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Simple Summary: Not all cancer patients receiving immunotherapy by immune checkpoint blockade experience a clinical benefit. Our study was aimed at identifying biomarkers that could guide the selection of immunotherapy-responsive patients. Immunotherapy targets two major populations of lymphocytes: CD8 T cells, which directly kill tumor cells, and CD4 T cells, which provide help to CD8 T cells, the role of which in clinical responsiveness to immunotherapy has been less explored. We identified, in the blood of cancer patients, a population of CD4 T cells expressing inhibitory receptors targeted by immunotherapy. We showed that these cells were activated and proliferating, indicating their potential involvement in ongoing immune responses. Accordingly, we showed that they were specific for tumor antigens. In a prospective cohort, we showed that high proportions of these cells prior to therapy were associated with a response to immunotherapy. Tumor-infiltrating exhausted PD-1hiCD39+ tumor-antigen (Ag)-specific CD4 T cells contribute to the response to immune checkpoint blockade (ICB), but their circulating counterparts, which could represent accessible biomarkers, have not been assessed. Here, we analyzed circulating PD-1+CD39+ CD4 T cells and show that this population was present at higher proportions in cancer patients than in healthy individuals and was enriched in activated HLA-DR+ and ICOS+ and proliferating KI67+ cells, indicative of their involvement in ongoing immune responses. Among memory CD4 T cells, this population contained the lowest proportions of cells producing effector cytokines, suggesting they were exhausted. In patients with HPV-induced malignancies, the PD-1+CD39+ population contained high proportions of HPV Ag-specific T cells. In patients treated by ICB for HPV-induced tumors, the proportion of circulating PD-1+CD39+ CD4 T cells was predictive of the clinical response. Our results identify CD39 expression as a surrogate marker of circulating helper tumor-Ag-specific CD4 T cells. [ABSTRACT FROM AUTHOR]

γδ T lymphocytes diverge from conventional T CD8 lymphocytes for ontogeny, homing, and antigen specificity, but whether their differentiation in tumors also deviates was unknown. Using innovative analyses of our original and ~150 published single-cell RNA sequencing datasets validated by phenotyping of human tumors and murine models, here we present the first high-resolution view of human γδ T cell differentiation in cancer. While γδ T lymphocytes prominently encompass TCRVγ9 cells more differentiated than T CD8 in healthy donor's blood, a different scenario is unveiled in tumors. Solid tumors and lymphomas are infiltrated by a majority of TCRVγnon9 γδ T cells which are quantitatively correlated and remarkably aligned with T CD8 for differentiation, exhaustion, gene expression profile, and response to immune checkpoint therapy. This cancer-wide association is critical for developing cancer immunotherapies. [ABSTRACT FROM AUTHOR]

Immunotherapies have achieved clinical benefit in many types of cancer but remain limited to a subset of patients in colorectal cancer (CRC). Resistance to immunotherapy can be attributed in part to tissue-specific factors constraining antitumor immunity. Thus, a better understanding of how the colon microenvironment shapes the immune response to CRC is needed to identify mechanisms of resistance to immunotherapies and guide the development of novel therapeutics. In an orthotopic mouse model of MC38-CRC, tumor progression was monitored by bioluminescence imaging and the immune signatures were assessed at a transcriptional level using NanoString and at a cellular level by flow cytometry. Despite initial tumor growth in all mice, only 25% to 35% of mice developed a progressive lethal CRC while the remaining animals spontaneously rejected their solid tumor. No tumor rejection was observed in the absence of adaptive immunity, nor when MC38 cells were injected in non-orthotopic locations, subcutaneously or into the liver. We observed that progressive CRC tumors exhibited a protumor immune response, characterized by a regulatory T-lymphocyte pattern, discernible shortly post-tumor implantation, as well as suppressive myeloid cells. In contrast, tumor-rejecting mice presented an early inflammatory response and an antitumor microenvironment enriched in CD8+ T cells. Taken together, our data demonstrate the role of the colon microenvironment in regulating the balance between anti or protumor immune responses. While emphasizing the relevance of the CRC orthotopic model, they set the basis for exploring the impact of the identified signatures in colon cancer response to immunotherapy. [ABSTRACT FROM AUTHOR]

