RESUMO
Multiple sclerosis (MS) is a T cell-driven inflammatory disease of the CNS. Research on T cell subsets involved in MS pathogenesis has mainly focused on classical CD4+ T cells, especially Th17 cells, as they produce the proinflammatory, MS-associated cytokine IL-17. However, the abundant unconventional mucosal-associated invariant T (MAIT) cells are also able to produce IL-17. MAIT cells are characterized by high CD161 expression and a semi-invariant Vα7.2 TCR, with which they recognize bacterial and yeast Ags derived from the riboflavin (vitamin B2) metabolism. In this study, we characterized MAIT cells from the peripheral blood of MS patients in comparison with healthy individuals with respect to their type-17 differentiation. We found a specific increase of IL-17+ MAIT cells as well as an increased expression of retinoic acid-related orphan receptor (ROR)γt and CCR6 in MAIT cells from MS patients, whereas the expression of T cell activation markers HLA-DR and CD38 was not different. IL-17 production by MAIT cells furthermore correlated with the surface expression level of the IL-7 receptor α-chain (CD127), which was significantly increased on MAIT cells from MS patients in comparison with healthy individuals. In summary, our findings indicate an augmented type-17 differentiation of MAIT cells in MS patients associated with their IL-7 receptor surface expression, implicating a proinflammatory role of these unconventional T cells in MS immunopathology.
Assuntos
Sistema Nervoso Central/patologia , Interleucina-17/biossíntese , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Células T Invariantes Associadas à Mucosa/imunologia , Esclerose Múltipla/patologia , ADP-Ribosil Ciclase 1/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Sistema Nervoso Central/imunologia , Antígenos HLA-DR/metabolismo , Humanos , Interferon gama/biossíntese , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/metabolismo , Células T Invariantes Associadas à Mucosa/metabolismo , Esclerose Múltipla/imunologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK/biossíntese , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/biossíntese , Receptores de Antígenos de Linfócitos T/imunologia , Receptores CCR6/biossíntese , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/imunologia , Proteínas Supressoras de Tumor/metabolismoRESUMO
While the functional plasticity of memory CD4(+) T cells has been studied extensively, less is known about this property in memory CD8(+) T cells. Here, we report the direct measurement of plasticity by paired daughter analysis of effector and memory OT-I CD8(+) T cells primed in vivo with ovalbumin. Naïve, effector, and memory OT-I cells were isolated and activated in single-cell culture; then, after the first division, their daughter cells were transferred to new cultures with and without IL-4; expression of IFN-γ and IL-4 mRNAs was measured 5 days later in the resultant subclones. Approximately 40% of clonogenic memory CD8(+) T cells were bipotential in this assay, giving rise to an IL-4(-) subclone in the absence of IL-4 and an IL-4(+) subclone in the presence of IL-4. The frequency of bipotential cells was lower among memory cells than naïve cells but markedly higher than among 8-day effectors. Separation based on high or low expression of CD62L, CD122, CD127, or Ly6C did not identify a phenotypic marker of the bipotential cells. Functional plasticity in memory CD8(+) T-cell populations can therefore reflect modulation at the level of a single memory cell and its progeny.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Plasticidade Celular/imunologia , Memória Imunológica/imunologia , Interleucina-4/farmacologia , Animais , Antígenos Ly/biossíntese , Biomarcadores/análise , Linhagem Celular , Interferon gama/biossíntese , Subunidade beta de Receptor de Interleucina-2/biossíntese , Interleucina-4/genética , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Selectina L/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/biossínteseRESUMO
The IL-7R plays critical roles in lymphocyte development and homeostasis. Although IL-7R expression is strictly regulated during lymphocyte differentiation and the immune response, little is known regarding its in vivo regulation. To address this issue, we established a mouse line with targeted deletion of the conserved non-coding sequence 1 (CNS1) element found 3.6 kb upstream of the IL-7Rα promoter. We report that IL-7Rα is expressed normally on T and B cells in thymus and bone marrow of CNS1(-/-) mice except for in regulatory T cells. In contrast, these mice show reduced IL-7Rα expression in conventional CD4 and CD8 T cells as well as regulatory T, NKT, and γδ T cells in the periphery. CD4 T cells of CNS1(-/-) mice showed IL-7Rα upregulation in the absence of growth factors and IL-7Rα downregulation by IL-7 or TCR stimulation, although the expression levels were lower than those in control mice. Naive CD4 and CD8 T cells of CNS1(-/-) mice show attenuated survival by culture with IL-7 and reduced homeostatic proliferation after transfer into lymphopenic hosts. CNS1(-/-) mice exhibit impaired maintenance of Ag-stimulated T cells. Furthermore, IL-7Rα upregulation by glucocorticoids and TNF-α was abrogated in CNS1(-/-) mice. This work demonstrates that the CNS1 element controls IL-7Rα expression and maintenance of peripheral T cells, suggesting differential regulation of IL-7Rα expression between central and peripheral lymphoid organs.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Elementos Facilitadores Genéticos , Subunidade alfa de Receptor de Interleucina-7/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Proliferação de Células/genética , Células Cultivadas , Dexametasona/farmacologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Deleção de Sequência/genética , Transdução de Sinais/imunologia , Transcrição Gênica/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
