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1.
Cancer Immunol Immunother ; 68(12): 1979-1993, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31686124

RESUMO

5T4 (trophoblast glycoprotein, TPBG) is a transmembrane tumor antigen expressed on more than 90% of primary renal cell carcinomas (RCC) and a wide range of human carcinomas but not on most somatic adult tissues. The favorable expression pattern has encouraged the development and clinical testing of 5T4-targeted antibody and vaccine therapies. 5T4 also represents a compelling and unexplored target for T-cell receptor (TCR)-engineered T-cell therapy. Our group has previously isolated high-avidity CD8+ T-cell clones specific for an HLA-A2-restricted 5T4 epitope (residues 17-25; 5T4p17). In this report, targeted single-cell RNA sequencing was performed on 5T4p17-specific T-cell clones to sequence the highly variable complementarity-determining region 3 (CDR3) of T-cell receptor α chain (TRA) and ß chain (TRB) genes. Full-length TRA and TRB sequences were cloned into lentiviral vectors and transduced into CD8+ T-cells from healthy donors. Redirected effector T-cell function against 5T4p17 was measured by cytotoxicity and cytokine release assays. Seven unique TRA-TRB pairs were identified. All seven TCRs exhibited high expression on CD8+ T-cells with transduction efficiencies from 59 to 89%. TCR-transduced CD8+ T-cells demonstrated redirected cytotoxicity and cytokine release in response to 5T4p17 on target-cells and killed 5T4+/HLA-A2+ kidney-, breast-, and colorectal-tumor cell lines as well as primary RCC tumor cells in vitro. TCR-transduced CD8+ T-cells also detected presentation of 5T4p17 in TAP1/2-deficient T2 target-cells. TCR-transduced T-cells redirected to recognize the 5T4p17 epitope from a broadly shared tumor antigen are of interest for future testing as a cellular immunotherapy strategy for HLA-A2+ subjects with 5T4+ tumors.


Assuntos
Linfócitos T CD8-Positivos/fisiologia , Vacinas Anticâncer/imunologia , Carcinoma de Células Renais/terapia , Epitopos de Linfócito T/metabolismo , Imunoterapia Adotiva/métodos , Neoplasias Renais/terapia , Glicoproteínas de Membrana/metabolismo , Linfócitos T CD8-Positivos/transplante , Carcinoma de Células Renais/imunologia , Células Clonais , Citotoxicidade Imunológica , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Neoplasias Renais/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética
2.
Mol Immunol ; 116: 174-179, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31704500

RESUMO

We considered the possibility that the greater the distance between an immune receptor V and J, the more likely the V usage. Such a hypothesis is supported by results from mouse experiments. And, such a hypothesis is consistent with the fundamental nature of recombination and genomic distance: the further the distance, the greater the chance of a DNA break. Thus, we exploited the vast dataset of V and J recombination reads available for the human TRA gene, particularly from cancer and blood specimens, to assess the frequency of TRAV usage with respect to distance from the TRAJ cluster. Results indicated that, indeed, over the entire TRAV cluster, there is a greater chance of V usage the further the distance from the J cluster. These results do not address causation, and are not consistent for certain individual V gene segments, but the results do indicate that overall, the larger the distance between the V and J gene segment cluster, the more likely the appearance of at least a subset of TRAV segments, particularly among tumor infiltrating lymphocytes. With a similar approach, the distal TRAV gene segments were also found to be more commonly associated with a subset of distal TRAJ segments. These results have implications for restrictions on the apparent TRA repertoire in disease settings.


Assuntos
Família Multigênica/genética , Neoplasias/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Humanos , Linfócitos do Interstício Tumoral/imunologia , Família Multigênica/imunologia , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia
3.
Cancer Sci ; 110(10): 3038-3048, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31385405

