Foxp3 Expressing CD4⁺CD25^high Regulatory T Cells Are Overrepresented in Human Metastatic Melanoma Lymph Nodes and Inhibit the Function of Infiltrating T Cells

Patients and samples

Twelve patients undergoing curative resections for LN metastatic melanoma before any immunotherapy or chemotherapy were included in this study after informed consent following human ethics committee procedures (Comité Consultatif pour la Protection des Personnes dans les Recherches Biomédicales; Hôpital Saint-Louis, Paris, France). Cancer stage was gathered according to the American Joint Committee on Cancer (41) (see Table I⇓). Peripheral blood and LNs were collected at the time of surgery. Diagnosis of metastatic LNs was obtained by histological examination and immunochemical staining with PS100 and HMB45 Abs. LNs with large tumor invasion as well as LNs with no or minimal tumor invasion (corresponding to micrometastasis or only sinusal invasion by tumor cells) could be obtained. Blood samples from healthy donors were obtained from the Etablissement Français du Sang (Hôpital Saint-Louis). LN cell suspensions, obtained after sterile mechanical dissociation, were filtered, washed, and cryopreserved for further analysis.

Abs and flow cytometric analysis

The following Abs were used: PE anti-CD25 (M-A251), allophycocyanin anti-CD28 (CD28.2), allophycocyanin anti-CD69 (L78), allophycocyanin anti-CTLA-4(BN13), allophycocyanin anti-CD45RO (UCHL1), biotinylated anti-cutaneous lymphocyte-associated Ag (HECA-452), biotinylated anti-CD45RA (HI100), allophycocyanin anti-HLA-DR (Tü36), and PerCP streptavidin (all from BD Pharmingen, San Diego, CA). We also used: FITC anti-CD4 (clone S3.5; Caltag Laboratories, Burlingame, CA) and allophycocyanin anti-CD27 Abs (O323; eBioscience, San Diego, CA). Anti-CCR7 and anti-CXCR5 were kindly provided by Dr. M. Lipp (Max-Delbrück-Centrum, Berlin, Germany). For CTLA-4 intracellular staining, the Cytofix/Cytoperm kit was used according to the manufacturer recommendations (BD Pharmingen). Lymphocytes were gated according to their forward and size scatter characteristics, and four-color FACSCalibur analysis was performed using the CellQuest software (BD Biosciences, San Jose, CA).

Purification of CD4⁺CD25^high, CD4⁺CD25⁻, and CD8⁺ tumor-infiltrating lymphocytes

LN cell suspensions were coated with CD4 and CD8 magnetic microbeads (Miltenyi Biotec, Auburn, CA), together with FITC anti-CD4 and allophycocyanin anti-CD8 Abs (BD Pharmingen), according to the manufacturer recommendations, and isolated using LS separation columns on VarioMACS (Miltenyi Biotec). The positive fraction was then labeled with PE anti-CD25 Ab and the T cell populations were purified using a MoFlo Cell Sorter (DakoCytomation, Fort Collins, CO). Purities of CD4⁺CD25^high, CD4⁺CD25⁻, and CD8⁺ T cell populations were 92.4 ± 8.4%, 99 ± 1.3%, and 92.6 ± 6.1% (mean ± SD), respectively.

