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1.
Cell ; 170(1): 142-157.e19, 2017 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-28648661

RESUMO

Immune-checkpoint-blockade (ICB)-mediated rejuvenation of exhausted T cells has emerged as a promising approach for treating various cancers and chronic infections. However, T cells that become fully exhausted during prolonged antigen exposure remain refractory to ICB-mediated rejuvenation. We report that blocking de novo DNA methylation in activated CD8 T cells allows them to retain their effector functions despite chronic stimulation during a persistent viral infection. Whole-genome bisulfite sequencing of antigen-specific murine CD8 T cells at the effector and exhaustion stages of an immune response identified progressively acquired heritable de novo methylation programs that restrict T cell expansion and clonal diversity during PD-1 blockade treatment. Moreover, these exhaustion-associated DNA-methylation programs were acquired in tumor-infiltrating PD-1hi CD8 T cells, and approaches to reverse these programs improved T cell responses and tumor control during ICB. These data establish de novo DNA-methylation programming as a regulator of T cell exhaustion and barrier of ICB-mediated T cell rejuvenation.


Assuntos
Linfócitos T CD8-Positivos/citologia , Epigênese Genética , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Adenocarcinoma/tratamento farmacológico , Animais , Linfócitos T CD8-Positivos/imunologia , Metilação de DNA , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias da Próstata/tratamento farmacológico , Viroses/tratamento farmacológico
2.
Nat Immunol ; 20(5): 613-625, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30778243

RESUMO

Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally and infect humans, with IAV and IBV causing the most severe disease. CD8+ T cells confer cross-protection against IAV strains, however the responses of CD8+ T cells to IBV and ICV are understudied. We investigated the breadth of CD8+ T cell cross-recognition and provide evidence of CD8+ T cell cross-reactivity across IAV, IBV and ICV. We identified immunodominant CD8+ T cell epitopes from IBVs that were protective in mice and found memory CD8+ T cells directed against universal and influenza-virus-type-specific epitopes in the blood and lungs of healthy humans. Lung-derived CD8+ T cells displayed tissue-resident memory phenotypes. Notably, CD38+Ki67+CD8+ effector T cells directed against novel epitopes were readily detected in IAV- or IBV-infected pediatric and adult subjects. Our study introduces a new paradigm whereby CD8+ T cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for the design of universal vaccines.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Reações Cruzadas/imunologia , Gammainfluenzavirus/imunologia , Vírus da Influenza A/imunologia , Vírus da Influenza B/imunologia , Influenza Humana/imunologia , Adolescente , Adulto , Idoso , Animais , Linfócitos T CD8-Positivos/virologia , Criança , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Vírus da Influenza A/fisiologia , Vírus da Influenza B/fisiologia , Vacinas contra Influenza/imunologia , Influenza Humana/virologia , Gammainfluenzavirus/fisiologia , Masculino , Camundongos , Pessoa de Meia-Idade , Adulto Jovem
3.
Immunity ; 49(3): 531-544.e6, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30170813

RESUMO

Compared to adults, infants suffer higher rates of hospitalization, severe clinical complications, and mortality due to influenza infection. We found that γδ T cells protected neonatal mice against mortality during influenza infection. γδ T cell deficiency did not alter viral clearance or interferon-γ production. Instead, neonatal influenza infection induced the accumulation of interleukin-17A (IL-17A)-producing γδ T cells, which was associated with IL-33 production by lung epithelial cells. Neonates lacking IL-17A-expressing γδ T cells or Il33 had higher mortality upon influenza infection. γδ T cells and IL-33 promoted lung infiltration of group 2 innate lymphoid cells and regulatory T cells, resulting in increased amphiregulin secretion and tissue repair. In influenza-infected children, IL-17A, IL-33, and amphiregulin expression were correlated, and increased IL-17A levels in nasal aspirates were associated with better clinical outcomes. Our results indicate that γδ T cells are required in influenza-infected neonates to initiate protective immunity and mediate lung homeostasis.


