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[ROR[gamma]t.sup.+] [T.sub.H]17 cells are a proinflammatory [CD4.sup.+] T-cell population associated with autoimmune tissue injury. In mice, priming of [T.sub.H]17 requires TGF-[beta], which alone directs the priming of [FOXP3.sup.+] regulatory T cells (Treg), in association with inflammatory cytokines. Priming of human [T.sub.H]17 cells from conventional naive [CD4.sup.+] T cells under similar conditions, however, has proved difficult to achieve. Here, we report that differentiation of human [T.sub.H]17 cells preferentially occurs from [FOXP3.sup.+] naive Treg (NTreg) in the presence of IL-2 and IL-1[beta] and is increased by IL-23 and TGF-[beta]. IL-1[beta]-mediated differentiation correlated with IL-1RI expression in stimulated NTreg and was accompanied by induction of [ROR[gamma]t along with down-regulation of FOXP3. IL-17-secreting cells in NTreg cultures cosecreted TNF-[alpha] and IL-2 and contained distinct subpopulations cosecreting or not cosecreting IFN-[gamma] and other [T.sub.H]17-associated cytokines. Polarized NTreg contained significant subpopulations of CCR6-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 [T.sub.H]17 from naive [CD4.sup.+] T cells preferentially takes place from [FOXP3.sup.+] Treg precursors in the presence of lineage-specific polarizing factors. IL-1 | IL-17 | IL-23 | TGF-[beta] | CD4 T cells doi/ 10.1073/pnas.1008247107

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

IL-17-producing CD4+ Th (Th17) cells are a unique subset of proinflammatory cells expressing the retinoic acid-related orphan receptor t and associated with different forms of inflammatory autoimmune pathologies. The development of Th17 cells, mediated by TGF-ß and IL-1, is closely related to that of FOXP3+ suppressor/regulatory T cells (Treg). In this study, we report that ex vivo expression of IL-1RI in human circulating CD4+ T cells identifies a subpopulation of FOXP3+ Treg that coexpress retinoic acid-related orphan receptor t, secrete IL-17, and are highly enriched among CCR7+ central memory cells. Consistent with the concept that IL-1RI expression in Treg identifies a subpopulation at an early stage of differentiation, we show that, in Th17 populations differentiated in vitro from natural naive FOXP3+ Treg, IL-1RI+ IL-17-secreting cells are central memory cells, whereas IL-1RI- cells secreting IL-17 are effector memory cells. Together with the absence of detectable IL-1RI and IL-17 expression in resting naive CD4+ T cells, these data identify circulating CCR7+ Treg expressing IL-1RI ex vivo as early intermediates along an IL-1-controlled differentiation pathway leading from naive FOXP3+ Treg to Th17 effectors. We further show that, whereas IL-1RI+ central memory Treg respond to stimulation in the presence of IL-1 by generating IL-17-secreting effectors, a significant fraction of them maintain FOXP3 expression, consistent with an important role of this population in maintaining the Treg/Th17 memory pool in vivo. [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]

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]

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 ESO-specific CD4(+) T cells in EOC patients with spontaneous immune responses to the antigen are prevalently T(H)1 type cells secreting IFN-γ but no IL-17 or IL-10 and are not suppressive. We detected tetramer(+) cells ex vivo, at an average frequency of 1:25,000 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 T(H)1 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.