Single-cell analysis of localized prostate cancer patients links high Gleason score with an immunosuppressive profile.

BACKGROUND: Evading immune surveillance is a hallmark for the development of multiple cancer types. Whether immune evasion contributes to the pathogenesis of high-grade prostate cancer (HGPCa) remains an area of active inquiry. METHODS: Through single-cell RNA sequencing and multicolor flow cytometry of freshly isolated prostatectomy specimens and matched peripheral blood, we aimed to characterize the tumor immune microenvironment (TME) of localized prostate cancer (PCa), including HGPCa and low-grade prostate cancer (LGPCa). RESULTS: HGPCa are highly infiltrated by exhausted CD8+ T cells, myeloid cells, and regulatory T cells (TRegs). These HGPCa-infiltrating CD8+ T cells expressed high levels of exhaustion markers including TIM3, TOX, TCF7, PD-1, CTLA4, TIGIT, and CXCL13. By contrast, a high ratio of activated CD8+  effector T cells relative to TRegs and myeloid cells infiltrate the TME of LGPCa. HGPCa CD8+  tumor-infiltrating lymphocytes (TILs) expressed more androgen receptor and prostate-specific membran antigen yet less prostate-specific antigen than the LGPCa CD8+  TILs. The PCa TME was infiltrated by macrophages but these did not clearly cluster by M1 and M2 markers. CONCLUSIONS: Our study reveals a suppressive TME with high levels of CD8+ T cell exhaustion in localized PCa, a finding enriched in HGPCa relative to LGPCa. These studies suggest a possible link between the clinical-pathologic risk of PCa and the associated TME. Our results have implications for our understanding of the immunologic mechanisms of PCa pathogenesis and the implementation of immunotherapy for localized PCa.

The Yin and Yang of Targeting KLRG1+ Tregs and Effector Cells.

The literature surrounding KLRG1 has primarily focused on NK and CD8+ T cells. However, there is evidence that the most suppressive Tregs express KLRG1. Until now, the role of KLRG1 on Tregs has been mostly overlooked and remains to be elucidated. Here we review the current literature on KLRG1 with an emphasis on the KLRG1+ Treg subset role during cancer development and autoimmunity. KLRG1 has been recently proposed as a new checkpoint inhibitor target, but these studies focused on the effects of KLRG1 blockade on effector cells. We propose that when designing anti-tumor therapies targeting KLRG1, the effects on both effector cells and Tregs will have to be considered.

Tumor-associated Tregs obstruct antitumor immunity by promoting T cell dysfunction and restricting clonal diversity in tumor-infiltrating CD8+ T cells.

BACKGROUND: Accumulation of regulatory T cells (Treg) has been described to often correlate with poor prognosis in many solid tumors. How Treg presence impinges on limited functionality and clonal composition of tumor-associated CD8 +T cells has important implications for their therapeutic targeting in the tumor microenvironment. In the present study, we investigated how accumulation of Tregs contributes to T cell dysfunction and clonal constriction of tumor-infiltrating CD8 +T cells. METHODS: Resected melanoma and lung adenocarcinoma tissues from tumor-bearing mice or patients were analyzed. The proportions and phenotype as well as clonal diversity of tumor-associated CD8 +T cells were evaluated by flow cytometry and single-cell T-cell receptor (TCR) sequencing, respectively, at early or advanced tumor stages or under Treg depletion conditions. Furthermore, antigen-specific T cells were evaluated on adoptive transfer into tumor-bearing mice in the presence or absence of anti-CTLA-4 antibody or CTLA-4 Ig. Lastly, tumor-bearing mice were treated with anti-KLRG1 antibody and/or bromodomain inhibitor JQ1 with interleukin (IL)-2 immune complexes to determine therapeutic efficacy. RESULTS: We demonstrate that the emergence of exhaustion-like phenotype and impaired effector functionality in tumor-associated CD8 +T cells is positively correlated with Treg accumulation in the tumor bed and this dysfunctional phenotype becomes reversed on Treg reduction in murine melanoma and lung cancer models. Heightened tumor-associated Treg-expressed CTLA-4 is key to emergence and sustenance of this phenotype. Furthermore, TCR sequencing revealed a clonal shrinkage of tumor-infiltrating CD8 +T cells as tumor progressed, which was associated with reduced survival profile concomitant to increasing Treg proportions. Limited IL-2 availability was a key mechanism contributing to this peripheral repertoire reshaping as Treg depletion improved IL-2 levels, rescued CD8 +T cell viability, and improved their clonal diversity. Finally, targeted reduction of tumor but not peripheral Tregs through JQ1 and/or anti-KLRG1 antibody significantly improved antitumor response in melanoma-bearing mice when supplemented with IL-2 immune complexes. CONCLUSION: Collectively, our study reveals a bimodal program enacted by Tregs to support T cell dysfunction in the tumor bed and highlights a promising therapeutic regimen for localized reprogramming of the tumor microenvironment to curb Treg impairment of antitumor CD8 +T cell response in favor of improved antitumor immunity.

