RESUMO
Like B cells, T cells can be immortalized through hybridization with lymphoma cells, a technique that has been particularly useful in the study of the T cell receptors (TCR) for antigen. In T cell hybridizations, the AKR mouse strain-derived thymus lymphoma BW5147 is by far the most popular fusion line. However, the full potential of this technology had to await inactivation of the productively rearranged TCR-α and -ß genes in the lymphoma. BWα-ß-, the TCR-gene deficient variant of the original lymphoma, which has become the fusion line of choice for αß T cells, is now available with numerous modifications, enabling the investigation of many aspects of TCR-mediated responses and TCR-structure. Unexpectedly, inactivating BW's functional TCR-α gene also rendered the lymphoma more permissive for the expression of TCR-γδ, facilitating the study of γδ T cells, their TCRs, and their TCR-mediated reactivity.
Assuntos
Linfoma de Células T/patologia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/fisiologia , Neoplasias do Timo/patologia , Animais , Fusão Celular , Linhagem Celular Tumoral , Humanos , Hibridomas , Linfoma de Células T/metabolismo , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismo , Neoplasias do Timo/metabolismoRESUMO
Regulatory effects of γδ T-cells on immune responses have been studied for years. We have investigated the regulatory effect of γδ T-cells on Th1 and Th17 autoimmune responses, and have studied molecular and cellular mechanisms by which γδ T-cells enhance or inhibit immune responses, exploiting a well-characterized murine model of experimental autoimmune uveitis (EAU). Our results show that (1) aberrant γδ T-cell activation is an important pathogenic event in EAU; (2) γδ T-cells have a unique regulatory effect on Th17 autoimmune responses, which is shaped by the activation status of γδ T-cells; and (3) γδ-mediated immunoregulation is closely linked with the extracellular adenosine metabolism. Reciprocal interactions between γδ T-cells and extracellular adenosine partially determine the development of EAU.
Assuntos
Doenças Autoimunes/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Uveíte/imunologia , Adenosina , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Modelos Animais , Receptores de Antígenos de Linfócitos T gama-delta/genéticaRESUMO
We previously reported that activated γδ T cells greatly enhance autoimmune responses, particularly the Th17 response. To determine the mechanisms involved, we made a series of comparisons between activated and non-activated γδ T cells. Our results showed that activated γδ T cells expressed greatly increased levels of A2A adenosine receptor (A2AR) and decreased amounts of CD73, as well as increased amounts of T cell activation markers such as CD69, CD44 and CD25. We show that A2AR is a major functional molecule in the enhancing activity of γδ T cells. A2AR-/- γδ T cells (isolated from A2AR-/- mouse), lost their Th17-enhancing activity as did A2AR+/+ γδ T cells (isolated from wt-B6 mouse) after treatment with an A2AR antagonist. Since γδ T cells possess either an enhancing or an inhibiting effect, we also tested whether A2AR expression on γδ T cells is essential to their inhibiting effect. Our results showed that the inhibiting effect of A2AR-/- γδ T cells was as potent as that of A2AR+/+ γδ T cells. In a previous report we showed that the expression of different levels of CD73 molecule allowed γδ T cells to adjust their suppressive activity; in the current study, we show that expression of increased amounts of A2AR allows γδ T cells to more effectively exert their enhancing function.
Assuntos
Doenças Autoimunes/imunologia , Autoimunidade/fisiologia , Receptor A2A de Adenosina/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Uveíte/imunologia , Animais , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor A2A de Adenosina/genética , Células Th17/imunologiaRESUMO
Whether γδ T cells inhibit or enhance the Foxp3 T cell response depends upon their activation status. The critical enhancing effector in the supernatant is adenosine. Activated γδ T cells express adenosine receptors at high levels, which enables them to deprive Foxp3+ T cells of adenosine, and to inhibit their expansion. Meanwhile, cell-free supernatants of γδ T cell cultures enhance Foxp3 T cell expansion. Thus, inhibition and enhancement by γδ T cells of Foxp3 T cell response are a reflection of the balance between adenosine production and absorption by γδ T cells. Non-activated γδ T cells produce adenosine but bind little, and thus enhance the Foxp3 T cell response. Activated γδ T cells express high density of adenosine receptors and have a greatly increased ability to bind adenosine. Extracellular adenosine metabolism and expression of adenosine receptor A2ARs by γδ T cells played a major role in the outcome of γδ and Foxp3 T cell interactions. A better understanding of the functional conversion of γδ T cells could lead to γδ T cell-targeted immunotherapies for related diseases.
