RESUMO
Recombinant adenovirus vectors are efficient at transferring genes into somatic tissues but are limited for use in clinical gene therapy by immunologic factors that result in the rapid loss of gene expression and inhibit secondary gene transfer. This study demonstrates that systemic coadministration of recombinant adenovirus with soluble CTLA4Ig, which is known to block co-stimulatory signals between T cells and antigen presenting cells, leads to persistent adenoviral gene expression in mice without long-term immunosuppression. This form of immunotherapy greatly enhances the likelihood that recombinant adenovirus vectors will be useful for human gene therapy.
Assuntos
Adenoviridae , Formação de Anticorpos , Células Apresentadoras de Antígenos/imunologia , Antígenos de Diferenciação/biossíntese , Expressão Gênica , Técnicas de Transferência de Genes , Imunoconjugados , Ativação Linfocitária , Linfócitos T/imunologia , Abatacepte , Animais , Antígenos CD , Antígenos de Diferenciação/análise , Antígenos de Diferenciação/genética , Antígeno CTLA-4 , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Terapia Genética/métodos , Vetores Genéticos , Humanos , Imunoglobulina G/biossíntese , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos C3H , Baço/imunologia , Fatores de Tempo , alfa 1-Antitripsina/biossínteseRESUMO
The isolation, characterization, and expression of a full-length cDNA encoding the human T cell glycoprotein CD6 is described. COS cells transfected with the CD6 clone express a 90-kD protein that reacts with all available anti-CD6 monoclonal antibodies. RNA blot hybridization analysis indicates that CD6 transcripts are predominantly restricted to cells in the T lineage. The predicted CD6 sequence is 468 amino acids long, with the typical features of a type I integral membrane protein. The cytoplasmic domain of CD6 contains two serine residues, one or both of which are substrates for phosphorylation during T cell activation. The extracellular domain of CD6 is significantly related to the extracellular domain of the human and mouse T cell antigen CD5, the cysteine-rich domain of the bovine and mouse type I macrophage scavenger receptor, the extracellular domain of the sea urchin spermatozoa protein that crosslinks the egg peptide speract, the mammalian complement factor 1, and the human lung tumor antigen L3. These molecules, therefore, constitute a new gene superfamily that is well conserved across species boundaries.
Assuntos
Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Superfície/genética , Glicoproteínas de Membrana/genética , Sequência de Aminoácidos , Animais , DNA de Neoplasias/genética , Biblioteca Gênica , Humanos , Linfócitos/imunologia , Dados de Sequência Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
Whereas dendritic cells (DC) are known to be potent activators of T cells both in vitro and in vivo, the critical costimulatory molecules expressed on DC are not well characterized. Using immunocytochemical and molecular techniques we find that splenic DC express B7/BB1, the counter-receptor for CD28. Moreover, expression of B7/BB1 is upregulated on epidermal Langerhans cells (LC) during their functional maturation into potent T cell stimulators. In blocking experiments, we find that participation of B7/BB1 is required for optimal proliferation of unprimed, allogeneic T cells in DC-driven, primary mixed leukocyte reactions. These data demonstrate that the regulated expression of B7/BB1 on DC may be important in the initiation of a primary T cell response.
Assuntos
Linfócitos B/imunologia , Células Dendríticas/imunologia , Receptores de Superfície Celular/biossíntese , Linfócitos T/imunologia , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Sequência de Bases , Antígenos CD28 , Células Dendríticas/citologia , Citometria de Fluxo , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genéticaRESUMO
A successful immune response requires intercellular contact between T and B lymphocytes. We recently showed that CD28, a T cell surface protein that regulates an activation pathway, could mediate intercellular adhesion with activated B cells by interaction with the B7 antigen. Here we show that CD28 is the primary receptor for B7 on activated peripheral blood T cells, that CD28 binds to B7 in the absence of other accessory molecules, and that interaction between CD28 and B7 is costimulatory for T cell activation. To characterize the binding of CD28 to B7, we have produced genetic fusions of the extracellular portions of B7 and CD28, and immunoglobulin (Ig) C gamma 1 chains. 125I-labeled B7 Ig bound to CD28-transfected Chinese hamster ovary (CHO) cells, and to immobilized CD28 Ig with a Kd approximately 200 nM. B7 Ig also inhibited CD28-mediated cellular adhesion. The function of CD28-B7 interactions during T cell activation was investigated with soluble fusion proteins and with B7-transfected CHO cells. Immobilized B7 Ig and B7+ CHO cells costimulated T cell proliferation. Stimulation of T cells with B7+ CHO cells also specifically increased levels of interleukin 2 transcripts. These results demonstrate that the CD28 signaling pathway could be activated by B7, resulting in increased T cell cytokine production and T cell proliferation. Cellular interactions mediated by B7 and CD28 may represent an important component of the functional interactions between T and B lymphoid cells.
