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1.
Nat Immunol ; 19(8): 809-820, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29967452

RESUMO

Regulatory factor X 7 (Rfx7) is an uncharacterized transcription factor belonging to a family involved in ciliogenesis and immunity. Here, we found that deletion of Rfx7 leads to a decrease in natural killer (NK) cell maintenance and immunity in vivo. Genomic approaches showed that Rfx7 coordinated a transcriptional network controlling cell metabolism. Rfx7-/- NK lymphocytes presented increased size, granularity, proliferation, and energetic state, whereas genetic reduction of mTOR activity mitigated those defects. Notably, Rfx7-deficient NK lymphocytes were rescued by interleukin 15 through engagement of the Janus kinase (Jak) pathway, thus revealing the importance of this signaling for maintenance of such spontaneously activated NK cells. Rfx7 therefore emerges as a novel transcriptional regulator of NK cell homeostasis and metabolic quiescence.


Assuntos
Interleucina-15/metabolismo , Células Matadoras Naturais/metabolismo , Fator Regulador X1/metabolismo , Animais , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Quimera , Metabolismo Energético , Redes Reguladoras de Genes , Imunidade Celular/genética , Imunidade Inata/genética , Janus Quinases/metabolismo , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator Regulador X1/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
2.
EMBO Rep ; 23(11): e55399, 2022 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-36194675

RESUMO

Anticancer T cells acquire a dysfunctional state characterized by poor effector function and expression of inhibitory receptors, such as PD-1. Blockade of PD-1 leads to T cell reinvigoration and is increasingly applied as an effective anticancer treatment. Recent work challenged the commonly held view that the phosphatase PTPN11 (known as SHP-2) is essential for PD-1 signaling in T cells, suggesting functional redundancy with the homologous phosphatase PTPN6 (SHP-1). Therefore, we investigated the effect of concomitant Ptpn6 and Ptpn11 deletion in T cells on their ability to mount antitumour responses. In vivo data show that neither sustained nor acute Ptpn6/11 deletion improves T cell-mediated tumor control. Sustained loss of Ptpn6/11 also impairs the therapeutic effects of anti-PD1 treatment. In vitro results show that Ptpn6/11-deleted CD8+ T cells exhibit impaired expansion due to a survival defect and proteomics analyses reveal substantial alterations, including in apoptosis-related pathways. These data indicate that concomitant ablation of Ptpn6/11 in polyclonal T cells fails to improve their anticancer properties, implying that caution shall be taken when considering their inhibition for immunotherapeutic approaches.


Assuntos
Linfócitos T CD8-Positivos , Receptor de Morte Celular Programada 1 , Linfócitos T CD8-Positivos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Transdução de Sinais
3.
J Immunol ; 196(7): 2939-46, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26944927

RESUMO

NLRC5, a member of the NOD-like receptor (NLR) protein family, has recently been characterized as the master transcriptional regulator of MHCI molecules in lymphocytes, in which it is highly expressed. However, its role in activated dendritic cells (DCs), which are instrumental to initiate T cell responses, remained elusive. We show in this study that, following stimulation of DCs with inflammatory stimuli, not only did NLRC5 level increase, but also its importance in directing MHCI transcription. Despite markedly reduced mRNA and intracellular H2-K levels, we unexpectedly observed nearly normal H2-K surface display in Nlrc5(-/-) DCs. Importantly, this discrepancy between a strong intracellular and a mild surface defect in H2-K levels was observed also in DCs with H2-K transcription defects independent of Nlrc5. Hence, alongside with demonstrating the importance of NLRC5 in MHCI transcription in activated DCs, we uncover a general mechanism counteracting low MHCI surface expression. In agreement with the decreased amount of neosynthesized MHCI, Nlrc5(-/-) DCs exhibited a defective capacity to display endogenous Ags. However, neither T cell priming by endogenous Ags nor cross-priming ability was substantially affected in activated Nlrc5(-/-) DCs. Altogether, these data show that Nlrc5 deficiency, despite significantly affecting MHCI transcription and Ag display, is not sufficient to hinder T cell activation, underlining the robustness of the T cell priming process by activated DCs.


Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Animais , Apresentação de Antígeno/imunologia , Linhagem Celular , Membrana Celular/metabolismo , Apresentação Cruzada/imunologia , Regulação da Expressão Gênica , Ativação Linfocitária/genética , Camundongos , Camundongos Knockout , Linfócitos T/metabolismo , Transcrição Gênica
4.
PLoS Genet ; 11(3): e1005088, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25811463

RESUMO

MHC class II (MHCII) genes are transactivated by the NOD-like receptor (NLR) family member CIITA, which is recruited to SXY enhancers of MHCII promoters via a DNA-binding "enhanceosome" complex. NLRC5, another NLR protein, was recently found to control transcription of MHC class I (MHCI) genes. However, detailed understanding of NLRC5's target gene specificity and mechanism of action remained lacking. We performed ChIP-sequencing experiments to gain comprehensive information on NLRC5-regulated genes. In addition to classical MHCI genes, we exclusively identified novel targets encoding non-classical MHCI molecules having important functions in immunity and tolerance. ChIP-sequencing performed with Rfx5(-/-) cells, which lack the pivotal enhanceosome factor RFX5, demonstrated its strict requirement for NLRC5 recruitment. Accordingly, Rfx5-knockout mice phenocopy Nlrc5 deficiency with respect to defective MHCI expression. Analysis of B cell lines lacking RFX5, RFXAP, or RFXANK further corroborated the importance of the enhanceosome for MHCI expression. Although recruited by common DNA-binding factors, CIITA and NLRC5 exhibit non-redundant functions, shown here using double-deficient Nlrc5(-/-)CIIta(-/-) mice. These paradoxical findings were resolved by using a "de novo" motif-discovery approach showing that the SXY consensus sequence occupied by NLRC5 in vivo diverges significantly from that occupied by CIITA. These sequence differences were sufficient to determine preferential occupation and transactivation by NLRC5 or CIITA, respectively, and the S box was found to be the essential feature conferring NLRC5 specificity. These results broaden our knowledge on the transcriptional activities of NLRC5 and CIITA, revealing their dependence on shared enhanceosome factors but their recruitment to distinct enhancer motifs in vivo. Furthermore, we demonstrated selectivity of NLRC5 for genes encoding MHCI or related proteins, rendering it an attractive target for therapeutic intervention. NLRC5 and CIITA thus emerge as paradigms for a novel class of transcriptional regulators dedicated for transactivating extremely few, phylogenetically related genes.


Assuntos
Genes MHC da Classe II , Genes MHC Classe I , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Transativadores/genética , Ativação Transcricional/genética , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Genoma , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas Nucleares/biossíntese , Proteínas Nucleares/imunologia , Regiões Promotoras Genéticas , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transativadores/biossíntese , Transativadores/imunologia , Ativação Transcricional/imunologia
5.
Sci Signal ; 16(780): eabq0752, 2023 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-37040441

RESUMO

Natural killer (NK) cells recognize virally infected cells and tumors. NK cell function depends on balanced signaling from activating receptors, recognizing products from tumors or viruses, and inhibitory receptors (such as KIR/Ly49), which recognize major histocompatibility complex class I (MHC-I) molecules. KIR/Ly49 signaling preserves tolerance to self but also conveys reactivity toward MHC-I-low target cells in a process known as NK cell education. Here, we found that NK cell tolerance and education were determined by the subcellular localization of the tyrosine phosphatase SHP-1. In mice lacking MHC-I molecules, uneducated, self-tolerant Ly49A+ NK cells showed accumulation of SHP-1 in the activating immune synapse, where it colocalized with F-actin and the signaling adaptor protein SLP-76. Education of Ly49A+ NK cells by the MHC-I molecule H2Dd led to reduced synaptic accumulation of SHP-1, accompanied by augmented signaling from activating receptors. Education was also linked to reduced transcription of Ptpn6, which encodes SHP-1. Moreover, synaptic SHP-1 accumulation was reduced in NK cells carrying the H2Dd-educated receptor Ly49G2 but not in those carrying the noneducating receptor Ly49I. Colocalization of Ly49A and SHP-1 outside of the synapse was more frequent in educated compared with uneducated NK cells, suggesting a role for Ly49A in preventing synaptic SHP-1 accumulation in NK cell education. Thus, distinct patterning of SHP-1 in the activating NK cell synapse may determine NK cell tolerance.


Assuntos
Antígenos Ly , Células Matadoras Naturais , Camundongos , Animais , Receptores Semelhantes a Lectina de Células NK/metabolismo , Antígenos Ly/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Sinapses/metabolismo
6.
J Exp Med ; 218(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33156338

RESUMO

Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in the tumor microenvironment (TME) for improved responses. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we describe optimized methods for the efficient retroviral transduction and expansion of murine T lymphocytes of a predominantly central memory T cell (TCM cell) phenotype. We present a bicistronic retroviral vector encoding both a tumor vasculature-targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages. The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in immunocompetent mice, an important step toward the acceleration of effective therapies reaching the clinic.