Proliferation assays

A total of 2.5 × 10⁴ purified CD4⁺CD25^high, CD4⁺CD25⁻, or CD8⁺ T cells were cultured in triplicate with 5 × 10⁴ allogeneic irradiated PBMCs in 96-well round-bottom plates in RPMI 1640 medium supplemented with 1 mM l-glutamine, 1% penicillin/streptomycin, nonessentials amino acid, sodium pyruvate (all from Invitrogen Life Technologies, Carlsbad, CA), and 10% FCS (Dominique Dutscher, Issy-les-Moulineaux, France). In inhibition assays, infiltrating CD4⁺CD25⁻ or CD8⁺ T cells were cocultured with different numbers of infiltrating autologous CD4⁺CD25^high T cells. [³H]Thymidine (ICN Biomedicals, Irvine, CA) was added at day 5 (1 μCi/well) for 16 additional hours before cell harvesting and counting in a Betaplate scintillation counter (LKB Pharmacia, Uppsala, Sweden). For blocking experiments, CD4⁺CD25^high were preincubated 20 min at 4°C with Abs before being cocultured in a 1 μg/ml Ab final concentration. The following blocking Abs were used: anti-CTLA-4 (BNI3; BD Pharmingen), anti-IL-10 (JES3-19F1; BD Pharmingen), anti-TGF-β_1–3 (1D11; R&D Systems, Minneapolis, MN), and anti-glucocorticoid-induced TNFR (GITR; clone 110416; R&D Systems), and rat IgG2a,κ, mouse IgG2a,κ, and IgG1,κ immunoglobulins isotype controls (BD Pharmingen). For TCR stimulation, T cells were incubated with soluble anti-CD28 (CD28.2, 1 μg/ml; BD Pharmingen) in 96-well round-bottom plates previously coated overnight at 4°C with anti-CD3 (UCHT1, 0.5–1 μg/ml; Cymbus Biotechnology, Hants, U.K.). For transwell experiments, CD4⁺CD25⁻ were placed at the bottom of a 96-well round-bottom plate together with irradiated allogeneic PBMCs, and were separated by a 0.2-μm size anopore membrane (Nunc, Roskilde, Denmark) from CD4⁺CD25^high T cells placed together with irradiated allogeneic PBMCs in the top chamber of the transwell. At day 5, the transwell was removed and [³H]thymidine incorporation was measured as described above.

Cytokines production

ELISA was used according to manufacturer recommendations (OptEIA; BD Pharmingen) to detect IL-2, IL-4, IL-10, TGF-β₁, and IFN-γ in supernatants of T cell cultures after 5 days of allogeneic stimulation or 3 days of anti-CD3/anti-CD28 stimulation. The minimal levels of detection were 3.9 pg/ml for IL-2, IL-4, and IL-10, 2.3 pg/ml for IFN-γ, and 31.2 pg/ml for TGF-β in four-time diluted supernatants.

Quantitative real-time PCR

RNA were first purified using the RNeasy kit (Qiagen, Carlsbad, CA), and oligo(dT) or random hexanucleotide-primed first strand cDNAs were synthesized using SuperScript II reverse transcriptase (Invitrogen Life Technologies). They were then used as a template for real-time PCR, with the TaqMan Universal Master Mix (Applied Biosystems, Foster City, CA). Specific primers for Foxp3 and human hypoxanthine ribosyltransferase 1 (HPRT) were used with the following TaqMan MGB probe (Assays-on-demand gene expression assays; Applied Biosystems): 5′-6FAM-ATCCGCTGGGCCATCCTGGAGGCTC-3′ (FoxP3), and 5′-6FAM-GGTCAAGGTCGCAAGCTTGCTGGTG (HPRT). Because sequences for the specific unlabelled PCR primers were not provided by the manufacturer, the real-time PCR products for Foxp3 were controlled by sequencing analysis,using an ABI Prism 3700 detection system (Applied Biosystems). Real-time PCR was conducted in a GeneAmp 5700 detection system (Applied Biosystems), with the following cycling conditions: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, and 1 min at 60°C. The mean values from duplicates were used for calculations. Data are expressed as normalized expression obtained by dividing the relative cDNA level for each sample by the relative cDNA level of HPRT for the same sample, where HPRT = 1.

