RESUMO
Mitotic proteins are well characterized and their cell cycle regulation roles studied extensively. Specific mitotic proteins can be key targets for controlling de regulated cell cycles. In the present study, cytosolic proteins of CHO (Chinese Hamster Ovary) cells were isolated and used for generating a range of monoclonal antibodies. Of the two antigenic doses utilized, 20 microg and 15 microg doses gave fusion efficiencies of 52.1% and 31.5% respectively. The specific efficiencies were found to be 24.5% for the 20 microg antigenic dose and was 20.45% for the 15 microg dose. Further screening showed 20 MAbs to common mitotic and interphase proteins, 5 specific to unique mitotic proteins and 3 to unique interphase proteins. MPFs do not exhibit species barriers and induce chromatin condensation and act as M- phase check point control molecules. Monoclonal antibodies to specific mitotic proteins can be very useful for various applications such as imaging tools, as possible mitotic inhibitors and also for affinity purification of specific proteins of interest. Antibodies specific to common mitotic and interphase proteins can also be of similar importance. Whole extract was used as immunogen also to present the mitotic proteins much better than isolated proteins.
Assuntos
Anticorpos Monoclonais/biossíntese , Interfase/imunologia , Mitose/imunologia , Proteínas/imunologia , Animais , Antígenos/isolamento & purificação , Células CHO , Cricetinae , Cricetulus , Citosol/imunologia , Feminino , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The location of immune activation is controversial during acute allograft rejection and unknown in xenotransplantation. To determine where immune activation to a xenograft occurs, we examined whether splenectomized alymphoplastic mice that possess no secondary lymphoid organs can reject porcine skin xenografts. Our results show that these mice rejected their xenografts, in a T cell-dependent fashion, at the same tempo as wild-type recipients, demonstrating that xenograft rejection is not critically dependent on secondary lymphoid organs. Furthermore, we provide evidence that immune activation in the bone marrow did not take place during xenograft rejection. Importantly, immunity to xenoantigens was only induced after xenotransplantation and not by immunization with porcine spleen cells, as xenografted mutant mice developed an effector response, whereas mutant mice immunized by porcine spleen cells via i.p. injection failed to do so. Moreover, we provide evidence that antixenograft immunity occurred via direct and indirect Ag presentation, as recipient T cells could be stimulated by either donor spleen cells or recipient APCs. Thus, our data provide evidence that direct and indirect Ag presentation by a xenograft induces immunity in the absence of secondary lymphoid organs. These results have important implications for developing relevant xenotransplantation protocols.
Assuntos
Apresentação de Antígeno/imunologia , Tecido Linfoide/imunologia , Transplante Heterólogo/imunologia , Animais , Formação de Anticorpos/genética , Apresentação de Antígeno/genética , Antígenos Heterófilos/administração & dosagem , Antígenos Heterófilos/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Divisão Celular/genética , Divisão Celular/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Rejeição de Enxerto/prevenção & controle , Imunidade Inata/genética , Memória Imunológica/genética , Injeções Intraperitoneais , Interfase/genética , Interfase/imunologia , Células Matadoras Naturais/imunologia , Tecido Linfoide/anormalidades , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos SCID , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transplante de Pele/imunologia , Transplante de Pele/patologia , Baço/citologia , Baço/imunologia , Baço/transplante , Esplenectomia , Suínos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/transplante , Transplante Heterólogo/métodos , Transplante Heterólogo/patologiaRESUMO
Plasmacytoid dendritic cells (PDCs) are key effectors in host innate immunity and orchestrate adaptive immune responses. CpG oligodeoxynucleotides (ODN) have potent immunostimulatory effects on PDCs through TLR9 recognition and signaling. Little is known about the effects of CpG ODN on human PDC-mediated T cell priming. Here we show that type B CpG ODN effectively promotes PDCs to prime allogeneic naive CD4(+)CD25(-) T cells to differentiate into CD4(+)CD25(+) regulatory T (Treg) cells. The CD4(+)CD25(+) T cells induced by CpG ODN-activated PDCs express forkhead transcription factor 3 and produce IL-10, TGF-beta, IFN-gamma, and IL-6, but low IL-2 and IL-4. These CD4(+)CD25(+) T cells are hyporesponsive to secondary alloantigen stimulation and strongly inhibit proliferation of autologous or allogeneic naive CD4(+) T cells in an Ag-nonspecific manner. CpG ODN-activated PDCs require direct contact with T cells to induce CD4(+)CD25(+) Treg cells. Interestingly, IL-10 and TGF-beta were undetectable in the supernatants of CpG ODN-stimulated PDC cultures. Both CpG-A and CpG-C ODN-activated PDCs similarly induced the generation of CD4(+)CD25(+) Treg cells with strong immune suppressive function. This study demonstrates that TLR9 stimulation can promote PDC-mediated generation of CD4(+)CD25(+) Treg cells and suggests PDCs may play an important role in the maintenance of immunological tolerance.