During infection or vaccination, only a small proportion of CD8(+) T cells differentiate into memory cells. The mechanisms underlying the differentiation of CD8(+) T cells into short-lived effector cells (SLECs) or memory precursor effector cells are poorly defined. It was recently shown in infectious models that the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp-1) enhances the formation of SLECs. The factors controlling Blimp-1 expression leading to the in vivo formation of SLECs are still not known. However, it has been shown that cytokines such as IL-2 induce Blimp-1 expression in vitro. In this study, we took advantage of the low-inflammation model of dendritic cell immunization to study the role of the IL-2/Blimp-1 axis in SLEC differentiation as well as the importance of Blimp-1 expression in memory precursor effector cells for proper CD8(+) memory generation. Our results show that Blimp-1 deficiency affects effector differentiation and function in the absence of inflammation. Unexpectedly, memory generation was not affected in Blimp-1-deficient OT-I cells responding to vaccination. In addition, modulation of the bioavailability of IL-2 by injection either of a blocking Ab or of the cytokine, demonstrates a link between IL-2, Blimp-1 induction, and SLEC formation in wild-type cells. Conversely, injection of IL-2 had less effect on Blimp-1-deficient CD8(+) T cells, indicating that the effect of IL-2 on in vivo SLEC differentiation is mediated by Blimp-1. In conclusion, IL-2 induction of Blimp-1 expression is a key regulator of SLEC differentiation in vivo.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Memória Imunológica , Interleucina-2/imunologia , Fatores de Transcrição/biossíntese , Animais , Anticorpos Bloqueadores/imunologia , Apoptose/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Feminino , Granzimas/biossíntese , Inflamação/imunologia , Interferon gama/biossíntese , Interleucina-2/biossíntese , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Subunidade alfa de Receptor de Interleucina-7/genética , Lectinas Tipo C , Listeria monocytogenes/genética , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , Fator 1 de Ligação ao Domínio I Regulador Positivo , Receptores Imunológicos/biossíntese , Receptores Imunológicos/genética , Fatores de Transcrição/genéticaRESUMO
The role of T-cell immunosenescence and functional CD8(+) T-cell responses in HIV/TB co-infection is unclear. We examined and correlated surrogate markers of HIV disease progression with immune activation, immunosenescence and differentiation using T-cell pools of HIV/TB co-infected, HIV-infected and healthy controls. Our investigations showed increased plasma viremia and reduced CD4/CD8 T-cell ratio in HIV/TB co-infected subjects relative to HIV-infected, and also a closer association with changes in the expression of CD38, a cyclic ADP ribose hydrolase and CD57, which were consistently expressed on late-senescent CD8(+) T cells. Up-regulation of CD57 and CD38 were directly proportional to lack of co-stimulatory markers on CD8(+) T cells, besides diminished expression of CD127 (IL-7Rα) on CD57(+)CD4(+) T cells. Notably, intracellular IFN-γ, perforin and granzyme B levels in HIV-specific CD8(+) T cells of HIV/TB co-infected subjects were diminished. Intracellular CD57 levels in HIV gag p24-specific CD8(+) T cells were significantly increased in HIV/TB co-infection. We suggest that HIV-TB co-infection contributes to senescence associated with chronic immune activation, which could be due to functional insufficiency of CD8(+) T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Senescência Celular/imunologia , Infecções por HIV/imunologia , Imunossenescência/imunologia , Tuberculose Pulmonar/imunologia , ADP-Ribosil Ciclase 1/biossíntese , Adulto , Relação CD4-CD8 , Antígenos CD57/biossíntese , Antígenos CD57/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/patologia , Diferenciação Celular/imunologia , Proliferação de Células , Coinfecção/imunologia , Progressão da Doença , Feminino , Granzimas/metabolismo , Cadeias alfa de HLA-DR/imunologia , Humanos , Interferon gama/metabolismo , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Ativação Linfocitária/imunologia , Masculino , Glicoproteínas de Membrana/biossíntese , Perforina/metabolismoRESUMO
