RESUMO
T cells possess an array of functional capabilities important for host defense against pathogens and tumors. T cell effector functions require the T cell antigen receptor (TCR). The TCR has no intrinsic enzymatic activity, and thus signal transduction from the receptor relies on additional signaling molecules. One such molecule is the cytoplasmic tyrosine kinase ZAP-70, which associates with the TCR complex and is required for initiating the canonical biochemical signal pathways downstream of the TCR. In this article, we describe recent structure-based insights into the regulation and substrate specificity of ZAP-70, and then we review novel methods for determining the role of ZAP-70 catalytic activity-dependent and -independent signals in developing and mature T cells. Lastly, we discuss the disease states in mouse models and humans, which range from immunodeficiency to autoimmunity, that are caused by mutations in ZAP-70.
Assuntos
Suscetibilidade a Doenças , Transdução de Sinais , Linfócitos T/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Animais , Autoimunidade , Biomarcadores , Catálise , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Regulação da Expressão Gênica , Humanos , Imunidade , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Fosforilação , Transporte Proteico , Relação Estrutura-Atividade , Especificidade por Substrato , Linfócitos T/imunologia , Proteína-Tirosina Quinase ZAP-70/antagonistas & inibidores , Proteína-Tirosina Quinase ZAP-70/química , Proteína-Tirosina Quinase ZAP-70/genéticaRESUMO
KRAS mutant pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic response that promotes hypovascularity, immunosuppression, and resistance to chemo- and immunotherapies. We show that a combination of MEK and CDK4/6 inhibitors that target KRAS-directed oncogenic signaling can suppress PDAC proliferation through induction of retinoblastoma (RB) protein-mediated senescence. In preclinical mouse models of PDAC, this senescence-inducing therapy produces a senescence-associated secretory phenotype (SASP) that includes pro-angiogenic factors that promote tumor vascularization, which in turn enhances drug delivery and efficacy of cytotoxic gemcitabine chemotherapy. In addition, SASP-mediated endothelial cell activation stimulates the accumulation of CD8+ T cells into otherwise immunologically "cold" tumors, sensitizing tumors to PD-1 checkpoint blockade. Therefore, in PDAC models, therapy-induced senescence can establish emergent susceptibilities to otherwise ineffective chemo- and immunotherapies through SASP-dependent effects on the tumor vasculature and immune system.
Assuntos
Envelhecimento/fisiologia , Carcinoma Ductal Pancreático/patologia , Remodelação Vascular/fisiologia , Animais , Linfócitos T CD8-Positivos/imunologia , Carcinoma Ductal Pancreático/microbiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Genes ras/genética , Humanos , Imunoterapia/métodos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Neoplasias Pancreáticas/patologia , Proteína do Retinoblastoma/imunologia , Transdução de Sinais/genética , Microambiente Tumoral , Remodelação Vascular/genéticaRESUMO
The interaction of the tumor necrosis factor receptor (TNFR) family member CD27 on naive CD8+ T (Tn) cells with homotrimeric CD70 on antigen-presenting cells (APCs) is necessary for T cell memory fate determination. Here, we examined CD27 signaling during Tn cell activation and differentiation. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 by a synthetic trimeric CD70 ligand triggered CD27 internalization and degradation, suggesting active regulation of this signaling axis. Internalized CD27 recruited the signaling adaptor TRAF2 and the phosphatase SHP-1, thereby modulating TCR and CD28 signals. CD27-mediated modulation of TCR signals promoted transcription factor circuits that induced memory rather than effector associated gene programs, which are induced by CD28 costimulation. CD27-costimulated chimeric antigen receptor (CAR)-engineered T cells exhibited improved tumor control compared with CD28-costimulated CAR-T cells. Thus, CD27 signaling during Tn cell activation promotes memory properties with relevance to T cell immunotherapy.
