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1.
Mol Immunol ; 127: 112-123, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32961421

RESUMO

A highly diverse repertoire of T cell antigen receptors (TCR) is created in the thymus by recombination of gene segments and the insertion or deletion of nucleotides at the junctions. Using next-generation TCR sequencing we define here the features of recombination and selection in the human TCRα and TCRß locus, and show that a strikingly high proportion of the repertoire is shared by unrelated individuals. The thymic TCRα nucleotide repertoire was more diverse than TCRß, with 4.1 × 106 vs. 0.81 × 106 unique clonotypes, and contained nonproductive clonotypes at a higher frequency (69.2% vs. 21.2%). The convergence of distinct nucleotide clonotypes to the same amino acid sequences was higher in TCRα than in TCRß repertoire (1.45 vs. 1.06 nucleotide sequences per amino acid sequence in thymus). The gene segment usage was biased, and generally all individuals favored the same genes in both TCRα and TCRß loci. Despite the high diversity, a large fraction of the repertoire was found in more than one donor. The shared fraction was bigger in TCRα than TCRß repertoire, and more common in in-frame sequences than in nonproductive sequences. Thus, both biases in rearrangement and thymic selection are likely to contribute to the generation of shared repertoire in humans.


Assuntos
Impressão Genômica , Linfócitos T/imunologia , Timo/citologia , Sequência de Bases , Células Clonais , Regiões Determinantes de Complementaridade/genética , Feminino , Variação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Mutagênese Insercional , Probabilidade , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Recombinação Genética/genética
2.
PLoS One ; 15(9): e0238875, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903281

RESUMO

To facilitate preclinical testing of T-cell receptors (TCRs) derived from tumor-reactive T-cell clones it is necessary to develop convenient and rapid cloning strategies for the generation of TCR expression constructs. Herein, we describe a pDONR™221 vector backbone allowing to generate Gateway™ compatible entry clones encoding optimized bicistronic αßTCR constructs. It harbors P2A-linked TCR constant regions and head-to-head-oriented recognition sites of the Type IIS restriction enzymes BsmBI and BsaI for seamless cloning of the TCRα and TCRß V(D)J regions, respectively. Additional well-established TCR optimizations were incorporated to enhance TCR functionality. This included replacing of the human αßTCR constant regions with their codon-optimized murine counterparts for chimerization, addition of a second interchain disulfide bond and arrangement of the TCR chains in the order ß-P2A-α. We exemplified the utility of our vector backbone by cloning and functional testing of three melanoma-reactive TCRs in primary human T cells.


Assuntos
Clonagem Molecular/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Retroviridae/genética , Linfócitos T/citologia , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Técnicas de Cocultura , Vetores Genéticos/genética , Células HEK293 , Humanos , Linfócitos T/imunologia , Linfócitos T/virologia , Transdução Genética , Recombinação V(D)J
3.
Nat Commun ; 11(1): 4767, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958743

RESUMO

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


Assuntos
Artrite Psoriásica/imunologia , Linfócitos T CD8-Positivos/imunologia , Seleção Clonal Mediada por Antígeno , Receptores de Retorno de Linfócitos/metabolismo , Líquido Sinovial/imunologia , Artrite Psoriásica/sangue , Linfócitos T CD8-Positivos/metabolismo , Perfilação da Expressão Gênica , Humanos , Memória Imunológica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores de Retorno de Linfócitos/genética , Análise de Célula Única , Membrana Sinovial/imunologia
4.
Nucleic Acids Res ; 48(17): 9621-9636, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32853367

RESUMO

The regulation of T cell receptor Tcra gene rearrangement has been extensively studied. The enhancer Eα plays an essential role in Tcra rearrangement by establishing a recombination centre in the Jα array and a chromatin hub for interactions between Vα and Jα genes. But the mechanism of the Eα and its downstream CTCF binding site (here named EACBE) in dynamic chromatin regulation is unknown. The Hi-C data showed that the EACBE is located at the sub-TAD boundary which separates the Tcra-Tcrd locus and the downstream region including the Dad1 gene. The EACBE is required for long-distance regulation of the Eα on the proximal Vα genes, and its deletion impaired the Tcra rearrangement. We also noticed that the EACBE and Eα regulate the genes in the downstream sub-TAD via asymmetric chromatin extrusion. This study provides a new insight into the role of CTCF binding sites at TAD boundaries in gene regulation.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Cromatina/genética , Regulação da Expressão Gênica , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Proteínas de Homeodomínio/genética , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Timo/citologia
5.
Nat Commun ; 11(1): 4089, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796830