CD8(+) T cells play a crucial role in the clearance of intracellular pathogens through the generation of cytotoxic effector cells that eliminate infected cells and long-lived memory cells that provide enhanced protection against reinfection. We have previously shown that the inhibitor of E protein transcription factors, Id2, is necessary for accumulation of effector and memory CD8(+) T cells during infection. In this study, we show that CD8(+) T cells lacking Id2 did not generate a robust terminally differentiated killer cell lectin-like receptor G1 (KLRG1)(hi) effector population, but displayed a cell-surface phenotype and cytokine profile consistent with memory precursors, raising the question as to whether loss of Id2 impairs the differentiation and/or survival of effector memory cells. We found that deletion of Bim rescued Id2-deficient CD8(+) cell survival during infection. However, the dramatic reduction in KLRG1(hi) cells caused by loss of Id2 remained in the absence of Bim, such that Id2/Bim double-deficient cells form an exclusively KLRG1(lo)CD127(hi) memory precursor population. Thus, we describe a role for Id2 in both the survival and differentiation of normal CD8(+) effector and memory populations.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteína 2 Inibidora de Diferenciação/fisiologia , Receptores Imunológicos/biossíntese , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/microbiologia , Linfócitos T CD8-Positivos/virologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Citocinas/biossíntese , Memória Imunológica/genética , Imunofenotipagem , Proteína 2 Inibidora de Diferenciação/deficiência , Proteína 2 Inibidora de Diferenciação/genética , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Lectinas Tipo C , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco/imunologia , Células-Tronco/microbiologia , Células-Tronco/virologia , Proteína bcl-X/deficiência , Proteína bcl-X/genéticaRESUMO
Whether differences between naive cell-derived primary (1°) and memory cell-derived secondary (2°) CD4(+) T-cell effectors contribute to protective recall responses is unclear. Here, we compare these effectors directly after influenza A virus infection. Both develop with similar kinetics, but 2° effectors accumulate in greater number in the infected lung and are the critical component of memory CD4(+) T-cell-mediated protection against influenza A virus, independent of earlier-acting memory-cell helper functions. Phenotypic, functional, and transcriptome analyses indicate that 2° effectors share organ-specific expression patterns with 1° effectors but are more multifunctional, with more multicytokine (IFN-γ(+)/IL-2(+)/TNF(+))-producing cells and contain follicular helper T-cell populations not only in the spleen and draining lymph nodes but also in the lung. In addition, they express more CD127 and NKG2A but less ICOS and Lag-3 than 1° effectors and express higher levels of several genes associated with survival and migration. Targeting two differentially expressed molecules, NKG2A and Lag-3, reveals differential regulation of 1° and 2° effector functions during pathogen challenge.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Memória Imunológica/imunologia , Animais , Antígenos CD/biossíntese , Galinhas , Citocinas/metabolismo , Perfilação da Expressão Gênica , Proteína Coestimuladora de Linfócitos T Induzíveis/biossíntese , Vírus da Influenza A/metabolismo , Interferon gama/metabolismo , Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Cinética , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Subfamília C de Receptores Semelhantes a Lectina de Células NK/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Baço/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
In a previous study, we identified TRIB1, a serine-threonine kinase-like molecule, as a biomarker of chronic antibody-mediated rejection of human kidneys when measured in peripheral blood mononuclear cells. Here, we focused our analysis on a specific subset of peripheral blood mononuclear cells that play a dominant role in regulating immune responses in health and disease, so-called CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). We isolated both human and murine Treg and non-Treg counterparts and analyzed TRIB1 and Foxp3 mRNA expression by quantitative PCR on the freshly isolated cells or following 24 h of activation. Physical interaction between the human TRIB1 and Foxp3 proteins was analyzed in live cell lines by protein complementation assay using both flow cytometry and microscopy and confirmed in primary freshly isolated human CD4(+)CD25(hi)CD127(-) Tregs by co-immunoprecipitation. Both TRIB1 and Foxp3 were expressed at significantly higher levels in Tregs than in their CD4(+)CD25(-) counterparts (p < 0.001). Moreover, TRIB1 and Foxp3 mRNA levels correlated tightly in Tregs (Spearman r = 1.0; p < 0.001, n = 7), but not in CD4(+)CD25(-) T cells. The protein complementation assay revealed a direct physical interaction between TRIB1 and Foxp3 in live cells. This interaction was impaired upon deletion of the TRIB1 N-terminal but not the C-terminal domain, suggesting an interaction in the nucleus. This direct interaction within the nucleus was confirmed in primary human Tregs by co-immunoprecipitation. These data show a direct relationship between TRIB1 and Foxp3 in terms of their expression and physical interaction and highlight Tribbles-1 as a novel binding partner of Foxp3 in Tregs.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Linfócitos T Reguladores/citologia , Animais , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/imunologia , Adesão Celular , Ciclo Celular , Proliferação de Células , Sobrevivência Celular , DNA Complementar/metabolismo , Teste de Complementação Genética , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucócitos Mononucleares/citologia , Camundongos , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Linfócitos T/citologia , Linfócitos T Reguladores/metabolismoRESUMO