Assuntos
Adjuvantes Imunológicos/farmacologia , Linfócitos T CD4-Positivos/imunologia , Ilhas de CpG/imunologia , Células Dendríticas/imunologia , Ativação Linfocitária/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Receptores de Interleucina-2/biossíntese , Linfócitos T Reguladores/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Divisão Celular/imunologia , Células Cultivadas , Citocinas/biossíntese , Proteínas de Ligação a DNA/biossíntese , Células Dendríticas/metabolismo , Relação Dose-Resposta Imunológica , Epitopos de Linfócito T/fisiologia , Fatores de Transcrição Forkhead , Humanos , Tolerância Imunológica , Interfase/imunologia , Teste de Cultura Mista de Linfócitos , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismoRESUMO
We have developed a critical test of the chromatin accessibility model of Ig isotype determination in which local unfolding of chromatin higher order structure (chromatin accessibility) in the region of specific germline genes in the H chain locus determines the Ab class to be expressed in the B cell. We show that multiple germline genes are constitutively transcribed in the majority of naive human B cells in a population. Thus, because chromatin in its higher order structure cannot be transcribed, the entire Ig H chain locus must be unfolded in naive B cells. We have also established that IL-4 and anti-CD40 act by enhancing transcription in the majority of cells, rather than by activating transcription in more of the cells. Transcriptional activity in the human H chain locus rules out the perturbation of chromatin higher order structure as a factor in isotype determination. We have also found that the levels of germline gene transcription cannot fully account for the levels of secretion of the different Ig isotypes, and that secretion of IgE, in particular, is suppressed relative to that of IgG.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Citocinas/fisiologia , Genes de Imunoglobulinas , Isotipos de Imunoglobulinas/biossíntese , Transcrição Gênica/imunologia , Separação Celular , Células Cultivadas , Citocinas/farmacologia , Regulação da Expressão Gênica/imunologia , Globinas/biossíntese , Globinas/genética , Humanos , Imunoglobulina E/biossíntese , Imunoglobulina G/biossíntese , Imunoglobulina G/classificação , Isotipos de Imunoglobulinas/genética , Cadeias épsilon de Imunoglobulina/biossíntese , Cadeias épsilon de Imunoglobulina/genética , Imunofenotipagem , Interleucina-4/farmacologia , Interfase/genética , Interfase/imunologia , Células K562 , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/imunologia , Ativação Linfocitária/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
The B7-1/2-CD28 system provides the critical signal for the generation of an efficient T cell response. We investigated the role played by B7-2 in influencing pathogenic autoimmunity from islet-reactive CD4 T cells in B7-2 knockout (KO) NOD mice which are protected from type 1 diabetes. B7-2 deficiency caused a profound diminishment in the generation of spontaneously activated CD4 T cells and islet-specific CD4 T cell expansion. B7-2 does not impact the effector phase of the autoimmune response as adoptive transfer of islet Ag-specific BDC2.5 splenocytes stimulated in vitro could easily induce disease in B7-2KO mice. CD4 T cells showed some hallmarks of hyporesponsiveness because TCR/CD28-mediated stimulation led to defective activation and failure to induce disease in NODscid recipients. Furthermore, CD4 T cells exhibited enhanced death in the absence of B7-2. Interestingly, we found that B7-2 is required to achieve normal levels of CD4+CD25+CD62L+ T regulatory cells because a significant reduction of these T regulatory cells was observed in the thymus but not in the peripheral compartments of B7-2KO mice. In addition, our adoptive transfer experiments did not reveal either pathogenic or regulatory potential associated with the B7-2KO splenocytes. Finally, we found that the lack of B7-2 did not induce a compensatory increase in the B7-1 signal on APC in the PLN compartment. Taken together these results clearly indicate that B7-2 plays a critical role in priming islet-reactive CD4 T cells, suggesting a simplified, two-cell model for the impact of this costimulatory molecule in autoimmunity against islets.
