See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/7838302 Roncador, G. et al. Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. Eur. J. Immunol. 35, 1681-1691 ARTICLE in EUROPEAN JOURNAL OF IMMUNOLOGY · JULY 2005 Impact Factor: 4.03 · DOI: 10.1002/eji.200526189 · Source: PubMed CITATIONS 407 READS 211 12 AUTHORS, INCLUDING: Giovanna Roncador Centro Nacional de Investigaciones Oncoló… 78 PUBLICATIONS 3,809 CITATIONS SEE PROFILE Lorena Maestre Centro Nacional de Investigaciones Oncoló… 23 PUBLICATIONS 951 CITATIONS SEE PROFILE Jorge Luis Martinez-Torrecuadrada Centro Nacional de Investigaciones Oncoló… 51 PUBLICATIONS 1,604 CITATIONS SEE PROFILE Alison H Banham University of Oxford 133 PUBLICATIONS 7,766 CITATIONS SEE PROFILE Available from: Jorge Luis Martinez-Torrecuadrada Retrieved on: 15 January 2016
Analysis of FOXP3 protein expression in humanCD4+CD25+ regulatory T cells at the single-cell level
Giovanna Roncador1, Philip J. Brown2, Lorena Maestre1, Sophie Hue3, Jorge L. Mart�nez-Torrecuadrada4, Khoon-Lin Ling4, Sarah Pratap5, Christy Toms3, Bridget C. Fox2,Vincenzo Cerundolo5, Fiona Powrie3 and Alison H. Banham2
1 Monoclonal Antibodies Unit, Biotechnology Program, Centro Nacional de Investigaciones Oncol�gicas (CNIO),Madrid, Spain
2 Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford, UK3 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK4 Protein Technology Unit, Biotechnology Program, Centro Nacional de Investigaciones Oncol�gicas (CNIO),Madrid, Spain
5 Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
The transcription factor FOXP3 plays a key role in CD4+CD25+ regulatory T cellfunction and represents a specific marker for these cells. Despite its strong associationwith regulatory T cell function, in humans little is known about the frequency ofCD4+CD25+ cells that express FOXP3 protein nor the distribution of these cells in vivo.Here we report the characterization of seven anti-FOXP3 monoclonal antibodiesenabling the detection of endogenous human FOXP3 protein by flow cytometry andimmunohistochemistry. Flow-cytometric analysis showed that FOXP3 was expressed bythe majority of CD4+CD25high T cells in peripheral blood. By contrast, less than half ofthe CD4+CD25int population were FOXP3+, providing an explanation for observationsin human T cells that regulatory activity is enriched within the CD4+CD25high pool.Although FOXP3 expression was primarily restricted to CD4+CD25+ cells, it wasinduced following activation of both CD4+ and CD8+ T cell clones. These findingsindicate that the frequency of FOXP3+ cells correlates with the level of expression ofCD25 in naturally arising regulatory Tcells and that FOXP3 protein is expressed by someactivated CD4+ and CD8+ T cell clones. These reagents represent valuable researchtools to further investigate FOXP3 function and are applicable for routine clinical use.
See accompanying Commentary: http://dx.doi.org/10.1002/eji.200526303
Introduction
FOXP3 is a member of the forkhead or winged helixfamily of transcription factors. The gene was firstdescribed as JM2 when its mutation was identified in anX-linked autoimmune and allergic disregulation syn-drome (XLAAD) [1]. Mutations in FOXP3 were subse-
Correspondence: Alison H. Banham, Nuffield Department ofClinical Laboratory Sciences, University of Oxford, Level 4Academic Block, John Radcliffe Hospital, Headington, Oxford,OX3 9DU, UKFax: +44-1865-220078e-mail: [email protected]
Received 11/3/05Accepted 20/4/05
[DOI 10.1002/eji.200526189]
Key words:FOXP3 � RegulatoryT cell � Monoclonal
antibody � Lymphoma
Abbreviation: TR cell: Regulatory T cell
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quently identified as the defect underlying the scurfymouse and the human X-linked neonatal diabetesmellitus, enteropathy and endocrinopathy syndrome(IPEX) [2–4]. CD4+ T cells are primarily responsible forthe pathology observed in scurfy mice [5, 6], which havea similar phenotype to that reported for CTLA-4knockout mice [7, 8]. Murine Foxp3 determines boththe number and functionality of peripheral T cells [9]and regulates T cell activation [10]. The absence ofFOXP3 in both mice and humans thus causes a fatalimmune proliferative disease, as a consequence ofchronic T cell activation, whereas Foxp3 overexpressionin transgenic mice results in a reduction in the mature Tcell population and diminished T cell function [9]. Morerecently FOXP3 has been identified as a masterregulatory gene for cell-lineage commitment or devel-opmental differentiation of CD4+CD25+ regulatory Tcells (TR cells). Gene transfer of Foxp3 converts naiveCD4+CD25– T cells towards a regulatory phenotype andthis molecule appears to represent an important markerof this TR cell population [11–13].
