Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level

Authors


Abstract

The transcription factor FOXP3 plays a key role in CD4+CD25+ regulatory T cell function and represents a specific marker for these cells. Despite its strong association with regulatory T cell function, in humans little is known about the frequency of CD4+CD25+ cells that express FOXP3 protein nor the distribution of these cells in vivo. Here we report the characterization of seven anti-FOXP3 monoclonal antibodies enabling the detection of endogenous human FOXP3 protein by flow cytometry and immunohistochemistry. Flow-cytometric analysis showed that FOXP3 was expressed by the majority of CD4+CD25high T cells in peripheral blood. By contrast, less than half of the CD4+CD25int population were FOXP3+, providing an explanation for observations in human T cells that regulatory activity is enriched within the CD4+CD25high pool. Although FOXP3 expression was primarily restricted to CD4+CD25+ cells, it was induced following activation of both CD4+ and CD8+ T cell clones. These findings indicate that the frequency of FOXP3+ cells correlates with the level of expression of CD25 in naturally arising regulatory T cells and that FOXP3 protein is expressed by some activated CD4+ and CD8+ T cell clones. These reagents represent valuable research tools to further investigate FOXP3 function and are applicable for routine clinical use.

See accompanying Commentary http://dx.doi.org/10.1002/eji.200526303

Abbreviation:
TR cell:

Regulatory T cell

Introduction

FOXP3 is a member of the forkhead or winged helix family of transcription factors. The gene was first described as JM2 when its mutation was identified in an X-linked autoimmune and allergic disregulation syndrome (XLAAD) 1. Mutations in FOXP3 were subsequently identified as the defect underlying the scurfy mouse and the human X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome (IPEX) 24. CD4+ T cells are primarily responsible for the pathology observed in scurfy mice 5, 6, which have a similar phenotype to that reported for CTLA-4 knockout mice 7, 8. Murine Foxp3 determines both the number and functionality of peripheral T cells 9 and regulates T cell activation 10. The absence of FOXP3 in both mice and humans thus causes a fatal immune proliferative disease, as a consequence of chronic T cell activation, whereas Foxp3 overexpression in transgenic mice results in a reduction in the mature T cell population and diminished T cell function 9. More recently FOXP3 has been identified as a master regulatory gene for cell-lineage commitment or developmental differentiation of CD4+CD25+ regulatory T cells (TR cells). Gene transfer of Foxp3 converts naive CD4+CD25 T cells towards a regulatory phenotype and this molecule appears to represent an important marker of this TR cell population 1113.

CD4+CD25+ TR cells comprise approximately 10% of peripheral CD4+ cells 1417. They play a crucial role in the control of T cell mediated autoimmunity by suppressing the proliferation and cytokine production of other T cells 1820. TR cells are implicated in a range of disease states including organ-specific autoimmune disease 21, inflammatory bowel disease 22, multiple sclerosis 23, allograft rejection 24, graft-versus-host disease 2527, allergy 28, and sterilizing immunity to infectious organisms 29. The accumulation of TR cells has been observed in the peripheral blood and tumor microenvironment of cancer patients 3034 and their removal has been reported to result in effective antitumor immunity, implicating these cells in immune evasion by cancer cells [35.

A problem hindering the study of TR cells has been the lack of a unique marker that defines all cells with regulatory activity. The activation marker CD25 has limitations in that its expression is not restricted to T cells and cannot distinguish TR cells from conventional activated T cells. Thus, although FOXP3 mRNA is expressed at high levels by CD4+CD25+ cells there is no information on the frequency of bona fide FOXP3+ TR cells within this population. Furthermore, both CD25 and other TR cell markers, such as GITR, CTLA-4, and CD45RB, are not expressed on all CD4+ T cells with regulatory activity. Neuropilin has recently been identified as a potentially useful surface marker of murine CD4+CD25+Foxp3+ TR cells 36. Because FOXP3 is a nuclear protein it is of limited value in the isolation of TR cells. However, unlike neuropilin, which is expressed in other cell types, the expression of FOXP3 is highly restricted to discrete TR cell populations and it is therefore valuable as a specific marker to detect TR cells in vivo.

