RESUMO
Foxp3+ T regulatory (Treg) cells promote immunological tumor tolerance, but how their immune-suppressive function is regulated in the tumor microenvironment (TME) remains unknown. Here, we used intravital microscopy to characterize the cellular interactions that provide tumor-infiltrating Treg cells with critical activation signals. We found that the polyclonal Treg cell repertoire is pre-enriched to recognize antigens presented by tumor-associated conventional dendritic cells (cDCs). Unstable cDC contacts sufficed to sustain Treg cell function, whereas T helper cells were activated during stable interactions. Contact instability resulted from CTLA-4-dependent downregulation of co-stimulatory B7-family proteins on cDCs, mediated by Treg cells themselves. CTLA-4-blockade triggered CD28-dependent Treg cell hyper-proliferation in the TME, and concomitant Treg cell inactivation was required to achieve tumor rejection. Therefore, Treg cells self-regulate through a CTLA-4- and CD28-dependent feedback loop that adjusts their population size to the amount of local co-stimulation. Its disruption through CTLA-4-blockade may off-set therapeutic benefits in cancer patients.
Assuntos
Antígeno CTLA-4/metabolismo , Retroalimentação Fisiológica , Neoplasias/imunologia , Linfócitos T Reguladores/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos CD28/metabolismo , Proliferação de Células , Células Dendríticas/imunologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia , Interleucina-2/metabolismo , Ligantes , Linfonodos/metabolismo , Ativação Linfocitária/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/metabolismo , Neoplasias/patologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Microambiente TumoralRESUMO
Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses.
Assuntos
Receptores CXCR6/metabolismo , Linfócitos T Citotóxicos/imunologia , Microambiente Tumoral , Animais , Antígeno B7-H1/metabolismo , Comunicação Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Quimiocina CXCL16 , Células Dendríticas/metabolismo , Interleucina-12/metabolismo , Interleucina-15/metabolismo , Ligantes , Linfonodos/metabolismo , Melanoma/imunologia , Melanoma/patologia , Camundongos Endogâmicos C57BLRESUMO
Solid tumours are infiltrated by effector T cells with the potential to control or reject them, as well as by regulatory T (Treg) cells that restrict the function of effector T cells and thereby promote tumour growth1. The anti-tumour activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of some forms of human cancer. However, weak tumour-associated inflammatory responses and the immune-suppressive function of Treg cells remain major hurdles to broader effectiveness of tumour immunotherapy2. Here we show that, after disruption of the CARMA1-BCL10-MALT1 (CBM) signalosome complex, most tumour-infiltrating Treg cells produce IFNγ, resulting in stunted tumour growth. Notably, genetic deletion of both or even just one allele of CARMA1 (also known as Card11) in only a fraction of Treg cells-which avoided systemic autoimmunity-was sufficient to produce this anti-tumour effect, showing that it is not the mere loss of suppressive function but the gain of effector activity by Treg cells that initiates tumour control. The production of IFNγ by Treg cells was accompanied by activation of macrophages and upregulation of class I molecules of the major histocompatibility complex on tumour cells. However, tumour cells also upregulated the expression of PD-L1, which indicates activation of adaptive immune resistance3. Consequently, blockade of PD-1 together with CARMA1 deletion caused rejection of tumours that otherwise do not respond to anti-PD-1 monotherapy. This effect was reproduced by pharmacological inhibition of the CBM protein MALT1. Our results demonstrate that partial disruption of the CBM complex and induction of IFNγ secretion in the preferentially self-reactive Treg cell pool does not cause systemic autoimmunity but is sufficient to prime the tumour environment for successful immune checkpoint therapy.
