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1.
Adv Exp Med Biol ; 1255: 29-50, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32949388

RESUMO

T cells recognize peptides bound to major histocompatibility complex (MHC) class I and class II molecules at the cell surface. This recognition is accomplished by the expression of T cell receptors (TCR) which are required to be diverse and adaptable in order to accommodate the various and vast number of antigens presented on the MHCs. Thus, determining TCR repertoires of effector T cells is necessary to understand the immunological process in responding to cancer progression, infection, and autoimmune development. Furthermore, understanding the TCR repertoires will provide a solid framework to predict and test the antigen which is more critical in autoimmunity. However, it has been a technical challenge to sequence the TCRs and provide a conceptual context in correlation to the vast number of TCR repertoires in the immunological system. The exploding field of single-cell sequencing has changed how the repertoires are being investigated and analyzed. In this review, we focus on the biology of TCRs, TCR signaling and its implication in autoimmunity. We discuss important methods in bulk sequencing of many cells. Lastly, we explore the most pertinent platforms in single-cell sequencing and its application in autoimmunity.


Assuntos
Receptores de Antígenos de Linfócitos T/genética , Análise de Sequência , Análise de Célula Única , Animais , Autoimunidade/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo
3.
Signal Transduct Target Ther ; 5(1): 156, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796814

RESUMO

The global Coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 has affected more than eight million people. There is an urgent need to investigate how the adaptive immunity is established in COVID-19 patients. In this study, we profiled adaptive immune cells of PBMCs from recovered COVID-19 patients with varying disease severity using single-cell RNA and TCR/BCR V(D)J sequencing. The sequencing data revealed SARS-CoV-2-specific shuffling of adaptive immune repertories and COVID-19-induced remodeling of peripheral lymphocytes. Characterization of variations in the peripheral T and B cells from the COVID-19 patients revealed a positive correlation of humoral immune response and T-cell immune memory with disease severity. Sequencing and functional data revealed SARS-CoV-2-specific T-cell immune memory in the convalescent COVID-19 patients. Furthermore, we also identified novel antigens that are responsive in the convalescent patients. Altogether, our study reveals adaptive immune repertories underlying pathogenesis and recovery in severe versus mild COVID-19 patients, providing valuable information for potential vaccine and therapeutic development against SARS-CoV-2 infection.


Assuntos
Linfócitos B/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/imunologia , Imunidade Celular , Imunidade Humoral , Pneumonia Viral/imunologia , Linfócitos T/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Linfócitos B/classificação , Linfócitos B/virologia , Betacoronavirus/imunologia , Estudos de Casos e Controles , China , Convalescença , Infecções por Coronavirus/genética , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Progressão da Doença , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno/imunologia , Humanos , Memória Imunológica , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Receptores de Antígenos de Linfócitos B/classificação , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos T/classificação , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Índice de Gravidade de Doença , Análise de Célula Única , Linfócitos T/classificação , Linfócitos T/virologia
4.
Nat Commun ; 11(1): 4166, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32820173

RESUMO

T cells engineered to express chimeric antigen receptors (CAR-T cells) have shown impressive clinical efficacy in the treatment of B cell malignancies. However, the development of CAR-T cell therapies for solid tumors is hampered by the lack of truly tumor-specific antigens and poor control over T cell activity. Here we present an avidity-controlled CAR (AvidCAR) platform with inducible and logic control functions. The key is the combination of (i) an improved CAR design which enables controlled CAR dimerization and (ii) a significant reduction of antigen-binding affinities to introduce dependence on bivalent interaction, i.e. avidity. The potential and versatility of the AvidCAR platform is exemplified by designing ON-switch CARs, which can be regulated with a clinically applied drug, and AND-gate CARs specifically recognizing combinations of two antigens. Thus, we expect that AvidCARs will be a highly valuable platform for the development of controllable CAR therapies with improved tumor specificity.


