RESUMO
Type 1 diabetes is caused by the destruction of insulin producing beta cells by the immune system. The p110δ isoform of PI3K is expressed primarily in cells of haematopoietic origin and the catalytic activity of p110δ is important for the activation of these cells. Targeting of this pathway offers an opportunity to reduce immune cell activity without unwanted side effects. We have explored the effects of a specific p110δ isoform inhibitor, IC87114, on diabetogenic T cells both in vitro and in vivo, and find that although pharmacological inhibition of p110δ has a considerable impact on the production of pro-inflammatory cytokines, it does not delay the onset of diabetes after adoptive transfer of diabetogenic cells. Further, we demonstrate that combination treatment with CTLA4-Ig does not improve the efficacy of treatment, but instead attenuates the protective effects seen with CTLA4-Ig treatment alone. Our results suggest that decreased IL-10 production by Foxp3+ CD4+ T cells in the presence of IC87114 negates individual anti-inflammatory effects of IC8114 and CTLA4-Ig.
Assuntos
Adenina/análogos & derivados , Diferenciação Celular/efeitos dos fármacos , Citocinas/biossíntese , Diabetes Mellitus Tipo 1/imunologia , Inibidores de Fosfoinositídeo-3 Quinase , Quinazolinas/farmacologia , Linfócitos T/efeitos dos fármacos , Adenina/farmacologia , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Linfócitos T/fisiologiaRESUMO
B cell development is controlled by a series of checkpoints that ensure that the immunoglobulin (Ig)-encoding genes produce a functional B cell receptor (BCR) and antibodies. As part of this process, recombination-activating gene (Rag) proteins regulate the in-frame assembly of the Ig-encoding genes. The BCR consists of Ig proteins in complex with the immunoreceptor tyrosine-based activation motif (ITAM)-containing Igalpha and Igbeta chains. Whereas the activation of the tyrosine kinases Src and Syk is essential for BCR signaling, the pathways that act downstream of these kinases are incompletely defined. Previous work has revealed a key role for the p110delta isoform of phosphatidylinositol 3-kinase (PI3K) in agonist-induced BCR signaling; however, early B cell development and mature B cell survival, which depend on agonist-independent or "tonic" BCR signaling, are not substantially affected by a deficiency in p110delta. Here, we show that p110alpha, but not p110beta, compensated in the absence of p110delta to promote early B cell development in the bone marrow and B cell survival in the spleen. In the absence of both p110alpha and p110delta activities, pre-BCR signaling failed to suppress the production of Rag proteins and to promote developmental progression of B cell progenitors. Unlike p110delta, however, p110alpha did not contribute to agonist-induced BCR signaling. These studies indicate that either p110alpha or p110delta can mediate tonic signaling from the BCR, but only p110delta can contribute to antigen-dependent activation of B cells.
Assuntos
Linfócitos B/citologia , Fosfatidilinositol 3-Quinases/imunologia , Receptores de Células Precursoras de Linfócitos B/metabolismo , Transdução de Sinais/imunologia , Análise de Variância , Animais , Linfócitos B/imunologia , Western Blotting , Classe I de Fosfatidilinositol 3-Quinases , Primers do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Camundongos , Camundongos Mutantes , Organismos Livres de Patógenos EspecíficosRESUMO
Activation of PI3K is among the earliest signaling events observed in T cells after conjugate formation with antigen-presenting cells (APCs). The relevant PI3K catalytic isoform and relative contribution of the TcR and CD28 to PI3K activity at the immune synapse have not been determined unequivocally. Using a quantitative imaging-based assay, we show that the PI3K activity at the T cell-APC contact area is dependent on the p110delta, but not the p110gamma, isoform of PI3K. CD28 enhanced PIP3 production at the T-cell synapse independently of its YMNM PI3K-recruitment motif that instead was required for efficient PKC recruitment. CD28 could partially compensate for the lack of p110delta activity during T-cell activation, which indicates that CD28 and p110delta act in parallel and complementary pathways to activate T cells. Consistent with this, CD28 and p110delta double-deficient mice were severely immune compromised. We therefore suggest that combined pharmaceutic targeting of p110delta activity and CD28 costimulation has potent therapeutic potential.
Assuntos
Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Ativação Linfocitária/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Formação de Anticorpos/imunologia , Antígenos CD28/genética , Proliferação de Células , Células Cultivadas , Quimiocina CXCL12/farmacologia , Dimerização , Ativação Enzimática/imunologia , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/deficiência , Fosfatidilinositol 3-Quinases/genética , Proteína Quinase C/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
Almost all responses of naive T cells require co-stimulation, i.e. engagement of the clonotypic TCR with relevant antigen/MHC and the co-stimulatory molecule CD28. How CD28 contributes to T-cell proliferation remains poorly understood, with widely conflicting reports existing which may reflect different methods of co-ligating receptors. Some CD28 mAb, however, can stimulate T-cell proliferation without the need for TCR co-ligation, and thus provide unique tools to dissect proliferative signals mediated through CD28 alone. Using primary peripheral T cells from CD28-transgenic mice, we show that both the YMNM and Lck-binding motifs, but not the Itk-binding motif, in CD28 are required for proliferation. Given that the YMNM motif recruits both phosphoinositide 3-kinase (PI3K) and the exchange factor Vav1, we investigated the role of these two molecules in CD28-mediated proliferation. In p110delta(D910A/D910A) transgenic T cells, which are defective in PI3K activation following CD28 ligation, proliferation was comparable to that in wild-type cells. By contrast, T-cell proliferation was abolished in Vav1(-/-) cells. Although we did not address the role of Grb2 in CD28 signalling, these results indicate that CD28 can mediate Lck- and Vav1-dependent proliferative signals independently of PI3K.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Antígenos CD28/imunologia , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Motivos de Aminoácidos , Animais , Antígenos CD28/química , Proliferação de Células , Ativação Linfocitária , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais , Linfócitos T/metabolismoRESUMO
Mouse gene-targeting studies have documented a central role of the p110delta isoform of phosphoinositide 3-kinase (PI3K) in B-cell development and function. A defect in B-cell antigen receptor (BCR) signaling is key to this B-cell phenotype. Here we further characterize this signaling defect and report that a p110delta-selective small molecule inhibitor mirrors the effect of genetic inactivation of p110delta in BCR signaling. p110delta activity is indispensable for BCR-induced DNA synthesis and phosphorylation of Akt/protein kinase B (PKB), forkhead transcription factor/forkhead box O3a (FOXO3a), and p70 S6 kinase (p70 S6K), with modest effects on the phosphorylation of glycogen synthase kinase 3 alpha/beta (GSK3alpha/beta) and extracellular signal-regulated kinase (Erk). The PI3K-dependent component of intracellular calcium mobilization also completely relies on p110delta catalytic activity. Resting B cells with inactive p110delta fail to enter the cell cycle, correlating with an incapacity to up-regulate the expression of cyclins D2, A, and E, and to phosphorylate the retinoblastoma protein (Rb). p110delta is also critical for interleukin 4 (IL-4)-induced phosphorylation of Akt/PKB and FOXO3a, and protection from apoptosis. Taken together, these data show that defects observed in p110delta mutant mice are not merely a consequence of altered B-cell differentiation, and emphasize the potential utility of p110delta as a drug target in autoimmune diseases in which B cells play a crucial role.