Assuntos
Interleucina-2/genética , Interleucina-4/metabolismo , Ativação Linfocitária , RNA Mensageiro/genética , Receptores Mitogênicos/imunologia , Linfócitos T/imunologia , Transcrição Gênica , Animais , Antígenos de Diferenciação de Linfócitos T/imunologia , Sequência de Bases , Antígenos CD28 , Linhagem Celular , Humanos , Cinética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Plasmídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Receptores de Interleucina-4 , Receptores Mitogênicos/genética , Receptores Mitogênicos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Optimal induction of clonal expansion by normal CD4 T cells requires a ligand that can engage the T cell receptor as well as functionally defined costimulatory activity on the same antigen-presenting cell surface. While the presence of effective costimulation induces proliferation, T cell receptor ligation in its absence renders T cells inactive or anergic. The molecular basis of this costimulatory activity remains to be defined. Here we describe a monoclonal antibody that can block the costimulatory activity of splenic accessory cells. Treatment with this antibody not only blocks the proliferation of CD4 T cells to a T cell receptor ligand, but also induces T cell nonresponsiveness to subsequent stimulation. Sequence analysis of the antigen recognized by this antibody indicates that it recognizes a protein that is identical to heat-stable antigen. Gene transfer experiments directly demonstrate that this protein has costimulatory activity. Thus, heat-stable antigen meets the criteria for a costimulator of T cell clonal expansion.
Assuntos
Antígenos CD , Antígenos de Diferenciação/imunologia , Linfócitos T CD4-Positivos/imunologia , Glicoproteínas de Membrana , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Linfócitos B/imunologia , Antígeno CD24 , Complexo CD3 , Células CHO , Cricetinae , Glicolipídeos/metabolismo , Glicosilfosfatidilinositóis , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Fosfatidilinositóis/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores Fc/imunologia , Transfecção , Fosfolipases Tipo C/farmacologiaRESUMO
CD80 and CD86 (B7-1 and B7-2) are the ligands on antigen-presenting cells (APCs) which bind CD28 and deliver the costimulatory signals necessary for T cell activation. The reasons for the existence of two CD28 binding molecules are not well understood. We created a mutant version of CTLA4-Ig that could selectively bind CD80 and block CD28-CD80 interaction but leave CD28-CD86 binding intact. CD80 blockade prevented antigen-induced accumulation of eosinophils and lymphocytes in the lung of immunized mice, but did not block antigen induced systemic blood eosinophilia or IgE antibody production. No preferential expression of CD80 could be demonstrated on a population of lung APC consisting mainly of macrophages. These results indicate that CD80 costimulation is not necessary for the induction of Th2 immune responses but rather for the maintenance or amplification of lung inflammatory responses.
Assuntos
Antígenos de Diferenciação/farmacologia , Antígeno B7-1/fisiologia , Eosinofilia/fisiopatologia , Eosinófilos/fisiologia , Imunoconjugados , Inflamação , Pneumopatias/fisiopatologia , Linfócitos/fisiologia , Abatacepte , Sequência de Aminoácidos , Animais , Antígenos CD , Antígeno B7-1/efeitos dos fármacos , Antígenos CD28/efeitos dos fármacos , Antígenos CD28/fisiologia , Células CHO , Antígeno CTLA-4 , Sequência Conservada , Cricetinae , Eosinofilia/prevenção & controle , Eosinófilos/efeitos dos fármacos , Citometria de Fluxo , Humanos , Cinética , Pneumopatias/imunologia , Pneumopatias/prevenção & controle , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Naive T cells require interleukin 4 (IL-4) to develop into IL-4-producing T cells and IL-4 blocks development of such cells into interferon gamma (IFN-gamma) producers. Prior studies in accessory cell-independent priming systems using antireceptor antibodies as agonists have demonstrated that IL-2 is also necessary for the development of IL-4-producing cells under these culture conditions. Here we have examined the role of IL-2 and the CD28 costimulation pathway in priming for IL-4 and IFN-gamma production using a more physiologic model. This involved antigen presentation by accessory cells to naive CD4+ T cells from transgenic mice whose cells express a T cell receptor (TCR) specific for a cytochrome c peptide in association with I-Ek. With splenic antigen-presenting cells (APCs), inhibition of CD28 costimulation by the fusion protein CTLA4-immunoglobulin (Ig) blocked effective priming. Similarly, transfected fibroblasts expressing both MHC class II and the CD28 ligand B7 could prime for IL-4 production and such priming also was blocked by CTLA4-Ig. However, APCs deficient in CD28 ligands also could prime TCR transgenic T cells to become IL-4 producers if an exogenous source of IL-2, as well as IL-4, was provided, and the inhibition of priming seen with splenic or transfected fibroblast APCs in the presence of CTLA4-Ig could be reversed by addition of IL-2. Likewise, priming for IFN-gamma production could be blocked by CTLA4-Ig and reversed by IL-2. Thus, we conclude that IL-2 plays a critical role in priming naive CD4+ T cells to become IL-4 or IFN-gamma producers. Engagement of the CD28 pathway, although normally important in such priming, is unnecessary in the presence of exogenous IL-2.