Assuntos
Proteína 10 de Linfoma CCL de Células B/antagonistas & inibidores , Antígeno B7-H1/antagonistas & inibidores , Proteínas Adaptadoras de Sinalização CARD/antagonistas & inibidores , Imunoterapia/métodos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Complexos Multiproteicos/antagonistas & inibidores , Neoplasias/terapia , Linfócitos T Reguladores/imunologia , Animais , Autoimunidade , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Feminino , Tolerância Imunológica , Interferon gama/biossíntese , Interferon gama/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Neoplasias/imunologia , Neoplasias/patologiaRESUMO
Interactions with antigen-presenting cells (APCs) interrupt T cell migration through tissues and trigger signaling pathways that converge on the activation of transcriptional regulators, including nuclear factor of activated T cells (NFAT), which control T cell function and differentiation. Both stable and unstable modes of cognate T cell-APC interactions have been observed in vivo, but the functional significance of unstable, serial contacts has remained unclear. Here we used multiphoton intravital microscopy in lymph nodes and tumors to show that while NFAT nuclear import was fast (t(1/2 max)â¼1 min), nuclear export was slow (t(1/2)â¼20 min) in T cells. During delayed export, nuclear NFAT constituted a short-term imprint of transient TCR signals and remained transcriptionally active for the T cell tolerance gene Egr2, but not for the effector gene Ifng, which required continuous TCR triggering for expression. This provides a potential mechanistic basis for the observation that a predominance of unstable APC interactions correlates with the induction of T cell tolerance.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Tolerância Imunológica , Memória Imunológica , Linfonodos/metabolismo , Fatores de Transcrição NFATC/genética , Linfócitos T/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/patologia , Comunicação Celular , Diferenciação Celular , Movimento Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/imunologia , Regulação da Expressão Gênica , Humanos , Interferon gama/genética , Interferon gama/imunologia , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica , Fatores de Transcrição NFATC/imunologia , Transporte Proteico , Receptores de Antígenos de Linfócitos T , Transdução de Sinais , Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Cellular immunotherapies for cancer represent a means by which a patient's immune system can be augmented with high numbers of tumor-specific T cells. Chimeric antigen receptor (CAR) therapy involves genetic engineering to 'redirect' peripheral T cells to tumor targets, showing remarkable potency in blood cancers. However, due to several resistance mechanisms, CAR-T cell therapies remain ineffective in solid tumors. We and others have shown the tumor microenvironment harbors a distinct metabolic landscape that produces a barrier to immune cell function. Further, altered differentiation of T cells within tumors induces defects in mitochondrial biogenesis, resulting in severe cell-intrinsic metabolic deficiencies. While we and others have shown murine T cell receptor (TCR)-transgenic cells can be improved through enhanced mitochondrial biogenesis, we sought to determine whether human CAR-T cells could be enabled through a metabolic reprogramming approach. MATERIALS AND METHODS: Anti-EGFR CAR-T cells were infused in NSG mice which bore A549 tumors. The tumor infiltrating lymphocytes were analyzed for exhaustion and metabolic deficiencies. Lentiviruses carrying PPAR-gamma coactivator 1α (PGC-1α), PGC-1αS571A and NT-PGC-1α constructs were used to co-transduce T cells with anti-EGFR CAR lentiviruses. We performed metabolic analysis via flow cytometry and Seahorse analysis in vitro as well as RNA sequencing. Finally, we treated therapeutically A549-carrying NSG mice with either PGC-1α or NT-PGC-1α anti-EGFR CAR-T cells. We also analyzed the differences in the tumor-infiltrating CAR-T cells when PGC-1α is co-expressed. RESULTS: Here, in this study, we show that an inhibition resistant, engineered version of PGC-1α, can metabolically reprogram human CAR-T cells. Transcriptomic profiling of PGC-1α-transduced CAR-T cells showed this approach effectively induced mitochondrial biogenesis, but also upregulated programs associated with effector functions. Treatment of immunodeficient animals bearing human solid tumors with these cells resulted in substantially improved in vivo efficacy. In contrast, a truncated version of PGC-1α, NT-PGC-1α, did not improve the in vivo outcomes. CONCLUSIONS: Our data further support a role for metabolic reprogramming in immunomodulatory treatments and highlight the utility of genes like PGC-1α as attractive candidates to include in cargo along with chimeric receptors or TCRs for cell therapy of solid tumors.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Animais , Camundongos , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T , Linfócitos T , Microambiente TumoralRESUMO
