RESUMO
The fetus is thought to be protected from exposure to foreign antigens, yet CD45RO+ T cells reside in the fetal intestine. Here we combined functional assays with mass cytometry, single-cell RNA sequencing and high-throughput T cell antigen receptor (TCR) sequencing to characterize the CD4+ T cell compartment in the human fetal intestine. We identified 22 CD4+ T cell clusters, including naive-like, regulatory-like and memory-like subpopulations, which were confirmed and further characterized at the transcriptional level. Memory-like CD4+ T cells had high expression of Ki-67, indicative of cell division, and CD5, a surrogate marker of TCR avidity, and produced the cytokines IFN-γ and IL-2. Pathway analysis revealed a differentiation trajectory associated with cellular activation and proinflammatory effector functions, and TCR repertoire analysis indicated clonal expansions, distinct repertoire characteristics and interconnections between subpopulations of memory-like CD4+ T cells. Imaging mass cytometry indicated that memory-like CD4+ T cells colocalized with antigen-presenting cells. Collectively, these results provide evidence for the generation of memory-like CD4+ T cells in the human fetal intestine that is consistent with exposure to foreign antigens.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Feto/imunologia , Memória Imunológica/imunologia , Intestinos/imunologia , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD5/genética , Antígenos CD5/imunologia , Antígenos CD5/metabolismo , Células Cultivadas , Feto/citologia , Feto/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Memória Imunológica/genética , Imunofenotipagem , Intestinos/citologia , Intestinos/embriologia , Antígeno Ki-67/genética , Antígeno Ki-67/imunologia , Antígeno Ki-67/metabolismoRESUMO
Follicular regulatory T cells (Tfr) can play opposite roles in the regulation of germinal center (GC) responses. Depending on the studies, Tfr suppress or support GC and B cell affinity maturation. However, which factors determine positive vs. negative effects of Tfr on the GC B cell is unclear. In this study, we show that GC centrocytes that express MYC up-regulate expression of CCL3 chemokine that is needed for both the positive and negative regulation of GC B cells by Tfr. B cell-intrinsic expression of CCL3 contributes to Tfr-dependent positive selection of foreign Ag-specific GC B cells. At the same time, expression of CCL3 is critical for direct Tfr-mediated suppression of GC B cells that acquire cognate to Tfr nuclear proteins. Our study suggests that CCR5 and CCR1 receptors promote Tfr migration to CCL3 and highlights Ccr5 expression on the Tfr subset that expresses Il10. Based on our findings and previous studies, we suggest a model of chemotactically targeted checkpoint control of B cells undergoing positive selection in GCs by Tfr, where Tfr directly probe and license foreign antigen-specific B cells to complete their positive selection in GCs but, at the same time, suppress GC B cells that present self-antigens cognate to Tfr.
Assuntos
Linfócitos B , Linfócitos T Reguladores , Centro Germinativo , Autoantígenos , Quimiocina CCL3RESUMO
HLA-DR-positive NK cells, found in both healthy individuals and patients with different inflammatory diseases, are characterized as activated cells. However, data on their capacity for IFNγ production or cytotoxic response vary between studies. Thus, more precise investigation is needed of the mechanisms related to the induction of HLA-DR expression in NK cells, their associations with NK cell differentiation stage, and functional or metabolic state. In this work, HLA-DR-expressing NK cell subsets were investigated using transcriptomic analysis, metabolic activity assays, and analysis of intercellular signaling cascades. We demonstrated that HLA-DR+CD56bright NK cells were characterized by a proliferative phenotype, while HLA-DR+CD56dim NK cells exhibited features of adaptive cells and loss of inhibitory receptors with increased expression of MHC class II trans-activator CIITA. The activated state of HLA-DR-expressing NK cells was confirmed by higher levels of ATP and mitochondrial mass observed in this subset compared to HLA-DR- cells, both ex vivo and after stimulation in culture. We showed that HLA-DR expression in NK cells in vitro can be induced both through stimulation by exogenous IL-2 and IL-21, as well as through auto-stimulation by NK-cell-produced IFNγ. At the intracellular level, HLA-DR expression depended on the activation of STAT3- and ERK1/2-mediated pathways, with subsequent activation of isoform 3 of the transcription factor CIITA. The obtained results broaden the knowledge about HLA-DR-positive NK cell appearance, diversity, and functions, which might be useful in terms of understanding the role of this subset in innate immunity and assessing their possible implications in NK cell-based therapy.
