Elevated frequencies of intratumoral CD4 + regulatory T (T reg ) cells have typically been associated with more rapid tumor progression, but the antigen specificity of these T reg cells within tumors is not well understood. Here, Ahmadzadeh et al. characterized the TCR repertoire of intratumoral T reg cells from patients with metastatic melanoma, gastrointestinal, and ovarian cancers. These T reg cells had a unique TCR repertoire different from other intratumoral CD4 + T cells, and dominant TCRs were specific to tumor/neoantigens. These tumor/neoantigen-specific T reg cells were also found in the periphery, indicating that these T reg cells may be expanding from both compartments. Together, these data provide insight into the tumor specificity of T reg cells and how they have the potential to be activated and expand within tumors.

CD4 + regulatory T (T reg ) cells have an essential function in maintaining self-tolerance; however, they may also play a detrimental role in antitumor immune responses. The presence of elevated frequencies of T reg cells in tumors correlates with disease progression and poor survival in patients with cancer. The antigen specificity of T reg cells that have expanded in the tumor microenvironment is poorly understood; answering this question may provide important insights for immunotherapeutic approaches. To address this, we used a novel combinatorial approach to characterizing the T cell receptor (TCR) profiles of intratumoral T reg cells from patients with metastatic melanoma, gastrointestinal, and ovarian cancers and elucidated their antigen specificities. The TCR repertoires of tumor-resident T reg cells were diverse yet displayed significant overlap with circulating T reg cells but not with conventional T cells in tumor or blood. TCRs isolated from T reg cells displayed specific reactivity against autologous tumors and mutated neoantigens, suggesting that intratumoral T reg cells act in a tumor antigen–selective manner leading to their activation and clonal expansion in the tumor microenvironment. Tumor antigen–specific T reg -derived TCRs resided in the tumor and in the circulation, suggesting that both T reg cell compartments may serve as a source for tumor-specific TCRs. These findings provide insights into the TCR specificity of tumor-infiltrating human T reg cells that may have potential implications for cancer immunotherapy.

We hypothesized that the elevated frequency of intratumoral T reg cells in human cancers may be due to oligoclonal expansion upon tumor antigen encounter. To explore this hypothesis, we studied the TCR repertoire of tumor-resident T reg cells in human metastatic melanoma, gastrointestinal, and ovarian cancers and elucidated their antigen specificity. We found that the TCR repertoire of intratumoral T reg cells was distinct from T conv cells in the tumor and PBL of patients; however, it overlapped significantly with circulating T reg cells. Furthermore, the most dominant TCRs derived from intratumoral T reg cells were shown to be tumor reactive and recognized mutated cancer neoantigens. The identified tumor antigen–specific T reg cells were also found in the circulation, suggesting that PBL may be used as an additional source of tumor-specific TCRs. These findings provide insights into the TCR specificity of tumor-infiltrating human T reg cells.

The antigen specificity of tumor-infiltrating T reg cells has thus far remained largely unexplored. Lack of an exclusive cell surface marker to unequivocally distinguish activated T reg cells from conventional T (T conv ) cells in tumors forms a major obstacle to isolate viable T reg cells, as staining for intracellular FOXP3 renders the cells nonviable. Using an antigen-specific tetramer against the cancer germline antigen MAGE-A3, Francois et al. ( 13 ) isolated and clonally expanded circulating human T cells with phenotypic and functional attributes of T reg cells. Additional studies also identified suppressive CD4 T cells from the peripheral blood (PBL) of patients with cancer with reactivity against nonmutated tumor antigens after stimulation with overlapping peptide libraries ( 14 , 15 ). Cloning of tumor-infiltrating lymphocytes from melanoma tumors identified CD4 + T cell clones specific for the cancer germline antigen LAGE1 protein ( 16 ) that were attributed to be T reg based on their phenotypic and functional characteristics of the clones. All these studies used T cell cloning and expansion techniques that could potentially alter the initial phenotypic and functional status of T cells. Furthermore, the frequency and dominance of these T reg -attributed clones in the tumor and circulation were not reported. Comparison of TCR repertoire of T reg and T conv cells in humans has been limited to PBL ( 17 , 18 ), and little is known about the TCR repertoire of intratumoral T reg cells in patients with cancer.