T cell repertoire diversity

TCR Vβ chain repertoire of sorted infiltrating CD4⁺CD25^high and CD4⁺CD25⁻ T cells was analyzed by quantitative Immunoscope (Applied Biosystems), as previously described (42, 43). cDNAs obtained after reverse transcription of total RNA with oligo(dT)₁₇ and SuperScript II reverse transcriptase (Invitrogen Life Technologies) were amplified with each of 24 TCR Vβ subfamily-specific primers and a nested fluorochrome-labeled TaqMan probe for TCR Cβ. Real-time PCR was conducted in a GeneAmp 5700 detection system (Applied Biosystems). PCR products were used as templates in run-off reactions using a nested TCR Cβ fluorescent primer producing labeled ssDNA fragments that were separated on a denaturing 6% acrylamide gel on a 373 DNA sequencer (Applied Biosystems) and analyzed with Immunoscope software (42).

Statistical analysis

Statistical analysis was performed using paired or unpaired Student’s t tests with the Kaleidagraph software (Synergy Software, Reading, PA).

Overrepresentation of CD4⁺CD25^high in metastatic melanoma LNs

Because regulatory CD4⁺ T cell activity has been identified in the CD4⁺CD25^high T cell subpopulation in human PBMCs and thymocytes (24, 25, 27, 28, 30, 31, 32, 33), we first estimated the proportion of such CD4⁺CD25^high T cells among lymphocytes infiltrating metastatic LNs in melanoma patients. Our study included 12 patients suffering from stage III melanoma before any treatment other than curative surgical resection (Table I⇑). Isolated lymphocytes were gated to estimate the proportion of CD4⁺ T cells that expressed high level of CD25 (IL-2R α-chain) and lower level of CD4 as previously described for CD4⁺CD25⁺ T_reg (Fig. 1⇓A).

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FIGURE 1.

CD4⁺CD25^high T cells are overrepresented in melanoma metastatic LNs and display a memory and activated phenotype. A, Infiltrating LN lymphocytes (TIL-LNs) and autologous PBMCs were stained with FITC anti-CD4 and PE anti-CD25 Abs, and CD4⁺CD25^high T cells were gated as indicated. A representative case is shown with increased percentage of CD4⁺CD25^high T cells among infiltrating CD4⁺ cells in LNs compared with PBMCs. B, FACS analysis of CD4⁺CD25^high (thick lines) and CD4⁺CD25⁻ T cells (thin lines) among TIL-LNs and autologous PBMCs showing that infiltrating CD4⁺CD25^high T cells display a memory and activated phenotype. C, Intracellular expression of CTLA-4 in CD4⁺CD25^high T cells (thick lines) and CD4⁺CD25⁻ T cells (thin lines) among TIL-LNs and autologous PBMCs. A representative case is shown revealing that infiltrating CD4⁺CD25^high T cells express higher levels of intracellular CTLA-4 compared with CD4⁺CD25^high T cells from PBMCs. Percentages of CTLA-4-positive T cells among CD4⁺CD25^high T cells and MFI are indicated.

The frequency of CD4⁺CD25^high T cells among CD4⁺ T cells was 11.06 ± 1.7% (mean ± SD) in metastatic LNs (range 8.3–14.05%; n = 13). We also studied tumor-free LNs and found that the frequency of CD4⁺CD25^high T cells was 6.2 ± 4.2% (n = 16). A global comparison between metastatic LNs and tumor-free LNs revealed almost a 2-fold increased frequency in metastatic LNs (p = 0.0006). The frequency of CD4⁺CD25^high T cells in metastatic LNs was also significantly increased when compared with the frequency observed in autologous PBMCs (4.9 ± 1.9%, range 2.6–8.7%, n = 8, p < 0.0001) or in healthy donor PBMCs (5.8 ± 1.7%, range 3–8.2%, n = 10, p < 0.0001). No statistical difference was found between frequencies of CD4⁺CD25^high T cells in PBMCs from metastatic melanoma patients and healthy donors (p = 0.37). When performing matched sample analysis (i.e., comparing metastatic LNs with either PBMCs or tumor-free LNs from the same patient), the difference remained highly significant (p = 0.014 and p = 0.016, respectively).