Assuntos
Diferenciação Celular , Antígenos HLA-DR , Interferon gama , Células Matadoras Naturais , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/imunologia , Humanos , Antígenos HLA-DR/metabolismo , Antígenos HLA-DR/genética , Interferon gama/metabolismo , Antígeno CD56/metabolismo , Ativação Linfocitária/imunologia , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Células Cultivadas , Proteínas Nucleares , TransativadoresRESUMO
T cell maturation and activation depend upon T cell receptor (TCR) interactions with a wide variety of antigenic peptides displayed in a given major histocompatibility complex (MHC) context. Complementarity-determining region 3 (CDR3) is the most variable part of the TCRα and -ß chains, which govern interactions with peptide-MHC complexes. However, it remains unclear how the CDR3 landscape is shaped by individual MHC context during thymic selection of naïve T cells. We established two mouse strains carrying distinct allelic variants of H2-A and analyzed thymic and peripheral production and TCR repertoires of naïve conventional CD4+ T (Tconv) and naïve regulatory CD4+ T (Treg) cells. Compared with tuberculosis-resistant C57BL/6 (H2-Ab) mice, the tuberculosis-susceptible H2-Aj mice had fewer CD4+ T cells of both subsets in the thymus. In the periphery, this deficiency was only apparent for Tconv and was compensated for by peripheral reconstitution for Treg We show that H2-Aj favors selection of a narrower and more convergent repertoire with more hydrophobic and strongly interacting amino acid residues in the middle of CDR3α and CDR3ß, suggesting more stringent selection against a narrower peptide-MHC-II context. H2-Aj and H2-Ab mice have prominent reciprocal differences in CDR3α and CDR3ß features, probably reflecting distinct modes of TCR fitting to MHC-II variants. These data reveal the mechanics and extent of how MHC-II shapes the naïve CD4+ T cell CDR3 landscape, which essentially defines adaptive response to infections and self-antigens.
Assuntos
Regiões Determinantes de Complementaridade/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Tuberculose/genética , Alelos , Animais , Linfócitos T CD4-Positivos/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Baço/imunologia , Linfócitos T Reguladores/química , Tuberculose/imunologiaRESUMO
Neocortical projection neurons are generated by neural progenitor cells (NPCs) within the ventricular and subventricular zone. While early NPCs can give rise to both deep and upper layer neurons, late progenitors are restricted to upper layer neurogenesis. The molecular mechanisms controlling the differentiation potential of early versus late NPCs are unknown. Here, we report a novel function for TrkC-T1, the non-catalytic isoform of the neurotrophin receptor TrkC, that is distinct from TrkC-TK+, the full-length isoform. We provide direct evidence that TrkC-T1 regulates the switch in NPC fate from deep to upper layer neuron production. Elevated levels of TrkC-T1 in early NPCs promote the generation of deep layer neurons. Conversely, downregulation of TrkC-T1 in these cells promotes upper layer neuron fate. Furthermore, we show that TrkC-T1 exerts this control by interaction with the signaling adaptor protein ShcA. TrkC-T1 prevents the phosphorylation of Shc and the downstream activation of the MAP kinase (Erk1/2) pathway. In vivo manipulation of the activity of ShcA or Erk1/2, directly affects cortical neuron cell fate. We thus show that the generation of upper layer neurons by late progenitors is dependent on the downregulation of TrkC-T1 in late progenitor cells and the resulting activation of the ShcA/Erk1/2 pathway.