Assuntos
Vírus da Influenza A/fisiologia , Influenza Humana/imunologia , Interleucina-17/metabolismo , Pulmão/imunologia , Infecções por Orthomyxoviridae/imunologia , Linfócitos T/imunologia , Células Th2/imunologia , Adulto , Anfirregulina/metabolismo , Animais , Células Cultivadas , Criança , Humanos , Imunidade , Recém-Nascido , Interleucina-33/metabolismo , Camundongos , Prognóstico , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo
4.
Nature ; 547(7661): 89-93, 2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28636592

RESUMO

T cells are defined by a heterodimeric surface receptor, the T cell receptor (TCR), that mediates recognition of pathogen-associated epitopes through interactions with peptide and major histocompatibility complexes (pMHCs). TCRs are generated by genomic rearrangement of the germline TCR locus, a process termed V(D)J recombination, that has the potential to generate marked diversity of TCRs (estimated to range from 1015 (ref. 1) to as high as 1061 (ref. 2) possible receptors). Despite this potential diversity, TCRs from T cells that recognize the same pMHC epitope often share conserved sequence features, suggesting that it may be possible to predictively model epitope specificity. Here we report the in-depth characterization of ten epitope-specific TCR repertoires of CD8+ T cells from mice and humans, representing over 4,600 in-frame single-cell-derived TCRαß sequence pairs from 110 subjects. We developed analytical tools to characterize these epitope-specific repertoires: a distance measure on the space of TCRs that permits clustering and visualization, a robust repertoire diversity metric that accommodates the low number of paired public receptors observed when compared to single-chain analyses, and a distance-based classifier that can assign previously unobserved TCRs to characterized repertoires with robust sensitivity and specificity. Our analyses demonstrate that each epitope-specific repertoire contains a clustered group of receptors that share core sequence similarities, together with a dispersed set of diverse 'outlier' sequences. By identifying shared motifs in core sequences, we were able to highlight key conserved residues driving essential elements of TCR recognition. These analyses provide insights into the generalizable, underlying features of epitope-specific repertoires and adaptive immune recognition.


Assuntos
Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Algoritmos , Animais , Linfócitos T CD8-Positivos/química , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Análise por Conglomerados , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Especificidade por Substrato , Recombinação V(D)J
5.
Nucleic Acids Res ; 46(D1): D419-D427, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28977646

RESUMO

The ability to decode antigen specificities encapsulated in the sequences of rearranged T-cell receptor (TCR) genes is critical for our understanding of the adaptive immune system and promises significant advances in the field of translational medicine. Recent developments in high-throughput sequencing methods (immune repertoire sequencing technology, or RepSeq) and single-cell RNA sequencing technology have allowed us to obtain huge numbers of TCR sequences from donor samples and link them to T-cell phenotypes. However, our ability to annotate these TCR sequences still lags behind, owing to the enormous diversity of the TCR repertoire and the scarcity of available data on T-cell specificities. In this paper, we present VDJdb, a database that stores and aggregates the results of published T-cell specificity assays and provides a universal platform that couples antigen specificities with TCR sequences. We demonstrate that VDJdb is a versatile instrument for the annotation of TCR repertoire data, enabling a concatenated view of antigen-specific TCR sequence motifs. VDJdb can be accessed at https://vdjdb.cdr3.net and https://github.com/antigenomics/vdjdb-db.


Assuntos
Antígenos/química , Bases de Dados de Proteínas , Anotação de Sequência Molecular , Receptores de Antígenos de Linfócitos T/química , Software , Sequência de Aminoácidos , Animais , Antígenos/imunologia , Antígenos/metabolismo , Sítios de Ligação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Internet , Macaca mulatta , Complexo Principal de Histocompatibilidade/genética , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Análise de Célula Única , Linfócitos T/citologia , Linfócitos T/imunologia
6.
Immunity ; 30(4): 566-75, 2009 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-19362023

RESUMO

Virus-induced interlukin-1beta (IL-1beta) and IL-18 production in macrophages are mediated via caspase-1 pathway. Multiple microbial components, including viral RNA, are thought to trigger assembly of the cryopyrin inflammasome resulting in caspase-1 activation. Here, we demonstrated that Nlrp3(-/-) and Casp1(-/-) mice were more susceptible than wild-type mice after infection with a pathogenic influenza A virus. This enhanced morbidity correlated with decreased neutrophil and monocyte recruitment and reduced cytokine and chemokine production. Despite the effect on innate immunity, cryopyrin-deficiency was not associated with any obvious defect in virus control or on the later emergence of the adaptive response. Early epithelial necrosis was, however, more severe in the infected mutants, with extensive collagen deposition leading to later respiratory compromise. These findings reveal a function of the cryopyrin inflammasome in healing responses. Thus, cryopyrin and caspase-1 are central to both innate immunity and to moderating lung pathology in influenza pneumonia.