CD4+CD25+ TR cells comprise approximately 10% ofperipheral CD4+ cells [14–17]. They play a crucial rolein the control of T cell mediated autoimmunity bysuppressing the proliferation and cytokine production ofother Tcells [18–20]. TR cells are implicated in a range ofdisease states including organ-specific autoimmunedisease [21], inflammatory bowel disease [22], multiplesclerosis [23], allograft rejection [24], graft-versus-hostdisease [25–27], allergy [28], and sterilizing immunityto infectious organisms [29]. The accumulation of TR
cells has been observed in the peripheral blood andtumor microenvironment of cancer patients [30–34]and their removal has been reported to result in effectiveantitumor immunity, implicating these cells in immuneevasion by cancer cells [35].
A problemhindering the study of TR cells has been thelack of a unique marker that defines all cells withregulatory activity. The activation marker CD25 haslimitations in that its expression is not restricted toTcellsand cannot distinguish TR cells from conventionalactivated T cells. Thus, although FOXP3 mRNA isexpressed at high levels by CD4+CD25+ cells there isno information on the frequency of bona fide FOXP3+ TR
cellswithin this population. Furthermore, bothCD25andotherTRcellmarkers,suchasGITR,CTLA-4,andCD45RB,are not expressed on all CD4+ T cells with regulatoryactivity. Neuropilin has recently been identified as apotentially useful surface marker of murineCD4+CD25+Foxp3+ TR cells [36]. Because FOXP3 is anuclear protein it is of limited value in the isolation of TR
cells. However, unlike neuropilin, which is expressed inother cell types, the expression of FOXP3 is highlyrestricted todiscreteTRcell populationsand it is thereforevaluable as a specific marker to detect TR cells in vivo.
Here we describe our production of a panel of mAbthat specifically recognize the human FOXP3 protein.Using these reagents we show that the majority ofFOXP3+ cells are CD4+CD25+ cells in secondarylymphoid tissue, although a minor population ofCD8+CD25+ and CD4+CD25– cells that express FOXP3is also present. In addition we describe the use of theseantibodies for flow-cytometric analysis of FOXP3 proteinexpression, enabling the first determination of thefrequency of TR cells within the CD4+CD25+ T cell poolin human PBL.
Results
The Abcam goat polyclonal FOXP3 Ab labels thehuman FOXP3 protein in frozen but not routinelyfixed tissues
The commercially available Abcam goat polyclonalFOXP3 Ab was evaluated as a reagent to detect theFOXP3 protein in human tissues. Immunohistochemis-try confirmed that the Ab recognized the human flag-tagged FOXP3 protein expressed in COS-1 cell transfec-tants and stained scattered interfollicular cells in tonsil(Fig. 1). However, we were unable to detect the FOXP3protein in routinely fixed paraffin-embedded tissueswith this reagent and thus additional reagents werenecessary to investigate archival biopsy material.
Production of FOXP3 mAb
An I.M.A.G.E. Consortium [LLNL] cDNA clone [37],encoding the start of the FOXP3 protein and having apolyA tail, was used to bacterially express a GST fusionprotein containing a full-length FOXP3 protein forimmunization. The hybridoma fusion generated sevenmAb that showed a similar staining pattern, of scatteredinterfollicular cells on frozen tonsil, to that observedwith the polyclonal FOXP3 Ab (Fig. 1). We observedmore FOXP3+ cells using the FOXP3mAb, which showeda stronger reactivity than was obtained with thepolyclonal Ab. The reactivity of these FOXP3 mAb wasfurther investigated.
The mAb specifically recognize FOXP3 and notother FOXP proteins
To confirm that the mAb recognized the human FOXP3protein their ability to detect the flag-tagged humanFOXP3 protein, expressed in COS-1 cells, was tested byimmunohistochemistry on frozen cytospin preparations.Labeling with the anti-FLAG Ab confirmed the efficiencyof transfection and the subcellular distribution of therecombinant FOXP3 protein. Cytoplasmic expression of
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proteins that are normally nuclear is commonlyobserved when cells are transfected using the describedFugene protocol and harvested after 24 h. All of themAbspecifically recognized the human FOXP3 protein byimmunohistochemistry (Fig. 1).
There is significant sequence similarity between thefour members of the FOXP family, FOXP1–4, particularlywithin the DNA binding forkhead domain. As a full-length FOXP3 protein was used as an antigen it wasimportant to ensure the specificity of the mAb for FOXP3and exclude the possibility of their cross-reactivity withother FOXP proteins. Expression of the FOXP1, FOXP2and FOXP4 proteins in transfected cells was confirmedusing the JC12 mAb to label FOXP1 and the anti-Xpressantibody to detect epitope-tagged FOXP2 and FOXP4(Fig. 1). All the FOXP3mAb specifically labeled the COS-1 cells expressing FOXP3 and not those expressing theother FOXP proteins (Fig. 1).
