia cells, resulting in cytokine secretion and NKG2DL expression by the transduced cells
(45). HSV possesses at least three molecular components capable of activating the innate
immune system: 1) dsRNA generated through self-hybridization of viral genes transcribed from
complementary DNA strands (46) 2) envelope glycoproteins recognized by toll-like receptor
(TLR) 2 (47), and 3) unmethylated CpG motifs encoded in the viral genome that activate TLR9
(48). Due to the fact that HSV amplicon DNA is concatamerized, the CpG effects on TLR9
could be quite potent. The enhanced capacity of transduced tumors to alert the innate immune
response may serve to facilitate a more specific adaptive response.
In certain settings, such as for human hematopoetic tumors, tissue may be available for
and amenable to HSV transduction (e.g. chronic lymphocytic leukemia cells) (45). Since HSV
vectors are theoretically safe and highly efficient means of gene transfer, the laboratory is
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pursuing pre-clinical development of these vectors for potential human use in chronic
lymphocytic leukemia.
Adoptive transfer of T cells permits screening of desirable functional and phenotypic
qualities of T cells for transfer. Recently, Gattinoni et al. provided a new criterion for the
generation and screening of optimal lymphocyte populations for adoptive immunotherapy (49).
Despite enhanced in vitro anti-tumor properties (IFN- γ secretion and CTL activity), more
differentiated effector T cells were less effective for in vivo tumor treatment than early effector
cells. Several reasons why late effectors may be less potent in vivo include: 1) downregulation of
homing molecules such as CD62L and/or costimulatory molecules, 2) inability to produce IL-2
and limited access to homeostatic cytokines, and 3) entry into a pro-apoptotic and replicative
senescent state. Previous studies using agonistic anti-4-1BB antibody and 4-1BBL-expressing
artificial APCs address the third point and indicate that use of 4-1BB signaling in combination
with CD28 costimulation in in vitro expansion may prevent apoptosis in culture-expanded clones
(9, 12, 17, 18, 43). We note that although in vitro 4-1BB-stimulated OT-1/GFP cells were highly
positive for CD44, they had higher levels of CD62L-expressing cells than OT-1/GFP cells
expanded using HSV.B7.1 (data not shown). Higher levels of CD62L may partially account for
the enhanced function of HSV.4-1BBL-stimulated OT-1/GFP following adoptive transfer and
may suggest that a higher proportion of therapeutically desirable “early effectors” arise as a
result of 4-1BB-mediated expansion.
Since we have not yet tested a combination of HSV.4-1BBL and HSV.B7.1, it is
unknown whether HSV.B7.1 will further augment effects seen with HSV.4-1BBL-stimulation
alone. HSV.B7.1-stimulated OT-1 failed to expand in vivo and inhibit LLC/OVA tumor growth.
In some cases, mice in this group appeared to have subtly larger tumor burden compared to the
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group given naïve OT-1 (not statistically significant). Since B7.1 serves as a ligand for both
CD28 and CTLA-4, B7.1 may have bound to CTLA-4 expressed on activated OT-1 cells,
inhibiting their clonal expansion and cytolytic activity. In addition, complete physical separation
of OT-1/GFP and transduced LLC/OVA following in vitro activation was difficult due to cell
debris. It is possible that the tumor-expressed B7.1 bound to CTLA-4 present on activated T cells
in vivo. If this is the case, use of anti-CD28 antibodies for in vitro expansion may be preferred to
use of the cognate B7.1 ligand.
In summary, our studies suggest that costimulation with 4-1BB may be employed to
enhance expansion and cytolytic activity of tumor-specific CD8+ T cells for the generation of
long-term tumor-specific immunity. Although the OT-1 model was useful in demonstrating
expansion of a tumor-specific response, results obtained with OT-1 may differ as compared to
what would be observed using a polyclonal T cell population as a source of expanded T cells.
Such a population might contain relatively fewer precursor T cells with anti-tumor activity, and
expansion of relevant effectors might prove more difficult. Adoptive transfer experiments using
polyclonal T cells from tumor-bearing mice are currently being performed in an effort to
ascertain utility of this approach in a non-transgenic setting. Nevertheless, the vigorous cytolytic
effector function as well as the increased expansion and persistence seen using HSV amplicon-
transduced tumor suggest this method should be explored further in the human setting.
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Figure 2. Overview of adoptive transfer experiment. LLC/OVA tumor cells were transduced
with HSV.4-1BBL or HSV.B7.1 at an MOI of 1 and cultured for one day. Tumor cells were then
washed and treated with mitomycin C. CD8+ OT-1/GFP cells were purified from spleen and co-
cultured with transduced tumor for three days. Stimulated OT-1/GFP were separated from tumor
cells using anti-CD8 antibody-magnetic beads. OT-1/GFP cells were administered i.v. into
C57BL/6 mice bearing LLC/OVA tumor. LLC/OVA tumor was palpable following 3-4 days of
s.c. injection. Peripheral blood and spleen were observed for the number of OT-1/GFP present.