Assuntos
Adenosina/farmacologia , Fatores de Transcrição Forkhead/imunologia , Receptor A2A de Adenosina/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Animais , Células Cultivadas , Feminino , Fatores de Transcrição Forkhead/genética , Camundongos , Camundongos Knockout , Receptor A2A de Adenosina/genética , Receptores de Antígenos de Linfócitos T gama-delta/genéticaRESUMO
Internal tandem duplication of the juxtamembrane domain of FMS-like tyrosine kinase 3 (FLT3-ITD) is the most prevalent genetic aberration present in 20-30% of acute myeloid leukaemia (AML) cases and is associated with a poor prognosis. FLT3-ITD expressing cells express elevated levels of NADPH oxidase 4 (NOX4)-generated pro-survival hydrogen peroxide (H2O2) contributing to increased levels of DNA oxidation and double strand breaks. NOX4 is constitutively active and has been found to have various isoforms expressed at multiple locations within a cell. The purpose of this study was to investigate the expression, localisation and regulation of NOX4 28 kDa splice variant, NOX4D. NOX4D has previously been shown to localise to the nucleus and nucleolus in various cell types and is implicated in the generation of reactive oxygen species (ROS) and DNA damage. Here, we demonstrate that FLT3-ITD expressing-AML patient samples as well as -cell lines express the NOX4D isoform resulting in elevated H2O2 levels compared to FLT3-WT expressing cells, as quantified by flow cytometry. Cell fractionation indicated that NOX4D is nuclear membrane-localised in FLT3-ITD expressing cells. Treatment of MV4-11 cells with receptor trafficking inhibitors, tunicamycin and brefeldin A, resulted in deglycosylation of NOX4 and NOX4D. Inhibition of the FLT3 receptor revealed that the FLT3-ITD oncogene is responsible for the production of NOX4D-generated H2O2 in AML. We found that inhibition of the PI3K/AKT and STAT5 pathways resulted in down-regulation of NOX4D-generated pro-survival ROS. Taken together these findings indicate that nuclear membrane-localised NOX4D-generated pro-survival H2O2 may be contributing to genetic instability in FLT3-ITD expressing AML.
RESUMO
γδ T cells can either enhance or inhibit an adaptive immune response, but the mechanisms involved are not fully understood. Given that CD73 is the main enzyme responsible for conversion of AMP into the immunosuppressive molecule adenosine, we investigated its role in the regulatory function of γδ T cells in experimental autoimmune uveitis (EAU). We found that γδ T cells expressed different amounts of CD73 during the different stages of EAU and that low CD73 expression on γδ T cells correlated with enhanced Th17 response-promoting activity. Functional comparison of CD73-deficient and wild-type B6 (CD73+/+) mice showed that failure to express CD73 decreased both the enhancing and suppressive effects of γδ T cells on EAU. We also demonstrated that γδ T cells expressed different amounts of CD73 when activated by different pathways, which enabled them to either enhance or inhibit an adaptive immune response. Our results demonstrate that targeting CD73 expression on γδ T cells may allow us to manipulate their pro- or anti-inflammatory effect on Th17 responses.
