Defective IgG2a/2b class switching in PKC alpha-/- mice.

Using model tumor T cell lines, protein kinase C (PKC) alpha has been implicated in IL-2 cytokine promoter activation in response to Ag receptor stimulation. In this study, for the first time, PKCalpha null mutant mice are analyzed and display normal T and B lymphocyte development. Peripheral CD3(+) PKCalpha-deficient T cells show unimpaired activation-induced IL-2 cytokine secretion, surface expression of CD25, CD44, and CD69, as well as transactivation of the critical transcription factors NF-AT, NF-kappaB, AP-1, and STAT5 in vitro. Nevertheless, CD3/CD28 Ab- and MHC alloantigen-induced T cell proliferation and IFN-gamma production are severely impaired in PKCalpha(-/-) CD3(+) T cells. Consistently, PKCalpha-deficient CD3(+) T cells from OVA-immunized PKCalpha-deficient mice exhibit markedly reduced recall proliferation to OVA in in vitro cultures. In vivo, PKCalpha-deficient mice give diminished OVA-specific IgG2a and IgG2b responses following OVA immunization experiments. In contrast, OVA-specific IgM and IgG1 responses and splenic PKCalpha(-/-) B cell proliferation are unimpaired. Our genetic data, thus, define PKCalpha as the physiological and nonredundant PKC isotype in signaling pathways that are necessary for T cell-dependent IFN-gamma production and IgG2a/2b Ab responses.

PKCtheta and PKA are antagonistic partners in the NF-AT transactivation pathway of primary mouse CD3+ T lymphocytes.

We here investigate the crosstalk of PKC and PKA signaling during primary CD3(+) T-lymphocyte activation using pharmacologic inhibitors and activators in combination with our established panel of PKC isotype-deficient mouse T cells in vitro. PKCtheta and PKA inversely affect the CD3/CD28-induced IL-2 expression, whereas other PKC isotypes are dispensable in this signaling pathway. Gene ablation of PKCtheta selectively results in a profound reduction of IL-2 production; however, complete abrogation of IL-2 production in these PKCtheta(-/-) T cells was achieved only by simultaneous coactivation of the cAMP/PKA pathway in CD3(+) T cells. Conversely, the reduced IL-2 production in PKC inhibitor-treated T cells can be rescued by inhibition of the cAMP/PKA pathway in wild-type but not in PKCtheta(-/-) T cells. Mechanistically, the cAMP/PKA and PKCtheta pathways converge at the level of NF-AT, as shown by DNA binding analysis. The combined increase in PKA and decrease in PKCtheta activity leads to an enhanced inhibition of nuclear NF-AT translocation. This PKCtheta/PKA crosstalk significantly affects neither the NF-kappaB, the AP-1, nor the CREB pathways. Taken together, this opposite effect between the positive PKCtheta and the negative cAMP/PKA signaling pathways appears rate limiting for NF-AT transactivation and IL-2 secretion responses of CD3(+) T lymphocytes.

PKCdelta is involved in signal attenuation in CD3+ T cells.

PKCdelta has been implicated in the signalling events after engagement of the antigen specific receptor on B cells and the Fc-epsilon receptor on mast cells. Employing our recently established PKCdelta null mice , we here investigate the physiological function of PKCdelta in CD3+ T cells. As result, administration of anti-CD3 antibodies in vivo induced markedly increased blood plasma IL-2 levels (but not IL-4, IFN-gamma, TNF-alpha and IL-6 levels) in the PKCdelta null mice, when compared to wild-type sibling controls. Additionally, in vitro T cell proliferative responses to allogenic MHC are significantly enhanced in PKCdelta-deficient CD3+ T cells. These findings suggest that PKCdelta serves a so far unrecognized role in TCR-induced negative regulation of IL-2 cytokine production and T cell proliferation.

Protein kinase C theta affects Ca2+ mobilization and NFAT cell activation in primary mouse T cells.

Protein kinase C (PKC)theta is an established component of the immunological synapse and has been implicated in the control of AP-1 and NF-kappaB. To study the physiological function of PKCtheta, we used gene targeting to generate a PKCtheta null allele in mice. Consistently, interleukin 2 production and T cell proliferative responses were strongly reduced in PKCtheta-deficient T cells. Surprisingly, however, we demonstrate that after CD3/CD28 engagement, deficiency of PKCtheta primarily abrogates NFAT transactivation. In contrast, NF-kappaB activation was only partially reduced. This NFAT transactivation defect appears to be secondary to reduced inositol 1,4,5-trisphosphate generation and intracellular Ca2+ mobilization. Our finding suggests that PKCtheta plays a critical and nonredundant role in T cell receptor-induced NFAT activation.