Altogether, these results revealed that the proportion of CD4⁺CD25^high T cells is clearly increased in metastatic LNs compared with tumor-free LNs and autologous PBMCs.

Infiltrating CD4⁺CD25^high T cells display a memory and activated phenotype

The majority of CD4⁺CD25^high T cells in 11 metastatic LNs from 10 patients expressed CD45RO (73.2 ± 11.8%) and few were CD45RA positive (18.9 ± 8.5%). They all expressed CD27 and CD28 molecules (Fig. 1⇑B) and were mostly CCR7 negative (87 ± 6.3%). Thus, most of these CD4⁺CD25^high T cells belong to the CD45RA⁻/CD27⁺ conventional memory CD4⁺ T cell population as defined by Campbell and colleagues (44). Few expressed HLA-DR (6.6 ± 2.5%) or the cutaneous homing molecule cutaneous lymphocyte-associated Ag (13.1 ± 11.2%), and the great majority were CXCR5 negative (95 ± 4.7%) (Fig. 1⇑B, and data not shown). As reported for human circulating T_reg, the majority of infiltrating CD4⁺CD25^high T cells intracellularly expressed the CTLA-4 molecule. However, more CD4⁺CD25^high T cells expressed CTLA-4 in metastasis (75.5 ± 15.8%) compared with autologous PBMCs (48.9 ± 15%) and their levels of expression were significantly higher (mean fluorescence intensity (MFI) = 44 ± 26 vs 15 ± 8, respectively, p = 0.01) (Fig. 1⇑C). Furthermore, surface expression of CTLA-4 was found on LN-infiltrating CD4⁺CD25^high T cells (4.5 ± 2.4%), but not on their circulating counterparts (0.7 ± 0.35%). Such an intracellular and surface up-regulation of CTLA-4 has been reported for human T_reg after activation in vitro (28), supporting the idea that infiltrating CD4⁺CD25^high T cells were activated at the tumor site.

Indeed, infiltrating CD4⁺CD25^high T cells expressed high levels of the activation molecule CD69 (43.8 ± 16%, range 19–71%) compared with only 1.2 ± 1.2% and 0.5 ± 0.4% in 8 autologous or 10 healthy donor PBMCs, respectively. Along the same lines, CD27, which is known to be up-regulated after TCR- or CD3-cross-linking (45), was expressed at a higher level on infiltrating CD4⁺CD25^high T cells compared with circulating CD4⁺CD25^high T cells (MFI = 754 ± 149 vs 489 ± 159, p = 0.002), also arguing for the activation of infiltrating CD4⁺CD25^high T cells. Moreover and as recently described for murine LNs (46), we also found activated CD4⁺CD25^high T cells expressing high levels of CTLA-4 (61.2 ± 18.9%, MFI = 32.7 ± 19) and CD69 (48.6 ± 16.9%) in 16 tumor-free LNs from seven different patients, as well as in two cutaneous draining LNs from healthy donors (data not shown).

Thus, CD4⁺CD25^high T cells present in metastatic as well as in tumor-free LNs display an activated phenotype.

Infiltrating CD4⁺CD25^high T cells express the Foxp3 transcription factor

Foxp3 was recently described as an important transcription factor involved in the development and function of murine CD4⁺CD25⁺ T_reg and appears to be the most specific molecular marker available to date (21). We first tested PBMCs from three healthy donors using real-time quantitative PCR. As expected, CD4⁺CD25^high T cells expressed high levels of Foxp3 while CD4⁺CD25⁻, CD4⁺CD25^int and CD8⁺ T cells expressed only very low levels (Fig. 2⇓, and data not shown). Similarly, when analyzing Foxp3 expression in nine melanoma metastatic LNs from seven different patients, we found that infiltrating CD4⁺CD25^high T cells highly expressed Foxp3 while CD4⁺CD25⁻ T cells expressed very low levels (Fig. 2⇓). The levels of Foxp3 expression in CD4⁺CD25^high T cells from metastatic LNs compared with autologous or healthy donor PBMCs were almost similar (Fig. 2⇓). In three tumor-free LNs from different patients, CD4⁺CD25^high T cells also expressed Foxp3 (data not shown). Altogether, these data revealed that CD4⁺CD25^high T cells infiltrating metastatic melanoma as well as tumor-free LNs express the Foxp3 transcription factor, strongly suggesting that this population includes T_reg.