Assuntos
Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Imunidade Inata , Vírus da Influenza A/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Proteínas de Transporte/genética , Caspase 1/genética , Células Cultivadas , Regulação da Expressão Gênica , Predisposição Genética para Doença , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR
7.
Curr Top Microbiol Immunol ; 386: 121-47, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25038940

RESUMO

The innate host response to influenza virus infection plays a critical role in determining the subsequent course of infection and the clinical outcome of disease. The host has a diverse array of detection and effector mechanisms that are able to recognize and initiate effective antiviral responses. In opposition, the virus utilizes a number of distinct mechanisms to evade host detection and effector activity in order to remain "stealthy" throughout its replication cycle. In this review, we describe these host and viral mechanisms, including the major pattern recognition receptor families (the TLRs, NLRs, and RLRs) in the host and the specific viral proteins such as NS1 that are key players in this interaction. Additionally, we explore nonreductive mechanisms of viral immune evasion and propose areas important for future inquiry.


Assuntos
Evasão da Resposta Imune , Influenza Humana/imunologia , Proteína DEAD-box 58 , RNA Helicases DEAD-box/fisiologia , Humanos , Imunidade Inata , Fenótipo , Receptores Imunológicos , Receptores Toll-Like/fisiologia
8.
J Immunol ; 193(1): 258-67, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24899501

RESUMO

The TCR:CD3 complex transduces signals that are critical for optimal T cell development and adaptive immunity. In resting T cells, the CD3ε cytoplasmic tail associates with the plasma membrane via a proximal basic-rich stretch (BRS). In this study, we show that mice lacking a functional CD3ε-BRS exhibited substantial reductions in thymic cellularity and limited CD4- CD8- double-negative (DN) 3 to DN4 thymocyte transition, because of enhanced DN4 TCR signaling resulting in increased cell death and TCR downregulation in all subsequent populations. Furthermore, positive, but not negative, T cell selection was affected in mice lacking a functional CD3ε-BRS, which led to limited peripheral T cell function and substantially reduced responsiveness to influenza infection. Collectively, these results indicate that membrane association of the CD3ε signaling domain is required for optimal thymocyte development and peripheral T cell function.


Assuntos
Complexo CD3/imunologia , Membrana Celular/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Timócitos/imunologia , Animais , Complexo CD3/genética , Membrana Celular/genética , Camundongos , Camundongos Knockout , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética , Timócitos/citologia
9.
Immunol Cell Biol ; 93(9): 804-14, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25804828

RESUMO

T-cell receptor (TCR) usage has an important role in determining the outcome of CD8(+) cytotoxic T-lymphocyte responses to viruses and other pathogens. However, the characterization of TCR usage from which such conclusions are drawn is based on exclusive analysis of either the TCRα chain or, more commonly, the TCRß chain. Here, we have used a multiplexed reverse transcription-PCR protocol to analyse the CDR3 regions of both TCRα and ß chains from single naive or immune epitope-specific cells to provide a comprehensive picture of epitope-specific TCR usage and selection into the immune response. Analysis of TCR repertoires specific for three influenza-derived epitopes (D(b)NP(366), D(b)PA(224) and D(b)PB1-F2(62)) showed preferential usage of particular TCRαß proteins in the immune repertoire relative to the naive repertoire, in some cases, resulting in a complete shift in TRBV preference or CDR3 length, and restricted repertoire diversity. The NP(366)-specific TCRαß repertoire, previously defined as clonally restricted based on TCRß analysis, was similarly diverse as the PA(224)- and PB1-F2(62)-specific repertoires. Intriguingly, preferred TCR characteristics (variable gene usage, CDR3 length and junctional gene usage) appeared to be able to confer specificity either independently or in concert with one another, depending on the epitope specificity. These data have implications for established correlations between the nature of the TCR repertoire and response outcomes after infection, and suggest that analysis of a subset of cells or a single TCR chain does not accurately depict the nature of the antigen-specific TCRαß repertoire.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Infecções por Orthomyxoviridae/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Células Clonais/imunologia , Células Clonais/metabolismo , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Citometria de Fluxo , Variação Genética/imunologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/virologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/virologia
10.
J Gen Virol ; 95(Pt 2): 350-362, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24243730