Here we describe our production of a panel of mAb that specifically recognize the human FOXP3 protein. Using these reagents we show that the majority of FOXP3+ cells are CD4+CD25+ cells in secondary lymphoid tissue, although a minor population of CD8+CD25+ and CD4+CD25 cells that express FOXP3 is also present. In addition we describe the use of these antibodies for flow-cytometric analysis of FOXP3 protein expression, enabling the first determination of the frequency of TR cells within the CD4+CD25+ T cell pool in human PBL.

Results

The Abcam goat polyclonal FOXP3 Ab labels the human FOXP3 protein in frozen but not routinely fixed tissues

The commercially available Abcam goat polyclonal FOXP3 Ab was evaluated as a reagent to detect the FOXP3 protein in human tissues. Immunohistochemistry confirmed that the Ab recognized the human flag-tagged FOXP3 protein expressed in COS-1 cell transfectants and stained scattered interfollicular cells in tonsil (Fig. 1). However, we were unable to detect the FOXP3 protein in routinely fixed paraffin-embedded tissues with this reagent and thus additional reagents were necessary to investigate archival biopsy material.

Figure 1.

Immunolabeling of tonsil and FOXP transfectants. The top and middle rows show immunoperoxidase labeling with the commercial goat polyclonal Ab or the 236A/E7 mouse FOXP3 mAb, respectively, on tonsil and FOXP3-COS-1 transfectants. The bottom row shows the lack of staining with the 236A/E7 FOXP3 mAb on COS-1 cells transfected with the related FOXP proteins. The insets at top right of the bottom row confirm the expression of these recombinant FOXP proteins using the indicated Ab.

Production of FOXP3 mAb

An I.M.A.G.E. Consortium [LLNL] cDNA clone 37, encoding the start of the FOXP3 protein and having a polyA tail, was used to bacterially express a GST fusion protein containing a full-length FOXP3 protein for immunization. The hybridoma fusion generated seven mAb that showed a similar staining pattern, of scattered interfollicular cells on frozen tonsil, to that observed with the polyclonal FOXP3 Ab (Fig. 1). We observed more FOXP3+ cells using the FOXP3 mAb, which showed a stronger reactivity than was obtained with the polyclonal Ab. The reactivity of these FOXP3 mAb was further investigated.

The mAb specifically recognize FOXP3 and not other FOXP proteins

To confirm that the mAb recognized the human FOXP3 protein their ability to detect the flag-tagged human FOXP3 protein, expressed in COS-1 cells, was tested by immunohistochemistry on frozen cytospin preparations. Labeling with the anti-FLAG Ab confirmed the efficiency of transfection and the subcellular distribution of the recombinant FOXP3 protein. Cytoplasmic expression of proteins that are normally nuclear is commonly observed when cells are transfected using the described Fugene protocol and harvested after 24 h. All of the mAb specifically recognized the human FOXP3 protein by immunohistochemistry (Fig. 1).

There is significant sequence similarity between the four members of the FOXP family, FOXP1–4, particularly within the DNA binding forkhead domain. As a full-length FOXP3 protein was used as an antigen it was important to ensure the specificity of the mAb for FOXP3 and exclude the possibility of their cross-reactivity with other FOXP proteins. Expression of the FOXP1, FOXP2 and FOXP4 proteins in transfected cells was confirmed using the JC12 mAb to label FOXP1 and the anti-Xpress antibody to detect epitope-tagged FOXP2 and FOXP4 (Fig. 1). All the FOXP3 mAb specifically labeled the COS-1 cells expressing FOXP3 and not those expressing the other FOXP proteins (Fig. 1).

While preparing this manuscript a FOXP3 murine mAb, hFOXY, has become commercially available (Cat No 14-5779, eBioscience, San Diego, CA, USA). We have investigated this reagent and compared its reactivity on tonsil with that obtained with our 236A/E7 FOXP3 mAb (data not shown). On frozen tonsil sections hFOXY stained scattered interfollicular cells at a dilution of 1/25. At this dilution some background staining was observed and there was a noticeably smaller population of nuclear+ cells than was stained with our FOXP3 mAb 236A/E7. Staining of COS-1 cell transfectants confirmed that hFOXY detected the FOXP3 protein and not the related FOXP1, FOXP2 or FOXP4 proteins. Staining of paraffin-embedded COS cell transfectants indicated that hFOXY recognizes a formalin-resistant epitope on FOXP3 and no cross-reactivity was observed under these conditions with other FOXP proteins. Thus hFOXY is suitable for detecting FOXP3 by immunohistochemistry on both frozen and paraffin-embedded tissues. However, this reagent may only detect a subpopulation of FOXP3+ cells.

