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1.
Anticancer Res ; 40(10): 5481-5487, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988870

RESUMO

BACKGROUND/AIM: γδ T cells mediate cytotoxicity against prostate cancer (PCa) cells in vitro; however, the clinical efficacy of γδ T cell-targeted immunotherapy for recurrent and metastatic PCa is unsatisfactory. We hypothesized that the resistance of recurrent and metastatic PCa to γδ T cells is related to the presence of prostate cancer stem cells (PCSCs), and we examined their relationship. MATERIALS AND METHODS: PCa spheres (prostaspheres) were generated from five PCa cell lines, and their susceptibility to cytotoxicity by γδ T cells was investigated. Expression of stemness-related markers was evaluated by qRT-PCR. RESULTS: Prostasphere-derived cancer cells were resistant to lysis by γδ T cells and expressed higher levels of several stemness markers, including CD133, NANOG, SOX2, and OCT4, than the parental PCa cell lines. CONCLUSION: Ex vivo-expanded γδ T cells are not effective against PCSCs.


Assuntos
Linfócitos Intraepiteliais/imunologia , Células-Tronco Neoplásicas/imunologia , Neoplasias da Próstata/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Antígeno AC133/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Masculino , Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Fatores de Transcrição SOXB1/genética , Linfócitos T
2.
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
3.
Proc Natl Acad Sci U S A ; 117(31): 18649-18660, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690687

RESUMO

Starting at birth, the immune system of newborns and children encounters and is influenced by environmental challenges. It is still not completely understood how γδ T cells emerge and adapt during early life. Studying the composition of T cell receptors (TCRs) using next-generation sequencing (NGS) in neonates, infants, and children can provide valuable insights into the adaptation of T cell subsets. To investigate how neonatal γδ T cell repertoires are shaped by microbial exposure after birth, we monitored the γ-chain (TRG) and δ-chain (TRD) repertoires of peripheral blood T cells in newborns, infants, and young children from Europe and sub-Saharan Africa. We identified a set of TRG and TRD sequences that were shared by all children from Europe and Africa. These were primarily public clones, characterized by simple rearrangements of Vγ9 and Vδ2 chains with low junctional diversity and usage of non-TRDJ1 gene segments, reminiscent of early ontogenetic subsets of γδ T cells. Further profiling revealed that these innate, public Vγ9Vδ2+ T cells underwent an immediate TCR-driven polyclonal proliferation within the first 4 wk of life. In contrast, γδ T cells using Vδ1+ and Vδ3+ TRD rearrangements did not significantly expand after birth. However, different environmental cues may lead to the observed increase of Vδ1+ and Vδ3+ TRD sequences in the majority of African children. In summary, we show how dynamic γδ TCR repertoires develop directly after birth and present important differences among γδ T cell subsets.


Assuntos
Receptores de Antígenos de Linfócitos T gama-delta , Subpopulações de Linfócitos T/imunologia , África ao Sul do Saara , Bactérias/imunologia , Criança , Pré-Escolar , Europa (Continente) , Rearranjo Gênico do Linfócito T/genética , Rearranjo Gênico do Linfócito T/imunologia , Humanos , Lactente , Recém-Nascido , Estudos Longitudinais , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia
4.
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
5.
Fish Shellfish Immunol ; 99: 587-593, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32112891