BACKGROUND & AIMS: Dysfunctional CD8(+) T cells are believed to contribute to the ability of hepatitis C virus (HCV) to evade the immune response. Most studies have focused on the effector functions of HCV-specific CD8(+) T cells or their surface expression of inhibitory receptors. There is currently no information available about the ex vivo ability of HCV-specific CD8(+) T cells to inhibit viral replication (antiviral efficacy). METHODS: To analyze the antiviral efficacy of virus-specific CD8(+) T cells ex vivo, we used an immunologic model based on a cell line that expresses HLA-A*02 and contains a stably replicating HCV reporter replicon. We isolated HCV-specific CD8(+) T cells from 18 HLA-A*02-positive patients with chronic HCV infection and 15 subjects with resolved HCV infection (7 spontaneous, 8 after therapy). Replicon cells were labeled with virus-specific peptides; inhibition of HCV replication was determined by measuring luciferase activity after 72 hours of coculture with virus-specific CD8(+) T cells. RESULTS: HCV-specific CD8(+) T cells from patients with chronic HCV infection had a significantly lower antiviral efficacy than influenza-, Epstein-Barr virus-, and cytomegalovirus-specific CD8(+) T cells. Antiviral efficacy was associated with the ability of virus-specific CD8(+) T cells to secrete interferon gamma. The antiviral efficacy of HCV-specific CD8(+) T cells was linked to surface expression of CD127 and PD-1. The cytokines interleukin-2, interleukin-7, and interleukin-15 increased the antiviral efficacy of CD127-positive but not of CD127-negative, HCV-specific CD8(+) T cells. Spontaneous, but not antiviral therapy-induced, viral clearance was associated with increased antiviral efficacy. CONCLUSIONS: The ability of CD8(+) T cells to inhibit HCV replication ex vivo is associated with their ability to secrete interferon gamma and their surface expression of CD127 and PD-1.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Imunidade Celular/imunologia , Replicação Viral/imunologia , Adulto , Idoso , Antivirais/uso terapêutico , Células Cultivadas , Técnicas de Cocultura , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Subunidade alfa de Receptor de Interleucina-7/imunologia , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/biossíntese , Carga Viral/imunologiaRESUMO
Treg cells are critical for the prevention of autoimmune diseases and are thus prime candidates for cell-based clinical therapy. However, human Treg cells are "plastic", and are able to produce IL-17 under inflammatory conditions. Here, we identify and characterize the human Treg subpopulation that can be induced to produce IL-17 and identify its mechanisms. We confirm that a subpopulation of human Treg cells produces IL-17 in vitro when activated in the presence of IL-1ß, but not IL-6. "IL-17 potential" is restricted to population III (CD4(+) CD25(hi) CD127(lo) CD45RA(-) ) Treg cells expressing the natural killer cell marker CD161. We show that these cells are functionally as suppressive and have similar phenotypic/molecular characteristics to other subpopulations of Treg cells and retain their suppressive function following IL-17 induction. Importantly, we find that IL-17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL-17. Finally, we show that CD161(+) population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL-17-producing Treg-cell population at these sites. As IL-17 production from this Treg-cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary.
Assuntos
Interleucina-17/biossíntese , Subfamília B de Receptores Semelhantes a Lectina de Células NK/metabolismo , Fator de Transcrição STAT3/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Adulto , Idoso , Antígenos CD4/biossíntese , Células Cultivadas , Feminino , Fatores de Transcrição Forkhead/biossíntese , Humanos , Interleucina-1beta/metabolismo , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Interleucina-6/metabolismo , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Antígenos Comuns de Leucócito/biossíntese , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Fator de Transcrição STAT3/genéticaRESUMO
The expression of CD127, the IL-7-binding subunit of the IL-7 R, is tightly regulated during the development and activation of T cells and is reduced during chronic viral infection. However, the molecular mechanism regulating the dynamic expression of CD127 is still poorly understood. In this study, we report that the transcription factor Ets-1 is required for maintaining the expression of CD127 in murine peripheral T cells. Ets-1 binds to and activates the CD127 promoter, and its absence leads to reduced CD127 expression, attenuated IL-7 signaling, and impaired IL-7-dependent homeostatic proliferation of T cells. The expression of CD127 and Ets-1 is strongly correlated in human T cells. Both CD127 and Ets-1 expression are decreased in CD8(+) T cells during HIV infection. In addition, HIV-associated loss of CD127 is only observed in Ets-1(low) effector memory and central memory but not in Ets-1(high) naive CD8(+) T cells. Taken together, our data identify Ets-1 as a critical regulator of CD127 expression in T cells.