The FOXP3 mAb recognize the FOXP3 protein in a variety of applications and two are cross-reactive with the murine Foxp3 protein

The FOXP3 mAb were tested for their ability to recognize formalin-resistant epitopes by immunohistochemistry using routinely fixed tonsil and formalin-fixed paraffin-embedded pellets of FOXP3-transfected COS-1 cells. All of the mAb recognized the FOXP3 protein in routinely fixed tissues and labeled FOXP3 transfectants. Some mAb gave less non-specific background staining than others, whereas some showed stronger labeling of positive cells, these data are summarized in Table 1. Ab 86D/D6 and 236A/E7 are recommended for immunohistochemistry on paraffin-embedded tissues.

Table 1. Reactivity of FOXP3 mAb
AntibodyIH FOXP3 transfectantsIH FOXP1,2 or 4 transfectantsIH tonsil frozenIH paraffin tonsilWestern blotting transfectantsFlow cytometry transfectantsMurine tissues
  1. a) Indicates that there was some nonspecific background staining. IH, immunohistochemistry.

86D/D6++++++
150D/E4++++++++
157B/F4++++a)+++
206D/B1++++++
221D/D3++++a)+++++
236A/E7++++++++a)
259D/C7++++a)++++a)

The ability of the mAb to recognize the FOXP3 protein by flow-cytometry and by Western blotting was also tested using FOXP3-transfected COS-1 cells. All the mAb recognized the recombinant FOXP3 protein to some extent in Western blotting and in flow cytometry (Table 1).

The human and murine FOXP3 proteins are approximately 87% identical, thus the FOXP3 mAb were also tested for reactivity with the murine Foxp3 protein. The mAb 150D/E4 and 221D/D3 were found to be cross-reactive with the murine Foxp3 protein and specifically labeled CD4+ T cells in murine spleen and lymph nodes (data not shown as these are consistent with the human data).

Characterization of FOXP3 protein expression in normal human tissues

FOXP3 protein expression was assessed by immunohistochemistry with mAb 236A/E7 on a routinely fixed normal-tissue microarray containing 39 different human tissues and on whole sections of lymphoid tissues. Tissues included tonsil, spleen, bone marrow, brain, 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 fetal thymus. There was no nuclear FOXP3 protein expression observed in the range of normal non-hematological tissues tested, with the exception of scattered positive lymphocytes in colon, stomach, and fallopian tube. These data are consistent with the reported restricted expression of FOXP3 within lymphocytes.

Within hematological tissues there were much higher numbers of lymphocytes expressing FOXP3. The FOXP3+ cells were scattered within the interfollicular areas of tonsil (Fig. 2A) and were occasionally seen within the follicular germinal centers (Fig. 2A). Reactive lymph node contained many FOXP3+ cells and these were distributed throughout the tissue, including within the mantle zone and germinal centers of the secondary follicles. In spleen there were occasional FOXP3+ cells in the red pulp and there were increased numbers in the T cell areas around vessels (Fig. 2A). There were many FOXP3+ cells in fetal thymus and in the mature thymus these were present predominantly in the medulla with only scattered FOXP3+ cells being present in the cortex (Fig. 2A). In bone marrow a small number of FOXP3+ cells were also observed.

Figure 2.

FOXP3 protein expression in normal human tissues. (A) Illustrates peroxidase immunolabeling of normal lymphoid tissues with the 236A/E7 FOXP3 Ab. (B) Illustrates double-immunofluorescent labeling of normal tonsil (g, green; r, red), confirming that the FOXP3 mAb recognizes predominantly CD4+CD25+ T cells. White arrows in the CD8 picture indicate the presence of rare CD8+FOXP3+ cells and in the CD25 picture these indicate that a proportion of the FOXP3+ cells are CD25. (C) Illustrates both activated and resting cells from the CD4+ T cell clone TB1 (left) and the CD8+ clone 2D10 (right) stained for FOXP3 protein expression showing that activation induces FOXP3 expression.