Assuntos
Antígenos CD/fisiologia , Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Ilhotas Pancreáticas/imunologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/fisiologia , Transferência Adotiva , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD/genética , Autoanticorpos/biossíntese , Antígeno B7-1/biossíntese , Antígeno B7-2 , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Divisão Celular/genética , Divisão Celular/imunologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Feminino , Interfase/genética , Interfase/imunologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , Ativação Linfocitária/genética , Contagem de Linfócitos , Linfopenia/genética , Linfopenia/imunologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Interleucina-2/biossíntese , Baço/metabolismo , Baço/patologia , Baço/transplante , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologiaRESUMO
The 2B4 molecule (CD244) has been described as a coreceptor in human NK cell activation. However, the behavior of 2B4 during the cytotoxic NK cell immune synapse (NK-IS) formation remains undetermined. In this study, we demonstrate the redistribution of 2B4 and the signaling adaptor molecule, signaling lymphocyte activation molecule-associated protein (SAP), to the cytotoxic NK-IS upon formation of conjugates between resting NK cells and EBV-infected 721.221 human cells. Confocal microscopy showed that 2B4 localized at the central supramolecular activation cluster, surrounded by a peripheral supramolecular activation cluster containing talin within NK cell and ICAM-1 on target cells. Videomicroscopy studies with 2B4-GFP-transfected NK cells revealed that 2B4 redistributed to cytotoxic NK-IS as soon as the cell contact occurred. Simultaneously, a SAP-GFP also clustered at the contact site, where it remained during the interaction period. The 2B4 molecular clusters remained bound to the target cell even after NK cell detachment. These results underscore the function of 2B4 as an adhesion molecule and suggest a relevant role in the initial binding, scanning of target cells, and formation of cytotoxic NK-IS. Finally, these findings are indicative of an important role of the activating 2B4/signaling lymphocyte activation molecule-associated protein complex during the recognition of EBV-infected cells.
Assuntos
Antígenos CD/metabolismo , Proteínas de Transporte/metabolismo , Comunicação Celular/imunologia , Citotoxicidade Imunológica , Peptídeos e Proteínas de Sinalização Intracelular , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Domínios de Homologia de src , Antígenos CD/sangue , Antígenos CD/fisiologia , Proteínas de Transporte/sangue , Proteínas de Transporte/fisiologia , Linhagem Celular Transformada , Transformação Celular Viral/imunologia , Testes Imunológicos de Citotoxicidade , Herpesvirus Humano 4/imunologia , Humanos , Interfase/imunologia , Células Matadoras Naturais/citologia , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/fisiologia , Receptores Imunológicos/sangue , Receptores Imunológicos/fisiologia , Transdução de Sinais/imunologia , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Família de Moléculas de Sinalização da Ativação Linfocitária , Frações Subcelulares/imunologia , Frações Subcelulares/metabolismo , Domínios de Homologia de src/imunologiaRESUMO
Studies of memory T cell differentiation are hampered by a lack of quantitative models to test hypotheses in silico before in vivo experimentation. We created a stochastic computer model of CD4+ memory T cell generation that can simulate and track 10(1)-10(8) individual lymphocytes over time. Parameters for the model were derived from experimental data using naive human CD4+ T cells stimulated in vitro. Using discrete event computer simulation, we identified two key variables that heavily influence effector burst size and the persistent memory pool size: the cell cycle dependent probability of apoptosis, and the postactivation mitosis at which memory T cells emerge. Multiple simulations were performed and varying critical parameters permitted estimates of how sensitive the model was to changes in all of the model parameters. We then compared two hypotheses of CD4+ memory T cell generation: maturation from activated naive to effector to memory cells (model I) vs direct progression from activated naive to memory cells (model II). We find that direct progression of naive to memory T cells does not explain published measurements of the memory cell mass unless postactivation expansion of the memory cell cohort occurs. We conclude that current models suggesting direct progression of activated naive cells to the persistent memory phenotype (model II) do not account for the experimentally measured size of the postactivation CD4+, Ag-specific, memory T cell cohort.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Ciclo Celular/imunologia , Simulação por Computador , Memória Imunológica , Modelos Imunológicos , Apoptose/imunologia , Morte Celular/imunologia , Divisão Celular/imunologia , Tamanho Celular/imunologia , Feminino , Humanos , Interfase/imunologia , Ativação Linfocitária , Masculino , Mitose/imunologia , Probabilidade , Sensibilidade e Especificidade , Processos EstocásticosRESUMO
IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation, induces proliferation of naive CD4+ T cells, and synergizes with IL-12 in IFN-gamma production. It has been recently reported that IL-27 induces T-bet and IL-12Rbeta2 expression through JAK1/STAT1 activation. In the present study, we further investigated the JAK/STAT signaling molecules activated by IL-27 and also the role of STAT1 in IL-27-mediated responses using STAT1-deficient mice. In addition to JAK1 and STAT1, IL-27-activated JAK2, tyrosine kinase-2, and STAT2, -3, and -5 in naive CD4+ T cells. The activation of STAT2 and STAT5, but not of STAT3, was greatly diminished in STAT1-deficient naive CD4+ T cells. Comparable proliferative response to IL-27 was observed between STAT1-deficient and wild-type naive CD4+ T cells. In contrast, IL-27 hardly induced T-bet and subsequent IL-12Rbeta2 expression, and synergistic IFN-gamma production by IL-27 and IL-12 was impaired in STAT1-deficient naive CD4+ T cells. Moreover, IL-27 augmented the expression of MHC class I on naive CD4+ T cells in a STAT1-dependent manner. These results suggest that IL-27 activates JAK1 and -2, tyrosine kinase-2, STAT1, -2, -3, and -5 in naive CD4+ T cells and that STAT1 plays an indispensable role in IL-27-induced T-bet and subsequent IL-12Rbeta2 expression and MHC class I expression as well but not proliferation, while STAT3 presumably plays an important role in IL-27-induced proliferation.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Proteínas de Ligação a DNA/fisiologia , Interleucinas/fisiologia , Interfase/imunologia , Transdução de Sinais/imunologia , Transativadores/fisiologia , Fatores de Transcrição/biossíntese , Adjuvantes Imunológicos/fisiologia , Animais , Linfócitos T CD4-Positivos/enzimologia , Divisão Celular/genética , Divisão Celular/imunologia , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/imunologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Interferon gama/antagonistas & inibidores , Interferon gama/biossíntese , Interleucina-12/fisiologia , Interfase/genética , Janus Quinase 1 , Janus Quinase 2 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Leite/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Interleucina/antagonistas & inibidores , Receptores de Interleucina/biossíntese , Receptores de Interleucina-12 , Fator de Transcrição STAT1 , Fator de Transcrição STAT2 , Fator de Transcrição STAT3 , Fator de Transcrição STAT5 , Transdução de Sinais/genética , Proteínas com Domínio T , TYK2 Quinase , Transativadores/deficiência , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/antagonistas & inibidoresRESUMO
Tissue T cells encounter Ag in a distinct microenvironment, where they are embedded in the interstitial extracellular matrix (ECM). In contrast, while naive T cells are exposed to Ag in the lymph node, immediately after naive T cells are activated they must extravasate into the ECM to function effectively. Because integrin-mediated adhesion to the ECM modulates cell cycle progression and survival in adherent nonimmune cells, we hypothesize that blood and tissue-derived T cells have similarly adapted their behavior to their first or continued encounter with ECM. T cells from peripheral blood (PBT) and tissue (the intestinal lamina propria T cell (LPT)) were stimulated with anti-CD3-coated beads in the presence or absence of native ECM derived from intestinal fibroblasts, plate-immobilized fibronectin, or collagen type I. Native ECM and collagen, but not fibronectin, induced in anti-CD3 activated PBT a 4- to 5-fold increase in the entry, progression, and completion of the cell cycle over that triggered by anti-CD3 alone. Neutralizing beta1 integrin Abs abrogated this increase. None of these ECM proteins stimulated cell cycle progression in LPT. In contrast, anti-CD3 activation of LPT in the presence of native ECM and fibronectin reduced activation-induced cell death by 40%. These results demonstrate that naive and effector/memory T cells respond differently upon exposure to specific ECM components. When naive PBT encounter Ag in the context of ECM, their progression through the cell cycle is enhanced, favoring clonal expansion; while tissue T cell longevity may be mediated by interactions with the ECM.
Assuntos
Adjuvantes Imunológicos/fisiologia , Apoptose/imunologia , Proteínas da Matriz Extracelular/fisiologia , Memória Imunológica , Interfase/imunologia , Subpopulações de Linfócitos T/citologia , Adesão Celular/imunologia , Agregação Celular/imunologia , Ciclo Celular/imunologia , Separação Celular , Sobrevivência Celular/imunologia , Células Cultivadas , Colágeno Tipo I/fisiologia , Fibronectinas/fisiologia , Inibidores do Crescimento/fisiologia , Humanos , Integrina beta1/biossíntese , Integrina beta1/imunologia , Integrina beta1/metabolismo , Integrina beta1/fisiologia , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Ativação Linfocitária/imunologia , Especificidade de Órgãos/imunologiaRESUMO
CD4(+)25(+) regulatory T (Treg) cells maintain immunological self-tolerance through mechanisms that are only in part understood. Previous studies suggest that the glucocorticoid-induced TNFR-related protein (GITR), which is preferentially expressed on the surface of Treg cells, potentially provides a signal that abrogates Treg suppression. In this study, we show that a soluble form of mouse GITR ligand (sGITR-L) induces GITR-dependent NF-kappaB activation and blocks in vitro suppression mediated by both resting and preactivated polyclonal and Ag-specific Treg cells. Since sGITR-L along with rIL-2 induces proliferation of CD4(+)25(+) cells, it appears that sGITR-L can break the anergic state of Treg cells. Because sGITR-L also up-regulates IL-2 secretion by activated CD4(+)25 (-)T cells, these two sGITR-L induced signals synergize to interfere with suppressor activity by CD4(+)25(+) Treg cells.