Coupling the immunomodulatory properties of the HDAC6 inhibitor ACY241 with Oxaliplatin promotes robust anti-tumor response in non-small cell lung cancer.

While HDAC inhibitors have shown promise in hematologic cancers, their efficacy remains limited in solid cancers. In the present study, we evaluated the immunomodulatory properties of the HDAC6 inhibitor, Citarinostat (ACY241) on lung tumor immune compartment and its therapeutic potential in combination with Oxaliplatin. As a single agent, ACY241 treatment promoted increased infiltration, activation, proliferation, and effector function of T cells in the tumors of lung adenocarcinoma-bearing mice. Furthermore, tumor-associated macrophages exhibited downregulated expression of inhibitory ligands in favor of increased MHC and co-stimulatory molecules in addition to higher expression of CCL4 that favored increased T cell numbers in the tumors. RNA-sequencing of tumor-associated T cells and macrophages after ACY241 treatment revealed significant genomic changes that is consistent with improved T cell viability, reduced inhibitory molecular signature, and enhancement of macrophage capacity for improved T cell priming. Finally, coupling these ACY241-mediated effects with the chemotherapy drug Oxaliplatin led to significantly enhanced tumor-associated T cell effector functionality in lung cancer-bearing mice and in patient-derived tumors. Collectively, our studies highlight the molecular underpinnings of the expansive immunomodulatory activity of ACY241 and supports its suitability as a partner agent in combination with rationally selected chemotherapy agents for therapeutic intervention in NSCLC.

Circulating Immunosuppressive Regulatory T Cells Predict Risk of Incident Cutaneous Squamous Cell Carcinoma.

Ultraviolet radiation exposure (UVR) is a risk factor for cutaneous squamous cell carcinoma (cuSCC) and has been shown to be positively associated with circulating immunosuppressive regulatory T cells ("Tregs"). However, the risk of cuSCC in association with circulating Tregs has not been studied. The aim of this study was to determine whether circulating Treg levels are associated with cuSCC development, particularly in the context of high UVR. Blood and spectrophotometer-based UVR measurements were obtained on 327 immunocompetent individuals undergoing routine skin cancer screenings at baseline and followed for up to 4 years for incident cuSCC development within a prospective cohort study. Proportions of phenotypically distinct Tregs, especially CCR4hi and CLA+ cells which are associated with activation and homing, respectively, were measured by flow cytometry. Tregs in cuSCC tumors were assessed using immunohistochemistry and graded for solar elastosis, a measure of cumulative UVR damage. Of several Treg phenotypes examined, higher levels of circulating CCR4hi Tregs at baseline were significantly associated with increased risk of subsequent cuSCC; those with higher levels of both CCR4hi and UVR were four times more likely to develop cuSCC compared to those with lower levels of both (Hazard Ratio = 4.11, 95% CI = 1.22-13.90). Within cuSCC tumors, CCR4hi Tregs were positively associated with solar elastosis. Results show that a higher proportion of CCR4hi peripheral Tregs predicts incident cuSCC up to 4 years, especially among highly UV-exposed individuals. Research of the underpinning biology of Tregs in UVR-associated skin damage may possibly reveal novel opportunities for screening, prevention, and treatment.

Targeted Therapy Given after Anti-PD-1 Leads to Prolonged Responses in Mouse Melanoma Models through Sustained Antitumor Immunity.