RESUMO

Retroperitoneal liposarcoma (RLPS) is one of the most common subtypes of retroperitoneal soft tissue sarcomas and lacks effective treatment. This study aimed to provide a thorough profile of immune characteristics of RLPS. This study included 56 RLPS patients. Multisite tumor tissues were collected from 16 patients. Immunohistochemistry was carried out to identify CD4+ , CD8+ , FoxP3+ , CD20+ , or programmed cell death-1 (PD-1)+ tumor infiltrating lymphocytes (TILs) and  Programmed cell death ligand-1 (PD-L1) expression in tumor tissues. Ultradeep sequencing of T-cell receptor (TCR) ß-chain gene was carried out in 42 tumor samples as well as peripheral blood samples collected from 6 patients. In RLPS, TILs were distributed in 3 patterns and T cells were more prevalent than B cells. Generally, the proportion of TILs decreased and PD-L1 expression increased with tumor progression. Patients with higher PD-1/PD-L1 expression tended to have poorer prognosis, whereas patients with tertiary lymphoid structure tended to have a favorable disease-free survival. Although T-cell clones in tumors were quite different from those in peripheral blood, TCR sequencing showed low TCR repertoire reads as well as polyclonal status within tumors, which indicated limited T cell response in the tumors. Both TILs distribution and TCR repertoires suggested spatial immune heterogeneity in RLPS. Our research described the immune landscape of RLPS, and suggested RLPS might be a kind of tumor with low T cell infiltration as well as great immune heterogeneity. Therefore, strategies that can facilitate lymphocytic infiltration and immune reactivity need to be developed in the future to improve the efficacy of immunotherapy.


Assuntos
Antígeno B7-H1/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Lipossarcoma/imunologia , Linfócitos do Interstício Tumoral/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Neoplasias Retroperitoneais/imunologia , Adulto , Idoso , Linfócitos B/imunologia , Intervalo Livre de Doença , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Lipossarcoma/genética , Lipossarcoma/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias Retroperitoneais/genética , Neoplasias Retroperitoneais/mortalidade , Análise de Sequência de DNA , Linfócitos T/imunologia , Regulação para Cima
4.
Nat Commun ; 10(1): 3353, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350389

RESUMO

The diverse repertoire of T cell receptors (TCR) and immunoglobulins is generated through the somatic rearrangement of respective V, D and J gene segments, termed V(D)J recombination, during early T or B cell development. However, epigenetic regulation of V(D)J recombination is still not fully understood. Here we show that the deficiency of Setd2, a histone methyltransferase that catalyzes lysine 36 trimethylation on histone 3 (H3K36me3) in mice, causes a severe developmental block of thymocytes at the CD4-CD8- DN3 stage. While H3K36me3 is normally enriched at the TCRß locus, Setd2 deficiency reduces TCRß H3K36me3 and suppresses TCRß V(D)J rearrangement by impairing RAG1 binding to TCRß loci and the DNA double-strand break repair. Similarly, Setd2 ablation also impairs immunoglobulin V(D)J rearrangement to induce B cell development block at the pro-B stage. Lastly, SETD2 is frequently mutated in patients with primary immunodeficiency. Our study thus demonstrates that Setd2 is required for optimal V(D)J recombination and normal lymphocyte development.


Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Recombinação V(D)J , Motivos de Aminoácidos , Animais , Linfócitos B/citologia , Linfócitos B/enzimologia , Diferenciação Celular , Pré-Escolar , Epigênese Genética , Histona-Lisina N-Metiltransferase/genética , Histonas/química , Histonas/metabolismo , Humanos , Lactente , Lisina/genética , Lisina/metabolismo , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , /genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/citologia
5.
Environ Toxicol Pharmacol ; 71: 103211, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31212134

RESUMO

We exploratively characterized T cell receptor (TCR) repertoires from occupational medicamentosa-like dermatitis due to trichloroethylene (OMDT) patients to better understand the underlying pathological mechanism. We used a combination of multiplex-PCR, Illumina sequencing and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR ß-chain complementarity-determining region 3 (CDR3) gene in 10 OMDT cases and 10 trichloroethylene-exposed healthy tolerant controls. Compared with the tolerant controls, OMDT cases showed no significant difference in TCR repertoire diversity including repertoire breadth, highly expanded clone, and CDR3 length distribution. However, we observed several differences in TRBV/TRBJ usage and combination between the two groups, as well as some shared and unique T cell clones in the cases. The pilot study delineated some features of TCR repertoire in OMDT patients that warrant further investigation.