While preparing this manuscript a FOXP3 murinemAb, hFOXY, has become commercially available (CatNo 14-5779, eBioscience, San Diego, CA, USA). We haveinvestigated this reagent and compared its reactivity ontonsil with that obtained with our 236A/E7 FOXP3 mAb
(data not shown). On frozen tonsil sections hFOXYstained scattered interfollicular cells at a dilution of 1/25. At this dilution some background staining wasobserved and there was a noticeably smaller populationof nuclear+ cells than was stained with our FOXP3 mAb236A/E7. Staining of COS-1 cell transfectants confirmedthat hFOXY detected the FOXP3 protein and not therelated FOXP1, FOXP2 or FOXP4 proteins. Staining ofparaffin-embedded COS cell transfectants indicated thathFOXY recognizes a formalin-resistant epitope onFOXP3 and no cross-reactivity was observed underthese conditions with other FOXP proteins. Thus hFOXYis suitable for detecting FOXP3 by immunohistochem-istry on both frozen and paraffin-embedded tissues.However, this reagent may only detect a subpopulationof FOXP3+ cells.
The FOXP3 mAb recognize the FOXP3 protein in avariety of applications and two are cross-reactivewith the murine Foxp3 protein
The FOXP3mAbwere tested for their ability to recognizeformalin-resistant epitopes by immunohistochemistryusing routinely fixed tonsil and formalin-fixed paraffin-embedded pellets of FOXP3-transfected COS-1 cells. Allof the mAb recognized the FOXP3 protein in routinelyfixed tissues and labeled FOXP3 transfectants. SomemAb gave less non-specific background staining thanothers, whereas some showed stronger labeling ofpositive cells, these data are summarized in Table 1.Ab 86D/D6 and 236A/E7 are recommended forimmunohistochemistry on paraffin-embedded tissues.
The ability of the mAb to recognize the FOXP3protein by flow-cytometry and by Western blotting wasalso tested using FOXP3-transfected COS-1 cells. All themAb recognized the recombinant FOXP3 protein tosome extent in Western blotting and in flow cytometry(Table 1).
The human and murine FOXP3 proteins are approxi-mately 87% identical, thus the FOXP3 mAb were alsotested for reactivity with the murine Foxp3 protein. ThemAb 150D/E4 and 221D/D3 were found to be cross-reactive with the murine Foxp3 protein and specificallylabeled CD4+ T cells in murine spleen and lymph nodes(data not shown as these are consistent with the humandata).
Characterization of FOXP3 protein expression innormal human tissues
FOXP3 protein expression was assessed by immunohis-tochemistry with mAb 236A/E7 on a routinely fixednormal-tissue microarray containing 39 different hu-man tissues and on whole sections of lymphoid tissues.Tissues included tonsil, spleen, bone marrow, brain,
Fig. 1. Immunolabeling of tonsil and FOXP transfectants. Thetop and middle rows show immunoperoxidase labeling withthe commercial goat polyclonal Ab or the 236A/E7 mouseFOXP3 mAb, respectively, on tonsil and FOXP3-COS-1 trans-fectants. The bottom row shows the lack of staining with the236A/E7 FOXP3mAbonCOS-1 cells transfectedwith the relatedFOXP proteins. The insets at top right of the bottom rowconfirm the expression of these recombinant FOXP proteinsusing the indicated Ab.
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larynx, parotid gland, thyroid, gall bladder, liver, lung,skin, skeletal muscle, kidney, pancreas, stomach, colon,duodenum, small intestine, bladder, ovary, uterus,breast, placenta, prostate, testis, fetal liver and fetalthymus. There was no nuclear FOXP3 protein expressionobserved in the range of normal non-hematologicaltissues tested, with the exception of scattered positivelymphocytes in colon, stomach, and fallopian tube.These data are consistent with the reported restrictedexpression of FOXP3 within lymphocytes.
Within hematological tissues there were much highernumbers of lymphocytes expressing FOXP3. TheFOXP3+ cells were scattered within the interfollicularareas of tonsil (Fig. 2A) and were occasionally seenwithin the follicular germinal centers (Fig. 2A). Reactivelymph node contained many FOXP3+ cells and thesewere distributed throughout the tissue, including withinthe mantle zone and germinal centers of the secondaryfollicles. In spleen there were occasional FOXP3+ cells inthe red pulp and there were increased numbers in the Tcell areas around vessels (Fig. 2A). There were manyFOXP3+ cells in fetal thymus and in the mature thymusthese were present predominantly in the medulla withonly scattered FOXP3+ cells being present in the cortex(Fig. 2A). In bone marrow a small number of FOXP3+
cells were also observed.To confirm the immunophenotype of the FOXP3+ cell
population in situwithin tissues, double-labeling studieswere performed by immunofluorescent (Fig. 2B) andimmunoenzymatic techniques (data not shown). Thesestudies confirmed that in tonsil the FOXP3 protein wasexpressed exclusively in the CD3+ T cell population andno double-labeling of CD20+ B cells was observed. Themajority of FOXP3+ cells were, as previously reported,both CD4+ and CD25+. However, double-labelingidentified a very small population of FOXP3+CD8+ cellsand indicated that a minority of FOXP3+ cells wereCD25– (Fig. 2B).
