RESUMO
T cells infiltrating neoplasms express surface molecules typical of chronically virus-stimulated T cells, often termed "exhausted" T cells. We compared the transcriptome of "exhausted" CD8 T cells infiltrating autochthonous melanomas to those of naïve and acutely stimulated CD8 T cells. Despite strong similarities between transcriptional signatures of tumor- and virus-induced exhausted CD8 T cells, notable differences appeared. Among transcriptional regulators, Nr4a2 and Maf were highly overexpressed in tumor-exhausted T cells and significantly upregulated in CD8 T cells from human melanoma metastases. Transduction of murine tumor-specific CD8 T cells to express Maf partially reproduced the transcriptional program associated with tumor-induced exhaustion. Upon adoptive transfer, the transduced cells showed normal homeostasis but failed to accumulate in tumor-bearing hosts and developed defective anti-tumor effector responses. We further identified TGFß and IL-6 as main inducers of Maf expression in CD8 T cells and showed that Maf-deleted tumor-specific CD8 T cells were much more potent to restrain tumor growth in vivo. Therefore, the melanoma microenvironment contributes to skewing of CD8 T cell differentiation programs, in part by TGFß/IL-6-mediated induction of Maf.
Assuntos
Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/imunologia , Melanoma/metabolismo , Proteínas Proto-Oncogênicas c-maf/metabolismo , Microambiente Tumoral/fisiologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Primers do DNA/genética , Citometria de Fluxo , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Interleucina-6/metabolismo , Luciferases , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-maf/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/metabolismoRESUMO
Cancer-germline genes in both humans and mice have been shown to encode antigens susceptible to targeting by cytotoxic CD8 T effector cells (CTL). We analysed the ability of CTL to kill different tumour cell lines expressing the same cancer-germline gene P1A (Trap1a). We previously demonstrated that CTL expressing a T-cell receptor specific for the P1A35-43 peptide associated with H-2Ld , although able to induce regression of P1A-expressing P815 mastocytoma cells, were much less effective against P1A-expressing melanoma cells. Here, we analysed parameters of the in vitro interaction between P1A-specific CTL and mastocytoma or melanoma cells expressing similar levels of the P1A gene and of surface H-2Ld . The mastocytoma cells were more sensitive to cytolysis than the melanoma cells in vitro. Analysis by video-microscopy of early events required for target cell killing showed that similar patterns of increase in cytoplasmic Ca2+ concentration ([Ca2+ ]i) were induced by both types of P1A-expressing tumour cells. However, the use of CTL expressing a fluorescent granzyme B (GZMB-Tom) showed a delay in the migration of cytotoxic granules to the tumour interaction site, as well as a partially deficient GZMB-Tom exocytosis in response to the melanoma cells. Among surface molecules possibly affecting tumour-CTL interactions, the mastocytoma cells were found to express intercellular adhesion molecule-1, the ligand for LFA-1, which was not detected on the melanoma cells.
Assuntos
Antígenos de Neoplasias/metabolismo , Exocitose , Mastocitoma/imunologia , Melanoma/imunologia , Fragmentos de Peptídeos/metabolismo , Vesículas Secretórias/metabolismo , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Neoplasias/genética , Sinalização do Cálcio , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Antígeno de Histocompatibilidade H-2D/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
The transcription factor NF-κB is central to inflammatory signaling and activation of innate and adaptive immune responses. Activation of the NF-κB pathway is tightly controlled by several negative feedback mechanisms, including A20, an ubiquitin-modifying enzyme encoded by the tnfaip3 gene. Mice with selective deletion of A20 in myeloid, dendritic, or B cells recapitulate some human inflammatory pathology. As we observed high expression of A20 transcripts in dysfunctional CD8 T cells in an autochthonous melanoma, we analyzed the role of A20 in regulation of CD8 T-cell functions, using mice in which A20 was selectively deleted in mature conventional T cells. These mice developed lymphadenopathy and some organ infiltration by T cells but no splenomegaly and no detectable pathology. A20-deleted CD8 T cells had increased sensitivity to antigen stimulation with production of large amounts of IL-2 and IFNγ, correlated with sustained nuclear expression of NF-κB components reticuloendotheliosis oncogene c-Rel and p65. Overexpression of A20 by retroviral transduction of CD8 T cells dampened their intratumor accumulation and antitumor activity. In contrast, relief from the A20 brake in NF-κB activation in adoptively transferred antitumor CD8 T cells led to improved control of melanoma growth. Tumor-infiltrating A20-deleted CD8 T cells had enhanced production of IFNγ and TNFα and reduced expression of the inhibitory receptor programmed cell death 1. As manipulation of A20 expression in CD8 T cells did not result in pathologic manifestations in the mice, we propose it as a candidate to be targeted to increase antitumor efficiency of adoptive T-cell immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Cisteína Endopeptidases/imunologia , Imunidade Celular , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Melanoma/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/patologia , Cisteína Endopeptidases/genética , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Melanoma/genética , Melanoma/patologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
