RESUMO
T cell responses display two key characteristics. First, a small population of epitope-specific naive T cells expands by several orders of magnitude. Second, the T cells within this proliferating population take on diverse functional and phenotypic properties that determine their ability to exert effector functions and contribute to T cell memory. Recent technological advances in lineage tracing allow us for the first time to study these processes in vivo at single-cell resolution. Here, we summarize resulting data demonstrating that although epitope-specific T cell responses are reproducibly similar at the population level, expansion potential and diversification patterns of the offspring derived from individual T cells are highly variable during both primary and recall immune responses. In spite of this stochastic response variation, individual memory T cells can serve as adult stem cells that provide robust regeneration of an epitope-specific tissue through population averaging. We discuss the relevance of these findings for T cell memory formation and clinical immunotherapy.
Assuntos
Células-Tronco Adultas/imunologia , Diferenciação Celular , Imunoterapia/métodos , Análise de Célula Única/métodos , Linfócitos T/imunologia , Animais , Biodiversidade , Linhagem da Célula , Proliferação de Células , Diversidade Cultural , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Humanos , Memória Imunológica , Ativação LinfocitáriaRESUMO
The kinase LCK and CD4/CD8 co-receptors are crucial components of the T cell antigen receptor (TCR) signaling machinery, leading to key T cell fate decisions. Despite decades of research, the roles of CD4-LCK and CD8-LCK interactions in TCR triggering in vivo remain unknown. In this study, we created animal models expressing endogenous levels of modified LCK to resolve whether and how co-receptor-bound LCK drives TCR signaling. We demonstrated that the role of LCK depends on the co-receptor to which it is bound. The CD8-bound LCK is largely dispensable for antiviral and antitumor activity of cytotoxic T cells in mice; however, it facilitates CD8+ T cell responses to suboptimal antigens in a kinase-dependent manner. By contrast, the CD4-bound LCK is required for efficient development and function of helper T cells via a kinase-independent stabilization of surface CD4. Overall, our findings reveal the role of co-receptor-bound LCK in T cell biology, show that CD4- and CD8-bound LCK drive T cell development and effector immune responses using qualitatively different mechanisms and identify the co-receptor-LCK interactions as promising targets for immunomodulation.
Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Linfócitos T Citotóxicos , Camundongos , Animais , Linfócitos T Citotóxicos/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Antígenos CD4 , Transdução de Sinais , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos CD8/metabolismoRESUMO
Multidimensional single-cell analyses of T cells have fueled the debate about whether there is extensive plasticity or 'mixed' priming of helper T cell subsets in vivo. Here, we developed an experimental framework to probe the idea that the site of priming in the systemic immune compartment is a determinant of helper T cell-induced immunopathology in remote organs. By site-specific in vivo labeling of antigen-specific T cells in inguinal (i) or gut draining mesenteric (m) lymph nodes, we show that i-T cells and m-T cells isolated from the inflamed central nervous system (CNS) in a model of multiple sclerosis (MS) are distinct. i-T cells were Cxcr6+, and m-T cells expressed P2rx7. Notably, m-T cells infiltrated white matter, while i-T cells were also recruited to gray matter. Therefore, we propose that the definition of helper T cell subsets by their site of priming may guide an advanced understanding of helper T cell biology in health and disease.