Assuntos
Neocórtex , Células-Tronco Neurais , Neocórtex/metabolismo , Células-Tronco Neurais/metabolismo , Isoformas de Proteínas/metabolismo , Receptor trkC , Transdução de Sinais/fisiologiaRESUMO
The mechanisms leading to autoimmune and inflammatory diseases in the CNS have not been elucidated. The environmental triggers of the aberrant presence of CD4+ T cells in the CNS are not known. In this article, we report that abnormal ß-catenin expression in T cells drives a fatal neuroinflammatory disease in mice that is characterized by CNS infiltration of T cells, glial activation, and progressive loss of motor function. We show that enhanced ß-catenin expression in T cells leads to aberrant and Th1-biased T cell activation, enhanced expression of integrin α4ß1, and infiltration of activated T cells into the spinal cord, without affecting regulatory T cell function. Importantly, expression of ß-catenin in mature naive T cells was sufficient to drive integrin α4ß1 expression and CNS migration, whereas pharmacologic inhibition of integrin α4ß1 reduced the abnormal T cell presence in the CNS of ß-catenin-expressing mice. Together, these results implicate deregulation of the Wnt/ß-catenin pathway in CNS inflammation and suggest novel therapeutic strategies for neuroinflammatory disorders.
Assuntos
Integrina alfa4beta1/imunologia , Doenças da Medula Espinal/imunologia , Medula Espinal/imunologia , Células Th1/imunologia , Via de Sinalização Wnt/imunologia , beta Catenina/imunologia , Animais , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Integrina alfa4beta1/genética , Camundongos , Camundongos Knockout , Medula Espinal/patologia , Doenças da Medula Espinal/genética , Doenças da Medula Espinal/patologia , Células Th1/patologia , Via de Sinalização Wnt/genética , beta Catenina/genéticaRESUMO
For understanding the rules and laws of adaptive immunity, high-throughput profiling of T-cell receptor (TCR) repertoires becomes a powerful tool. The structure of TCR repertoires is instructive even before the antigen specificity of each particular receptor becomes available. It embodies information about the thymic and peripheral selection of T cells; the readiness of an adaptive immunity to withstand new challenges; the character, magnitude and memory of immune responses; and the aetiological and functional proximity of T-cell subsets. Here, we describe our current analytical approaches for the comparative analysis of murine TCR repertoires, and show several examples of how these approaches can be applied for particular experimental settings. We analyse the efficiency of different metrics used for estimation of repertoire diversity, repertoire overlap, V-gene and J-gene segments usage similarity, and amino acid composition of CDR3. We discuss basic differences of these metrics and their advantages and limitations in different experimental models, and we provide guidelines for choosing an efficient way to lead a comparative analysis of TCR repertoires. Applied to the various known and newly developed mouse models, such analysis should allow us to disentangle multiple sophisticated puzzles in adaptive immunity.
Assuntos
Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Imunidade Celular/fisiologia , Subpopulações de Linfócitos T/imunologia , Animais , Camundongos , Subpopulações de Linfócitos T/citologiaRESUMO
Objective: The risk of AS is associated with genomic variants related to antigen presentation and specific cytokine signalling pathways, suggesting the involvement of cellular immunity in disease initiation/progression. The aim of the present study was to explore the repertoire of TCR sequences in healthy donors and AS patients to uncover AS-linked TCR variants. Methods: Using quantitative molecular-barcoded 5'-RACE, we performed deep TCR ß repertoire profiling of peripheral blood (PB) and SF samples for 25 AS patients and 108 healthy donors. AS-linked TCR variants were identified using a new computational approach that relies on a probabilistic model of the VDJ rearrangement process. Results: Using the donor-agnostic probabilistic model, we reveal a TCR ß motif characteristic for PB of AS patients, represented by eight highly homologous amino acid sequence variants. Some of these variants were previously reported in SF and PB of patients with ReA and in PB of AS patients. We demonstrate that identified AS-linked clones have a CD8+ phenotype, present at relatively low frequencies in PB, and are significantly enriched in matched SF samples of AS patients. Conclusion: Our results suggest the involvement of a particular antigen-specific subset of CD8+ T cells in AS pathogenesis, confirming and expanding earlier findings. The high similarity of the clonotypes with the ones found in ReA implies common mechanisms for the initiation of the diseases.