RESUMO

Chlorpyrifos is an insecticide that is widely used in agricultural production. However, little is known about how chlorpyrifos disrupts lymphocyte homeostasis in common carp. Herein, we identified TCRγ through the results of transcriptome analysis. Subsequently, we established TCR γ knockdown and overexpression models in carp head kidney lymphocyte respectively using RNA interference and the pcDNA3.1 plasmid, respectively. Real-time PCR, fluorescent staining, ultrastructure observation and flow cytometry were used to detect the levels of the PI3K/AKT pathway, autophagy and apoptosis. Our results demonstrated that chlorpyrifos significantly decreased the expression of TCR γ, TCR γ suppression thereby induced increased mRNA expression of TNF-α, Bax, caspase-3, caspase-8, caspase-9 and significantly inhibited the expression of Bcl-2, which indicated that apoptosis was triggered. This conclusion was supported by our flow cytometry and ultrastructure observation results. In addition, the control and TCR γ overexpression groups had normal cell morphology. Moreover, TCR γ suppression activated the expression of Becline-1, ATG5, ATG10, ATG12, ATG16 and reduced the expression of mTOR, with the opposite results observed in the TCR γ overexpression group. Together, these results suggested that TCR γ imbalance triggers apoptosis and autophagy in lymphocyte. Moreover, we found that TCR γ knockdown significantly increased the mRNA expression of JNK and decreased the expression of PI3K and AKT, which indicated that the PI3K/AKT/JNK pathway was activated. Our results reported here indicated that chlorpyrifos induces apoptosis and autophagy in head kidney lymphocyte through the inhibition of TCR γ.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carpas/imunologia , Clorpirifos/toxicidade , Linfócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Carpas/genética , Células Cultivadas , Perfilação da Expressão Gênica , Inseticidas/toxicidade , Linfócitos/imunologia , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia
6.
J Leukoc Biol ; 107(6): 1069-1079, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32022317

RESUMO

γδT cells play an important role in cancer immunosurveillance and are able to distinguish malignant cells from their healthy counterparts via their γδTCR. This characteristic makes γδT cells an attractive candidate for therapeutic application in cancer immunotherapy. Previously, we have identified a novel CD8α-dependent tumor-specific allo-HLA-A*24:02-restricted Vγ5Vδ1TCR with potential therapeutic value when used to engineer αßT cells from HLA-A*24:02 harboring individuals. αßT cells engineered to express this defined Vγ5Vδ1TCR (TEG011) have been suggested to recognize spatial changes in HLA-A*24:02 present selectively on tumor cells but not their healthy counterparts. However, in vivo efficacy and toxicity studies of TEG011 are still limited. Therefore, we extend the efficacy and toxicity studies as well as the dynamics of TEG011 in vivo in a humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mouse model to allow the preparation of a first-in-men clinical safety package for adoptive transfer of TEG011. Mice treated with TEG011 did not exhibit any graft-versus-host disease-like symptoms and extensive analysis of pathologic changes in NSG-A24:02 mice did not show any off-target toxicity of TEG011. However, loss of persistence of TEG011 in tumor-bearing mice was associated with the outgrowth of extramedullary tumor masses as also observed for mock-treated mice. In conclusion, TEG011 is well tolerated without harming HLA-A*24:02+ expressing healthy tissues, and TEG011 persistence seems to be crucial for long-term tumor control in vivo.


Assuntos
Antígeno HLA-A24/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Neoplasias de Tecidos Moles/prevenção & controle , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Engenharia Celular , Expressão Gênica , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Antígeno HLA-A24/imunologia , Humanos , Imunoterapia/métodos , Células K562 , Masculino , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Transdução de Sinais , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/imunologia , Neoplasias de Tecidos Moles/patologia , Linfócitos T Reguladores/patologia , Linfócitos T Reguladores/transplante , Transdução Genética , Irradiação Corporal Total
7.
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
8.
J Leukoc Biol ; 107(6): 1023-1032, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32064671

RESUMO

The Vγ9Vδ2 T cell subset is the major γδ T cell subset in human peripheral blood and has the unique ability to contribute to immune surveillance by detecting pyrophosphorylated metabolites of isoprenoid synthesis, termed phosphoantigens (pAgs). Vγ9Vδ2 T cells are first detected at midgestation and show postnatal expansion. Interestingly, neonatal Vγ9Vδ2 T cells display a higher TCR repertoire diversity with more public clonotypes and lower pAg responsiveness than in adults. Notably, it is not known whether postnatal changes occur by TCR-dependent reactivity to pAg exposure. Here, we applied next-generation sequencing of γδ TCR repertoires to understand potential differences in the pAg-mediated response of neonatal and adult Vγ9Vδ2 T cells at the level of the expressed γδ TCR. We observed a polyclonal pAg-induced response of neonatal and adult Vγ9Vδ2 T cells, albeit neonatal γδ T cells showed less in vitro pAg responsiveness. Neonatal Vγ9Vδ2 T cells displayed a less pronounced bias for Jδ1 usage and a more frequent use of Jδ2 or Jδ3 that remained stable after pAg exposure. In addition, public and private Vδ2 TRD clones took part in the polyclonal pAg-induced response in neonates and adults. In conclusion, adult and neonatal Vγ9Vδ2 T cells both undergo polyclonal pAg-induced proliferation, whereas especially adult Vγ9Vδ2 T cells display a high stability at the level of the expressed TCR repertoire.