Assuntos
Antígenos CD28 , Redes Reguladoras de Genes , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Antígenos CD28/metabolismo , Transdução de Sinais , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Ligante CD27/genética , Ligante CD27/metabolismo , Linfócitos T CD8-PositivosRESUMO
Knowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We profiled 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph nodes, using single-cell RNA-seq. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data. Despite significant similarity between normal and tumor tissue-resident immune cells, we observed continuous phenotypic expansions specific to the tumor microenvironment. Analysis of paired single-cell RNA and T cell receptor (TCR) sequencing data from 27,000 additional T cells revealed the combinatorial impact of TCR utilization on phenotypic diversity. Our results support a model of continuous activation in T cells and do not comport with the macrophage polarization model in cancer. Our results have important implications for characterizing tumor-infiltrating immune cells.
Assuntos
Neoplasias da Mama/imunologia , Regulação Neoplásica da Expressão Gênica , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Microambiente Tumoral/imunologia , Teorema de Bayes , Neoplasias da Mama/patologia , Análise por Conglomerados , Biologia Computacional , Feminino , Perfilação da Expressão Gênica , Humanos , Sistema Imunitário , Imunoterapia/métodos , Linfonodos , Linfócitos do Interstício Tumoral , Macrófagos/metabolismo , Fenótipo , TranscriptomaRESUMO
Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, single guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA sequencing (RNA-seq) revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.
Assuntos
Sistemas CRISPR-Cas , Genoma Humano , Linfócitos T/imunologia , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas de Inativação de Genes , Estudo de Associação Genômica Ampla , Humanos , Linfócitos T/citologiaRESUMO
Dendritic cells (DCs) patrol tissues and transport antigens to lymph nodes to initiate adaptive immune responses. Within tissues, DCs constitute a complex cell population composed of distinct subsets that can exhibit different activation states and functions. How tissue-specific cues orchestrate DC diversification remains elusive. Here, we show that the small intestine included two pools of cDC2s originating from common pre-DC precursors: (1) lamina propria (LP) CD103+CD11b+ cDC2s that were mature-like proinflammatory cells and (2) intraepithelial cDC2s that exhibited an immature-like phenotype as well as tolerogenic properties. These phenotypes resulted from the action of food-derived retinoic acid (ATRA), which enhanced actomyosin contractility and promoted LP cDC2 transmigration into the epithelium. There, cDC2s were imprinted by environmental cues, including ATRA itself and the mucus component Muc2. Hence, by reaching distinct subtissular niches, DCs can exist as immature and mature cells within the same tissue, revealing an additional mechanism of DC functional diversification.
Assuntos
Células Dendríticas/imunologia , Inflamação/imunologia , Mucosa Intestinal/patologia , Linfócitos T/imunologia , Actomiosina/metabolismo , Animais , Apresentação de Antígeno , Antígenos CD/metabolismo , Antígeno CD11b/metabolismo , Diferenciação Celular , Movimento Celular , Células Cultivadas , Tolerância Imunológica , Cadeias alfa de Integrinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucina-2/imunologia , Tretinoína/metabolismoRESUMO
Memory T cells are thought to rely on oxidative phosphorylation and short-lived effector T cells on glycolysis. Here, we investigated how T cells arrive at these states during an immune response. To understand the metabolic state of rare, early-activated T cells, we adapted mass cytometry to quantify metabolic regulators at single-cell resolution in parallel with cell signaling, proliferation, and effector function. We interrogated CD8+ T cell activation in vitro and in response to Listeria monocytogenes infection in vivo. This approach revealed a distinct metabolic state in early-activated T cells characterized by maximal expression of glycolytic and oxidative metabolic proteins. Cells in this transient state were most abundant 5 days post-infection before rapidly decreasing metabolic protein expression. Analogous findings were observed in chimeric antigen receptor (CAR) T cells interrogated longitudinally in advanced lymphoma patients. Our study demonstrates the utility of single-cell metabolic analysis by mass cytometry to identify metabolic adaptations of immune cell populations in vivo and provides a resource for investigations of metabolic regulation of immune responses across a variety of applications.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária/imunologia , Transdução de Sinais/imunologia , Animais , Proliferação de Células/fisiologia , Feminino , Glicólise/imunologia , Memória Imunológica/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação Oxidativa , Receptores de Antígenos Quiméricos/imunologia , Análise de Célula Única/métodosRESUMO