Assuntos
Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica/imunologia , Humanos , Ativação Linfocitária/imunologia , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismo
5.
BMC Bioinformatics ; 21(1): 314, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677886

RESUMO

BACKGROUND: Recent advances in DNA sequencing technologies have enabled significant leaps in capacity to generate large volumes of DNA sequence data, which has spurred a rapid growth in the use of bioinformatics as a means of interrogating antibody variable gene repertoires. Common tools used for annotation of antibody sequences are often limited in functionality, modularity and usability. RESULTS: We have developed PyIR, a Python wrapper and library for IgBLAST, which offers a minimal setup CLI and API, FASTQ support, file chunking for large sequence files, JSON and Python dictionary output, and built-in sequence filtering. CONCLUSIONS: PyIR offers improved processing speed over multithreaded IgBLAST (version 1.14) when spawning more than 16 processes on a single computer system. Its customizable filtering and data encapsulation allow it to be adapted to a wide range of computing environments. The API allows for IgBLAST to be used in customized bioinformatics workflows.


Assuntos
Imunoglobulinas/genética , Receptores de Antígenos de Linfócitos T/genética , Alinhamento de Sequência , Software , Sequência de Bases , Humanos , Análise de Sequência de DNA , Fatores de Tempo , Interface Usuário-Computador
6.
Proc Natl Acad Sci U S A ; 117(24): 13730-13739, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32482869

RESUMO

Merkel cell carcinoma (MCC) is a lethal skin cancer that metastasizes rapidly. Few effective treatments are available for patients with metastatic MCC. Poor intratumoral T cell infiltration and activation are major barriers that prevent MCC eradication by the immune system. However, the mechanisms that drive the immunologically restrictive tumor microenvironment remain poorly understood. In this study, we discovered that the innate immune regulator stimulator of IFN genes (STING) is completely silenced in MCCs. To reactivate STING in MCC, we developed an application of a human STING mutant, STINGS162A/G230I/Q266I, which we found to be readily stimulated by a mouse STING agonist, DMXAA. This STING molecule was efficiently delivered to MCC cells via an AAV vector. Introducing STINGS162A/G230I/Q266I expression and stimulating its activity by DMXAA in MCC cells reactivates their antitumor inflammatory cytokine/chemokine production. In response to MCC cells with restored STING, cocultured T cells expressing MCPyV-specific T cell receptors (TCRs) show increased cytokine production, migration toward tumor cells, and tumor cell killing. Our study therefore suggests that STING deficiency contributes to the immune suppressive nature of MCCs. More importantly, DMXAA stimulation of STINGS162A/G230I/Q266I causes robust cell death in MCCs as well as several other STING-silenced cancers. Because tumor antigens and DNA released by dying cancer cells have the potential to amplify innate immune response and activate antitumor adaptive responses, our finding indicates that targeted delivery and activation of STINGS162A/G230I/Q266I in tumor cells holds great therapeutic promise for the treatment of MCC and many other STING-deficient cancers.


Assuntos
Carcinoma de Célula de Merkel/imunologia , Proteínas de Membrana/imunologia , Neoplasias Cutâneas/imunologia , Carcinoma de Célula de Merkel/genética , Linhagem Celular Tumoral , Humanos , Imunidade Inata , Proteínas de Membrana/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/genética , Xantonas/farmacologia
7.
Proc Natl Acad Sci U S A ; 117(24): 13659-13669, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32482872

RESUMO

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/imunologia
8.
Anticancer Res ; 40(4): 2043-2051, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32234895

RESUMO

BACKGROUND/AIM: While there has been a rapid development in genomic data mining approaches for T-cell receptor recombinations (TcR), less emphasis has been placed on B-cell receptor (BcR) recombinations. MATERIALS AND METHODS: We obtained lung cancer exome files from the cancer genome atlas (TCGA) and mined the files for TcR and BcR recombination reads. RESULTS: There was a robust detection of BcR light chain recombination reads in lung adenocarcinoma (TCGA-LUAD) samples, and there was a correlation between the detection of light chain recombination reads and a more favorable outcome. This result was supported by analyses of the expression of B-cell markers as indicated by LUAD RNASeq files. CONCLUSION: BcR and TcR recombination reads recovered from LUAD WXS files, either alone or in combination with the human leukocyte antigen (HLA) type, are likely to have prognostic value.