Assuntos
Sangue Fetal/imunologia , Fosfoproteínas/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Proliferação de Células/efeitos dos fármacos , Células Clonais , Sangue Fetal/citologia , Sangue Fetal/efeitos dos fármacos , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recém-Nascido , Interleucina-2/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Organofosfatos/farmacologia , Fosfoproteínas/genética , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T gama-delta/genética , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Ácido Zoledrônico/farmacologia
9.
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
10.
Cancer Immunol Res ; 8(4): 530-543, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019779

RESUMO

γδ T cells in human solid tumors remain poorly defined. Here, we describe molecular and functional analyses of T-cell receptors (TCR) from tumor-infiltrating γδ T lymphocytes (γδ TIL) that were in direct contact with tumor cells in breast cancer lesions from archival material. We observed that the majority of γδ TILs harbored a proinflammatory phenotype and only a minority associated with the expression of IL17. We characterized TCRγ or TCRδ chains of γδ TILs and observed a higher proportion of Vδ2+ T cells compared with other tumor types. By reconstructing matched Vδ2- TCRγ and TCRδ pairs derived from single-cell sequencing, our data suggest that γδ TILs could be active against breast cancer and other tumor types. The reactivity pattern against tumor cells depended on both the TCRγ and TCRδ chains and was independent of additional costimulation through other innate immune receptors. We conclude that γδ TILs can mediate tumor reactivity through their individual γδ TCR pairs and that engineered T cells expressing TCRγ and δ chains derived from γδ TILs display potent antitumor reactivity against different cancer cell types and, thus, may be a valuable tool for engineering immune cells for adoptive cell therapies.


Assuntos
Imunoterapia Adotiva/métodos , Leucócitos Mononucleares/imunologia , Linfócitos do Interstício Tumoral/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Neoplasias de Mama Triplo Negativas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Voluntários Saudáveis , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T gama-delta/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/terapia
11.
J Leukoc Biol ; 107(6): 1033-1044, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31943366

RESUMO

Although γδTCRs were discovered more than 30 yr ago, principles of antigen recognition by these receptors remain unclear and the nature of these antigens is largely elusive. Numerous studies reported that T cell hybridomas expressing several Vγ1-containing TCRs, including the Vγ1Vδ6 TCR of γδNKT cells, spontaneously secrete cytokines. This property was interpreted as recognition of a self-ligand expressed on the hybridoma cells themselves. Here, we revisited this finding using a recently developed reporter system and live single cell imaging. We confirmed strong spontaneous signaling by Vγ1Vδ6 and related TCRs, but not by TCRs from several other γδ or innate-like αß T cells, and demonstrated that both γ and δ chains contributed to this reactivity. Unexpectedly, live single cell imaging showed that activation of this signaling did not require any interaction between cells. Further investigation revealed that the signaling is instead activated by interaction with negatively charged surfaces abundantly present under regular cell culture conditions and was abrogated when noncharged cell culture vessels were used. This mode of TCR signaling activation was not restricted to the reporter cell lines, as interaction with negatively charged surfaces also triggered TCR signaling in ex vivo Vγ1 γδ T cells. Taken together, these results explain long-standing observations on the spontaneous reactivity of Vγ1Vδ6 TCR and demonstrate an unexpected antigen presentation-independent mode of TCR activation by a spectrum of chemically unrelated polyanionic ligands.