To confirm the immunophenotype of the FOXP3+ cell population in situ within tissues, double-labeling studies were performed by immunofluorescent (Fig. 2B) and immunoenzymatic techniques (data not shown). These studies confirmed that in tonsil the FOXP3 protein was expressed exclusively in the CD3+ T cell population and no double-labeling of CD20+ B cells was observed. The majority of FOXP3+ cells were, as previously reported, both CD4+ and CD25+. However, double-labeling identified a very small population of FOXP3+CD8+ cells and indicated that a minority of FOXP3+ cells were CD25 (Fig. 2B).

FOXP3 is expressed in activated T cells

There have been reports that FOXP3 expression is induced in activated T cells. Cytospin preparations of both resting and activated CD8+ and CD4+ human T cell clones were immunostained with FOXP3 mAb 236A/E7. FOXP3 protein expression was restricted to the nuclei of activated CD4+ and CD8+ T cell clones and was absent in the resting cells (Fig. 2C).

Frequency of FOXP3 expression in the human CD4+ population

CD4+CD25+ and CD4+CD25 populations were purified from peripheral blood taken from three individuals without any known disease as described in the "Materials and Methods" section. Immunofluorescent labeling of FOXP3 expression was performed and the numbers of FOXP3+ and FOXP3 cells were scored in each 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.

Figure 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 from peripheral blood and observed using immunofluorescent labeling. No staining of the CD4+CD25+ cells was observed with the secondary Ab alone (right). (B) The top left panel shows the FACS plots and gating of PBL labeled with CD4 and CD25. The top right panel represents the FOXP3 expression according to CD25 expression in the whole CD4 population. The FOXP3 expression in the gated populations is illustrated in the histograms. Quadrants are drawn based on isotype-matched control antibodies that gave <1% background. Representative of three separate experiments.

Despite early difficulties with detecting endogenous FOXP3 protein by flow cytometry, a modified staining protocol that included a DNAse digestion step enabled FACS analysis of endogenous FOXP3 protein expression in the CD4+ population and correlation with CD25 expression (Fig. 3B). In peripheral blood, 95.7% (94.7–95.8) of CD4+CD25high T cells expressed FOXP3 whereas only 34.9% (22.3–47.4) of CD4+CD25int T cells stained positive for FOXP3. No detectable staining was observed in the CD25 population or in resting CD8+ T cells (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 commercially available hFOXY mAb is also suitable for FACS analysis of FOXP3 expression. However, we were unable to reveal FOXP3 expression amongst peripheral blood CD4+CD25+ cells using this reagent, suggesting it is not suitable for this application (data not shown).

The FOXP3 mAb label functional CD4+CD25+T cells

Although the double-labeling studies indicate that the FOXP3 mAb label predominantly CD4+CD25+ T cells, their ability to detect T cells with functional regulatory activity was also confirmed. The immunophenotype of CD4+CD25+ T cells purified from peripheral blood using magnetic-bead selection was confirmed by flow cytometry (Fig. 4A). Nuclear expression of the FOXP3 protein was confirmed by immunofluorescent labeling (Fig. 4B), only the occasional FOXP3+ cell was observed in the CD25 population and no staining was observed when using the isotype-matched MR12 control mAb (data not shown). These FOXP3+ cells were able to suppress the proliferation of CD4+CD25 T cells (Fig. 4C) confirming that the mAb label functional CD4+CD25+FOXP3+ TR cells.

Figure 4.

The FOXP3 236A/E7 mAb labels functional suppressor T cells. (A) Illustrates FACS confirmation of the CD4+CD25+ phenotype of the bead-sorted suppressor cells. (B) Shows the labeling of these CD4+CD25+ cells with the 236A/E7 FOXP3 mAb. (C) The CD4+CD25+FOXP3+ cells are functionally able to suppress the proliferation of CD4+CD25FOXP3 cells in a dose-dependent manner.

Discussion

A critical role of immunological tolerance is to enable the immune system to discriminate self from non-self antigens. This occurs through central tolerance in the thymus during the development of the immune system and through peripheral tolerance to eliminate self-reactive T cells that have escaped thymic selection or arisen de novo. The FOXP3 forkhead transcription factor plays a critical role in this process through its central role in the generation of immunoregulatory T cells. The molecular mechanisms governing CD4+CD25+ TR cell development and function are currently highly topical because of their importance in preventing the development of autoimmunity and their therapeutic potential.