Through recurrent bouts synchronous with the hair cycle, quiescent melanocyte stem cells (McSCs) become activated to generate proliferative progeny that differentiate into pigment-producing melanocytes. The signaling factors orchestrating these events remain incompletely understood. Here, we use single-cell RNA sequencing with comparative gene expression analysis to elucidate the transcriptional dynamics of McSCs through quiescence, activation, and melanocyte maturation. Unearthing converging signs of increased WNT and BMP signaling along this progression, we endeavored to understand how these pathways are integrated. Employing conditional lineage-specific genetic ablation studies in mice, we found that loss of BMP signaling in the lineage leads to hair graying due to a block in melanocyte maturation. We show that interestingly, BMP signaling functions downstream from activated McSCs and maintains WNT effector, transcription factor LEF1. Employing pseudotime analysis, genetics, and chromatin landscaping, we show that following WNT-mediated activation of McSCs, BMP and WNT pathways collaborate to trigger the commitment of proliferative progeny by fueling LEF1- and MITF-dependent differentiation. Our findings shed light upon the signaling interplay and timing of cues that orchestrate melanocyte lineage progression in the hair follicle and underscore a key role for BMP signaling in driving complete differentiation.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Diferenciação Celular/genética , Melanócitos/citologia , Transdução de Sinais/genética , Células-Tronco/citologia , Animais , Linhagem da Célula/genética , Perfilação da Expressão Gênica , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia/metabolismo , Análise de Célula ÚnicaRESUMO
PKCß-null (Prkcb-/-) mice are severely immunodeficient. Here we show that mice whose B cells lack PKCß failed to form germinal centers and plasma cells, which undermined affinity maturation and antibody production in response to immunization. Moreover, these mice failed to develop plasma cells in response to viral infection. At the cellular level, we have shown that Prkcb-/- B cells exhibited defective antigen polarization and mTORC1 signaling. While altered antigen polarization impaired antigen presentation and likely restricted the potential of GC development, defective mTORC1 signaling impaired metabolic reprogramming, mitochondrial remodeling, and heme biosynthesis in these cells, which altogether overwhelmingly opposed plasma cell differentiation. Taken together, our study reveals mechanistic insights into the function of PKCß as a key regulator of B cell polarity and metabolic reprogramming that instructs B cell fate.
Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Ativação Linfocitária/imunologia , Plasmócitos/imunologia , Proteína Quinase C beta/imunologia , Animais , Heme/biossíntese , Camundongos , Camundongos Knockout , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Plasmócitos/citologiaRESUMO
Initial molecular details of cellular activation following αßT cell antigen receptor (TCR) ligation by peptide-major histocompatibility complexes (pMHC) remain unexplored. We determined the nuclear magnetic resonance (NMR) structure of the TCRα subunit transmembrane (TM) domain revealing a bipartite helix whose segmentation fosters dynamic movement. Positively charged TM residues Arg251 and Lys256 project from opposite faces of the helix, with Lys256 controlling immersion depth. Their modification caused stepwise reduction in TCR associations with CD3ζζ homodimers and CD3εγ plus CD3εδ heterodimers, respectively, leading to an activated transcriptome. Optical tweezers revealed that Arg251 and Lys256 mutations altered αßTCR-pMHC bond lifetimes, while mutations within interacting TCRα connecting peptide and CD3δ CxxC motif juxtamembrane elements selectively attenuated signal transduction. Our findings suggest that mechanical forces applied during pMHC ligation initiate T cell activation via a dissociative mechanism, shifting disposition of those basic sidechains to rearrange TCR complex membrane topology and weaken TCRαß and CD3 associations.