FOXP3 is expressed in activated T cells
There have been reports that FOXP3 expression isinduced in activated T cells. Cytospin preparations ofboth resting and activated CD8+ and CD4+ humanTcellclones were immunostained with FOXP3 mAb 236A/E7.FOXP3 protein expression was restricted to the nuclei ofactivated CD4+ and CD8+ T cell clones and was absentin the resting cells (Fig. 2C).
Frequency of FOXP3 expression in the humanCD4+ population
CD4+CD25+ and CD4+CD25– populations were purifiedfrom peripheral blood taken from three individualswithout any known disease as described in the"Materials and Methods" section. Immunofluorescentlabeling of FOXP3 expression was performed and thenumbers of FOXP3+ and FOXP3– cells were scored ineach sample. Approximately half of the CD4+CD25+
population was FOXP3+ with a frequency of 55.7�5.2%(Fig. 3A). In contrast, only 3.6�0.9% of the CD4+CD25–
cells expressed FOXP3.Despite early difficulties with detecting endogenous
FOXP3 protein by flow cytometry, a modified stainingprotocol that included a DNAse digestion step enabledFACS analysis of endogenous FOXP3 protein expressionin the CD4+ population and correlation with CD25expression (Fig. 3B). In peripheral blood, 95.7%(94.7–95.8) of CD4+CD25high T cells expressed FOXP3whereas only 34.9% (22.3–47.4) of CD4+CD25int T cellsstained positive for FOXP3. No detectable staining wasobserved in the CD25– population or in resting CD8+ Tcells (data not shown). As in the PBL, the majority(>95%) of thymic CD4+CD25high T cells express FOXP3(data not shown). It is claimed that the commerciallyavailable hFOXYmAb is also suitable for FACS analysis ofFOXP3 expression. However, we were unable to revealFOXP3 expression amongst peripheral blood
Table 1. Reactivity of FOXP3 mAb
Antibody IHFOXP3transfectants
IH FOXP1,2 or 4transfectants
IHtonsilfrozen
IHparaffintonsil
Westernblottingtransfectants
Flowcytometrytransfectants
Murine tissues
86D/D6 + – + ++ + + –
150D/E4 + – + + + +++ +
157B/F4 + – + ++a) + ++ –
206D/B1 + – + + + ++ –
221D/D3 + – + ++a) + +++ +
236A/E7 + – + ++ + +++a) –
259D/C7 + – + ++a) + +++a) –
a) Indicates that there was some nonspecific background staining. IH, immunohistochemistry.
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CD4+CD25+ cells using this reagent, suggesting it is notsuitable for this application (data not shown).
The FOXP3 mAb label functional CD4+CD25+
T cells
Although the double-labeling studies indicate that theFOXP3 mAb label predominantly CD4+CD25+ T cells,their ability to detect T cells with functional regulatoryactivity was also confirmed. The immunophenotype of
CD4+CD25+ Tcells purified from peripheral blood usingmagnetic-bead selection was confirmed by flow cyto-metry (Fig. 4A). Nuclear expression of the FOXP3protein was confirmed by immunofluorescent labeling(Fig. 4B), only the occasional FOXP3+ cell was observedin the CD25– population and no staining was observedwhen using the isotype-matched MR12 control mAb(data not shown). These FOXP3+ cells were able tosuppress the proliferation of CD4+CD25– T cells(Fig. 4C) confirming that the mAb label functionalCD4+CD25+FOXP3+ TR cells.
Fig. 3. Frequency of FOXP3 expression in CD4+ T cells. (A)Illustrates the frequency of FOXP3+ cells within the CD4+CD25+
(left) and CD4+CD25– (middle) populations purified fromperipheral blood and observed using immunofluorescentlabeling. No staining of the CD4+CD25+ cells was observedwith the secondary Ab alone (right). (B) The top left panelshows the FACS plots and gating of PBL labeled with CD4 andCD25. The top right panel represents the FOXP3 expressionaccording to CD25 expression in the whole CD4 population.The FOXP3 expression in the gated populations is illustrated inthe histograms. Quadrants are drawn based on isotype-matched control antibodies that gave <1% background.Representative of three separate experiments.
Fig. 2. FOXP3 protein expression in normal human tissues. (A)Illustrates peroxidase immunolabeling of normal lymphoidtissues with the 236A/E7 FOXP3 Ab. (B) Illustrates double-immunofluorescent labeling of normal tonsil (g, green; r, red),confirming that the FOXP3 mAb recognizes predominantlyCD4+CD25+ T cells. White arrows in the CD8 picture indicatethe presence of rare CD8+FOXP3+ cells and in the CD25 picturethese indicate that a proportion of the FOXP3+ cells are CD25–.(C) Illustrates both activated and resting cells from the CD4+ Tcell clone TB1 (left) and the CD8+ clone 2D10 (right) stained forFOXP3 protein expression showing that activation inducesFOXP3 expression.