The lack of suitable antibodies to detect endogenous human FOXP3 protein expression means that the majority of TR studies to date have examined FOXP3 mRNA expression by quantitative real-time PCR. However, this will not address post-transcriptional control of FOXP3 protein levels and mRNA expression is measured largely in vitro in purified cell populations with no information as to the relative proportions of FOXP3+ versus FOXP3 cells or their distribution and abundance in tissues. One of the few studies comparing both Foxp3 mRNA and protein expression, in response to the activation of murine wild-type CD4+CD25+ T cells, reported the up-regulation of Foxp3 protein despite a reduction in Foxp3 mRNA expression 11. This lack of concordance between Foxp3 mRNA and protein levels indicates that further investigation of the expression and functional role of FOXP3 should include studies of FOXP3 protein expression.

To investigate the expression of FOXP3 protein in human tissues we have raised a panel of seven mAb that specifically recognize the human FOXP3 protein and do not cross-react with the other FOXP proteins. The mAb have been tested in a variety of commonly used immunological techniques and can be used for immunohistochemistry on frozen and paraffin-embedded tissues, Western blotting, flow cytometry and to detect the murine Foxp3 protein. With the identification of FOXP3 expression in adult T cell leukemia/lymphoma 38, the mAb that are reactive on paraffin-embedded tissue also be beneficial in the routine diagnosis of this malignancy.

Published studies have consistently reported that FOXP3 is predominantly expressed in both human and murine CD4+CD25+ TR cells. We analyzed FOXP3 protein expression in both lymphoid tissues and in peripheral blood. Our double-labeling studies confirm that the majority of FOXP3+ cells are indeed CD4+CD25+.

These mAb have enabled, for the first time, an investigation of the frequency of FOXP3+ cells within the CD25+ and CD25 populations of CD4+ T cells. In peripheral blood from healthy individuals, approximately half of the whole CD4+CD25+ population express the FOXP3 protein whereas only a minority of CD4+CD25 cells are FOXP3+ by immunofluorescent labeling of cytospins. FACS analysis revealed a strong correlation between the level of expression of CD25 and the frequency of FOXP3+ cells. Thus there was a much higher frequency of FOXP3+ cells amongst CD4+CD25high PBL (96%) when compared with CD4+CD25int PBL (35%), consistent with the findings that in humans TR cell activity is restricted to the CD4+CD25high population 39. The CD4+CD25int population, on the other hand, is a more heterogeneous mix containing not only regulatory T cells, but also activated CD25-expressing effector T cells 39. Future studies will address whether the frequencies of FOXP3+ cells are altered in cancer patients and/or patients with autoimmune diseases.

A more contested issue has been whether murine and human CD8+ TR cells express FOXP3. A distinct population of CD8+ antigen-primed T cells displays regulatory functions in human transplant recipients and in a murine autoimmune disease model 40, 41. Unlike other TR cells, these are antigen-specific, MHC class I-restricted, and interact directly with antigen-presenting cells 4245. Although low-level Foxp3 mRNA expression has been reported in murine CD8+ T cells 3, 13, this finding was not confirmed by subsequent studies of either the Foxp3 protein or mRNA expression 11, 12. However, unlike wild-type mice, Foxp3-transgenic mice expressed large amounts of Foxp3 mRNA in both CD4+CD25 and CD8+ cells and these cell populations were found to have suppressor activity 13. Recently a population 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 from peripheral blood expressed FOXP3 protein, although at lower levels than was observed in CD4+ cells 47. However, a subsequent study reported FOXP3 mRNA expression in human CD8+CD28 suppressor T cell lines but not in CD8+CD28 and CD8+CD28+ T cells from fresh peripheral blood 48. Our double-labeling studies have allowed us to immunophenotype individual FOXP3+ cells in situ and have clearly demonstrated the existence of a small naturally occurring population of CD8+ cells in the secondary lymphoid tissue that express the FOXP3 protein. However, FACS analysis did not reveal detectable CD8+FOXP3+ cells in PBL. The presence of CD8+FOXP3+ cells in secondary lymphoid tissue but not peripheral blood may reflect differences in the anatomical location of these cells or be a consequence of reduced sensitivity of FACS analysis compared with immunohistochemistry.