Human CD4 + regulatory T (T reg ) cells comprise a small subset of circulating CD4 + T cells with potent suppressive function in vitro and in vivo ( 1 ). They play a vital role in regulating immune responses and maintaining self-tolerance; however, they also impede antitumor immunity [reviewed in ( 2 , 3 )]. Human T reg cells express high levels of the interleukin-2 receptor α chain (CD25) and the forkhead winged-helix transcription factor (FOXP3), which is pivotal for their development and function [reviewed in ( 4 )]. Elevated frequencies of T reg cells have been reported in many types of tumors, including melanoma ( 5 ), breast ( 6 ), lung ( 7 ), and ovarian carcinoma ( 8 ), and their high frequencies correlate with poor prognosis [reviewed in ( 9 )]. In contrast to circulating T reg cells, tumor-resident T reg cells display an activated profile ( 5 - 7 ). Given that T cell receptor (TCR) stimulation is required for the activation and acquisition of suppressive function in T reg cells ( 10 - 12 ), the activated profile of intratumoral T reg cells suggests that antigen stimulation may play an important role in the activation and accumulation of T reg cells in the tumor microenvironment.

RESULTS

The TCRB repertoires of FOXP3+T reg cells were distinct from FOXP3−T conv cells in tumors To study the TCR clonotypic repertoire of intratumoral T reg cells of patients with cancer, we performed TCR β (TCRB) chain deep sequencing of T reg cells isolated by flow cytometric sorting based on the expression of FOXP3. Because the enzymatic digestion of tumor samples diminished direct staining for CD4 coreceptor, samples were stained for CD8 and CD3 cell surface markers, followed by intracellular staining for FOXP3 as previously performed (5), and FOXP3+ and FOXP3− CD4 T cell subsets were sorted from CD8−CD3+ T cell populations (Fig. 1A). Previous studies have shown that FOXP3 expression was confined to CD4 T reg cells in vivo, and in vitro activation of T conv cells can lead to up-regulation of FOXP3 in non-T reg cells in both CD4 and CD8 T cells (19-21); thus, our strategy for the isolation of bona fide intratumoral T reg cells included staining and sorting cells immediately after thawing to not alter the expression of FOXP3. Lack of FOXP3 expression in the intratumoral CD3+CD8+ T cells ex vivo served as a negative control and assurance that the expression of FOXP3 by a subset of intratumoral CD4 T cells was likely confined to CD4 T reg cells as previously reported (5). Functional and epigenetic analyses could not be performed on the sorted intratumoral FOXP3+ cells due to lack of cell viability upon intracellular staining for FOXP3. Moreover, these analyses would be limited on a bulk population and would not be informative for individual cells expressing TCRs of interest. Fig. 1 TCRB repertoire of intratumoral FOXP 3+ T reg cells was primarily distinct from FOXP3− T conv cells. (A) Representation of sorting strategy to isolate FOXP3+ T reg cells and FOXP3− T conv cells from freshly thawed single-cell suspension of a patient’s (3107) tumor digest. The dot plot was gated on CD3+ lymphocytes. The TCRB immunosequencing was performed on each sorted population to determine the rank and frequency of TCRB clonotypes. (B) The frequency of all productive TCRB sequences in FOXP3+ and FOXP3− subsets for each patient (pt.) was plotted along the x axis and y axis, respectively. Each dot represents a unique TCRB clonotype. The number of overlapping clonotypes and the percentage per total FOXP3+ clonotypes are indicated in red. The number of unique TCRB clonotypes for FOXP3+ (x axis) and FOXP3− (y axis) is indicated next to each axis, respectively. N.D., not detected. A summary of cell