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Discussion
A critical role of immunological tolerance is to enablethe immune system to discriminate self from non-selfantigens. This occurs through central tolerance in thethymus during the development of the immune systemand through peripheral tolerance to eliminate self-reactive T cells that have escaped thymic selection orarisen de novo. The FOXP3 forkhead transcription factorplays a critical role in this process through its central rolein the generation of immunoregulatory T cells. Themolecular mechanisms governing CD4+CD25+ TR celldevelopment and function are currently highly topicalbecause of their importance in preventing the develop-ment of autoimmunity and their therapeutic potential.
The lack of suitable antibodies to detect endogenoushuman FOXP3 protein expression means that themajority of TR studies to date have examined FOXP3mRNA expression by quantitative real-time PCR. How-ever, this will not address post-transcriptional control ofFOXP3 protein levels and mRNA expression is measuredlargely in vitro in purified cell populations with noinformation as to the relative proportions of FOXP3+
versus FOXP3– cells or their distribution and abundancein tissues. One of the few studies comparing both Foxp3mRNA and protein expression, in response to theactivation of murine wild-type CD4+CD25+ T cells,reported the up-regulation of Foxp3 protein despite areduction in Foxp3 mRNA expression [11]. This lack of
concordance between Foxp3 mRNA and protein levelsindicates that further investigation of the expression andfunctional role of FOXP3 should include studies ofFOXP3 protein expression.
To investigate the expression of FOXP3 protein inhuman tissues we have raised a panel of seven mAb thatspecifically recognize the human FOXP3 protein and donot cross-react with the other FOXP proteins. The mAbhave been tested in a variety of commonly usedimmunological techniques and can be used for im-munohistochemistry on frozen and paraffin-embeddedtissues, Western blotting, flow cytometry and to detectthe murine Foxp3 protein. With the identification ofFOXP3 expression in adult T cell leukemia/lymphoma[38], the mAb that are reactive on paraffin-embeddedtissue also be beneficial in the routine diagnosis of thismalignancy.
Published studies have consistently reported thatFOXP3 is predominantly expressed in both human andmurine CD4+CD25+ TR cells. We analyzed FOXP3protein expression in both lymphoid tissues and inperipheral blood. Our double-labeling studies confirmthat the majority of FOXP3+ cells are indeedCD4+CD25+.
These mAb have enabled, for the first time, aninvestigation of the frequency of FOXP3+ cells withinthe CD25+ and CD25– populations of CD4+ T cells. Inperipheral blood from healthy individuals, approxi-mately half of the whole CD4+CD25+ populationexpress the FOXP3 protein whereas only a minority ofCD4+CD25– cells are FOXP3+ by immunofluorescentlabeling of cytospins. FACS analysis revealed a strongcorrelation between the level of expression of CD25 andthe frequency of FOXP3+ cells. Thus there was a muchhigher frequency of FOXP3+ cells amongstCD4+CD25high PBL (96%) when compared withCD4+CD25int PBL (35%), consistent with the findingsthat in humans TR cell activity is restricted to theCD4+CD25high population [39]. The CD4+CD25int
population, on the other hand, is a more heterogeneousmix containing not only regulatory T cells, but alsoactivated CD25-expressing effector T cells [39]. Futurestudies will address whether the frequencies of FOXP3+
cells are altered in cancer patients and/or patients withautoimmune diseases.
A more contested issue has been whether murine andhuman CD8+ TR cells express FOXP3. A distinctpopulation of CD8+ antigen-primed T cells displaysregulatory functions in human transplant recipients andin a murine autoimmune disease model [40, 41]. Unlikeother TR cells, these are antigen-specific, MHC class I-restricted, and interact directly with antigen-presentingcells [42–45]. Although low-level Foxp3 mRNA expres-sion has been reported in murine CD8+ T cells [3, 13],this finding was not confirmed by subsequent studies of
Fig. 4. The FOXP3 236A/E7 mAb labels functional suppressor Tcells. (A) Illustrates FACS confirmation of the CD4+CD25+
phenotype of the bead-sorted suppressor cells. (B) Shows thelabeling of theseCD4+CD25+ cellswith the 236A/E7 FOXP3mAb.(C) The CD4+CD25+FOXP3+ cells are functionally able tosuppress the proliferation of CD4+CD25–FOXP3– cells in adose-dependent manner.
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either the Foxp3 protein or mRNA expression [11, 12].However, unlike wild-type mice, Foxp3-transgenic miceexpressed large amounts of Foxp3 mRNA in bothCD4+CD25– and CD8+ cells and these cell populationswere found to have suppressor activity [13]. Recently apopulation of functionally active rat CD8+CD45RClow TR
cells, isolated from normal lymph nodes and spleen,have been reported to express Foxp3 mRNA [46].