RESUMO

Type I alveolar epithelial cells are a replicative niche for influenza in vivo, yet their response to infection is not fully understood. To better characterize their cellular responses, we have created an immortalized murine lung epithelial type I cell line (LET1). These cells support spreading influenza virus infection in the absence of exogenous protease and thus permit simultaneous analysis of viral replication dynamics and host cell responses. LET1 cells can be productively infected with human, swine and mouse-adapted strains of influenza virus and exhibit expression of an antiviral transcriptional programme and robust cytokine secretion. We characterized influenza virus replication dynamics and host responses of lung type I epithelial cells and identified the capacity of epithelial cell-derived type I IFN to regulate specific modules of antiviral effectors to establish an effective antiviral state. Together, our results indicate that the type I epithelial cell can play a major role in restricting influenza virus infection without contribution from the haematopoietic compartment.


Assuntos
Células Epiteliais/imunologia , Células Epiteliais/virologia , Imunidade Inata , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Replicação Viral , Animais , Linhagem Celular , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
11.
Proc Natl Acad Sci U S A ; 108(35): 14602-7, 2011 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-21873213

RESUMO

It is currently thought that T cells with specificity for self-peptide/MHC (pMHC) ligands are deleted during thymic development, thereby preventing autoimmunity. In the case of CD4(+) T cells, what is unclear is the extent to which self-peptide/MHC class II (pMHCII)-specific T cells are deleted or become Foxp3(+) regulatory T cells. We addressed this issue by characterizing a natural polyclonal pMHCII-specific CD4(+) T-cell population in mice that either lacked or expressed the relevant antigen in a ubiquitous pattern. Mice expressing the antigen contained one-third the number of pMHCII-specific T cells as mice lacking the antigen, and the remaining cells exhibited low TCR avidity. In mice lacking the antigen, the pMHCII-specific T-cell population was dominated by phenotypically naive Foxp3(-) cells, but also contained a subset of Foxp3(+) regulatory cells. Both Foxp3(-) and Foxp3(+) pMHCII-specific T-cell numbers were reduced in mice expressing the antigen, but the Foxp3(+) subset was more resistant to changes in number and TCR repertoire. Therefore, thymic selection of self-pMHCII-specific CD4(+) T cells results in incomplete deletion within the normal polyclonal repertoire, especially among regulatory T cells.


Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Fatores de Transcrição Forkhead/análise , Antígenos de Histocompatibilidade Classe II/imunologia , Timo/imunologia , Animais , Deleção Clonal , Tolerância Imunológica , Camundongos , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T Reguladores/imunologia
12.
J Immunother Cancer ; 12(4)2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38609317