Assuntos
Adjuvantes Imunológicos/fisiologia , Proteínas de Transporte/fisiologia , Anergia Clonal/imunologia , Receptores de Fator de Crescimento Neural/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Adjuvantes Imunológicos/metabolismo , Animais , Proteínas de Transporte/metabolismo , Divisão Celular/imunologia , Linhagem Celular , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Humanos , Interleucina-2/metabolismo , Interleucina-2/fisiologia , Interfase/imunologia , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , NF-kappa B/fisiologia , Receptores de Interleucina-2/biossíntese , Transdução de Sinais/imunologia , Solubilidade , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Fatores de Necrose Tumoral , Regulação para Cima/imunologiaRESUMO
Regulatory CD4(+) T cells are known to develop during the induction of donor-specific peripheral tolerance to transplanted tissues; it is proposed that such tolerance is a consequence of persistent, danger-free stimulation by Ag. To test this hypothesis, male RAG-1(-/-) mice were recolonized with small numbers of monospecific CD4(+) T cells specific for the male H-2E(k)-restricted Ag Dby. After 6 wk in the male environment, the monospecific CD4(+) T cells, having recolonized the host, had become anergic to stimulation in vitro and had acquired a regulatory capacity. CD4(+) T cells in these mice expressed higher levels of CTLA-4 and glucocorticoid-induced TNF-related receptor than naive CD4(+) T cells, but only 3% of the recolonizing cells were CD25(+) and did not express significant foxP3 mRNA. In vivo, these tolerant T cells could censor accumulation of, and IFN-gamma production by, naive T cells, with only a slight inhibition of proliferation. This suppressive effect was not reversed by the addition of fresh bone marrow-derived male dendritic cells. These results suggest that persistent exposure to Ag in conditions that fail to evoke proinflammatory stimuli leads to the development of T cells that are both anergic and regulatory.
Assuntos
Antígenos/administração & dosagem , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Anergia Clonal/imunologia , Proteínas/administração & dosagem , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Transferência Adotiva , Sequência de Aminoácidos , Animais , Antígenos/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Divisão Celular/genética , Divisão Celular/imunologia , Anergia Clonal/genética , Técnicas de Cocultura , RNA Helicases DEAD-box , Feminino , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Proteínas de Homeodomínio/genética , Imunofenotipagem , Interfase/genética , Interfase/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Proteínas/imunologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Baço/citologia , Baço/transplanteRESUMO
Normal adult human liver (AHL) contains populations of unconventional lymphocytes that have been shown in the mouse to mature locally. The presence of lymphoid progenitors together with IL-7, recombinase-activating gene, and pre-TCR-alpha expression in AHL suggests similar local T cell development activity in humans. Flow cytometry was used to characterize potentially naive hepatic alphabeta-T cells. We looked for evidence of TCR-alphabeta cell development in AHL by quantifying delta deletion TCR excision circles (TRECs) in CD3(pos) populations isolated from the liver and matched blood of eight individuals. Phenotypic analysis of hepatic T cells suggests the presence of Ag-inexperienced populations. TRECs were detected in all blood samples (mean, 164.10 TRECs/ micro g DNA), whereas only two hepatic samples were positive at low levels (59.40 and 1.92). The relatively high level of CD8(pos) T cells in these livers with a naive phenotype suggests that in addition to its role as a graveyard for Ag-specific activated CD8(pos) T cells, naive CD8(pos) T cells may enter the liver without prior activation. The almost complete absence of TRECs suggests that normal AHL is not a site for the development of conventional alphabeta T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Imunofenotipagem , Fígado/imunologia , Fígado/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Feminino , Citometria de Fluxo , Rearranjo Gênico do Linfócito T , Humanos , Interfase/imunologia , Fígado/citologia , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/sangue , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/biossínteseRESUMO
Intrathymic expression of tissue-specific self-Ags can mediate tolerance of self-reactive T cells. However, in this study we define circumstances by which thymic expression of a tissue-specific autoepitope enhances positive selection of disease-causing, self-reactive T cells. An immunodominant gastritogenic epitope, namely the gastric H/K ATPase beta subunit(253-277) (H/Kbeta(253-277)), was attached to the C terminus of the invariant chain (Ii) and the hybrid Ii (Ii-H/Kbeta(253-277)) expressed in mice under control of the Ii promoter. The Ii-H/Kbeta(253-277) fusion protein was localized to MHC class II-expressing cells in the thymus and periphery of Ii-H/Kbeta(253-277) transgenic mice. In one transgenic line the level of presentation in the periphery (spleen) was insufficient to activate naive, low affinity H/Kbeta(253-277)-specific transgenic T cells (1E4-TCR), whereas thymic presentation of H/Kbeta(253-277) enhanced positive selection of 1E4-TCR cells in Ii-H/Kbeta(253-277)/1E4-TCR double-transgenic mice. Furthermore, Ii-H/Kbeta(253-277)/1E4-TCR double-transgenic mice had an increased incidence of autoimmune gastritis compared with 1E4-TCR single-transgenic mice, demonstrating that the 1E4 T cells that seeded the periphery of Ii-H/Kbeta(253-277) mice were pathogenic. Therefore, low levels of tissue-specific Ags in the thymus can result in positive selection of low avidity, self-reactive T cells. These findings also suggest that the precise level of tissue-specific Ags in the thymus may be an important consideration in protection against autoimmune disease and that perturbation of the levels of self-Ags may be detrimental.