RESUMO

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


Assuntos
Antirretrovirais/uso terapêutico , Infecções por HIV/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Adulto , Células Cultivadas , Feminino , Citometria de Fluxo , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-1/patogenicidade , Humanos , Masculino , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Carga Viral , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
6.
PLoS One ; 15(7): e0236366, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702062

RESUMO

Deep sequencing of T-cell receptor (TCR) genes is powerful at profiling immune repertoire. To prepare a TCR sequencing library, multiplex polymerase chain reaction (mPCR) is widely applied and is highly efficient. That is, most mPCR products contain the region critical for antigen recognition, which also indicates regular V(D)J recombination. Multiplex PCR, however, may suffer from primer bias. A promising alternative is 5'-RACE, which avoids primer bias by applying only one primer pair. In 5'-RACE data, however, non-regular V(D)J recombination (e.g., TCR sequences without a V gene segment) has been observed and the frequency varies (30-80%) between studies. This suggests that the cause of or how to reduce non-regular TCR sequences is not yet well known by the science community. Although it is possible to speculate the cause by comparing the 5'-RACE protocols, careful experimental confirmation is needed and such a systematic study is still not available. Here, we examined the 5'-RACE protocol of a commercial kit and demonstrated how a modification increased the fraction of regular TCR-ß sequences to >85%. We also found a strong linear correlation between the fraction of short DNA fragments and the percentage of non-regular TCR-ß sequences, indicating that the presence of short DNA fragments in the library was the main cause of non-regular TCR-ß sequences. Therefore, thorough removal of short DNA fragments from a 5'-RACE library is the key to high data efficiency. We highly recommend conducting a fragment length analysis before sequencing, and the fraction of short DNA fragments can be used to estimate the percentage of non-regular TCR sequences. As deep sequencing of TCR genes is still relatively expensive, good quality control should be valuable.


Assuntos
Sequência de Aminoácidos/genética , DNA/genética , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Sequência de Bases , Fragmentação do DNA , DNA Complementar/genética , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/imunologia , Humanos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia
7.
Proc Natl Acad Sci U S A ; 117(25): 14342-14353, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513716

RESUMO

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


Assuntos
Antígenos CD5/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Inibidor de NF-kappaB alfa/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Linfócitos T/imunologia , Transferência Adotiva , Animais , Apresentação do Antígeno/imunologia , Antígenos CD5/genética , Linhagem Celular Tumoral , Separação Celular , Sobrevivência Celular/imunologia , Feminino , Citometria de Fluxo , Lipopolissacarídeos/imunologia , Ativação Linfocitária , Camundongos , Camundongos Knockout , Modelos Animais , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Timo/citologia , Timo/crescimento & desenvolvimento , Timo/imunologia , Fator de Transcrição RelA/metabolismo , Regulação para Cima
8.
Proc Natl Acad Sci U S A ; 117(27): 15809-15817, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571924

RESUMO

Src family kinase Lck plays critical roles during T cell development and activation, as it phosphorylates the TCR/CD3 complex to initiate TCR signaling. Lck is present either in coreceptor-bound or coreceptor-unbound (free) forms, and we here present evidence that the two pools of Lck have different molecular properties. We discovered that the free Lck fraction exhibited higher mobility than CD8α-bound Lck in OT-I T hybridoma cells. The free Lck pool showed more activating Y394 phosphorylation than the coreceptor-bound Lck pool. Consistent with this, free Lck also had higher kinase activity, and free Lck mediated higher T cell activation as compared to coreceptor-bound Lck. Furthermore, the coreceptor-Lck coupling was independent of TCR activation. These findings give insights into the initiation of TCR signaling, suggesting that changes in coreceptor-Lck coupling constitute a mechanism for regulation of T cell sensitivity.


Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/metabolismo , Quinases da Família src/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/genética , Hibridomas/imunologia , Ativação Linfocitária/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Camundongos , Fosforilação/genética , Ligação Proteica/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T/imunologia
9.
Scand J Immunol ; 92(2): e12912, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32458431

RESUMO

Immune processes in liver transplantation remain poorly understood. Acute allograft rejection in liver transplantation is a kind of T cell-mediated inflammatory disease accompanied by inflammatory cell infiltration. However, the effect of acute allograft rejection on the immunological characteristics of TCRs in peripheral blood mononuclear cell is unknown. In this study, we characterized the pattern of the human T cell receptor beta chain (TRB) and immunoglobulin heavy chain (IGH) complementarity-determining region 3 (CDR3) repertoires via high-throughput sequencing in 11 acute allograft rejection (AG) cases, 23 patients with stable allograft liver function (ST) who had liver transplantation performed and 20 healthy controls (HC). The diversity of TRB-CDR3 was significantly reduced in the AG group compared with the ST group and healthy controls (HC). The CDR3 and N-addition length distribution were not significantly different between the AG and ST groups. However, N-addition length distribution was significantly changed compared to HC. It seemed that AG used more short N-additions and healthy people used more long N-additions in TRB-CDR3 repertoire. Our findings suggested that the TRB-CDR3 region of AG had distinctive V gene use compared with that of HC. The characteristics of ST seemed to be in between those of AG and HC although the difference is not significant. Cluster analysis showed that the TRB repertoire could not effectively distinguish AG from ST. This research might give to a better understanding of the immune process of liver transplantation.


Assuntos
Regiões Determinantes de Complementaridade/imunologia , Rejeição de Enxerto/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Transplante de Fígado , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Adulto , Regiões Determinantes de Complementaridade/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T alfa-beta/genética
10.
J Leukoc Biol ; 107(6): 1057-1067, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32362028

RESUMO

γδ T cells contribute to the immune response against many cancers, notably through their powerful effector functions that lead to the elimination of tumor cells and the recruitment of other immune cells. However, their presence in the tumor microenvironment has been associated with poor prognosis in breast, colon, and pancreatic cancer, suggesting that γδ T cells may also display pro-tumor activities. Here, we identified in blood from healthy donors a subpopulation of Vδ1T cells that represents around 20% of the whole Vδ1 population, expresses CD73, and displays immunosuppressive phenotype and functions (i.e., production of immunosuppressive molecules, such as IL-10, adenosine, and the chemotactic factor IL-8, and inhibition of αß T cell proliferation). We then found that in human breast tumors, γδ T cells were present particularly in late stage breast cancer samples, and that ∼20% of tumor-infiltrating γδ T cells expressed CD73. Taken together, these results suggest that regulatory γδ T cells are present in the breast cancer microenvironment and may display immunosuppressive functions through the production of immunosuppressive molecules, such as IL-10, IL-8, and adenosine, thus promoting tumor growth.


Assuntos
5'-Nucleotidase/imunologia , Neoplasias da Mama/imunologia , Linhagem da Célula/imunologia , Regulação Neoplásica da Expressão Gênica , Linfócitos do Interstício Tumoral/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T Reguladores/imunologia , 5'-Nucleotidase/genética , Adenosina/imunologia , Adenosina/metabolismo , Adolescente , Adulto , Idoso , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Diferenciação Celular , Linhagem da Célula/genética , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Ativação Linfocitária , Linfócitos do Interstício Tumoral/patologia , Pessoa de Meia-Idade , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Transdução de Sinais , Linfócitos T Reguladores/patologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
11.
Nat Commun ; 11(1): 1522, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251280

RESUMO

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


Assuntos
Infecções por Arenaviridae/imunologia , Autoimunidade , Linfócitos T CD8-Positivos/imunologia , Colite/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Linfócitos T Reguladores/imunologia , Animais , Infecções por Arenaviridae/complicações , Linhagem Celular , Colite/microbiologia , Colo/imunologia , Colo/microbiologia , Fatores de Transcrição Forkhead/metabolismo , Microbioma Gastrointestinal/imunologia , Voluntários Saudáveis , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T Reguladores/metabolismo
12.
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
13.
PLoS Comput Biol ; 16(3): e1007714, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32163410

RESUMO

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


Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta , Aminoácidos , Animais , Antígenos/química , Antígenos/metabolismo , Biologia Computacional , Bases de Dados de Proteínas , Humanos , Camundongos , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia
14.
Proc Natl Acad Sci U S A ; 117(10): 5453-5462, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32098847