Assuntos
Linfócitos T CD8-Positivos/imunologia , DNA/genética , Fatores do Domínio POU/genética , Espondilite Anquilosante/genética , Líquido Sinovial/metabolismo , Feminino , Humanos , Masculino , Fatores do Domínio POU/metabolismo , Reação em Cadeia da Polimerase , Proibitinas , Espondilite Anquilosante/imunologia , Espondilite Anquilosante/metabolismo , Líquido Sinovial/imunologiaRESUMO
The diversity of T-cell receptors recognizing foreign pathogens is generated through a highly stochastic recombination process, making the independent production of the same sequence rare. Yet unrelated individuals do share receptors, which together constitute a "public" repertoire of abundant clonotypes. The TCR repertoire is initially formed prenatally, when the enzyme inserting random nucleotides is downregulated, producing a limited diversity subset. By statistically analyzing deep sequencing T-cell repertoire data from twins, unrelated individuals of various ages, and cord blood, we show that T-cell clones generated before birth persist and maintain high abundances in adult organisms for decades, slowly decaying with age. Our results suggest that large, low-diversity public clones are created during pre-natal life, and survive over long periods, providing the basis of the public repertoire.
Assuntos
Envelhecimento/genética , Rearranjo Gênico do Linfócito T/genética , Variação Genética/genética , Receptores de Antígenos de Linfócitos T/fisiologia , Especificidade do Receptor de Antígeno de Linfócitos T/genética , Gêmeos Monozigóticos/genética , Envelhecimento/imunologia , Sequência de Bases , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Humanos , Dados de Sequência Molecular , Recombinação GenéticaRESUMO
The diversity, architecture, and dynamics of the TCR repertoire largely determine our ability to effectively withstand infections and malignancies with minimal mistargeting of immune responses. In this study, we have employed deep TCRß repertoire sequencing with normalization based on unique molecular identifiers to explore the long-term dynamics of T cell immunity. We demonstrate remarkable stability of repertoire, where approximately half of all T cells in peripheral blood are represented by clones that persist and generally preserve their frequencies for 3 y. We further characterize the extremes of lifelong TCR repertoire evolution, analyzing samples ranging from umbilical cord blood to centenarian peripheral blood. We show that the fetal TCR repertoire, albeit structurally maintained within regulated borders due to the lower numbers of randomly added nucleotides, is not limited with respect to observed functional diversity. We reveal decreased efficiency of nonsense-mediated mRNA decay in umbilical cord blood, which may reflect specific regulatory mechanisms in development. Furthermore, we demonstrate that human TCR repertoires are functionally more similar at birth but diverge during life, and we track the lifelong behavior of CMV- and EBV-specific T cell clonotypes. Finally, we reveal gender differences in dynamics of TCR diversity constriction, which come to naught in the oldest age. Based on our data, we propose a more general explanation for the previous observations on the relationships between longevity and immunity.
Assuntos
Envelhecimento , Sangue Fetal/citologia , Sangue Fetal/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Células Clonais , Feminino , Humanos , Epitopos Imunodominantes , Longevidade , Masculino , Pessoa de Meia-Idade , Simulação de Dinâmica Molecular , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Software , Linfócitos T/fisiologia , Fatores de Tempo , Adulto JovemRESUMO
Deep profiling of antibody and T cell-receptor repertoires by means of high-throughput sequencing has become an attractive approach for adaptive immunity studies, but its power is substantially compromised by the accumulation of PCR and sequencing errors. Here we report MIGEC (molecular identifier groups-based error correction), a strategy for high-throughput sequencing data analysis. MIGEC allows for nearly absolute error correction while fully preserving the natural diversity of complex immune repertoires.
