RESUMEN
High-affinity class-switched Abs and memory B cells are products of the germinal center (GC). The CD4+ T cell help required for the development and maintenance of the GC is delivered by follicular Th cells (T(FH)), a CD4+ Th cell subset characterized by expression of Bcl-6 and secretion of IL-21. The cellular interactions that mediate differentiation of TFH and GC B cells remain an important area of investigation. We previously showed that MHC class II (MHCII)-dependent dendritic cell Ag presentation is sufficient for the differentiation of a T(FH) intermediate (termed pre-T(FH)), characterized by Bcl-6 expression but lacking IL-21 secretion. In this article, we examine the contributions of MHCII Ag presentation by B cells to T(FH) differentiation and GC responses in several contexts. B cells alone do not efficiently prime naive CD4+ T cells or induce T(FH) after protein immunization; however, during lymphocytic choriomeningitis virus infection, B cells induce T(FH) differentiation despite the lack of effector CD4+ T cell generation. Still, MHCII+ dendritic cells and B cells cooperate for optimal T(FH) and GC B cell differentiation in response to both model Ags and viral infection. This study highlights the roles for B cells in both CD4+ T cell priming and T(FH) differentiation, and demonstrates that different APC subsets work in tandem to mediate the GC response.
Asunto(s)
Presentación de Antígeno/inmunología , Linfocitos B/inmunología , Diferenciación Celular/inmunología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Antígenos/inmunología , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/genética , Células Dendríticas/inmunología , Antígenos H-2/genética , Antígenos H-2/inmunología , Antígenos H-2/metabolismo , Inmunización , Ratones , Ratones Noqueados , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Virosis/inmunología , Virosis/metabolismoRESUMEN
Ag presentation by dendritic cells (DC) in vivo is essential to the initiation of primary and secondary T cell responses. We have reported that DC presenting Ag in the context of MHC I molecules also become targets of specific CTL and are rapidly killed in mice. However, activated DC up-regulate expression of serine protease inhibitor (SPI)-6, a specific blocker of the cytotoxic granule protein granzyme B, which modulates their susceptibility to CTL-mediated killing in vitro. We wanted to determine whether susceptibility to CTL-mediated killing in vivo is also modulated by DC activation. As was previously reported by others, DC treated with different doses of LPS expressed higher levels of SPI-6 mRNA than did untreated DC. The increased expression of SPI-6 was functionally relevant, as LPS-treated DC became less susceptible to CTL-mediated killing in vitro. However, when these LPS-treated DC were injected in vivo, they remained sensitive to CTL-mediated killing regardless of whether the CTL activity was elicited in host mice via active immunization or was passively transferred via injection of in vitro-activated CTL. LPS-treated DC were also sensitive to killing in lymph node during the reactivation of memory CTL. We conclude that increased SPI-6 expression is not sufficient to confer DC with resistance to direct killing in vivo. However, SPI-6 expression may provide DC with a survival advantage in some conditions, such as those modeled by in vitro cytotoxicity assays.
Asunto(s)
Citotoxicidad Inmunológica , Células Dendríticas/enzimología , Células Dendríticas/inmunología , Lipopolisacáridos/farmacología , Proteínas de la Membrana/biosíntesis , Serina Endopeptidasas/biosíntesis , Serpinas/biosíntesis , Linfocitos T Citotóxicos/enzimología , Linfocitos T Citotóxicos/inmunología , Regulación hacia Arriba/inmunología , Animales , Muerte Celular/inmunología , Células Cultivadas , Células Dendríticas/trasplante , Inmunidad Innata , Activación de Linfocitos/inmunología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células 3T3 NIH , Serina Endopeptidasas/genética , Serpinas/genética , Linfocitos T Citotóxicos/citologíaRESUMEN
Immune suppression by Treg has been demonstrated in a number of models, but the mechanisms of this suppression are only partly understood. Recent work has suggested that Tregs may suppress by directly killing immune cell populations in vivo in a perforin- and granzyme B-dependent manner. To establish whether perforin is necessary for the regulation of immune responses in vivo, we examined OVA-specific CD8(+) T cell responses in WT and PKO mice immunized with OVA and α-GalCer and the expansion of WT OT-I CD8(+) T cells adoptively transferred into WT or PKO mice immunized with DC-OVA. We observed similar expansion, phenotype, and effector function of CD8(+) T cells in WT and PKO mice, suggesting that CD8(+) T cells were subjected to a similar amount of regulation in the two mouse strains. In addition, when WT and PKO mice were depleted of Tregs by anti-CD25 mAb treatment before DC-OVA immunization, CD8(+) T cell proliferation, cytotoxicity, and cytokine production were increased similarly, suggesting a comparable involvement of CD25(+) Tregs in controlling T cell proliferation and effector function in these two mouse strains. These data suggest that perforin expression is not required for normal immune regulation in these models of in vivo CD8(+) T cell responses induced by immunization with OVA and α-GalCer or DC-OVA.