Cbp deficiency alters Csk localization in lipid rafts but does not affect T-cell development.

The ubiquitously expressed transmembrane adaptor Csk-binding protein (Cbp) recruits Csk to lipid rafts, where the latter exerts its negative regulatory effect on the Src family of protein tyrosine kinases. We have inactivated Cbp in the mouse germ line. In contrast to Csk gene inactivation, which leads to embryonic lethality and impaired T-cell development, Cbp-deficient mice were viable and exhibited normal T-cell development but with an increased thymocyte population. In the absence of Cbp, the amount of Csk that localizes to the lipid rafts was greatly reduced. Interestingly, this altered lipid raft localization of Csk did not lead to any detectable biochemical or functional defect in T cells. The T-cell receptor-induced intracellular calcium flux, cell proliferation, and cytokine secretion were not affected by the absence of Cbp. Peripheral T-cell tolerance to superantigen SEB was also largely intact in Cbp-deficient mice. Thus, Cbp is dispensable for T-cell development and activation.

Stochastic pairing of Ig heavy and light chains frequently generates B cell antigen receptors that are subject to editing in vivo.

We examined the generation and selection of the B cell antibody repertoire through crossing of mice bearing distinct Ig heavy (H) and light (L) chain rearranged variable region transgenes. Ig gene knock-in and transgenic mice whose H and L chains pair to form a non-autoreactive, functional B cell antigen receptor (BCR) have significantly reduced pre-B cells in the bone marrow as their B cell progenitors rapidly differentiate into surface IgM(+) B cells. The presence of a pre-B cell compartment in these Ig transgenic mice, however, indicates the induction of receptor editing. Here, 18 distinct combinations of H and L chains were generated that we showed could pair in vitro to form BCRs of unknown specificities. Of these, nine induced receptor editing in vivo as evidenced by the presence of pre-B cells and endogenous L chain rearrangements in mice bearing these H and L chain transgenes. These data thus suggest that about half of the emerging antibody repertoire is negatively selected during B lymphopoiesis due to the likely encoding of autoreactive or non-functional BCRs.

Peritoneal CD5+ B-1 cells have signaling properties similar to tolerant B cells.

CD5(+) B (or B-1) cells are the normal precursors of B cell chronic lymphocytic leukemia. They differ from conventional B (B-2) cells with respect to their phenotype and mitogenic responses and are often secretors of the natural polyreactive antibodies in the serum. The origin of B-1 cells remains controversial, and the relationship between B-1 cells and autoreactive B cells is unclear. Here, we compare the signaling pathways that are activated by the engagement of the B cell antigen receptor (BCR) in B-1 and B-2 cells. Stimulation of the BCR leads to the induced activation of the three major classes of mitogen-activated protein kinases (MAPKs), ERK, JNK, and p38 MAPK, as well as the Akt kinase and the transcription factors nuclear factor of activated T cells (NF-AT) and NF-kappaB in B-2 cells. In contrast, B-1 cells have constitutive activation of ERK and NF-AT but exhibit delayed JNK and lack p38 MAPK and NF-kappaB induction upon BCR cross-linking. The lack of NF-kappaB activation in B-1 cells may be due to a lack of Akt activation in these cells. Furthermore, our study using specific inhibitors reveals that the extended survival of B-1 cells in culture is not due to the constitutive activation of ERK; nor is it due to Akt signaling or Bcl-x(L) up-regulation, since these are not induced in B-1 cells. The current findings of altered MAPK and NF-AT activation and lack of NF-kappaB induction in B-1 cells indicate that these cells have signaling properties similar to tolerant B cells that are chronically exposed to self-antigens. Indeed, BCR stimulation of B-1 cells does not lead to their full activation as indicated by their lack of maximal up-regulation of specific markers such as CD25, CD69, and CD86.