Productive T cell activation requires efficient reorganization of the actin cytoskeleton. We showed previously that the actin-regulatory protein, hematopoietic lineage cell-specific protein 1 (HS1), is required for the stabilization of F-actin and Vav1 at the immunological synapse and for efficient calcium responses. The Tec family kinase IL-2-inducible T cell kinase (Itk) regulates similar aspects of T cell activation, suggesting that these proteins act in the same pathway. Using video microscopy, we show that T cells lacking Itk or HS1 exhibited similar defects in actin responses, extending unstable lamellipodial protrusions upon TCR stimulation. HS1 and Itk could be coimmunoprecipitated from T cell lysates, and GST-pulldown studies showed that Itk's Src homology 2 domain binds directly to two phosphotyrosines in HS1. In the absence of Itk, or in T cells overexpressing an Itk Src homology 2 domain mutant, HS1 failed to localize to the immunological synapse, indicating that Itk serves to recruit HS1 to sites of TCR engagement. Because Itk is required for phospholipase C (PLC)gamma1 phosphorylation and calcium store release, we examined the calcium signaling pathway in HS1(-/-) T cells in greater detail. In response to TCR engagement, T cells lacking HS1 exhibited diminished calcium store release, but TCR-dependent PLCgamma1 phosphorylation was intact, indicating that HS1's role in calcium signaling is distinct from that of Itk. HS1-deficient T cells exhibited defective cytoskeletal association of PLCgamma1 and altered formation of PLCgamma1 microclusters. We conclude that HS1 functions as an effector of Itk in the T cell actin-regulatory pathway, and directs the spatial organization of PLCgamma1 signaling complexes.
Assuntos
Fator Estimulador de Colônias de Granulócitos/imunologia , Sinapses Imunológicas/imunologia , Fosfolipase C gama/imunologia , Proteínas Tirosina Quinases/imunologia , Linfócitos T/imunologia , Actinas/metabolismo , Animais , Western Blotting , Sinalização do Cálcio/imunologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Sinapses Imunológicas/metabolismo , Imunoprecipitação , Células Jurkat , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Fosfolipase C gama/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Pseudópodes/metabolismo , Pseudópodes/patologia , Interferência de RNA , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , TransfecçãoRESUMO
BACKGROUND: Organisms that are sensitive to nitrofurantoin express a nitroreductase. Since bacterial resistance to this compound results primarily from mutations in the gene encoding nitroreductase, the resulting loss of function of nitroreductase results in a selectable phenotype; resistance to nitrofurantoin. We exploited this direct selection for mutation to study the frequency at which spontaneous mutations arise (transitions and transversions, insertions and deletions). RESULTS: A nitroreductase- encoding gene was identified in the N. gonorrhoeae FA1090 genome by using a bioinformatic search with the deduced amino acid sequence derived from the Escherichia coli nitroreductase gene, nfsB. Cell extracts from N. gonorrhoeae were shown to possess nitroreductase activity, and activity was shown to be the result of NfsB. Spontaneous nitrofurantoin-resistant mutants arose at a frequency of approximatelty 3 x 10(-6) - 8 x 10(-8) among the various strains tested. The nfsB sequence was amplified from various nitrofurantoin-resistant mutants, and the nature of the mutations determined. Transition, transversion, insertion and deletion mutations were all readily detectable with this reporter gene. CONCLUSION: We found that nfsB is a useful reporter gene for measuring spontaneous mutation frequencies. Furthermore, we found that mutations were more likely to arise in homopolymeric runs rather than as base substitutions.