Assuntos
5'-Nucleotidase/fisiologia , Doença Autoimune do Sistema Nervoso Experimental/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Uveíte/imunologia , 5'-Nucleotidase/biossíntese , 5'-Nucleotidase/deficiência , 5'-Nucleotidase/genética , Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Células Dendríticas/imunologia , Proteínas do Olho/imunologia , Proteínas do Olho/toxicidade , Feminino , Regulação da Expressão Gênica/imunologia , Interferon gama/sangue , Interferon gama/deficiência , Interleucina-17/sangue , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença Autoimune do Sistema Nervoso Experimental/enzimologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/toxicidade , Receptores de Antígenos de Linfócitos T gama-delta/análise , Receptores de Antígenos de Linfócitos T gama-delta/deficiência , Proteínas de Ligação ao Retinol/imunologia , Proteínas de Ligação ao Retinol/toxicidade , Subpopulações de Linfócitos T/enzimologia , Linfócitos T Reguladores/enzimologia , Células Th1/imunologia , Células Th17/imunologia , Uveíte/enzimologiaRESUMO
We previously reported that selective ablation of certain γδ T cell subsets, rather than removal of all γδ T cells, strongly affects serum Ab levels in nonimmunized mice. This type of manipulation also changed T cells, including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4(+) and Vγ6(+) γδ T cells (B6.TCR-Vγ4(-/-)/6(-/-)), we observed expanded Vγ1(+) cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αß T cells as well as spontaneous germinal center formation in the spleen, and elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4(-/-)/6(-/-) mice and other peripheral B cell populations were diminished, most of all splenic marginal zone (MZ) B cells. However, relative frequencies and absolute numbers of Ab-producing cells, as well as serum levels of Abs, IL-4, and BAFF, were increased. Cell transfers confirmed that these changes are directly dependent on the altered γδ T cells in this strain and on their enhanced potential of producing IL-4. Further evidence suggests the possibility of direct interactions between γδ T cells and B cells in the splenic MZ. Taken together, these data demonstrate the capability of γδ T cells of modulating size and productivity of preimmune peripheral B cell populations.
Assuntos
Linfócitos B/imunologia , Interleucina-4/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Baço/imunologia , Subpopulações de Linfócitos T/imunologia , Transferência Adotiva , Animais , Anticorpos/sangue , Autoanticorpos/sangue , Fator Ativador de Células B/sangue , Células Cultivadas , Técnicas de Cocultura , Centro Germinativo/imunologia , Imunoglobulina G/sangue , Interleucina-4/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/genética , Baço/citologia , Subpopulações de Linfócitos T/transplanteRESUMO
The insulin peptide B:9-23 is a natural antigen in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). In addition to αß T cells and B cells, γδ T cells recognize the peptide and infiltrate the pancreatic islets where the peptide is produced within ß cells. The peptide contains a cysteine in position 19 (Cys19), which is required for the γδ but not the αß T cell response, and a tyrosine in position 16 (Tyr16), which is required for both. A peptide-specific mAb, tested along with the T cells, required neither of the two amino acids to bind the B:9-23 peptide. We found that γδ T cells require Cys19 because they recognize the peptide antigen in an oxidized state, in which the Cys19 thiols of two peptide molecules form a disulfide bond, creating a soluble homo-dimer. In contrast, αß T cells recognize the peptide antigen as a reduced monomer, in complex with the MHCII molecule I-A(g7). Unlike the unstructured monomeric B:9-23 peptide, the γδ-stimulatory homo-dimer adopts a distinct secondary structure in solution, which differs from the secondary structure of the corresponding portion of the native insulin molecule. Tyr16 is required for this adopted structure of the dimerized insulin peptide as well as for the γδ response to it. This observation is consistent with the notion that γδ T cell recognition depends on the secondary structure of the dimerized insulin B:9-23 antigen.
