RESUMO
Chimeric antigen receptor T cells are an effective therapy for B-lineage malignancies. However, many patients relapse and this therapeutic has yet to show strong efficacy in other hematologic or solid tumors. One opportunity for improvement lies in the ability to generate T cells with desirable functional characteristics. Here, we dissect the biology of CD8+ CAR T cells (CAR8) by controlling whether the T cell has encountered cognate TCR antigen prior to CAR generation. We find that prior antigen experience influences multiple aspects of in vitro and in vivo CAR8 functionality, resulting in superior effector function and leukemia clearance in the setting of limiting target antigen density compared to antigen-inexperienced T cells. However, this comes at the expense of inferior proliferative capacity, susceptibility to phenotypic exhaustion and dysfunction, and inability to clear wildtype leukemia in the setting of limiting CAR+ cell dose. Epigenomic and transcriptomic comparisons of these cell populations identified overexpression of the Runx2 transcription factor as a novel strategy to enhance CAR8 function, with a differential impact depending on prior cell state. Collectively, our data demonstrate that prior antigen experience determines functional attributes of a CAR T cell, as well as amenability to functional enhancement by transcription factor modulation.
RESUMO
Adoptive T cell therapies (ACTs) hold great promise in cancer treatment, but low overall response rates in patients with solid tumors underscore remaining challenges in realizing the potential of this cellular immunotherapy approach. Promoting CD8+ T cell adaptation to tissue residency represents an underutilized but promising strategy to improve tumor-infiltrating lymphocyte (TIL) function. Here, we report that deletion of the HIF negative regulator von Hippel-Lindau (VHL) in CD8+ T cells induced HIF-1α/HIF-2α-dependent differentiation of tissue-resident memory-like (Trm-like) TILs in mouse models of malignancy. VHL-deficient TILs accumulated in tumors and exhibited a core Trm signature despite an exhaustion-associated phenotype, which led to retained polyfunctionality and response to αPD-1 immunotherapy, resulting in tumor eradication and protective tissue-resident memory. VHL deficiency similarly facilitated enhanced accumulation of chimeric antigen receptor (CAR) T cells with a Trm-like phenotype in tumors. Thus, HIF activity in CD8+ TILs promotes accumulation and antitumor activity, providing a new strategy to enhance the efficacy of ACTs.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Linfócitos T CD8-Positivos/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Imunidade Celular , Memória Imunológica , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Experimentais/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Linfócitos do Interstício Tumoral/patologia , Camundongos , Camundongos Knockout , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/imunologiaRESUMO
T cells expressing chimeric antigen receptors (CAR T cells) targeting human CD19 (hCD19) have shown clinical efficacy against B cell malignancies1,2. CAR T cells have been less effective against solid tumours3-5, in part because they enter a hyporesponsive ('exhausted' or 'dysfunctional') state6-9 triggered by chronic antigen stimulation and characterized by upregulation of inhibitory receptors and loss of effector function. To investigate the function of CAR T cells in solid tumours, we transferred hCD19-reactive CAR T cells into hCD19+ tumour-bearing mice. CD8+CAR+ tumour-infiltrating lymphocytes and CD8+ endogenous tumour-infiltrating lymphocytes expressing the inhibitory receptors PD-1 and TIM3 exhibited similar profiles of gene expression and chromatin accessibility, associated with secondary activation of nuclear receptor transcription factors NR4A1 (also known as NUR77), NR4A2 (NURR1) and NR4A3 (NOR1) by the initiating transcription factor NFAT (nuclear factor of activated T cells)10-12. CD8+ T cells from humans with cancer or chronic viral infections13-15 expressed high levels of NR4A transcription factors and displayed enrichment of NR4A-binding motifs in accessible chromatin regions. CAR T cells lacking all three NR4A transcription factors (Nr4a triple knockout) promoted tumour regression and prolonged the