RESUMO

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy target receptor tyrosine kinase-like orphan receptor 1 (ROR1) is broadly expressed in hematologic and solid tumors, however clinically-characterized ROR1-CAR T cells with single chain variable fragment (scFv)-R12 targeting domain failed to induce durable remissions, in part due to the immunosuppressive tumor microenvironment (TME). Herein, we describe the development of an improved ROR1-CAR with a novel, fully human scFv9 targeting domain, and augmented with TGFßRIIDN armor protective against a major TME factor, transforming growth factor beta (TGFß). METHODS: CAR T cells were generated by lentiviral transduction of enriched CD4+ and CD8+ T cells, and the novel scFv9-based ROR1-CAR-1 was compared with the clinically-characterized ROR1-R12-scFv-based CAR-2 in vitro and in vivo. RESULTS: CAR-1 T cells exhibited greater CAR surface density than CAR-2 when normalized for %CAR+, and produced more interferon (IFN)-γ tumor necrosis factor (TNF)-α and interleukin (IL)-2 in response to hematologic (Jeko-1, RPMI-8226) and solid (OVCAR-3, Capan-2, NCI-H226) tumor cell lines in vitro. In vivo, CAR-1 and CAR-2 both cleared hematologic Jeko-1 lymphoma xenografts, however only CAR-1 fully rejected ovarian solid OVCAR-3 tumors, concordantly with greater expansion of CD8+ and CD4+CAR T cells, and enrichment for central and effector memory phenotype. When equipped with TGFß-protective armor TGFßRIIDN, CAR-1 T cells resisted TGFß-mediated pSmad2/3 phosphorylation, as compared with CAR-1 alone. When co-cultured with ROR-1+ AsPC-1 pancreatic cancer line in the presence of TGFß1, armored CAR-1 demonstrated improved recovery of killing function, IFN-γ, TNF-α and IL-2 secretion. In mouse AsPC-1 pancreatic tumor xenografts overexpressing TGFß1, armored CAR-1, in contrast to CAR-1 alone, achieved complete tumor remissions, and yielded accelerated expansion of CAR+ T cells, diminished circulating active TGFß1, and no apparent toxicity or weight loss. Unexpectedly, in AsPC-1 xenografts without TGFß overexpression, TGFß1 production was specifically induced by ROR-1-CAR T cells interaction with ROR-1 positive tumor cells, and the TGFßRIIDN armor conferred accelerated tumor clearance. CONCLUSIONS: The novel fully human TGFßRIIDN-armored ROR1-CAR-1 T cells are highly potent against ROR1-positive tumors, and withstand the inhibitory effects of TGFß in solid TME. Moreover, TGFß1 induction represents a novel, CAR-induced checkpoint in the solid TME, which can be circumvented by co-expressing the TGßRIIDN armor on T cells.


Assuntos
Neoplasias Ovarianas , Neoplasias Pancreáticas , Humanos , Animais , Feminino , Camundongos , Apoptose , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Fator de Crescimento Transformador beta , Microambiente Tumoral , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética
13.
J Virol ; 86(8): 4151-7, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22318144

RESUMO

RNA interference (RNAi) is a critical component of many cellular antiviral responses in plants, invertebrates, and mammals. However, its in vivo role in host protection from the negative-sense RNA virus influenza virus type A (flu) is unclear. Here we have examined the role of RNAi in host defense to flu by analyzing Argonaute 1 and 3 double-knockout mice deficient in components of the RNA-induced silencing complex. Compared to littermate controls, flu-infected double-knockout mice exhibited increased mortality, consistent with more severe alveolitis and pneumonitis. These data indicate that optimal resistance to flu requires Argonaute 1 and/or 3. Enhanced mortality of double-knockout mice was not associated either with increased viral replication or with differential pulmonary recruitment or function of innate and adaptive immune cells. Given the absence of detectable immune defects, our results support the notion that the enhanced flu susceptibility of double-knockout mice arises from an intrinsic impairment in the ability of lung cells to tolerate flu-elicited inflammation.


Assuntos
Proteínas Argonautas/genética , Fatores de Iniciação em Eucariotos/genética , Predisposição Genética para Doença , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/genética , Animais , Humanos , Pulmão/imunologia , Pulmão/virologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/imunologia , Interferência de RNA , Replicação Viral
14.
PLoS Pathog ; 7(12): e1002377, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22144888

