Germinal centers (GCs) generate memory B and plasma cells, essential for long-lived humoral immunity. GC B cells with high affinity B cell receptors (BCRs) are selectively expanded. To enable this selection, BCRs of such cells are thought to signal differently from those with lower affinity. We show that, surprisingly, most proliferating GC B cells did not demonstrate active BCR signaling. Rather, spontaneous and induced signaling was limited by increased phosphatase activity. Accordingly, both SHP-1 and SHIP-1 were hyperphosphorylated in GC cells and remained colocalized with BCRs after ligation. Furthermore, SHP-1 was required for GC maintenance. Intriguingly, GC B cells in the cell cycle G2 period regained responsiveness to BCR stimulation. These data have implications for how higher affinity B cells are selected in the GC.

Rationale GDF11 (Growth Differentiation Factor 11) is a member of the transforming growth factor β (TGFβ) super family of secreted factors. A recent study showed that reduced GDF11 blood levels with aging was associated with pathological cardiac hypertrophy (PCH), and restoring GDF11 to normal levels in old mice rescued PCH. Objective To determine if and by what mechanism GDF11 rescues aging dependent PCH. Methods and Results 24-month-old C57BL/6 mice were given a daily injection of either recombinant (r) GDF11 at 0.1mg/kg or vehicle for 28 days. rGDF11 bioactivity was confirmed in-vitro. After treatment, rGDF11 levels were significantly increased but there was no significant effect on either heart weight (HW) or body weight (BW). HW/BW ratios of old mice were not different from 8 or 12 week-old animals, and the PCH marker ANP was not different in young versus old mice. Ejection fraction, internal ventricular dimension, and septal wall thickness were not significantly different between rGDF11 and vehicle treated animals at baseline and remained unchanged at 1, 2 and 4 weeks of treatment. There was no difference in myocyte cross-sectional area rGDF11 versus vehicle-treated old animals. In vitro studies using phenylephrine-treated neonatal rat ventricular myocytes (NRVM), to explore the putative anti-hypertrophic effects of GDF11, showed that GDF11 did not reduce NRVM hypertrophy, but instead induced hypertrophy. Conclusions Our studies show that there is no age-related PCH in disease free 24-month-old C57BL/6 mice and that restoring GDF11 in old mice has no effect on cardiac structure or function.

Humoral memory to an antigen (Ag) is maintained for several decades in the form of memory B cells and serum Ab. In fact, plasma cells (PCs) that secrete Ab are known to be long-lived and could be solely responsible for maintaining the long-lived Ab titers. Alternatively, it has been proposed that the PC compartment is maintained for long periods by the differentiation of memory cells into long-lived PCs as a result of nonspecific stimulation. This model predicts accelerated decay of PC numbers in the absence of memory cells for the same Ag. To address this prediction, we have developed a mouse model system that combined the ability to deplete B cells with the ability to detect Ag-specific memory and PCs. After establishing an immune response, we depleted Agspecific memory B cells with an anti-hCD20 mAb and determined the effect on the PC compartment over 16 weeks. Using a combination of surface markers, we demonstrated that memory B cells remained depleted over the course of the experiment. However, despite this absence of memory cells for an extended duration, PC numbers in spleen and bone marrow did not decline, which indicates that the PC compartment does not require a significant contribution from memory B cells for its maintenance and instead that PCs are sufficiently long-lived to maintain Ab titers over a long period without renewal. This observation settles an important controversy in B cell biology and has implications for the design of vaccines and for B cell depletion therapy in patients.CD20 ͉ antibody-forming cell ͉ serum antibody ͉ B cell depletion

B lymphocytes producing high affinity antibodies (Abs) are critical for protection from extracellular pathogens, such as bacteria and parasites. The process by which high affinity B cells are selected during the immune response has never been elucidated. Though it has been shown that high affinity cells directly outcompete low affinity cells in the germinal center (GC)2, whether there are also intrinsic differences between these cells has not been addressed. It could be that higher affinity cells proliferate more rapidly or are more likely to enter cell cycle, thereby outgrowing lower affinity cells. Alternatively, higher affinity cells could be relatively more resistant to cell death in the germinal center. By comparing high and low affinity B cells for the same antigen, we show here that low affinity cells have an intrinsically higher death rate than cells of higher affinity, even in the absence of competition. This suggests that selection in the GC reaction is due at least in part to the control of survival of higher affinity B cells and not by a proliferative advantage conferred upon these cells compared to lower affinity B cells. Control over survival rather than proliferation of low and high affinity B cells in the GC allows greater diversity not only in the primary response but also in the memory response.

SUMMARY To simulate transient B cell activation that is the likely initiator of T-dependent responses, we examined the molecular and functional consequences of a single-round of immunoglobulin M (IgM) signaling. This form of activation triggered early cytosolic signaling and the transcription factor NF-κB activation indistinguishably from conventional continuous IgM cross-linking, but did not induce G1 progression. However, single-round IgM signaling changed the expression of chemokine and chemokine receptor genes implicated in initiating T-dependent responses, as well as accentuated responsiveness to CD40 signaling. Several features of single-round IgM signaling in vitro were recapitulated in B cells after short-term exposure to antigen in vivo. We propose that transient BCR signals prime B cells to receive T cell help by increasing the probability of B-T encounter and creating a cellular environment that is hyper-responsive to CD40 signaling.