Assuntos
Autoimunidade , Encéfalo/imunologia , Linhagem da Célula , Encefalomielite Autoimune Experimental/imunologia , Intestinos/imunologia , Pele/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Transferência Adotiva , Animais , Autoimunidade/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Sinalização do Cálcio , Líquido Cefalorraquidiano/imunologia , Líquido Cefalorraquidiano/metabolismo , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Cloridrato de Fingolimode/farmacologia , Perfilação da Expressão Gênica , Genes Codificadores dos Receptores de Linfócitos T , Células HEK293 , Humanos , Imunossupressores/farmacologia , Intestinos/efeitos dos fármacos , Microscopia Intravital , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Esclerose Múltipla Recidivante-Remitente/genética , Esclerose Múltipla Recidivante-Remitente/imunologia , Esclerose Múltipla Recidivante-Remitente/metabolismo , Fenótipo , Estudos Prospectivos , RNA-Seq , Receptores CXCR6/genética , Receptores CXCR6/metabolismo , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Análise de Célula Única , Pele/efeitos dos fármacos , Pele/metabolismo , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/transplante , TranscriptomaRESUMO
Chronic cytomegalovirus (CMV) infection leads to long-term maintenance of extraordinarily large CMV-specific T cell populations. The magnitude of this so-called 'memory inflation' is thought to mainly depend on antigenic stimulation during the chronic phase of infection. However, by mapping the long-term development of CD8+ T cell families derived from single naive precursors, we find that fate decisions made during the acute phase of murine CMV infection can alter the level of memory inflation by more than 1,000-fold. Counterintuitively, a T cell family's capacity for memory inflation is not determined by its initial expansion. Instead, those rare T cell families that dominate the chronic phase of infection show an early transcriptomic signature akin to that of established T central memory cells. Accordingly, a T cell family's long-term dominance is best predicted by its early content of T central memory precursors, which later serve as a stem-cell-like source for memory inflation.
Assuntos
Evolução Clonal/imunologia , Interações Hospedeiro-Patógeno/imunologia , Memória Imunológica , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Viroses/etiologia , Viroses/metabolismo , Doença Aguda , Animais , Biomarcadores , Doença Crônica , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Camundongos , Muromegalovirus/imunologiaRESUMO
Adaptive evolution is a key feature of T cell immunity. During acute immune responses, T cells harboring high-affinity T cell antigen receptors (TCRs) are preferentially expanded, but whether affinity maturation by clonal selection continues through the course of chronic infections remains unresolved. Here we investigated the evolution of the TCR repertoire and its affinity during the course of infection with cytomegalovirus, which elicits large T cell populations in humans and mice. Using single-cell and bulk TCR sequencing and structural affinity analyses of cytomegalovirus-specific T cells, and through the generation and in vivo monitoring of defined TCR repertoires, we found that the immunodominance of high-affinity T cell clones declined during the chronic infection phase, likely due to cellular senescence. These data showed that under conditions of chronic antigen exposure, low-affinity TCRs preferentially expanded within the TCR repertoire, with implications for immunotherapeutic strategies.
Assuntos
Infecções por Citomegalovirus/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Senescência Celular/imunologia , Citomegalovirus/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Repetitive pathogen exposure leads to the dominant outgrowth of T cell clones with high T cell receptor (TCR) affinity to the relevant pathogen-associated antigens. However, low-affinity clones are also known to expand and form immunological memory. While these low-affinity clones contribute less immunity to the original pathogen, their role in protection against pathogens harboring immune escape mutations remains unclear. Based on identification of the TCR repertoire and functionality landscape of naive epitope-specific CD8+ T cells, we reconstructed defined repertoires that could be followed as polyclonal populations during immune responses in vivo. We found that selective clonal expansion is governed by clear TCR avidity thresholds. Simultaneously, initial recruitment of broad TCR repertoires provided a polyclonal niche from which flexible secondary responses to mutant epitopes could be recalled. Elucidating how T cell responses develop "from scratch" is informative for the development of enhanced immunotherapies and vaccines.
Assuntos
Linfócitos T CD8-Positivos , Reinfecção , Humanos , Epitopos , Receptores de Antígenos de Linfócitos T/genética , Células Clonais , Mutação/genéticaRESUMO
Upon viral infection, natural killer (NK) cells expressing certain germline-encoded receptors are selected, expanded, and maintained in an adaptive-like manner. Currently, these are thought to differentiate along a common pathway. However, by fate mapping of single NK cells upon murine cytomegalovirus (MCMV) infection, we identified two distinct NK cell lineages that contributed to adaptive-like responses. One was equivalent to conventional NK (cNK) cells while the other was transcriptionally similar to type 1 innate lymphoid cells (ILC1s). ILC1-like NK cells showed splenic residency and strong cytokine production but also recognized and killed MCMV-infected cells, guided by activating receptor Ly49H. Moreover, they induced clustering of conventional type 1 dendritic cells and facilitated antigen-specific T cell priming early during MCMV infection, which depended on Ly49H and the NK cell-intrinsic expression of transcription factor Batf3. Thereby, ILC1-like NK cells bridge innate and adaptive viral recognition and unite critical features of cNK cells and ILC1s.
