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1.
Nat Commun ; 11(1): 4767, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958743

RESUMO

Psoriatic arthritis (PsA) is a debilitating immune-mediated inflammatory arthritis of unknown pathogenesis commonly affecting patients with skin psoriasis. Here we use complementary single-cell approaches to study leukocytes from PsA joints. Mass cytometry demonstrates a 3-fold expansion of memory CD8 T cells in the joints of PsA patients compared to peripheral blood. Meanwhile, droplet-based and plate-based single-cell RNA sequencing of paired T cell receptor alpha and beta chain sequences show pronounced CD8 T cell clonal expansions within the joints. Transcriptome analyses find these expanded synovial CD8 T cells to express cycling, activation, tissue-homing and tissue residency markers. T cell receptor sequence comparison between patients identifies clonal convergence. Finally, chemokine receptor CXCR3 is upregulated in the expanded synovial CD8 T cells, while two CXCR3 ligands, CXCL9 and CXCL10, are elevated in PsA synovial fluid. Our data thus provide a quantitative molecular insight into the cellular immune landscape of psoriatic arthritis.


Assuntos
Artrite Psoriásica/imunologia , Linfócitos T CD8-Positivos/imunologia , Seleção Clonal Mediada por Antígeno , Receptores de Retorno de Linfócitos/metabolismo , Líquido Sinovial/imunologia , Artrite Psoriásica/sangue , Linfócitos T CD8-Positivos/metabolismo , Perfilação da Expressão Gênica , Humanos , Memória Imunológica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores de Retorno de Linfócitos/genética , Análise de Célula Única , Membrana Sinovial/imunologia
2.
Proc Natl Acad Sci U S A ; 117(35): 21336-21345, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32796106

RESUMO

Each [Formula: see text]T cell receptor (TCR) functions as a mechanosensor. The TCR is comprised of a clonotypic TCR[Formula: see text] ligand-binding heterodimer and the noncovalently associated CD3 signaling subunits. When bound by ligand, an antigenic peptide arrayed by a major histocompatibility complex molecule (pMHC), the TCR[Formula: see text] has a longer bond lifetime under piconewton-level loads. The atomistic mechanism of this "catch bond" behavior is unknown. Here, we perform molecular dynamics simulation of a TCR[Formula: see text]-pMHC complex and its variants under physiologic loads to identify this mechanism and any attendant TCR[Formula: see text] domain allostery. The TCR[Formula: see text]-pMHC interface is dynamically maintained by contacts with a spectrum of occupancies, introducing a level of control via relative motion between Vα and Vß variable domains containing the pMHC-binding complementarity-determining region (CDR) loops. Without adequate load, the interfacial contacts are unstable, whereas applying sufficient load suppresses Vα-Vß motion, stabilizing the interface. A second level of control is exerted by Cα and Cß constant domains, especially Cß and its protruding FG-loop, that create mismatching interfaces among the four TCR[Formula: see text] domains and with a pMHC ligand. Applied load enhances fit through deformation of the TCR[Formula: see text] molecule. Thus, the catch bond involves the entire TCR[Formula: see text] conformation, interdomain motion, and interfacial contact dynamics, collectively. This multilayered architecture of the machinery fosters fine-tuning of cellular response to load and pMHC recognition. Since the germline-derived TCR[Formula: see text] ectodomain is structurally conserved, the proposed mechanism can be universally adopted to operate under load during immune surveillance by diverse [Formula: see text]TCRs constituting the T cell repertoire.


Assuntos
Complexo Principal de Histocompatibilidade , Simulação de Dinâmica Molecular , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Humanos , Ligantes , Mecanotransdução Celular , Linfócitos T/metabolismo
3.
Nat Commun ; 11(1): 4089, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796830

RESUMO

Clonal expansions occur in the persistent HIV reservoir as shown by the duplication of proviral integration sites. However, the source of the proliferation of HIV-infected cells remains unclear. Here, we analyze the TCR repertoire of single HIV-infected cells harboring translation-competent proviruses in longitudinal samples from eight individuals on antiretroviral therapy (ART). When compared to uninfected cells, the TCR repertoire of reservoir cells is heavily biased: expanded clonotypes are present in all individuals, account for the majority of reservoir cells and are often maintained over time on ART. Infected T cell clones are detected at low frequencies in the long-lived central memory compartment and overrepresented in the most differentiated memory subsets. Our results indicate that clonal expansions highly contribute to the persistence of the HIV reservoir and suggest that reservoir cells displaying a differentiated phenotype are the progeny of infected central memory cells undergoing antigen-driven clonal expansion during ART.