survival of tumour-bearing mice. Nr4a triple knockout CAR tumour-infiltrating lymphocytes displayed phenotypes and gene expression profiles characteristic of CD8+ effector T cells, and chromatin regions uniquely accessible in Nr4a triple knockout CAR tumour-infiltrating lymphocytes compared to wild type were enriched for binding motifs for NF-κB and AP-1, transcription factors involved in activation of T cells. We identify NR4A transcription factors as having an important role in the cell-intrinsic program of T cell hyporesponsiveness and point to NR4A inhibition as a promising strategy for cancer immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias/genética , Neoplasias/imunologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Fatores de Transcrição/metabolismo , Transferência Adotiva , Animais , Antígenos CD19/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Neoplasias/patologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/deficiência , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/deficiência , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Receptores de Esteroides/deficiência , Receptores de Esteroides/metabolismo , Receptores dos Hormônios Tireóideos/deficiência , Receptores dos Hormônios Tireóideos/metabolismo , Taxa de Sobrevida , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/deficiênciaRESUMO
The ten-eleven-translocation (TET) proteins oxidize 5-methylcytosine in DNA. Alterations in TET protein function have been linked to cancer, but TETs have also been observed to influence many cell differentiation processes. Here we review recent work assessing the contribution of TET proteins to natural and induced differentiation. Altogether these analyses have helped characterize how TETs and their enzymatic products influence DNA methylation patterns, regulatory element activity, DNA binding protein specificity and gene expression.
Assuntos
Diferenciação Celular/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética , Proteínas Proto-Oncogênicas/genética , 5-Metilcitosina/metabolismo , Dioxigenases , Humanos , Família Multigênica/genéticaRESUMO
T-cell exhaustion is a progressive loss of effector function and memory potential due to persistent antigen exposure, which occurs in chronic viral infections and cancer. Here we investigate the relation between gene expression and chromatin accessibility in CD8+ tumor-infiltrating lymphocytes (TILs) that recognize a model tumor antigen and have features of both activation and functional exhaustion. By filtering out accessible regions observed in bystander, nonexhausted TILs and in acutely restimulated CD8+ T cells, we define a pattern of chromatin accessibility specific for T-cell exhaustion, characterized by enrichment for consensus binding motifs for Nr4a and NFAT transcription factors. Anti-PD-L1 treatment of tumor-bearing mice results in cessation of tumor growth and partial rescue of cytokine production by the dysfunctional TILs, with only limited changes in gene expression and chromatin accessibility. Our studies provide a valuable resource for the molecular understanding of T-cell exhaustion in cancer and other inflammatory settings.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Cromatina/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/imunologia , Sequências Reguladoras de Ácido NucleicoRESUMO
TET proteins oxidize 5-methylcytosine in DNA to 5-hydroxymethylcytosine and other oxidation products. We found that simultaneous deletion of Tet2 and Tet3 in mouse CD4+CD8+ double-positive thymocytes resulted in dysregulated development and proliferation of invariant natural killer T cells (iNKT cells). Tet2-Tet3 double-knockout (DKO) iNKT cells displayed pronounced skewing toward the NKT17 lineage, with increased DNA methylation and impaired expression of genes encoding the key lineage-specifying factors T-bet and ThPOK. Transfer of purified Tet2-Tet3 DKO iNKT cells into immunocompetent recipient mice resulted in an uncontrolled expansion that was dependent on the nonclassical major histocompatibility complex (MHC) protein CD1d, which presents lipid antigens to iNKT cells. Our data indicate that TET proteins regulate iNKT cell fate by ensuring their proper development and maturation and by suppressing aberrant proliferation mediated by the T cell antigen receptor (TCR).