Assuntos
Autoantígenos/biossíntese , Doenças Autoimunes/imunologia , Epitopos de Linfócito T/biossíntese , Gastrite/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Timo/imunologia , Timo/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos de Diferenciação de Linfócitos B/biossíntese , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Doenças Autoimunes/enzimologia , Doenças Autoimunes/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Cruzamentos Genéticos , Epitopos de Linfócito T/metabolismo , Gastrite/enzimologia , Gastrite/genética , ATPase Trocadora de Hidrogênio-Potássio/biossíntese , ATPase Trocadora de Hidrogênio-Potássio/genética , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Interfase/genética , Interfase/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Subunidades Proteicas/biossíntese , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Baço/citologia , Baço/enzimologia , Baço/imunologia , Subpopulações de Linfócitos T/enzimologia , Timo/patologiaRESUMO
Naive T cells can be tolerized in the periphery by diverse mechanisms. However, the extent to which memory T cells are susceptible to tolerance induction is less well defined. Vaccination of mice with a minimal CTL epitope derived from human adenovirus type 5 E1A in IFA s.c. readily tolerizes naive as well as recently activated CD8(+) T cells due to the overwhelming systemic and persistent presence of the peptide. We have now studied the effect of this peptide on established memory cells, which were induced at least 50 days before by virus vaccination. Memory cells did not undergo peripheral deletion and kept their ability to produce IFN-gamma as well as their cytolytic activity in response to Ag directly ex vivo. However, memory CTL responses in virus vaccinated mice injected with peptide ceased to control tumor outgrowth. Interestingly, functional capacities were regained when T cells were transferred to an Ag-free environment in vivo as determined by their ability to reject an otherwise lethal tumor challenge. Together, these findings indicate that memory CTL responses can be functionally incapacitated, but are not, in contrast to naive or recently activated T cells, irreversibly tolerized by persistent systemic Ag, as memory T cells quickly regain effector function upon disappearance of the Ag.
Assuntos
Proteínas E1A de Adenovirus/imunologia , Proteínas E1A de Adenovirus/metabolismo , Apresentação de Antígeno , Citotoxicidade Imunológica , Memória Imunológica , Linfócitos T Citotóxicos/imunologia , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/terapia , Proteínas E1A de Adenovirus/administração & dosagem , Transferência Adotiva , Animais , Apresentação de Antígeno/imunologia , Divisão Celular/imunologia , Células Cultivadas , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Tolerância Imunológica/imunologia , Injeções Subcutâneas , Interfase/imunologia , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/transplante , Infecções Tumorais por Vírus/patologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Thymus-derived, natural CD4(+)CD25(+) regulatory T cells can educate peripheral CD4(+)CD25(-) cells to develop suppressive activity by poorly understood mechanisms. TGF-beta has IL-2-dependent costimulatory effects on alloactivated naive, human CD4(+) T cells and induces them ex vivo to become potent contact-dependent, cytokine-independent suppressor cells. In this study, we report that CD4(+)CD25(+) cells are the targets of the costimulatory effects of IL-2 and TGF-beta. These cells do not divide, but, instead, greatly increase the numbers of CD4(+)CD25(-) cells that become CD25(+) cytokine-independent suppressor cells. These CD4(+)CD25(+) regulatory cells, in turn, induce other alloactivated CD4(+)CD25(-) cells to become potent suppressor cells by mechanisms that, surprisingly, require both cell contact and TGF-beta and IL-10. The suppressive effects of these secondary CD4(+)CD25(+) cells depend upon TGF-beta and IL-10. Moreover, both the naive CD4(+) cells induced by IL-2 and TGF-beta to become suppressor cells, and the subsequent CD4(+)CD25(-) cells educated by them to become suppressors express FoxP3. We suggest that the long-term effects of adoptively transferred natural-like CD4(+)CD25(+) regulatory cells induced ex vivo are due to their ability to generate new cytokine-producing CD4(+) regulatory T cells in vivo.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Interleucina-10/fisiologia , Interleucina-2/fisiologia , Ativação Linfocitária/imunologia , Receptores de Interleucina-2/biossíntese , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/citologia , Fator de Crescimento Transformador beta/fisiologia , Adjuvantes Imunológicos/fisiologia , Adulto , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição Forkhead , Humanos , Imunidade Inata , Interfase/imunologia , Modelos Imunológicos , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