Assuntos
Antirretrovirais/uso terapêutico , Infecções por HIV/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Adulto , Células Cultivadas , Feminino , Citometria de Fluxo , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-1/patogenicidade , Humanos , Masculino , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Carga Viral , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
4.
Proc Natl Acad Sci U S A ; 117(25): 14342-14353, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513716

RESUMO

Immature T cells undergo a process of positive selection in the thymus when their new T cell receptor (TCR) engages and signals in response to self-peptides. As the T cell matures, a slew of negative regulatory molecules, including the inhibitory surface glycoprotein CD5, are up-regulated in proportion to the strength of the self-peptide signal. Together these regulators dampen TCR-proximal signaling and help avoid any subsequent peripheral activation of T cells by self-peptides. Paradoxically, antigen-specific T cells initially expressing more CD5 (CD5hi) have been found to better persist as effector/memory cells after a peripheral challenge. The molecular mechanisms underlying such a duality in CD5 function is not clear. We found that CD5 alters the basal activity of the NF-κB signaling in resting peripheral T cells. When CD5 was conditionally ablated, T cells were unable to maintain higher expression of the cytoplasmic NF-κB inhibitor IκBα. Consistent with this, resting CD5hi T cells expressed more of the NF-κB p65 protein than CD5lo cells, without significant increases in transcript levels, in the absence of TCR signals. This posttranslationally stabilized cellular NF-κB depot potentially confers a survival advantage to CD5hi T cells over CD5lo ones. Taken together, these data suggest a two-step model whereby the strength of self-peptide-induced TCR signal lead to the up-regulation of CD5, which subsequently maintains a proportional reserve of NF-κB in peripheral T cells poised for responding to agonistic antigen-driven T cell activation.


Assuntos
Antígenos CD5/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Inibidor de NF-kappaB alfa/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Linfócitos T/imunologia , Transferência Adotiva , Animais , Apresentação do Antígeno/imunologia , Antígenos CD5/genética , Linhagem Celular Tumoral , Separação Celular , Sobrevivência Celular/imunologia , Feminino , Citometria de Fluxo , Lipopolissacarídeos/imunologia , Ativação Linfocitária , Camundongos , Camundongos Knockout , Modelos Animais , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Timo/citologia , Timo/crescimento & desenvolvimento , Timo/imunologia , Fator de Transcrição RelA/metabolismo , Regulação para Cima
5.
Nat Commun ; 11(1): 2859, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503973

RESUMO

Mature double negative (DN) T cells are a population of αß T cells that lack CD4 and CD8 coreceptors and contribute to systemic lupus erythematosus (SLE). The splenic marginal zone macrophages (MZMs) are important for establishing immune tolerance, and loss of their number or function contributes to the progression of SLE. Here we show that loss of MZMs impairs the tolerogenic clearance of apoptotic cells and alters the serum cytokine profile, which in turn provokes the generation of DN T cells from self-reactive CD8+ T cells. Increased Ki67 expression, narrowed TCR V-beta repertoire usage and diluted T-cell receptor excision circles confirm that DN T cells from lupus-prone mice and patients with SLE undergo clonal proliferation and expansion in a self-antigen dependent manner, which supports the shared mechanisms for their generation. Collectively, our results provide a link between the loss of MZMs and the expansion of DN T cells, and indicate possible strategies to prevent the development of SLE.