Immunotherapy (IT) and targeted therapy (TT) are both effective against melanoma, but their combination is frequently toxic. Here, we investigated whether the sequence of IT (anti-PD-1)→ TT (ceritinib-trametinib or dabrafenib-trametinib) was associated with improved antitumor responses in mouse models of BRAF- and NRAS-mutant melanoma. Mice with NRAS-mutant (SW1) or BRAF-mutant (SM1) mouse melanomas were treated with either IT, TT, or the sequence of IT→TT. Tumor volumes were measured, and samples from the NRAS-mutant melanomas were collected for immune-cell analysis, single-cell RNA sequencing (scRNA-seq), and reverse phase protein analysis (RPPA). scRNA-seq demonstrated that the IT→TT sequence modulated the immune environment, leading to increased infiltration of T cells, monocytes, dendritic cells and natural killer cells, and decreased numbers of tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells. Durable responses to the IT→TT sequence were dependent on T-cell activity, with depletion of CD8+, but not CD4+, T cells abrogating the therapeutic response. An analysis of transcriptional heterogeneity in the melanoma compartment showed the sequence of IT→TT enriched for a population of melanoma cells with increased expression of MHC class I and melanoma antigens. RPPA analysis demonstrated that the sustained immune response induced by IT→TT suppressed tumor-intrinsic signaling pathways required for therapeutic escape. These studies establish that upfront IT improves the responses to TT in BRAF- and NRAS-mutant melanoma models.

Integrative multi-omics analysis of muscle-invasive bladder cancer identifies prognostic biomarkers for frontline chemotherapy and immunotherapy.

Only a subgroup of patients with muscle-invasive bladder cancer (MIBC) are responders toward cisplatin-based chemotherapy and PD-L1 blockade immunotherapy. There is a clinical need to identify MIBC molecular subtypes and biomarkers for patient stratification toward the therapies. Here, we performed an integrative clustering analysis of 388 MIBC samples with multi-omics data and identified basal and luminal/differentiated integrative subtypes and derived a 42 gene panel for classification of MIBC. Using nine additional gene expression data (n = 844), we demonstrated the prognostic value of the 42 basal-luminal genes. The basal subtype was associated with worse overall survival in patients receiving no neoadjuvant chemotherapy (NAC), but better overall survival in patients receiving NAC in two clinical trials. Each of the subtypes could be further divided into chr9 p21.3 normal or loss subgroup. The patients with low expression of MTAP/CDKN2A/2B (indicative of chr9 p21.3 loss) had a significantly lower response rate to anti-PD-L1 immunotherapy and worse survival than the patients with high expression of MTAP/CDKN2A/2B. This integrative analysis reveals intrinsic MIBC subtypes and biomarkers with prognostic value for the frontline therapies.

Innate αß T Cells Mediate Antitumor Immunity by Orchestrating Immunogenic Macrophage Programming.

Unconventional T-lymphocyte populations are emerging as important regulators of tumor immunity. Despite this, the role of TCRαß+CD4-CD8-NK1.1- innate αß T cells (iαßT) in pancreatic ductal adenocarcinoma (PDA) has not been explored. We found that iαßTs represent ∼10% of T lymphocytes infiltrating PDA in mice and humans. Intratumoral iαßTs express a distinct T-cell receptor repertoire and profoundly immunogenic phenotype compared with their peripheral counterparts and conventional lymphocytes. iαßTs comprised ∼75% of the total intratumoral IL17+ cells. Moreover, iαßT-cell adoptive transfer is protective in both murine models of PDA and human organotypic systems. We show that iαßT cells induce a CCR5-dependent immunogenic macrophage reprogramming, thereby enabling marked CD4+ and CD8+ T-cell expansion/activation and tumor protection. Collectively, iαßTs govern fundamental intratumoral cross-talk between innate and adaptive immune populations and are attractive therapeutic targets. SIGNIFICANCE: We found that iαßTs are a profoundly activated T-cell subset in PDA that slow tumor growth in murine and human models of disease. iαßTs induce a CCR5-dependent immunogenic tumor-associated macrophage program, T-cell activation and expansion, and should be considered as novel targets for immunotherapy.See related commentary by Banerjee et al., p. 1164.This article is highlighted in the In This Issue feature, p. 1143.

Differential control of human Treg and effector T cells in tumor immunity by Fc-engineered anti-CTLA-4 antibody.

