Functional, Non-Clonal IgMa-Restricted B Cell Receptor Interactions with the HIV-1 Envelope gp41 Membrane Proximal External Region

The membrane proximal external region (MPER) of HIV-1 gp41 has several features that make it an attractive antibody-based vaccine target, but eliciting an effective gp41 MPER-specific protective antibody response remains elusive. One fundamental issue is whether the failure to make gp41 MPER-specific broadly neutralizing antibodies like 2F5 and 4E10 is due to structural constraints with the gp41 MPER, or alternatively, if gp41 MPER epitope-specific B cells are lost to immunological tolerance. An equally important question is how B cells interact with, and respond to, the gp41 MPER epitope, including whether they engage this epitope in a non-canonical manner i.e., by non-paratopic recognition via B cell receptors (BCR). To begin understanding how B cells engage the gp41 MPER, we characterized B cell-gp41 MPER interactions in BALB/c and C57BL/6 mice. Surprisingly, we found that a significant (∼7%) fraction of splenic B cells from BALB/c, but not C57BL/6 mice, bound the gp41 MPER via their BCRs. This strain-specific binding was concentrated in IgMhi subsets, including marginal zone and peritoneal B1 B cells, and correlated with enriched fractions (∼15%) of gp41 MPER-specific IgM secreted by in vitro-activated splenic B cells. Analysis of Igha (BALB/c) and Ighb (C57BL/6) congenic mice demonstrated that gp41 MPER binding was controlled by determinants of the Igha locus. Mapping of MPER gp41 interactions with IgMa identified MPER residues distinct from those to which mAb 2F5 binds and demonstrated the requirement of Fc CH regions. Importantly, gp41 MPER ligation produced detectable BCR-proximal signaling events, suggesting that interactions between gp41 MPER and IgMa determinants may elicit partial B cell activation. These data suggest that low avidity, non-paratopic interactions between the gp41 MPER and membrane Ig on naïve B cells may interfere with or divert bnAb responses

Developmental failure of chimeric embryos expressing high levels of H-2Dd transplantation antigens.

The absence of expression of class I products of the major histocompatibility complex at early stages of development is thought to play a key role in maternal tolerance of the fetal allograft. To test this, we developed a strategy that would allow us to describe the consequences of overexpression of the H-2Dd transplantation antigen in the developing embryo. A construct containing the H-2Dd gene under control of the human beta-actin promoter was transfected into pluripotent embryonic stem (ES) cells. Particularly in this case, since overexpression of major histocompatibility complex class I gene products may profoundly affect embryonic development, an important advantage of the ES cell system is the ability to analyze gene expression and study effects on cell growth and differentiation in vitro. ES cells do not constitutively express beta 2-microglobulin. Consistent with this, H-2Dd H chains expressed by ES cell transformants were not associated with beta 2-microglobulin or transported to the cell surface. Significant levels of beta 2-microglobulin and H-2Dd membrane glycoproteins were expressed following differentiation in vitro. H-2Dd-transfected ES cells gave rise to a wide range of differentiated cell types, and there was no evidence to suggest that expression of the introduced H-2Dd gene affects the differentiation abilities of ES cells in vitro. When introduced into blastocysts, H-2Dd-transfected ES cells extensively contribute to embryonic and extraembryonic tissues, but this results in the failure of chimeric conceptuses at midgestation. Considering that transgenic chimeras cannot be rescued by transfer into syngeneic foster females, it seems likely that nonimmunological mechanisms are responsible for these prenatal lethalities

The Ii41 isoform of invariant chain mediates both positive and negative selection events in T-cell receptor transgenic mice.

The functional role of invariant chain in T-cell selection events and antigen presentation is well established. The invariant chain gene encodes differentially spliced isoforms, Ii31 and Ii41. The Ii41 isoform has been described to increase the efficiency of antigen presentation. We have analysed the effect of the Ii41 isoform on positive and negative selection of transgenic CD4 T cells with specificity for a natural self antigen (C5) which are crucially dependent on invariant chain for their development and functional antigen recognition. The data show that Ii41 fully substitutes for wild-type invariant chain in both positive and negative selection events during functional maturation of T cells with specificity for a natural, blood-borne self antigen

Association with BiP and aggregation of class II MHC molecules synthesized in the absence of invariant chain.

Class II molecules of the major histocompatibility complex (MHC) are composed of two polymorphic glycoprotein chains (alpha and beta), that associate in the ER with a third, non-polymorphic glycoprotein known as the invariant chain (Ii). We have examined the relationship between the intracellular transport and physico-chemical characteristics of various combinations of murine alpha, beta and Ii chains. Biochemical and morphological analyses of transfected fibroblasts expressing class II MHC chains show that both unassembled alpha and beta chains, as well as a large fraction of alpha+beta complexes synthesized in the absence of Ii chain, are retained in the ER in association with the immunoglobulin heavy chain binding protein, BiP. Analyses by sedimentation velocity on sucrose gradients show that most incompletely assembled class II MHC species exist as high molecular weight aggregates in both transfected fibroblasts and spleen cells from mice carrying a disruption of the Ii chain gene. This is in contrast to the sedimentation properties of alpha beta Ii complexes from normal mice, which migrate as discrete, stoichiometric complexes of M(r) approximately 200,000-300,000. These observations suggest that assembly with the Ii chain prevents accumulation of aggregated alpha and beta chains in the ER, which might relate to the known ability of the Ii chain to promote exit of class II MHC molecules from the ER

The PR/SET domain zinc finger protein Prdm4 regulates gene expression in embryonic stem cells but plays a nonessential role in the developing mouse embryo

Prdm4 is a highly conserved member of the Prdm family of PR/SET domain zinc finger proteins. Many well-studied Prdm family members play critical roles in development and display striking loss-of-function phenotypes. Prdm4 functional contributions have yet to be characterized. Here, we describe its widespread expression in the early embryo and adult tissues. We demonstrate that DNA binding is exclusively mediated by the Prdm4 zinc finger domain, and we characterize its tripartite consensus sequence via SELEX (systematic evolution of ligands by exponential enrichment) and ChIP-seq (chromatin immunoprecipitation-sequencing) experiments. In embryonic stem cells (ESCs), Prdm4 regulates key pluripotency and differentiation pathways. Two independent strategies, namely, targeted deletion of the zinc finger domain and generation of a EUCOMM LacZ reporter allele, resulted in functional null alleles. However, homozygous mutant embryos develop normally and adults are healthy and fertile. Collectively, these results strongly suggest that Prdm4 functions redundantly with other transcriptional partners to cooperatively regulate gene expression in the embryo and adult animal