Assuntos
Autoantígenos/imunologia , Interleucina-17/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Subpopulações de Linfócitos T/imunologia , Transferência Adotiva , Animais , Autoantígenos/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Tolerância Imunológica , Antígeno Ki-67/imunologia , Antígeno Ki-67/metabolismo , Lúpus Eritematoso Sistêmico/sangue , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(19): 10465-10475, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32341160

RESUMO

The antigen-presenting molecule MR1 presents riboflavin-based metabolites to Mucosal-Associated Invariant T (MAIT) cells. While MR1 egress to the cell surface is ligand-dependent, the ability of small-molecule ligands to impact on MR1 cellular trafficking remains unknown. Arising from an in silico screen of the MR1 ligand-binding pocket, we identify one ligand, 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoic acid, DB28, as well as an analog, methyl 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoate, NV18.1, that down-regulate MR1 from the cell surface and retain MR1 molecules in the endoplasmic reticulum (ER) in an immature form. DB28 and NV18.1 compete with the known MR1 ligands, 5-OP-RU and acetyl-6-FP, for MR1 binding and inhibit MR1-dependent MAIT cell activation. Crystal structures of the MAIT T cell receptor (TCR) complexed with MR1-DB28 and MR1-NV18.1, show that these two ligands reside within the A'-pocket of MR1. Neither ligand forms a Schiff base with MR1 molecules; both are nevertheless sequestered by a network of hydrophobic and polar contacts. Accordingly, we define a class of compounds that inhibits MR1 cellular trafficking.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Células T Invariáveis Associadas à Mucosa/metabolismo , Apresentação do Antígeno , Linhagem Celular , Membrana Celular/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica/genética , Humanos , Ligantes , Ativação Linfocitária , Transporte Proteico , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Riboflavina/metabolismo , Células THP-1
7.
Nat Commun ; 11(1): 1522, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251280

RESUMO

Foxp3+ regulatory T (Treg) cells are essential for maintaining peripheral tolerance and preventing autoimmunity. While genetic factors may predispose for autoimmunity, additional environmental triggers, such as viral infections, are usually required to initiate the onset of disease. Here, we show that viral infection with LCMV results in type I IFN-dependent Treg cell loss that is rapidly compensated by the conversion and expansion of Vß5+ conventional T cells into iTreg cells. Using Vß5-deficient mice, we show that these Vß5+ iTreg cells are dispensable for limiting anti-viral immunity. Rather, the delayed replenishment of Treg cells in Vß5-deficient mice compromises suppression of microbiota-dependent activation of CD8+ T cells, resulting in colitis. Importantly, recovery from clinical symptoms in IBD patients is marked by expansion of the corresponding Vß2+ Treg population in humans. Collectively, we provide a link between a viral trigger and an impaired Treg cell compartment resulting in the initiation of immune pathology.


Assuntos
Infecções por Arenaviridae/imunologia , Autoimunidade , Linfócitos T CD8-Positivos/imunologia , Colite/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Linfócitos T Reguladores/imunologia , Animais , Infecções por Arenaviridae/complicações , Linhagem Celular , Colite/microbiologia , Colo/imunologia , Colo/microbiologia , Fatores de Transcrição Forkhead/metabolismo , Microbioma Gastrointestinal/imunologia , Voluntários Saudáveis , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T Reguladores/metabolismo
8.
Cancer Immunol Immunother ; 69(7): 1279-1292, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32185408

RESUMO

The presence of activated T cells in colorectal cancer tissues is a strong predictor of patient survival. Our previous studies have shown that regulatory T cells (Treg) are able to reduce T cell transendothelial migration in vitro and accumulation of effector T cells in intestinal tumors in vivo in the murine APCMin/+ model for microsatellite stable intestinal tumors. In this study, we investigated the effect of Treg depletion on the density and effector functions of different TCRαß+ and TCRγδ+ T cell populations in intestinal tumors. We used the APCMin/+\DEREG mouse model, which harbor a diphtheria toxin receptor under the control of the FOXP3 promoter, to deplete Treg in tumor bearing mice. We found that the density of conventional TCRαß+CD8αß+ T cells was significantly increased in Treg-depleted tumors in comparison with Treg-proficient tumors. Furthermore, TCRαß+CD8αß+ T cells showed increased proliferation and activation as well as increased Granzyme B and IFN-γ production in Treg-depleted tumors. In sharp contrast, the densities and effector functions of TCRαß+CD8αα+ T cells and TCRγδ+ T cells remained unchanged by Treg depletion. We also documented a distinct population of IL-17A+TNF+ TCRγδ+CD8- T cells in tumors, which were not affected by Treg depletion. We conclude that Treg depletion affects only conventional TCRαß+CD8αß+ T cells in intestinal tumors, while unconventional T cells and T cells in unaffected tissue are not altered. Immunotherapies aimed at depleting Treg from tumors may thus be a viable option for reinvigoration of conventional cytotoxic T cells with a Th1 cytokine profile.


