Editors and Editing of Anti-DNA Receptors

AbstractReceptor editing is a means by which immature bone marrow B cells can become self-tolerant. Rearrangements of heavy (H) and/or light (L) chain genes are induced by encounter with autoantigens to change the specificity from self to nonself. We have developed site-directed transgenic mice (sd-tg) whose transgenes code for the H chain of antibodies that bind DNA. B cells that express the transgenic H chain associate mainly with four of the 93 functional Vκ genes of the mouse. Numerous aspartate residues that might inhibit DNA binding by the VH domain distinguish these L chain Vκ sequences, but engaging these Vκ editors often requires multiple rearrangements. Among the edited B cells is a subset of multispecific cells that express multiple receptors. One consequence of multispecificity is partial autoreactivity; these multispecific B cells may contribute to autoimmunity

The Future of Blood Testing Is the Immunome

It is increasingly clear that an extraordinarily diverse range of clinically important conditions-including infections, vaccinations, autoimmune diseases, transplants, transfusion reactions, aging, and cancers-leave telltale signatures in the millions of V(D)J-rearranged antibody and T cell receptor [TR per the Human Genome Organization (HUGO) nomenclature but more commonly known as TCR] genes collectively expressed by a person's B cells (antibodies) and T cells. We refer to these as the immunome. Because of its diversity and complexity, the immunome provides singular opportunities for advancing personalized medicine by serving as the substrate for a highly multiplexed, near-universal blood test. Here we discuss some of these opportunities, the current state of immunome-based diagnostics, and highlight some of the challenges involved. We conclude with a call to clinicians, researchers, and others to join efforts with the Adaptive Immune Receptor Repertoire Community (AIRR-C) to realize the diagnostic potential of the immunome

DNA Damage and L1 Retrotransposition

Barbara McClintock was the first to suggest that transposons are a source of genome instability and that genotoxic stress assisted in their mobilization. The generation of double-stranded DNA breaks (DSBs) is a severe form of genotoxic stress that threatens the integrity of the genome, activates cell cycle checkpoints, and, in some cases, causes cell death. Applying McClintock's stress hypothesis to humans, are L1 retrotransposons, the most active autonomous mobile elements in the modern day human genome, mobilized by DSBs? Here, evidence that transposable elements, particularly retrotransposons, are mobilized by genotoxic stress is reviewed. In the setting of DSB formation, L1 mobility may be affected by changes in the substrate for L1 integration, the DNA repair machinery, or the L1 element itself. The review concludes with a discussion of the potential consequences of L1 mobilization in the setting of genotoxic stress

Regulation of Anti-Phosphatidylserine Antibodies

AbstractThe degree of heavy chain (H) editing, the types of Vκ editors, and the pattern of Jκ usage are correlated with a range of the affinity of anti-DNA. This range was determined by the number and location of arginine (R) residues in the VH. We, here, changed a key arginine residue in the VH of anti-DNA transgene to glycine, which sharply reduces the affinity for dsDNA. However, complete reversion of this anti-DNA to germline enhances the affinity for phosphatidylserine (PS). The B cells of this low-affinity anti-DNA and anti-PS transgenic mouse are tightly regulated by receptor editing. Thus, anti-PS B cells are another example of a constitutive self-antigen regulated in the bone marrow

Gamma radiation increases endonuclease-dependent L1 retrotransposition in a cultured cell assay

Long Interspersed Elements (LINE-1s, L1s) are the most active mobile elements in the human genome and account for a significant fraction of its mass. The propagation of L1 in the human genome requires disruption and repair of DNA at the site of integration. As Barbara McClintock first hypothesized, genotoxic stress may contribute to the mobilization of transposable elements, and conversely, element mobility may contribute to genotoxic stress. We tested the ability of genotoxic agents to increase L1 retrotransposition in a cultured cell assay. We observed that cells exposed to gamma radiation exhibited increased levels of L1 retrotransposition. The L1 retrotransposition frequency was proportional to the number of phosphorylated H2AX foci, an indicator of genotoxic stress. To explore the role of the L1 endonuclease in this context, endonuclease-deficient tagged L1 constructs were produced and tested for their activity in irradiated cells. The activity of the endonuclease-deficient L1 was very low in irradiated cells, suggesting that most L1 insertions in irradiated cells still use the L1 endonuclease. Consistent with this interpretation, DNA sequences that flank L1 insertions in irradiated cells harbored target site duplications. These results suggest that increased L1 retrotransposition in irradiated cells is endonuclease dependent. The mobilization of L1 in irradiated cells potentially contributes to genomic instability and could be a driving force for secondary mutations in patients undergoing radiation therapy