Assuntos
Imunidade Adaptativa/imunologia , Linhagem da Célula/imunologia , Infecções por Herpesviridae/imunologia , Imunidade Inata/imunologia , Células Matadoras Naturais/imunologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MuromegalovirusRESUMO
Neuromyelitis optica is a paradigmatic autoimmune disease of the central nervous system, in which the water-channel protein AQP4 is the target antigen1. The immunopathology in neuromyelitis optica is largely driven by autoantibodies to AQP42. However, the T cell response that is required for the generation of these anti-AQP4 antibodies is not well understood. Here we show that B cells endogenously express AQP4 in response to activation with anti-CD40 and IL-21 and are able to present their endogenous AQP4 to T cells with an AQP4-specific T cell receptor (TCR). A population of thymic B cells emulates a CD40-stimulated B cell transcriptome, including AQP4 (in mice and humans), and efficiently purges the thymic TCR repertoire of AQP4-reactive clones. Genetic ablation of Aqp4 in B cells rescues AQP4-specific TCRs despite sufficient expression of AQP4 in medullary thymic epithelial cells, and B-cell-conditional AQP4-deficient mice are fully competent to raise AQP4-specific antibodies in productive germinal-centre responses. Thus, the negative selection of AQP4-specific thymocytes is dependent on the expression and presentation of AQP4 by thymic B cells. As AQP4 is expressed in B cells in a CD40-dependent (but not AIRE-dependent) manner, we propose that thymic B cells might tolerize against a group of germinal-centre-associated antigens, including disease-relevant autoantigens such as AQP4.
Assuntos
Aquaporina 4 , Autoanticorpos , Autoantígenos , Linfócitos B , Tolerância Imunológica , Neuromielite Óptica , Animais , Humanos , Camundongos , Proteína AIRE , Aquaporina 4/deficiência , Aquaporina 4/genética , Aquaporina 4/imunologia , Aquaporina 4/metabolismo , Autoanticorpos/imunologia , Autoantígenos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Antígenos CD40/imunologia , Centro Germinativo/citologia , Centro Germinativo/imunologia , Neuromielite Óptica/imunologia , Neuromielite Óptica/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Timo/citologia , Timo/imunologia , Células Epiteliais da Tireoide/imunologia , Células Epiteliais da Tireoide/metabolismo , TranscriptomaRESUMO
Cancer-specific TCF1+ stem-like CD8+ T cells can drive protective anticancer immunity through expansion and effector cell differentiation1-4; however, this response is dysfunctional in tumours. Current cancer immunotherapies2,5-9 can promote anticancer responses through TCF1+ stem-like CD8+ T cells in some but not all patients. This variation points towards currently ill-defined mechanisms that limit TCF1+CD8+ T cell-mediated anticancer immunity. Here we demonstrate that tumour-derived prostaglandin E2 (PGE2) restricts the proliferative expansion and effector differentiation of TCF1+CD8+ T cells within tumours, which promotes cancer immune escape. PGE2 does not affect the priming of TCF1+CD8+ T cells in draining lymph nodes. PGE2 acts through EP2 and EP4 (EP2/EP4) receptor signalling in CD8+ T cells to limit the intratumoural generation of early and late effector T cell populations that originate from TCF1+ tumour-infiltrating CD8+ T lymphocytes (TILs). Ablation of EP2/EP4 signalling in cancer-specific CD8+ T cells rescues their expansion and effector differentiation within tumours and leads to tumour elimination in multiple mouse cancer models. Mechanistically, suppression of the interleukin-2 (IL-2) signalling pathway underlies the PGE2-mediated inhibition of TCF1+ TIL responses. Altogether, we uncover a key mechanism that restricts the IL-2 responsiveness of TCF1+ TILs and prevents anticancer T cell responses that originate from these cells. This study identifies the PGE2-EP2/EP4 axis as a molecular target to restore IL-2 responsiveness in anticancer TILs to achieve cancer immune control.