Assuntos
Proteína da Polipose Adenomatosa do Colo/fisiologia , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias Intestinais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Feminino , Humanos , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Ativação Linfocitária , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo
9.
Sci Rep ; 10(1): 4472, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161287

RESUMO

We investigated T-cell receptor variable ß chains binding to the superantigen staphylococcal enterotoxin C3 (SEC 3) with structure information in different stages of affinity maturation. Metadynamics in combination with molecular dynamics simulations allow to access the micro-to-millisecond timescale and reveal a strong effect of energetically significant mutations on the flexibility of the antigen-binding site. The observed changes in dynamics of the complementarity determining region (CDR) loops, especially the CDR 2, and HV 4 loop on this specific pathway of affinity maturation are reflected in their structural diversity, thermodynamics of conformations and kinetics of structural transitions. In addition, this affinity maturation pathway follows the concept of conformational selection, because even without the presence of the antigen the binding competent state is present in this pre-existing ensemble of conformations. In all stages of this affinity maturation process we observe a link between specificity and reduced flexibility.


Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Sítios de Ligação , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade
10.
PLoS Comput Biol ; 16(3): e1007714, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32163410

RESUMO

Antigen recognition by T-cells is guided by the T-cell receptor (TCR) heterodimer formed by α and ß chains. A huge diversity of TCR sequences should be maintained by the immune system in order to be able to mount an effective response towards foreign pathogens, so, due to cooperative binding of α and ß chains to the pathogen, any constraints on chain pairing can have a profound effect on immune repertoire structure, diversity and antigen specificity. By integrating available structural data and paired chain sequencing results we were able to show that there are almost no constraints on pairing in TCRαß complexes, allowing naive T-cell repertoire to reach the highest possible diversity. Additional analysis reveals that the specific choice of contacting amino acids can still have a profound effect on complex conformation. Moreover, antigen-driven selection can distort the uniform landscape of chain pairing, while small, yet significant, differences in the pairing can be attributed to various specialized T-cell subsets such as MAIT and iNKT T-cells, as well as other TCR sets specific to certain antigens.


Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta , Aminoácidos , Animais , Antígenos/química , Antígenos/metabolismo , Biologia Computacional , Bases de Dados de Proteínas , Humanos , Camundongos , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia
11.
Cytotherapy ; 22(3): 149-157, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32089448

RESUMO

Forkhead box P3 (FOXP3)+ regulatory T cell (Treg) reconstitution after unrelated donor umbilical cord blood transplantation in chemotherapy-naïve children is incompletely characterized. We studied 21 children with nonmalignant diseases receiving an identical alemtuzumab-containing regimen. We hypothesized that Treg recovery may be perturbed in patients not only by acute graft-versus-host disease (aGVHD) but also by viremia. Tregs and their memory and naïve subsets were serially monitored for proliferation and apoptosis along with conventional T cells (Tcon). A "reconstitution index" (RI) was calculated relative to pretransplantation values for each parameter. At 3 months post-UCBT, the RI of Tregs was faster compared with other immune components tested and was most rapid in patients free of aGVHD and viremia. There were significantly fewer Tregs in patients experiencing grade I-II aGVHD and/or viremia, leading to an imbalance between Tregs-Tcon ratios. Central and effector memory Tregs were most affected at this time point when they dominated in the circulation. Impaired Treg proliferation without increased apoptosis accounted for the reduced Treg-Tcon ratio. In patients affected with grade II aGVHD and viremia, the overall reduction in circulating Treg pool were associated with a more oligoclonal T-cell receptor ß repertoire. Taken together, aGVHD and viremia can lead to defective Treg expansion homeostasis.