Assuntos
Adenocarcinoma de Pulmão/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T/genética , Recombinação Genética/genética , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Intervalo Livre de Doença , Exoma/genética , Feminino , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , Estimativa de Kaplan-Meier , Masculino , Prognóstico , Interferência de RNA , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Recombinação Genética/imunologia
9.
Biol Blood Marrow Transplant ; 26(7): 1239-1246, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32298807

RESUMO

The SARS-CoV-2 coronavirus (COVID-19) pandemic has significantly impacted the delivery of cellular therapeutics, including chimeric antigen receptor (CAR) T cells. This impact has extended beyond patient care to include logistics, administration, and distribution of increasingly limited health care resources. Based on the collective experience of the CAR T-cell Consortium investigators, we review and address several questions and concerns regarding cellular therapy administration in the setting of COVID-19 and make general recommendations to address these issues. Specifically, we address (1) necessary resources for safe administration of cell therapies; (2) determinants of cell therapy utilization; (3) selection among patients with B cell non-Hodgkin lymphomas and B cell acute lymphoblastic leukemia; (4) supportive measures during cell therapy administration; (5) use and prioritization of tocilizumab; and (6) collaborative care with referring physicians. These recommendations were carefully formulated with the understanding that resource allocation is of the utmost importance, and that the decision to proceed with CAR T cell therapy will require extensive discussion of potential risks and benefits. Although these recommendations are fluid, at this time it is our opinion that the COVID-19 pandemic should not serve as reason to defer CAR T cell therapy for patients truly in need of a potentially curative therapy.


Assuntos
Infecções por Coronavirus/epidemiologia , Imunoterapia Adotiva/métodos , Linfoma de Células B/terapia , Pandemias , Pneumonia Viral/epidemiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Linfócitos T/transplante , Anticorpos Monoclonais Humanizados/uso terapêutico , Controle de Doenças Transmissíveis , Infecções por Coronavirus/imunologia , Alocação de Recursos para a Atenção à Saúde/ética , Alocação de Recursos para a Atenção à Saúde/organização & administração , Humanos , Imunoterapia Adotiva/ética , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Pneumonia Viral/imunologia , Guias de Prática Clínica como Assunto , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Doadores de Tecidos/provisão & distribução , Estados Unidos/epidemiologia
10.
Cancer Sci ; 111(7): 2223-2233, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32324315

RESUMO

Invariant natural killer T (iNKT) cells are innate-like CD1d-restricted T cells that express the invariant T cell receptor (TCR) composed of Vα24 and Vß11 in humans. iNKT cells specifically recognize glycolipid antigens such as α-galactosylceramide (αGalCer) presented by CD1d. iNKT cells show direct cytotoxicity toward CD1d-positive tumor cells, especially when CD1d presents glycolipid antigens. However, iNKT cell recognition of CD1d-negative tumor cells is unknown, and direct cytotoxicity of iNKT cells toward CD1d-negative tumor cells remains controversial. Here, we demonstrate that activated iNKT cells recognize leukemia cells in a CD1d-independent manner, however still in a TCR-mediated way. iNKT cells degranulated and released Th1 cytokines toward CD1d-negative leukemia cells (K562, HL-60, REH) as well as αGalCer-loaded CD1d-positive Jurkat cells. The CD1d-independent cytotoxicity was enhanced by natural killer cell-activating receptors such as NKG2D, 2B4, DNAM-1, LFA-1 and CD2, but iNKT cells did not depend on these receptors for the recognition of CD1d-negative leukemia cells. In contrast, TCR was essential for CD1d-independent recognition and cytotoxicity. iNKT cells degranulated toward patient-derived leukemia cells independently of CD1d expression. iNKT cells targeted myeloid malignancies more than acute lymphoblastic leukemia. These findings reveal a novel anti-tumor mechanism of iNKT cells in targeting CD1d-negative tumor cells and indicate the potential of iNKT cells for clinical application to treat leukemia independently of CD1d.


Assuntos
Antígenos CD1d/metabolismo , Leucemia/imunologia , Leucemia/metabolismo , Ativação Linfocitária/imunologia , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Animais , Antígenos CD1d/genética , Biomarcadores , Degranulação Celular , Linhagem Celular Tumoral , Receptores Coestimuladores e Inibidores de Linfócitos T/metabolismo , Citocinas/metabolismo , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Edição de Genes , Xenoenxertos , Humanos , Imunofenotipagem , Leucemia/genética , Leucemia/patologia , Ativação Linfocitária/genética , Camundongos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores Desencadeadores da Citotoxicidade Natural/metabolismo
11.
PLoS Comput Biol ; 16(4): e1007873, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32348312