Assuntos
Linfócitos T CD8-Positivos , Proliferação de Células , Dinoprostona , Linfócitos do Interstício Tumoral , Neoplasias , Células-Tronco , Evasão Tumoral , Animais , Feminino , Humanos , Masculino , Camundongos , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Dinoprostona/metabolismo , Modelos Animais de Doenças , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Interleucina-2 , Linfonodos/citologia , Linfonodos/imunologia , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Neoplasias/prevenção & controle , Receptores de Prostaglandina E Subtipo EP2/deficiência , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/deficiência , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/imunologia , Células-Tronco/metabolismo , Evasão Tumoral/imunologiaRESUMO
Exhausted CD8+ T cells adopt a functionally attenuated state but still confer a certain degree of pathogen control. Chen et al. (2019), Hudson et al. (2019), and Zander et al. (2019) assign the lasting maintenance of this restrained pathogen control to an equilibrium of effector-like, transitory, terminal, and memory-like exhausted T cells.
Assuntos
Linfócitos T CD8-Positivos , Redes Reguladoras de GenesRESUMO
ABSTRACT: Relapse after anti-CD19 chimeric antigen receptor (CD19-CAR) occurs in a substantial proportion of patients with lymphoid malignancies. We assessed the potential benefits of co-administering CD20-targeting bispecific antibodies (CD20-BsAbs) with CD19-CAR T cells with the aim of enhancing immunotherapeutic efficacy. Addition of CD20-BsAbs to cocultures of CD19-CARs and primary samples of B-cell malignancies, comprising malignant B cells and endogenous T cells, significantly improved killing of malignant cells and enhanced the expansion of both endogenous T cells and CD19-CAR T cells. In an immunocompetent mouse model of chronic lymphocytic leukemia, relapse after initial treatment response frequently occurred after CD19-CAR T-cell monotherapy. Additional treatment with CD20-BsAbs significantly enhanced the treatment response and led to improved eradication of malignant cells. Higher efficacy was accompanied by improved T-cell expansion with CD20-BsAb administration and led to longer survival with 80% of the mice being cured with no detectable malignant cell population within 8 weeks of therapy initiation. Collectively, our in vitro and in vivo data demonstrate enhanced therapeutic efficacy of CD19-CAR T cells when combined with CD20-BsAbs in B-cell malignancies. Activation and proliferation of both infused CAR T cells and endogenous T cells may contribute to improved disease control.
Assuntos
Anticorpos Biespecíficos , Antígenos CD19 , Antígenos CD20 , Imunoterapia Adotiva , Leucemia Linfocítica Crônica de Células B , Animais , Camundongos , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/terapia , Leucemia Linfocítica Crônica de Células B/patologia , Humanos , Antígenos CD19/imunologia , Antígenos CD20/imunologia , Anticorpos Biespecíficos/uso terapêutico , Imunoterapia Adotiva/métodos , Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Engagement of the inhibitory T cell receptor programmed cell death protein 1 (PD-1) associates with dysfunctional states of pathogen- or tumor-specific T cells. Accordingly, systemic antibody-mediated blockade of PD-1 has become a central target for immunotherapies but is also associated with severe toxicities due to loss of peripheral tolerance. Therefore, selective ablation of PD-1 expression on adoptively transferred T cells through direct genetic knockout (KO) is currently being explored as an alternative therapeutic approach. However, since PD-1 might also be required for the regulation of physiological T cell function and differentiation, the suitability of PD-1 as an engineering target is controversial. In this study, we systematically investigated the maintenance of T cell functionality after CRISPR/Cas9-mediated PD-1 KO in vivo during and after acute and chronic antigen encounter. Under all tested conditions, PD-1 ablation preserved the persistence, differentiation, and memory formation of adoptively transferred receptor transgenic T cells. Functional PD-1 KO T cells expressing chimeric antigen receptors (CARs) targeting CD19 could be robustly detected for over 390 d in a syngeneic immunocompetent mouse model, in which constant antigen exposure was provided by continuous B cell renewal, representing the longest in vivo follow-up of CAR-T cells described to date. PD-1 KO CAR-T cells showed no evidence for malignant transformation during the entire observation period. Our data demonstrate that genetic ablation of PD-1 does not impair functionality and longevity of adoptively transferred T cells per se and therefore may be pursued more generally in engineered T cell-based immunotherapy to overcome a central immunosuppressive axis.
