An immunoglobulin Cκ-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system

Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly normal immune system. Here we demonstrate an advance in in vivo analysis of immune tolerance that overcomes these shortcomings. We show that custom superantigens generated by single chain antibody technology permit the study of tolerance in a normal, polyclonal immune system. In the present study we generated a membrane-tethered anti-Igκ–reactive single chain antibody chimeric gene and expressed it as a transgene in mice. B cell tolerance was directly characterized in the transgenic mice and in radiation bone marrow chimeras in which ligand-bearing mice served as recipients of nontransgenic cells. We find that the ubiquitously expressed, Igκ-reactive ligand induces efficient B cell tolerance primarily or exclusively by receptor editing. We also demonstrate the unique advantages of our model in the genetic and cellular analysis of immune tolerance

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

Regulation studies of the human recombination activating genes, RAG-1 and RAG-2

grantor: University of TorontoIt has been suggested that a signal transduced through the B-cell antigen receptor complex (BCR) may be required for regulating the expression of the recombination activating genes. To study this possibility, we utilized a unique set of cell variants from the human mature B cell line OCI LY8 which vary in expression of both the BCR and of the RAG-1 and RAG-2 genes. Two forms of stimulation were employed to generate a signal: either a soluble F(ab) '2 anti-[mu] fragment or the combination of PMA and ionomycin. The finding that RAG expression can be upregulated by BCR ligation may be relevant in the context of its potential role in situations where BCR + B cells may undergo secondary rearrangements for the purpose of "editing" their sIg receptors. To better understand the molecular mechanisms that may be involved in this inducible upregulation as well as in the constitutively expressed increases in RAG gene expression in RAGhi OCI LY8 variants (as compared to the parental RAGlo clone), we determined to what extent various levels of regulation were contributing to these effects. (Abstract shortened by UMI.)M.Sc

Molecular cloning and characterization of hBRAG and other genes as potential regulators of RAG1 in the B cell lineage

grantor: University of TorontoThe RAG genes are critical in lymphocyte development and in antigen receptor rearrangement. Finding other factors associated with RAG in expression will allow insight into the B cell developmental program and possibly into the regulation of lymphoid-specific components of the V(D)J recombination reaction. The goal of my study was to isolate and characterize novel genes by virtue of their co-expression with RAG1 and RAG2 mRNAs. Genes of this sort this would be of considerable general interest because they may potentially fall into one of the following three non-mutually exclusive categories: (1) genes involved in the regulation of human RAG1 and RAG2, (2) unidentified components of the recombinase machinery or, (3) other novel pre-lymphocyte specific genes (based on their temporally associated pattern of expression with the RAGs). In this thesis, I develop a differential display-based screening approach to identify and characterize genes that are co-expressed with human RAG1 in the context of the B cell lineage. Using this approach, I have identified several candidate genes, both known and novel, that co-express with and possibly regulate RAG1 in a B cell-specific context. I show that one of these is a novel, B cell-associated type II transmembrane glycoprotein that may be important in B cell signaling. Based on these studies, it may be possible to identify several factors that are not only important factors in RAG regulation, but also in B cell development.Ph.D

Immune System Regulation in the Induction of Broadly Neutralizing HIV-1 Antibodies

In this brief review, we discuss immune tolerance as a factor that determines the magnitude and quality of serum antibody responses to HIV-1 infection and vaccination in the context of recent work. We propose that many conserved, neutralizing epitopes of HIV-1 are weakly immunogenic because they mimic host antigens. In consequence, B cells that strongly bind these determinants are removed by the physiological process of immune tolerance. This structural mimicry may represent a significant impediment to designing protective HIV-1 vaccines, but we note that several vaccine strategies may be able to mitigate this evolutionary adaptation of HIV and other microbial pathogens

B-cell lineage-based approach to vaccine design.

<p>Mature bnAbs can be isolated from HIV-1–infected donors using modern methods such as memory B-cell culture or sorting of antigen-specific B-cells. Based on the known bnAb sequence, next generation sequencing can be used to find numerous clonal relatives of the mature bnAb. If appropriate longitudinal samples are available, it is possible to infer the full antibody lineage, including the UA and IAs. The expressed UA and IA sequences can then be used as templates for the design of HIV-1 immunogens with high-affinity binding. As the antibody lineage is known to evolve in response to viral evolution, it may be possible to design sequential immunogens with high-affinity binding for the UA and IA, thus guiding the antibody response toward the mature antibody with broad neutralizing activity (with permission from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004073#ppat.1004073-Mascola1" target="_blank">[4]</a>).</p