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FIGURE 2.

Infiltrating CD4⁺CD25^high T cells constitutively express Foxp3. cDNAs obtained from sorted CD4⁺CD25^high and CD4⁺CD25⁻ T cells were submitted to quantitative real-time PCR with specific primers and probes for Foxp3 and HPRT. Relative Foxp3 expression for each sample is shown after normalization to HPRT expression, where HPRT = 1. Results obtained from representative healthy donor PBMCs, autologous PBMCs, and metastatic LNs are shown, enlightening that in all compartments CD4⁺CD25^high T cells, but not CD4⁺CD25⁻ T cells, express substantial level of Foxp3. The healthy donors and melanoma patients are identified by the abbreviation HD or SLM, respectively.

Infiltrating CD4⁺CD25^high T cells suppress in vitro the proliferation and cytokine production of infiltrating CD4⁺CD25⁻ as well as CD8⁺ T cells

We next evaluated the proliferative capacities and the regulatory function of CD4⁺CD25^high T cells from metastatic melanoma LNs. For this purpose, we sorted CD4⁺CD25^high as well as CD4⁺CD25⁻ and CD8⁺ T cells, and tested them in in vitro proliferation assays. In all the nine metastatic LNs tested from six different patients, the sorted CD4⁺CD25^high T cells proliferated poorly and did not produce IL-2 or IFN-γ in response to allogeneic stimulation (Figs. 3⇓, A, D, and E), thus exhibiting an anergic profile similar to what has been described for human T_reg (24, 25, 27, 28, 30, 31, 32, 33). In contrast, CD4⁺CD25⁻ T cells proliferated vigorously and produced both IL-2 and IFN-γ in response to allogeneic stimulation (Fig. 3⇓, A and D, and data not shown).

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FIGURE 3.

Infiltrating CD4⁺CD25^high T cells inhibit the proliferation and cytokine production of corresponding CD4⁺CD25⁻ and CD8⁺ T cells. A, Sorted infiltrating CD4⁺CD25^high and CD4⁺CD25⁻ T cells were cultured either alone or at different CD4⁺CD25^high:CD4⁺CD25⁻ ratios under allogeneic stimulation. [³H]Thymidine incorporations at day 5 are shown for one representative patient. B, Infiltrating CD4⁺CD25^high T cells inhibit the proliferation of autologous infiltrating CD4⁺CD25⁻ (filled symbols) or CD8⁺ T cells (open symbols) in a dose-dependent manner. Percentages of inhibition of the proliferation at different ratios are shown for five distinct metastatic LNs from different patients. C, Sorted CD4⁺CD25^high T cells infiltrating tumor-free LN also display some suppressive activity but at a lesser extent than in metastatic LN from the same patient (shown in A). IL-2 production of infiltrating CD4⁺CD25⁻ T cells (D) and IFN-γ production of infiltrating CD8⁺ T cells (E) are suppressed when cocultured with CD4⁺CD25^high T cells at different ratios. Data obtained by ELISA on culture supernatants for a representative metastatic LN are shown.