In humans, data obtained using a rabbit anti-human-FOXP3 antibody showed that CD8+ T cells isolated fromperipheral blood expressed FOXP3 protein, although atlower levels than was observed in CD4+ cells [47].However, a subsequent study reported FOXP3 mRNAexpression in human CD8+CD28– suppressor T cell linesbut not in CD8+CD28– and CD8+CD28+ T cells fromfresh peripheral blood [48]. Our double-labeling studieshave allowed us to immunophenotype individualFOXP3+ cells in situ and have clearly demonstratedthe existence of a small naturally occurring populationof CD8+ cells in the secondary lymphoid tissue thatexpress the FOXP3 protein. However, FACS analysis didnot reveal detectable CD8+FOXP3+ cells in PBL. Thepresence of CD8+FOXP3+ cells in secondary lymphoidtissue but not peripheral bloodmay reflect differences inthe anatomical location of these cells or be aconsequence of reduced sensitivity of FACS analysiscompared with immunohistochemistry.
There are also reports of Foxp3 expression in murineB220+ B cells [3, 13], whereas CD19+ B cells werereported to be negative for Foxp3 mRNA expression[12]. Increased levels of Foxp3 mRNA and proteinexpression have also been reported in B cells from thelymph nodes and spleen of the Foxp3-transgenic mouse[13, 49]. However, these Foxp3+ B cells were not foundto have a suppressor function in vitro [13]. In the currentstudy there was no detectable expression of FOXP3protein in CD20+ tonsillar B cells or in naive CD20+ cellsor activated (by IgM or by rIL-2 plus SAC) CD20+ cellsthat had been isolated from peripheral blood (data notshown).
There is a reported difference in the human andmouse immune systems as to whether FOXP3 expressionis induced by T cell activation. On activation, murineCD4+CD25– T cells acquire surface CD25 but do notexpress high levels of Foxp3 mRNA or protein [11, 13].Nevertheless, forced overexpression of Foxp3 convertsnaive murine CD4+CD25– T cells to TR cells [11, 12]. Incontrast, the activation of human CD4+CD25– T cellsthrough the TCR leads to the increased expression ofFOXP3 mRNA, the surface expression of CD25 and gainof regulatory function [47]. Both resting and activatedCD4+ and CD8+ T cell clones were examined for FOXP3protein expression, which was found exclusively in theactivated cell populations. These findings are consistentwith those from a recent study that reported the
induction of FOXP3 mRNA expression in activated T cellclones [50]. These findings raise the possibility thatFOXP3 plays a functional role not only inTR cells but alsofollowing activation of conventional T cells.
This panel of mAb provides an essential tool withwhich to further investigate the expression and functionof the FOXP3 transcription factor and to label FOXP3+
TR cells in both frozen and routinely fixed humantissues. The application of these mAb for FACS analysisopens new avenues to investigate the correlationbetween the frequency and level of expression of FOXP3in distinct T cell subsets in health and disease.
Materials and methods
Bacterial expression of GST–FOXP3 fusion protein
An I.M.A.G.E. Consortium [LLNL] cDNA clone [37] containingthe FOXP3 cDNA (IMAGE:5747723) was obtained from theMRC geneservice. A cDNA fragment encoding the full-lengthhuman FOXP3 protein was introduced to pDEST15 (Invitro-gen) by means of GatewayJ technology. The GST–FOXP3fusion protein was then expressed in Esherichia coli, strainBL21(DE3), and purified by affinity chromatography.
Antibodies
Three BALB/c mice were injected intraperitoneally (threetimes at 14-day intervals) with 100 lg GST–FOXP3 fusionprotein plus Freund's adjuvant. A 150-lg booster of therecombinant GST–FOXP3 protein was injected intraperitone-ally, and a fusion was carried out 3 days later usingconventional techniques [51]. Hybridoma supernatants werescreened by immunohistochemistry on frozen normal humantonsil sections as described below. The seven mouse mAb thatwere raised against FOXP3 (86D/D6, 150D/E4, 157B/F4,206D/B1, 221D/D3, 236A/E7, 259D/C7) were cloned by thelimiting dilution technique. Other Ab used include a goatFOXP3 polyclonal (Ab2481, Abcam) and mAb against FOXP1(JC12, "in house"), CD25 (Novocastra, Newcastle upon Tyne,UK), anti-XpressTM (Invitrogen), anti-FLAG M2 (Sigma), andanti-CD4, -CD8, -CD3 or -CD20 (all from DakoCytomation,Glostrup, Denmark).
Tissue samples
Normal and neoplastic human tissues were obtained from theDepartment of Pathology at the John Radcliffe Hospital,Oxford, UK and the tissue archives of the CNIO Tumor Bank,with local ethical committee approval. Samples were fixed inbuffered formalin and embedded in paraffin according toroutine procedures [52]. Frozen sections of spleen and lymphnode were also obtained from normal mice (Balb/c or B6).
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Production of FOXP transfectants and theirimmunolabeling
Eukaryotic expression of the FOXP proteins in COS-1 cells
To confirm that the FOXP3 mAb were specifically reactive withthe FOXP3 protein, their reactivity was tested on COS-1 cellsexpressing FOXP3 and on COS-1 cells expressing the closelyrelated FOXP1, FOXP2 or FOXP4 proteins. FOXP1, FOXP2 andFOXP4 expression constructs have been previously described[53, 54]. The Flag-tagged FOXP3 cDNAwas kindly provided byDr Mary Brunkow, Celltech.