CD4 T cells secreting IL-10 or IL-17 are frequent at mucosal sites, where their equilibrium is important for simultaneously maintaining tolerance and immunity to the resident microbiota. The mode of action of these cells, however, is as yet incompletely understood. In this study, we have combined ex vivo analysis of CD4 T cells producing IL-10 or/and IL-17 with assessment of clonal populations isolated ex vivo using a cytokine catch assay. We found that circulating CD4 T cells secreting IL-10 or/and IL-17 ex vivo include both conventional FOXP3- CD4 T cells and FOXP3+ Helios- Treg. Upon assessment of clonal populations derived from single ex vivo isolated cytokine secreting cells, we found that IL-10 or/and IL-17 secreting cells prevalently secrete one or the other cytokine depending on the type of stimulation, the time after stimulation and the presence of microbial products. Namely, IL-10 secretion by clonal cells was prevalent at early time points after TCR mediated stimulation, was independent of co-stimulation and was increased in the presence of the microbial fermentation product butyrate. In contrast, IL-17 secretion was higher at later time points after TCR mediated stimulation and in the presence of co-stimulatory signals. Taken together, these results provide insights into the mechanisms that, through modulation of cytokine secretion depending on conditions, allow IL-10 and IL-17 producing CD4 T cells to contribute to maintain tolerance to microbes locally, while retaining the ability to participate in protective immune responses at distant sites. [ABSTRACT FROM AUTHOR]

Spontaneous CD4+ T-cell responses to the tumor-specific antigen NY-ESO-1 (ESO) are frequently found in patients with epithelial ovarian cancer (EOC). If these responses are of effector or/and Treg type, however, has remained unclear. Here, we have used functional approaches together with recently developed MHC class II/ESO tetramers to assess the frequency, phenotype and function of ESO-specific cells in circulating lymphocytes from EOC patients. We found that circulating ESOspecific CD4+ T cells in EOC patients with spontaneous immune responses to the antigen are prevalently TH1 type cells secreting IFN-c but no IL-17 or IL-10 and are not suppressive. We detected tetramer+ cells ex vivo, at an average frequency of 1:25000 memory cells, that is, significantly lower than in patients immunized with an ESO vaccine. ESO tetramer+ cells were mostly effector memory cells at advanced stages of differentiation and were not detected in circulating CD25+FOXP3+Treg. Thus, spontaneous CD4+ T-cell responses to ESO in cancer patients are prevalently of TH1 type and not Treg. Their relatively low frequency and advanced differentiation stage, however, may limit their efficacy, that may be boosted by immunogenic ESO vaccines [ABSTRACT FROM AUTHOR]

RORγt+ TH17 cells are a proinflammatory CD4+ T-cell population associated with autoimmune tissue injury. In mice, priming of TH17 requires TGF-β, which alone directs the priming of FOXP3+ regulatory T cells (Treg), in association with inflammatory cytokines. Priming of human TH17 cells from conventional naive CD4+ I cells under similar conditions, however, has proved difficult to achieve. Here, we report that differentiation of human TH17 cells preferentially occurs from FOXP3+ naive Treg (NTreg) in the presence of lL-2 and !L-1β and is increased by IL-23 and TGF-β. lL-1β- mediated differentiation correlated with IL-1Rl expression in stimulated NTreg and was accompanied by induction of ROR1t along with down-regulation of FOXP3. IL-17-secreting cells in NTreg cultures cosecreted TNF-α and Il-2 and contained distinct subpopulations cosecreting or not cosecreting IFN-γ and other TH17-associated cytokines. Polarized Nlreg contained significant subpopulations of (CR6-expressing cells that were highly enriched in IL-17-secreting cells. Finally, analysis of CCR6 expression with respect to that of IL-1RI identified distinct IL-17-secreting subpopulations that had maintained or lost their suppressive functions. Together our results support the concept that priming of human TH17 from naive CD4+ T cells preferentially takes place from FOXP3+ Treg precursors in the presence of lineage-specific polarizing factors. [ABSTRACT FROM AUTHOR]