Assuntos
Antígenos/imunologia , Insulina/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Compostos de Sulfidrila/imunologia , Animais , Diabetes Mellitus Tipo 1/imunologia , Dimerização , Feminino , Camundongos , Camundongos Endogâmicos NOD , OxirreduçãoRESUMO
PURPOSE: We determined the mechanism by which all-trans retinoic acid (ATRA) inhibits experimental autoimmune uveitis (EAU) and determined the role of γδ T cells in this autoimmune disease. METHODS: C57BL/6 (B6) mice were immunized with the uveitogenic, interphotoreceptor retinoid-binding protein1-20 peptide (IRBP1-20) in complete Freund's adjuvant (CFA), with or without a preceding ATRA treatment. Responses and pathogenic activity of Th1- and Th17-autoreactive T cells were compared, and the effects of ATRA on γδ T cells and CD25(+) dendritic cell (DC) subset were determined. Interactions among uveitogenic T cells, DC subsets, and γδ T cells were investigated. RESULTS: Administration of ATRA to B6 mice in which EAU was induced suppressed the response of Th17 autoreactive T cells, which was associated with decreased generation of the CD25(+) DC subset and suppressed activation of γδ T cells. Adoptively transferred γδ T cells isolated from ATRA-treated mice showed a diminished ability to promote the activation of Th17 autoreactive T cells in vitro and in vivo compared to γδ T cells from untreated donors. CONCLUSIONS: ATRA inhibits the expansion of CD25(+) DCs and γδ T-cell activation, thereby restraining the Th17 autoreactive T-cell response.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Células Dendríticas/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Tretinoína/farmacologia , Uveíte/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Interleucina-17/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/metabolismo , Uveíte/imunologia , Uveíte/patologiaRESUMO
It has been demonstrated that the two main subsets of peripheral γδ T cells, Vγ1 and Vγ4, have divergent functions in many diseases models. Recently, we reported that Vγ4 γδ T cells played a protective role in tumor immunity through eomesodermin-controlled mechanisms. However, the precise roles of Vγ1 γδ T cells in tumor immunity, especially whether Vγ1 γδ T cells have any interaction with Vγ4 γδ T cells, remain unknown. We demonstrated in this paper that Vγ1 γδ T cells suppressed Vγ4 γδ T cell-mediated antitumor function both in vitro and in vivo, and this suppression was cell contact independent. Using neutralizing anti-IL-4 Ab or IL-4(-/-) mice, we determined the suppressive factor derived from Vγ1 γδ T cells was IL-4. Indeed, treatment of Vγ4 γδ T cells with rIL-4 significantly reduced expression levels of NKG2D, perforin, and IFN-γ. Finally, Vγ1 γδ T cells produced more IL-4 and expressed significantly higher level of GATA-3 upon Th2 priming in comparison with Vγ4 γδ T cells. Therefore, to our knowledge, our results established for the first time a negative regulatory role of Vγ1 γδ T cells in Vγ4 γδ T cell-mediated antitumor immunity through cell contact-independent and IL-4-mediated mechanisms. Selective depletion of this suppressive subset of γδ T cells may be beneficial for tumor immune therapy.
Assuntos
Regulação para Baixo/imunologia , Interleucina-4/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/biossíntese , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia , Animais , Comunicação Celular/genética , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Regulação para Baixo/genética , Interleucina-4/deficiência , Interleucina-4/genética , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/prevenção & controle , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T gama-delta/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Microambiente Tumoral/genéticaRESUMO
γδ T cells express adaptive antigen receptors encoded by rearranging genes. Their diversity is highest in the small region of TCR V-J junctions, especially in the δ chain, which should enable the γδ TCRs to distinguish differences in small epitopes. Indeed, recognition of small molecules, and of an epitope on a larger protein has been reported. Responses to small non-peptides known as phospho-antigens are multi-clonal yet limited to a single γδ T cell subset in humans and non-human primates. Responses to small peptides are multi-clonal or oligo-clonal, include more than one subset of γδ T cells, and occur in rodents and primates. However, less effort has been devoted to investigate the peptide responses. To settle the questions of whether peptides can be ligands for the γδ TCRs, and whether responses to small peptides might occur normally, peptide binding will have to be demonstrated, and natural peptide ligands identified.
