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1.
Nat Commun ; 11(1): 5243, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067472

RESUMO

SARS-CoV-2 is the novel coronavirus responsible for the current COVID-19 pandemic. Severe complications are observed only in a small proportion of infected patients but the cellular mechanisms underlying this progression are still unknown. Comprehensive flow cytometry of whole blood samples from 54 COVID-19 patients reveals a dramatic increase in the number of immature neutrophils. This increase strongly correlates with disease severity and is associated with elevated IL-6 and IP-10 levels, two key players in the cytokine storm. The most pronounced decrease in cell counts is observed for CD8 T-cells and VD2 γδ T-cells, which both exhibit increased differentiation and activation. ROC analysis reveals that the count ratio of immature neutrophils to VD2 (or CD8) T-cells predicts pneumonia onset (0.9071) as well as hypoxia onset (0.8908) with high sensitivity and specificity. It would thus be a useful prognostic marker for preventive patient management and improved healthcare resource management.


Assuntos
Betacoronavirus/imunologia , Linfócitos T CD8-Positivos/imunologia , Neutrófilos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Biomarcadores/sangue , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/patologia , Citometria de Fluxo , Humanos , Imunofenotipagem/métodos , Interleucina-10/sangue , Interleucina-6/sangue , Contagem de Linfócitos , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Índice de Gravidade de Doença
2.
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
3.
Proc Natl Acad Sci U S A ; 117(31): 18638-18648, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32665435

RESUMO

Vγ9Vδ2 T cells are a major human blood γδ T cell population that respond in a T cell receptor (TCR)-dependent manner to phosphoantigens which are generated by a variety of microorganisms. It is not clear how Vγ9Vδ2 T cells react toward the sudden microbial exposure early after birth. We found that human Vγ9Vδ2 T cells with a public/shared fetal-derived TCR repertoire expanded within 10 wk postpartum. Such an expansion was not observed in non-Vγ9Vδ2 γδ T cells, which possessed a private TCR repertoire. Furthermore, only the Vγ9Vδ2 T cells differentiated into potent cytotoxic effector cells by 10 wk of age, despite their fetal origin. Both the expansion of public fetal Vγ9Vδ2 T cells and their functional differentiation were not affected by newborn vaccination with the phosphoantigen-containing bacillus Calmette-Guérin (BCG) vaccine. These findings suggest a strong and early priming of the public fetal-derived Vγ9Vδ2 T cells promptly after birth, likely upon environmental phosphoantigen exposure.


Assuntos
Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Vacina BCG/imunologia , Células Cultivadas , Citocinas/metabolismo , Feminino , Feto/imunologia , Humanos , Lactente , Recém-Nascido , Gravidez
4.
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
5.
Nat Commun ; 11(1): 3769, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32724083

RESUMO

Butyrophilin-like (Btnl) genes are emerging as major epithelial determinants of tissue-associated γδ T cell compartments. Thus, the development of signature, murine TCRγδ+ intraepithelial lymphocytes (IEL) in gut and skin depends on Btnl family members, Btnl1 and Skint1, respectively. In seeking mechanisms underlying these profound effects, we now show that normal gut and skin γδ IEL development additionally requires Btnl6 and Skint2, respectively, and furthermore that different Btnl heteromers can seemingly shape different intestinal γδ+ IEL repertoires. This formal genetic evidence for the importance of Btnl heteromers also applied to the steady-state, since sustained Btnl expression is required to maintain the signature TCR.Vγ7+ IEL phenotype, including specific responsiveness to Btnl proteins. In sum, Btnl proteins are required to select and to maintain the phenotypes of tissue-protective γδ IEL compartments, with combinatorially diverse heteromers having differential impacts on different IEL subsets.