Assuntos
Complexo CD3/metabolismo , Membrana Celular/metabolismo , Domínios Proteicos , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Sequência de Aminoácidos , Biomarcadores , Complexo CD3/química , Sequência Conservada , Perfilação da Expressão Gênica , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Transdução de Sinais , TranscriptomaRESUMO
It is unclear how quiescence is enforced in naive T cells, but activation by foreign antigens and self-antigens is allowed, despite the presence of inhibitory signals. We showed that active transforming growth factor ß (TGF-ß) signaling was present in naive T cells, and T cell receptor (TCR) engagement reduced TGF-ß signaling during T cell activation by downregulating TGF-ß type 1 receptor (TßRI) through activation of caspase recruitment domain-containing protein 11 (CARD11) and nuclear factor κB (NF-κB). TGF-ß prevented TCR-mediated TßRI downregulation, but this was abrogated by interleukin-6 (IL-6). Mitigation of TCR-mediated TßRI downregulation through overexpression of TßRI in naive and activated T cells rendered T cells less responsive and suppressed autoimmunity. Naive T cells in autoimmune patients exhibited reduced TßRI expression and increased TCR-driven proliferation compared to healthy subjects. Thus, TCR-mediated regulation of TßRI-TGF-ß signaling acts as a crucial criterion to determine T cell quiescence and activation.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linfócitos T CD4-Positivos/imunologia , Guanilato Ciclase/metabolismo , Ativação Linfocitária/imunologia , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Autoimunidade/imunologia , Proteínas Adaptadoras de Sinalização CARD/genética , Linhagem Celular , Proliferação de Células , Colite/imunologia , Colite/patologia , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Guanilato Ciclase/genética , Células HEK293 , Humanos , Interleucina-6/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/biossíntese , Transdução de Sinais/imunologia , Fator de Crescimento Transformador beta1/biossínteseRESUMO
We developed a highly sensitive assay for detecting protein-protein interaction using chimeric receptors comprising two molecules of interest in the extracellular domain and interferon alpha and beta receptor subunit 1 or 2 (IFNAR1/2) in the intracellular domain. This intracellular IFNAR1/2 reconstitution system (IFNARRS) proved markedly more sensitive than the NanoBiT system, currently considered one of the best detection systems for protein interaction. Employing chimeric receptors with extracellular domains from the IFNγ or IL-2 receptor and the intracellular domains of IFNAR1/2, the IFNARRS system effectively identifies low IFNγ or IL-2 levels. Cells stably expressing these chimeric receptors responded to IFNγ secreted by activated T cells following various stimuli, including a specific peptide-antigen. The activation signals were further enhanced by the expression of relevant genes, such as costimulators, via IFN-stimulated response elements in the promoters. Besides IFNγ or IL-2, the IFNARRS system demonstrated the capability to detect other cytokines by using the corresponding extracellular domains from these target cytokine receptors.