Assuntos
Alemtuzumab/uso terapêutico , Transplante de Células-Tronco de Sangue do Cordão Umbilical/efeitos adversos , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Linfócitos T Reguladores/imunologia , Condicionamento Pré-Transplante , Viremia/imunologia , Adolescente , Proliferação de Células , Criança , Pré-Escolar , Feminino , Homeostase , Humanos , Lactente , Subpopulações de Linfócitos/imunologia , Masculino , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/imunologia , Viremia/patologia
12.
Immunopharmacol Immunotoxicol ; 42(2): 110-118, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32066303

RESUMO

Objective: This paper aims to investigate the dynamic changes of the T-cell receptor (TCR) ß complementarity-determining region 3 (CDR3) repertoire during cyclophosphamide or Cytoxan (CTX) damage or inhibition of bone marrow hematopoiesis caused by a reduction of peripheral blood white blood cells (WBCs) in BALB/c mice.Methods: We analyze TCR CDR3 repertoire of BALB/c mice including (1) NS control group (2) CTX damage group (3) CTX damage + GM-CSF recovery group (4) CTX damage + auto-recovery group.Results: The number of WBCs in the CTX group is significantly lower than that in the NS group and after GM-CSF injection, the GM-CSF group is higher than that in the NS group. The diversity of the CTX damage group is the highest and there is a significant difference in high-frequency clonal proliferation between the CTX damage group and CTX damage + GM-CSF recovery group compared with the NS control group. In addition, the numbers of unique productive CDR3 overlapping numbers in the four experimental groups are similar.Conclusions: These data reveal that CTX significantly reduced the number of WBCs and ratio of high-frequency TCR CDR3 sequences, and indirectly increased the diversity of the TCR CDR3 repertoire. GM-CSF quickly restored the number of WBCs, and partially restored changes in the TCR CDR3 repertoire induced by CTX. Results from monitoring the dynamic changes of the TCR CDR3 repertoire can be used to assess the effects of CTX and GM-CSF on the function of peripheral blood T cells and to explore the possible underlying mechanisms.


Assuntos
Medula Óssea/efeitos dos fármacos , Regiões Determinantes de Complementaridade/metabolismo , Ciclofosfamida/toxicidade , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Hematopoese/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Animais , Medula Óssea/patologia , Relação Dose-Resposta a Droga , Feminino , Contagem de Leucócitos , Leucócitos/patologia , Camundongos Endogâmicos BALB C
13.
J Immunol ; 204(4): 858-867, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31924652

RESUMO

Thymic regulatory T cells (tTreg) are critical in the maintenance of normal T cell immunity and tolerance. The role of TCR in tTreg selection remains incompletely understood. In this study, we assessed TCRα and TCRß sequences of mouse tTreg and thymic conventional CD4+ T cells (Tconv) by high-throughput sequencing. We identified αß TCR sequences that were unique to either tTreg or Tconv and found that these were distinct as recognized by machine learning algorithm and by preferentially used amino acid trimers in αß CDR3 of tTreg. In addition, a proportion of αß TCR sequences expressed by tTreg were also found in Tconv, and machine learning classified the great majority of these shared αß TCR sequences as characteristic of Tconv and not tTreg. These findings identify two populations of tTreg, one in which the regulatory T cell fate is associated with unique properties of the TCR and another with TCR properties characteristic of Tconv for which tTreg fate is determined by factors beyond TCR sequence.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Aprendizado de Máquina , Camundongos , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T Reguladores/metabolismo
14.
Cancer Res ; 80(4): 643-654, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31888887