Assuntos
Impressões Digitais de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Receptores de Antígenos de Linfócitos T/genética , Projetos de Pesquisa , Impressões Digitais de DNA/normas , Reação em Cadeia da Polimerase/normasRESUMO
Emerging high-throughput sequencing methods for the analyses of complex structure of TCR and BCR repertoires give a powerful impulse to adaptive immunity studies. However, there are still essential technical obstacles for performing a truly quantitative analysis. Specifically, it remains challenging to obtain comprehensive information on the clonal composition of small lymphocyte populations, such as Ag-specific, functional, or tissue-resident cell subsets isolated by sorting, microdissection, or fine needle aspirates. In this study, we report a robust approach based on unique molecular identifiers that allows profiling Ag receptors for several hundred to thousand lymphocytes while preserving qualitative and quantitative information on clonal composition of the sample. We also describe several general features regarding the data analysis with unique molecular identifiers that are critical for accurate counting of starting molecules in high-throughput sequencing applications.
Assuntos
Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Contagem de Linfócitos , Linfócitos/metabolismo , Biologia Computacional/métodos , DNA Complementar , Perfilação da Expressão Gênica/métodos , Humanos , Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , Matrizes de Pontuação de Posição Específica , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
BACKGROUND: The repertoire of T- and B-cell receptor sequences encodes the antigen specificity of adaptive immunity system, determines its present state and guides its ability to mount effective response against encountered antigens in future. High throughput sequencing of immune repertoires (Rep-Seq) is a promising technique that allows to profile millions of antigen receptors of an individual in a single experiment. While a substantial number of tools for mapping and assembling Rep-Seq data were published recently, the field still lacks an intuitive and flexible tool that can be used by researchers with little or no computational background for in-depth analysis of immune repertoire profiles. RESULTS: Here we report VDJviz, a web tool that can be used to browse, analyze and perform quality control of Rep-Seq results generated by various pre-processing software. On a set of real data examples we show that VDJviz can be used to explore key repertoire characteristics such as spectratype, repertoire clonality, V-(D)-J recombination patterns and to identify shared clonotypes. We also demonstrate the utility of VDJviz in detection of critical Rep-Seq biases such as artificial repertoire diversity and cross-sample contamination. CONCLUSIONS: VDJviz is a versatile and lightweight tool that can be easily employed by biologists, immunologists and immunogeneticists for routine analysis and quality control of Rep-Seq data. The software is freely available for non-commercial purposes, and can be downloaded from: https://github.com/antigenomics/vdjviz .
Assuntos
Genômica/métodos , Software , Recombinação V(D)J , Linfócitos B/imunologia , Linfócitos B/metabolismo , Evolução Clonal/genética , Análise por Conglomerados , Regiões Determinantes de Complementaridade/genética , Biologia Computacional/métodos , Biologia Computacional/normas , Genômica/normas , Humanos , Linfócitos T/imunologia , Linfócitos T/metabolismo , NavegadorRESUMO
Despite the growing number of immune repertoire sequencing studies, the field still lacks software for analysis and comprehension of this high-dimensional data. Here we report VDJtools, a complementary software suite that solves a wide range of T cell receptor (TCR) repertoires post-analysis tasks, provides a detailed tabular output and publication-ready graphics, and is built on top of a flexible API. Using TCR datasets for a large cohort of unrelated healthy donors, twins, and multiple sclerosis patients we demonstrate that VDJtools greatly facilitates the analysis and leads to sound biological conclusions. VDJtools software and documentation are available at https://github.com/mikessh/vdjtools.
Assuntos
Biologia Computacional/métodos , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Análise de Sequência de DNA/métodos , Software , Adolescente , Adulto , Criança , Análise por Conglomerados , Transplante de Células-Tronco Hematopoéticas , Humanos , Esclerose Múltipla/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Gêmeos/genética , Adulto JovemRESUMO
The decrease of TCR diversity with aging has never been studied by direct methods. In this study, we combined high-throughput Illumina sequencing with unique cDNA molecular identifier technology to achieve deep and precisely normalized profiling of TCR ß repertoires in 39 healthy donors aged 6-90 y. We demonstrate that TCR ß diversity per 10(6) T cells decreases roughly linearly with age, with significant reduction already apparent by age 40. The percentage of naive T cells showed a strong correlation with measured TCR diversity and decreased linearly up to age 70. Remarkably, the oldest group (average age 82 y) was characterized by a higher percentage of naive CD4(+) T cells, lower abundance of expanded clones, and increased TCR diversity compared with the previous age group (average age 62 y), suggesting the influence of age selection and association of these three related parameters with longevity. Interestingly, cross-analysis of individual TCR ß repertoires revealed a set >10,000 of the most representative public TCR ß clonotypes, whose abundance among the top 100,000 clones correlated with TCR diversity and decreased with aging.
