Developmental checkpoints eliminate B cells synthesizing defective immunoglobulin heavy (HC) and light (LC) chains. The first checkpoint tests for formation of a VpreB/λ5/µHC-containing preB-cell receptor (preBCR) and predicts whether µHCs will bind conventional LCs to form membrane IgM. VpreB and λ5 also create a sensing site that interacts with µHC antigen-binding region CDR-H3, but whether it plays a role in immunoglobulin repertoire selection and function is unknown. On a position-by-position basis, we analyzed the amino acid content of CDR-H3s from H chains cloned from living and apoptotic preB cells and from IgG:Antigen structures. Using a panel of DH gene-targeted mice, we show that progressively reducing CDR-H3 tyrosine content increasingly impairs preBCR checkpoint passage. Counting from cysteine at Framework 3 position 96, we found that VpreB particularly selects for tyrosine at CDR-H3 position 101, and that Y101 also binds antigen in IgG:Antigen structures. VpreB thus acts as an early invariant antigen. It selects for particular CDR-H3 amino acids and shapes the specificity of the IgG humoral response. This helps explain why some neutralizing antibodies against pathogens are readily produced while others are rare.

The Drosophila adipose tissue, or fat body, and the bodywall muscle are two major tissues derived from the mesoderm. Although much is known about the lineage of muscle cells, little is known about the development of the fat body. Using known genes and an enhancer trap (29D), we have begun to trace the lineage of the cells comprising the fat body. The genes Adh (alcohol dehydrogenase) and DCg1 (type IV collagen) code for gene products involved in fat-cell metabolism and therefore serve as terminal fat-cell differentiation markers. The expression of these genes was used to identify the fat body at stage 17 and to identify the start of terminal fat-cell differentiation at stage 15. We found that the steroid-hormone receptor gene, svp (seven-up), was expressed transiently within the fat-cell lineage from stages 12 to 14. We suggest that stage 12 marks the beginning of early fat-cell differentiation and that the svp-positive cells within the mesoderm are early precursor fat cells. To confirm the identity of these cells and to establish the role of svp in the developing fat cell, we examined svp mutant embryos for alterations in the expression of the two terminal fat-cell differentiation markers, Adh and DCg1. Loss of svp function resulted in the loss of Adh transcript and a reduction of DCg1 expression specifically in the fat body. Thus, svp plays a role in fat-body-specific expression of at least two terminal fat-cell differentiation genes. In contrast to svp, we found no evidence that the steroid receptor HNF-4(D) gene was expressed in the fat body nor that it was involved in the development of this tissue. Using an enhancer-trap line (29D), we further traced the fat-cell lineage to nine bilateral clusters of cells within the mesoderm at germ-band extension. We suggest these 29D-positive cells represent the progenitor fat cells. In stage-12 embryos, the 29D-positive cell clusters can be identified within the mesoderm internal to nautilus-expressing cells. These data suggest that the precursor fat cells may be derived from the inner mesoderm, or spanchnopleura. Embryos deficient for the DNA region surrounding the site of the 29D enhancer trap lack most, if not all, of the cells in the fat-cell lineage. These embryos exhibit the loss of svp-positive precursor fat cells and concomitant loss of fat-body-specific expression of Adh and DCg1.(ABSTRACT TRUNCATED AT 400 WORDS)

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The complement system is now a therapeutic target for the management of serious and life-threatening conditions such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, glomerulonephritis and other diseases caused by complement deficiencies or genetic variants. As complement therapeutics expand into more clinical conditions, monitoring complement activation is increasingly important, as is the baseline levels of complement activation fragments in blood or other body fluid levels. Although baseline complement levels have been reported in the literature, the majority of these data were generated using non-standard assays and with variable sample handling, potentially skewing results. In this study, we examined the plasma and serum levels of the soluble membrane attack complex of complement (sMAC). sMAC is formed in the fluid phase when complement is activated through the terminal pathway. It binds the regulatory proteins vitronectin and/or clusterin and cannot insert into cell membranes, and can serve as a soluble diagnostic marker in infectious disease settings, as previously shown for intraventricular shunt infections. Here we show that in healthy adults, serum sMAC levels were significantly higher than those in plasma, that plasma sMAC levels were similar between in African Americans and Caucasians and that plasma sMAC levels increase with age. Plasma sMAC levels were significantly higher in virally suppressed people living with HIV (PLWH) compared to non-HIV infected healthy donors. More specifically, PLWH with CD4 + T cell counts below 200 had even greater sMAC levels, suggesting diagnostic value in monitoring sMAC levels in this group.

FCRLA is homologous to receptors for the Fc portion of IgG (FcγR) and is located in the same region of human chromosome one, but has several unusual and unique features. It is a soluble resident ER protein retained in this organelle by unknown mechanisms involving the N-terminal domain, a disordered domain with three Cys residues in close proximity in the human protein. Unlike the FcγRs, FCRLA is not glycosylated and has no transmembrane region. FCRLA is included in this CTMI volume on IgM-binding proteins because it binds IgM in the ER, but quite surprisingly, given the isotype-restricted ligand specificity of the other FcRs, it also binds all other Ig isotypes so far tested, IgG and IgA. In the case of IgM, there is even preferential binding of the secretory and not the transmembrane form. Among B cells, FCRLA is most highly expressed in the germinal center and shows little expression in plasma cells. Based on these observations, we propose that one human FCRLA function is to stop GC B cells from secreting IgM, which would act as a decoy receptor, thus preventing the B cells from capturing antigen, processing it, and presenting the antigen-derived peptides to T follicular helper cells. Without help from these T cells, there would be limited B cell isotype switching, proliferation, and differentiation. On the other hand, FCRLA is downregulated in plasma cells, where IgM secretion is an essential function. FCRLA may also act as a chaperone involved by unknown mechanisms in the proper assembly of Ig molecules of all isotypes.