Characterization of the roles of PLCgamma and Tak1 in lymphocyte antigen receptor signaling

T and B lymphocytes share both a common precursor in their development and many of the same molecular mechanisms that lead to their functions. The antigen receptors of T cells (TCR) and B cells (BCR) are responsible for the recognition of foreign antigen and the initiation of signaling pathways that ultimately decide the cell's response to the antigen. Though many of the interactions of signaling molecules downstream of the TCR and BCR have been elucidated, the roles of PLCgamma in iNKT cells and Tak1 in B cells remain undescribed or controversial. Here we utilize several mouse models to study PLCgamma and Tak1 in a physiologically relevant setting.;One of the downstream targets of BCR cross-linking is the transcription factor NF-kappaB, which is directly activated by the IKK complex and is essential for an appropriate B cell response. Though much of the BCR signaling pathway from membrane-proximal events to activation of the CARMA1/Bcl-10/Malt1 (CBM) complex has been characterized, a mechanism for IKK activation by the CBM complex in B cells is lacking. We hypothesize that Tak1 is important in the activation of B cells by functioning to link the CBM complex to the IKK complex. To address this, we generated mice that were conditionally deficient for Tak1 in the B cell lineage. Early B cell development in the bone marrow of these mice was largely unaffected while splenic B cells were significantly decreased, with the effects of Tak1 deficiency having a greater affect with maturation. The mature follicular, marginal zone and B1 B cells were all drastically reduced and Tak1-deficient splenic B cells had reduced proliferation and decreased survival. Western blot analysis showed that activation of MAPK family members and NF-kappaB was impaired after LPS stimulation and that JNK and NF-kappaB activity were impaired following BCR stimulation. Following stimulation through the BCR, we saw decreased expression of the NF-kappaB target genes c-Myc and Bcl-XL. These findings support our hypothesis that Tak1 is vital for BCR-induced activation of NF-kappaB by linking the CBM complex to IKK.;The lipid antigen-recognizing iNKT cells follow a distinct developmental pathway and rely on signaling events that differ from conventional T cells. Much about the signaling pathways downstream of the TCR in iNKT cells has not been described, though some events can be assumed to be consistent with what is known of conventional T cell receptor signaling. Based on conventional T cell and NK cell signaling studies, we hypothesize that both isoforms of PLCgamma played a role in iNKT cell development and activation. To address this, we generated mice that were conditionally or inducibly deficient for PLCgamma1 and mice that were deficient for PLCgamma2. Using tetramers specific for invariant TCR expression in the thymus and spleen, we were able to show that PLCgamma1 is essential for iNKT cell development, disrupting maturation at the CD44-NK1.1- stage. This developmental defect was shown to be iNKT cell intrinsic through assessment of thymic CD1d expression and by generating bone marrow chimeric mice that provided a thymic environment that was less disrupted by PLCgamma1 deletion. PLCgamma1-deficient iNKT cells showed a decreased capacity to generate both TH1 and TH2 cytokines when stimulated through their TCR. Further, mobilization of Ca2+ from intracellular stores was decreased in PLCgamma1-deficient iNKT cells. PLCgamma2 was shown to play a limited role in iNKT cell development that was only detectable when PLCgamma1 was also deleted. Thus we have demonstrated that PLCgamma1 is essential for iNKT cell development and function while PLCgamma2 plays an additive role in iNKT cell development.