Assuntos
Envelhecimento/imunologia , Variação Genética/imunologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Sequência de Aminoácidos , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Criança , Regiões Determinantes de Complementaridade/genética , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adulto JovemRESUMO
First insights into the molecular programs orchestrating the progression from neural stem cells to cortical projection neurons are emerging. Loss of the transcriptional regulator Ski has been linked to the human 1p36 deletion syndrome, which includes central nervous system defects. Here, we report critical roles for Ski in the maintenance of the neural stem cell pool and the specification of callosal neurons. Ski-deficient callosal neurons lose their identity and ectopically express the transcription factor Ctip2. The misspecified callosal neurons largely fail to form the corpus callosum and instead redirect their axons toward subcortical targets. We identify the chromatin-remodeling factor Satb2 as a partner of Ski, and show that both proteins are required for transcriptional repression of Ctip2 in callosal neurons. We propose a model in which Satb2 recruits Ski to the Ctip2 locus, and Ski attracts histone deacetylases, thereby enabling the formation of a functional nucleosome remodeling and deacetylase repressor complex. Our findings establish a central role for Ski-Satb2 interactions in regulating transcriptional mechanisms of callosal neuron specification.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Corpo Caloso/citologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Ligação à Região de Interação com a Matriz/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Repressoras/biossíntese , Fatores de Transcrição/fisiologia , Proteínas Supressoras de Tumor/biossíntese , Agenesia do Corpo Caloso/embriologia , Agenesia do Corpo Caloso/genética , Agenesia do Corpo Caloso/patologia , Animais , Axônios/ultraestrutura , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Histona Desacetilases/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/deficiência , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Camundongos Knockout , Camundongos Mutantes Neurológicos , Modelos Genéticos , Complexos Multiproteicos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neurogênese/genética , Nucleossomos/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Our ability to analyze adaptive immunity and engineer its activity has long been constrained by our limited ability to identify native pairs of heavy-light antibody chains and alpha-beta T-cell receptor (TCR) chains--both of which comprise coupled "halves of a key", collectively capable of recognizing specific antigens. Here, we report a cell-based emulsion RT-PCR approach that allows the selective fusion of the native pairs of amplified TCR alpha and beta chain genes for complex samples. A new type of PCR suppression technique was developed that makes it possible to amplify the fused library with minimal noise for subsequent analysis by high-throughput paired-end Illumina sequencing. With this technique, single analysis of a complex blood sample allows identification of multiple native TCR chain pairs. This approach may be extended to identify native antibody chain pairs and, more generally, pairs of mRNA molecules that are coexpressed in the same living cells.