Assuntos
Interleucina-15/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Engenharia Genética/métodos , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Memória Imunológica/genética , Memória Imunológica/imunologia , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
7.
J Immunother Cancer ; 9(8)2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34389616

RESUMO

BACKGROUND: The adoptive transfer of chimeric antigen receptor (CAR)-T cells has emerged as a potent immunotherapy against some hematological malignancies but not yet for epithelial-derived solid tumors. One critical issue is the paucity of broadly expressed solid tumor antigens (TAs), and another is the presence of suppressive mechanisms in the tumor microenvironment (TME) that can impair CAR-T cell homing, extravasation and effector functions. TAs expressed by endothelial cells of the tumor vasculature are of clinical interest for CAR therapy because of their genomic stability and accessibility to circulating T cells, as well as their expression across multiple tumor types. In this study, we sought to explore limitations to the efficacy of second-generation (2G) murine CAR-T cells redirected against the vascular endothelial growth factor receptor-2 (VEGFR-2) with the well-characterized single-chain variable fragment DC101. METHODS: Primary murine T cells were retrovirally transduced to express a 2G anti-VEGFR-2-CAR, and the in vitro binding to VEGFR-2, as well as reactivity against TA-expressing cells, was evaluated in the absence versus presence of exogenous VEGF-A. The CAR-T cells were further tested in vivo for tumor control alone and in combination with anti-VEGF-A antibody. Finally, we performed ex vivo phenotypic analyses of tumor-infiltrating CAR-T cells for the two treatment groups. RESULTS: In line with previous reports, we observed poor control of B16 melanoma by the 2G anti-VEGFR-2 CAR-T cells as a monotherapy. We further showed that VEGFR-2 is not downregulated by B16 melanoma tumors post treatment, but that its soluble ligand VEGF-A is upregulated and furthermore competes in vitro with the CAR-T cells for binding to VEGFR-2. This competition resulted in impaired CAR-T cell adhesion and effector function in vitro that could be restored in the presence of anti-VEGF-A antibody. Finally, we demonstrated that coadministration of anti-VEGF-A antibody in vivo promoted CAR-T cell persistence and tumor control and was associated with reduced frequencies of PD-1+ Ki67- and LAG-3+ Ki67- CAR-T cells in the TME. CONCLUSIONS: This study represents the first example of impaired function of a vasculature-targeted CAR by an angiogenic ligand and rationalizes the use of combinatorial therapies that target the tumor vasculature and augment CAR-T cell effector function.


Assuntos
Neoplasias/imunologia , Receptores de Antígenos Quiméricos/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Humanos , Camundongos
8.
Sci Adv ; 7(29)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34272244

RESUMO

The mechanisms maintaining adult lymphatic vascular specialization throughout life and their role in coordinating inter-organ communication to sustain homeostasis remain elusive. We report that inactivation of the mechanosensitive transcription factor Foxc2 in adult lymphatic endothelium leads to a stepwise intestine-to-lung systemic failure. Foxc2 loss compromised the gut epithelial barrier, promoted dysbiosis and bacterial translocation to peripheral lymph nodes, and increased circulating levels of purine metabolites and angiopoietin-2. Commensal microbiota depletion dampened systemic pro-inflammatory cytokine levels, corrected intestinal lymphatic dysfunction, and improved survival. Foxc2 loss skewed the specialization of lymphatic endothelial subsets, leading to populations with mixed, pro-fibrotic identities and to emergence of lymph node-like endothelial cells. Our study uncovers a cross-talk between lymphatic vascular function and commensal microbiota, provides single-cell atlas of lymphatic endothelial subtypes, and reveals organ-specific and systemic effects of dysfunctional lymphatics. These effects potentially contribute to the pathogenesis of diseases, such as inflammatory bowel disease, cancer, or lymphedema.