Anti-CTLA-4 mAb is efficacious in enhancing tumor immunity in humans. CTLA-4 is expressed by conventional T cells upon activation and by naturally occurring FOXP3+CD4+ Treg cells constitutively, raising a question of how anti-CTLA-4 mAb can differentially control these functionally opposing T cell populations in tumor immunity. Here we show that FOXP3high potently suppressive effector Treg cells were abundant in melanoma tissues, expressing CTLA-4 at higher levels than tumor-infiltrating CD8+ T cells. Upon in vitro tumor-antigen stimulation of peripheral blood mononuclear cells from healthy individuals or melanoma patients, Fc-region-modified anti-CTLA-4 mAb with high antibody-dependent cell-mediated cytotoxicity (ADCC) and cellular phagocytosis (ADCP) activity selectively depleted CTLA-4+FOXP3+ Treg cells and consequently expanded tumor-antigen-specific CD8+T cells. Importantly, the expansion occurred only when antigen stimulation was delayed several days from the antibody treatment to spare CTLA-4+ activated effector CD8+T cells from mAb-mediated killing. Similarly, in tumor-bearing mice, high-ADCC/ADCP anti-CTLA-4 mAb treatment with delayed tumor-antigen vaccination significantly prolonged their survival and markedly elevated cytokine production by tumor-infiltrating CD8+ T cells, whereas antibody treatment concurrent with vaccination did not. Anti-CTLA-4 mAb modified to exhibit a lesser or no Fc-binding activity failed to show such timing-dependent in vitro and in vivo immune enhancement. Thus, high ADCC anti-CTLA-4 mAb is able to selectively deplete effector Treg cells and evoke tumor immunity depending on the CTLA-4-expressing status of effector CD8+ T cells. These findings are instrumental in designing cancer immunotherapy with mAbs targeting the molecules commonly expressed by FOXP3+ Treg cells and tumor-reactive effector T cells.

RIP1 Kinase Drives Macrophage-Mediated Adaptive Immune Tolerance in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by immune tolerance and immunotherapeutic resistance. We discovered upregulation of receptor-interacting serine/threonine protein kinase 1 (RIP1) in tumor-associated macrophages (TAMs) in PDA. To study its role in oncogenic progression, we developed a selective small-molecule RIP1 inhibitor with high in vivo exposure. Targeting RIP1 reprogrammed TAMs toward an MHCIIhiTNFα+IFNγ+ immunogenic phenotype in a STAT1-dependent manner. RIP1 inhibition in TAMs resulted in cytotoxic T cell activation and T helper cell differentiation toward a mixed Th1/Th17 phenotype, leading to tumor immunity in mice and in organotypic models of human PDA. Targeting RIP1 synergized with PD1-and inducible co-stimulator-based immunotherapies. Tumor-promoting effects of RIP1 were independent of its co-association with RIP3. Collectively, our work describes RIP1 as a checkpoint kinase governing tumor immunity.

Intron retention is a source of neoepitopes in cancer.

We present an in silico approach to identifying neoepitopes derived from intron retention events in tumor transcriptomes. Using mass spectrometry immunopeptidome analysis, we show that retained intron neoepitopes are processed and presented on MHC I on the surface of cancer cell lines. RNA-derived neoepitopes should be considered for prospective personalized cancer vaccine development.

BET Bromodomain Inhibition Cooperates with PD-1 Blockade to Facilitate Antitumor Response in Kras-Mutant Non-Small Cell Lung Cancer.

KRAS mutation is present in approximately 30% of human lung adenocarcinomas. Although recent advances in targeted therapy have shown great promise, effective targeting of KRAS remains elusive, and concurrent alterations in tumor suppressors render KRAS-mutant tumors even more resistant to existing therapies. Contributing to the refractoriness of KRAS-mutant tumors are immunosuppressive mechanisms, such as increased presence of suppressive regulatory T cells (Treg) in tumors and elevated expression of the inhibitory receptor PD-1 on tumor-infiltrating T cells. Treatment with BET bromodomain inhibitors is beneficial for hematologic malignancies, and they have Treg-disruptive effects in a non-small cell lung cancer (NSCLC) model. Targeting PD-1-inhibitory signals through PD-1 antibody blockade also has substantial therapeutic impact in lung cancer, although these outcomes are limited to a minority of patients. We hypothesized that the BET bromodomain inhibitor JQ1 would synergize with PD-1 blockade to promote a robust antitumor response in lung cancer. In the present study, using Kras+/LSL-G12D ; Trp53L/L (KP) mouse models of NSCLC, we identified cooperative effects between JQ1 and PD-1 antibody. The numbers of tumor-infiltrating Tregs were reduced and activation of tumor-infiltrating T cells, which had a T-helper type 1 (Th1) cytokine profile, was enhanced, underlying their improved effector function. Furthermore, lung tumor-bearing mice treated with this combination showed robust and long-lasting antitumor responses compared with either agent alone, culminating in substantial improvement in the overall survival of treated mice. Thus, combining BET bromodomain inhibition with immune checkpoint blockade offers a promising therapeutic approach for solid malignancies such as lung adenocarcinoma. Cancer Immunol Res; 6(10); 1234-45. ©2018 AACR.