RESUMO

Lymphocytes play a critical role in antitumor immune responses. They are directly targeted by some therapies, and the composition and spatial organization of intratumor T-cell populations is prognostic in some cancer types. A better understanding of lymphocyte population dynamics over the course of disease and in response to therapy is urgently needed to guide therapy decisions and to develop new therapy targets. Deep sequencing of the repertoire of antigen receptor-encoding genes expressed in a lymphocyte population has become a widely used approach for profiling the population's immune status. Lymphocyte antigen receptor repertoire deep sequencing data can be used to assess the clonal richness and diversity of lymphocyte populations; to track clone members over time, between tissues, and across lymphocyte subsets; to detect clonal expansion; and to detect the recruitment of new clones into a tissue. Repertoire sequencing is thus a critical complement to other methods of lymphocyte and immune profiling in cancer. This review describes the current state of knowledge based on repertoire sequencing studies conducted on human cancer patients, with a focus on studies of the T-cell receptor beta chain locus. The review then outlines important questions left unanswered and suggests future directions for the field.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Imunidade Adaptativa/efeitos dos fármacos , Antineoplásicos Imunológicos/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Perfilação da Expressão Gênica , Heterogeneidade Genética , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Neoplasias/genética , Prognóstico , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
15.
Proc Natl Acad Sci U S A ; 117(1): 532-540, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31879353

RESUMO

The T cell repertoire in each individual includes T cell receptors (TCRs) of enormous sequence diversity through the pairing of diverse TCR α- and ß-chains, each generated by somatic recombination of paralogous gene segments. Whether the TCR repertoire contributes to susceptibility to infectious or autoimmune diseases in concert with disease-associated major histocompatibility complex (MHC) polymorphisms is unknown. Due to a lack in high-throughput technologies to sequence TCR α-ß pairs, current studies on whether the TCR repertoire is shaped by host genetics have so far relied only on single-chain analysis. Using a high-throughput single T cell sequencing technology, we obtained the largest paired TCRαß dataset so far, comprising 965,523 clonotypes from 15 healthy individuals including 6 monozygotic twin pairs. Public TCR α- and, to a lesser extent, TCR ß-chain sequences were common in all individuals. In contrast, sharing of entirely identical TCRαß amino acid sequences was very infrequent in unrelated individuals, but highly increased in twins, in particular in CD4 memory T cells. Based on nucleotide sequence identity, a subset of these shared clonotypes appeared to be the progeny of T cells that had been generated during fetal development and had persisted for more than 50 y. Additional shared TCRαß in twins were encoded by different nucleotide sequences, implying that genetic determinants impose structural constraints on thymic selection that favor the selection of TCR α-ß pairs with entire sequence identities.


Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/genética , Gêmeos Monozigóticos/genética , Adulto , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Linfócitos T CD4-Positivos/metabolismo , Conjuntos de Dados como Assunto , Feminino , Antígenos HLA/genética , Antígenos HLA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Teste de Histocompatibilidade , Humanos , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Análise de Sequência de DNA , Análise de Célula Única
16.
Nat Commun ; 10(1): 5579, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31811120

RESUMO

Although influenza viruses lead to severe illness in high-risk populations, host genetic factors associated with severe disease are largely unknown. As the HLA-A*68:01 allele can be linked to severe pandemic 2009-H1N1 disease, we investigate a potential impairment of HLA-A*68:01-restricted CD8+ T cells to mount robust responses. We elucidate the HLA-A*68:01+CD8+ T cell response directed toward an extended influenza-derived nucleoprotein (NP) peptide and show that only ~35% individuals have immunodominant A68/NP145+CD8+ T cell responses. Dissecting A68/NP145+CD8+ T cells in low vs. medium/high responders reveals that high responding donors have A68/NP145+CD8+ memory T cells with clonally expanded TCRαßs, while low-responders display A68/NP145+CD8+ T cells with predominantly naïve phenotypes and non-expanded TCRαßs. Single-cell index sorting and TCRαß analyses link expansion of A68/NP145+CD8+ T cells to their memory potential. Our study demonstrates the immunodominance potential of influenza-specific CD8+ T cells presented by a risk HLA-A*68:01 molecule and advocates for priming CD8+ T cell compartments in HLA-A*68:01-expressing individuals for establishment of pre-existing protective memory T cell pools.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígenos HLA-A/imunologia , Antígenos HLA-A/metabolismo , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Apresentação do Antígeno , Antígenos Virais/química , Linhagem Celular , Proteção Cruzada , Reações Cruzadas/imunologia , Epitopos de Linfócito T/imunologia , Antígenos HLA-A/química , Antígenos HLA-A/genética , Humanos , Memória Imunológica/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Modelos Moleculares , Nucleoproteínas/química , Orthomyxoviridae/genética , Orthomyxoviridae/imunologia , Fragmentos de Peptídeos/química , Fenótipo , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Proteínas do Core Viral/genética
17.
Nat Commun ; 10(1): 4967, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31672972