Spleen was also analyzed for cytolytic activity and the phenotype of OT-1/GFP cells.
Figure 3. Presence of OT-1/GFP cells in peripheral blood and spleen following adoptive
transfer of expanded OT-1/GFP. A) Blood was collected 6 days post transfer of expanded OT-
1/GFP. Percentage of OT-1/GFP in blood was assessed using flow cytometry and used to
calculate cell number from the volume of blood collected. N=5, 6, and 6 for the tumor bearing
group. N=3 for each of the non-tumor bearing groups. Numbers are statistical results from a
Student’s t-Test. B and C) Splenocytes from mice were collected on day 17 post OT-1/GFP
adoptive transfer. They were stained with anti-CD8-Cy-Chrome and analyzed by flow
cytometry. B) Blue numbers indicate percentage of GFP+ cells in the CD8+ gate. C) Total
number of splenocytes x percentage of GFP+ cells in spleen = Number of GFP+ cells per spleen.
Number of GFP+ cells per spleen on day 17 is shown. D) Frozen spleen harvested 32 days
following adoptive transfer of OT-1/GFP cells were sectioned, fixed by formaldehyde vapor, and
counterstained with Hoechst 33342 to show nuclei. GFP+ areas indicate infiltration by
adoptively transferred CD8+ OT-1/GFP cells. Images were taken at 20x magnification. Upper
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left: GFP+ spleen (positive control); upper right: transfer of naïve OT-1/GFP; lower left: transfer
of HSV.4-1BBL-expanded OT-1/GFP; lower right: transfer of HSV.B7.1-expanded OT-1/GFP.
Figure 4. Tumor volumes and survival in mice following adoptive transfer of OT-1 T cells.
CD8+ OT-1 T cells were activated in vitro with transduced LLC/OVA at an E:T ratio of 3:2 for 3
days. A) Activated OT-1/GFP (O/G) or naïve O/G (2 x 106) were administered into the tail vein
of mice injected with tumor s.c. 4 days previously. Tumors were measured at the time points
indicated. Error bars indicate SEM. P values were determined from regression analysis. O/G
HSV.4-1BBL= 10 mice; O/G HSV.B7.1 = 5 mice; naïve O/G = 5 mice; No O/G = 3 mice. B)
Activated OT-1 or naïve OT-1 (106) were administered into the tail vein of mice injected with
LLC/OVA tumor (1.5 x 106) s.c. 3 days previously. Mice were sacrificed when tumor diameter
reached 15 mm. Mice surviving by day 45 are tumor-free.
Figure 5. Tumor-specific cytolytic activity of splenocytes following administration of OT-1
T cells expanded ex vivo. At 22 days following tumor injection, splenocytes were harvested and
cultured with recombinant human IL-2 (10 U/ml) and mitomycin C-treated LLC/OVA at an E:T
ratio of 10:1 for 6 days. LLC/OVA cells were used as targets in an 8-h 51Cr release assay.
Percent lysis with standard deviation is shown.
Figure 6. CD44 and Ly-6C expression of OT-1/GFP in splenocytes. Splenocytes from mice
were collected on day 17 following OT-1 adoptive transfer. They were stained with anti-CD8-
Cy-Chrome and A) anti-CD44-PE or B) biotinylated-anti-Ly-6C followed by streptavidin-PE
and analyzed by flow cytometry. Numbers indicate percentages in the CD8+ gate.
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HSV IE 4/5HSV IE 4/5
Promoter Promoter
HSVHSV OriOriss
HSVHSV pacpac
sitesiteSV40 poly ASV40 poly A
MurineMurine
44--1BBL1BBLAmpr
Transfection Harvest
HSV-BAC
parCparC
parBparB
parAparA
repErepE
OriSOriSÄÄaa
ÄÄaaUUSS
UULL
pBS-vhs
HSV
Amplicon
B LLC
HSV.B7.1
LLC/OVA
HSV.B7.1
100 101 102 103 104
FITC100 101 102 103 104
FITC
100 101 102 103 104
PE
100 101 102 103 10PE
B7.1
4-1BBL
LLC
HSV.4-1BBL
LLC/OVA
HSV.4-1BBL
A
Yi--Figure 1
C
Day 3
0
10
2030
40
50
60
70
80
T cells only T + LLC/OVA
(-)
T + LLC/OVA
HSV.B7.1
T + LLC/OVA
HSV.4-1BBL
T + LLC/OVA
HSV.LacZ
T h y m i d i n
e I n c o r p o r a t i o n
( c p
m x
1 0 - 3 )
Day 5
0
5
10
15
20
25
T cells only T + LLC/OVA
(-)
T + LLC/OVA
HSV.B7.1
T + LLC/OVA
HSV.4-1BBL
T + LLC/OVA
HSV.LacZ
T h y m i d i n e I n c o r p o r a t i o n
( c p m x
1 0 - 3 )
A
A
A
A A
10 0 10 1 10 2 10 3 10 4
ANTI-CD44-PE
100
101
102
103
104
ANTI-CD25-PE
D
100
101
102
103
104
ANTI-CD107A-PE
CD44
CD25
CD107a
100
101
102
103
104
ANTI-GRANZYME B-PE,I
Granzyme B,
intracellular
100
101
102
103
104
ANTI-LY6C-PE
Ly-6C
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LLC/OVA
HSV.4-1BBL
or
HSV.B7.1
1 day
OT-1/GFP
3 days
Stimulated
OT-1/GFP
(1-2 x 106) i.v.