There are also reports of Foxp3 expression in murine B220+ B cells 3, 13, whereas CD19+ B cells were reported to be negative for Foxp3 mRNA expression 12. Increased levels of Foxp3 mRNA and protein expression have also been reported in B cells from the lymph nodes and spleen of the Foxp3-transgenic mouse 13, 49. However, these Foxp3+ B cells were not found to have a suppressor function in vitro13. In the current study there was no detectable expression of FOXP3 protein in CD20+ tonsillar B cells or in naive CD20+ cells or activated (by IgM or by rIL-2 plus SAC) CD20+ cells that had been isolated from peripheral blood (data not shown).

There is a reported difference in the human and mouse immune systems as to whether FOXP3 expression is induced by T cell activation. On activation, murine CD4+CD25 T cells acquire surface CD25 but do not express high levels of Foxp3 mRNA or protein 11, 13. Nevertheless, forced overexpression of Foxp3 converts naive murine CD4+CD25 T cells to TR cells 11, 12. In contrast, the activation of human CD4+CD25 T cells through the TCR leads to the increased expression of FOXP3 mRNA, the surface expression of CD25 and gain of regulatory function 47. Both resting and activated CD4+ and CD8+ T cell clones were examined for FOXP3 protein expression, which was found exclusively in the activated cell populations. These findings are consistent with those from a recent study that reported the induction of FOXP3 mRNA expression in activated T cell clones 50. These findings raise the possibility that FOXP3 plays a functional role not only in TR cells but also following activation of conventional T cells.

This panel of mAb provides an essential tool with which to further investigate the expression and function of the FOXP3 transcription factor and to label FOXP3+ TR cells in both frozen and routinely fixed human tissues. The application of these mAb for FACS analysis opens new avenues to investigate the correlation between the frequency and level of expression of FOXP3 in 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 containing the FOXP3 cDNA (IMAGE:5747723) was obtained from the MRC geneservice. A cDNA fragment encoding the full-length human FOXP3 protein was introduced to pDEST15 (Invitrogen) by means of Gateway® technology. The GST–FOXP3 fusion protein was then expressed in Esherichia coli, strain BL21(DE3), and purified by affinity chromatography.

Antibodies

Three BALB/c mice were injected intraperitoneally (three times at 14-day intervals) with 100 µg GST–FOXP3 fusion protein plus Freund's adjuvant. A 150-µg booster of the recombinant GST–FOXP3 protein was injected intraperitoneally, and a fusion was carried out 3 days later using conventional techniques 51. Hybridoma supernatants were screened by immunohistochemistry on frozen normal human tonsil sections as described below. The seven mouse mAb that were raised against FOXP3 (86D/D6, 150D/E4, 157B/F4, 206D/B1, 221D/D3, 236A/E7, 259D/C7) were cloned by the limiting dilution technique. Other Ab used include a goat FOXP3 polyclonal (Ab2481, Abcam) and mAb against FOXP1 (JC12, "in house"), CD25 (Novocastra, Newcastle upon Tyne, UK), anti-XpressTM (Invitrogen), anti-FLAG M2 (Sigma), and anti-CD4, -CD8, -CD3 or -CD20 (all from DakoCytomation, Glostrup, Denmark).

Tissue samples

Normal and neoplastic human tissues were obtained from the Department 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 in buffered formalin and embedded in paraffin according to routine procedures 52. Frozen sections of spleen and lymph node were also obtained from normal mice (Balb/c or B6).

Production of FOXP transfectants and their immunolabeling

Eukaryotic expression of the FOXP proteins in COS-1 cells

To confirm that the FOXP3 mAb were specifically reactive with the FOXP3 protein, their reactivity was tested on COS-1 cells expressing FOXP3 and on COS-1 cells expressing the closely related FOXP1, FOXP2 or FOXP4 proteins. FOXP1, FOXP2 and FOXP4 expression constructs have been previously described 53, 54. The Flag-tagged FOXP3 cDNA was kindly provided by Dr Mary Brunkow, Celltech.