Assuntos
Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Sequência de Bases , Amplificação de Genes , Humanos , Cadeias Pesadas de Imunoglobulinas/análise , Cadeias Leves de Imunoglobulina/análise , Leucócitos Mononucleares , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Suppressive function of regulatory T cells (Treg) is dependent on signaling of their antigen receptors triggered by cognate self, dietary, or microbial peptides presented on MHC II. However, it remains largely unknown whether distinct or shared repertoires of Treg TCRs are mobilized in response to different challenges in the same tissue or the same challenge in different tissues. Here we use a fixed TCRß chain FoxP3-GFP mouse model to analyze conventional (eCD4) and regulatory (eTreg) effector TCRα repertoires in response to six distinct antigenic challenges to the lung and skin. This model shows highly 'digital' repertoire behavior with easy-to-track challenge-specific TCRα CDR3 clusters. For both eCD4 and eTreg subsets, we observe challenge-specific clonal expansions yielding homologous TCRα clusters within and across animals and exposure sites, which are also reflected in the draining lymph nodes but not systemically. Some CDR3 clusters are shared across cancer challenges, suggesting a response to common tumor-associated antigens. For most challenges, eCD4 and eTreg clonal response does not overlap. Such overlap is exclusively observed at the sites of certain tumor challenges, and not systematically, suggesting transient and local tumor-induced eCD4=>eTreg plasticity. This transition includes a dominant tumor-responding eCD4 CDR3 motif, as well as characteristic iNKT TCRα CDR3. In addition, we examine the homeostatic tissue residency of clonal eTreg populations by excluding the site of challenge from our analysis. We demonstrate that distinct CDR3 motifs are characteristic of eTreg cells residing in particular lymphatic tissues, regardless of the challenge. This observation reveals the tissue-resident, antigen-specific clonal Treg populations.
Assuntos
Linfócitos T CD4-Positivos , Linfócitos T Reguladores , Camundongos , Animais , Receptores de Antígenos de Linfócitos T/genética , Peptídeos , Células ClonaisRESUMO
Mycobacterium tuberculosis (Mtb) remains a major threat worldwide, although only a fraction of infected individuals develops tuberculosis (TB). TB susceptibility is shaped by multiple genetic factors, and we performed comparative immunological analysis of two mouse strains to uncover relevant mechanisms underlying susceptibility and resistance. C57BL/6 mice are relatively TB-resistant, whereas I/St mice are prone to develop severe TB, partly due to the MHC-II allelic variant that shapes suboptimal CD4+ T cell receptor repertoire. We investigated the repertoires of lung-infiltrating helper T cells and B cells at the progressed stage in both strains. We found that lung CD4+ T cell repertoires of infected C57BL/6 but not I/St mice contained convergent TCR clusters with functionally confirmed Mtb specificity. Transcriptomic analysis revealed a more prominent Th1 signature in C57BL/6, and expression of pro-inflammatory IL-16 in I/St lung-infiltrating helper T cells. The two strains also showed distinct Th2 signatures. Furthermore, the humoral response of I/St mice was delayed, less focused, and dominated by IgG/IgM isotypes, whereas C57BL/6 mice generated more Mtb antigen-focused IgA response. We conclude that the inability of I/St mice to produce a timely and efficient anti-Mtb adaptive immune responses arises from a suboptimal helper T cell landscape that also impacts the humoral response, leading to diffuse inflammation and severe disease.
Assuntos
Imunidade Adaptativa , Predisposição Genética para Doença , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis , Tuberculose , Animais , Camundongos , Mycobacterium tuberculosis/imunologia , Imunidade Adaptativa/genética , Tuberculose/imunologia , Tuberculose/genética , Pulmão/imunologia , Pulmão/patologia , Linfócitos B/imunologia , Modelos Animais de Doenças , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
BACKGROUND: Some peptide anticancer vaccines elicit a strong T-cell memory response but fail to suppress tumor growth. To gain insight into tumor resistance, we compared two peptide vaccines, p20 and p30, against B16 melanoma, with both exhibiting good in vitro T-cell responses but different tumor suppression abilities. METHODS: We compared activation markers and repertoires of T-lymphocytes from tumor-draining (dLN) and non-draining (ndLN) lymph nodes for the two peptide vaccines. RESULTS: We showed that the p30 vaccine had better tumor control as opposed to p20. p20 vaccine induced better in vitro T-cell responsiveness but failed to suppress tumor growth. Efficient antitumor vaccination is associated with a higher clonality of cytotoxic T-cells (CTLs) in dLNs compared with ndLNs and the convergence of most of the enriched clones. With the inefficient p20 vaccine, the most expanded and converged were clones of the bystander T-cells without an LN preference. CONCLUSIONS: Here, we show that the clonality and convergence of the T-cell response are the hallmarks of efficient antitumor vaccination. The high individual and methodological dependencies of these parameters can be avoided by comparing dLNs and ndLNs.