Assuntos
Interleucina-7/biossíntese , Proteína Proto-Oncogênica c-ets-1/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Células Cultivadas , Feminino , HIV-1/imunologia , Humanos , Interleucina-7/genética , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Subunidade alfa de Receptor de Interleucina-7/genética , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Masculino , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/imunologia , Ligação Proteica/imunologia , Proteína Proto-Oncogênica c-ets-1/deficiência , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Subpopulações de Linfócitos T/virologiaRESUMO
The use of hypocalcemic vitamin D analogs is an appealing strategy to exploit the immunomodulatory actions of active vitamin D in vivo while circumventing its calcemic side effects. The functional modulation of dendritic cells by these molecules is regarded as the key mechanism underlying their ability to regulate T cell reactivity. In this article, we demonstrate the capacity of the vitamin D analog, TX527, to target T cells directly. Microarray analysis of purified human CD3(+) T cells, cultured in the presence of TX527, revealed differential expression of genes involved in T cell activation, proliferation, differentiation, and migratory capacity. Accordingly, functional analysis showed a TX527-mediated suppression of the T cell proliferative capacity and activation status, accompanied by decreased expression of effector cytokines (IFN-γ, IL-4, and IL-17). Furthermore, TX527 triggered the emergence of CD4(+)CD25(high)CD127(low) regulatory T cells featuring elevated levels of IL-10, CTLA-4, and OX40 and the functional capacity to suppress activation and proliferation of effector T cells. Moreover, the vitamin D analog profoundly altered the homing receptor profile of T cells and their migration toward chemokine ligands. Remarkably, TX527 not only modulated skin-homing receptors as illustrated for the parent compound, but also reduced the expression of lymphoid organ-homing receptors (CD62L, CCR7, and CXCR4) and uniquely promoted surface expression of inflammatory homing receptors (CCR5, CXCR3, and CXCR6) on T cells. We conclude that TX527 directly affects human T cell function, thereby inhibiting effector T cell reactivity while inducing regulatory T cell characteristics, and imprints them with a specific homing signature favoring migration to sites of inflammation.
Assuntos
Quimiotaxia de Leucócito/imunologia , Colecalciferol/análogos & derivados , Colecalciferol/fisiologia , Mediadores da Inflamação/fisiologia , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Alcinos , Complexo CD3/biossíntese , Complexo CD3/genética , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Epitopos de Linfócito T/imunologia , Regulação da Expressão Gênica/imunologia , Inibidores do Crescimento/farmacologia , Inibidores do Crescimento/fisiologia , Humanos , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Ativação Linfocitária/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Receptores CCR10/biossíntese , Receptores CCR4/biossíntese , Receptores CCR5/biossíntese , Receptores CXCR3/biossíntese , Receptores CXCR6 , Receptores de Quimiocinas/biossíntese , Receptores Virais/biossíntese , Linfócitos T Reguladores/metabolismo , Transcrição Gênica/imunologiaRESUMO
Potent and broad cellular immune responses against the nonstructural (NS) proteins of hepatitis C virus (HCV) are associated with spontaneous viral clearance. In this study, we have improved the immunogenicity of an adenovirus (Ad)-based HCV vaccine by fusing NS3 from HCV (Strain J4; Genotype 1b) to the MHC class II chaperone protein invariant chain (Ii). We found that, after a single vaccination of C57BL/6 or BALB/c mice with Ad-IiNS3, the HCV NS3-specific CD8(+) T cell responses were significantly enhanced, accelerated, and prolonged compared with the vaccine encoding NS3 alone. The AdIiNS3 vaccination induced polyfunctional CD8(+) T cells characterized by coproduction of IFN-γ, TNF-α and IL-2, and this cell phenotype is associated with good viral control. The memory CD8(+) T cells also expressed high levels of CD27 and CD127, which are markers of long-term survival and maintenance of T cell memory. Functionally, the AdIiNS3-vaccinated mice had a significantly increased cytotoxic capacity compared with the AdNS3 group. The AdIiNS3-induced CD8(+) T cells protected mice from infection with recombinant vaccinia virus expressing HCV NS3 of heterologous 1b strains, and studies in knockout mice demonstrated that this protection was mediated primarily through IFN-γ production. On the basis of these promising results, we suggest that this vaccination technology should be evaluated further in the chimpanzee HCV challenge model.