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Depleción Linfocítica , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Células Dendríticas/metabolismo , Galactosilceramidas/metabolismo , Inmunización , Lectinas Tipo C , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/inmunología , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptores Inmunológicos/metabolismo , Linfocitos T ReguladoresRESUMEN
In this study, we examine whether recognition of α-GalCer presented on CD1d-expressing DCs and B cells in vivo elicits the cytotoxic activity of iNKT cells and elimination of α-GalCer-presenting cells. We report that i.v. injection of α-GalCer induced a decrease in the percentage and number of splenic CD8(+)Langerin(+) DCs, while CD8(-) DCs were not affected. The decline in CD8(+) DC numbers was clearly detectable by 15 h after α-GalCer injection, was maximal at 24-48 h, returned to normal by day 7, and was accompanied by a reduced cross-presentation of OVA protein given i.v. to specific CD8(+) T cells in vitro. The decrease in the numbers of CD8(+) DCs required iNKT cells but was independent of perforin, Fas, or IFN-γ, as it was observed in mice deficient in each of these molecules. In contrast, treatment with a TNF-α-neutralizing antibody was effective at reducing the decline in CD8(+) DC numbers and DC activation. Treatment with immunostimulatory CpG ODN also resulted in DC activation and a decreased number of CD8(+) DCs; however, the decline in DC number was a result of down-regulation of CD11c and CD8 and did not require iNKT cells or TNF-α. Although CD8(+)Langerin(+) DCs appeared to be selectively affected by α-GalCer treatment, they were not required for early iNKT cell responses, as their prior depletion did not prevent the increase in serum TNF-α and IL-4 observed after α-GalCer treatment. Thus, iNKT cells regulate the survival of CD8(+) DCs through a mechanism that does not appear to involve direct cell killing.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Galactosilceramidas/farmacología , Células T Asesinas Naturales/efectos de los fármacos , Bazo/efectos de los fármacos , Animales , Antígenos CD1d/fisiología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Reactividad Cruzada/efectos de los fármacos , Reactividad Cruzada/inmunología , Células Dendríticas/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes/metabolismo , Interferón gamma/fisiología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células T Asesinas Naturales/metabolismo , Bazo/citología , Bazo/metabolismo , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Transfer of antigen between antigen-presenting cells (APCs) is potentially a physiologically relevant mechanism to spread antigen to cells with specialized stimulatory functions. Here we show that specific CD8+ T cell responses induced in response to intravenous administration of antigen-loaded bone marrow-derived dendritic cells (BM-DCs), were ablated in mice selectively depleted of endogenous lymphoid-resident langerin+ CD8α+ dendritic cells (DCs), suggesting that the antigen is transferred from the injected cells to resident APCs. In contrast, antigen-specific CD4+ T cells were primed predominantly by the injected BM-DCs, with only very weak contribution of resident APCs. Crucially, resident langerin+ CD8α+ DCs only contributed to the priming of CD8+ T cells in the presence of maturation stimuli such as intravenous injection of TLR ligands, or by loading the BM-DCs with the glycolipid α-galactosylceramide (α-GalCer) to recruit the adjuvant activity of activated invariant natural killer-like T (iNKT) cells. In fact, injection of α-GalCer-loaded CD1d-/- BM-DCs resulted in potent iNKT cell activation, suggesting that this glycolipid antigen can also be transferred to resident CD1d+ APCs. While iNKT cell activation per se was independent of langerin+ CD8α+ DCs, some iNKT cell-mediated activities were reduced, notably release of IL-12p70 and transactivation of NK cells. We conclude that both protein and glycolipid antigens can be exchanged between distinct DC species. These data suggest that the efficacy of DC-based vaccination strategies may be improved by the incorporation of a systemic maturation signal aimed to engage resident APCs in CD8+ T cell priming, and α-GalCer may be particularly well suited to this purpose.