Assuntos
Receptor de Morte Celular Programada 1 , Linfócitos T , Animais , Camundongos , Receptor de Morte Celular Programada 1/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais Geneticamente Modificados , Anticorpos BloqueadoresRESUMO
Rapid clonal expansion of antigen-specific T cells is a fundamental feature of adaptive immune responses. It enables the outgrowth of an individual T cell into thousands of clonal descendants that diversify into short-lived effectors and long-lived memory cells. Clonal expansion is thought to be programmed upon priming of a single naive T cell and then executed by homogenously fast divisions of all of its descendants. However, the actual speed of cell divisions in such an emerging "T cell family" has never been measured with single-cell resolution. Here, we utilize continuous live-cell imaging in vitro to track the division speed and genealogical connections of all descendants derived from a single naive CD8+ T cell throughout up to ten divisions of activation-induced proliferation. This comprehensive mapping of T cell family trees identifies a short burst phase, in which division speed is homogenously fast and maintained independent of external cytokine availability or continued T cell receptor stimulation. Thereafter, however, division speed diversifies, and model-based computational analysis using a Bayesian inference framework for tree-structured data reveals a segregation into heritably fast- and slow-dividing branches. This diversification of division speed is preceded already during the burst phase by variable expression of the interleukin-2 receptor alpha chain. Later it is accompanied by selective expression of memory marker CD62L in slower dividing branches. Taken together, these data demonstrate that T cell clonal expansion is structured into subsequent burst and diversification phases, the latter of which coincides with specification of memory versus effector fate.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linhagem da Célula , Animais , Antígenos CD/imunologia , Biomarcadores , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Divisão Celular , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND & AIMS: Infection with Helicobacter pylori strongly affects global health by causing chronic gastritis, ulcer disease, and gastric cancer. Although extensive research into the strong immune response against this persistently colonizing bacterium exists, the specific role of CD8+ T cells remains elusive. METHODS: We comprehensively characterize gastric H pylori-specific CD8+ T-cell responses in mice and humans by flow cytometry, RNA-sequencing, immunohistochemistry, and ChipCytometry, applying functional analyses including T-cell depletion, H pylori eradication, and ex vivo restimulation. RESULTS: We define CD8+ T-cell populations bearing a tissue-resident memory (TRM) phenotype, which infiltrate the gastric mucosa shortly after infection and mediate pathogen control by executing antigen-specific effector properties. These induced CD8+ tissue-resident memory T cells (TRM cells) show a skewed T-cell receptor beta chain usage and are mostly specific for cytotoxin-associated gene A, the distinctive oncoprotein injected by H pylori into host cells. As the infection progresses, we observe a loss of the TRM phenotype and replacement of CD8+ by CD4+ T cells, indicating a shift in the immune response during the chronic infection phase. CONCLUSIONS: Our results point toward a hitherto unknown role of CD8+ T-cell response in this bacterial infection, which may have important clinical implications for treatment and vaccination strategies against H pylori.