Assuntos
Genes Reporter/genética , Mutação/genética , Neisseria gonorrhoeae/enzimologia , Neisseria gonorrhoeae/genética , Nitrorredutases/genética , Nitrorredutases/metabolismo , Sequência de Aminoácidos , Anti-Infecciosos Urinários/farmacologia , Farmacorresistência Bacteriana/genética , Dados de Sequência Molecular , Neisseria gonorrhoeae/efeitos dos fármacos , Nitrofurantoína/farmacologia , Nitrorredutases/químicaRESUMO
Epithelial resident memory T (eTRM) cells serve as sentinels in barrier tissues to guard against previously encountered pathogens. How eTRM cells are generated has important implications for efforts to elicit their formation through vaccination or prevent it in autoimmune disease. Here, we show that during immune homeostasis, the cytokine transforming growth factor ß (TGF-ß) epigenetically conditions resting naïve CD8+ T cells and prepares them for the formation of eTRM cells in a mouse model of skin vaccination. Naïve T cell conditioning occurs in lymph nodes (LNs), but not in the spleen, through major histocompatibility complex class I-dependent interactions with peripheral tissue-derived migratory dendritic cells (DCs) and depends on DC expression of TGF-ß-activating αV integrins. Thus, the preimmune T cell repertoire is actively conditioned for a specialized memory differentiation fate through signals restricted to LNs.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Memória Imunológica , Fator de Crescimento Transformador beta/metabolismo , Animais , Movimento Celular , Epiderme/imunologia , Integrina alfaV/genética , Integrina alfaV/metabolismo , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pele/imunologiaRESUMO
BACKGROUND: To understand the ability of gliomas to manipulate their microenvironment, we visualized the transfer of vesicles and the effects of tumor-released extracellular RNA on the phenotype of microglia in culture and in vivo. METHODS: Extracellular vesicles (EVs) released from primary human glioblastoma (GBM) cells were isolated and microRNAs (miRNAs) were analyzed. Primary mouse microglia were exposed to GBM-EVs, and their uptake and effect on proliferation and levels of specific miRNAs, mRNAs, and proteins were analyzed. For in vivo analysis, mouse glioma cells were implanted in the brains of mice, and EV release and uptake by microglia and monocytes/macrophages were monitored by intravital 2-photon microscopy, immunohistochemistry, and fluorescence activated cell sorting analysis, as well as RNA and protein levels. RESULTS: Microglia avidly took up GBM-EVs, leading to increased proliferation and shifting of their cytokine profile toward immune suppression. High levels of miR-451/miR-21 in GBM-EVs were transferred to microglia with a decrease in the miR-451/miR-21 target c-Myc mRNA. In in vivo analysis, we directly visualized release of EVs from glioma cells and their uptake by microglia and monocytes/macrophages in brain. Dissociated microglia and monocytes/macrophages from tumor-bearing brains revealed increased levels of miR-21 and reduced levels of c-Myc mRNA. CONCLUSIONS: Intravital microscopy confirms the release of EVs from gliomas and their uptake into microglia and monocytes/macrophages within the brain. Our studies also support functional effects of GBM-released EVs following uptake into microglia, associated in part with increased miRNA levels, decreased target mRNAs, and encoded proteins, presumably as a means for the tumor to manipulate its environs.
Assuntos
Neoplasias Encefálicas/metabolismo , Vesículas Extracelulares/metabolismo , Glioblastoma/metabolismo , Macrófagos/metabolismo , MicroRNAs/metabolismo , Microglia/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
Tregs control various functions of effector T cells; however, where and how Tregs exert their immunomodulatory effects remain poorly understood. Here we developed a murine model of adoptive T cell therapy and found that Tregs induce a dysfunctional state in tumor-infiltrating CTLs that resembles T cell exhaustion and is characterized by low expression of effector cytokines, inefficient cytotoxic granule release, and coexpression of coinhibitory receptors PD-1 and TIM-3. Induction of CTL dysfunction was an active process, requiring local TCR signals in tumor tissue. Tregs infiltrated tumors only subsequent to Ag-dependent activation and expansion in tumor-draining LNs; however, Tregs also required local Ag reencounter within tumor tissue to induce CTL dysfunction and prevent tumor rejection. Multiphoton intravital microscopy revealed that in contrast to CTLs, Tregs only rarely and briefly interrupted their migration in tumor tissue in an Ag-dependent manner and formed unstable tethering-interactions with CD11c+ APCs, coinciding with a marked reduction of CD80 and CD86 on APCs. Activation of CTLs by Treg-conditioned CD80/86lo DCs promoted enhanced expression of both TIM-3 and PD-1. Based on these data, we propose that Tregs locally change the costimulatory landscape in tumor tissue through transient, Ag-dependent interactions with APCs, thus inducing CTL dysfunction by altering the balance of costimulatory and coinhibitory signals these cells receive.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/imunologia , Imunidade Adaptativa , Animais , Antígenos de Neoplasias , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Imunoterapia Adotiva , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Modelos Imunológicos , Transdução de Sinais/imunologia , Evasão TumoralRESUMO