Assuntos
Antígenos CD28/metabolismo , Interferon gama/biossíntese , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Linfócitos T/metabolismo , Animais , Anticorpos/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Técnicas de Cultura , Humanos , Camundongos , Camundongos Transgênicos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
Functional interactions between T and B lymphocytes are necessary for optimal activation of an immune response. Recently, the T lymphocyte receptor CD28 was shown to bind the B7 counter-receptor on activated B lymphocytes, and subsequently to costimulate interleukin 2 production and T cell proliferation. CTLA-4 is a predicted membrane receptor from cytotoxic T cells that is homologous to CD28 and whose gene maps to the same chromosomal band as the gene for CD28. It is not known, however, if CD28 and CTLA-4 also share functional properties. To investigate functional properties of CTLA-4, we have produced a soluble genetic fusion between the extracellular domain of CTLA-4 and an immunoglobulin C gamma chain. Here, we show that the fusion protein encoded by this construct, CTLA4Ig, bound specifically to B7-transfected Chinese hamster ovary cells and to lymphoblastoid cells. CTLA4Ig also immunoprecipitated B7 from cell surface 125I-labeled extracts of these cells. The avidity of 125I-labeled B7Ig fusion protein for immobilized CTLA4Ig was estimated (Kd approximately 12 nM). Finally, we show that CTLA4Ig was a potent inhibitor of in vitro immune responses dependent upon cellular interactions between T and B lymphocytes. These findings provide direct evidence that, like its structural homologue CD28, CTLA-4 is able to bind the B7 counter-receptor on activated B cells. Lymphocyte interactions involving the B7 counter-receptor are functionally important for alloantigen responses in vitro.
Assuntos
Antígenos de Diferenciação/imunologia , Antígenos de Superfície/metabolismo , Linfócitos B/imunologia , Imunoconjugados , Ativação Linfocitária , Receptores Imunológicos , Linfócitos T/imunologia , Abatacepte , Sequência de Aminoácidos , Animais , Antígenos CD , Antígenos de Diferenciação/genética , Antígeno B7-1 , Sequência de Bases , Antígeno CTLA-4 , Linhagem Celular , Técnicas In Vitro , Ligantes , Cooperação Linfocítica , Dados de Sequência Molecular , Oligonucleotídeos/química , Testes de Precipitina , Proteínas Recombinantes de FusãoRESUMO
Allograft rejection is a T cell-dependent process. Productive T cell activation by antigen requires antigen engagement of the T cell receptor as well as costimulatory signals delivered through other T cell surface molecules such as CD28. Engagement of CD28 by its natural ligand B7 can be blocked using a soluble recombinant fusion protein, CTLA4Ig. Administration of CTLA4Ig blocks antigen-specific immune responses in vitro and in vivo, and we have shown that treatment of rats with a 7-d course of CTLA4Ig at the time of transplantation leads to prolonged survival of cardiac allografts (median 30 d), although most grafts are eventually rejected. Here, we have explored additional strategies employing CTLA4Ig in order to achieve long-term allograft survival. Our data indicate that donor-specific transfusion (DST) plus CTLA4Ig can provide effective antigen-specific immunosuppression. When DST is administered at the time of transplantation followed by a single dose of CTLA4Ig 2 d later, all animals had long-term graft survival (> 60 d). These animals had delayed responses to donor-type skin transplants, compared with normal rejection responses to third-party skin transplants. Furthermore, donor-matched second cardiac allografts were well tolerated with minimal histologic evidence of rejection. These data indicate that peritransplant use of DST followed by subsequent treatment with CTLA4Ig can induce prolonged, often indefinite, cardiac allograft acceptance. These results may be clinically applicable for cadaveric organ and tissue transplantation in humans.