RESUMO

Following respiratory syncytial virus infection of adult CB6F1 hybrid mice, a predictable CD8+ T cell epitope hierarchy is established with a strongly dominant response to a K(d)-restricted peptide (SYIGSINNI) from the M2 protein. The response to K(d)M2(82-90) is ∼5-fold higher than the response to a subdominant epitope from the M protein (NAITNAKII, D(b)M(187-195)). After infection of neonatal mice, a distinctly different epitope hierarchy emerges with codominant responses to K(d)M2(82-90) and D(b)M(187-195). Adoptive transfer of naïve CD8+ T cells from adults into congenic neonates prior to infection indicates that intrinsic CD8+ T cell factors contribute to age-related differences in hierarchy. Epitope-specific precursor frequency differs between adults and neonates and influences, but does not predict the hierarchy following infection. Additionally, dominance of K(d)M2(82-90)-specific cells does not correlate with TdT activity. Epitope-specific Vß repertoire usage is more restricted and functional avidity is lower in neonatal mice. The neonatal pattern of codominance changes after infection at 10 days of age, and rapidly shifts to the adult pattern of extreme K(d)M2(82-90)-dominance. Thus, the functional properties of T cells are selectively modified by developmental factors in an epitope-specific and age-dependent manner.


Assuntos
Envelhecimento/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Proteínas Virais/imunologia , Transferência Adotiva , Animais , Animais Recém-Nascidos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C
15.
J Immunol ; 187(2): 835-41, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21690324

RESUMO

The immune system has evolved to use sophisticated mechanisms to recruit lymphocytes to sites of pathogen exposure. Trafficking pathways are precise. For example, lymphocytes that are primed by gut pathogens can, in some cases, be imprinted with CCR9 membrane receptors, which can influence migration to the small intestine. Currently, little is known about T cell trafficking to the upper respiratory tract or the relationship between effectors that migrate to the diffuse nasal-associated lymphoid tissue (d-NALT), the lower airways, and the lung. To determine whether a T cell primed by Ag from a respiratory pathogen is imprinted for exclusive trafficking to the upper or lower respiratory tract or whether descendents from that cell have the capacity to migrate to both sites, we inoculated mice by the intranasal route with Sendai virus and conducted single-cell-sequencing analyses of CD8(+) T lymphocytes responsive to a K(b)-restricted immunodominant peptide, FAPGNYPAL (Tet(+)). Cells from the d-NALT, lung airways (bronchoalveolar lavage), lung, and mediastinal lymph node were examined 10 d postinfection to determine TCR usage and clonal relationships. We discovered that 1) Tet(+) cells were heterogeneous but preferentially used TCR elements TRAV6, TRAV16, and TRBD1; 2) both N and C termini of Vα and Vß TCR junctions frequently encompassed charged residues, perhaps facilitating TCR αß pairing and interactions with a neutral target peptide; and 3) T cells in the d-NALT were often clonally related to cells in the lower respiratory tract.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Movimento Celular/imunologia , Pulmão/imunologia , Tecido Linfoide/imunologia , Mucosa Nasal/imunologia , Infecções Respiratórias/imunologia , Infecções por Respirovirus/imunologia , Vírus Sendai/imunologia , Administração Intranasal , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Células Clonais , Modelos Animais de Doenças , Epitopos de Linfócito T/metabolismo , Feminino , Antígenos H-2/administração & dosagem , Antígenos H-2/imunologia , Antígenos H-2/metabolismo , Epitopos Imunodominantes/metabolismo , Região Variável de Imunoglobulina/metabolismo , Pulmão/patologia , Pulmão/virologia , Tecido Linfoide/patologia , Tecido Linfoide/virologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mucosa Nasal/patologia , Mucosa Nasal/virologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Infecções por Respirovirus/patologia , Infecções por Respirovirus/virologia , Vírus Sendai/isolamento & purificação
16.
J Biol Chem ; 286(6): 4829-41, 2011 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-21118816