MHC-peptide tetramers have become essential tools for T-cell analysis, but few MHC class II tetramers incorporating peptides from human tumorand self-antigens have been developed. Among limiting factors are the high polymorphism of class II molecules and the low binding capacity of the peptides. Here, we report the generation of molecularly defined tetramers using His-tagged peptides and isolation of folded MHC/peptide monomers by affinity purification. Using this strategy we generated tetramers of DR52b (DRB3*0202), an allele expressed by approximately half of Caucasians, incorporating an epitope from the tumor antigen NY-ESO-1. Molecularly defined tetramers avidly and stably bound to specific CD4+ T cells with negligible background on nonspecific cells. Using molecularly defined DR52b/NY-ESO-1 tetramers, we could demonstrate that in DR52b+ cancer patients immunized with a recombinant NY-ESO-1 vaccine, vaccine-induced tetramer-positive cells represent ex vivo in average 1:5,000 circulating CD4+T cells, include central and transitional memory polyfunctional populations, and do not include CD4+CD25+CD127- regulatory T cells. This approach may significantly accelerate the development of reliable MHC class II tetramers to monitor immune responses to tumor and self-antigens. [ABSTRACT FROM AUTHOR]

Recent studies have suggested a close relationship between CD4+FOXP3+ regulatory T cells (Tregs) and proinflammatory IL-17-producing I helper cells (TH17) expressing the lineage-specific transcription factor RORγt. We report here the unexpected finding that human memory Tregs secrete lL-17 ex vivo and constitutively express RORγt. IL-17-secreting Tregs share some phenotypic and functional features with conventional TH17 cells, expressing high levels of CCR4 and CCR6 and low levels of CXCR3. However, unlike conventional TH17 cells, they express low levels of CD161 and mostly fail to cosecrete lL-22 and TNF-α ex vivo. Ex vivo secretion of IL-17 and constitutive expression of RORγt by human memory Tregs suggest that, in addition to their well-known suppressive functions, these cells likely play additional, as yet undescribed, proinflammatory functions. [ABSTRACT FROM AUTHOR]

Proteins encoded by genes of the SSX family are specifically expressed in tumors and are therefore relevant targets for cancer immunotherapy. One of the first identified family members, SSX-1, is expressed in a large fraction of synovial sarcomas as a fusion protein together with the product of the SYT gene. In addition, the full-length SSX-1 antigen is frequently expressed in tumors of several other histological types such as sarcoma, melanoma, hepatocellular carcinoma, ovarian cancer and myeloma. To date, however, SSX-1 specific T cell responses have not been investigated and no SSX-1 derived T cell epitopes have been described. Here, we have assessed the presence of CD4+ T cells directed against the SSX-1 antigen in circulating lymphocytes of cancer-free individuals. After a single in vitro stimulation with a pool of peptides spanning the entire SSX-1 protein we could detect and isolate SSX-1-specific CD4+ T cells from 5/5 donors analyzed. SSX-1-specific polyclonal populations isolated from these cultures recognized peptides located in three distinct regions of the protein containing clusters of sequences with significant predicted binding to frequently expressed MHC class II alleles. Characterization of specific clonal CD4+ T cell populations derived from one donor allowed the identification of several naturally processed epitopes recognized in association with HLA-DR. These data document the existence of a significant repertoire of CD4+ T cells specific for SSX-1 derived sequences in circulating lymphocytes of any individual that can be exploited for the development of both passive and active immunotherapeutic approaches to control disease evolution in cancer patients. [ABSTRACT FROM AUTHOR]

Tumor-specific gene products, such as cancer/testis (CT) antigens, constitute promising targets for the development of T cell vaccines. Whereas CT antigens are frequently expressed in melanoma, their expression in colorectal cancers (CRC) remains poorly characterized. Here, we have studied the expression of the CT antigens MAGE-A3, MAGE-A4, MAGE-A10, NY-ESO-1 and SSX2 in CRC because of the presence of well-described HLA-A2-restricted epitopes in their sequences. Our analyses of 41 primary CRC and 14 metastatic liver lesions confirmed the low frequency of expression of these CT antigens. No increased expression frequencies were observed in metastatic tumors compared to primary tumors. Histological analyses of CRC samples revealed heterogeneous expression of individual CT antigens. Finally, evidence of a naturally acquired CT antigen-specific CD8+ T cell response could be demonstrated. These results show that the expression of CT antigens in a subset of CRC patients induces readily detectable T cell responses. [ABSTRACT FROM AUTHOR]