Importantly, the proliferation of CD4⁺CD25⁻ T cells was strongly inhibited in a dose-dependent manner when cocultured with CD4⁺CD25^high T cells (Fig. 3⇑, A and B). The median percentage of inhibition at a 1:1 CD4⁺CD25^high:CD4⁺CD25⁻ T cell ratio obtained in nine metastatic LNs was 77.5% (range 51–98%) and remained detectable even at a 1:8 or 1:10 ratio in most of the five cases where all ratios were tested (Fig. 3⇑, A and B). In addition, CD4⁺CD25^high T cells strongly inhibited IL-2 and IFN-γ production of CD4⁺CD25⁻ T cells (Fig. 3⇑D, and data not shown) corresponding to a 95.5 ± 6.6% of inhibition for IL-2 and 98.6 ± 3.8% for IFN-γ at a 1:1 ratio. It is noteworthy that at 1:8 or 1:10 ratios, which almost correspond to the ratios observed in metastatic LNs, the inhibition of cytokine production was still major (∼90% inhibition; Fig. 3⇑C, and data not shown), while the inhibition of proliferation was weaker (∼30% inhibition; Fig. 3⇑A). This suggests that infiltrating CD4⁺CD25^high T cells may be more suppressive on T cell functions than on T cell proliferation. In four metastatic LNs, the suppressive activity of CD4⁺CD25^high T cells was also tested at a 1:1 ratio against sorted infiltrating CD8⁺ T cells. Although these cells proliferated to a lower extent than CD4⁺CD25⁻ T cells in response to allogeneic stimulation, their proliferation was also strongly inhibited by CD4⁺CD25^high T cells in a dose-dependent manner with a median percentage of inhibition of 62% at a 1:1 ratio (Fig. 3⇑B, and data not shown). Similarly, the IFN-γ production of CD8⁺ T cells was also strongly inhibited by CD4⁺CD25^high T cells (Fig. 3⇑E), with a median percentage of inhibition of 85% at a 1:1 ratio. In two LNs with minimal tumor invasion, the inhibition of proliferation of both CD4⁺CD25⁻ and CD8⁺ T cells was weak, around 50% at 1:1 ratio (data not shown). In addition, as expected from recent studies on murine LNs (46), CD4⁺CD25^high T cells isolated from four tumor-free LNs from three different patients also displayed some suppressive activity, albeit not as potent as in metastatic LNs, because suppression was not detected at a lower ratio than 1:2 or 1:4 (Fig. 3⇑C).

Taken together, CD4⁺CD25^high T cells infiltrating melanoma metastatic LNs, and to a lesser extent tumor-free LNs, suppressed in vitro the proliferation and cytokine production of both infiltrating CD4⁺CD25⁻ as well as CD8⁺ T cells.

Suppression by tumor-infiltrating CD4⁺CD25^high T cells is mediated through a cell contact mechanism

Although controversial, the mechanism of action of human CD4⁺CD25^high T_reg appears to be largely cell contact dependent (24, 25, 27, 28, 30, 31, 32, 33). The role of cytokines, like IL-10 or TGF-β, as well as cell surface molecules, like CTLA-4 or GITR, is still a matter of debate (14). Therefore, we investigated whether infiltrating CD4⁺CD25^high T cells inhibited cell proliferation in vitro through a cell-contact- and/or cytokine-dependent mechanism. Transwell experiments performed in four metastatic LNs revealed that CD4⁺CD25^high T cells required cell contact to suppress proliferation of CD4⁺CD25⁻ T cells, because in all cases the inhibition of proliferation was either completely or partially abolished by the transwell (Fig. 4⇓A, and data not shown). Preincubation of CD4⁺CD25^high T cells with blocking anti-IL-10 or anti-TGF-β_1–3 Abs significantly reverted the suppression in experiments performed in three cases, while isotype control blocking mAbs did not (Fig. 4⇓A, and data not shown). As control, the preincubation of CD4⁺CD25⁻ T cells alone with these blocking Abs did not affect their proliferation (data not shown). Thus, the suppressive activity of LN-infiltrating CD4⁺CD25^high T cells was found to be both contact and cytokine dependent. Interestingly, we found that preincubation of infiltrating CD4⁺CD25^high T cells with blocking anti-CTLA-4 Ab also significantly reverted the suppression (Fig. 4⇓A). In all the cases tested, the anti-GITR Ab did not significantly alter the suppressive activity of the CD4⁺CD25^high T cells (Fig. 4⇓A). Finally and as previously described (27, 30, 31, 32), addition of IL-2 (50 or 100 IU/ml) allowed CD4⁺CD25^high T cells to proliferate and abolished their suppressive activity (data not shown).