Plasmid DNA was prepared for transfection using thePlasmid Midi Kit according to the manufacturer's instructions(Qiagen). COS-1 cells were transfected with pcDNA4/HisMax/FOXP2, FOXP3, pcDNA4/HisMax/FOXP4, pcDNA4/HisMaxalone or pAB195 (FOXP1) using Fugene 6 transfection reagent,following the protocol described by the manufacturer (RocheApplied Science). Approximately 24 h post-transfection, thecells were washed with sterile PBS and harvested bytrypsinisation. Cell pellets were snap-frozen and stored at–70oC whereas cytocentrifuge preparations were made forimmunocytochemical staining and stored at –20oC [55].Paraffin-embedded cell pellets were prepared by fixingtransfectants for 48 h in neutral buffered formalin (10%formalin in PBS), before centrifugation into 2% agar in neutralbuffered formalin; the agar pellet was then embedded inparaffin and sectioned as for tissues. Transfected COS-1 cellswere indirectly immunoenzymatically labeled using anti-XpressTM Ab at 5.5 lg ml–1, anti-FLAG Ab at 10 lg ml–1,JC12 hybridoma supernatant diluted 1/10 in PBS plus 10%human serum or in FOXP3 hybridoma supernatants. Abbinding was detected using the DAKO EnVisionTM + System,HRP and diaminobenzidine (DAB) as directed by themanufacturer (DakoCytomation).
FACS staining of FOXP3-transfected COS cells
COS cells were permeabilised with FACS Perm 2 solution (BDBiosciences). Cells were stained with each FOXP3 mAb and asecondary goat-anti-mouse–PE (Southern Biotech). They wereanalyzed using FACS Calibur (BD Biosciences) and CELLQuestsoftware.
Immunohistochemistry
Frozen tonsil tissue sections were incubated for 30 min withprimary Ab, washed in PBS and incubated with either HRP-conjugated goat anti-mouse-Ig (diluted 1/50 in PBS) (Dako-Cytomation) or HRP-conjugated rabbit anti-goat-Ig (DakoCy-tomation). The peroxidase reaction was developed using DAB(DakoCytomation) for 5 min and washed with distilled water.Murine tissue sections were fixed in acetone, blocked with 10%normal donkey serum, incubated with each FOXP3 mAb(diluted 1/2) followed by anti-mouse–Texas-red (diluted 1/100) and anti-CD4–FITC (diluted 1/75).
Four-micron sections were cut from paraffin blocks andcaptured on electrically charged slides (Snowcoat X-traTM,Surgipath). Sections were dewaxed in citroclear (HD Supplies,Aylesbury,UK)andantigenretrievalwasperformedbymicrowave
pressurecookingfor3 minatfullpressurein50 mMTrisand2 mMEDTA,pH 9.Beforestainingthesections,endogenousperoxidasewasblocked,theslideswereincubatedfor40 minwiththeprimaryAb, washed with PBS and the immunodetection was performedwithbiotinylatedanti-mousesecondaryAb(25 min),followedbyperoxidase-labeledstreptavidin(25 min)andDABchromogenassubstrate. Immunostaining performed in Oxford used theEnVisionTM system (DakoCytomation). All immunostaining inMadrid was performed using the Techmate 500 automaticimmunostaining device and reagents from DakoCytomation.Incubations either omitting the specific Ab or containingunrelated Ab were used as a control of the technique. Sectionswere counterstained with hematoxylin Gill No. 3 (Sigma) andmounted in Aquamount (BDH, UK).
Double immunoenzymatic labeling
Double enzymatic immunostaining for FOXP3 and othermarkers including CD3, CD4, CD25, CD8 and CD20 wasperformed on tonsil. The details of the methodology used forthe immunoenzymatic and immunofluorescent double im-munostaining has been described previously [56].
Western blotting
Approximately 1�107 FOXP3-transfected COS-1 cells werelysed in 90 ll RIPA buffer, then 10 ll of EBB (1.5 M NaCl,0.1 M CaCl2, 0.1 M MgCl2, 0.2 mg ml–1 DNAse I) was added.Following a 10-min incubation at room temperature, lysateswere mixed with 100 ll 2� SDS gel loading buffer, resolved on12% SDSPAGE gels, then transferred to Immobilon-P mem-branes (Millipore) using semidry apparatus. The membraneswere blocked in 5%marvel in PBS at 4�C overnight. FOXP3 andMR12 (mouse anti-rabbit-Ig, negative control) hybridomasupernatants were used neat and anti-FLAG M2 at 10 lg ml–1
and applied for 90 min. Following three 10-min washes in PBScontaining 0.02% Tween, membranes were incubated with a1/1000 dilution of secondary Ab, rabbit anti-mouse-Ig–HRPconjugate (DakoCytomation) for 90 min. Wash steps wererepeated and Ab binding was detected using ECL reagent(Amersham Biosciences, Uppsala, Sweden). To confirm sampleloading and transfer, membranes were incubated in strippingbuffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM TrisHCl, pH 6.8) for 30 min at 50�C, reblocked for 1 h, then re-probed using anti-b-actin (AB6276, Abcam) diluted 1/25000in blocking buffer following the same procedure.