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Células T Matadoras Naturais/fisiologia , Células Precursoras de Linfócitos T/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Antígenos CD1d/genética , Antígenos CD1d/metabolismo , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Dioxigenases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In response to acute infection, naive CD8+ T cells expand, differentiate into effector cells, and then contract to a long-lived pool of memory cells after pathogen clearance. During chronic infections or in tumors, CD8+ T cells acquire an "exhausted" phenotype. Here we present genome-wide comparisons of chromatin accessibility and gene expression from endogenous CD8+ T cells responding to acute and chronic viral infection using ATAC-seq and RNA-seq techniques. Acquisition of effector, memory, or exhausted phenotypes was associated with stable changes in chromatin accessibility away from the naive T cell state. Regions differentially accessible between functional subsets in vivo were enriched for binding sites of transcription factors known to regulate these subsets, including E2A, BATF, IRF4, T-bet, and TCF1. Exhaustion-specific accessible regions were enriched for consensus binding sites for NFAT and Nr4a family members, indicating that chronic stimulation confers a unique accessibility profile on exhausted cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Montagem e Desmontagem da Cromatina/imunologia , Expressão Gênica/imunologia , Memória Imunológica/imunologia , Ativação Linfocitária/imunologia , Animais , Infecções por Arenaviridae/imunologia , Cromatina , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Vírus da Coriomeningite Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The differentiation of helper T cells into effector subsets is critical to host protection. Transcription factors of the E-protein and Id families are important arbiters of T cell development, but their role in the differentiation of the TH1 and TFH subsets of helper T cells is not well understood. Here, TH1 cells showed more robust Id2 expression than that of TFH cells, and depletion of Id2 via RNA-mediated interference increased the frequency of TFH cells. Furthermore, TH1 differentiation was blocked by Id2 deficiency, which led to E-protein-dependent accumulation of effector cells with mixed characteristics during viral infection and severely impaired the generation of TH1 cells following infection with Toxoplasma gondii. The TFH cell-defining transcriptional repressor Bcl6 bound the Id2 locus, which provides a mechanism for the bimodal Id2 expression and reciprocal development of TH1 cells and TFH cells.
Assuntos
Infecções por Arenaviridae/imunologia , Diferenciação Celular , Proteína 2 Inibidora de Diferenciação/metabolismo , Vírus da Coriomeningite Linfocítica/imunologia , Células Th1/fisiologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Cultivadas , Feminino , Centro Germinativo/imunologia , Proteína 2 Inibidora de Diferenciação/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , RNA Interferente Pequeno/genética , Células Th1/parasitologia , Células Th1/virologiaRESUMO
We have hypothesized that in the prenegative selection TCR repertoire, many somatically generated complementary-determining region (CDR) 3 loops combine with evolutionarily selected germline Valpha/Vbeta CDR1/CDR2 loops to create highly MHC/peptide cross-reactive T cells that are subsequently deleted by negative selection. Here, we present a mutational analysis of the Vbeta CDR3 of such a cross-reactive T-cell receptor (TCR), YAe62. Most YAe62 TCRs with the mutant CDR3s became less MHC promiscuous. However, others with CDR3s unrelated in sequence to the original recognized even more MHC alleles than the original TCR. Most importantly, this recognition was still dependent on the conserved CDR1/CDR2 residues. These results bolster the idea that germline TCR V elements are inherently reactive to MHC but that this reactivity is fine-tuned by the somatically generated CDR3 loops.
Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Alelos , Animais , Regiões Determinantes de Complementaridade , Análise Mutacional de DNA , Citometria de Fluxo/métodos , Deleção de Genes , Mutação em Linhagem Germinativa , Interleucina-2/metabolismo , Ligantes , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos NOD , Conformação Molecular , Peptídeos/químicaRESUMO
An alphabeta T-cell response depends on the recognition of antigen plus major histocompatibility complex (MHC) proteins by its antigen receptor (TCR). The ability of peripheral alphabeta T cells to recognize MHC is at least partly determined by MHC-dependent thymic selection, by which an immature T cell survives only if its TCR can recognize self MHC. This process may allow MHC-reactive TCRs to be selected from a repertoire with completely random and unbiased specificities. However, analysis of thymocytes before positive selection indicated that TCR proteins might have a predetermined ability to bind MHC. Here we show that specific germline-encoded amino acids in the TCR promote 'generic' MHC recognition and control thymic selection. In mice expressing single, rearranged TCR beta-chains, individual mutation of amino acids in the complementarity-determining region (CDR) 2beta to Ala reduced development of the entire TCR repertoire. Altogether, these results show that thymic selection is controlled by germline-encoded MHC contact points in the alphabeta TCR and indicate that the diversity of the peripheral T-cell repertoire is enhanced by this 'built-in' specificity.