Assuntos
Regiões Determinantes de Complementaridade/sangue , Erupção por Droga/sangue , Doenças Profissionais/sangue , Exposição Ocupacional/análise , Receptores de Antígenos de Linfócitos T alfa-beta/sangue , Tricloroetileno/toxicidade , Adulto , Estudos de Casos e Controles , Regiões Determinantes de Complementaridade/genética , Erupção por Droga/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Exposição Ocupacional/efeitos adversos , Projetos Piloto , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/efeitos dos fármacos
6.
PLoS One ; 14(5): e0216815, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31071196

RESUMO

Using induced pluripotent stem cells (iPSCs) to derive chimeric antigen receptor-modified T (CAR-T) cells has great industrial potential. A previous study used αß T cell-derived CAR-modified iPSCs to produce CAR-T cells. However, these αß T cells are restricted to autologous use and only recognize single cancer antigen. To make CAR-T alternative for allogeneic use, we reprogrammed γδ T cell into iPSCs (γδ T-iPSCs) to circumvent the risk of graft-versus-host disease. To target multiple cancer-associated antigens, we used an "NK cell-promoting" protocol to differentiate γδ T-iPSCs and to induce expression of natural killer receptors (NKRs). Through such two-step strategy, mimetic γδ T cells endowed with an array of NKRs and thus designated as "γδ natural killer T (γδ NKT) cells" were derived. With no/low-level expression of inhibitory killer cell immunoglobulin-like receptors (KIRs) and immune checkpoint receptors, γδ NKT cells may provide a potent "off-the-shelf" cytotoxic cell source to recognize multiple ubiquitous antigens in a broad spectrum of cancers.


Assuntos
Antígenos de Neoplasias/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Células T Matadoras Naturais/imunologia , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos Quiméricos/imunologia , Antígenos de Neoplasias/genética , Células HCT116 , Células Hep G2 , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Células K562 , Células MCF-7 , Células T Matadoras Naturais/patologia , Neoplasias/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos Quiméricos/genética , Células THP-1
7.
Cancer Immunol Immunother ; 68(7): 1095-1106, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31104075

RESUMO

Checkpoint blockade immunotherapy is now a first-line treatment option for patients with melanoma. Despite achieving objective responses in about half of patients, the exact immune mechanisms elicited and those required for therapeutic success have not been clearly identified. Insight into these mechanisms is key for improving outcomes in a broader range of cancer patients. We used a murine melanoma model to track responses by different subsets of tumor-infiltrating lymphocytes (TIL) during checkpoint blockade immunotherapy. Tumors from treated mice had increased frequencies of both CD4+ and CD8+ T cells, which also showed evidence of functional reinvigoration and elevated effector cytokine production after immunotherapy. We predicted that increased T cell numbers and function within tumors reflected either infiltration by new T cells or clonal expansion by a few high-affinity tumor-reactive T cells. To address this, we compared TIL diversity before and after immunotherapy by sequencing the complementarity determining region 3 (CDR3) of all T cell receptor beta (TCRß) genes. While checkpoint blockade effectively slowed tumor progression and increased T cell frequencies, the diversity of intratumoral T cells remained stable. This was true when analyzing total T cells and when focusing on smaller subsets of effector CD4+ and CD8+ TIL as well as regulatory T cells. Our study suggests that checkpoint blockade immunotherapy does not broaden the T cell repertoire within murine melanoma tumors, but rather expands existing T cell populations and enhances effector capabilities.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Neoplasias Cutâneas/tratamento farmacológico , Animais , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Antígeno CTLA-4/antagonistas & inibidores , Linhagem Celular Tumoral , Regiões Determinantes de Complementaridade/genética , Humanos , Imunoterapia/métodos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia
8.
Nat Commun ; 10(1): 2243, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113973

RESUMO

Mucosal-associated invariant T (MAIT) cells express an invariant TRAV1/TRAJ33 TCR-α chain and are restricted to the MHC-I-like molecule, MR1. Whether MAIT cell development depends on this invariant TCR-α chain is unclear. Here we generate Traj33-deficient mice and show that they are highly depleted of MAIT cells; however, a residual population remains and can respond to exogenous antigen in vitro or pulmonary Legionella challenge in vivo. These residual cells include some that express Trav1+ TCRs with conservative Traj-gene substitutions, and others that express Trav1- TCRs with a broad range of Traj genes. We further report that human TRAV1-2- MR1-restricted T cells contain both MAIT-like and non-MAIT-like cells, as judged by their TCR repertoire, antigen reactivity and phenotypic features. These include a MAIT-like population that expresses a public, canonical TRAV36+ TRBV28+ TCR. Our findings highlight the TCR diversity and the resulting potential impact on antigen recognition by MR1-restricted T cells.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Legionelose/imunologia , Antígenos de Histocompatibilidade Menor/metabolismo , Células T Invariáveis Associadas à Mucosa/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Animais , Apresentação do Antígeno/imunologia , Modelos Animais de Doenças , Células HEK293 , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Legionella/imunologia , Legionelose/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/imunologia , Células T Invariáveis Associadas à Mucosa/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia
9.
Scand J Immunol ; 90(2): e12790, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31127959