In adoptive therapy, CD8 T cells expressing active STAT5 (STAT5CA) transcription factors were found to be superior to unmanipulated counterparts in long-term persistence, capacity to infiltrate autochthonous mouse melanomas, thrive in their microenvironment, and induce their regression. However, the molecular mechanisms sustaining these properties were undefined. In this study, we report that STAT5CA induced sustained expression of genes controlling tissue homing, cytolytic granule composition, type 1 CD8 cytotoxic T cell-associated effector molecules granzyme B(+), IFN-γ(+), TNF-α(+), and CCL3(+), but not IL-2, and transcription factors T-bet and eomesodermin (Eomes). Chromatin immunoprecipitation sequencing analyses identified the genes possessing regulatory regions to which STAT5 bound in long-term in vivo maintained STAT5CA-expressing CD8 T cells. This analysis identified 34% of the genes differentially expressed between STAT5CA-expressing and nonexpressing effector T cells as direct STAT5CA target genes, including those encoding T-bet, Eomes, and granzyme B. Additionally, genes encoding the IL-6R and TGFbRII subunits were stably repressed, resulting in dampened IL-17-producing CD8 T cell polarization in response to IL-6 and TGF-ß1. The absence of T-bet did not affect STAT5CA-driven accumulation of the T cells in tissue or their granzyme B expression but restored IL-2 secretion and IL-6R and TGFbRII expression and signaling, as illustrated by IL-17 induction. Therefore, concerted STAT5/T-bet/Eomes regulation controls homing, long-term maintenance, recall responses, and resistance to polarization towards IL-17-producing CD8 T cells while maintaining expression of an efficient type 1 CD8 cytotoxic T cell program (granzyme B(+), IFN-γ(+)).
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Interleucina-6/metabolismo , Fator de Transcrição STAT5/metabolismo , Proteínas com Domínio T/genética , Fator de Crescimento Transformador beta1/metabolismo , Animais , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Interleucina-17/metabolismo , Camundongos , Camundongos Transgênicos , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Fator de Transcrição STAT5/genética , Transdução de Sinais , Proteínas com Domínio T/deficiência , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Transcrição GênicaRESUMO
Central tolerance toward tissue-restricted Ags is considered to rely on ectopic expression in the thymus, which was also observed for tumor Ags encoded by cancer-germline genes. It is unknown whether endogenous expression shapes the T cell repertoire against the latter Ags and explains their weak immunogenicity. We addressed this question using mouse cancer-germline gene P1A, which encodes antigenic peptide P1A(35-43) presented by H-2L(d). We made P1A-knockout (P1A-KO) mice and asked whether their anti-P1A(35-43) immune responses were stronger than those of wild-type mice and whether P1A-KO mice responded to other P1A epitopes, against which wild-type mice were tolerized. We observed that both types of mice mounted similar P1A(35-43)-specific CD8 T cell responses, although the frequency of P1A(35-43)-specific CD8 T cells generated in response to P1A-expressing tumors was slightly higher in P1A-KO mice. This higher reactivity allowed naive P1A-KO mice to reject spontaneously P1A-expressing tumors, which progressed in wild-type mice. TCR-Vß usage of P1A(35-43)-specific CD8 cells was slightly modified in P1A-KO mice. Peptide P1A(35-43) remained the only P1A epitope recognized by CD8 T cells in both types of mice, which also displayed similar thymic selection of a transgenic TCR recognizing P1A(35-43). These results indicate the existence of a minimal tolerance to an Ag encoded by a cancer-germline gene and suggest that its endogenous expression only slightly affects diversification of the T cell repertoire against this Ag.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Tolerância Imunológica , Neoplasias/imunologia , Peptídeos/imunologia , Animais , Antígenos de Neoplasias/genética , Linhagem Celular Tumoral , Epitopos/genética , Camundongos , Camundongos Knockout , Neoplasias/genética , Peptídeos/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologiaRESUMO
In mice expressing a transgenic T-cell receptor (TCR; TCRP1A) of DBA/2 origin with reactivity towards a cancer-germline antigen P1A, the number of TCRP1A CD8+ T cells in lymphoid organs is lower in DBA/2 than in B10.D2 or B10.D2(x DBA/2)F1 mice. This reduction results from haemopoietic cell autonomous differences in the differentiation of the major histocompatibility complex class I-restricted TCRP1A thymocytes controlled by DBA/2 versus B10.D2-encoded genes. We report here that the lower number of TCRP1A CD8+ T cells in DBA/2 mice correlated with their poor resistance to P1A-expressing mastocytoma solid tumours. Functional potency of CD8+ cytolytic T lymphocytes (CTL) from the above strains was not compromised, but their number after expansion appeared to be influenced by their genetic background. Intriguingly, non-transgenic DBA/ 2 mice resisted P1A+ tumours more efficiently despite poor representation of P1A-specific CTL. This was partly the result of their more heterogeneous TCR repertoire, including reactivity to non-P1A tumour antigens because mice that had rejected a P1A+ tumour became resistant to a P1A) variant of the tumour. Such 'cross-resistance' did not develop in the TCRP1A transgenic mice. Nonetheless, reconstitution of RAGo/o mice with TCRP1A CD8+ T cells, with or without CD4+ T cells, or exclusive representation of TCRP1A CD8+ T cells in RAGo/o TCRP1A transgenic mice efficiently resisted the growth of P1A-expressing tumours. Natural killer cells present at a higher number in RAGo/o mice also contributed to tumour resistance, in part through an NKG2D-dependent mechanism. Hence, in the absence of a polyclonal T-cell repertoire, precursor frequencies of natural killer cells and tumour-specific CTL affect tumour resistance.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Contagem de Células , Células Matadoras Naturais/metabolismo , Mastocitoma/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Transferência Adotiva , Animais , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Processos de Crescimento Celular , Deleção Clonal/genética , Imunidade Celular , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Cooperação Linfocítica , Mastocitoma/patologia , Camundongos , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Transplante de Neoplasias , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T/genéticaRESUMO
Tumors with reduced expression of MHC class I (MHC-I) molecules may be unrecognized by tumor antigen-specific CD8+ T cells and thus constitute a challenge for cancer immunotherapy. Here we monitored development of autochthonous melanomas in TiRP mice that develop tumors expressing a known tumor antigen as well as a red fluorescent protein (RFP) reporter knock in gene. The latter permits non-invasive monitoring of tumor growth by biofluorescence. One developing melanoma was deficient in cell surface expression of MHC-I, but MHC-I expression could be rescued by exposure of these cells to IFNγ. We show that CD8+ T cells specific for tumor antigen/MHC-I were efficient at inducing regression of the MHC-I-deficient melanoma, provided that the T cells were endowed with properties permitting their migration into the tumor and their efficient production of IFNγ. This was the case for CD8+ T cells transfected to express an active form of STAT5 (STAT5CA). The amount of IFNγ produced ex vivo from T cells present in tumors after adoptive transfer of the CD8+ T cells was correlated with an increase in surface expression of MHC-I molecules by the tumor cells. We also show that these CD8+ T cells expressed PD-1 and upregulated its ligand PDL-1 on melanoma cells within the tumor. Despite upregulation of this immunosuppressive pathway, efficient IFNγ production in the melanoma microenvironment was found associated with resistance of STAT5CA-expressing CD8+ T cells to inhibition both by PD-1/PDL-1 engagement and by TGFß1, two main immune regulatory mechanisms hampering the efficiency of immunotherapy in patients.
RESUMO
Discovery of tumor antigen (TA) recognized by autologous T cells (TCs) in patients with melanoma has led to clinical protocols using either vaccination or adoptive transfer of TA-specific TCs. However, efficacy of these treatments has been hampered by inhibitory effects exerted on tumor-infiltrating TCs by tumor-intrinsic mediators or by recruitment of immunosuppressive cells. A mouse model of autochthonous melanoma recapitulates some aspects of inflammatory melanoma development in patients. These include a systemic Th2-/Th17-oriented chronic inflammation, recruitment of immunosuppressive myeloid cells and acquisition by tumor-infiltrating TCs of an 'exhausted' phenotype characterized by expression of multiple inhibitory receptors including programmed death-1, also expressed on patients' melanoma-infiltrating TCs. Rather than using extracellular blocking reagents to inhibitory surface molecules on TCs, we sought to dampen negative signaling exerted on them. Adoptively transferred TCs presenting increased cytokine receptor signaling due to expression of an active Stat5 transcription factor were efficient at inducing melanoma regression in the preclinical melanoma model. These transferred TCs thrived and retained expression of effector molecules in the melanoma microenvironment, defining a protocol endowing TCs with the ability to resist melanoma-induced immunosuppression.
