Summary Immunity to the intestinal parasite, Heligomosomoides polygyrus, is dependent on the successful generation of Th2 memory cells. We show that B cells contribute to immunity against H. polygyrus by producing antibody and by promoting expansion and differentiation of primary and memory Th2 cells. We also demonstrate that cytokine-producing “effector” B cells are essential for effective immunity to H. polygyrus. TNFα production by B cells is necessary for sustained Ab production, while IL-2 production by B cells is necessary for Th2 expansion and differentiation. These results show that B cells mediate protection to pathogens not only by presenting antigen and secreting antibody but also by producing cytokines that regulate the quality and magnitude of humoral and cellular immune responses.

The ectoenzyme CD38 catalyzes the production of cyclic ADP-ribose (cADPR) and ADP-ribose (ADPR) from its substrate, NAD+. Both products of the CD38 enzyme reaction play important roles in signal transduction, as cADPR regulates calcium release from intracellular stores and ADPR controls cation entry through the plasma membrane channel TRPM2. We previously demonstrated that CD38 and the cADPR generated by CD38 regulate calcium signaling in leukocytes stimulated with some, but not all, chemokines and controls leukocyte migration to inflammatory sites. However, it is not known whether the other CD38 product, ADPR, also regulates leukocyte trafficking In this study we characterize 8-bromo (8Br)-ADPR, a novel compound that specifically inhibits ADPR-activated cation influx without affecting other key calcium release and entry pathways. Using 8Br-ADPR, we demonstrate that ADPR controls calcium influx and chemotaxis in mouse neutrophils and dendritic cells activated through chemokine receptors that rely on CD38 and cADPR for activity, including mouse FPR1, CXCR4, and CCR7. Furthermore, we show that the calcium and chemotactic responses of leukocytes are not dependent on poly-ADP-ribose polymerase 1 (PARP-1), another potential source of ADPR in some leukocytes. Finally, we demonstrate that NAD+ analogues specifically block calcium influx and migration of chemokine-stimulated neutrophils without affecting PARP-1-dependent calcium responses. Collectively, these data identify ADPR as a new and important second messenger of mouse neutrophil and dendritic cell migration, suggest that CD38, rather than PARP-1, may be an important source of ADPR in these cells, and indicate that inhibitors of ADPR-gated calcium entry, such as 8Br-ADPR, have the potential to be used as anti-inflammatory agents.

We recently cloned a partial cDNA (35H) for a protein kinase C (PKC) binding protein from a rat kidney cDNA library and demonstrated that it is a PKC substrate in vitro (Chapline, C., Ramsay, K., Klauck, T., and Jaken, S. (1993) J. Biol. Chem. 268, 6858 -6861). Additional library screening and 5 rapid amplification of cDNA ends were used to obtain the complete open reading frame. Amino acid sequence analysis, DNA sequence analysis, and Northern analysis indicate that 35H is a unique cDNA related to ␣-and ␤-adducins. Antisera prepared to the 35H bacterial fusion protein recognized two polypeptides of 80 and 90 kDa on immunoblots of kidney homogenates and cultured renal proximal tubule epithelial cell extracts. The 35H-related proteins were similar to ␣-and ␤-adducins in that they were preferentially recovered in the Triton X-100-insoluble (cytoskeletal, CSK) fraction of cell extracts and were predominantly localized to cell borders. Phorbol esters stimulated phosphorylation of CSK 35H proteins, thus emphasizing that sequences isolated according to PKC binding activity in vitro are also PKC substrates in vivo. The phosphorylated forms of the 35H proteins were preferentially recovered in the soluble fraction, thus demonstrating that phosphorylation regulates their CSK association and, thereby, their function in regulating cytoskeletal assemblies. We have isolated another PKC binding protein partial cDNA (clone 45) from a rat fibroblast library with substantial homology to ␣-adducin. Antisera raised against this expressed sequence recognized a protein of 120 kDa, the reported size of ␣-adducin, on immunoblots of renal proximal tubule epithelial cell extracts. A 120-kDa protein that cross-reacts with the clone 45 (␣-adducin) antisera coprecipitated with 35H immunecomplexes, indicating that ␣-adducin associates with 35H proteins in vivo. Taken together, these results indicate that 35H is a new, widely expressed form of adducin capable of forming heterodimers with ␣-adducin. We propose naming this adducin homologue ␥-adducin.Protein kinase Cs are a heterogeneous group of phospholipiddependent kinases important for cell growth and differentiated functions (reviewed in Ref. 1). The family can be divided into three categories based on enzymatic properties. The conventional or Group A PKCs 1 are calcium-dependent kinases whose activities are stimulated by diacylglycerol or phorbol esters. The novel or Group B PKCs are calcium-independent but still diacylglycerol-stimulatable. The atypical or Group C PKCs are calcium-and diacylglycerol-independent. Most cells express more than one type of PKC, which implies that PKCs have unique rather than overlapping functions.Activation of Group A and B PKCs is regulated by receptormediated production of diacylglycerol through phospholipase C or D pathways (1). In many cases, activation correlates with PKC redistribution from soluble to particulate fractions. However, in other cases, evidence for activation in the absence of measurable translocation has been noted (2-5). The correlation ...

Although B cells expressing the IFNγR or the IFNγ-inducible transcription factor T-bet promote autoimmunity in Systemic Lupus Erythematosus (SLE)-prone mouse models, the role for IFNγ signaling in human antibody responses is unknown. We show that elevated levels of IFNγ in SLE patients correlate with expansion of the T-bet expressing IgDnegCD27negCD11c+CXCR5neg (DN2) pre-antibody secreting cell (pre-ASC) subset. We demonstrate that naïve B cells form T-bethi pre-ASCs following stimulation with either Th1 cells or with IFNγ, IL-2, anti-Ig and TLR7/8 ligand and that IL-21 dependent ASC formation is significantly enhanced by IFNγ or IFNγ-producing T cells. IFNγ promotes ASC development by synergizing with IL-2 and TLR7/8 ligands to induce genome-wide epigenetic reprogramming of B cells, which results in increased chromatin accessibility surrounding IRF4 and BLIMP1 binding motifs and epigenetic remodeling of IL21R and PRDM1 loci. Finally, we show that IFNγ signals poise B cells to differentiate by increasing their responsiveness to IL-21.

Although viral infections elicit robust interferon-g (IFN-g) and long-lived antibody-secreting cell (ASC) responses, the roles for IFN-g and IFN-g-induced transcription factors (TFs) in ASC development are unclear. We showed that B cell intrinsic expression of IFN-gR and the IFN-g-induced TF T-bet were required for T-helper 1 cell-induced differentiation of B cells into ASCs. IFN-gR signaling induced Blimp1 expression in B cells but also initiated an inflammatory gene program that, if not restrained, prevented ASC formation. T-bet did not affect Blimp1 upregulation in IFN-g-activated B cells but instead regulated chromatin accessibility within the Ifng and Ifngr2 loci and repressed the IFN-g-induced inflammatory gene program. Consistent with this, B cell intrinsic T-bet was required for formation of long-lived ASCs and secondary ASCs following viral, but not nematode, infection. Therefore, T-bet facilitates differentiation of IFN-g-activated inflammatory effector B cells into ASCs in the setting of IFN-g-, but not IL-4-, induced inflammatory responses.