During their migration into inflammatory sites, immune cells, such as T cells, secrete extracellular matrix (ECM)-degrading enzymes, such as heparanase, which, under mildly acidic conditions, degrade heparan sulfate proteoglycans (HSPG). We have previously shown that at pH 7.2, human placental heparanase loses its enzymatic activity, while retaining its ability to bind HSPG and promote T cell adhesion to unfractionated ECM. We now demonstrate that the 65-kDa recombinant human heparanase, which is devoid of enzymatic activity, but can still bind HSPG, captures T cells under shear flow conditions and mediates their rolling and arrest, in the absence or presence of stromal cell-derived factor 1 alpha (SDF-1 alpha; CXCL12), in an alpha(4)beta(1)-VCAM-1-dependent manner. Furthermore, heparanase binds to and induces T cell adhesion to key ECM components, like fibronectin and hyaluronic acid, in beta(1) integrin- and CD44-specific manners, respectively, via the activation of the protein kinase C and phosphatidylinositol 3-kinase intracellular signaling machineries. Although the nature of the putative T cell heparanase-binding moiety is unknown, it appears that heparanase exerts its proadhesive activity by interacting with the T cells' surface HSPG, because pretreatment of the cells with heparinase abolished their subsequent response to heparanase. Also, heparanase augmented the SDF-1 alpha-triggered phosphorylation of Pyk-2 and extracellular signal-regulated kinase-2 implicated in integrin functioning. Moreover, heparanase, which had no chemotactic effect on T cells on its own, augmented the SDF-1 alpha-induced T cell chemotaxis across fibronectin. These findings add another dimension to the known versatility of heparanase as a key regulator of T cell activities during inflammation, both in the context of the vasculature and at extravascular sites.
Assuntos
Adjuvantes Imunológicos/fisiologia , Comunicação Celular/imunologia , Matriz Extracelular/enzimologia , Matriz Extracelular/imunologia , Glucuronidase/fisiologia , Proteínas Recombinantes/farmacologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/fisiologia , Molécula 1 de Adesão de Célula Vascular/fisiologia , Adjuvantes Imunológicos/metabolismo , Adesão Celular/imunologia , Comunicação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/farmacologia , Quimiotaxia de Leucócito/imunologia , Colágeno Tipo IV/fisiologia , Matriz Extracelular/fisiologia , Fibronectinas/metabolismo , Fibronectinas/fisiologia , Quinase 2 de Adesão Focal , Glucuronidase/metabolismo , Humanos , Ácido Hialurônico/fisiologia , Interfase/imunologia , Migração e Rolagem de Leucócitos/imunologia , Ativação Linfocitária , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais/imunologia , Especificidade por Substrato/imunologia , Subpopulações de Linfócitos T/enzimologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The development of T cell tolerance directed toward tumor-associated Ags can limit the repertoire of functional tumor-reactive T cells, thus impairing the ability of vaccines to elicit effective antitumor immunity. Adoptive immunotherapy strategies using ex vivo expanded tumor-reactive effector T cells can bypass this problem; however, the susceptibility of effector T cells to undergoing tolerization suggests that tolerance might also negatively impact adoptive immunotherapy. Nonetheless, adoptive immunotherapy strategies can be effective, particularly those utilizing the drug cyclophosphamide (CY) and/or exogenous IL-2. In the current study, we used a TCR-transgenic mouse adoptive transfer system to assess whether CY plus IL-2 treatment rescues effector CD4 cell function in the face of tolerizing Ag (i.e., cognate parenchymal self-Ag). CY plus IL-2 treatment not only enhances proliferation and accumulation of effector CD4 cells, but also preserves the ability of these cells to express the effector cytokine IFN-gamma (and to a lesser extent TNF-alpha) in proportion to the level of parenchymal self-Ag expression. When administered individually, CY but not IL-2 can markedly impede tolerization, although their combination is the most effective. Although effector CD4 cells in CY plus IL-2-treated self-Ag-expressing mice eventually succumb to tolerization, this delay results in an increased level of in situ IFN-gamma expression in cognate Ag-expressing parenchymal tissues as well as death via a mechanism that requires direct parenchymal Ag presentation. These results suggest that one potential mechanism by which CY and IL-2 augment adoptive immunotherapy strategies to treat cancer is by impeding the tolerization of tumor-reactive effector T cells.