RESUMO

High-throughput sequencing of B- and T-cell receptors makes it possible to track immune repertoires across time, in different tissues, and in acute and chronic diseases or in healthy individuals. However, quantitative comparison between repertoires is confounded by variability in the read count of each receptor clonotype due to sampling, library preparation, and expression noise. Here, we present a general Bayesian approach to disentangle repertoire variations from these stochastic effects. Using replicate experiments, we first show how to learn the natural variability of read counts by inferring the distributions of clone sizes as well as an explicit noise model relating true frequencies of clones to their read count. We then use that null model as a baseline to infer a model of clonal expansion from two repertoire time points taken before and after an immune challenge. Applying our approach to yellow fever vaccination as a model of acute infection in humans, we identify candidate clones participating in the response.


Assuntos
RNA-Seq/métodos , Receptores de Antígenos de Linfócitos B , Receptores de Antígenos de Linfócitos T , Teorema de Bayes , Biologia Computacional , Humanos , Modelos Imunológicos , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transcriptoma/imunologia , Vacina contra Febre Amarela/imunologia
12.
Nat Immunol ; 21(5): 567-577, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32284593

RESUMO

Unprimed mice harbor a substantial population of 'memory-phenotype' CD8+ T cells (CD8-MP cells) that exhibit hallmarks of activation and innate-like functional properties. Due to the lack of faithful markers to distinguish CD8-MP cells from bona fide CD8+ memory T cells, the developmental origins and antigen specificities of CD8-MP cells remain incompletely defined. Using deep T cell antigen receptor (TCR) sequencing, we found that the TCRs expressed by CD8-MP cells are highly recurrent and distinct from the TCRs expressed by naive-phenotype CD8+ T cells. CD8-MP clones exhibited reactivity to widely expressed self-ligands. T cell precursors expressing CD8-MP TCRs showed upregulation of the transcription factor Eomes during maturation in the thymus, prior to induction of the full memory phenotype, which is suggestive of a unique program triggered by recognition of self-ligands. Moreover, CD8-MP cells infiltrate oncogene-driven prostate tumors and express high densities of PD-1, which suggests potential roles in antitumor immunity and the response to immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias da Próstata/imunologia , Receptores de Antígenos de Linfócitos T/genética , Proteínas com Domínio T/metabolismo , Timo/fisiologia , Animais , Autoantígenos/imunologia , Diferenciação Celular , Seleção Clonal Mediada por Antígeno , Células Clonais , Memória Imunológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/metabolismo , Proteínas com Domínio T/genética , Regulação para Cima
13.
Int J Nanomedicine ; 15: 483-495, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32158206

RESUMO

Background: The complex preparation procedures and severe toxicities are two major obstacles facing the wide use of chimeric antigen receptor-modified T (CAR-T) cells in clinical cancer immunotherapy. The nanotechnology-based T cell temporary CAR modification may be a potential approach to solve these problems and make the CAR-T cell-based tumor therapy feasible and broadly applicable. Methods: A series of plasmid DNA-loaded self-assembled nanoparticles (pDNA@SNPsx/y) prepared from adamantane-grafted polyamidoamine (Ad-PAMAM) dendrimers of different generations (G1 or G5) and cyclodextrin-grafted branched polyethylenimine (CD-PEI) of different molecular weights (800, 2000, or 25,000 Da) were characterized and evaluated. The detailed physicochemical properties, cellular interaction, and cytotoxicity of selected pDNA@SNPG1/800 were systematically investigated. Thereafter, the epidermal growth factor receptor variant III (EGFRvIII) CAR-expression plasmid vector (pEGFRvIII-CAR) was constructed and encapsulated into SNPG1/800. The resulting pEGFRvIII-CAR@SNPG1/800 was used for Jurkat cell transient transfection, and the EGFRvIII-CAR expressed in transfected cells was measured by flow cytometry and Western blot. Finally, the response of EGFRvIII CAR-positive Jurkat T cell to target tumor cell was evaluated. Results: The pDNA@SNPG1/800 showed the highest efficacy in Jurkat cell gene transfection and exhibited low cytotoxicity. pEGFRvIII-CAR@SNPG1/800 can efficiently deliver pEGFRvIII-CAR into Jurkat T cells, thereby resulting in transient EGFRvIII-CAR expression in transfected cells. EGFRvIII-CAR that is present on the cell membrane enabled Jurkat T cells to recognize and bind specifically with EGFRvIII-positive tumor cells. Conclusion: These results indicated that pEGFRvIII-CAR@SNPG1/800 can effectively achieve T-cell transient CAR modification, thereby demonstrating considerable potential in CAR-T cancer therapy.