Assuntos
Aminoácidos/genética , Aminoácidos/metabolismo , Células Germinativas/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Timo/citologia , Timo/imunologia , Animais , Medula Óssea/imunologia , Medula Óssea/metabolismo , Linhagem Celular , Quimera/imunologia , Quimera/metabolismo , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Hibridomas/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T alfa-beta/deficiência , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
Mycobacterium tuberculosis (Mtb) frequently establishes persistent infections that may be facilitated by mechanisms that dampen immunity. T regulatory (T reg) cells, a subset of CD4(+) T cells that are essential for preventing autoimmunity, can also suppress antimicrobial immune responses. We use Foxp3-GFP mice to track the activity of T reg cells after aerosol infection with Mtb. We report that during tuberculosis, T reg cells proliferate in the pulmonary lymph nodes (pLNs), change their cell surface phenotype, and accumulate in the pLNs and lung at a rate parallel to the accumulation of effector T cells. In the Mtb-infected lung, T reg cells accumulate in high numbers in all sites where CD4(+) T cells are found, including perivascular/peribronchiolar regions and within lymphoid aggregates of granulomas. To determine the role of T reg cells in the immune response to tuberculosis, we generated mixed bone marrow chimeric mice in which all cells capable of expressing Foxp3 expressed Thy1.1. When T reg cells were depleted by administration of anti-Thy1.1 before aerosol infection with Mtb, we observed approximately 1 log less of colony-forming units of Mtb in the lungs. Thus, after aerosol infection, T reg cells proliferate and accumulate at sites of infection, and have the capacity to suppress immune responses that contribute to the control of Mtb.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Tuberculose/imunologia , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Movimento Celular , Proliferação de Células , Quimera , Contagem de Colônia Microbiana , Granuloma/imunologia , Granuloma/patologia , Interleucina-10/biossíntese , Pulmão/microbiologia , Pulmão/patologia , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/isolamento & purificação , Fenótipo , Regulação para Cima/genéticaRESUMO
A mouse model of gastrointestinal stromal tumor (GIST) has been developed by a knock-in gene targeting strategy, which introduced a Kit gene K641E mutation, originally identified in sporadic human GISTs and in the germ line of familial GIST syndrome patients. Homozygous and heterozygous Kit K641E mice develop gastrointestinal pathology with complete penetrance and all Kit K641E homozygotes die by age 30 weeks due to gastrointestinal obstruction by hyperplastic interstitial cells of Cajal (ICC) or GISTs. Heterozygous mice have less extensive ICC hyperplasia and smaller GISTs, suggesting a dose-response relationship between oncogenically activated Kit and ICC proliferation. Immunoprecipitation and Western blotting reveal GISTs to contain abundant phosphorylated/activated Kit. In addition to ICC hyperplasia and GISTs, homozygous Kit K641E mice exhibit loss-of-function Kit phenotypes, including white coat color, decreased numbers of dermal mast cells, and sterility, indicating that despite its oncogenic activity the mutant form cannot accomplish many activities of the wild-type gene. Kit K641E reproduces the pathology associated with the familial GIST syndrome and thus is an excellent model to study Kit pathway activation, ICC biology, GIST pathogenesis, and preclinical validations of GIST therapies and mechanisms of drug resistance.