Cutting edge: differential signaling requirements for activation of assembled cyclin D3-cdk4 complexes in B-1 and B-2 lymphocyte subsets.

B-1 lymphocytes represent a distinct B cell subset with unusual mitogenic responses. PMA alone promotes proliferation in B-1 cells, but not in splenic B-2 cells. Although cyclin D2-cyclin-dependent kinase 4 (cdk4) complexes mediate early retinoblastoma gene product (pRb) phosphorylation in B-1 cells, the transient nature of their accumulation cannot account for the continued increase in pRb phosphorylation, which is maximal at 24 h. We show herein that PMA promotes the accumulation of functional cyclin D3-cdk4 complexes in B-1 cells following loss of cyclin D2. PMA also induces accumulation of cyclin D3-cdk4 complexes in B-2 cells; however, these complexes do not phosphorylate pRb. Thus, PMA is sufficient to induce synthesis and assembly of cyclin D3-cdk4 complexes in B-1 and B-2 cells; however, PMA triggers cyclin D3-cdk4 activation only in B-1 cells. These results reveal a novel regulatory step that controls activation of cyclin D3-cdk4 complexes whose function segregates differentially in B cell subsets.

Immunologic recovery after autologous blood stem cell transplantation in patients with AL-amyloidosis.

We prospectively studied absolute lymphocyte (ALC) and monocyte counts (AMC), lymphocyte subsets and proliferative in vitro responses to mitogen and antigen in 12 patients with AL-amyloidosis (AL) undergoing autologous blood stem cell transplantation (SCT) with high-dose i.v. melphalan. Myeloid and lymphoid recovery (>500 per microl) occurred in a median of 10 days post SCT. While there was a continuous decline in the number of CD4+ T cells at 3 months, ALC, AMC, B cells (CD19+), CD8+ T cells, and NK cells (CD16+/56+) returned to baseline. While T cell proliferative responses to phytohemagglutinin (PHA) remained depressed, B cell function measured by the proliferative response to staphylococcal antigen returned to baseline by 3 months. To supplement our findings, we retrospectively evaluated ALC, AMC and serum immunoglobulin levels in a separate group of patients treated with the same protocol at our institution. ALC and AMC recovery was similar to the pattern observed in the initial study group. Immunoglobulin levels remained within normal ranges at 3 and 12 months after SCT. Of 50 patients who were followed for a minimum of 1 year following SCT, seven (14%) developed shingles and one (2%) had PCP pneumonia. In conclusion, cellular immune function, reflected by absolute numbers of CD4+ T cells and PHA responsive T cell proliferation, is significantly suppressed at 3 months after SCT in patients with AL, and this post-transplant immunosuppression is associated with a low but clinically meaningful occurrence of opportunistic infections typical of T cell immunosuppression.

Early induction of cyclin D2 expression in phorbol ester-responsive B-1 lymphocytes.

B-1 lymphocytes represent a distinct B cell subset with characteristic features that include self-renewing capacity and unusual mitogenic responses. B-1 cells differ from conventional B cells in terms of the consequences of phorbol ester treatment: B-1 cells rapidly enter S phase in response to phorbol ester alone, whereas B-2 cells require a calcium ionophore in addition to phorbol ester to trigger cell cycle progression. To address the mechanism underlying the varied proliferative responses of B-1 and B-2 cells, we evaluated the expression and activity of the G1 cell cycle regulator, cyclin D2, and its associated cyclin-dependent kinases (Cdks). Cyclin D2 expression was upregulated rapidly, within 2-4 h, in phorbol ester-stimulated B-1 cells, in a manner dependent on intact transcription/translation, but was not increased in phorbol ester- stimulated B-2 cells. Phorbol ester-stimulated cyclin D2 expression was accompanied by the formation of cyclin D2-Cdk4, and, to a lesser extent, cyclin D2-Cdk6, complexes; cyclin D2- containing complexes were found to be catalytically functional, in terms of their ability to phosphorylate exogenous Rb in vitro and to specifically phosphorylate endogenous Rb on serine780 in vivo. These results strongly suggest that the rapid induction of cyclin D2 by a normally nonmitogenic phorbol ester stimulus is responsible for B-1 cell progression through G1 phase. The ease and rapidity with which cyclin D2 responds in B-1 cells may contribute to the proliferative features of this subset.

A novel gene coding for a Fas apoptosis inhibitory molecule (FAIM) isolated from inducibly Fas-resistant B lymphocytes.

The sensitivity of primary splenic B cells to Fas-mediated apoptosis is modulated in a receptor-specific fashion. Here we used a differential display strategy to detect cDNAs present in B cells rendered Fas resistant but absent in those rendered Fas sensitive. This led to the cloning and characterization of a novel 1.2-kb gene that encodes a Fas apoptosis inhibitory molecule (FAIM). faim-transfected BAL-17 B lymphoma cells were less sensitive by half or more to Fas-mediated apoptosis than were vector-transfected controls, using Fas ligand-bearing T cells or a cytotoxic anti-Fas antibody to trigger Fas, and this was associated with inhibition of Fas- induced poly-ADP ribose polymerase (PARP) cleavage. In primary B cells, the time course of faim mRNA and FAIM protein expression correlated with the induction of Fas resistance by surface (s)Ig engagement. Thus, FAIM is an inducible effector molecule that mediates Fas resistance produced by sIg engagement in B cells. However, faim is broadly expressed in various tissues and the faim sequence is highly conserved evolutionarily, suggesting that its role extends beyond lymphocyte homeostasis. As FAIM has no significant regions of homology to other gene products that modulate Fas killing, it appears to represent a distinct, new class of antiapoptotic protein.

Unmethylated CpG motifs protect murine B lymphocytes against Fas-mediated apoptosis.

CD40 ligand (CD40L) has been shown to increase surface Fas expression and induce B cell sensitivity to Fas-dependent CD4+ Th1 cell-mediated cytotoxicity (Th1-CMC). We investigated the role of unmethylated mitogenic CpG motifs in regulating Fas-mediated apoptosis in primary murine B cells. Unmethylated CpG motifs protected CD40L-stimulated B cells from Th1-CMC and apoptosis mediated by Fas-specific antibody. Mitogenic CpG motifs downregulated Fas expression on CD40L-stimulated B cells in a time-dependent fashion. These observations suggest that Fas-mediated apoptosis requires minimum upregulation of surface Fas expression and that CpG motifs protect B cells from Fas-mediated apoptosis by decreasing surface Fas expression. Thus, these results suggest a novel mechanism for induction of Fas resistance in B cells.