Genomic correlates of response to immune checkpoint blockade in microsatellite-stable solid tumors.

Tumor mutational burden correlates with response to immune checkpoint blockade in multiple solid tumors, although in microsatellite-stable tumors this association is of uncertain clinical utility. Here we uniformly analyzed whole-exome sequencing (WES) of 249 tumors and matched normal tissue from patients with clinically annotated outcomes to immune checkpoint therapy, including radiographic response, across multiple cancer types to examine additional tumor genomic features that contribute to selective response. Our analyses identified genomic correlates of response beyond mutational burden, including somatic events in individual driver genes, certain global mutational signatures, and specific HLA-restricted neoantigens. However, these features were often interrelated, highlighting the complexity of identifying genetic driver events that generate an immunoresponsive tumor environment. This study lays a path forward in analyzing large clinical cohorts in an integrated and multifaceted manner to enhance the ability to discover clinically meaningful predictive features of response to immune checkpoint blockade.

Assessing Therapeutic Efficacy of MEK Inhibition in a KRASG12C-Driven Mouse Model of Lung Cancer.

Purpose: Despite the challenge to directly target mutant KRAS due to its high GTP affinity, some agents are under development against downstream signaling pathways, such as MEK inhibitors. However, it remains controversial whether MEK inhibitors can boost current chemotherapy in KRAS-mutant lung tumors in clinic. Considering the genomic heterogeneity among patients with lung cancer, it is valuable to test potential therapeutics in KRAS mutation-driven mouse models.Experimental Design: We first compared the pERK1/2 level in lung cancer samples with different KRAS substitutions and generated a new genetically engineered mouse model whose tumor was driven by KRAS G12C, the most common KRAS mutation in lung cancer. Next, we evaluated the efficacy of selumetinib or its combination with chemotherapy, in KRASG12C tumors compared with KRASG12D tumors. Moreover, we generated KRASG12C/p53R270H model to explore the role of a dominant negative p53 mutation detected in patients in responsiveness to MEK inhibition.Results: We determined higher pERK1/2 in KRASG12C lung tumors compared with KRASG12D Using mouse models, we further identified that KRASG12C tumors are significantly more sensitive to selumetinib compared with KrasG12D tumors. MEK inhibition significantly increased chemotherapeutic efficacy and progression-free survival of KRASG12C mice. Interestingly, p53 co-mutation rendered KRASG12C lung tumors less sensitive to combination treatment with selumetinib and chemotherapy.Conclusions: Our data demonstrate that unique KRAS mutations and concurrent mutations in tumor-suppressor genes are important factors for lung tumor responses to MEK inhibitor. Our preclinical study supports further clinical evaluation of combined MEK inhibition and chemotherapy for lung cancer patients harboring KRAS G12C and wild-type p53 status. Clin Cancer Res; 24(19); 4854-64. ©2018 AACR.

NK Cells Mediate Synergistic Antitumor Effects of Combined Inhibition of HDAC6 and BET in a SCLC Preclinical Model.

Small-cell lung cancer (SCLC) has the highest malignancy among all lung cancers, exhibiting aggressive growth and early metastasis to distant sites. For 30 years, treatment options for SCLC have been limited to chemotherapy, warranting the need for more effective treatments. Frequent inactivation of TP53 and RB1 as well as histone dysmodifications in SCLC suggest that transcriptional and epigenetic regulations play a major role in SCLC disease evolution. Here we performed a synthetic lethal screen using the BET inhibitor JQ1 and an shRNA library targeting 550 epigenetic genes in treatment-refractory SCLC xenograft models and identified HDAC6 as a synthetic lethal target in combination with JQ1. Combined treatment of human and mouse SCLC cell line-derived xenograft tumors with the HDAC6 inhibitor ricolinostat (ACY-1215) and JQ1 demonstrated significant inhibition of tumor growth; this effect was abolished upon depletion of NK cells, suggesting that these innate immune lymphoid cells play a role in SCLC tumor treatment response. Collectively, these findings suggest a potential new treatment for recurrent SCLC.Significance: These findings identify a novel therapeutic strategy for SCLC using a combination of HDAC6 and BET inhibitors. Cancer Res; 78(13); 3709-17. ©2018 AACR.

MUC1-C promotes the suppressive immune microenvironment in non-small cell lung cancer.