Assuntos
Apresentação do Antígeno , Polímeros/farmacologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Timócitos/efeitos dos fármacos , Animais , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Expressão Gênica , Hibridomas/química , Imunofenotipagem , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Polímeros/química , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Transdução de Sinais , Eletricidade Estática , Timócitos/citologia , Timócitos/imunologia , Timo/citologia , Timo/imunologia
12.
J Leukoc Biol ; 107(6): 1097-1105, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31967358

RESUMO

Dissection of the role and function of human γδ T cells and their heterogeneous subsets in cancer, inflammation, and auto-immune diseases is a growing and dynamic research field of increasing interest to the scientific community. Therefore, harmonization and standardization of techniques for the characterization of peripheral and tissue-resident γδ T cells is crucial to facilitate comparability between published and emerging research. The application of commercially available reagents to classify γδ T cells, in particular the combination of multiple Abs, is not always trouble-free, posing major demands on researchers entering this field. Occasionally, even entire γδ T cell subsets may remain undetected when certain Abs are combined in flow cytometric analysis with multicolor Ab panels, or might be lost during cell isolation procedures. Here, based on the recent literature and our own experience, we provide an overview of methods commonly employed for the phenotypic and functional characterization of human γδ T cells including advanced polychromatic flow cytometry, mass cytometry, immunohistochemistry, and magnetic cell isolation. We highlight potential pitfalls and discuss how to circumvent these obstacles.


Assuntos
Citometria de Fluxo/normas , Separação Imunomagnética/normas , Receptores de Antígenos de Linfócitos T gama-delta/análise , Subpopulações de Linfócitos T/imunologia , Anticorpos/química , Carcinoma/diagnóstico , Carcinoma/imunologia , Carcinoma/patologia , Estudos de Casos e Controles , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Expressão Gênica , Doença de Hodgkin/diagnóstico , Doença de Hodgkin/imunologia , Doença de Hodgkin/patologia , Humanos , Imuno-Histoquímica , Separação Imunomagnética/métodos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/patologia
13.
J Leukoc Biol ; 107(6): 1045-1055, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31994778

RESUMO

There are 2 populations of T lymphocytes, αß T and γδ T cells, that can be distinguished by the expression of either an αß TCR or a γδ TCR, respectively. Pairing of the Ag binding heterodimer, which consists of TCR-α/TCR-ß (TCRαß) or TCR-γ/TCR-δ (TCRγδ), with proteins of the CD3 complex forms the complete αß or γδ TCR. Despite some similarities in the structure of TCRαß and TCRγδ and the shared subunits of the CD3 complex, the 2 receptors differ in important aspects. These include the assembly geometry of the complex, the glycosylation pattern, the plasma membrane organization, as well as the accessibility of signaling motifs in the CD3 intracellular tails. These differences are reflected in the different demands and outcomes of ligand-induced signaling. It was shown that exposure of the proline-rich sequence (PRS) in CD3ε occurs with all activating αß TCR ligands and is required to induce αß TCR signaling. In sharp contrast, CD3ε PRS exposure was not induced by binding of those ligands to the γδ TCR that have been studied. Further, signaling by the γδ TCR occurs independently of CD3ε PRS exposure. Interestingly, it can be enhanced by anti-CD3ε Ab-induced enforcement of CD3ε PRS exposure. This review contrasts these two similar, but different immune receptors.


Assuntos
Complexo CD3/imunologia , Linhagem da Célula/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Complexo CD3/antagonistas & inibidores , Complexo CD3/química , Complexo CD3/genética , Diferenciação Celular , Linhagem da Célula/genética , Expressão Gênica , Glicosilação , Humanos , Ligantes , Camundongos , Ligação Proteica/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/química , Receptores de Antígenos de Linfócitos T gama-delta/genética , Transdução de Sinais , Linfócitos T/classificação , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Timo/citologia , Timo/imunologia
14.
Immunohorizons ; 4(1): 33-46, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992577