RESUMO

To build or dissect complex pathways in bacteria and mammalian cells, it is often necessary to recur to at least two plasmids, for instance harboring orthogonal inducible promoters. Here we present SiMPl, a method based on rationally designed split enzymes and intein-mediated protein trans-splicing, allowing the selection of cells carrying two plasmids with a single antibiotic. We show that, compared to the traditional method based on two antibiotics, SiMPl increases the production of the antimicrobial non-ribosomal peptide indigoidine and the non-proteinogenic aromatic amino acid para-amino-L-phenylalanine from bacteria. Using a human T cell line, we employ SiMPl to obtain a highly pure population of cells double positive for the two chains of the T cell receptor, TCRα and TCRß, using a single antibiotic. SiMPl has profound implications for metabolic engineering and for constructing complex synthetic circuits in bacteria and mammalian cells.


Assuntos
Antibacterianos , Bactérias/enzimologia , Farmacorresistência Bacteriana , Inteínas , Engenharia Metabólica/métodos , Plasmídeos/genética , Processamento de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/metabolismo , Resistência a Ampicilina , Linhagem Celular , Resistência ao Cloranfenicol , Cinamatos , Humanos , Higromicina B/análogos & derivados , Piperidonas , Puromicina , Trans-Splicing
18.
Anticancer Res ; 39(11): 5911-5918, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31704815

RESUMO

BACKGROUND/AIM: Double-negative T (DNT) cells are phenotypically CD3+CD4-CD8-T cells. This study aimed to investigate the anti-cancer activity of DNT cells against pancreatic cancer cells. MATERIALS AND METHODS: DNT cells were isolated from human peripheral blood. The effect of DNT cells on proliferation and invasion of the human pancreatic cell line Panc-1 was assessed. Expression of Nrf2 and Fas in Panc-1 cells co-cultured with DNT cells was analyzed with RT-PCR. The supernatants of Panc-1 and DNT co-cultures were analyzed with ELISA for IFN-r and FasL levels. RESULTS: The isolated DNT cell phenotype was CD4-CD8-CD56- CD3+TCR (T cell receptor) α/ß+ T cells with more than 90% purity. Panc-1 cell proliferation was significantly inhibited by co-culture with DNT cells. Panc-1 cells co-cultured with DNT cells showed significantly reduced cell invasion. Panc-1 cells co-cultured with DNT cells showed increased Nrf2 and Fas mRNA expression. Increased INF-r and FasL levels were detected in the supernatants of co-cultures of DNT and pancreatic cells. CONCLUSION: DNT cells inhibited proliferation and invasion of human pancreatic cancer cells. The INF-r, Fas/FasL pathway and Nrf2 may be involved in the anti-cancer effect of DNT cells against human pancreatic cancer.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Técnicas de Cocultura/métodos , Ativação Linfocitária/imunologia , Neoplasias Pancreáticas/prevenção & controle , Apoptose , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Proteína Ligante Fas/metabolismo , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Interferon/metabolismo , Células Tumorais Cultivadas , Receptor fas/metabolismo
19.
Proc Natl Acad Sci U S A ; 116(51): 25923-25931, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31772015