The cancer immune microenvironment is of importance for the effectiveness of immunotherapy; however, its dysregulation is poorly understood. The MUC1-C oncoprotein is aberrantly overexpressed in non-small cell lung cancer (NSCLC) and has been linked to the induction of PD-L1. The present work investigated the effects of targeting MUC1-C in an immuno-competent MUC1 transgenic (MUC1.Tg) mouse model. We show that Lewis Lung Carcinoma cells expressing MUC1-C (LLC/MUC1) exhibit upregulation of PD-L1 and suppression of interferon-γ (IFN-γ). In studies of LLC/MUC1 cells growing in vitro and as tumors in MUC1.Tg mice, treatment with the MUC1-C inhibitor, GO-203, was associated with the downregulation of PD-L1 and induction of IFN-γ. The results further demonstrate that targeting MUC1-C results in enhanced effector function of CD8+ tumor-infiltrating lymphocytes (TILs) as evidenced by increased expression of the activation marker CD69, the degranulation marker CD107α, and granzyme B. Notably, targeting MUC1-C was also associated with marked increases in TIL-mediated killing of LLC/MUC1 cells. Analysis of gene expression data sets further showed that overexpression of MUC1 in NSCLCs correlates negatively with CD8, IFNG and GZMB, and that decreases in CD8 and IFNG are associated with poor clinical outcomes. These findings in LLC/MUC1 tumors and in NSCLCs indicate that MUC1-C→PD-L1 signaling promotes the suppression of CD8+ T-cell activation and that MUC1-C is a potential target for reprogramming of the tumor microenvironment.

Synergistic Immunostimulatory Effects and Therapeutic Benefit of Combined Histone Deacetylase and Bromodomain Inhibition in Non-Small Cell Lung Cancer.

Effective therapies for non-small cell lung cancer (NSCLC) remain challenging despite an increasingly comprehensive understanding of somatically altered oncogenic pathways. It is now clear that therapeutic agents with potential to impact the tumor immune microenvironment potentiate immune-orchestrated therapeutic benefit. Herein, we evaluated the immunoregulatory properties of histone deacetylase (HDAC) and bromodomain inhibitors, two classes of drugs that modulate the epigenome, with a focus on key cell subsets that are engaged in an immune response. By evaluating human peripheral blood and NSCLC tumors, we show that the selective HDAC6 inhibitor ricolinostat promotes phenotypic changes that support enhanced T-cell activation and improved function of antigen-presenting cells. The bromodomain inhibitor JQ1 attenuated CD4+FOXP3+ T regulatory cell suppressive function and synergized with ricolinostat to facilitate immune-mediated tumor growth arrest, leading to prolonged survival of mice with lung adenocarcinomas. Collectively, our findings highlight the immunomodulatory effects of two epigenetic modifiers that, together, promote T cell-mediated antitumor immunity and demonstrate their therapeutic potential for treatment of NSCLC.Significance: Selective inhibition of HDACs and bromodomain proteins modulates tumor-associated immune cells in a manner that favors improved T-cell function and reduced inhibitory cellular mechanisms. These effects facilitated robust antitumor responses in tumor-bearing mice, demonstrating the therapeutic potential of combining these epigenetic modulators for the treatment of NSCLC. Cancer Discov; 7(8); 852-67. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 783.

The Lower Limit of Regulatory CD4+ Foxp3+ TCRß Repertoire Diversity Required To Control Autoimmunity.

The TCR repertoire of regulatory T cells (Tregs) is highly diverse. The relevance of this diversity to maintain self-tolerance remains unknown. We established a model where the TCR repertoire of normal polyclonal Tregs was limited by serial transfers into IL-2Rß-/- mice, which lack functional Tregs. After a primary transfer, the donor Treg TCR repertoire was substantially narrowed, yet the recipients remained autoimmune-free. Importantly, upon purification and transfer of donor-derived Tregs from an individual primary recipient into neonatal IL-2Rß-/- mice, the secondary recipients developed autoimmunity. In this study, the Treg TCRß repertoire was reshaped and further narrowed. In contrast, secondary IL-2Rß recipients showed fewer symptoms of autoimmunity when they received donor Tregs that were premixed from several primary recipients to increase their TCRß repertoire diversity. About 8-11% of the Treg TCRß repertoire was estimated to be the minimum required to establish and maintain tolerance in primary IL-2Rß-/- recipients. Collectively, these data quantify where limitations imposed on the Treg TCRß repertoire results in a population of Tregs that cannot fully suppress polyclonal autoreactive T cells. Our data favor a model where the high diversity of the Treg TCR provides a mechanism for Tregs to actively adapt and effectively suppress autoreactive T cells, which are not fixed, but are evolving as they encounter self-antigens.