RESUMO

The TCR is consisted of four chains: α (TCRα), ß (TCRß), γ (TCRγ), and δ (TCRδ) that are present in all jawed vertebrates. Birds are very important in terms of evolutionary aspects of the adaptive immune system, in which it bridges the evolutionary gap between mammals and other vertebrates. To gain better understanding into the genomic organization and complexity of birds' TCR loci, we applied cross-reference error-correction sequencing approach by using Illumina and single-molecule real-time sequencing technology to resequence genomic regions of chicken TCR loci based on 10 mapped bacterial artificial chromosome clones. We did de novo classification of V and J genes for all four chains of the TCR loci according to our sequencing results using the Immunogenetics nomenclature. In sum, we identified 85, 8, and 37 TCR V gene segments in the chicken TCRα/TCRδ, TCRß, and TCRγ loci, respectively. The phylogenetic analysis showed the Vα 7 and Vα family 4 gene sequences shared greater sequence similarity with mammalian species, whereas the other Vα segment sequences are evolutionary closer with sequences from bony fishes. The organization of chicken TCRß locus is more similar to fish TCRß locus over mammalian species, as chicken TCRß locus has a single translocon of its V-D-J-C and exhibits significantly fewer Vß gene segments. In this study, we present a highly precise genomic map for chicken TCR loci and phylogenetic relationships of TCR variable gene segments against other animal species and verified the relative stability of the receptor structure during evolutional process.


Assuntos
Genes Codificadores dos Receptores de Linfócitos T , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T/genética , Animais , Evolução Biológica , Galinhas , Mapeamento Cromossômico , Evolução Molecular , Genômica , Filogenia , Análise de Sequência de DNA
15.
Clin Cancer Res ; 26(2): 408-418, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31636100

RESUMO

PURPOSE: Mycosis fungoides is the most common subtype of cutaneous T-cell lymphoma. Skin-directed treatments often improve but do not cure mycosis fungoides skin lesions. The purpose of this study was to (i) assess whether remission was associated with malignant T-cell clone depletion at treated sites using either low-dose radiotherapy (LDRT, 8 Gy) or topical steroids and (ii) assess whether a clone-ablative therapy, like LDRT, is associated with overall survival in patients with high-risk early-stage CTCL. EXPERIMENTAL DESIGN: Pre- and posttreatment biopsies from 20 lesional skin samples of 18 patients with mycosis fungoides who received either 8 Gy LDRT (n = 16) or topical steroids (n = 4) underwent high-throughput T-cell receptor sequencing of the TCRB gene to quantify the malignant T-cell clone. For the retrospective chart review, overall survival of 47 high-risk early-stage patients was compared between patients who did or did not receive radiation. RESULTS: LDRT eradicated the clone in 5 of 16 lesions and reduced it >90% in 11 of 16; there were no recurrences in these lesions. Patients treated with topical steroids appeared to clinically improve, but the malignant clone persisted. We found that the number of residual malignant T cells predicted lesion recurrence. A retrospective review showed that early-stage high-risk patients who received radiation as part of their treatment regimen had prolonged overall survival compared with patients who did not. CONCLUSIONS: These findings demonstrate that LDRT can eradicate malignant T cells in mycosis fungoides, provides robust disease control, and is associated with improved survival in high-risk early-stage patients.


Assuntos
Biomarcadores Tumorais/análise , Linfoma Cutâneo de Células T/mortalidade , Micose Fungoide/mortalidade , Recidiva Local de Neoplasia/mortalidade , Radioterapia/mortalidade , Neoplasias Cutâneas/mortalidade , Subpopulações de Linfócitos T/efeitos da radiação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Linfoma Cutâneo de Células T/patologia , Linfoma Cutâneo de Células T/radioterapia , Masculino , Pessoa de Meia-Idade , Micose Fungoide/patologia , Micose Fungoide/radioterapia , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/radioterapia , Estudos Prospectivos , Receptores de Antígenos de Linfócitos T gama-delta/genética , Estudos Retrospectivos , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/radioterapia , Taxa de Sobrevida , Resultado do Tratamento , Adulto Jovem
16.
Immunol Cell Biol ; 98(1): 79-87, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31680329