T cell clones and activation
The CD8+ clones, 2D10 and 3F6, were generated by tetramerstaining and FACS cloning and maintained as describedpreviously [57]. The CD4+ clone TB1 generated from a patientwith a thymoma, as described previously [58], was kindlyprovided by Professor Nick Willcox (Oxford). The cells weremaintained in culture in RPMI-1640 containing 10% fetal calfserum (Invitrogen, Paisley, Scotland) at 37oC in 5% CO2. Cellswere activated, within 10–14 days, by stimulation with 20 lg/ml phytohemagglutinin (Murex) and irradiated allogeneicperipheral blood lymphocytes (50 Gy). Cells were consideredto be resting after more than 21 days post-stimulation.
Giovanna Roncador et al. Eur. J. Immunol. 2005. 35: 1681–16911688
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Cell isolation and immunofluorescent labeling of sortedcells
Human mononuclear cells were isolated from fresh blood byLymphoprep (Nycomed, Oslo) gradient centrifugation. TheCD4+ cells were obtained by negatively sorting the mono-nuclear cells with CD8 and CD14 magnetic beads (MiltenyiBiotech, GmbH, Germany). These were then positively sortedfor CD4+CD25+ cells using CD25 magnetic beads (MiltenyiBiotec). In some instances, CD4+CD25+ cells were isolatedusing the CD4+CD25+ regulatory isolation kit according to themanufacturer's instructions (Miltenyi Biotech). MACS-sortedCD4+CD25+ and CD4+CD25– cells isolated from humanperipheral blood were spotted onto slides (Henley-Essex) at5�104/spot. Slides were air-dried and then frozen at –20�C.Cells were fixed in pre-chilled acetone (–20�C) for 5 min andblocked with 10% normal goat serum. Primary Ab (clone221D/D3) at 10 lg ml–1 was added overnight at 4�C, followedby goat anti-mouse–AlexaFluor-488 (1/500) (MolecularProbes, The Netherlands).
Cytocentrifuge preparations of T cells purified for thesuppression assay were air-dried overnight, fixed in 50:50acetone:methanol for 60 sec at room temperature, rinsed inPBS, then incubated with primary mAb (clone 236A/E7) for50 min. Following a 5-min wash in PBS the secondary Ab (goatanti-mouse-IgG1–AlexaFluor-546, Molecular Probes) diluted1/400, was applied for 30 min.
FACS methodology for detecting endogenous FOXP3
ForFACSanalysis, 5�105 cellswere fixed in1 mlofPBSwith1%paraformaldehyde and 0.05%Tween-20 overnight at 4�C. Cellswere treated twice with 0.5 ml of DNAse at 100 Kunitz/mlaccording to the manufacturer's instructions (Sigma-Aldrich).Staining steps were performed at room temperature for onehour. Cells were incubated with mouse anti-human-FOXP3(clone150D/E4),washedwithFACSbuffer(PBS1�,3.00%fetalcalf serum, 0.50% Tween-20, 0.05% azide). FOXP3 Ab bindingwas detected using Alexa Fluor-488J goat anti-mouse-IgG(Molecular Probes) and washed as above. Cell surface stainingwas then performed using the mAb Cy-Chrome–anti-human-CD4(Pharmingen)andPE–anti-human-CD25(MiltenyiBiotec)for 20 min at room temperature followed by washing in PBS/BSA. Cells were analyzed using a FACSCaliburTM withCELLQuestTM software (Becton Dickinson).
Cell proliferation assay
Cells were cultured in RPMI-1640 medium supplemented with5% human AB serum, 2 mM L-glutamine (Gibco/Invitrogen),100 U/lg/ml penicillin/streptomycin (Gibco/Invitrogen),0.5 mM sodium pyruvate (Gibco/Invitrogen) and 0.05 mMnonessential amino acids (Gibco/Invitrogen) in 96-well plates(NalgeNunc,Rochester,NY,USA).Plate-boundanti-CD3(cloneUCHT1,at5 lgml–1)andsoluble anti-CD28(clone28.2, at5 lgml–1) were purchased from Pharmingen (BD BiosciencesPharmingen). The CD4+CD25– responder cells were used at5�104/well and a variable number of CD4+CD25+ regulatorycells were added. [3H]thymidine at 0.5 lCi per well was addedfor the final 16 h of a 5-day assay.
Acknowledgements: This work was supported byfunding from the Leukaemia Research Fund, theAssociation for International Cancer Research, theWellcome Trust, Cancer Research UK (C399-A2291),the EU (LSHB-CT-2003-503410), the US Cancer ResearchInstitute and the UK Medical Research Council.
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