Assuntos
Antígenos/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Animais , Antígenos/metabolismo , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Humanos , Peptídeos/metabolismo , Ligação Proteica/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/metabolismoRESUMO
Although the TNF receptor family member CD27 has been known for some time, its functional role as a coreceptor on T and B cells remains poorly understood. Recent reports have shown that CD27 and its ligand CD70 play a critical role in the development and function of γδ T cells in mice. In this issue of the European Journal of Immunology, a study now extends these findings to the Vγ9Vδ2(+) subset of human γδ T cells. This subset, whose responses are readily elicited by phosphoantigens, plays an important role in anti-tumor immune responses. This study shows that most Vγ9Vδ2(+) cells express CD27, and signaling via the CD27-CD70 axis is needed for their survival, proliferation and cytokine secretion. Moreover, CD27 functions as a coreceptor, which promotes, in conjunction with TCR-mediated signals, expansion of Th1-biased Vγ9Vδ2(+) cells. This new information underscores the significance of CD27 in γδ T-cell functional differentiation, and is likely to facilitate the development of γδ T-cell-based clinical immunotherapy.
Assuntos
Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Antígenos de Neoplasias/imunologia , Ligante CD27/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Ativação Linfocitária/imunologia , Camundongos , Fosfoproteínas/imunologia , Células Th1/imunologiaRESUMO
We previously demonstrated that gammadelta T cells played an important role in tumor immune surveillance by providing an early source of IFN-gamma. The precise role of different subsets of gammadelta T cells in the antitumor immune response, however, is unknown. Vgamma1 and Vgamma4 gammadelta T cells are the principal subsets of peripheral lymphoid gammadelta T cells and they might play distinct roles in tumor immunity. In support of this, we observed that reconstitution of TCRdelta(-/-) mice with Vgamma4, but not Vgamma1, gammadelta T cells restored the antitumor response. We also found that these effects were exerted by the activated (CD44(high)) portion of Vgamma4 gammadelta T cells. We further determined that IFN-gamma and perforin are critical elements in the Vgamma4-mediated antitumor immune response. Indeed, CD44(high) Vgamma4 gammadelta T cells produced significantly more IFN-gamma and perforin on activation, and showed greater cytolytic activity than did CD44(high) Vgamma1 gammadelta T cells, apparently due to the high level of eomesodermin (Eomes) in these activated Vgamma4 gammadelta T cells. Consistently, transfection of dominant-negative Eomes in Vgamma4 gammadelta T cells diminished the level of IFN-gamma secretion, indicating a critical role of Eomes in the effector function of these gammadelta T cells. Our results thus reveal distinct functions of Vgamma4 and Vgamma1 gammadelta T cells in antitumor immune response, and identify a protective role of activated Vgamma4 gammadelta T cells, with possible implications for tumor immune therapy.
Assuntos
Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Proteínas com Domínio T/biossíntese , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Regulação para Cima/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Citotoxicidade Imunológica , Receptores de Hialuronatos/biossíntese , Receptores de Hialuronatos/fisiologia , Interferon gama/biossíntese , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Perforina/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Proteínas com Domínio T/genéticaRESUMO
Granulomas play an essential role in the sequestration and killing of mycobacteria in the lung; however, the mechanisms of their development and maturation are still not clearly understood. IL-17A is involved in mature granuloma formation in the mycobacteria-infected lung. Therefore, IL-17A gene-knockout (KO) mice fail to develop mature granulomas in the Mycobacterium bovis bacille Calmette-Guérin (BCG)-infected lung. This study analyzed the mechanism of IL-17A-dependent mature granuloma formation in the mycobacteria-infected lung. The IL-17A KO mice showed a normal level of nascent granuloma formation on day 14 but failed to develop mature granulomas on day 28 after the BCG infection in the lung. The observation implies that IL-17A is required for the maturation of granuloma from the nascent to mature stage. TCR gammadelta T cells expressing TCR Vgamma4 or Vgamma6 were identified as the major IL-17A-producing cells that resided in the BCG-induced lung granuloma. The adoptive transfer of the IL-17A-producing TCR gammadelta T cells reconstituted granuloma formation in the IL-17A KO mice. The expression of ICAM-1 and LFA-1, which are adhesion molecules important in granuloma formation, decreased in the lung of the BCG-infected IL-17A KO mice, and their expression was induced on BCG-infected macrophages in coculture with IL-17A-producing TCR gammadelta T cells. Furthermore, IL-17A KO mice showed not only an impaired mature granuloma formation, but also an impaired protective response to virulent Mycobacterium tuberculosis. Therefore, IL-17A produced by TCR gammadelta T cells plays a critical role in the prevention of M. tuberculosis infection through the induction of mature granuloma formation.