Assuntos
Antígenos de Diferenciação/imunologia , Transplante de Coração/imunologia , Teste de Histocompatibilidade , Imunoconjugados , Transfusão de Linfócitos , Complexo Principal de Histocompatibilidade , Abatacepte , Animais , Antígenos CD , Antígeno CTLA-4 , Sobrevivência de Enxerto/imunologia , Transplante de Coração/patologia , Terapia de Imunossupressão/métodos , Masculino , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Transplante de Pele/imunologia , Baço , Transplante Homólogo/imunologia , Transplante Homólogo/patologiaRESUMO
The specificity of T lymphocyte activation is determined by engagement of the T cell receptor (TCR) by peptide/major histocompatibility complexes expressed on the antigen-presenting cell (APC). Lacking costimulation by accessory molecules on the APC, T cell proliferation does not occur and unresponsiveness to subsequent antigenic stimulus is induced. The B7/BB1 receptor on APCs binds CD28 and CTLA-4 on T cells, and provides a costimulus for T cell proliferation. Here, we show that prolonged, specific T cell hyporesponsiveness to antigenic restimulation is achieved by blocking the interaction between CD28 and B7/BB1 in human mixed leukocyte culture (MLC). Secondary T cell proliferative responses to specific alloantigen were inhibited by addition to the primary culture of monovalent Fab fragments of anti-CD28 monoclonal antibody (mAb) 9.3, which block interaction of CD28 with B7/BB1 without activating T cells. Hypo-responsiveness was also induced in MLC by CTLA4Ig, a chimeric immunoglobulin fusion protein incorporating the extracellular domain of CTLA-4 with high binding avidity for B7/BB1. Cells previously primed could also be made hyporesponsive, if exposed to alloantigen in the presence of CTLA4Ig. Maximal hyporesponsiveness was achieved in MLC after 2 d of incubation with CTLA4Ig, and was maintained for at least 27 d after removal of CTLA4Ig. Accumulation of interleukin 2 (IL-2) and interferon gamma but not IL-4 mRNA was blocked by CTLA4Ig in T cells stimulated by alloantigen. Antigen-specific responses could be restored by addition of exogenous IL-2 at the time of the secondary stimulation. Addition to primary cultures of the intact bivalent anti-CD28 mAb 9.3, or B7/BB1+ transfected CHO cells or exogenous IL-2, abrogated induction of hyporesponsiveness by CTLA4Ig. These data indicate that interaction of CD28 with B7/BB1 during TCR engagement with antigen is required to maintain T cell competence and that blocking such interaction can result in a state of T cell hyporesponsiveness.
Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos T/fisiologia , Antígenos de Superfície/fisiologia , Isoantígenos/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígeno B7-1 , Antígenos CD28 , Antígenos CD4/análise , Células CHO , Cricetinae , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Interleucina-2/farmacologia , Camundongos , Linfócitos T Citotóxicos/fisiologiaRESUMO
T cell surface receptors CD28 and CTLA-4 are homologous members of the immunoglobulin superfamily (IgSF), each comprising a single V-like extracellular domain. CD28 and CTLA-4 bind to the B7-1 and B7-2 counter-receptors on antigen presenting cells (APCs), thereby triggering a costimulatory pathway important for optimal T cell activation in vitro and in vivo. Soluble forms of CD28 and CTLA-4 in which the V-like extracellular domains were fused to Ig constant domains (CD28Ig and CTLA4Ig), have been used to study their interactions with B7-1 and B7-2, with CTLA4Ig binding B7-1 more strongly than CD28Ig (approximately 20-fold higher avidity). We have now, by site-specific and homologue mutagenesis, identified regions in CTLA4Ig important for strong binding to B7-1. A hexapeptide motif (MYPPPY) in the complementarity determining region 3 (CDR3)-like region is fully conserved in all CD28 and CTLA-4 family members. Alanine scanning mutagenesis through the motif in CTLA4Ig and at selected residues in CD28Ig reduced or abolished binding to B7-1. Chimeric molecules HS4, HS4-A, and HS4-B were constructed in which CDR3-like regions of CTLA-4, COOH-terminally extended to include nonconserved residues, were grafted onto CD28Ig. These homologue mutants showed stronger binding to B7-1 than did CD28Ig. Grafting of the CDR1-like region of CTLA-4, which is not conserved in CD28 and is predicted to be spatially adjacent to CDR3, into HS4 and HS4-A, resulted in chimeric molecules (HS7 and HS8) which bound B7-1 even better. Inclusion of the CDR2-like domain of CTLA-4 into HS7 and HS8 did not further increase binding. Thus, the MYPPPY motifs of CTLA4Ig and CD28Ig are important for their binding to B7-1, but the increased strength of this binding by CTLA4Ig is mediated by nonconserved residues in the CDR1- and CDR3-analogous regions.