Assuntos
Adenoviridae/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Vetores Genéticos/imunologia , Hepacivirus/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Vacinas contra Hepatite Viral/imunologia , Proteínas não Estruturais Virais/imunologia , Adenoviridae/genética , Animais , Antígenos de Diferenciação de Linfócitos B/administração & dosagem , Antígenos de Diferenciação de Linfócitos B/genética , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Testes Imunológicos de Citotoxicidade/métodos , Modelos Animais de Doenças , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Vetores Genéticos/administração & dosagem , Hepacivirus/genética , Hepatite C/imunologia , Hepatite C/patologia , Hepatite C/prevenção & controle , Antígenos de Histocompatibilidade Classe II/administração & dosagem , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/genética , Memória Imunológica/genética , Imunofenotipagem , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Subunidade alfa de Receptor de Interleucina-7/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Vaccinia virus/genética , Vaccinia virus/imunologia , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/genética , Proteínas não Estruturais Virais/administração & dosagem , Proteínas não Estruturais Virais/genéticaRESUMO
In this study, we expanded regulatory T cells (Tregs) ex vivo from CD4(+) CD25(+) T cells from cord blood (CB) and CD4(+) CD25(+) CD127(-) T cells from adult peripheral blood (APB) and compared the suppressive functions of the newly generated Tregs. The Tregs from CB and APB were expanded either in two cycles with a polyclonal stimulus or in two cycles with an alloantigen stimulus in the first cycle and a polyclonal stimulus in the second cycle. Cell yield after Treg expansion with polyclonal stimulation was greater than that of Tregs expanded with combined alloantigen and polyclonal stimulation. The expanded Tregs expressed high levels of Foxp3, CD39 and cytotoxic T-lymphocyte antigen-4 and low levels of CD127, interleukin-2 and interferon-γ. After two cycles of expansion, the CB Tregs maintained expression of the GARP gene and showed greater suppressive function than APB Tregs. The CB Tregs that were expanded with two cycles of polyclonal stimulation suppressed not only the polyclonal antigen-driven responder T (T(resp)) cell proliferation but also the HLA mismatched dendritic cell-driven T(resp) cell proliferation. When CB and APB Tregs were expanded with a primary alloantigen stimulus followed by a secondary polyclonal stimulus, the Tregs showed a potent, antigen-specific suppressive capacity. The Tregs expanded with two cycles of polyclonal stimulation from both CB and APB alleviated acute graft-versus-host disease symptoms and prolonged survival in a murine model of graft-versus-host disease. In conclusion, CB Tregs expanded with two cycles of polyclonal stimulation had a stronger immunosuppressive function than APB Tregs. It is feasible to obtain human functional alloantigen-specific Tregs expanded ex vivo from CB and APB in large numbers.
Assuntos
Ativação Linfocitária/imunologia , Linfócitos T Reguladores/metabolismo , Adulto , Animais , Antígenos CD/biossíntese , Antígenos CD/imunologia , Apirase/biossíntese , Apirase/imunologia , Sangue/imunologia , Sangue/metabolismo , Antígeno CTLA-4/biossíntese , Antígeno CTLA-4/imunologia , Sangue Fetal/imunologia , Sangue Fetal/metabolismo , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/imunologia , Humanos , Terapia de Imunossupressão , Recém-Nascido , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-2/biossíntese , Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Subunidade alfa de Receptor de Interleucina-7/imunologia , Isoantígenos/imunologia , Isoantígenos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Linfócitos T Reguladores/imunologiaRESUMO
Human thymus contains major dendritic cell (DC) subsets, myeloid DCs (mDCs), and plasmacytoid DCs (pDCs). We previously showed that mDCs, educated by thymic stromal lymphopoietin (TSLP) produced by the epithelial cells of the Hassall's corpuscles, induced differentiation of CD4(+)CD25(-) thymocytes into Forkhead Box P3(+) (FOXP3(+)) regulatory T cells (T(R)) within the medulla of human thymus. In this study, we show that pDCs expressed the TSLP receptor and IL-7 receptor alpha complexes upon activation and became responsive to TSLP. TSLP-activated human pDCs secrete macrophage-derived chemokine CCL-22 and thymus- and activation-regulated chemokine CCL-17 but not Th1- or Th2-polarizing cytokines. TSLP-activated pDCs induced the generation of FOXP3(+) T(R) from CD4(+)CD8(-)CD25(-) thymocytes, which could be strongly inhibited by Th1-polarizing cytokine IL-12 or Th2-polarizing cytokine IL-4. Interestingly, the FOXP3(+) T(R) induced by the TSLP-pDCs expressed more IL-10 but less TGF-beta than that induced by the TSLP-mDCs. These data suggest that TSLP expressed by thymic epithelial cells can activate mDCs and pDCs to positively select the FOXP3(+) T(R) with different cytokine production potential in human thymus. The inability of TSLP to induce DC maturation without producing Th1- or Th2-polarizing cytokines may provide a thymic niche for T(R) development.