Assuntos
Receptores ErbB/genética , Técnicas de Transferência de Genes , Imunoterapia Adotiva/métodos , Nanopartículas/química , Linfócitos T/fisiologia , Linhagem Celular Tumoral , Dendrímeros/química , Vetores Genéticos , Humanos , Iminas/química , Imunoterapia , Células Jurkat , Peso Molecular , Polietilenos/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/genética , Transfecção/métodos
14.
Pediatr Cardiol ; 41(4): 809-815, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32166410

RESUMO

In the fetus, the cardiac neural crest gives rise to both the thymus and the conotruncus of the heart. In newborn screening for severe T-cell lymphopenia neonates with congenital heart defects may be detected. In this study, we investigated the occurrence of T-cell lymphopenia in neonates with or without 22q11.2 deletion syndrome (del) suffering from heart defects. This retrospective cohort study included 125 patients with heart defects. T-cell receptor excision circles (TRECs), a measure for T-cell lymphopenia, were quantified by RT-PCR using stored newborn screening blood spots. Three patient groups were compared: non-conotruncal defects (n = 57), conotruncal defects (n = 42), and 22q11.2 del with conotruncal defects (n = 26). Significantly lower TREC values were detected in patients with 22q11.2 del and conotruncal heart defects compared to those with non-syndromic conotruncal (p < 0.001) and non-conotruncal (p < 0.001) defects. In contrast, no significant difference was found between patients with non-syndromic conotruncal and non-conotruncal heart defects (p = 0.152). Low TREC levels were obtained in neonates treated with heart surgery/intervention within 2 weeks after birth and in those with a fatal outcome (p = 0.02) independent of patient group. A correlation was found between low TREC numbers and oxygen saturation, SpO2 below 95% (p = 0.017). The SpO2 was significantly lower in the non-syndromic conotruncal group compared to non-conotruncal (p < 0.001) and 22q11.2 del group (p = 0.015). No correlation was found between low neonatal TRECs and infections needing hospitalization later in life (p = 0.135). Patients with 22q11.2 del and conotruncal defects have significantly lower TREC levels compared to patients with heart defects without this syndrome.


Assuntos
Síndrome de DiGeorge/diagnóstico , Cardiopatias Congênitas/diagnóstico , Cardiopatias Congênitas/genética , Receptores de Antígenos de Linfócitos T/imunologia , Síndrome de DiGeorge/complicações , Síndrome de DiGeorge/genética , Feminino , Cardiopatias Congênitas/complicações , Humanos , Recém-Nascido , Masculino , Triagem Neonatal , Receptores de Antígenos de Linfócitos T/genética , Estudos Retrospectivos
15.
PLoS Biol ; 18(3): e3000648, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32182234

RESUMO

The memory CD8 T-cell pool must select for clones that bind immunodominant epitopes with high affinity to efficiently counter reinfection. At the same time, it must retain a level of clonal diversity to allow recognition of pathogens with mutated epitopes. How the level of diversity within the memory pool is controlled is unclear, especially in the context of a selective drive for antigen affinity. We find that preservation of clones that bind the activating antigen with low affinity depends on expression of the transcription factor Eomes in the first days after antigen encounter. Eomes is induced at low activating signal strength and directly drives transcription of the prosurvival protein Bcl-2. At higher signal intensity, T-bet is induced which suppresses Bcl-2 and causes a relative survival advantage for cells of low affinity. Clones activated with high-affinity antigen form memory largely independent of Eomes and have a proliferative advantage over clones that bind the same antigen with low affinity. This causes high-affinity clones to prevail in the memory pool, despite their relative survival deficit. Genetic or therapeutic targeting of the Eomes/Bcl-2 axis reduces the clonal diversity of the memory pool, which diminishes its ability to respond to pathogens carrying mutations in immunodominant epitopes. Thus, we demonstrate on a molecular level how sufficient diversity of the memory pool is established in an environment of affinity-based selection.