RESUMO

γδ T cells are unconventional T cells that function on the border of innate and adaptive immunity. They are suggested to play important roles in neonatal and infant immunity, although their phenotype and function are not fully characterized in early childhood. We aimed to investigate γδ T cells in relation to age, prematurity and cytomegalovirus (CMV) infection. Therefore, we used flow cytometry to characterize the γδ T-cell compartment in cord blood and peripheral blood cells from 14-day-, 2-year- and 5-year-old children, as well as in peripheral blood samples collected at several time points during the first months of life from extremely premature neonates. γδ T cells were phenotypically similar at 2 and 5 years of age, whereas cord blood was divergent and showed close proximity to γδ T cells from 14-day-old neonates. Interestingly, 2-year-old children and adults showed comparable Vδ2+ γδ T-cell functionality toward both microbial and polyclonal stimulations. Importantly, extreme preterm birth compromised the frequencies of Vδ1+ cells and affected the functionality of Vδ2+ γδ T cells shortly after birth. In addition, CMV infection was associated with terminal differentiation of the Vδ1+ compartment at 2 years of age. Our results show an adult-like functionality of the γδ T-cell compartment already at 2 years of age. In addition, we demonstrate an altered γδ T-cell phenotype early after birth in extremely premature neonates, something which could possible contribute to the enhanced risk for infections in this vulnerable group of children.


Assuntos
Envelhecimento , Desenvolvimento Infantil , Receptores de Antígenos de Linfócitos T gama-delta , Linfócitos T/imunologia , Adulto , Envelhecimento/genética , Envelhecimento/imunologia , Pré-Escolar , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro/crescimento & desenvolvimento , Recém-Nascido Prematuro/imunologia , Masculino , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia
17.
J Leukoc Biol ; 107(6): 1081-1095, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31833593

RESUMO

Epithelial ovarian cancer displays the highest mortality of all gynecological tumors. A relapse of the disease even after successful surgical treatment is a significant problem. Resistance against the current platinum-based chemotherapeutic standard regime requires a detailed ex vivo immune profiling of tumor-infiltrating cells and the development of new therapeutic strategies. In this study, we phenotypically and functionally characterize tumor cells and autologous tumor-derived αß and γδ T lymphocyte subsets. Tumor-infiltrating (TIL) and tumor-ascites lymphocytes (TAL) were ex vivo isolated out of tumor tissue and ascites, respectively, from high-grade ovarian carcinoma patients (FIGO-stage IIIa-IV). We observed an increased γδ T cell percentage in ascites compared to tumor-tissue and blood of these patients, whereas CD8+ αß T cells were increased within TAL and TIL. The number of Vδ1 and non-Vδ1/Vδ2-expressing γδ T cells was increased in the ascites and in the tumor tissue compared to the blood of the same donors. Commonly in PBL, the Vγ9 chain of the γδ T cell receptor is usually associated exclusively with the Vδ2 chain. Interestingly, we detected Vδ1 and non-Vδ1/Vδ2 T cells co-expressing Vγ9, which is so far not described for TAL and TIL. Importantly, our data demonstrated an expression of human epidermal growth factor receptor (HER)-2 on high-grade ovarian tumors, which can serve as an efficient tumor antigen to target CD3 TIL or selectively Vγ9-expressing γδ T cells by bispecific antibodies (bsAbs) to ovarian cancer cells. Our bsAbs efficiently enhance cytotoxicity of TIL and TAL against autologous HER-2-expressing ovarian cells.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Carcinoma Epitelial do Ovário/imunologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Neoplasias Ovarianas/imunologia , Receptor ErbB-2/genética , Linfócitos T Citotóxicos/efeitos dos fármacos , Adulto , Ascite/genética , Ascite/imunologia , Ascite/patologia , Ascite/cirurgia , Complexo CD3/genética , Complexo CD3/imunologia , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/patologia , Carcinoma Epitelial do Ovário/cirurgia , Técnicas de Cocultura , Citotoxicidade Imunológica/efeitos dos fármacos , Feminino , Expressão Gênica , Humanos , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/cirurgia , Cultura Primária de Células , Receptor ErbB-2/imunologia , 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 , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia
19.
Front Immunol ; 10: 2717, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824502