RESUMO

There is a sharp difference in how one views TCR structure-function-behaviour dependent on whether its recognition of major histocompatibility complex-encoded restriction elements (R) is germline selected or somatically generated. The generally accepted or Standard model is built on the assumption that recognition of R is by the V regions of the αß TCR, which is not driven by allele specificity, whereas the competing model posits that recognition of R is allele-specific. The establishing of allele-specific recognition of R by the TCR would rule out the Standard model and clear the road to a consideration of a competing construct, the Tritope model. Here, the case for allele-specific recognition (germline selected) is detailed making it obvious that the Standard model is untenable.


Assuntos
Alelos , Complexo Principal de Histocompatibilidade/imunologia , Modelos Biológicos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Haplótipos/genética , Humanos , Polimorfismo Genético/genética
10.
Methods Mol Biol ; 1979: 197-224, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028640

RESUMO

Single-cell sequencing of TCR alleles enables determination of T cell specificity. Here we describe a sensitive protocol for targeted amplification of TCR CDR3 regions from single-cell full-length cDNA libraries. By exploiting the specificity of RNase H-dependent PCR (rhPCR), the protocol achieves amplification of TCR alleles and addition of cell barcodes in a single PCR step.


Assuntos
Alelos , Biblioteca Gênica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Análise de Célula Única/métodos , Sequência de Bases , Regiões Determinantes de Complementaridade/genética , Primers do DNA/genética , Humanos , Reação em Cadeia da Polimerase/métodos
11.
PLoS Comput Biol ; 15(3): e1006874, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30830899

RESUMO

The T-cell (TCR) repertoire relies on the diversity of receptors composed of two chains, called α and ß, to recognize pathogens. Using results of high throughput sequencing and computational chain-pairing experiments of human TCR repertoires, we quantitively characterize the αß generation process. We estimate the probabilities of a rescue recombination of the ß chain on the second chromosome upon failure or success on the first chromosome. Unlike ß chains, α chains recombine simultaneously on both chromosomes, resulting in correlated statistics of the two genes which we predict using a mechanistic model. We find that ∼35% of cells express both α chains. Altogether, our statistical analysis gives a complete quantitative mechanistic picture that results in the observed correlations in the generative process. We learn that the probability to generate any TCRαß is lower than 10(-12) and estimate the generation diversity and sharing properties of the αß TCR repertoire.


Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Cromossomos Humanos , Humanos , Probabilidade , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Recombinação Genética
13.
Immunol Lett ; 208: 44-51, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30905825

RESUMO

T cell immunity is dependent on T cell receptor (TCR) recognition of large numbers of antigenic peptides presented in the context of major histocompatibility complex (MHC) class I and II molecules. To explore whether the TCR ß-chain repertoire changes with different ages and genders in healthy Chinese individuals, we analyzed the TCR ß-chain repertoire in 154 healthy Chinese individuals by High-throughput sequencing (HTS) with age (age range: 6-70) and each age group contains about 20 individuals (male and female). Here we report that the extent of TCR diversity was not dependent on gender but it was significantly different between age groups. We found that the rearrangement of the V with J genes in T cell receptor ß-chain was highest at the age of 21-30. Moreover, we found that the combination of V6-1 and J2-1 gene had the highest expression over a human lifespan. We further identified J2-1 and V7-2 gene expressed higher at the age of 6-10 and 11-20 than other age groups. However, D gene in TCR ß-chain was not significantly diverse in different age groups. In addition, the T cell receptor ß complementarity-determining region 3 (CDR3) amino acid sequence expressed the highest between the age of 21-30. These results showed different features of TCR ß-chain repertoire in different groups of healthy Chinese individuals. In conclusion, it was expected that these TCR repertoires could serve as a useful tool for investigating the role of immune profiling in healthy Chinese individuals. These results stress the importance of considering age as a factor for immune response.


Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adolescente , Adulto , Fatores Etários , Sequência de Aminoácidos , Criança , Feminino , Variação Genética , Voluntários Saudáveis , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Família Multigênica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Fatores Sexuais , Recombinação V(D)J , Adulto Jovem
14.
PLoS One ; 14(3): e0213684, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30870493

RESUMO

Current methods to quantify T-cell clonal expansion only account for variance due to random sampling from a highly diverse repertoire space. We propose a beta-binomial model to incorporate time-dependent variance into the assessment of differentially abundant T-cell clones, identified by unique T Cell Receptor (TCR) ß-chain rearrangements, and show that this model improves specificity for detecting clinically relevant clonal expansion. Using blood samples from ten healthy donors, we modeled the variance of T-cell clones within each subject over time and calibrated the dispersion parameters of the beta distribution to fit this variance. As a validation, we compared pre- versus post-treatment blood samples from urothelial cancer patients treated with atezolizumab, where clonal expansion (quantified by the earlier binomial model) was previously reported to correlate with benefit. The beta-binomial model significantly reduced the false-positive rate for detecting differentially abundant clones over time compared to the earlier binomial method. In the urothelial cancer cohort, the beta-binomial model enriched for tumor infiltrating lymphocytes among the clones detected as expanding in the peripheral blood in response to therapy compared to the binomial model and improved the overall correlation with clinical benefit. Incorporating time-dependent variance into the statistical framework for measuring differentially abundant T-cell clones improves the model's specificity for T-cells that correlate more strongly with the disease and treatment setting of-interest. Reducing background-level clonal expansion, therefore, improves the quality of clonal expansion as a biomarker for assessing the T cell immune response and correlations with clinical measures.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Linfócitos T/citologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/imunologia , Urotélio/patologia , Adulto , Biomarcadores Tumorais , Reações Falso-Positivas , Feminino , Humanos , Linfócitos do Interstício Tumoral/citologia , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Reprodutibilidade dos Testes , Resultado do Tratamento
16.
Diabetes ; 68(5): 1002-1013, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30796028

RESUMO

Insulin is a major autoantigen in type 1 diabetes, targeted by both CD8 and CD4 T cells. We studied an insulin-reactive T-cell receptor (TCR) α-chain transgenic NOD mouse on a TCRCα and proinsulin 2 (PI2)-deficient background, designated as A22Cα-/-PI2-/- NOD mice. These mice develop a low incidence of autoimmune diabetes. To test the role of gut microbiota on diabetes development in this model system, we treated the A22Cα-/-PI2-/- NOD mice with enrofloxacin, a broad-spectrum antibiotic. The treatment led to male mice developing accelerated diabetes. We found that enrofloxacin increased the frequency of the insulin-reactive CD8+ T cells and activated the cells in the Peyer's patches and pancreatic lymph nodes, together with induction of immunological effects on the antigen-presenting cell populations. The composition of gut microbiota differed between the enrofloxacin-treated and untreated mice and also between the enrofloxacin-treated mice that developed diabetes compared with those that remained normoglycemic. Our results provide evidence that the composition of the gut microbiota is important for determining the expansion and activation of insulin-reactive CD8+ T cells.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Microbioma Gastrointestinal/fisiologia , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Enrofloxacina/uso terapêutico , Microbioma Gastrointestinal/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Proinsulina/genética , Proinsulina/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo
17.
Int J Cancer ; 145(5): 1423-1431, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30664810

RESUMO

Lung cancer is one of the greatest threats to human health, and is initially detected and attacked by the immune system through tumor-reactive T cells. The aim of this study was to determine the basic characteristics and clinical significance of the peripheral blood T-cell receptor (TCR) repertoire in patients with advanced lung cancer. To comprehensively profile the TCR repertoire, high-throughput sequencing was used to identify hypervariable rearrangements of complementarity determining region 3 (CDR3) of the TCR ß chain in peripheral blood samples from 64 advanced lung cancer patients and 31 healthy controls. We found that the TCR repertoire differed substantially between lung cancer patients and healthy controls in terms of CDR3 clonotype, diversity, V/J segment usage, and sequence. Specifically, baseline diversity correlated with several clinical characteristics, and high diversity reflected a better immune status. Dynamic detection of the TCR repertoire during anticancer treatment was useful for prognosis. Both increased diversity and high overlap rate between the pre- and post-treatment TCR repertoires indicated clinical benefit. Combination of the diversity and overlap rate was used to categorize patients into immune improved or immune worsened groups and demonstrated enhanced prognostic significance. In conclusion, TCR repertoire analysis served as a useful indicator of disease development and prognosis in advanced lung cancer and may be utilized to direct future immunotherapy.