Assuntos
Apoptose/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Proteínas com Domínio T/imunologia , Animais , Variação Antigênica/imunologia , Sobrevivência Celular/imunologia , Células Cultivadas , Seleção Clonal Mediada por Antígeno/genética , Seleção Clonal Mediada por Antígeno/imunologia , Regulação da Expressão Gênica/imunologia , Ativação Linfocitária , Camundongos , Células Precursoras de Linfócitos T/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Proteínas com Domínio T/genética
16.
Cancer Treat Res ; 180: 251-279, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32215873

RESUMO

Immune checkpoint inhibitors (ICIs) targeting the programed cell-death protein 1 (PD-1) or its ligand PD-L1 and cytotoxic T-lymphocyte antigen 4 (CTLA-4) pathways have improved the survival for patients with solid tumors. Unfortunately, durable clinical responses are seen in only 10-40% of patients at the cost of potential immune-related adverse events. In the tumor microenvironment (TME), tumor cells can influence the microenvironment by releasing extracellular signals and generating peripheral immune tolerance, while the immune cells can affect the initiation, growth, proliferation, and evolution of cancer cells. Currently, translational biomarkers that predict responses to ICIs include high PD-L1 tumor proportion score, defective DNA mismatch repair, high microsatellite instability, and possibly high tumor mutational burden. Characterization of immune cells in the TME, such as tumor-infiltrating lymphocytes, T-cell gene expression profile, T-cell receptor sequencing, and peripheral blood biomarkers are being explored as promising biomarkers. Recent neoadjuvant studies have integrated the real-time assessment of both molecular and immune biomarkers using the tissue and blood specimens simultaneously and longitudinally. This review summarizes the current knowledge and progress in developing translational biomarkers and rational combinational strategies to improve the efficacy of ICIs tailored to individual cancer patients.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais , Resistencia a Medicamentos Antineoplásicos , Neoplasias , Antígeno B7-H1/antagonistas & inibidores , Antígeno CTLA-4/antagonistas & inibidores , Reparo de Erro de Pareamento de DNA , Humanos , Linfócitos do Interstício Tumoral , Instabilidade de Microssatélites , Receptores de Antígenos de Linfócitos T/genética , Microambiente Tumoral
17.
Transfusion ; 60(5): 1004-1014, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32167176

RESUMO

BACKGROUND: Most chimeric antigen receptor T (CAR-T) cells and other adoptive T-cell therapies (ACTs) are currently manufactured by ex vivo expansion of patient lymphocytes in culture media supplemented with human plasma from group AB donors. As lymphocytes do not express A or B antigens, the isoagglutinins of non-AB plasmas are unlikely to cause deleterious effects on lymphocytes in culture. STUDY DESIGN AND METHODS: Seeding cultures with peripheral blood mononuclear cell (PBMNC) concentrates from group A1 donors and using a CAR-T culture protocol, parallel cultures were performed, each with unique donor plasmas as media supplements (including group O plasmas with high-titer anti-A and group AB plasmas as control). An additional variable, a 3% group A1 red blood cell (RBC) spike, was added to simulate a RBC-contaminated PBMNC collection. Cultures were monitored by cell count, viability, flow cytometric phenotype, gene expression analysis, and supernatant chemokine analysis. RESULTS: There was no difference in lymphocyte expansion or phenotype when cultured with AB plasma or O plasma with high-titer anti-A. Compared to controls, the presence of contaminating RBCs in lymphocyte culture led to poor lymphocyte expansion and a less desirable phenotype-irrespective of the isoagglutinin titer of the plasma supplement used. CONCLUSIONS: This study suggests that ABO incompatible plasma may be used as a media supplement when culturing cell types that do not express ABO antigens-such as lymphocytes for CAR-T or other ACT. The presence of contaminating RBCs in culture was disadvantageous independent of isoagglutinin titer.