Assuntos
Autoantígenos/fisiologia , Linfócitos T CD4-Positivos/imunologia , Ciclofosfamida/administração & dosagem , Imunoterapia Adotiva , Interleucina-2/administração & dosagem , Tolerância a Antígenos Próprios/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/mortalidade , Doenças Autoimunes/patologia , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD4-Positivos/virologia , Células Clonais , Ciclofosfamida/toxicidade , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Imunoterapia Adotiva/métodos , Interfase/imunologia , Linfopenia/induzido quimicamente , Linfopenia/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tolerância a Antígenos Próprios/efeitos dos fármacosRESUMO
Immune evasion mechanisms of human CMV are known; however, the immune control of infection remains poorly elucidated. We show that interaction between the viral protein UL18 on infected cells and the invariant receptor CD85j/LIR-1/ILT2 expressed on CTL is relevant for the control of infection. Resting and activated CD8(+) T cells lysed UL18 expressing cells, whereas cells infected with CMV defective for UL18 were not killed. Lysis was not dependent on CD8(+) T cell Ag specificity, MHC-unrestricted and specifically blocked by anti-CD85j and anti-UL18 mAb. Moreover, soluble recombinant UL18Fc immunoprecipitated CD85j from T cells. Activation is mediated by CD85j and its pathway is unrelated to CD3/TCR engagement. UL18 is detected in immunocompromised patients with productive infection and the mechanism used in vivo by human CMV to ensure survival of the immunocompetent host may be mediated by activation signals delivered by infected cells to T lymphocytes via UL18/CD85j interactions.
Assuntos
Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/imunologia , Proteínas do Capsídeo/metabolismo , Citomegalovirus/imunologia , Citotoxicidade Imunológica , Antígenos de Histocompatibilidade Classe I/fisiologia , Receptores Imunológicos/metabolismo , Adulto , Animais , Antígenos CD/biossíntese , Antígenos CD/fisiologia , Linfócitos T CD8-Positivos/metabolismo , Proteínas do Capsídeo/biossíntese , Linhagem Celular Tumoral , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Testes Imunológicos de Citotoxicidade , Fibroblastos/imunologia , Fibroblastos/virologia , Células HeLa , Humanos , Interfase/imunologia , Células Jurkat , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Camundongos , Pessoa de Meia-Idade , Receptores Imunológicos/biossíntese , Receptores Imunológicos/fisiologiaRESUMO
There are conflicting reports on the requirements for the IL-27-WSX-1 pathway in the development of Th type 1 responses and resistance to intracellular pathogens; although early IFN-gamma production and resistance to Leishmania major are impaired in the absence of WSX-1 signaling, WSX-1(-/-) mice generate robust IFN-gamma responses and control infection with other intracellular protozoan pathogens. In this report, we resolve these conflicting observations and demonstrate that, in the absence of IL-4, WSX-1 is not required for early IFN-gamma production and control of L. major. Thus, the requirement for WSX-1 signaling in Th type 1 cell differentiation is restricted to conditions in which IL-4 is produced.
Assuntos
Citocinas/biossíntese , Interleucina-4/biossíntese , Interleucinas/fisiologia , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Receptores de Citocinas/fisiologia , Transdução de Sinais/imunologia , Células Th1/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Interferon gama/biossíntese , Interleucina-12/biossíntese , Interleucina-4/antagonistas & inibidores , Interleucina-4/imunologia , Interleucina-4/fisiologia , Interfase/genética , Interfase/imunologia , Leishmania major/crescimento & desenvolvimento , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Citocinas/deficiência , Receptores de Citocinas/genética , Receptores de Interleucina , Células Th1/citologia , Células Th1/metabolismo , Células Th1/parasitologia , Fatores de TempoRESUMO
Results from several mouse tolerance models indicate that autoreactive B cells in peripheral lymphoid organs develop an anergic phenotype, migrate to the boundary between the T cell zone and the B cell follicle (T/B boundary), and undergo rapid cell death. We have used B cells from mice that are double-transgenic for soluble hen egg lysozyme (HEL) and an Ig that recognizes HEL with a high affinity to characterize the mechanisms underlying the migration and elimination of autoreactive B cells. In contrast to the situation for acutely activated B cells, we find that anergic B cells have reduced levels of CXCR5, the receptor for the follicular chemokine, CXCL13, and this contributes to their exclusion from follicles. CCR7 expression is required for follicular exclusion of anergic cells, although up-regulation of the receptor does not appear to be necessary. By TUNEL analysis, we observe that excluded anergic cells die in situ at the T/B boundary. We also show that this elimination occurs via a Fas-independent mechanism. Using CCR7(-/-)Ig(HEL)-transgenic B cells we find that localization to the T/B boundary is not a necessary event to achieve the competitive elimination of autoantigen-binding B cells. These findings characterize the mechanism for follicular exclusion of autoantigen-binding B cells and they indicate that B cells compete for survival by mechanisms that are separate from competition for the follicular niche.