The use of recombinant tumor antigen proteins is a realistic approach for the development of generic cancer vaccines, but the potential of this type of vaccines to induce specific CD8+ T cell responses, through in vivo cross-priming, has remained unclear. In this article, we report that repeated vaccination of cancer patients with recombinant NY-ESO-1 protein, Montanide ISA-51, and CpG ODN 7909, a potent stimulator of B cells and T helper type 1 (Th1)-type immunity, resulted in the early induction of specific integrated CD4+ Th cells and antibody responses in most vaccinated patients, followed by the development of later CD8+ T cell responses in a fraction of them. The correlation between antibody and T cell responses, together with the ability of vaccine-induced antibodies to promote in vitro cross-presentation of NY-ESO-1 by dendritic cells to vaccine-induced CD8+ T cells, indicated that elicitation of NY-ESO-1-specific CD8+ T cell responses by cross-priming in vivo was associated with the induction of adequate levels of specific antibodies. Together, our data provide clear evidence of in viva cross-priming of specific cytotoxic T lymphocytes by a recombinant tumor antigen vaccine, underline the importance of specific antibody induction for the cross-priming to occur, and support the use of this type of formulation for the further development of efficient cancer vaccines. [ABSTRACT FROM AUTHOR]

Abstract: Because of its frequent expression in tumors and spontaneous immunogenicity in advanced cancer patients, NY-ESO-1 is presently viewed as a prototype tumor antigen for the development of cancer vaccines. A prerequisite for the analysis of NY-ESO-1-specific T cell responses in vaccinated patients is the assessment of the complete T cell repertoire available for the antigen. Here, we have assessed frequency and fine specificity of CD4+ T cells reactive against NY-ESO-1-derived sequences in circulating lymphocytes from cancer patients with spontaneous responses to the antigen. We found that, relative to healthy donors, this frequency was only moderately increased in cancer patients. The reactivity of these cells, however, was directed against the same immunodominant regions previously identified for healthy donors. On account of these data, we developed an approach for the immune monitoring of NY-ESO-1-specific CD4+ T cell responses based on the assessment of CD4+ T cell populations of defined phenotype. Using this approach, a similar frequency of NY-ESO-1-specific CD4+ T cells was found among naive T cells of healthy donors and cancer patients. In contrast, among antigen-experienced T cells, NY-ESO-1-specific CD4+ T cells were exclusively detectable in cancer patients. We anticipate that this phenotype-based approach will be useful for the immune monitoring of vaccine-induced responses in vaccination trials using NY-ESO-1 as well as other tumor antigens. [Copyright &y& Elsevier]

Abstract: Because of their specific expression in tumors of different histological types, the products of the SSX genes are important candidate targets for development of cancer vaccines. We have previously identified two immunodominant SSX-2-derived T cell epitopes recognized by HLA-A2-restricted CD8+ T cells (SSX-2 41–49) and HLA-DR11-restricted CD4+ T cells (SSX-2 45–59), respectively. In this study, we report the identification of an HLA-DR3-restricted epitope mapping to the 37–51 region of SSX-2, overlapping both previously identified epitopes. As about one fifth of individuals from several major ethnic groups express HLA-DR3, the identification of this epitope significantly increases the percent of patients that are expected to mount specific CD4+ T cell responses following vaccination with peptides in this region of SSX-2. Retrieval of multiple overlapping epitopes in a defined region of SSX-2 protein suggests the presence of a “hot spot” for T cell recognition that may prove sufficient for the induction of immune responses. [Copyright &y& Elsevier]

Discusses the use of autologous tumor reactive CD8 cytolytic T lymphocyte and CD4 T-cell clones and antibodies from cancer patients for the identification of genes encoding the target antigens. Characteristics of the melanoma antigen A (Melan-A) or melanoma antigen recognized by T cells 1; Optimization of the antigenicity and immunogenecity of antigenic peptide; Effect of Melan-A peptide-based vaccination in specific immunotherapy of melanoma.