Assuntos
Sistema ABO de Grupos Sanguíneos/sangue , Meios de Cultura/química , Imunoterapia Adotiva , Plasma/fisiologia , Cultura Primária de Células/métodos , Linfócitos T/citologia , Células Cultivadas , Citometria de Fluxo , Antígenos HLA-A/sangue , Antígenos HLA-A/imunologia , Humanos , Imunoterapia Adotiva/métodos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/transplante , Ativação Linfocitária , Plasma/química , Cultura Primária de Células/normas , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismo , Linfócitos T/transplante , Doadores de Tecidos
18.
PLoS One ; 15(2): e0228112, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32040512

RESUMO

Neoantigens can be predicted and in some cases identified using the data obtained from the whole exome sequencing and transcriptome sequencing of tumor cells. These sequencing data can be coupled with single-cell RNA sequencing for the direct interrogation of the transcriptome, surfaceome, and pairing of αß T-cell receptors (TCRαß) from hundreds of single T cells. Using these 2 large datasets, we established a platform for identifying antigens recognized by TCRαßs obtained from single T cells. Our approach is based on the rapid expression of cloned TCRαß genes as Sleeping Beauty transposons and the determination of the introduced TCRαßs' antigen specificity and avidity using a reporter cell line. The platform enables the very rapid identification of tumor-reactive TCRs for the bioengineering of T cells with redirected specificity.


Assuntos
Engenharia Celular/métodos , Clonagem Molecular/métodos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Expressão Gênica , Biblioteca Gênica , Genes MHC Classe I/genética , Genes MHC da Classe II/genética , Células HEK293 , Humanos , Cinética , Receptores de Antígenos de Linfócitos T alfa-beta/genética
19.
Nat Med ; 26(2): 193-199, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32042196

RESUMO

Immune checkpoint blockade (ICB) of PD-1 and CTLA-4 to treat metastatic melanoma (MM) has variable therapeutic benefit. To explore this in peripheral samples, we characterized CD8+ T cell gene expression across a cohort of patients with MM receiving anti-PD-1 alone (sICB) or in combination with anti-CTLA-4 (cICB). Whereas CD8+ transcriptional responses to sICB and cICB involve a shared gene set, the magnitude of cICB response is over fourfold greater, with preferential induction of mitosis- and interferon-related genes. Early samples from patients with durable clinical benefit demonstrated overexpression of T cell receptor-encoding genes. By mapping T cell receptor clonality, we find that responding patients have more large clones (those occupying >0.5% of repertoire) post-treatment than non-responding patients or controls, and this correlates with effector memory T cell percentage. Single-cell RNA-sequencing of eight post-treatment samples demonstrates that large clones overexpress genes implicated in cytotoxicity and characteristic of effector memory T cells, including CCL4, GNLY and NKG7. The 6-month clinical response to ICB in patients with MM is associated with the large CD8+ T cell clone count 21 d after treatment and agnostic to clonal specificity, suggesting that post-ICB peripheral CD8+ clonality can provide information regarding long-term treatment response and, potentially, facilitate treatment stratification.


Assuntos
Linfócitos T CD8-Positivos/citologia , Antígeno CTLA-4/imunologia , Imunoterapia/métodos , Melanoma/imunologia , Melanoma/terapia , Adulto , Anticorpos/uso terapêutico , Antígenos de Diferenciação de Linfócitos T/genética , Proliferação de Células , Quimiocina CCL4/genética , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica , Humanos , Sistema Imunitário , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Metástase Neoplásica , Receptor de Morte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Análise de Célula Única , Adulto Jovem
20.
Nature ; 579(7798): 274-278, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32103181

RESUMO

Despite the resounding clinical success in cancer treatment of antibodies that block the interaction of PD1 with its ligand PDL11, the mechanisms involved remain unknown. A major limitation to understanding the origin and fate of T cells in tumour immunity is the lack of quantitative information on the distribution of individual clonotypes of T cells in patients with cancer. Here, by performing deep single-cell sequencing of RNA and T cell receptors in patients with different types of cancer, we survey the profiles of various populations of T cells and T cell receptors in tumours, normal adjacent tissue, and peripheral blood. We find clear evidence of clonotypic expansion of effector-like T cells not only within the tumour but also in normal adjacent tissue. Patients with gene signatures of such clonotypic expansion respond best to anti-PDL1 therapy. Notably, expanded clonotypes found in the tumour and normal adjacent tissue can also typically be detected in peripheral blood, which suggests a convenient approach to patient identification. Analyses of our data together with several external datasets suggest that intratumoural T cells, especially in responsive patients, are replenished with fresh, non-exhausted replacement cells from sites outside the tumour, suggesting continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response.


Assuntos
Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/patologia , Variantes Farmacogenômicos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Células Clonais , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Linfócitos T/metabolismo , Transcriptoma
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