RESUMO

CD8+ T cell responses are important for recognizing and resolving viral infections. To better understand the selection and hierarchy of virus-specific T cell responses, we compared the T cell receptor (TCR) clonotype in parent and hybrid strains of respiratory syncytial virus-infected mice. K(d)M2(82-90) (SYIGSINNI) in BALB/c and D(b)M(187-195) (NAITNAKII) in C57Bl/6 are both dominant epitopes in parent strains but assume a distinct hierarchy, with K(d)M2(82-90) dominant to D(b)M(187-195) in hybrid CB6F1/J mice. The dominant K(d)M2(82-90) response is relatively public and is restricted primarily to the highly prevalent Vß13.2 in BALB/c and hybrid mice, whereas D(b)M(187-195) responses in C57BL/6 mice are relatively private and involve multiple Vß subtypes, some of which are lost in hybrids. A significant frequency of TCR CDR3 sequences in the D(b)M(187-195) response have a distinct "(D/E)WG" motif formed by a limited number of recombination strategies. Modeling of the dominant epitope suggested a flat, featureless structure, but D(b)M(187-195) showed a distinctive structure formed by Lys(7). The data suggest that common recombination events in prevalent Vß genes may provide a numerical advantage in the T cell response and that distinct epitope structures may impose more limited options for successful TCR selection. Defining how epitope structure is interpreted to inform T cell function will improve the design of future gene-based vaccines.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Epitopos de Linfócito T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Motivos de Aminoácidos , Animais , Linfócitos T CD8-Positivos/metabolismo , Quimera/genética , Quimera/imunologia , Quimera/metabolismo , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Modelos Imunológicos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/metabolismo , Especificidade da Espécie
17.
Biomedicines ; 10(12)2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36551771

RESUMO

The microbiome shapes the mature T cell receptor (TCR) repertoire and thereby influences pathogen control. To investigate microbiome influences on T cells at an earlier, immature stage, we compared single-cell TCR transcript sequences between CD4+CD8+ (double-positive) thymocytes from gnotobiotic [E. coli mono-associated (Ec)] and germ-free (GF) mice. Identical TCRß transcripts (termed repeat, REP) were more often shared between cells of individual Ec mice compared to GF mice (Fishers Exact test, p < 0.0001). Among Ec REPs, a cluster of Vß genes (Vß12-1, 12-2, 13-1, and 13-2, termed 12-13) was well represented, whereas 12-13 sequences were not detected among GF REPs (Fishers Exact test, p = 0.046). Vα genes located in the distal region of the TCRα locus were more frequently expressed in Ec mice compared to GF mice, both among REPs and total sequences (Fishers Exact test, p = 0.009). Results illustrate how gut bacteria shape the TCR repertoire, not simply among mature T cells, but among immature CD4+CD8+ thymocytes.

18.
Front Immunol ; 13: 832645, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35222421

RESUMO

CAR T-cell therapies targeting the B-cell maturation antigen eliminate tumors in relapsed/refractory multiple myeloma patients, however durable remissions remain difficult to attain. Transforming growth factor beta (TGF-ß) is a multifunctional cytokine abundantly expressed in the multiple myeloma bone marrow niche, where it promotes an immunosuppressive tumor microenvironment. We hypothesized that BCMA CAR T-cells armored to resist the suppressive effects of TGF-ß will provide an advantage in treating multiple myeloma. The armored B2ARM CAR T cells, co-expressing BCMA targeting CAR with TGF-ß dominant-negative receptor II, were generated by lentiviral transduction of primary human CD4+ and CD8+ T cells. The B2ARM CAR T cells eliminated MM.1S multiple myeloma targets in long-term cytotoxicity assays, even under TGF-ß-high conditions, whereas cytotoxic function of the non-armored B2 CAR -T cells was inhibited by TGF-ß. Concordantly, after long-term exposure to targets in the presence of TGF-ß, the B2ARM CAR T cells were enriched for Granzyme B, CD107a, Ki67 and polyfunctional cells T-cells (double or triple-positive for IFN-γ, IL-2 and/or TNF-α), as determined by flow cytometry. In addition, the B2ARM CAR T-cells, but not the conventional B2 CAR T-cells, resisted the TGF-ß-mediated suppression of activation (CD25), exhaustion (PD-1, LAG3), and differentiation to T effectors (CD45RA+ CD45RO-CD62L-). In NSG mice bearing RPMI-8226 tumors overexpressing TGF-ß, the B2ARM CAR mediated 100% tumor rejection and survival, superior infiltration of tumors on day 7 post CAR T treatment (%CD3+CAR+), and greater expression of IFN-γ, TNF-α, Ki67, Granzyme B, and PD-1, as compared to tumor-infiltrating non-armored B2 CAR T-cells. In NSG RPMI-8226 xenograft model in which tumors were additionally supplemented with TGF-ß injections on days -1 through 11 of CAR T treatment, the B2ARM CAR T cells rejected tumors faster than the non-armored B2 CARs, and showed greater numbers of CD3+ and CD3+CAR+, central memory (CD45RO+CD62L+) and effector memory (CD45RO+CD62L-) T cells in the peripheral blood 18 days after treatment. In summary, the armored B2ARM CAR T cells mediate superior persistence, proliferation, multi-functionality, effector differentiation and anti-tumor function in pre-clinical models of multiple myeloma, while abrogating TGF-ß-mediated suppression.