Plasmid DNA was prepared for transfection using the Plasmid Midi Kit according to the manufacturer's instructions (Qiagen). COS-1 cells were transfected with pcDNA4/HisMax/FOXP2, FOXP3, pcDNA4/HisMax/FOXP4, pcDNA4/HisMax alone or pAB195 (FOXP1) using Fugene 6 transfection reagent, following the protocol described by the manufacturer (Roche Applied Science). Approximately 24 h post-transfection, the cells were washed with sterile PBS and harvested by trypsinisation. Cell pellets were snap-frozen and stored at –70oC whereas cytocentrifuge preparations were made for immunocytochemical staining and stored at –20oC 55. Paraffin-embedded cell pellets were prepared by fixing transfectants for 48 h in neutral buffered formalin (10% formalin in PBS), before centrifugation into 2% agar in neutral buffered formalin; the agar pellet was then embedded in paraffin and sectioned as for tissues. Transfected COS-1 cells were indirectly immunoenzymatically labeled using anti-XpressTM Ab at 5.5 µg ml–1, anti-FLAG Ab at 10 µg ml–1, JC12 hybridoma supernatant diluted 1/10 in PBS plus 10% human serum or in FOXP3 hybridoma supernatants. Ab binding was detected using the DAKO EnVision™ + System, HRP and diaminobenzidine (DAB) as directed by the manufacturer (DakoCytomation).

FACS staining of FOXP3-transfected COS cells

COS cells were permeabilised with FACS Perm 2 solution (BD Biosciences). Cells were stained with each FOXP3 mAb and a secondary goat-anti-mouse–PE (Southern Biotech). They were analyzed using FACS Calibur (BD Biosciences) and CELLQuest software.

Immunohistochemistry

Frozen tonsil tissue sections were incubated for 30 min with primary Ab, washed in PBS and incubated with either HRP-conjugated goat anti-mouse-Ig (diluted 1/50 in PBS) (DakoCytomation) or HRP-conjugated rabbit anti-goat-Ig (DakoCytomation). 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 and captured on electrically charged slides (Snowcoat X-traTM, Surgipath). Sections were dewaxed in citroclear (HD Supplies, Aylesbury, UK) and antigen retrieval was performed by microwave pressure cooking for 3 min at full pressure in 50 mM Tris and 2 mM EDTA, pH 9. Before staining the sections, endogenous peroxidase was blocked, the slides were incubated for 40 min with the primary Ab, washed with PBS and the immunodetection was performed with biotinylated anti-mouse secondary Ab (25 min), followed by peroxidase-labeled streptavidin (25 min) and DAB chromogen as substrate. Immunostaining performed in Oxford used the EnVisionTM system (DakoCytomation). All immunostaining in Madrid was performed using the Techmate 500 automatic immunostaining device and reagents from DakoCytomation. Incubations either omitting the specific Ab or containing unrelated Ab were used as a control of the technique. Sections were counterstained with hematoxylin Gill No. 3 (Sigma) and mounted in Aquamount (BDH, UK).

Double immunoenzymatic labeling

Double enzymatic immunostaining for FOXP3 and other markers including CD3, CD4, CD25, CD8 and CD20 was performed on tonsil. The details of the methodology used for the immunoenzymatic and immunofluorescent double immunostaining has been described previously 56.

Western blotting

Approximately 1×107 FOXP3-transfected COS-1 cells were lysed in 90 μl RIPA buffer, then 10 μl 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, lysates were mixed with 100 µl 2× SDS gel loading buffer, resolved on 12% SDSPAGE gels, then transferred to Immobilon-P membranes (Millipore) using semidry apparatus. The membranes were blocked in 5% marvel in PBS at 4°C overnight. FOXP3 and MR12 (mouse anti-rabbit-Ig, negative control) hybridoma supernatants were used neat and anti-FLAG M2 at 10 μg ml–1 and applied for 90 min. Following three 10-min washes in PBS containing 0.02% Tween, membranes were incubated with a 1/1000 dilution of secondary Ab, rabbit anti-mouse-Ig–HRP conjugate (DakoCytomation) for 90 min. Wash steps were repeated and Ab binding was detected using ECL reagent (Amersham Biosciences, Uppsala, Sweden). To confirm sample loading and transfer, membranes were incubated in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris HCl, pH 6.8) for 30 min at 50°C, reblocked for 1 h, then re-probed using anti-β-actin (AB6276, Abcam) diluted 1/25000 in blocking buffer following the same procedure.