We found that hematopoietic cell-specific Lyn substrate 1 (HCLS1 or HS1) is highly expressed in human myeloid cells and that stimulation with granulocyte colony-stimulating factor (G-CSF) leads to HCLS1 phosphorylation. HCLS1 binds the transcription factor lymphoid-enhancer binding factor 1 (LEF-1), transporting LEF-1 into the nucleus upon G-CSF stimulation and inducing LEF-1 autoregulation. In patients with severe congenital neutropenia, inherited mutations in the gene encoding HCLS1-associated protein X-1 (HAX1) lead to profound defects in G-CSF-triggered phosphorylation of HCLS1 and subsequently to reduced autoregulation and expression of LEF-1. Consistent with these results, HCLS1-deficient mice are neutropenic. In bone marrow biopsies of the majority of tested patients with acute myeloid leukemia, HCLS1 protein expression is substantially elevated, associated with high levels of G-CSF synthesis and, in some individuals, a four-residue insertion in a proline-rich region of HCLS1 protein known to accelerate intracellular signaling. These data demonstrate the importance of HCLS1 in myelopoiesis in vitro and in vivo.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Sanguíneas/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Mielopoese , Neutropenia/congênito , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sequência de Bases , Proteínas Sanguíneas/genética , Linhagem Celular , Proliferação de Células , Síndrome Congênita de Insuficiência da Medula Óssea , Feminino , Granulócitos/metabolismo , Células HEK293 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Mielopoese/genética , Neutropenia/genética , Neutropenia/fisiopatologia , Fosforilação , Interferência de RNA , RNA Interferente Pequeno , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Análise de Sequência de DNA , Transdução de SinaisRESUMO
T cell cytoarchitecture differs dramatically depending on whether the cell is circulating within the bloodstream, migrating through tissues, or interacting with antigen-presenting cells. The transition between these states requires important signaling-dependent changes in actin cytoskeletal dynamics. Recently, analysis of actin-regulatory proteins associated with T cell activation has provided new insights into how T cells control actin dynamics in response to external stimuli and how actin facilitates downstream signaling events and effector functions. Among the actin-regulatory proteins that have been identified are nucleation-promoting factors such as WASp, WAVE2, and HS1; severing proteins such as cofilin; motor proteins such as myosin II; and linker proteins such as ezrin and moesin. We review the current literature on how signaling pathways leading from diverse cell surface receptors regulate the coordinated activity of these and other actin-regulatory proteins and how these proteins control T cell function.
Assuntos
Actinas/metabolismo , Citoesqueleto/fisiologia , Ativação Linfocitária , Linfócitos T/fisiologia , Animais , Polaridade Celular/fisiologia , Humanos , Modelos Biológicos , Receptores Imunológicos/fisiologia , Transdução de Sinais/fisiologia , Linfócitos T/citologiaRESUMO
HS1, the leukocyte-specific homolog of cortactin, regulates F-actin in vitro and is phosphorylated in response to TCR ligation, but its role in lymphocyte activation has not been addressed. We demonstrate that HS1-deficient T cells fail to accumulate F-actin at the immune synapse (IS) and, upon TCR ligation, form actin-rich structures that are disordered and unstable. Early TCR activation events are intact in these cells, but Ca2+ influx and IL-2 gene transcription are defective. Importantly, HS1 tyrosine phosphorylation is required for its targeting to the IS and for its function in regulating actin dynamics and IL-2 promoter activity. Phosphorylation also links HS1 to multiple signaling proteins, including Lck, PLCgamma1, and Vav1, and is essential for the stable recruitment of Vav1 to the IS. Taken together, our studies show that HS1 is indispensable for signaling events leading to actin assembly and IL-2 production during T cell activation.