RESUMO

Developing lymphocytes diversify their antigen receptor (AgR) loci by variable (diversity) joining (V[D]J) recombination. Here, using the micrococcal nuclease (MNase)-based chromatin accessibility (MACC) assay with low-cell count input, we profile both small-scale (kilobase) and large-scale (megabase) changes in chromatin accessibility and nucleosome occupancy in primary cells during lymphoid development, tracking the changes as different AgR loci become primed for recombination. The three distinct chromatin structures identified in this work define unique features of immunoglobulin H (IgH), Igκ, and T cell receptor-α (TCRα) loci during B lymphopoiesis. In particular, we find locus-specific temporal changes in accessibility both across megabase-long AgR loci and locally at the recombination signal sequences (RSSs). These changes seem to be regulated independently and can occur prior to lineage commitment. Large-scale changes in chromatin accessibility occur without significant change in nucleosome density and represent key features of AgR loci not previously described. We further identify local dynamic repositioning of individual RSS-associated nucleosomes at IgH and Igκ loci while they become primed for recombination during B cell commitment. These changes in chromatin at AgR loci are regulated in a locus-, lineage-, and stage-specific manner during B lymphopoiesis, serving either to facilitate or to impose a barrier to V(D)J recombination. We suggest that local and global changes in chromatin openness in concert with nucleosome occupancy and placement of histone modifications facilitate the temporal order of AgR recombination. Our data have implications for the organizing principles that govern assembly of these large loci as well as for mechanisms that might contribute to aberrant V(D)J recombination and the development of lymphoid tumors.


Assuntos
Linfócitos B/fisiologia , Cromatina/metabolismo , Rearranjo Gênico do Linfócito B , Linfopoese/genética , Receptores de Antígenos/genética , Recombinação V(D)J , Animais , Cromatina/química , Loci Gênicos , Testes Genéticos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/genética , Linfoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Nuclease do Micrococo , Nucleossomos , Receptores de Antígenos de Linfócitos T alfa-beta/genética
15.
J Leukoc Biol ; 107(6): 1009-1022, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32034803

RESUMO

Vitamin C (VitC) is an essential vitamin that needs to be provided through exogenous sources. It is a potent anti-oxidant, and an essential cofactor for many enzymes including a group of enzymes that modulate epigenetic regulation of gene expression. Moreover, VitC has a significant influence on T-cell differentiation, and can directly interfere with T-cell signaling. Conventional CD4 and CD8 T cells express the αß TCR and recognize peptide antigens in the context of MHC presentation. The numerically small population of γδ T cells recognizes antigens in an MHC-independent manner. γδ T cells kill a broad variety of malignant cells, and because of their unique features, are interesting candidates for cancer immunotherapy. In this review, we summarize what is known about the influence of VitC on T-cell activation and differentiation with a special focus on γδ T cells. The known mechanisms of action of VitC on αß T cells are discussed and extrapolated to the effects observed on γδ T-cell activation and differentiation. Overall, VitC enhances proliferation and effector functions of γδ T cells and thus may help to increase the efficacy of γδ T cells applied as cancer immunotherapy in adoptive cell transfer.


Assuntos
Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Epigênese Genética/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Epigênese Genética/imunologia , Humanos , Imunoterapia/métodos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Transdução de Sinais , Linfócitos T/classificação , Linfócitos T/citologia , Linfócitos T/imunologia , Timo/citologia , Timo/imunologia
16.
J Leukoc Biol ; 107(6): 993-1007, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32068302

RESUMO

Cutting-edge questions in αß T cell biology were addressed by investigating a range of different genetically modified mouse models. In comparison, the γδ T cell field lacks behind on the availability of such models. Nevertheless, transgenic mouse models proved useful for the investigation of γδ T cell biology and their stepwise development in the thymus. In general, animal models and especially mouse models give access to a wide range of opportunities of modulating γδ T cells, which is unachievable in human beings. Because of their complex biology and specific tissue tropism, it is especially challenging to investigate γδ T cells in in vitro experiments since they might not reliably reflect their behavior and phenotype under physiologic conditions. This review aims to provide a comprehensive historical overview about how different transgenic mouse models contributed in regards of the understanding of γδ T cell biology, whereby a special focus is set on studies including the elusive role of the γδTCR. Furthermore, evolutionary and translational remarks are discussed under the aspect of future implications for the field. The ultimate full understanding of γδ T cells will pave the way for their usage as a powerful new tool in immunotherapy.