numbers, total productive reads, unique TCRB sequences, and TCRB clonality for each sorted FOXP3 subsets from tumor and PBL is listed in table S2. The median values for the number of sorted cells for FOXP3+ TUM, FOXP3− TUM, FOXP3+ PBL, and FOXP3− PBL were 10,500 cells (range, 3000 to 22,000), 30,000 cells (range, 5000 to 50,000), 55,000 cells (range, 10,000 to 100,000), and 500,000 cells (range, 300,000 to 1,500,000), respectively. We collected all the possible events for the FOXP3+ subsets for each sample because this population was limiting both in the tumor and PBL. The TCR repertoire of each sorted population was analyzed using productive reads (in-frame and no stop codons). The total number of productive reads was not statistically different among the sorted populations (fig. S1), verifying that each population received a comparable sequencing coverage. Although both FOXP3 subsets in the tumor had lower numbers of unique TCRB sequences than their counterparts in PBL, their differences were not statistically significant (fig. S2). This difference is likely associated with the lower number of cells sorted from the tumor as compared with PBL. The TCRB repertoire of intratumoral T reg cells (FOXP3+ TUM) appeared diverse and exhibited a distinct and unique TCRB clonotypic repertoire compared with T conv cells (FOXP3− TUM) for all the six patients studied (Fig. 1B). Only a small fraction of the TCRB clonotypes was shared between the FOXP3+ and FOXP3− subsets, accounting for 0.5 to 13.2% (mean of 7.9%, n = 6 patients) of the FOXP3+ population, consistent with a previous report on the comparison of circulating T reg and T conv subsets in the PBL of healthy adults (18). These findings reveal that the TCRB repertoires of intratumoral FOXP3+ T reg cells were primarily distinct from intratumoral T conv cells with low clonal overlaps, consistent with the findings in the PBL (17, 18), suggesting that the accumulation of intratumoral T reg cells might be mediated by antigen-specific clonal expansion in the tumor microenvironment.

The TCRB clonotypes of intratumoral FOXP3+T reg cells overlapped with circulating FOXP3+ T reg cells Because the TCRB repertoire analyses of intratumoral FOXP3+ T reg cells revealed that they were principally distinct compared with intratumoral T conv cells, we subsequently compared the intratumoral FOXP3+ T reg cells repertoire with circulating T reg cells. The most dominant TCRB clonotypes of intratumoral T reg cells (FOXP3+ TUM) overlapped significantly (P < 0.05) with circulating T reg cells (FOXP3+ PBL) but not with FOXP3− T conv subsets in the tumor (FOXP3− TUM) or in the circulation (FOXP3− PBL; Fig. 2A). There were no overlapping clonotypes detected between FOXP3+ TUM and FOXP3− PBL in two patients (4067 and 4060; Fig. 2A). In contrast, FOXP3− TUM displayed a significant (P < 0.05) overlap with FOXP3− PBL and a minimal overlap with FOXP3+ subsets in the tumor and PBL (Fig 2B). These findings indicate that the most dominant TCRB repertoire of tumor-resident FOXP3+ T reg cells resembles the circulating T reg cells rather than the T conv cells. Fig. 2 The dominant TCRB clonotypes in intratumoral FOXP 3+ T reg cells significantly overlapped with circulating FOXP3+ T reg cells. Each symbol represents a patient’s total number of overlapping TCRB clonotypes among the top 100 TCRB sequences from intratumoral FOXP3+ (A) or FOXP3− (B) subset compared with the other T cell compartments. The total samples were n = 6 and n = 5 for tumor and for PBL, respectively. The dominant TCRB clonotypes of FOXP3+ TUM did not share any clonotypes with FOXP3− PBL for patients 4060 and 4067 (A). *P < 0.05 using Wilcoxon signed-rank test; n.s., nonsignificant values.