Assuntos
Butirofilinas/metabolismo , Imunidade Celular , Linfócitos Intraepiteliais/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Animais , Butirofilinas/genética , Butirofilinas/imunologia , Perfilação da Expressão Gênica , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Linfócitos Intraepiteliais/metabolismo , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T gama-delta/imunologia
6.
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
7.
Clin Exp Immunol ; 201(1): 40-57, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32255193

RESUMO

Hypoxia within the tumor microenvironment (TME) is a key factor contributing to immunosuppression in tumors, co-relating with poor treatment outcome and decreased overall survival in advanced oral cancer (OC) patients. Vδ2 is a dominant subset of gamma delta T cells (γδT cells) present in the peripheral blood which exhibits potent anti-tumor cytotoxicity and is evolving as a key player of anti-cancer cellular therapy. However, the fate of γδT cells in hypoxic oral tumors remains elusive. In the present study, we compared the effect of hypoxia (1% O2 ) and normoxia (21% O2 ) on the expansion, proliferation, activation status, cytokine secretion and cytotoxicity of γδT cells isolated from OC patients and healthy individuals. Hypoxia-exposed γδT cells exhibited reduced cytotoxicity against oral tumor cells. Our data demonstrated that hypoxia reduces the calcium efflux and the expression of degranulation marker CD107a in γδT cells, which explains the decreased anti-tumor cytotoxicity of γδT cells observed under hypoxia. Hypoxia-exposed γδT cells differentiated to γδT17 [γδ T cells that produce interleukin (IL)-17] cells, which corroborated our observations of increased γδT17 cells observed in the oral tumors. Co-culture of γδT cells with CD8 T cells in the presence of hypoxia showed that programmed cell death ligand 1 (PD-L1)high γδT cells brought about apoptosis of programmed cell death 1 (PD-1)high CD8 T cells which could be significantly reversed upon blocking PD-1. Thus, future immunotherapeutic treatment modality for oral cancer may use a combined approach of blocking the PD-1/PD-L1 signaling and targeting hypoxia-inducible factor 1α, which may help in reversing hypoxia-induced immunosuppression.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Imunidade Celular , Neoplasias Bucais/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Células Th17/imunologia , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/patologia , Hipóxia Celular/imunologia , Técnicas de Cocultura , Feminino , Humanos , Masculino , Neoplasias Bucais/patologia , Células Th17/patologia
8.
Immunity ; 52(3): 487-498.e6, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32155411

RESUMO

Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.


Assuntos
Antígenos/imunologia , Butirofilinas/imunologia , Células Germinativas/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Animais , Antígenos/metabolismo , Antígenos CD/química , Antígenos CD/imunologia , Antígenos CD/metabolismo , Butirofilinas/química , Butirofilinas/metabolismo , Células CHO , Cricetinae , Cricetulus , Células Germinativas/metabolismo , Células HEK293 , Humanos , Fosforilação , Ligação Proteica , Multimerização Proteica , Receptores de Antígenos de Linfócitos T gama-delta/química , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/metabolismo
9.
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
10.
Proc Natl Acad Sci U S A ; 117(12): 6697-6707, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32139608

RESUMO

Vγ9Vδ2 T cells are a major γδ T cell population in the human blood expressing a characteristic Vγ9JP rearrangement paired with Vδ2. This cell subset is activated in a TCR-dependent and MHC-unrestricted fashion by so-called phosphoantigens (PAgs). PAgs can be microbial [(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, HMBPP] or endogenous (isopentenyl pyrophosphate, IPP) and PAg sensing depends on the expression of B7-like butyrophilin (BTN3A, CD277) molecules. IPP increases in some transformed or aminobisphosphonate-treated cells, rendering those cells a target for Vγ9Vδ2 T cells in immunotherapy. Yet, functional Vγ9Vδ2 T cells have only been described in humans and higher primates. Using a genome-based study, we showed in silico translatable genes encoding Vγ9, Vδ2, and BTN3 in a few nonprimate mammalian species. Here, with the help of new monoclonal antibodies, we directly identified a T cell population in the alpaca (Vicugna pacos), which responds to PAgs in a BTN3-dependent fashion and shows typical TRGV9- and TRDV2-like rearrangements. T cell receptor (TCR) transductants and BTN3-deficient human 293T cells reconstituted with alpaca or human BTN3 or alpaca/human BTN3 chimeras showed that alpaca Vγ9Vδ2 TCRs recognize PAg in the context of human and alpaca BTN3. Furthermore, alpaca BTN3 mediates PAg recognition much better than human BTN3A1 alone and this improved functionality mapped to the transmembrane/cytoplasmic part of alpaca BTN3. In summary, we found remarkable similarities but also instructive differences of PAg-recognition by human and alpaca, which help in better understanding the molecular mechanisms controlling the activation of this prominent population of γδ T cells.


