RESUMO
Syk and Zap-70 constitute a closely related nonreceptor protein tyrosine kinase family, of which both members are functionally indispensable for conferring their respective antigen receptors with enzymatic activity. In this study, we analyze the impact of altering BCR signaling output on B-cell germinal center (GC) fate selection by constitutive, as well as inducible, monoallelic Syk kinase loss in the presence of a Zap-70 knock-in rescue allele. Cre-mediated Syk deletion in Syk(flox/Zap-70) B cells lowers pErk, but not pAkt-mediated signaling. Surprisingly, the use of a B-cell-specific constitutive mb1-cre deleter mouse model showed that a small cohort of peripheral Syk(flox/Zap-70);mb1-cre B cells efficiently circumvents deletion, which ultimately favors these Syk-sufficient cells to contribute to the GC reaction. Using a developmentally unbiased Syk(flox/Zap-70);mb1-creER(T2) approach in combination with an inducible tdRFP allele, we further demonstrate that this monoallelic deletion escape is not fully explained by leakiness of Cre expression, but is possibly the result of differential Syk locus accessibility in maturing B cells. Altogether, this underscores the importance of proper Syk kinase function not only during central and peripheral selection processes, but also during GC formation and maintenance.
Assuntos
Linfócitos B/metabolismo , Centro Germinativo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Alelos , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Teste de Complementação Genética , Centro Germinativo/citologia , Centro Germinativo/imunologia , Integrases/genética , Integrases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Camundongos , Camundongos Transgênicos , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais , Quinase Syk , Proteína-Tirosina Quinase ZAP-70/genética , Proteína-Tirosina Quinase ZAP-70/imunologiaRESUMO
CD1d-restricted invariant natural killer T cells (iNKT) constitute an important immunoregulatory T-cell subset that can be activated by the synthetic glycolipid α-galactosylceramide (α-GalCer) and play a dominant role in antitumor immunity. Clinical trials with α-GalCer-pulsed monocyte-derived dendritic cells (moDC) have shown anecdotal antitumor activity in advanced cancer. It was reported that phosphoantigen (pAg)-activated Vγ9Vδ2-T cells can acquire characteristics of professional antigen-presenting cells (APC). Considering the clinical immunotherapeutic applications, Vγ9Vδ2-T APC can offer important advantages over moDC, potentially constituting an attractive novel APC platform. Here, we demonstrate that Vγ9Vδ2-T APC can present antigens to iNKT. However, this does not result from de novo synthesis of CD1d by Vγ9Vδ2-T, but critically depends on trogocytosis of CD1d-containing membrane fragments from pAg-expressing cells. CD1d-expressing Vγ9Vδ2-T cells were able to activate iNKT in a CD1d-restricted and α-GalCer-dependent fashion. Although α-GalCer-loaded moDC outperformed Vγ9Vδ2-T APC on a per cell basis, Vγ9Vδ2-T APC possess unique features with respect to clinical immunotherapeutic application that make them an interesting platform for consideration in future clinical trials.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Antígenos CD1d/imunologia , Células T Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Apresentação de Antígeno , Linhagem Celular , Galactosilceramidas/farmacologia , Células HeLa , Humanos , Leucócitos Mononucleares/citologiaRESUMO
The spleen tyrosine kinase family members Syk and Zap-70 are pivotal signal transducers downstream of antigen receptors and exhibit overlapping expression patterns at early lymphocytic developmental stages. To assess their differential kinase fitness in vivo, we generated mice, which carry a Zap-70 cDNA knock-in controlled by intrinsic Syk promoter elements that disrupts wild-type Syk expression. Kinase replacement severely compromised Erk1/2-mediated survival and proper selection of developing B cells at central and peripheral checkpoints, demonstrating critical dependence on BCR signalling quality. Furthermore, ITAM- and hemITAM-mediated activation of platelets and neutrophils was completely blunted, while surprisingly FcγR-mediated phagocytosis in macrophages was retained. The alteration in BCR signalling quality resulted in preferential development and survival of marginal zone B cells and prominent autoreactivity, causing the generation of anti-insulin antibodies and age-related glomerulonephritis. Development of concomitant fasting glucose intolerance in knock-in mice highlights aberrant B cell selection as a potential risk factor for type 1 diabetes, and suggests altered BCR signalling as a mechanism to cause biased cellular and Ig repertoire selection, ultimately contributing to B cell-mediated autoimmune predisposition.