Assuntos
Linhagem da Célula/imunologia , Efeito Fundador , Camundongos Transgênicos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T/imunologia , Animais , Diferenciação Celular , Linhagem da Célula/genética , Movimento Celular , Expressão Gênica , Humanos , Imunoterapia/métodos , Camundongos , Camundongos Transgênicos/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Transdução de Sinais , Especificidade da Espécie , Linfócitos T/classificação , Linfócitos T/citologia , Timo/citologia , Timo/imunologia
17.
J Pathol ; 251(1): 26-37, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32073142

RESUMO

The co-evolving tumour cells and the systemic immune environment are mutually dysregulated. Tumours affect the immune response in a complex manner. For example, although lymphocytes are mobilized in response to tumours, their function is impaired by tumour progression. This study aimed to explore how the baseline and dynamic renal cell carcinoma (RCC) tumour burdens affect the T-cell repertoire, and whether the baseline T-cell receptor ß-chain (TCRB) diversity predicts prognosis. To characterise the TCRB repertoire, the baseline and follow-up peripheral TCRB repertoires of 45 patients with RCC and 2 patients with benign renal disease patients were examined using high-throughput TCRB sequencing. To explain the significance of TCRB diversity, 56 peripheral leukocyte samples from 28 patients before and after surgery were subjected to transcriptome sequencing. To validate the results, an advanced RCC patient's sample was subjected to single-cell RNA sequencing (scRNA, 10x Genomics). Higher TCRB diversity was found to be correlated with a higher lymphocyte-to-neutrophil ratio, especially indicating more naïve T cells. High-baseline TCRB diversity predicted a better prognosis for stage IV patients, and different tumour burdens exerted distinct effects on the immune status. The pre-operative TCRB diversity was significantly higher in benign and stage I (low tumour burden) RCC patients than in stage IV (high tumour burden) patients. After the tumour burden of advanced patients was mostly relieved, we observed that the TCRB diversity was restored, T-cell exhaustion was reduced, and naïve T-cells were mobilized. It was demonstrated that the circulating TCRB repertoire could reflect the immune status and predict prognosis, and to some extent that cytoreductive nephrectomy (CN) reduces the burden of the immune system in advanced patients, which might provide a good opportunity for immunotherapy. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Assuntos
Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/patologia , Adulto , Carcinoma de Células Renais/genética , Feminino , Humanos , Neoplasias Renais/genética , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Masculino , Pessoa de Meia-Idade , Linfócitos T/imunologia
18.
Science ; 367(6481)2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32029687

RESUMO

CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRß (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.


Assuntos
Transferência Adotiva , Sistemas CRISPR-Cas , Edição de Genes , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Linfócitos T/transplante , Idoso , Proteína 9 Associada à CRISPR , Engenharia Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/genética , Transgenes
19.
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
20.
Immunology ; 159(4): 404-412, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31909831

RESUMO

Dendritic cells (DCs) are potent immune cells that control innate and adaptive immune responses. Previous studies have shown that the DCs are required for protection against Staphylococcus aureus infection. However, the role of conventional DC (cDC) subsets during S. aureus infection in vivo has not been well investigated. In this study, we examined the function of spleen DC subsets in the activation of immunity against S. aureus infection. C57BL/6 mice were infected intravenously with S. aureus and DC and T-cell activation were analyzed in vivo. We found that the spleen CD8α- cDCs phagocytosed S. aureus more efficiently than type-1 conventional DCs (cDC1s) did. Moreover, the CD8α- cDCs contributed to the production of pro-inflammatory cytokines in response to S. aureus infection, whereas the cDC1s did not. In addition, infection with S. aureus promoted an increase in the number of Vß T cells. The CD4+ and CD8+ Vß T cells up-regulated the production of interferon-γ (IFN-γ) and interleukin-17 (IL-17) in response to S. aureus infection. Importantly, the induction of IFN-γ and IL-17 production in CD4+ and CD8+ Vß T cells was mediated by S. aureus-stimulated CD8α- cDCs, whereas cDC1s failed to promote IFN-γ and IL-17 production in the cells. Therefore, these data suggested that the spleen CD8α- cDCs are the main DC subsets for induction of S. aureus superantigen-specific immunity.


Assuntos
Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD4-Positivos/patologia , Antígenos CD8/deficiência , Antígenos CD8/genética , Linfócitos T CD8-Positivos/microbiologia , Linfócitos T CD8-Positivos/patologia , Linhagem da Célula/imunologia , Células Dendríticas/microbiologia , Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Imunidade Celular , Interferon gama/genética , Interferon gama/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Baço/imunologia , Baço/microbiologia , Baço/patologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/patogenicidade
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