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FIGURE 4.

Cell-contact- and cytokine-dependent suppression activity of infiltrating CD4⁺CD25^high T cells could be identified. A, [³H]Thymidine incorporation of infiltrating CD4⁺CD25^high and CD4⁺CD25⁻ T cells is shown for a representative metastatic LN under allogeneic stimulation at a 1:1 ratio separated or not by a transwell (TW) or in the presence of blocking mAbs: anti-TGF-β_1–3 (aTGFβ_1–3), anti-IL-10 (aIL-10), anti-CTLA-4 (aCTLA-4), or anti-GITR (aGITR). Suppressive activity of infiltrating CD4⁺CD25^high T cells was found to be inhibited by both TW and blocking Abs. Statistical significative p values when compared with the 1:1 ratio are indicated. IL-10 (B) and TGF-β₁ (C) production of infiltrating CD4⁺CD25^high and CD4⁺CD25⁻ T cells were assessed by ELISA on supernatants after allogeneic stimulation. Infiltrating CD4⁺CD25^high but also CD4⁺CD25⁻ T cells from LNs with advanced tumor involvement produced IL-10 and occasionally TGF-β₁, while cells from less-invaded LNs did not. The percentage of inhibition of the proliferation of CD4⁺CD25⁻ T cells by infiltrating CD4⁺CD25^high T cells obtained at a 1:1 ratio as shown in Fig. 3⇑B is indicated above each tumor name in B.

Therefore, CD4⁺CD25^high T cells infiltrating melanoma metastatic LN suppress CD4⁺CD25⁻ T cells in a cell-contact- and CTLA-4-dependent mechanism. In addition, a cytokine-dependent mechanism could also be involved.

Tumor-infiltrating CD4⁺CD25^high but also CD4⁺CD25⁻ T cells secrete IL-10 and/or TGF-β

Because the suppressive activity of CD4⁺CD25^high T cells was abolished by anti-IL-10 or anti-TGF-β_1–3 blocking Abs, we investigated whether infiltrating CD4⁺CD25^high and CD4⁺CD25⁻ T cells indeed produced these cytokines upon in vitro stimulation. We found that CD4⁺CD25^high T cells from metastatic LNs with large tumor invasion produced IL-10 upon allogeneic stimulation (36.6 up to 279.2 pg/ml; mean, 110.8 pg/ml), while in cases of minimal tumor invasion such a production was not detected (Fig. 4⇑B). Because in these latter cases a lower level of inhibition of CD4⁺CD25⁻ T cell proliferation was also observed compared with more advanced disease, this suggests that IL-10 production may be correlated to the strength of suppression detected in vitro (Figs. 3⇑B and 4⇑B). However, stronger TCR-mediated stimulation using coated anti-CD3 and soluble anti-CD28 Abs revealed that these cells also displayed the capacity to secrete IL-10 (data not shown). In contrast, infiltrating CD4⁺CD25⁻ T cells also secrete IL-10 (27.9 up to 265.9 pg/ml; mean, 130.9 pg/ml) upon allogeneic stimulation, mostly in case of large tumor invasion (Fig. 4⇑B). In tumor-free LNs, we similarly observed that both CD4⁺CD25^high and CD4⁺CD25⁻ T cells could produce IL-10, albeit at slightly lower levels (mean, 84 and 69 pg/ml, respectively).

In few cases, infiltrating CD4⁺CD25^high T cells and CD4⁺CD25⁻ T cells were found to produce TGF-β₁ at a substantial level (>2000 and up to 8500 pg/ml) (Fig. 4⇑C). In the case of minimal tumor invasion, such a TGF-β₁ secretion was not observed and could not be induced by stimulation with anti-CD3 and anti-CD28 Abs (data not shown). Finally, TGF-β₁ production was not detected when analyzing T cells from tumor-free LNs (data not shown).