Assuntos
Interferon gama , Interleucina-2 , Receptor de Interferon alfa e beta , Linfócitos T , Humanos , Receptor de Interferon alfa e beta/metabolismo , Receptor de Interferon alfa e beta/genética , Linfócitos T/metabolismo , Linfócitos T/imunologia , Interleucina-2/metabolismo , Interferon gama/metabolismo , Receptores de Interleucina-2/metabolismo , Receptores de Interleucina-2/genética , Ligação Proteica , Ativação Linfocitária , Células HEK293RESUMO
Adoptive T-cell transfer (ACT) therapies, including of tumor infiltrating lymphocytes (TILs) and T cells gene-modified to express either a T cell receptor (TCR) or a chimeric antigen receptor (CAR), have demonstrated clinical efficacy for a proportion of patients and cancer-types. The field of ACT has been driven forward by the clinical success of CD19-CAR therapy against various advanced B-cell malignancies, including curative responses for some leukemia patients. However, relapse remains problematic, in particular for lymphoma. Moreover, for a variety of reasons, relative limited efficacy has been demonstrated for ACT of non-hematological solid tumors. Indeed, in addition to pre-infusion challenges including lymphocyte collection and manufacturing, ACT failure can be attributed to several biological processes post-transfer including, (i) inefficient tumor trafficking, infiltration, expansion and retention, (ii) chronic antigen exposure coupled with insufficient costimulation resulting in T-cell exhaustion, (iii) a range of barriers in the tumor microenvironment (TME) mediated by both tumor cells and suppressive immune infiltrate, (iv) tumor antigen heterogeneity and loss, or down-regulation of antigen presentation machinery, (v) gain of tumor intrinsic mechanisms of resistance such as to apoptosis, and (vi) various forms of toxicity and other adverse events in patients. Affinity-optimized TCRs can improve T-cell function and innovative CAR designs as well as gene-modification strategies can be used to coengineer specificity, safety, and function into T cells. Coengineering strategies can be designed not only to directly support the transferred T cells, but also to block suppressive barriers in the TME and harness endogenous innate and adaptive immunity. Here, we review a selection of the remarkable T-cell coengineering strategies, including of tools, receptors, and gene-cargo, that have been developed in recent years to augment tumor control by ACT, more and more of which are advancing to the clinic.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Linfócitos T , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Imunoterapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Antígenos de Neoplasias , Microambiente TumoralRESUMO
The multifunctional RNA-binding protein hnRNPL is implicated in antibody class switching but its broader function in B cells is unknown. Here, we show that hnRNPL is essential for B cell activation, germinal center formation, and antibody responses. Upon activation, hnRNPL-deficient B cells show proliferation defects and increased apoptosis. Comparative analysis of RNA-seq data from activated B cells and another eight hnRNPL-depleted cell types reveals common effects on MYC and E2F transcriptional programs required for proliferation. Notably, while individual gene expression changes are cell type specific, several alternative splicing events affecting histone modifiers like KDM6A and SIRT1, are conserved across cell types. Moreover, hnRNPL-deficient B cells show global changes in H3K27me3 and H3K9ac. Epigenetic dysregulation after hnRNPL loss could underlie differential gene expression and upregulation of lncRNAs, and explain common and cell type-specific phenotypes, such as dysfunctional mitochondria and ROS overproduction in mouse B cells. Thus, hnRNPL is essential for the resting-to-activated B cell transition by regulating transcriptional programs and metabolism, at least in part through the alternative splicing of several histone modifiers.
Assuntos
Processamento Alternativo , Linfócitos B , Epigênese Genética , Ativação Linfocitária , Animais , Humanos , Camundongos , Apoptose/genética , Linfócitos B/metabolismo , Linfócitos B/imunologia , Proliferação de Células/genética , Regulação da Expressão Gênica , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Histonas/metabolismo , Ativação Linfocitária/genéticaRESUMO
In circulation, T cells are spherical with selectin enriched dynamic microvilli protruding from the surface. Following extravasation, these microvilli serve another role, continuously surveying their environment for antigen in the form of peptide-MHC (pMHC) expressed on the surface of antigen presenting cells (APCs). Upon recognition of their cognate pMHC, the microvilli are initially stabilized and then flatten into F-actin dependent microclusters as the T cell spreads over the APC. Within 1-5 min, clathrin is recruited by the ESCRT-0 component Hrs to mediate release of T cell receptor (TCR) loaded vesicles directly from the plasma membrane by clathrin and ESCRT-mediated ectocytosis (CEME). After 5-10 min, Hrs is displaced by the endocytic clathrin adaptor epsin-1 to induce clathrin-mediated trans-endocytosis (CMTE) of TCR-pMHC conjugates. Here we discuss some of the functional properties of the clathrin machinery which enables it to control these topologically opposite modes of membrane transfer at the immunological synapse, and how this might be regulated during T cell activation.