Assuntos
Anticorpos Monoclonais/imunologia , Butirofilinas/metabolismo , Hemiterpenos/farmacologia , Ativação Linfocitária/imunologia , Compostos Organofosforados/farmacologia , Subpopulações de Linfócitos T/imunologia , Animais , Butirofilinas/antagonistas & inibidores , Butirofilinas/genética , Butirofilinas/imunologia , Sistemas CRISPR-Cas , Camelídeos Americanos , Feminino , Células HEK293 , Humanos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Receptores de Antígenos de Linfócitos T gama-delta/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismo
12.
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
13.
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
14.
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
15.
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
16.
J Immunol ; 204(7): 1968-1981, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32102904

RESUMO

Intestinal intraepithelial lymphocytes (IEL) comprise a diverse population of cells residing in the epithelium at the interface between the intestinal lumen and the sterile environment of the lamina propria. Because of this anatomical location, IEL are considered critical components of intestinal immune responses. Indeed, IEL are involved in many different immunological processes, ranging from pathogen control to tissue stability. However, despite their critical importance in mucosal immune responses, very little is known about the homeostasis of different IEL subpopulations. The phosphoprotein osteopontin is important for critical physiological processes, including cellular immune responses, such as survival of Th17 cells and homeostasis of NK cells among others. Because of its impact in the immune system, we investigated the role of osteopontin in the homeostasis of IEL. In this study, we report that mice deficient in the expression of osteopontin exhibit reduced numbers of the IEL subpopulations TCRγδ+, TCRß+CD4+, TCRß+CD4+CD8α+, and TCRß+CD8αα+ cells in comparison with wild-type mice. For some IEL subpopulations, the decrease in cell numbers could be attributed to apoptosis and reduced cell division. Moreover, we show in vitro that exogenous osteopontin stimulates the survival of murine IEL subpopulations and unfractionated IEL derived from human intestines, an effect mediated by CD44, a known osteopontin receptor. We also show that iCD8α IEL but not TCRγδ+ IEL, TCRß+ IEL, or intestinal epithelial cells, can promote survival of different IEL populations via osteopontin, indicating an important role for iCD8α cells in the homeostasis of IEL.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Homeostase/imunologia , Intestinos/imunologia , Linfócitos Intraepiteliais/imunologia , Osteopontina/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Epitélio/imunologia , Feminino , Humanos , Receptores de Hialuronatos/imunologia , Células Matadoras Naturais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Células Th17/imunologia
17.
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
18.
J Immunol ; 204(7): 1798-1809, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32066596

RESUMO

Plasmodium spp., the causative agent of malaria, have a complex life cycle. The exponential growth of the parasites during the blood stage is responsible for almost all malaria-associated morbidity and mortality. Therefore, tight immune control of the intraerythrocytic replication of the parasite is essential to prevent clinical malaria. Despite evidence that the particular lymphocyte subset of γδ T cells contributes to protective immunity during the blood stage in naive hosts, their precise inhibitory mechanisms remain unclear. Using human PBMCs, we confirmed in this study that γδ T cells specifically and massively expanded upon activation with Plasmodium falciparum culture supernatant. We also demonstrate that these activated cells gain cytolytic potential by upregulating cytotoxic effector proteins and IFN-γ. The killer cells bound to infected RBCs and killed intracellular P. falciparum via the transfer of the granzymes, which was mediated by granulysin in a stage-specific manner. Several vital plasmodial proteins were efficiently destroyed by granzyme B, suggesting proteolytic degradation of these proteins as essential in the lymphocyte-mediated death pathway. Overall, these data establish a granzyme- and granulysin-mediated innate immune mechanism exerted by γδ T cells to kill late-stage blood-residing P. falciparum.


Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Granzimas/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Antígenos de Protozoários/imunologia , Células Cultivadas , Eritrócitos/imunologia , Humanos , Imunidade Inata/imunologia , Interferon gama/imunologia , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/imunologia , Estágios do Ciclo de Vida/imunologia , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/imunologia , Regulação para Cima/imunologia
19.
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
20.
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
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