Clonal expansion of tumor-infiltrating FOXP3+T reg cells In contrast to circulating T reg cells, intratumoral T reg cells were previously shown to exhibit phenotypic and functional characteristics of activated T reg cells particularly with higher expression level of CTLA-4, OX40, TIGIT, 4-1BB, and CD45RO (5-7, 22). Consistent with these previous studies, the frequency of FOXP3+ T reg was higher in tumors (TUM) than in circulation (PBL) by several folds (Fig. 3A). Directly ex vivo, a fraction (19.4%) of intratumoral FOXP3+ T reg cells expressed Ki67, a marker for recently dividing cells, indicating that intratumoral T reg such as T conv (FOXP3−, lower quadrants) cells were actively dividing within the tumor microenvironment (Fig. 3A, top panel). Intratumoral T reg cells were reported to be more proliferative than T conv cells in breast tumors (6). We also compared the clonality for each sorted population in the tumor and blood (fig. S3 and table S2) as previously reported (23). The median values of clonality for FOXP3+ TUM, FOXP3− TUM, FOXP3+ PBL, and FOXP3− PBL were 0.088 (range, 0.049 to 0.161), 0.080 (range, 0.063 to 0.104), 0.059 (range, 0.054 to 0.092), and 0.068 (range, 0.035 to 0.184), respectively. No significant difference was found in the clonality of the entire sorted population among FOXP3 subsets isolated from tumor or PBL. In contrast, the top 10 TCRB clonotypes in the intratumoral T reg (FOXP3+ TUM) subset constituted a significantly (P < 0.005) higher fraction than the circulating T reg (FOXP3+ PBL) subset in all studied patients, regardless of their tumor histology (Fig. 3B). In contrast to FOXP3+ TUM, no significant difference was detected in the frequency of the top 10 TCRB clonotypes between FOXP3− subsets in the tumor and PBL (Fig. 3B). Overall, these findings suggest that the oligoclonality observed within the intratumoral FOXP3+ population may be the result of clonal expansion in response to tumor antigen stimulation. Fig. 3 Clonal expansion of tumor-infiltrating FOXP 3+ T reg cells. (A) A single-cell suspension of tumor digest and PBL from the same patient (3107) was stained for CD3, CD8, FOXP3, and Ki67; the dot plots were gated on CD8−CD3+ T cells. The values within each quadrant represent the percentage of cells in that quadrant. The fraction of dividing cells within the T reg (Ki67+FOXP3+/total FOXP3+) and T conv (Ki67+FOXP3−/total FOXP3−) is depicted as percentage values in the upper corner and lower corner outside the dot plots, respectively. The quadrants were set on the basis of negative control. (B) The fraction of the top 10 TCRB clonotypes was calculated by taking the sum of their TCRB frequencies divided by the total TCRB frequencies per each FOXP3+ or FOXP3− subset in tumor or PBL for each patient. Each symbol represents one patient. The total samples were n = 6 and n = 5 for tumor and for PBL, respectively. ***P < 0.0005 using Wilcoxon signed-rank test.