Assuntos
Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/química , Antígenos CD28/biossíntese , Antígenos CD28/química , Sequência Conservada , Imunoconjugados , Linfócitos T/imunologia , Abatacepte , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos CD , Sítios de Ligação , Células CHO , Antígeno CTLA-4 , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Humanos , Ativação Linfocitária , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Receptores de Antígenos de Linfócitos T/biossíntese , Receptores de Antígenos de Linfócitos T/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
T cell costimulation by molecules on the antigen presenting cell (APC) is required for optimal T cell proliferation. The B7 molecule on APC binds the T lymphocyte receptor CD28, triggering increased interleukin 2 (IL-2) production and subsequent T cell proliferation. CTLA-4 is a predicted T cell membrane receptor homologous to CD28, which also binds the B7 counter receptor, but whose distribution and function are unknown. Here we have developed monoclonal antibodies (mAbs) specific for CTLA-4 and have investigated these questions. mAbs were produced that bound CTLA-4 but not CD28, and that blocked binding of CTLA-4 to B7. CTLA-4 expression as measured by these mAbs was virtually undetectable on resting T cells, but was increased several hundred-fold during T cell activation. On activated lymphocytes, CTLA-4 was expressed equally on CD4+ and CD8+ T cell subsets and was coexpressed with CD25, CD28, and CD45RO. CTLA-4 expression was lower than that of CD28, reaching a maximum of approximately 1/30-50 the level of CD28. Despite its lower expression, CTLA-4 was responsible for much of the B7 binding by large activated T cells. Anti-CTLA-4 mAb 11D4 and anti-CD28 mAb 9.3 acted cooperatively to inhibit T cell adhesion to B7, and to block T cell proliferation in primary mixed lymphocyte culture. When coimmobilized with anti T cell receptor (TCR) mAb, anti-CTLA-4 mAbs were less effective than anti-CD28 mAb 9.3 at costimulating proliferation of resting or activated T cells. However, coimmobilized combinations of anti-CD28 and anti-CTLA-4 were synergistic in their ability to augment anti-TCR-induced proliferation of preactivated CD4+ T cells. These results indicate that CTLA-4 is coexpressed with CD28 on activated T lymphocytes and cooperatively regulates T cell adhesion and activation by B7.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos de Diferenciação/biossíntese , Imunoconjugados , Ativação Linfocitária , Linfócitos T/imunologia , Abatacepte , Animais , Anticorpos Monoclonais , Antígenos CD/análise , Antígenos CD/imunologia , Antígenos de Diferenciação/análise , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos CD28 , Antígenos CD4/análise , Antígenos CD4/imunologia , Células CHO , Antígeno CTLA-4 , Adesão Celular/imunologia , Células Cultivadas , Cricetinae , Cinética , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Plasmídeos , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/imunologia , Subpopulações de Linfócitos T/imunologia , TransfecçãoRESUMO
Blocking the CD28-B7 T cell costimulatory pathway with the fusion protein CTLA4Ig inhibits alloimmune responses in vitro and in vivo and induces tolerance to cardiac allografts in mice and rats, but the mechanisms mediating the tolerant state in vivo are unknown. Here, we report the effects and potential mechanisms of CTLA4Ig in the rat renal allograft model. LEW rats were nephrectomized and received renal allografts from major histocompatibility complex-incompatible WF rats. While all untreated and control immunoglobulin (Ig)-treated animals acutely rejected their allografts and died, 86% of rats that received a single injection of CTLA4Ig on day 2 after transplantation had prolonged survival (> 60-100 days) with preserved renal function. By contrast, only 29% of animals that received CTLA4Ig on the day of engraftment had prolonged survival. Long-term survivors (> 100 days) exhibited donor-specific tolerance, accepting donor-matched WF but acutely rejecting third-party BN cardiac allografts. Immunohistological analysis of grafts sampled at 1 week after transplantation showed that both control and CTLA4Ig-treated animals had mononuclear cell infiltrates, with a higher percentage of CD4+ cells in the CTLA4Ig-treated group. However, while this was associated with vasculitis and tubulitis in control grafts, there was no evidence of tissue injury in CTLA4Ig-treated animals. The immune response leading to graft rejection in control animals was characterized by expression of the T helper (Th) type 1 cytokines interleukin (IL)-2 and interferon-gamma. In contrast, the persistent CD4+ infiltrate without graft rejection in CTLA4Ig-treated animals was associated with increased staining for the Th2-related cytokines IL-4 and IL-10. Furthermore, grafts from CTLA4Ig-treated animals had marked upregulation of intragraft staining for IgG1, but not IgG2a or IgG2b. Administration of rIL-2 to CTLA4Ig-treated animals restored allograft rejection in 50% of animals tested. These results confirm that blockade of the CD28-B7 pathway after alloantigenic challenge induces donor-specific acceptance of vascularized organ allografts, and indicates in this model that CTLA4Ig inhibits Th1 but spares Th2 cytokines in vivo.