4-8 days
LLC/OVA
(106) s.c.
Bleed
Add to
Spleen:
#/% OT-1/GFP
CTL assay
CD44/Ly-6C
Granzyme B
Yi--Figure 2
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0
200
400
600
800
000
200
0.007 0.017
N a ï v e
O T
- 1 / G F P
H S V
. 4 - 1 B B L
- s t i m u l a t e
d
O T
- 1 / G F P
H
S V
. B 7 .
1 - s t i m
u l a t e
d
O T
- 1 / G F P
N a ï v e
O T
- 1 / G F P
H S V
. 4 - 1 B B L
- s t i m u l a t e
d
O T
- 1 / G F P
H S V
. B 7 .
1 - s t i m
u l a t e
d
O T
- 1 / G F P
tumor bearing mice non-tumor bearing mice
A B
N a ï v e
O T - 1 / G F P
O T - 1 / G F P +
L / O H
S V . 4 - 1 B B L
O T - 1 / G F P +
L / O H
S V
. B 7 . 1
LLC/OVA-tumor
bearing mice
non-tumor
bearing mice
10
0
10
1
10
2
10
3
10
4
GFP
M1
100
101
102
103
104
GFP
M1
100
101
102
103
104
GFP
M1
10
0
10
1
10
2
10
3
GFP
M1
100
101
102
103
GFP
M1
100
101
102
103
GFP
M1
0.4
7.0
0.3
1.1
19.8
0.1
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1 2 3 4 5 6 7
O T
- 1 / G F P
+
L / O
H S V
. B 7 . 1
O T
- 1 / G F P
+
L / O
H S V
. 4 - 1 B B L
N a ï v e
O T
- 1 / G F P
N o O T
- 1 / G F P
DC
HSV.4-1BBL-stimulated
OT-1/GFP Spleen
GFP+ Spleen Naïve OT-1/GFP Spleen
HSV.B7.1-stimulated
OT-1/GFP Spleen
Yi--Figure 3
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0
200
400
600
800
1000
1200
1400
1600
5 7 9 11 13 15
Days following tumor injection
T u m o r v o l u m e ( m m ^
3 )
O/G HSV.4-1BBL
O/G HSV.B7.1
naïve OT-1
No O/G
P
= 0 . 0 0 0 3
P
= < 0 . 0 0 0 1
A
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50
Days post OT-1 transfer
% S u r v i v a l L/O only
(n=9)
L/O + naïve OT-1
(n=10)
L/O + OT-1/HSV.4-1BBL
(n=5)
B
Yi--Figure 4
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0
5
10
15
20
25
30
35
40
45
0.4:12:110:150:1
E:T ratio
% Lysis
L/O + O/G HSV.4-1BB
L/O + O/G HSV.B7.1
L/O + naïve O/G
L/O only
Yi--Figure 5
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tumor bearing mice non-tumor bearing mice
A
10
0
10
1
10
2
10
3
10
4
GFP10
0
10
1
10
2
10
3
10
4
GFP
100
101
102
103
104
GFP
100
101
102
103
104
GFP
0.4
0.8
12.7
7.1
10
0
10
1
10
2
10
3
10
4
GFP10
0
10
1
10
2
10
3
10
4
GFP
100
101
102
103
104
GFP
100
101
102
103
104
GFP
0.1
0.2
3.8
3.0
Naive OT-1/GFP
OT-1/GFP +
L/O HSV.4-1BBL
100
101
102
103
104
GFP
100
101
102
103
104
GFP
100
101
102
103
104
GFP
100
101
102
103
104
GFP
OT-1/GFP +L/O HSV.B7.1
0.1
0.2
0.1
0.2
100
101
102
103
104
GFP
100
101
102
103
104
GFP
100
101
102
103
104
GFP
100
101
102
103
104
GFP
100
101
102
103
104
GFP
100
101
102
103
104
GFP
100
101
102
103
104
GFP
100
101
102
103
104
GFP
0.7
0.4
9.7
0.2
0.0
0 1
0.1
0.1
3.8
0.1
0.1
B
Naive OT-1/GFP
OT-1/GFP +
L/O HSV.4-1BBL
OT-1/GFP +
L/O HSV.B7.1