Tumor reactivity of the most dominant FOXP3+T reg -derived TCRs in the tumor To determine the antigen specificity of the most frequent FOXP3+ T reg cells in tumors of patients, we identified the paired TCRB and TCR α (TCRA) chain sequences from the top-ranking TCRB clonotypes in tumors using pairSEQ, a statistical model for pairing TCRA and TCRB sequences (24). These paired TCRA and TCRB sequences were used to reconstruct TCRs, which were subsequently cloned into retroviral vectors (23, 25). Next, retroviral supernatants generated from T reg -derived TCRs were used to transduce autologous PBL (patients 3107 and 4066) or human leukocyte antigen (HLA) class II–matched donor PBL (patient 3919) when PBLs were not available. Subsequently, T reg -derived TCR-transduced T cells were examined for T cell reactivity against autologous and allogeneic tumor cell (TC) lines using interferon-γ (IFN-γ) production assays and up-regulation of the T cell activation marker CD137 (4-1BB) by flow cytometry. Eleven TCRs were constructed from the most dominant intratumoral T reg cells from a metastatic melanoma tumor (patient 3107), ranging from ranks 1 to 39. Six of these TCRs (TCRs 1, 9, 10, 13, 23, and 34) exhibited specific tumor recognition of the autologous TC as measured by IFN-γ production (Fig. 4A) and up-regulation of 4-1BB on TCR-transduced cells (Fig. 4B). The production of IFN-γ was more profound against TC transduced with class II major histocompatibility complex transactivator (CIITA), presumably due to the higher expression levels of HLA class II molecules on TC (fig. S4). No or minimal recognition was detected of CIITA-transduced allogeneic melanoma (MEL) and renal cell carcinoma (RCC) TC (Fig. 4, A and B). Overall, 6 of 11 intratumoral T reg -derived TCRs from patient 3107 exhibited specific tumor recognition, and 3 of these TCRs (TCRs 1, 9, and 10) ranked among the top 10 clonotypes in the intratumoral T reg cell population. Fig. 4 FOXP 3+ T reg -derived TCRs exhibited tumor reactivity. (A) The most dominant TCR clonotypes derived from the intratumoral FOXP3+ T reg cells (patient 3107) were transduced into autologous PBL and subsequently cocultured overnight with autologous (Auto MEL) or allogenic (Allo MEL or Allo RCC) TC lines, and the IFN-γ in the supernatant was quantified by ELISA. (B) The cocultured cells were also stained with anti-CD3, anti-CD4, anti-mTCRB, and anti−4-1BB antibodies to quantify the percentage of 4-1BB up-regulation on mTCRB+ T cells by FACS. The dot plots were gated on CD4+CD3+ propidium iodine (PI)− T cells. (C) The most dominant TCR clonotypes derived from the intratumoral FOXP3+ T reg cells (patient 3919) were transduced into HLA class II–matched donor PBL and cocultured overnight with autologous or allogenic TC, and the IFN-γ in the supernatant was quantified by ELISA. Data are representative of at least two independent experiments. We also constructed nine TCRs from FOXP3− T conv cells isolated from the same metastatic melanoma tumor (patient 3107), ranging from ranks 1 to 20. Seven of these TCRs exhibited specific tumor recognition against the autologous TC, as demonstrated by the production of IFN-γ (fig. S5A) and up-regulation of 4-1BB (fig. S5B). The recognition of autologous TC was enhanced by CIITA transduction. In addition to the reactivity against autologous MEL-3107, FOXP3− TCR 20 recognized allogeneic RCC-1764 and not MEL-2630. Because the HLA class II expression is partially matched among these TCs (table S3), it is not clear whether this recognition pattern by TCR 20 reflects either on the reactivity against a shared antigen expressed by RCC-1764 and MEL-3107 and not MEL-2630 or on the lack of expression of the HLA restriction element of TCR 20 by MEL-2630. Overall, seven of nine intratumoral T conv -derived TCRs exhibited tumor reactivity, and three of seven ranked among the top 10 clonotypes in the intratumoral T conv cell population. TCRs from intratumoral FOXP3+ T reg cells from two additional patients with metastatic melanoma (patients 4066 and 3919) were also evaluated using a similar approach. For patient 4066, all the top 10 FOXP3+ TCRs were constructed and screened; however, TCR-transduced T cells showed no discernible specific reactivity against the autologous TC or the mutated neoantigens tested. For patient 3919, six FOXP3+ TCRs were constructed, ranging from ranks 3 to 34 (TCRs 3, 7, 14, 23, 25, and 34), and screened against autologous TC. One of these six FOXP3+ TCRs (TCR 14) exhibited tumor reactivity specific to autologous TC (Fig. 4C). Three FOXP3− TCRs (TCRs 1, 17, and 25) were also reconstructed, cloned, and screened. None of these three TCRs recognized autologous TC (fig. S5C). In summary, TCRs from intratumoral T reg cells in the two evaluable patients displayed specific tumor reactivity.