Assuntos
Infecções por Helicobacter , Helicobacter pylori , Humanos , Animais , Camundongos , Linfócitos T CD8-Positivos , Linfócitos T CD4-Positivos , Estômago , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/microbiologia , Antígenos de Bactérias , Proteínas de BactériasRESUMO
T cell ignorance is a specific form of immunological tolerance. It describes the maintenance of naivety in antigen-specific T cells in vivo despite the presence of their target antigen. It is thought to mainly play a role during the steady state, when self-antigens are presented in absence of costimulatory signals and at low density or to T cells of low affinity. In how far antigen-specific T cells can also remain clonally ignorant to foreign antigens, presented in the inflammatory context of systemic infection, remains unclear. Using single-cell in vivo fate mapping and high throughput flow cytometric enrichment, we find that high-affinity antigen-specific CD8+ T cells are efficiently recruited upon systemic infection. In contrast, most low-affinity antigen-specific T cells ignore the priming antigen and persist in the naïve state while remaining fully responsive to subsequent immunization with a high-affinity ligand. These data establish the widespread clonal ignorance of low-affinity T cells as a major factor shaping the composition of antigen-specific CD8+ T cell responses to systemic infection.
Assuntos
Autoantígenos , Linfócitos T CD8-Positivos , Tolerância Imunológica , Diferenciação CelularRESUMO
CD8+ T cells recognizing their cognate antigen are typically recruited as a polyclonal population consisting of multiple clonotypes with varying T-cell receptor (TCR) affinity to the target peptide-major histocompatibility complex (pMHC) complex. Advances in single-cell sequencing have increased accessibility toward identifying TCRs with matched antigens. Here we present the discovery of a monoclonal CD8+ T-cell population with specificity for a hepatitis C virus (HCV)-derived human leukocyte antigen (HLA) class I epitope (HLA-B*07:02 GPRLGVRAT) which was isolated directly ex vivo from an individual with an episode of acutely resolved HCV infection. This population was absent before infection and underwent expansion and stable maintenance for at least 2 years after infection as measured by HLA-multimer staining. Furthermore, the monoclonal clonotype was characterized by an unusually long dissociation time (half-life = 794 s and koff = 5.73 × 10-4) for its target antigen when compared with previously published results. A comparison with related populations of HCV-specific populations derived from the same individual and a second individual suggested that high-affinity TCR-pMHC interactions may be inherent to epitope identity and shape the phenotype of responses which has implications for rational TCR selection and design in the age of personalized immunotherapies.
Assuntos
Linfócitos T CD8-Positivos , Células Clonais , Hepacivirus , Hepatite C , Receptores de Antígenos de Linfócitos T , Humanos , Linfócitos T CD8-Positivos/imunologia , Hepatite C/imunologia , Hepatite C/virologia , Hepacivirus/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Epitopos de Linfócito T/imunologiaRESUMO
T-cell therapy has emerged as an effective approach for treating viral infections and cancers. However, a significant challenge is the selection of T-cell receptors (TCRs) that exhibit the desired functionality. Conventionally in vitro techniques, such as peptide sensitivity measurements and cytotoxicity assays, provide valuable insights into TCR potency but are labor-intensive. In contrast, measuring ligand binding properties (z-Movi technology) could provide an accelerated processing while showing robust correlations with T-cell functions. In this study, we assessed whether cell avidity can predict functionality also in the context of TCR-engineered T cells. To this end, we developed a flexible system for TCR re-expression by generating a Jurkat-derived T cell clone lacking TCR and CD3 expression through CRISPR-Cas9-mediated TRBC knockout. The knockin of a transgenic TCR into the TRAC locus restored TCR/CD3 expression, allowing for CD3-based purification of TCR-engineered T cells. Subsequently, we characterized these engineered cell lines by functional readouts, and assessment of binding properties through the z-Movi technology. Our findings revealed a strong correlation between the cell avidities and functional sensitivities of Jurkat TCR-T cells. Altogether, by integrating cell avidity measurements with our versatile T cell engineering platform, we established an accelerated system for enhancing the in vitro selection of clinically relevant TCRs.
Assuntos
Receptores de Antígenos de Linfócitos T , Humanos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Células Jurkat , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/citologia , Sistemas CRISPR-Cas/genética , Complexo CD3/metabolismo , Complexo CD3/imunologiaRESUMO
It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution due to effects on aspects of speech and language. Here, we introduce these substitutions into the endogenous Foxp2 gene of mice. Although these mice are generally healthy, they have qualitatively different ultrasonic vocalizations, decreased exploratory behavior and decreased dopamine concentrations in the brain suggesting that the humanized Foxp2 allele affects basal ganglia. In the striatum, a part of the basal ganglia affected in humans with a speech deficit due to a nonfunctional FOXP2 allele, we find that medium spiny neurons have increased dendrite lengths and increased synaptic plasticity. Since mice carrying one nonfunctional Foxp2 allele show opposite effects, this suggests that alterations in cortico-basal ganglia circuits might have been important for the evolution of speech and language in humans.