Assuntos
Antígeno B7-1/fisiologia , Antígenos CD28/fisiologia , Citocinas/biossíntese , Imunoconjugados , Isoantígenos/imunologia , Células Th1/fisiologia , Células Th2/fisiologia , Abatacepte , Animais , Antígenos CD , Antígenos de Diferenciação/imunologia , Antígeno CTLA-4 , Tolerância Imunológica , Interleucina-2/farmacologia , Transplante de Rim , Masculino , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Transplante HomólogoRESUMO
During the differentiation of thymocytes to mature T cells the processes of positive and negative selection result in signals that either protect thymocytes from cell death, or delete, through apoptosis, thymocytes with self-reactive T cell receptors (TCR). Glucocorticoids have been shown to induce thymocyte apoptosis and are produced within the thymic microenvironment. Furthermore, steroid-induced apoptosis of thymocytes has been suggested as a potential mechanism for removal of nonselected thymocytes. In this report, we demonstrate that thymocytes can be rescued from glucocorticoid-induced apoptosis by incubation with cells that express high levels of B7-1 or B7-2. In addition, the ability to be rescued by B7-1 and/or B7-2 can precede expression of the TCR. We demonstrate that CD3(+)-depleted or CD3+/ TCR-beta(+)-doubly depleted thymocytes can be rescued from glucocorticoid-induced apoptosis through the interaction of CD28 or CTLA-4 on thymocytes with cells bearing high levels of B7-1 or B7-2. Furthermore, these transfected cells are major histocompatibility complex (MHC) class II negative and, while they may express MHC class I, there is no preferential rescue of CD8+ thymocytes in the presence of glucocorticoids. Together, these data suggest that the rescue of thymocytes from glucocorticoids can be independent of the TCR. We also demonstrate that, in addition to CD28, CTLA-4 is expressed on thymocytes, suggesting that rescue from glucocorticoid-induced cell death can be mediated by both CD28 and CTLA-4. A CTLA-4Ig fusion protein which binds to both B7-1 and B7-2 was shown to completely block the rescue of thymocytes from glucocorticoid-induced cell death. Therefore, we conclude that interactions between B7-1/B7-2 and CD28/CTLA-4 are sufficient and necessary for rescue of thymocytes from glucocorticoid-induced cell death.
Assuntos
Antígenos CD/imunologia , Apoptose , Glucocorticoides/farmacologia , Imunoconjugados , Timo/citologia , Timo/imunologia , Abatacepte , Animais , Antígenos CD/genética , Antígenos de Diferenciação/imunologia , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígeno B7-2 , Antígenos CD28/imunologia , Antígeno CTLA-4 , Sobrevivência Celular , Antígenos de Histocompatibilidade Classe I , Antígenos de Histocompatibilidade Classe II , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos , Receptores de Antígenos de Linfócitos T , Timo/efeitos dos fármacos , TransfecçãoRESUMO
The zona pellucida (ZP), an ovarian extracellular structure, contains three major glycoproteins: ZP1, ZP2, and ZP3. A ZP3 peptide contains both an autoimmune oophoritis-inducing T cell epitope and a B cell epitope that induces autoantibody to ZP. This study investigates two major T cell costimulation pathways in this disease model. Herein we show that blockage of glycoprotein (gp)39 and CD40 interaction with gp39 monoclonal antibody (mAb) results in the failure to induce both autoimmune oophoritis and autoantibody production. Inhibition of ligand binding to the CD28 receptor with the fusion protein, murine CTLA4-immunoglobulin (Ig), also results in failure to generate antibody to ZP and significantly reduces disease severity and prevalence. Surprisingly, the frequencies of antigen-specific T cells in anti-gp39 mAb-treated mice, CTLA4-Ig treated mice, and in mice given control hamster IgG or control fusion protein L6, were equivalent as determined by limiting dilution analysis (approximately equals 1:5,000). These T cells, which produced comparable amounts of interleukin 4 and interferon gamma in vitro, were able to transfer oophoritis to normal recipients. When anti-gp39 mAb and CTLA4-Ig were given together, the effect was additive, leading to inhibition of T cell activation as determined by in vitro proliferation and limiting dilution analysis (approximately equals 1:190,000); disease and antibody responses were absent in these mice. By studying these two costimulatory pathways in parallel, we have shown that autoimmune disease and autoantibody production are inhibitable by blocking either the gp39 or the CD28 pathway, whereas inhibition of clonal expansion of the effector T cell population occurs only when both pathways are blocked.