Assuntos
Granuloma/imunologia , Granuloma/patologia , Interleucina-17/fisiologia , Mycobacterium bovis/imunologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/patologia , Animais , Movimento Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Granuloma/microbiologia , Interleucina-17/deficiência , Interleucina-17/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium bovis/patogenicidade , Receptores de Antígenos de Linfócitos T gama-delta/biossíntese , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/transplante , Tuberculose Pulmonar/prevenção & controle , Virulência/imunologiaRESUMO
Successful application of gammadelta T cells in adoptive cell therapies depends upon our ability to maintain these cells in vivo. Using an adoptive transfer model to study lymphopenia-induced homeostatic expansion, we show that CD8(+) and NK1.1(+) gammadelta T cell subsets are differentially regulated. While CD8(+) gammadelta T cells have an early and sustained advantage following transfer into TCRbeta(-/-)/delta(-/-) mice, NK1.1(+) gammadelta T cells proliferate slowly and are maintained at low numbers. The advantage of the CD8(+) subset could not be explained by increased bcl-2 or cytokine receptor expression but did correlate with Vgamma4(+) and Vdelta5(+) expression. Despite the role of CD8 in MHC class I recognition by alphabeta T cells, beta(2)-microglobulin (beta(2)m)-associated MHC class I molecules were not required for CD8(+) gammadelta T cell homeostatic expansion. Surprisingly, all gammadelta T cells, including the CD8(+) subset, exhibited enhanced proliferation following adoptive transfer into Rag1(-/-)/beta(2)m(-/-) compared with Rag1(-/-) recipients. This effect was most notable for the NK1.1(+) subset, which expresses high levels of NKG2A/CD94 and Ly49. Although expression of these inhibitory receptors correlated with poor homeostatic expansion in the presence of beta(2)m, gammadelta T cell homeostatic proliferation in TCRbeta(-/-)/delta(-/-) mice was not altered in the presence of Ly49C/I- and NKG2-blocking Abs. While the mechanism by which beta(2)m negatively regulates gammadelta T cell homeostasis remains to be determined, this observation is unique to gammadelta T cells and confirms that multiple mechanisms are in place to maintain strict regulation of both the size and the composition of the gammadelta T cell pool.
Assuntos
Homeostase/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Microglobulina beta-2/imunologia , Transferência Adotiva , Animais , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores de Interleucina/biossíntese , Subpopulações de Linfócitos T/metabolismo , Microglobulina beta-2/metabolismoRESUMO
gammadelta T cells regulate airway reactivity, but their role in ozone (O3)-induced airway hyperresponsiveness (AHR) is not known. Our objective was to determine the role of gammadelta T cells in O3-induced AHR. Different strains of mice, including those that were genetically manipulated or antibody-depleted to render them deficient in total gammadelta T cells or specific subsets of gammadelta T cells, were exposed to 2.0 ppm of O3 for 3 hours. Airway reactivity to inhaled methacholine, airway inflammation, and epithelial cell damage were monitored. Exposure of C57BL/6 mice to O3 resulted in a transient increase in airway reactivity, neutrophilia, and increased numbers of epithelial cells in the lavage fluid. TCR-delta(-/-) mice did not develop AHR, although they exhibited an increase in neutrophils and epithelial cells in the lavage fluid. Similarly, depletion of gammadelta T cells in wild-type mice suppressed O3-induced AHR without influencing airway inflammation or epithelial damage. Depletion of Vgamma1+, but not of Vgamma4+ T cells, reduced O3-induced AHR, and transfer of total gammadelta T cells or Vgamma1+ T cells to TCR-delta(-/-) mice restored AHR. After transfer of Vgamma1+ cells to TCR-delta(-/-) mice, restoration of AHR after O3 exposure was blocked by anti-TNF-alpha. However, AHR could be restored in TCR-delta(-/-)mice by transfer of gammadelta T cells from TNF-alpha-deficient mice, indicating that another cell type was the source of TNF-alpha. These results demonstrate that TNF-alpha and activation of Vgamma1+ gammadelta T cells are required for the development of AHR after O3 exposure.