A letter to the editor is presented in response to the article "Helios+ and Helios- Cells Coexist within the Natural FOXP3+T Regulatory Cell Subset in Humans" by M. E. Himmel in 2013 issue is presented.

In the past 20 years, the immune system has increasingly been recognized as a major player in tumor cell control, leading to considerable advances in cancer treatment. While promising with regards to melanoma, renal cancer and non-small cell lung cancer, immunotherapy provides, for the time being, limited success in other cancers, including ovarian cancer, potentially due to insufficient immunogenicity or to a particularly immunosuppressive microenvironment. In this review, we provide a global description of the immune context of ovarian cancer, in particular epithelial ovarian cancer (EOC). We describe the adaptive and innate components involved in the EOC immune response, including infiltrating tumor-specific T lymphocytes, B lymphocytes, and natural killer and myeloid cells. In addition, we highlight the rationale behind the use of EOC preclinical mouse models to assess resistance to immunotherapy, and we summarize the main preclinical studies that yielded anti-EOC immunotherapeutic strategies. Finally, we focus on major published or ongoing immunotherapy clinical trials concerning EOC. [ABSTRACT FROM AUTHOR]

In a recent study, we have demonstrated that T helper type 1 (TH1) cells specific for the tumor antigen NY-ESO-1 are amplified at ovarian tumor sites but are not “perverted” into immunosuppressive FOXP3+regulatory T cells (Tregs). These findings encourage the development of protocols aiming to eliminate, or inactivate, FOXP3+Tregs and reinforce Type I anticancer immunity, to improve clinical outcomes. [ABSTRACT FROM AUTHOR]

Antitumor type I T-cell responses involving IFN-γ production are critical to control cancer, but the efficacy of this response is limited by a variety of immunosuppressive mechanisms that promote tumoral immune escape. One critical mechanism involves the accumulation of FOXP3+ T regulatory cells (Treg), a class of suppressive T cells that prevent excessive tissue destruction caused by unchecked immune responses. Recent studies have revealed that FOXP3+ Treg include distinct subsets specifically controlling over the corresponding effector subset. In particular, CXCR3+ Treg have been described as a subset specialized in the control of type I T-cell responses in vivo. Here, we show that CXCR3+ Treg are highly enriched in human ovarian carcinomas, particularly in solid tumor masses, where they represent the majority of Treg. Tumor-associated CXCR3+. Treg coexpress Tbet but do not secrete IFN-γ ex vivo and suppress proliferation and IFN-g secretion of T effectors. In addition, they coexpress Helios, suggesting that they originate from natural Treg. Finally, we show that the proportion of CXCR3+ Treg at tumor sites is directly correlated with that of CXCR3+ T effectors, consistent with expression of CXCR3 ligands. Together, our findings support the concept that natural CXCR3+ T-bet+ Treg selectively accumulate in ovarian tumors to control type I T-cell responses, resulting in the collateral limitation of efficient antitumor immunity. [ABSTRACT FROM AUTHOR]

The role of TH17 cells in cancer is being investigated, but the existence of tumor antigen-specific TH17 cells has yet to be ascertained. Here, we report the first description of a spontaneous TH17 (IL-17+) response to the important tumor antigen MAGE-A3, which occurred concurrently with a TH1 (IFN-γ+) response in a lung cancer patient. MAGE-A3-specific interleukin (IL)-17+) T cells were mainly CCR7+) central memory T cells, whereas IFN-γ+) cells were enriched for CCR7-) effector memory T cells. An assessment of the fine specificity of antigen recognition by these T cells indicated that the CCR6+) CCR4+) and CCR6+)CXCR3+) fractions contained the same TH17/TH1 population at early and late differentiation stages, respectively, whereas the CCR6-) CXCR3+) fraction contained a distinct TH1 population. These findings are important because they suggest a differentiation model in which tumor antigen-specific CD4+) T cells that are primed under TH17 polarizing conditions will progressively convert into IFN-γ-secreting cells in vivo as they differentiate into effector T cells that can effectively attack tumors. [ABSTRACT FROM AUTHOR]