Neoantigen reactivity of the most dominant FOXP3+TCRs in the tumor We also asked whether FOXP3+ T reg TCRs were specific for patient-specific mutated neoantigens. For patient 3107, tumor-reactive TCRs were screened for reactivity against 163 identified somatic mutations identified from the patient’s tumor using whole-exome and RNA sequencing (RNA-seq). Autologous dendritic cells (DCs) were pulsed with pools of mutant peptides, with each peptide representing one mutated neoantigen. One of 11 FOXP3+ TCRs screened exhibited reactivity to peptide pool (pp) 9 (pp9) and not to any of the other 13 peptide pools, as assessed by the up-regulation of OX40 and 4-1BB (fig. S6). Subsequent screening of individual mutant peptides within pp9 revealed reactivity to mutated annexin A1 (ANXA1; Fig. 5A). For patient 3919, FOXP3+ TCR 34 exhibited recognition of DC pulsed with pp13 (Fig. 5B). The sequences of mutant and wild-type peptides are shown in table S4. Subsequent screening of individual mutant peptides within pp13 identified reactivity to mutated CCL-5 (CC chemokine ligand 5, also known as RANTES; Fig. 5B). The other remaining five FOXP3+ TCRs and three FOXP3− TCRs did not display reactivity against any of the screened peptide pools. The reactivity of FOXP3+ TCR 34 was specific to mutated CCL5, as assessed by IFN-γ production (Fig. 5C) and the up-regulation of 4-1BB (Fig. 5D). Recognition of as low as 1 nM of mutated CCL5 peptide was observed, indicating high functional avidity (Fig. 5C). No or minimal recognition of wild-type CCL5 peptide was detected (Fig. 5, C and D). FOXP3+ TCR 34 did not display recognition of the autologous TC (Fig. 4C). At the genomic level, mutated CCL5 was detected both in the tumor and in the TC (patient 3919) based on whole-exome sequencing, and TC exhibited loss of heterozygosity at this site. However, RNA-seq revealed that TC lacked expression of CCL5 (table S5), providing a plausible explanation for the lack of TC recognition by FOXP3+ TCR 34 despite its specific reactivity against the mutated CCL5 peptide. Similarly, FOXP3+ TCR 7 recognized mutated ANXA1 peptide pulsed on DC but not the autologous TC. The low expression of mutated ANXA1 by TC (table S5) may explain its lack of recognition by this TCR. Overall, two intratumoral T reg -derived TCRs isolated from two patients displayed reactivity against mutated cancer neoantigens. Fig. 5 FOXP 3+ T reg -derived TCRs exhibited specific reactivity against mutant neoantigens. (A) FOXP3+ TCR 7 (patient 3107) was cocultured overnight with autologous DC pulsed with individual long mutated peptides from pp9, and IFN-γ production was quantified by ELISA. (B) HLA class II–matched donor PBL was transduced with FOXP3+ TCR 34 (patient 3919) and subsequently cocultured overnight with the donor-derived DCs that were pulsed with pp13 or its individual peptides, and IFN-γ production was quantified by ELISA. (C) FOXP3+ TCR 34 was cocultured with titrated amount of high-performance liquid chromatography (HPLC) purified mutated (mut-) or wild-type (wt-) CCL5 (initially identified as pp13-5), and IFN-γ in the supernatant was quantified by ELISA. (D) The cocultured cells from (B) were stained similarly as in Fig. 4B to assess the up-regulation of 4-1BB on FOXP3+ TCR 34–transduced T cells (mTCRB+ cells) after an overnight coculture with DC pulsed with mut- or wt-CCL5 peptides (HPLC). The dot plots were gated on CD4+CD3+ PI T cells. All data are representative of at least two independent experiments.