Assuntos
Clatrina , Linfócitos T , Clatrina/metabolismo , Células Apresentadoras de Antígenos/metabolismo , Receptores de Antígenos de Linfócitos T , Endocitose/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , ComunicaçãoRESUMO
T cell activation is a complex biological process of naive cells maturing into effector cells. Proteomic and phospho-proteomic approaches have provided critical insights into this process, yet it is not always clear how changes in individual proteins or phosphorylation sites have functional significance. Here, we developed the Phosphorylation Integrated Thermal Shift Assay (PITSA) that combines the measurement of protein or phosphorylation site abundance and thermal stability into a single tandem mass tags experiment and apply this method to study T cell activation. We quantified the abundance and thermal stability of over 7500 proteins and 5000 phosphorylation sites and identified significant differences in chromatin-related, TCR signaling, DNA repair, and proliferative phosphoproteins. PITSA may be applied to a wide range of biological contexts to generate hypotheses as to which proteins or phosphorylation sites are functionally regulated in a given system as well as the mechanisms by which this regulation may occur.
Assuntos
Ativação Linfocitária , Proteômica , Linfócitos T , Fosforilação , Linfócitos T/metabolismo , Proteômica/métodos , Fosfoproteínas/metabolismo , Animais , Humanos , Estabilidade Proteica , Transdução de Sinais , Espectrometria de Massas em Tandem , CamundongosRESUMO
To mount appropriate responses, T cells integrate complex sequences of receptor stimuli perceived during transient interactions with antigen-presenting cells. Although it has been hypothesized that the dynamics of these interactions influence the outcome of T cell activation, methodological limitations have hindered its formal demonstration. Here, we have engineered the Light-inducible T cell engager (LiTE) system, a recombinant optogenetics-based molecular tool targeting the T cell receptor (TCR). The LiTE system constitutes a reversible molecular switch displaying exquisite reactivity. As proof of concept, we dissect how specific temporal patterns of TCR stimulation shape T cell activation. We established that CD4+ T cells respond to intermittent TCR stimulation more efficiently than their CD8+ T cells counterparts and provide evidence that distinct sequences of TCR stimulation encode different cytokine programs. Finally, we show that the LiTE system could be exploited to create light-activated bispecific T cell engagers and manipulate tumor cell killing. Overall, the LiTE system provides opportunities to understand how T cells integrate TCR stimulations and to trigger T cell cytotoxicity with high spatiotemporal control.
Assuntos
Células Apresentadoras de Antígenos , Linfócitos T CD8-Positivos , Citocinas , Células Epiteliais , Ativação LinfocitáriaRESUMO
T cell activation stimulates substantially increased protein synthesis activity to accumulate sufficient biomass for cell proliferation. The protein synthesis is fueled by the amino acids transported from the environment. Steroid nuclear receptor coactivator 2 (SRC2) is a member of a family of transcription coactivators. Here, we show that SRC2 recruited by c-Myc enhances CD4+ T cell activation to stimulate immune responses via upregulation of amino acid transporter Slc7a5. Mice deficient of SRC2 in T cells (SRC2fl/fl/CD4Cre) are resistant to the induction of experimental autoimmune encephalomyelitis (EAE) and susceptible to Citrobacter rodentium (C. rodentium) infection. Adoptive transfer of naive CD4+ T cells from SRC2fl/fl/CD4Cre mice fails to elicit EAE and colitis in Rag1/ recipients. Further, CD4+ T cells from SRC2fl/fl/CD4Cre mice display defective T cell proliferation, cytokine production, and differentiation both in vitro and in vivo. Mechanically, SRC2 functions as a coactivator to work together with c-Myc to stimulate the expression of amino acid transporter Slc7a5 required for T cell activation. Slc7a5 fails to be up-regulated in CD4+ T cells from SRC2fl/fl/CD4Cre mice, and forced expression of Slc7a5 rescues proliferation, cytokine production, and the ability of SRC2fl/fl/CD4Cre CD4+ T cells to induce EAE. Therefore, SRC2 is essential for CD4+ T cell activation and, thus, a potential drug target for controlling CD4+ T cell-mediated autoimmunity.