Assuntos
Transferência Adotiva , Terapia de Imunossupressão , Inflamação/patologia , Melanoma/imunologia , Melanoma/terapia , Linfócitos T/imunologia , Linfócitos T/patologia , Imunidade Adaptativa/imunologia , Animais , Antígenos de Neoplasias/imunologia , Humanos , Imunoterapia Adotiva , Linfócitos do Interstício Tumoral/metabolismo , Melanoma/patologia , Camundongos , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapiaRESUMO
Immunotherapy based on adoptive transfer of tumor antigen-specific CD8(+) T cell (TC) is generally limited by poor in vivo expansion and tumor infiltration. In this study, we report that activated STAT5 transcription factors (STAT5CA) confer high efficiency on CD8(+) effector T cells (eTC) for host colonization after adoptive transfer. Engineered expression of STAT5CA in antigen-experienced TCs with poor replicative potential was also sufficient to convert them into long-lived antigen-responsive eTCs. In transplanted mastocytoma- or melanoma-bearing hosts, STAT5CA greatly enhanced the ability of eTCs to accumulate in tumors, become activated by tumor antigens, and to express the cytolytic factor granzyme B. Taken together, these properties contributed to an increase in tumor regression by STAT5CA-transduced, as compared with untransduced, TCs including when the latter control cells were combined with infusion of interleukin (IL)-2/anti-IL-2 complexes. In tumors arising in the autochthonous TiRP transgenic model of melanoma associated with systemic chronic inflammation, endogenous CD8(+) TCs were nonfunctional. In this setting, adoptive transfer of STAT5CA-transduced TCs produced superior antitumor effects compared with nontransduced TCs. Our findings imply that STAT5CA expression can render TCs resistant to the immunosuppressive environment of melanoma tumors, enhancing their ability to home to tumors and to maintain high granzyme B expression, as well as their capacity to stimulate granzyme B expression in endogenous TCs.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Melanoma/fisiopatologia , Fator de Transcrição STAT5/fisiologia , Transferência Adotiva , Animais , Citometria de Fluxo , Melanoma/patologia , Melanoma/terapia , Camundongos , Transplante HeterólogoRESUMO
Innate immunity is considered to initiate adaptive antitumor responses. We demonstrate that monoclonal CD8 T lymphocytes reactive to tumor Ag P1A on P815 mastocytoma cells provide essential "help" to NK cells for rejection of P1A-deficient tumors. RAG-deficient mice have normal NK cells but do not reject either tumor. Reconstitution of these mice with P1A-specific T cells conferred resistance to both P1A-expressing and -deficient tumor cells provided they were present at the same site. Elimination of Ag-negative tumor variants required both activated T and NK cells. Gene expression profiling of NK cells infiltrating P1A-positive tumors in mice with specific CD8 T cells demonstrated an activated effector phenotype. However, CD8 T cell help to NK cells appeared ineffective for P1A-negative variants separated from the P1A-positive tumor. Local tumor Ag-specific T cell-NK cell collaboration results in the elimination of tumor cells whether they express or not the T cell tumor Ag epitope, thus containing the emergence of tumor escape variants before metastasis.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas de Homeodomínio/imunologia , Imunidade Inata , Células Matadoras Naturais/imunologia , Sarcoma de Mastócitos/imunologia , Animais , Antígenos de Neoplasias/genética , Linfócitos T CD8-Positivos/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/imunologia , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Proteínas de Homeodomínio/genética , Imunidade Inata/genética , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Sarcoma de Mastócitos/genética , Camundongos , Camundongos Mutantes , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Evasão Tumoral/imunologiaRESUMO
CD8(+) cytotoxic T lymphocyte (CTL) clones are able to exert both perforin- and Fas-dependent cytotoxicity. We show in the present work that phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 prevent TCR/CD3-induced functional Fas ligand (FasL) expression, but not perforin-dependent cytotoxicity. The specific inhibitor of classical protein kinase C (PKC) isoforms, Gö6976, completely inhibited perforin-dependent cytotoxicity and only affected slightly TCR/CD3-induced FasL expression, while the opposite was observed using rottlerin, an inhibitor with higher specificity for PKCtheta. To address further the dependence of FasL expression on PI3K, a luciferase reporter controlled by the FasL promoter was used. Reporter gene induction by anti-CD3 mAb was abolished in cells transfected with dominant-negative PI3K (PI3K-DN) and increased in cells transfected with constitutively active PI3K (PI3K*). Transfection with constitutively active mutants (A/E) of PKCepsilon, and especially of PKCtheta, improved anti-CD3 mAb-induced reporter expression and completely abolished inhibition by wortmannin, while transfection with dominant-negative (K/R) PKCtheta prevented the induction of the reporter. Finally, transfection with PKCalpha A/E, but not with PKCtheta A/E, cooperated with ionomycin to induce degranulation in the CTL line 1.3E6SN. Altogether, the results suggest that TCR/CD3-induced FasL gene transcription is controlled by PI3K and PKCtheta activation, while this signaling pathway is not implicated in CTL degranulation, which is rather dependent on the activation of classical PKC isoforms.