Abstract: Natural CD4+CD25+ regulatory T cells (Treg) and interleukin 17 (IL-17)-producing T helper cells (TH17) carry out opposite functions, the former maintaining self-tolerance and the latter being involved in inflammation and autoimmunity. We report here that stimulation of human Natural Treg under TH17 polarizing conditions in the presence of IL-2 converts them into TH17 cells. Conversion of Tregs into TH17 cells occurs both from natural naïve Tregs (NnTregs) and, to a higher extent, from memory Tregs (MTregs). Among antigen presenting cells, fresh monocytes activated by microbial stimuli were the most efficient inducers of TH17 cells from Tregs. Conversion of Treg into TH17 cells was induced by IL-1β and involved down-regulation of the Treg lineage transcription factor FOXP3 and suppressive functions. Our results indicate that, under inflammatory conditions, in the presence of IL-2, Treg can be converted into pro-inflammatory TH17 cells and establish a functional link between inflammation and autoimmunity. [Copyright &y& Elsevier]

Abstract: Identification of immunodominant CD8+ T cell responses to frequently expressed tumor antigens across MHC class I polymorphism is essential for the implementation of cancer immunotherapy. However, the key factors that determine immunodominance are not fully understood. Because of its frequent expression in tumors and its spontaneous immunogenicity, NY-ESO-1 is a prime target of cancer vaccines and an ideal model antigen for elucidating the molecular basis of immunodominant tumor-specific CD8+ T cell responses. Here, we have assessed CD8+T cell responses to full-length NY-ESO-1 in cancer patients. We identified 3 immunodominant regions of the protein located within 3 distinct clusters of MHC class I binding sequences that co-localize with previously defined clusters of MHC class II binding sequences, are predicted to be hydrophobic and undergo efficient proteasomal processing. Our results support the concept that epitope clustering within defined protein regions identifies tumor antigen immunodominant regions and suggest a general strategy for their identification. [Copyright &y& Elsevier]

Abstract: Because of its expression pattern restricted to cells of the melanocytic lineage and to melanoma cells, Melan-A is an important target of immunotherapeutic approaches for the treatment of melanoma. Identification of Melan-A derived sequences recognized by specific T cells is therefore of great interest for the development of these therapeutic strategies. Using circulating CD4+ T cells from healthy donors, we identified two Melan-A-derived CD4+ T cell epitopes mapping to the 1–20 and 91–110 regions of the protein and restricted by HLA-DR11 and HLA-DR52 molecules, respectively. CD4+ T cells specific for the identified epitopes were able to recognize the native antigen when endogenously expressed by antigen presenting cells and tumor cells. In addition, CD4+ T cells specific for Melan-A 91–110 recognized the epitope after exogenous processing and presentation of Melan-A recombinant protein. Identification of these epitopes will be instrumental for the evaluation of the immune response to Melan-A in cancer patients. [Copyright &y& Elsevier]

CD4+CD25+ Tregs play a central role in the maintenance of peripheral self tolerance by keeping autoreactive T cells in check. Whereas the thymic origin of CD4+CD25+ Tregs, as a distinct lineage, has been inferred, understanding of their developmental pathways has remained elusive. In both mice and humans, peripheral CD4+CD25+ Treg populations have been described as composed of antigen-experienced T cells that fail to significantly proliferate following TCR stimulation but suppress proliferation and effector functions of CD25- T cells. Here we show that analysis of CD25 expression in human circulating CD4+ T lymphocytes with respect to their in vivo differentiation stages identifies a distinct subset of CD25+CCR7+CD62L+CTLA-4+FOXP3+ cells contained in the CD45RA+/RO- naive fraction. The subset, which we have named natural naive Tregs (NnTregs), is prominent in young adults and decreases with age together with the total naive CD4+ population. NnTregs are anergic following stimulation in the absence of IL-2 and exert ex vivo cell-cell contact-mediated suppressor functions. In addition, they proliferate in response to stimulation with autologous APCs, which indicates a high enrichment in T cells bearing self-reactive TCRs. The definition of this subset has important implications for the analysis of human naturally occurring Tregs and for their targeting in therapeutic immune interventions. [ABSTRACT FROM AUTHOR]