Assuntos
Vasos Linfáticos , Linfedema , Células Endoteliais/metabolismo , Endotélio Linfático/metabolismo , Endotélio Linfático/patologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Vasos Linfáticos/metabolismo , Linfedema/metabolismo , Linfedema/patologia
9.
Nat Commun ; 10(1): 1444, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926899

RESUMO

The phosphatase Shp-2 was implicated in NK cell development and functions due to its interaction with NK inhibitory receptors, but its exact role in NK cells is still unclear. Here we show, using mice conditionally deficient for Shp-2 in the NK lineage, that NK cell development and responsiveness are largely unaffected. Instead, we find that Shp-2 serves mainly to enforce NK cell responses to activation by IL-15 and IL-2. Shp-2-deficient NK cells have reduced proliferation and survival when treated with high dose IL-15 or IL-2. Mechanistically, Shp-2 deficiency hampers acute IL-15 stimulation-induced raise in glycolytic and respiration rates, and causes a dramatic defect in ERK activation. Moreover, inhibition of the ERK and mTOR cascades largely phenocopies the defect observed in the absence of Shp-2. Together, our data reveal a critical function of Shp-2 as a molecular nexus bridging acute IL-15 signaling with downstream metabolic burst and NK cell expansion.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Matadoras Naturais/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Receptores de Interleucina-15/metabolismo , Animais , Antígenos Ly/metabolismo , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Integrases/metabolismo , Interleucina-15/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muromegalovirus/fisiologia , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/deficiência , Serina-Treonina Quinases TOR/metabolismo
10.
Cell Rep ; 23(1): 39-49, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29617671

RESUMO

In chronic infection and cancer, T cells acquire a dysfunctional state characterized by the expression of inhibitory receptors. In vitro studies implicated the phosphatase Shp-2 downstream of these receptors, including PD-1. However, whether Shp-2 is responsible in vivo for such dysfunctional responses remains elusive. To address this, we generated T cell-specific Shp-2-deficient mice. These mice did not show differences in controlling chronic viral infections. In this context, Shp-2-deleted CD8+ T lymphocytes expanded moderately better but were less polyfunctional than control cells. Mice with Shp-2-deficient T cells also showed no significant improvement in controlling immunogenic tumors and responded similarly to controls to α-PD-1 treatment. We therefore showed that Shp-2 is dispensable in T cells for globally establishing exhaustion and for PD-1 signaling in vivo. These results reveal the existence of redundant mechanisms downstream of inhibitory receptors and represent the foundation for defining these relevant molecular events.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias Experimentais/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Transdução de Sinais , Viroses/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/imunologia
11.
Nat Commun ; 7: 10554, 2016 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-26861112

RESUMO

NLRC5 is a transcriptional regulator of MHC class I (MHCI), which maintains high MHCI expression particularly in T cells. Recent evidence highlights an important NK-T-cell crosstalk, raising the question on whether NLRC5 specifically modulates this interaction. Here we show that NK cells from Nlrc5-deficient mice exhibit moderate alterations in inhibitory receptor expression and responsiveness. Interestingly, NLRC5 expression in T cells is required to protect them from NK-cell-mediated elimination upon inflammation. Using T-cell-specific Nlrc5-deficient mice, we show that NK cells surprisingly break tolerance even towards 'self' Nlrc5-deficient T cells under inflammatory conditions. Furthermore, during chronic LCMV infection, the total CD8(+) T-cell population is severely decreased in these mice, a phenotype reverted by NK-cell depletion. These findings strongly suggest that endogenous T cells with low MHCI expression become NK-cell targets, having thus important implications for T-cell responses in naturally or therapeutically induced inflammatory conditions.


Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Células Matadoras Naturais/imunologia , Tolerância a Antígenos Próprios/imunologia , Linfócitos T/imunologia , Animais , Animais Congênicos , Infecções por Arenaviridae/imunologia , Chlorocebus aethiops , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Indutores de Interferon/toxicidade , Peptídeos e Proteínas de Sinalização Intracelular/genética , Vírus da Coriomeningite Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Poli I-C/toxicidade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/citologia , Baço/imunologia , Linfócitos T/efeitos dos fármacos , Células Vero
12.
Curr Opin Microbiol ; 16(3): 296-302, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23659869

RESUMO

Pattern recognition receptors (PRRs) are commonly known as sensor proteins crucial for the early detection of microbial or host-derived stress signals by innate immune cells. Interestingly, some PRRs are also expressed and functional in cells of the adaptive immune system. These receptors provide lymphocytes with innate sensing abilities; for example, B cells express Toll-like receptors, which are important for the humoral response. Strikingly, certain other NOD-like receptors are not only highly expressed in adaptive immune cells, but also exert functions related specifically to adaptive immune system pathways, such as regulating antigen presentation. In this review, we will focus particularly on the current understanding of PRR functions intrinsic to B and T lymphocytes; a developing aspect of PRR biology.


Assuntos
Imunidade Adaptativa , Linfócitos B/imunologia , Imunidade Inata , Receptores de Reconhecimento de Padrão/metabolismo , Linfócitos T/imunologia , Animais , Humanos
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