The fact that infiltrating CD4⁺CD25⁻ T cells secreted IL-10 and/or TGF-β₁ suggested that they may also display some suppressive activity. Indeed, when tested functionally in coculture experiments with infiltrating CD8⁺ T cells, irradiated CD4⁺CD25⁻ T cells suppressed the proliferation and IFN-γ production of CD8⁺ T cells (data not shown).

Altogether, these data indicate that both infiltrating CD4⁺CD25^high and CD4⁺CD25⁻ T cells can produce the immunosuppressive cytokines IL-10 and/or TGF-β₁, and that IL-10 may play a role in the suppressive activity of CD4⁺CD25^high T cells. They also provide evidence that cells with a Tr1/Th3-like phenotype (22, 23) are also present in melanoma metastatic LNs, mostly in cases of large tumor invasion. Finally, because both infiltrating CD4⁺CD25^high and CD4⁺CD25⁻ T cells did not produce IL-4 upon stimulation (data not shown), the presence of Th2 cells is unlikely.

Tumor-infiltrating CD4⁺CD25^high T cells display a largely polyclonal TCR Vβ (TCRBV) repertoire

It has been largely described in mice and humans that oligoclonality of TCR usage and characteristic features of complementarity-determining regions 3 (CDR3) regions may reveal Ag-expanded T cells in a given T lymphocyte population even if the Ag is not known (47). Thus, to obtain insight into the potential Ag-driven expansion of CD4⁺CD25^high T cells infiltrating melanoma metastatic LNs, we performed a detailed Immunoscope analysis of their TCRBV repertoire by, first, quantifying the BV family usage through real-time PCR, and, second, by determining their CDR3β chain length distributions through Immunoscope analysis (42, 43). We found that both CD4⁺CD25^high and CD4⁺CD25⁻ T cell populations from three metastatic LNs from different patients and from one tumor-free LN expressed an highly diverse set of BV families and that these populations differed quantitatively only on a limited number of BV families for a given patient. Similar results were obtained for the four LNs studied and a representative case is shown in Fig. 5⇓A. In each case, including the tumor-free LN, similar BV families (as BV15, 20, or 23, for example) were poorly or not detected in both CD4⁺CD25^high and CD4⁺CD25⁻ T cell populations. They correspond to infrequently used BV families in the normal human TCR repertoire (42). When looking at the CDR3β chain length profiles (Fig. 5⇓B), both populations showed an almost Gaussian-like curve distribution for each BV family studied with no evidence for oligoclonal expansion even in case of differential usage of a given BV (see for example the BV2 profile among the representative BV profiles shown in Fig. 5⇓B).

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FIGURE 5.

Infiltrating CD4⁺CD25^high T cells display a polyclonal TCRBV repertoire. A, Real-time PCR amplifications analyzing the BV usage of TCR β-chains expressed by sorted infiltrating CD4⁺CD25^high and CD4⁺CD25⁻ T cells. The percentages obtained for BV1 to BV24 subfamily usage show that both populations express a highly diverse set of BV subfamilies with few differences from each other. B, CDR3 length distribution of TCRBV chains expressed by infiltrating CD4⁺CD25^high (upper line) and CD4⁺CD25⁻ T cells (lower line) for selected representative BV subfamilies. For each Immunoscope profile, fluorescent intensity in arbitrary units, represented as a function of the CDR3 length measured in codons, reveals an almost Gaussian distribution with no evidence for oligoclonal expansion. The peak of the 10th codon is marked on the abscissa axis. Percentages indicate the usage of given BV, as derived from quantitative amplifications. Analysis of a representative metastatic LN is shown in A and B.

Overall, these data do not argue for a restricted Ag-driven expansion of CD4⁺CD25^high T cells infiltrating melanoma metastatic LNs.