Assuntos
Antígenos CD28/fisiologia , Anergia Clonal , Imunoconjugados , Glicoproteínas de Membrana/fisiologia , Linfócitos T/imunologia , Zona Pelúcida/fisiologia , Abatacepte , Animais , Anticorpos Monoclonais , Formação de Anticorpos , Antígenos CD , Antígenos de Diferenciação/imunologia , Autoanticorpos/biossíntese , Ligante de CD40 , Antígeno CTLA-4 , Células Cultivadas , Citocinas/análise , Citocinas/biossíntese , Feminino , Interferon gama/biossíntese , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Ooforite/imunologia , Ovário/imunologia , Peptídeos/química , Peptídeos/imunologia , Zona Pelúcida/imunologiaRESUMO
B7-2 is a recently discovered, second ligand for the CTLA-4/CD28, T cell signaling system. Using the GL-1 rat monoclonal antibody (mAb), we monitored expression of B7-2 on mouse leukocytes with an emphasis on dendritic cells. By cytofluorography, little or no B7-2 was detected on most cell types isolated from spleen, thymus, peritoneal cavity, skin, marrow, and blood. However, expression of B7-2 could be upregulated in culture. In the case of epidermal and spleen dendritic cells, which become highly immunostimulatory for T cells during a short period of culture, the upregulation of B7-2 was dramatic and did not require added stimuli. Lipopolysaccharide did not upregulate B7-2 levels on dendritic cells, in contrast to macrophages and B cells. By indirect immunolabeling, the level of staining with GL-1 mAb exceeded that seen with rat mAbs to several other surface molecules including intercellular adhesion molecule 1, B7-1, CD44, and CD45, as well as new hamster mAbs to CD40, CD48, and B7-1/CD80. Of these accessory molecules, B7-2 was a major species that increased in culture, implying a key role for B7-2 in the functional maturation of dendritic cells. B7-2 was the main (> 90%) CTLA-4 ligand on mouse dendritic cells. When we applied GL-1 to tissue sections of a dozen different organs, clear-cut staining with B7-2 antigen was found in many. B7-2 staining was noted on liver Kupffer cells, interstitial cells of heart and lung, and profiles in the submucosa of the esophagus. B7-2 staining was minimal in the kidney and in the nonlymphoid regions of the gut, and was not observed at all in the brain. In the tongue, only rare dendritic cells in the oral epithelium were B7-2+, but reactive cells were scattered about the interstitial spaces of the muscle. In all lymphoid tissues, Gl-1 strongly stained certain distinct regions that are occupied by dendritic cells and by macrophages. For dendritic cells, these include the thymic medulla, splenic periarterial sheaths, and lymph node deep cortex; for macrophages, the B7-2-rich regions included the splenic marginal zone and lymph node subcapsular cortex. Splenic B7-2+ cells were accessible to labeling with GL-1 mAb given intravenously. Dendritic cell stimulation of T cells (DNA synthesis) during the mixed leukocyte reaction was significantly (35-65%) blocked by GL-1.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Antígenos CD , Antígeno B7-1/análise , Células Dendríticas/química , Imunoconjugados , Glicoproteínas de Membrana , Abatacepte , Animais , Antígenos de Diferenciação/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2 , Antígeno CTLA-4 , Células Dendríticas/fisiologia , Feminino , Lipopolissacarídeos/farmacologia , Tecido Linfoide/química , Camundongos , Camundongos Endogâmicos , Distribuição TecidualRESUMO
The specificity of antibody (Ab) responses depends on focusing helper T (Th) lymphocyte signals to suitable B lymphocytes capable of binding foreign antigens (Ags), and away from nonspecific or self-reactive B cells. To investigate the molecular mechanisms that prevent the activation of self-reactive B lymphocytes, the activation requirements of B cells specific for the Ag hen egg lysozyme (HEL) obtained from immunoglobulin (Ig)-transgenic mice were compared with those of functionally tolerant B cells isolated from Ig-transgenic mice which also express soluble HEL. To eliminate the need for surface (s)Ig-mediated Ag uptake and presentation and allow the effects of sIg signaling to be studied in isolation, we assessed the ability of allogeneic T cells from bm12 strain mice to provide in vivo help to C57BL/6 strain-transgenic B cells. Interestingly, non-tolerant Ig-transgenic B cells required both allogeneic Th cells and binding of soluble HEL for efficient activation and Ab production. By contrast, tolerant self-reactive B cells from Ig/HEL double transgenic mice responded poorly to the same combination of allogeneic T cells and soluble HEL. The tolerant B cells were nevertheless normally responsive to stimulation with interleukin 4 and anti-CD40 Abs in vitro, suggesting that they retained the capacity to respond to mediators of T cell help. However, the tolerant B cells exhibited a proximal block in the sIg signaling pathway which prevented activation of receptor-associated tyrosine kinases in response to the binding of soluble HEL. The functional significance of this sIg signaling defect was confirmed by using a more potent membrane-bound form of HEL capable of triggering sIg signaling in tolerant B cells, which markedly restored their ability to collaborate with allogeneic Th cells and produce Ab. These findings indicate that Ag-specific B cells require two signals for mounting a T cell-dependent Ab response and identify regulation of sIg signaling as a mechanism for controlling self-reactive B cells.