Assuntos
Diferenciação Celular/imunologia , Citocinas/fisiologia , Células Dendríticas/imunologia , Fatores de Transcrição Forkhead/biossíntese , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Timo/imunologia , Timo/metabolismo , Adulto , Antígeno B7-1/biossíntese , Antígeno B7-2/biossíntese , Células Cultivadas , Quimiocina CCL17/metabolismo , Quimiocina CCL22/metabolismo , Pré-Escolar , Técnicas de Cocultura , Células Dendríticas/metabolismo , Humanos , Lactente , Recém-Nascido , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Receptores de Citocinas/biossíntese , Células Estromais/imunologia , Células Estromais/metabolismo , Linfócitos T Reguladores/citologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Timo/citologia , Linfopoietina do Estroma do TimoRESUMO
Regulatory T cells (Treg) are commonly identified by CD25 (IL-2R alpha) surface expression and/or intracellular expression of the FOXP3 transcription factor. In addition, Treg are also characterized by low CD127 (IL-7R alpha) expression when compared to conventional T cells and their biology in the periphery is considered essentially independent of IL-7. We further investigated CD127 expression on Treg and we demonstrated differential CD127 expression depending on Treg subsets considered. Notably, we observed high CD127 expression on inducible costimulatory molecule (ICOS)- and CD103-expressing Treg subsets. Since these two markers reflect activation status, we addressed whether Treg activation modulated CD127 expression. We demonstrated that in contrast to conventional T cells, Treg significantly upregulated CD127 expression during in vitro and in vivo activation using adoptive transfer and contact dermatitis models. High CD127 expression on Treg was also predominantly detected ex vivo in some specific sites, notably bone marrow and skin. Importantly, higher CD127 expression on Treg correlated with higher phosphorylation of STAT5 upon IL-7 exposure. High CD127 expression on Treg also provided survival advantage upon in vitro incubation with IL-7. We thus demonstrated that low CD127 expression is not an intrinsic characteristic of Treg and we identified activated Treg as a potential target of endogenous or therapeutic IL-7.
Assuntos
Dermatite de Contato/imunologia , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Interleucina-7/metabolismo , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Transferência Adotiva , Animais , Antígenos CD4/biossíntese , Sobrevivência Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Dermatite de Contato/patologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica , Interleucina-7/imunologia , Subunidade alfa de Receptor de Interleucina-7/genética , Subunidade alfa de Receptor de Interleucina-7/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
Signaling via the IL-7 receptor complex (IL-7Ralpha/CD127 and IL-2Rgamma/CD132) is required for T-cell development and survival. Decreased CD127 expression has been associated with persistent viral infections (e.g. HIV, HCV) and cancer. Many IL-2Rgamma-sharing (gammaC) cytokines decrease CD127 expression on CD4+ and CD8+ T cells in mice (IL-2, IL-4, IL-7, IL-15) and in humans (IL-2, IL-7), suggesting a common function. IL-4 is of particular interest as it is upregulated in HIV infection and in thyroid and colon cancers. The role of IL-4 in regulating CD127 expression and IL-7 activity in human thymocytes and mature CD8+ T cells is unknown and was therefore investigated. IL-4 decreased CD127 expression on all thymocyte subsets tested and only on naïve (CD45RA+) CD8+ T cells, without altering membrane-bound CD127 mRNA expression. Pre-treatment of thymocytes or CD8+ T cells with IL-4 inhibited IL-7-mediated phosphorylation of STAT5 and decreased proliferation of CD8+ T cells. By downregulating CD127 expression and signaling on developing thymocytes and CD8+ T cells, IL-4 is a potential contributor to impaired CD8+ T-cell function in some anti-viral and anti-tumor responses. These findings are of particular consequence to diseases such as HIV, HCV, RSV, measles and cancer, in which CD127 expression is decreased, IL-7 activity is impaired and IL-4 concentrations are elevated.