Assuntos
Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Hipersensibilidade Respiratória/imunologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Transferência Adotiva , Animais , Anticorpos/farmacologia , Contagem de Células , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Ozônio , Pneumonia/complicações , Pneumonia/imunologia , Pneumonia/patologia , Receptores de Antígenos de Linfócitos T gama-delta/deficiência , Hipersensibilidade Respiratória/induzido quimicamente , Hipersensibilidade Respiratória/complicações , Hipersensibilidade Respiratória/patologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Linfócitos T/efeitos dos fármacos , Fator de Necrose Tumoral alfa/deficiênciaRESUMO
gammadelta T cells respond rapidly following West Nile virus (WNV) infection, limiting viremia and invasion of the central nervous system and thereby protecting the host from lethal encephalitis. Here, we investigated the role of two major subpopulations of peripheral gammadelta T cells, Vgamma1(+) and Vgamma4(+) cells, in host immunity against WNV infection. We found initially that aged mice were more susceptible to WNV infection than young mice. Following WNV challenge, Vgamma1(+) cells in young mice expanded significantly whereas Vgamma4(+) cells expanded modestly. In contrast, aged mice exhibited a slower and reduced response of Vgamma1(+) cells but maintained a higher content of Vgamma4(+) cells. Vgamma1(+) cells were the major gammadelta subset producing IFN-gamma during WNV infection. Mice depleted of Vgamma1(+) cells had an enhanced viremia and higher mortality to WNV encephalitis. Vgamma4(+) cells had a higher potential for producing tumor necrosis factor-alpha (TNF-alpha), a cytokine known to be involved in blood-brain barrier compromise and WNV entry into the brain. Depletion of Vgamma4(+) cells reduced TNF-alpha level in the periphery, accompanied by a decreased viral load in the brain and a lower mortality to WN encephalitis. These results suggest that Vgamma1(+) and Vgamma4(+) cells play distinct roles in protection and pathogenesis during WNV infection.
Assuntos
Receptores de Antígenos de Linfócitos T gama-delta/análise , Subpopulações de Linfócitos T/imunologia , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/imunologia , Fatores Etários , Animais , Interferon gama/biossíntese , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sobrevida , Fator de Necrose Tumoral alfa/biossíntese , Viremia/imunologia , Febre do Nilo Ocidental/mortalidadeRESUMO
The present study was designed to elucidate the role of Vgamma4(+) gammadelta T cells, a major subset of pulmonary gammadelta T cells, in host defense against infection with Streptococcus pneumoniae. The proportion and number of whole gammadelta T cells, identified as CD3(+) and TCR-delta(+) cells, and Vgamma4(+) gammadelta T cells, identified as CD3(+) and TCR-Vgamma4(+) cells, increased in the lungs at 3, 6 and 12h post-infection. Survival of infected mice and lung bacterial clearance were severely impaired in TCR-Vgamma4(-/-) mice compared with control wild-type (WT) mice. The impaired host protection in TCR-Vgamma4(-/-) mice correlated well with attenuated recruitment of neutrophils in lungs. MIP-2 and TNF-alpha synthesis in the infected tissues was significantly reduced in TCR-Vgamma4(-/-) mice compared with WT mice. Similar results were noted in the synthesis of TNF-alpha, but not clearly of MIP-2, by lung leukocytes stimulated with live bacteria. Our results demonstrate that Vgamma4(+) gammadelta T cells play an important role in the neutrophil-mediated host defense against S. pneumoniae infection by promoting the synthesis of TNF-alpha and possibly of MIP-2 in the lungs.