Assuntos
Linfócitos B/fisiologia , Tolerância Imunológica , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Linfócitos T/fisiologia , Animais , Antígenos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Comunicação Celular , Membrana Celular/enzimologia , Células Cultivadas , Imunoglobulina D/imunologia , Imunoglobulina M/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Muramidase/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Efficient T cell activation is dependent on the intimate contact between antigen-presenting cells (APCs) and T cells. The engagement of the B7 family of molecules on APCs with CD28 and CD152 (cytotoxic T lymphocyte-associated antigen 4 [CTLA-4]) receptors on T cells delivers costimulatory signal(s) important in T cell activation. We investigated the dependence of pathologic cellular activation in psoriatic plaques on B7-mediated T cell costimulation. Patients with psoriasis vulgaris received four intravenous infusions of the soluble chimeric protein CTLA4Ig (BMS-188667) in a 26-wk, phase I, open label dose escalation study. Clinical improvement was associated with reduced cellular activation of lesional T cells, keratinocytes, dendritic cells (DCs), and vascular endothelium. Expression of CD40, CD54, and major histocompatibility complex (MHC) class II HLA-DR antigens by lesional keratinocytes was markedly reduced in serial biopsy specimens. Concurrent reductions in B7-1 (CD80), B7-2 (CD86), CD40, MHC class II, CD83, DC-lysosomal-associated membrane glycoprotein (DC-LAMP), and CD11c expression were detected on lesional DCs, which also decreased in number within lesional biopsies. Skin explant experiments suggested that these alterations in activated or mature DCs were not the result of direct toxicity of CTLA4Ig for DCs. Decreased lesional vascular ectasia and tortuosity were also observed and were accompanied by reduced presence of E-selectin, P-selectin, and CD54 on vascular endothelium. This study highlights the critical and proximal role of T cell activation through the B7-CD28/CD152 costimulatory pathway in maintaining the pathology of psoriasis, including the newly recognized accumulation of mature DCs in the epidermis.
Assuntos
Antígenos de Diferenciação/farmacologia , Células Dendríticas/fisiologia , Endotélio Vascular/citologia , Imunoconjugados , Imunossupressores/farmacologia , Queratinócitos/fisiologia , Psoríase/terapia , Linfócitos T/imunologia , Abatacepte , Antígenos CD , Antígenos de Diferenciação/fisiologia , Antígenos de Diferenciação/uso terapêutico , Antígenos CD28/fisiologia , Antígeno CTLA-4 , Endotélio Vascular/fisiologia , Citometria de Fluxo , Humanos , Integrina alfaXbeta2/análise , Integrinas/análise , Ativação Linfocitária , Neutrófilos/fisiologia , Psoríase/imunologia , Psoríase/patologia , Selectinas/análiseRESUMO
The interaction of B7-related molecules on antigen-presenting cells with CD28 or CTLA-4 antigens on T cells provides a second signal for T cell activation. Selection inhibition of the B7-CD28 or B7-CTLA-4 interactions produces antigen-specific T cell unresponsiveness in vitro and suppresses immune function in vivo. To determine whether selective inhibition of the B7-CD28 or B7-CTLA-4 interactions could suppress spontaneous autoimmune disease, a B7-binding protein was generated by genetic fusion of the extracellular domain of murine CTLA-4 to the Fc portion of a mouse immunoglobulin G2a monoclonal antibody (muCTLA4Ig). In lupus-prone NZB/NZW filial generation (F1) mice, treatment with muCTLA4Ig blocked autoantibody production and prolonged life, even when treatment was delayed until the most advanced stage of clinical illness. These findings suggest a possible role for human CTLA4Ig in the treatment of autoimmune diseases in humans.
Assuntos
Antígenos de Diferenciação/uso terapêutico , Imunoconjugados , Lúpus Eritematoso Sistêmico/terapia , Abatacepte , Animais , Anticorpos Antinucleares/biossíntese , Anticorpos Monoclonais , Antígenos CD , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Linfócitos B/imunologia , Antígeno B7-1/metabolismo , Antígeno CTLA-4 , Feminino , Humanos , Imunoterapia , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos NZB , Camundongos Endogâmicos , Proteínas Recombinantes de Fusão/uso terapêutico , Linfócitos T/imunologiaRESUMO
Oncostatin M, a cytokine produced by activated lymphoid cells, regulates the growth and differentiation of a number of tumor and normal cells. In contrast to its effects on normal endothelial and aortic smooth muscle cell cultures, Oncostatin M was a potent mitogen for cells derived from acquired immunodeficiency syndrome-related Kaposi's sarcoma (AIDS-KS). After exposure to Oncostatin M, AIDS-KS cells assumed a spindle morphology, had an increased ability to proliferate in soft agar, and secreted increased amounts of interleukin-6. Oncostatin M RNA and immunoreactive Oncostatin M protein were found in AIDS-KS-derived cell isolates. These results suggest that Oncostatin M may play a role in the pathogenesis of AIDS-KS.