RS rearrangement frequency as a marker of receptor editing in lupus and type 1 diabetes

Continued antibody gene rearrangement, termed receptor editing, is an important mechanism of central B cell tolerance that may be defective in some autoimmune individuals. We describe a quantitative assay for recombining sequence (RS) rearrangement that we use to estimate levels of antibody light chain receptor editing in various B cell populations. RS rearrangement is a recombination of a noncoding gene segment in the κ antibody light chain locus. RS rearrangement levels are highest in the most highly edited B cells, and are inappropriately low in autoimmune mouse models of systemic lupus erythematosus (SLE) and type 1 diabetes (T1D), including those without overt disease. Low RS rearrangement levels are also observed in human subjects with SLE or T1D

Lenalidomide Therapy in Treatment Refractory Cutaneous Lupus Erythematosus: Histologic and Circulating Leukocyte Profile and Potential Risk of a Systemic Lupus Flare

Background Lenalidomide is a thalidomide analogue that may serve as an adjunctive therapy for treatment refractory cutaneous lupus erythematosus (CLE). Objectives We evaluate the use of lenalidomide in CLE and describe the skin and circulating leukocyte profile of treatment refractory patients before and after treatment. Patients/Methods Five subjects were treated with lenalidomide in an unblinded open-label study. Immunohistochemistry of skin was performed for T-cell markers, glycosaminoglycans and CXCL10, an interferon (IFN)-inducible chemokine, before and after treatment. Immunophenotyping and measurement of IFN-inducible genes from peripheral blood mononuclear cells was also performed before and after treatment. Results Four subjects demonstrated clinical improvement of their skin, however one of these responders subsequently developed symptoms of systemic lupus erythematosus. Small changes in rare circulating leukocyte subsets, plasmacytoid dendritic cells and regulatory T-cells, were observed with treatment and may correlate with clinical response. Treatment was associated with increased circulating HLA-DR expression and decreased markers of IFN-mediated pathways, regardless of clinical response. Limitations Our results are limited by small sample size and the measurement of rare populations of circulating cell subsets. Conclusions Lenalidomide may have utility as therapy for severe, treatment refractory CLE. However, our preliminary data suggest that lenalidomide may activate T-cells and trigger systemic disease in some CLE patients. We also saw a unique histologic and circulating leukocyte phenotype in the nonresponding subject. Further characterization of the skin and circulating leukocyte profile of treatment refractory patients will improve our understanding of CLE

ImmuneDB, a Novel Tool for the Analysis, Storage, and Dissemination of Immune Repertoire Sequencing Data

ImmuneDB is a system for storing and analyzing high-throughput immune receptor sequencing data. Unlike most existing tools, which utilize flat-files, ImmuneDB stores data in a well-structured MySQL database, enabling efficient data queries. It can take raw sequencing data as input and annotate receptor gene usage, infer clonotypes, aggregate results, and run common downstream analyses such as calculating selection pressure and constructing clonal lineages. Alternatively, pre-annotated data can be imported and analyzed data can be exported in a variety of common Adaptive Immune Receptor Repertoire (AIRR) file formats. To validate ImmuneDB, we compare its results to those of another pipeline, MiXCR. We show that the biological conclusions drawn would be similar with either tool, while ImmuneDB provides the additional benefits of integrating other common tools and storing data in a database. ImmuneDB is freely available on GitHub at https://github.com/arosenfeld/immunedb, on PyPi at https://pypi.org/project/ImmuneDB, and a Docker container is provided at https://hub.docker.com/r/arosenfeld/immunedb. Full documentation is available at http://immunedb.com