Assuntos
Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Animais , Antígeno de Maturação de Linfócitos B , Granzimas , Humanos , Antígeno Ki-67 , Camundongos , Receptor de Morte Celular Programada 1 , Receptores de Antígenos Quiméricos/genética , Fator de Crescimento Transformador beta , Microambiente Tumoral , Fator de Necrose Tumoral alfa
19.
J Virol ; 84(18): 9149-60, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20592089

RESUMO

Three-dimensional (3D) porcine nasal mucosal and tracheal mucosal epithelial cell cultures were developed to analyze foot-and-mouth disease virus (FMDV) interactions with mucosal epithelial cells. The cells in these cultures differentiated and polarized until they closely resemble the epithelial layers seen in vivo. FMDV infected these cultures predominantly from the apical side, primarily by binding to integrin alphav beta6, in an Arg-Gly-Asp (RGD)-dependent manner. However, FMDV replicated only transiently without any visible cytopathic effect (CPE), and infectious progeny virus could be recovered only from the apical side. The infection induced the production of beta interferon (IFN-beta) and the IFN-inducible gene Mx1 mRNA, which coincided with the disappearance of viral RNA and progeny virus. The induction of IFN-beta mRNA correlated with the antiviral activity of the supernatants from both the apical and basolateral compartments. IFN-alpha mRNA was constitutively expressed in nasal mucosal epithelial cells in vitro and in vivo. In addition, FMDV infection induced interleukin 8 (IL-8) protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES mRNA in the infected epithelial cells, suggesting that it plays an important role in modulating the immune response.


Assuntos
Células Epiteliais/virologia , Vírus da Febre Aftosa/fisiologia , Replicação Viral , Animais , Antígenos de Neoplasias/metabolismo , Quimiocina CCL5/biossíntese , Efeito Citopatogênico Viral , Feminino , Vírus da Febre Aftosa/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Integrinas/metabolismo , Interferon-alfa/biossíntese , Interferon beta/biossíntese , Interleucina-8/biossíntese , Masculino , Técnicas de Cultura de Órgãos , RNA Mensageiro/biossíntese , Mucosa Respiratória/virologia , Suínos , Ativação Transcricional , Ligação Viral
20.
J Exp Med ; 217(5)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32106283

RESUMO

T cells are classically recognized as distinct subsets that express αß or γδ TCRs. We identify a novel population of T cells that coexpress αß and γδ TCRs in mice and humans. These hybrid αß-γδ T cells arose in the murine fetal thymus by day 16 of ontogeny, underwent αß TCR-mediated positive selection into CD4+ or CD8+ thymocytes, and constituted up to 10% of TCRδ+ cells in lymphoid organs. They expressed high levels of IL-1R1 and IL-23R and secreted IFN-γ, IL-17, and GM-CSF in response to canonically restricted peptide antigens or stimulation with IL-1ß and IL-23. Hybrid αß-γδ T cells were transcriptomically distinct from conventional γδ T cells and displayed a hyperinflammatory phenotype enriched for chemokine receptors and homing molecules that facilitate migration to sites of inflammation. These proinflammatory T cells promoted bacterial clearance after infection with Staphylococcus aureus and, by licensing encephalitogenic Th17 cells, played a key role in the development of autoimmune disease in the central nervous system.


Assuntos
Inflamação/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/imunologia , Animais , Biomarcadores/metabolismo , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Humanos , Inflamação/patologia , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Fenótipo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/fisiologia , Transcrição Gênica , Transcriptoma/genética
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