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Interleucina-4/farmacologia , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Subpopulações de Linfócitos T/efeitos dos fármacos , Timo/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Técnicas de Cocultura , Humanos , Interleucina-7/metabolismo , Subunidade alfa de Receptor de Interleucina-7/genética , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/biossíntese , Fator de Transcrição STAT5/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127(+) perforin(-)/CD127(-) perforin(+), and CD127(-)/perforin(-) CD8 T cells, respectively. CD127(-)/perforin(-) and CD127(-)/perforin(+) cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin(+)/IL-2(-) or perforin(-)/IL-2(+) cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P < 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127(+)/IL-2-secreting) and cytotoxic (perforin(+)) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Citotoxicidade Imunológica , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Perforina/biossíntese , Subpopulações de Linfócitos T/imunologia , Viroses/imunologia , Degranulação Celular , Células Cultivadas , Citometria de Fluxo , Humanos , Interferon gama/biossíntese , Interleucina-2/biossíntese , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Advanced chronic lymphocytic leukaemia (CLL) is associated with profound immunodeficiency, including changes in T regulatory cells (T(regs)). We determined the pattern of expression of forkhead box P3 (FoxP3), CD25, CD27 and CD127 and showed that the frequency of CD4+ FoxP3+ T cells was increased in CLL patients (12% versus 8% in controls). This increase was seen only in advanced disease, with selective expansion of FoxP3-expressing cells in the CD4+ CD25(low) population, whereas the number of CD4+ CD25(high) FoxP3+ cells was unchanged. CD4+ CD25(low) cells showed reduced expression of CD127 and increased CD27, and this regulatory phenotype was also seen on all CD4 T cells subsets in CLL patients, irrespective of CD25 or FoxP3 expression. Incubation of CD4+ T cells with primary CLL tumours led to a sixfold increase in the expression of FoxP3 in CD4+ CD25- T cells. Patients undergoing treatment with fludarabine demonstrated a transient increase in the percentage of CD4+ FoxP3+ T cells, but this reduced to normal levels post-treatment. This work demonstrates that patients with CLL exhibit a systemic T cell dysregulation leading to the accumulation of CD4+ FoxP3+ T cells. This appears to be driven by interaction with malignant cells, and increased understanding of the mechanisms that are involved could provide novel avenues for treatment.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Fatores de Transcrição Forkhead/biossíntese , Leucemia Linfocítica Crônica de Células B/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Técnicas de Cocultura , Citometria de Fluxo , Humanos , Imunomodulação , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Contagem de Linfócitos , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Vidarabina/análogos & derivados , Vidarabina/uso terapêuticoRESUMO
We have identified a novel interleukin (IL)-7-responsive T cell population [forkhead box P3 (FoxP3(+) ) CD4(+) CD25(+) CD127(+) ] that is comparably functionally suppressive to conventional FoxP3(+) CD4(+) CD25(+) regulatory T cells (T(regs) ). Although IL-2 is the most critical cytokine for thymic development of FoxP3(+) T(regs) , in the periphery other cytokines can be compensatory. CD25(+) CD127(+) T cells treated with IL-7 phenotypically 'matured' into the known 'classical' FoxP3(+) CD4(+) CD25(high) CD127(-) FoxP3(+) T(regs) . In freshly isolated splenocytes, the highest level of FoxP3 expression was found in CD127(+) CD25(+) T cells when compared with CD127(-) CD25(+) or CD127(+) CD25(-) cells. IL-7 treatment of CD4(+) CD25(+) T cells induced an increase in the accumulation of FoxP3 in the nucleus in vitro. IL-7-mediated CD25 cell surface up-regulation was accompanied by a concurrent down-regulation of CD127 in vitro. IL-7 treatment of the CD127(+) CD25(+) FoxP3(+) cells also resulted in up-regulation of cytotoxic T lymphocyte antigen 4 without any changes in CD45RA at the cell surface. Collectively, these data support emerging evidence that FoxP3(+) T cells expressing CD127 are comparably functionally suppressive to CD25(+) CD127(-) FoxP3(+) T cells. This IL-7-sensitive regulation of FoxP3(+) T(reg) phenotype could underlie one peripheral non-IL-2-dependent compensatory mechanism of T(reg) survival and functional activity, particularly for adaptive T(regs) in the control of autoimmunity or suppression of activated effector T cells.