Assuntos
Substituição de Aminoácidos , Gânglios da Base/metabolismo , Evolução Biológica , Fatores de Transcrição Forkhead/metabolismo , Vocalização Animal , Animais , Dendritos/metabolismo , Dopamina/metabolismo , Expressão Gênica , Heterozigoto , Humanos , Idioma , Depressão Sináptica de Longo Prazo , Camundongos , Vias Neurais , Plasticidade Neuronal , FalaRESUMO
OBJECTIVE: Helicobacter pylori infection is the most prevalent bacterial infection worldwide. Besides being the most important risk factor for gastric cancer development, epidemiological data show that infected individuals harbour a nearly twofold increased risk to develop colorectal cancer (CRC). However, a direct causal and functional connection between H. pylori infection and colon cancer is lacking. DESIGN: We infected two Apc-mutant mouse models and C57BL/6 mice with H. pylori and conducted a comprehensive analysis of H. pylori-induced changes in intestinal immune responses and epithelial signatures via flow cytometry, chip cytometry, immunohistochemistry and single cell RNA sequencing. Microbial signatures were characterised and evaluated in germ-free mice and via stool transfer experiments. RESULTS: H. pylori infection accelerated tumour development in Apc-mutant mice. We identified a unique H. pylori-driven immune alteration signature characterised by a reduction in regulatory T cells and pro-inflammatory T cells. Furthermore, in the intestinal and colonic epithelium, H. pylori induced pro-carcinogenic STAT3 signalling and a loss of goblet cells, changes that have been shown to contribute-in combination with pro-inflammatory and mucus degrading microbial signatures-to tumour development. Similar immune and epithelial alterations were found in human colon biopsies from H. pylori-infected patients. Housing of Apc-mutant mice under germ-free conditions ameliorated, and early antibiotic eradication of H. pylori infection normalised the tumour incidence to the level of uninfected controls. CONCLUSIONS: Our studies provide evidence that H. pylori infection is a strong causal promoter of colorectal carcinogenesis. Therefore, implementation of H. pylori status into preventive measures of CRC should be considered.
Assuntos
Neoplasias do Colo , Infecções por Helicobacter , Helicobacter pylori , Microbiota , Neoplasias Gástricas , Humanos , Camundongos , Animais , Helicobacter pylori/genética , Infecções por Helicobacter/complicações , Infecções por Helicobacter/microbiologia , Camundongos Endogâmicos C57BL , Carcinogênese/patologia , Neoplasias Gástricas/patologia , Neoplasias do Colo/patologia , Muco , Mucosa Gástrica/patologiaRESUMO
The avidity of TCRs for peptide-major histocompatibility complexes (pMHCs) is a governing factor in how T cells respond to antigen. TCR avidity is generally linked to T-cell functionality and there is growing evidence for distinct roles of low and high avidity T cells in different phases of immune responses. While physiological immune responses and many therapeutic T-cell products targeting infections or cancers consist of polyclonal T-cell populations with a wide range of individual avidities, the role of T-cell avidity is usually investigated only in monoclonal experimental settings. In this report, we induced polyclonal T-cell responses with a wide range of avidities toward a model epitope by altered peptide ligands, and benchmarked global avidity of physiological polyclonal populations by investigation of TCR-pMHC koff -rates. We then investigated how varying sizes and avidities of monoclonal subpopulations translate into global koff -rates. Global koff -rates integrate subclonal avidities in a predictably weighted manner and robustly correlate with the functionality of murine polyclonal T-cell populations in vitro and in vivo. Surveying the full avidity spectrum is essential to accurately assess polyclonal immune responses and inform the design of polyclonal T-cell therapeutics.