RESUMO

Streptococcal toxic shock syndrome (STSS) is a rapidly progressing, life-threatening, systemic reaction to invasive infection caused by group A streptococci (GAS). GAS superantigens are key mediators of STSS through their potent activation of T cells leading to a cytokine storm and consequently vascular leakage, shock, and multiorgan failure. Mucosal-associated invariant T (MAIT) cells recognize MR1-presented antigens derived from microbial riboflavin biosynthesis and mount protective innate-like immune responses against the microbes producing such metabolites. GAS lack de novo riboflavin synthesis, and the role of MAIT cells in STSS has therefore so far been overlooked. Here we have conducted a comprehensive analysis of human MAIT cell responses to GAS, aiming to understand the contribution of MAIT cells to the pathogenesis of STSS. We show that MAIT cells are strongly activated and represent the major T cell source of IFNγ and TNF in the early stages of response to GAS. MAIT cell activation is biphasic with a rapid TCR Vß2-specific, TNF-dominated response to superantigens and a later IL-12- and IL-18-dependent, IFNγ-dominated response to both bacterial cells and secreted factors. Depletion of MAIT cells from PBMC resulted in decreased total production of IFNγ, IL-1ß, IL-2, and TNFß. Peripheral blood MAIT cells in patients with STSS expressed elevated levels of the activation markers CD69, CD25, CD38, and HLA-DR during the acute compared with the convalescent phase. Our data demonstrate that MAIT cells are major contributors to the early cytokine response to GAS, and are therefore likely to contribute to the pathological cytokine storm underlying STSS.


Assuntos
Citocinas/metabolismo , Células T Invariáveis Associadas à Mucosa/imunologia , Choque Séptico/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Adulto , Idoso , Citocinas/sangue , Antígenos HLA-DR/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-12/metabolismo , Interleucina-18/metabolismo , Interleucina-1alfa/metabolismo , Interleucina-2/metabolismo , Linfotoxina-alfa/metabolismo , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Riboflavina/biossíntese , Streptococcus pyogenes/patogenicidade , Superantígenos/metabolismo
20.
PLoS Pathog ; 15(11): e1008122, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31765434

RESUMO

The T cell receptor (TCR) repertoire is an essential component of the CD8 T-cell immune response. Here, we seek to investigate factors that drive selection of TCR repertoires specific to the HLA-A2-restricted immunodominant epitope BRLF1109-117 (YVLDHLIVV) over the course of primary Epstein Barr virus (EBV) infection. Using single-cell paired TCRαß sequencing of tetramer sorted CD8 T cells ex vivo, we show at the clonal level that recognition of the HLA-A2-restricted BRLF1 (YVL-BR, BRLF-1109) epitope is mainly driven by the TCRα chain. For the first time, we identify a CDR3α (complementarity determining region 3 α) motif, KDTDKL, resulting from an obligate AV8.1-AJ34 pairing that was shared by all four individuals studied. This observation coupled with the fact that this public AV8.1-KDTDKL-AJ34 TCR pairs with multiple different TCRß chains within the same donor (median 4; range: 1-9), suggests that there are some unique structural features of the interaction between the YVL-BR/MHC and the AV8.1-KDTDKL-AJ34 TCR that leads to this high level of selection. Newly developed TCR motif algorithms identified a lysine at position 1 of the CDR3α motif that is highly conserved and likely important for antigen recognition. Crystal structure analysis of the YVL-BR/HLA-A2 complex revealed that the MHC-bound peptide bulges at position 4, exposing a negatively charged aspartic acid that may interact with the positively charged lysine of CDR3α. TCR cloning and site-directed mutagenesis of the CDR3α lysine ablated YVL-BR-tetramer staining and substantially reduced CD69 upregulation on TCR mutant-transduced cells following antigen-specific stimulation. Reduced activation of T cells expressing this CDR3 motif was also observed following exposure to mutated (D4A) peptide. In summary, we show that a highly public TCR repertoire to an immunodominant epitope of a common human virus is almost completely selected on the basis of CDR3α and provide a likely structural basis for the selection. These studies emphasize the importance of examining TCRα, as well as TCRß, in understanding the CD8 T cell receptor repertoire.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Proteínas Imediatamente Precoces/imunologia , Epitopos Imunodominantes/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T Citotóxicos/imunologia , Transativadores/imunologia , Sequência de Aminoácidos , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Epitopos de Linfócito T/imunologia , Infecções por Vírus Epstein-Barr/virologia , Antígeno HLA-A2/imunologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transativadores/genética , Transativadores/metabolismo
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