RESUMO

In the last years, important progresses have been registered in the treatment of patients suffering from oncological/haematological malignancies, but more still needs to be done to reduce toxicity and side effects, improve outcome and offer new strategies for relapsed or refractory disease. A remarkable part of these clinical benefits is due to advances in immunotherapy. Here, we investigate the generation of a novel, universal and ready-to-use immunotherapeutic product based on γδ-T lymphocytes. These cells are part of the innate immune system, exerting potent natural cytotoxicity against bacteria, viruses and tumours. This ability, coupled with their negligible alloreactivity, makes them attractive for adoptive immunotherapy approaches. To achieve a cell product suitable for clinical use, we developed a strategy capable to generate polyclonal γδ-T cells with predominant memory-Vδ1 phenotype in good manufacturing practice (GMP) procedures with the additional possibility of gene-modification to improve their anti-tumour activity. Irradiated, engineered artificial antigen-presenting cells (aAPCs) expressing CD86/41BBL/CD40L and the cytomegalovirus (CMV)-antigen-pp65 were used. The presence of CMV-pp65 and CD40L proved to be crucial for expansion of the memory-Vδ1 subpopulation. To allow clinical translation and guarantee patient safety, aAPCs were stably transduced with an inducible suicide gene. Expanded γδ-T cells showed high expression of activation and memory markers, without signs of exhaustion; they maintained polyclonality and potent anti-tumour activity both in vitro (against immortalised and primary blasts) and in in vivo studies without displaying alloreactivity signals. The molecular characterisation (phophoproteomic and gene-expression) of these cell products underlines their unique properties. These cells can further be armed with chimeric antigen receptors (CAR) to improve anti-tumour capacity and persistence. We demonstrate the feasibility of establishing an allogeneic third-party, off-the-shelf and ready-to-use, γδ-T-cell bank. These γδ-T cells may represent an attractive therapeutic option endowed with broad clinical applications, including treatment of viral infections in highly immunocompromised patients, treatment of aggressive malignancies refractory to conventional approaches, bridging therapy to more targeted immunotherapeutic approaches and, ultimately, an innovative platform for the development of off-the-shelf CAR-T-cell products.


Assuntos
Transferência Adotiva , Memória Imunológica , Ativação Linfocitária/imunologia , Neoplasias Experimentais/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T , Animais , Humanos , Células K562 , Ativação Linfocitária/genética , Camundongos , Neoplasias Experimentais/genética , Neoplasias Experimentais/terapia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T/imunologia , Linfócitos T/transplante , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Front Immunol ; 10: 2282, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31608066

RESUMO

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection is a serious threat to human health. γδT cells, which are characterized by major histocompatibility complex (MHC) non-restriction, are rapidly activated and initiate anti-infectious immune responses in the early stages of Mtb infection. However, the mechanism underlying the recognition of Mtb by γδT cells remains unclear. In this study, we characterized the pattern of the human T-cell receptor (TCR) γδ complementary determinant region 3 (CDR3) repertoire in TB patients by using high-throughput immune repertoire sequencing. The results showed that the diversity of CDR3δ was significantly reduced and that the frequency of different gene fragments (V/J), particularly the V-segment of the δ-chain, was substantially altered, which indicate that TB infection-related γδT cells, especially the δ genes, were activated and amplified in TB patients. Then, we screened the Mtb-associated epitopes/proteins recognized by γδT cells using an Mtb proteome chip with dominant CDR3δ peptides as probes. We identified the Mtb protein Rv0002 as a potential ligand capable of stimulating the activation and proliferation of γδT cells. Our findings provide a further understanding of the mechanisms underlying γδT cell-mediated immunity against Mtb infection.


Assuntos
Mycobacterium tuberculosis/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Tuberculose/imunologia , Adulto , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Epitopos/genética , Epitopos/imunologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunidade Celular/imunologia , Ligantes , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/fisiologia , Peptídeos/genética , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/metabolismo , Tuberculose/genética , Tuberculose/microbiologia , Adulto Jovem
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