Assuntos
Neoplasias Pulmonares/sangue , Receptores de Antígenos de Linfócitos T/sangue , Linfócitos T/imunologia , Estudos de Casos e Controles , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/sangue , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/patologia
18.
J Immunol ; 202(3): 637-644, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30670579

RESUMO

Dual TCR T cells are a common and natural product of TCR gene rearrangement and thymocyte development. As much as one third of the T cell population may have the capability to express two different TCR specificities on the cell surface. This discovery provoked a reconsideration of the classic model of thymic selection. Many potential roles for dual TCR T cells have since been hypothesized, including posing an autoimmune hazard, dominating alloreactive T cell responses, inducing allergy, and expanding the TCR repertoire to improve protective immunity. Yet, since the initial wave of publications following the discovery of dual TCR T cells, research in the area has slowed. In this study, we aim to provide a brief but comprehensive history of dual TCR T cell research, re-evaluate past observations in the context of current knowledge of the immune system, and identify key issues for future study.


Assuntos
Ativação Linfocitária , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Alelos , Animais , Humanos , Camundongos , Transdução de Sinais , Timo/citologia , Timo/imunologia
19.
Mol Immunol ; 106: 170-177, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30623817

RESUMO

Stevens-Johnson syndrome (SJS) /toxic epidermal necrolysis (TEN) are life-threatening severe cutaneous adverse drug reactions characterized by widespread epidermal necrosis. Recent studies have indicated that SJS/TEN is a specific immune reaction regulated by T cells. Certain drug serves as foreign antigens that are presented by major histocompatibility complex (MHC) and recognized by T cell receptors (TCRs), inducing adaptive immune responses. However, few studies have performed detailed characterization of TCR repertoire in SJS/TEN, and it remains unclear whether the particular types of TCRs expanded clonally are drug-specific, which would provide a potential underlying mechanism of SJS/TEN. In this study, using high-throughput sequencing, we comprehensively assessed the diversity, composition and molecular characteristics of the TCRß repertoires in 17 SJS/TEN patients associated with three different causative drugs including methazolamide (MZ), carbamazepine (CBZ) and allopurinol (ALP). Systematic analysis of the TCRß sequences revealed that SJS/TEN patients had more highly expanded clones and less TCR repertoire diversity, and the TCR repertoire diversity of these patients showed certain associations with the clinical severity of disease. Similar predominant clonotypes, shared-usage TRBV/TRBJ subtypes and combinations thereof were observed among different subjects with the same causative agent. Our observations provide enhanced understanding of the role of T lymphocytes in the pathogenesis of SJS/TEN and enumerate potential therapeutic targets.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Síndrome de Stevens-Johnson/genética , Alopurinol/administração & dosagem , Alopurinol/efeitos adversos , Carbamazepina/administração & dosagem , Carbamazepina/efeitos adversos , Feminino , Humanos , Masculino , Metazolamida/administração & dosagem , Metazolamida/efeitos adversos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Síndrome de Stevens-Johnson/imunologia , Síndrome de Stevens-Johnson/patologia
20.
Hum Immunol ; 80(3): 195-203, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30576702

RESUMO

T cell receptors (TCRs) are a class of T cell surface molecules that recognize the antigen-derived peptides presented by the major histocompatibility complex (MHC) and are able to trigger a series of immune responses. TCRs are important members of the adaptive immune system that arose in the jawed fish 500 million years ago. T cell receptor beta variable (TRBV) genes have been widely used to characterize TCR repertoires. Studying the evolution of TRBV may help us to better understand the adaptive immune system. To investigate TRBV evolution and its impacts on the usages of TRBV genes in human populations, we compared the TRBV genes and their homologous sequences among humans, mouse, rhesus and chimpanzee, analyzed the single-nucleotide polymorphisms (SNPs) located at TRBV loci, and sequenced TCR repertoires in the peripheral blood of 97 healthy donors. We found that functional TRBVs are more evolutionarily conserved but possess more SNPs in human populations than do nonfunctional (pseudo) TRBVs. Based on the conservation levels in the four species, we classified the functional TRBVs into 2 groups: old (conserved between mouse and humans) and new (conserved only in primates). The new TRBVs evolve faster and possess more SNPs than the old TRBVs. The variations in TRBV genes frequencies in the peripheral blood of healthy donors are negatively correlated with SNP density. These observations suggest that TRBV usages may be influenced by TCR-MHC co-evolution.


Assuntos
Evolução Molecular , Variação Genética , Genética Populacional , Polimorfismo de Nucleotídeo Único , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Animais , Voluntários Saudáveis , Humanos , Camundongos , Filogenia , Primatas , Linfócitos T/metabolismo
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