Assuntos
Encefalomielite Autoimune Experimental , Linfócitos T , Animais , Camundongos , Linfócitos T CD4-Positivos , Citocinas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coativador 2 de Receptor Nuclear/metabolismo , Regulação para CimaRESUMO
Dendritic cell (DC) activation by viral RNA sensors such as TLR3 and MDA-5 is critical for initiating antiviral immunity. Optimal DC activation is promoted by type I interferon (IFN) signaling which is believed to occur in either autocrine or paracrine fashion. Here, we show that neither autocrine nor paracrine type I IFN signaling can fully account for DC activation by poly(I:C) in vitro and in vivo. By controlling the density of type I IFN-producing cells in vivo, we establish that instead a quorum of type I IFN-producing cells is required for optimal DC activation and that this process proceeds at the level of an entire lymph node. This collective behavior, governed by type I IFN diffusion, is favored by the requirement for prolonged cytokine exposure to achieve DC activation. Furthermore, collective DC activation was found essential for the development of innate and adaptive immunity in lymph nodes. Our results establish how collective rather than cell-autonomous processes can govern the initiation of immune responses.
Assuntos
Células Dendríticas/fisiologia , Interferon Tipo I/metabolismo , Linfonodos/citologia , Percepção de Quorum/fisiologia , Animais , Linfócitos T CD8-Positivos/fisiologia , Contagem de Células , Células Dendríticas/efeitos dos fármacos , Imunidade Inata/imunologia , Inflamação/patologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Interferon Tipo I/farmacologia , Linfonodos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Poli I-C/farmacologiaRESUMO
BACKGROUND AND AIMS: Intestinal epithelial cell (IEC) damage is a hallmark of celiac disease (CeD); however, its role in gluten-dependent T-cell activation is unknown. We investigated IEC-gluten-T cell interactions in organoid monolayers expressing human MHC class II (HLA-DQ2.5), which facilitates gluten antigen recognition by CD4+ T cells in CeD. METHODS: Epithelial MHC class II (MHCII) was determined in active and treated CeD, and in non-immunized and gluten-immunized DR3-DQ2.5 transgenic mice, lacking mouse MHCII molecules. Organoid monolayers from DR3-DQ2.5 mice were treated with or without IFN-γ, and MHCII expression was evaluated by flow cytometry. Organoid monolayers and CD4+ T cell co-cultures were incubated with gluten, pre-digested, or not by elastase-producing Pseudomonas aeruginosa or its lasB mutant. T cell function was assessed based on proliferation, expression of activation markers, and cytokine release in the co-culture supernatants. RESULTS: Active CeD patients and gluten-immunized DR3-DQ2.5 mice demonstrated epithelial MHCII expression. Organoid monolayers derived from gluten-immunized DR3-DQ2.5 mice expressed MHCII, which was upregulated by IFN-γ. In organoid monolayer-T cell co-cultures, gluten increased the proliferation of CD4+ T cells, expression of T cell activation markers, and the release of IL-2, IFN-γ, and IL-15 in co-culture supernatants. Gluten metabolized by P. aeruginosa, but not the lasB mutant, enhanced CD4+ T cell proliferation and activation. CONCLUSIONS: Gluten antigens are efficiently presented by MHCII-expressing IECs, resulting in the activation of gluten-specific CD4+ T cells, which is enhanced by gluten pre-digestion with microbial elastase. Therapeutics directed at IECs may offer a novel approach for modulating both adaptive and innate immunity in CeD patients.