Assuntos
Pulmão/imunologia , Neutrófilos/imunologia , Pneumonia Pneumocócica/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Streptococcus pneumoniae/patogenicidade , Linfócitos T/imunologia , Animais , Quimiocina CXCL2 , Quimiocinas/metabolismo , Humanos , Pulmão/citologia , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/imunologia , Pneumonia Pneumocócica/microbiologia , Fator de Necrose Tumoral alfa/metabolismo , VirulênciaRESUMO
BACKGROUND: Previous work from our center has suggested a correlation between increased donor-derived Vdelta1+ gammadelta T cells and long-term relapse-free survival following bone marrow transplantation for leukemia. Questions remain, however, as to whether this observation can be explained by a gammadelta T cell-based immune response against primary leukemia. METHODS: We examined gammadelta T cell receptor (TCR) phenotype, cell proliferation, and cytolytic activity following culture with irradiated primary leukemia blasts from a haploidentical first-degree relative. Subsequently, we also studied the gammadelta TCR phenotype and complimentarity determining region 3 (CDR3) cDNA sequences from 17 newly diagnosed leukemia patients. RESULTS: In 17/28 (61%) of in vitro cultures, gammadelta T cells proliferated in culture with primary blasts. Vdelta1+ T cells were proportionally increased in all cultures and were the predominant cell population in 6/17. In the 7 cultures where cytotoxicity could be assessed, 6 (86%) showed some degree of cytotoxicity to the primary leukemia. Vdelta1+ T cells were also the predominant gammadelta T cell subtype in pre-treatment leukemia patients principally due to loss of Vdelta2+ T cells rather than expansion of Vdelta1+ cells. The Vdelta1 CDR3-region cDNA sequence from these patients revealed exclusive use of the Jdelta1 constant region and sequence conservation in 4/11 patients. CONCLUSIONS: gammadelta T cells exhibit an in vitro response to primary leukemia blasts that is manifested by proliferation, an increased proportion of Vdelta1+ T cells, and cytotoxicity to the primary leukemia blasts. The Vdelta1+ T cell population is also predominant in newly diagnosed leukemia patients likely due to a loss of circulating Vdelta2+ T cells. A small proportion of newly diagnosed patients showed Vdelta1 CDR3 region similarity. These findings suggest a role for gammadelta T cells in the immune response to leukemia.
Assuntos
Imunofenotipagem , Leucemia Mieloide Aguda/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/biossíntese , Linfócitos T/imunologia , Sequência de Bases , Proliferação de Células , Células Cultivadas , Regiões Determinantes de Complementaridade/genética , Testes Imunológicos de Citotoxicidade , Feminino , Humanos , Teste de Cultura Mista de Linfócitos , Masculino , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T gama-delta/genética , Análise de Sequência de DNA , Taxa de SobrevidaRESUMO
The natural ligands recognized by gammadelta TCRs are still largely unknown, in part because immunization does not normally result in Ag-specific gammadelta T cell responses. Taking advantage of an established ligand for a particular gammadelta TCR, we demonstrated that a multimerized recombinant form of this gammadelta TCR can be used like a mAb to specifically detect its own ligand. Using the same approach for more common gammadelta TCRs whose ligands remain unknown, we detected on certain cell lines molecules that appear to be ligands for three additional gammadelta TCRs. One of these represents the mouse Vgamma6/Vdelta1 invariant gammadelta TCR, which predominates in the female reproductive tract, the tongue, and the lung, and other tissues during inflammation. The second represents the closely related Vgamma5/Vdelta1 invariant gammadelta TCR expressed by most epidermal T cells. The third is a Vgamma1/Vdelta6.3 TCR, representative of a variable type frequently found on lymphoid gammadelta T cells. We found evidence that ligands for multiple gammadelta TCRs may be simultaneously expressed on a single cell line, and that at least some of the putative ligands are protease sensitive. This study suggests that soluble versions of gammadelta TCRs can be as tools to identify and characterize the natural ligands of gammadelta T cells.