T cell clones and activation

The CD8+ clones, 2D10 and 3F6, were generated by tetramer staining and FACS cloning and maintained as described previously 57. The CD4+ clone TB1 generated from a patient with a thymoma, as described previously 58, was kindly provided by Professor Nick Willcox (Oxford). The cells were maintained in culture in RPMI-1640 containing 10% fetal calf serum (Invitrogen, Paisley, Scotland) at 37oC in 5% CO2. Cells were activated, within 10–14 days, by stimulation with 20 µg/ml phytohemagglutinin (Murex) and irradiated allogeneic peripheral blood lymphocytes (50 Gy). Cells were considered to be resting after more than 21 days post-stimulation.

Cell isolation and immunofluorescent labeling of sorted cells

Human mononuclear cells were isolated from fresh blood by Lymphoprep (Nycomed, Oslo) gradient centrifugation. The CD4+ cells were obtained by negatively sorting the mononuclear cells with CD8 and CD14 magnetic beads (Miltenyi Biotech, GmbH, Germany). These were then positively sorted for CD4+CD25+ cells using CD25 magnetic beads (Miltenyi Biotec). In some instances, CD4+CD25+ cells were isolated using the CD4+CD25+ regulatory isolation kit according to the manufacturer's instructions (Miltenyi Biotech). MACS-sorted CD4+CD25+ and CD4+CD25 cells isolated from human peripheral blood were spotted onto slides (Henley-Essex) at 5×104/spot. Slides were air-dried and then frozen at –20°C. Cells were fixed in pre-chilled acetone (–20°C) for 5 min and blocked with 10% normal goat serum. Primary Ab (clone 221D/D3) at 10 µg ml1 was added overnight at 4°C, followed by goat anti-mouse–AlexaFluor-488 (1/500) (Molecular Probes, The Netherlands).

Cytocentrifuge preparations of T cells purified for the suppression assay were air-dried overnight, fixed in 50:50 acetone:methanol for 60 sec at room temperature, rinsed in PBS, then incubated with primary mAb (clone 236A/E7) for 50 min. Following a 5-min wash in PBS the secondary Ab (goat anti-mouse-IgG1–AlexaFluor-546, Molecular Probes) diluted 1/400, was applied for 30 min.

FACS methodology for detecting endogenous FOXP3

For FACS analysis, 5×105 cells were fixed in 1 ml of PBS with 1% paraformaldehyde and 0.05% Tween-20 overnight at 4°C. Cells were treated twice with 0.5 ml of DNAse at 100 Kunitz/ml according to the manufacturer's instructions (Sigma-Aldrich). Staining steps were performed at room temperature for one hour. Cells were incubated with mouse anti-human-FOXP3 (clone 150D/E4), washed with FACS buffer (PBS 1×, 3.00% fetal calf serum, 0.50% Tween-20, 0.05% azide). FOXP3 Ab binding was detected using Alexa Fluor-488® goat anti-mouse-IgG (Molecular Probes) and washed as above. Cell surface staining was then performed using the mAb Cy-Chrome–anti-human-CD4 (Pharmingen) and PE–anti-human-CD25 (Miltenyi Biotec) for 20 min at room temperature followed by washing in PBS/BSA. Cells were analyzed using a FACSCalibur™ with CELLQuest™ software (Becton Dickinson).

Cell proliferation assay

Cells were cultured in RPMI-1640 medium supplemented with 5% human AB serum, 2 mM L-glutamine (Gibco/Invitrogen), 100 U/µg/ml penicillin/streptomycin (Gibco/Invitrogen), 0.5 mM sodium pyruvate (Gibco/Invitrogen) and 0.05 mM nonessential amino acids (Gibco/Invitrogen) in 96-well plates (Nalge Nunc, Rochester, NY, USA). Plate-bound anti-CD3 (clone UCHT1, at 5 µg ml–1) and soluble anti-CD28 (clone 28.2, at 5 µg ml–1) were purchased from Pharmingen (BD Biosciences Pharmingen). The CD4+CD25 responder cells were used at 5×104/well and a variable number of CD4+CD25+ regulatory cells were added. [3H]thymidine at 0.5 µCi per well was added for the final 16 h of a 5-day assay.

Acknowledgements

This work was supported by funding from the Leukaemia Research Fund, the Association for International Cancer Research, the Wellcome Trust, Cancer Research UK (C399-A2291), the EU (LSHB-CT-2003-503410), the US Cancer Research Institute and the UK Medical Research Council.

Footnotes

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