Chemokine Receptor Activation Enhances Memory B Cell Class Switching Linked to IgE Sensitization to Alpha Gal and Cardiovascular Disease

Background: Recent studies have suggested that IgE sensitization to α-gal is associated with coronary artery disease (CAD). However, the B cell subtype(s) responsible for production of IgE to α-gal and mechanisms mediating this production remain elusive.Methods: Single cell multi-omics sequencing, was utilized to phenotype B cells obtained from 60 subjects that had undergone coronary angiography in whom serum IgE was evaluated by ImmunoCAP. Bioinformatics approaches were used to identify B cell subtype(s) and transcriptomic signatures associated with α-gal sensitization. In vitro characterization of chemokine/chemokine receptor pairs on switched memory B cells associated with IgE to α-gal was performed.Results: Of the 60 patients, 17 (28%) were positive for IgE to α-gal. CITESeq identified CCR6+ class-switched memory (SWM) B cells and CXCR4 expresssion on these CCR6+ SWM B cells as significantly associated with IgE sensitization to α-gal but not to other common allergens (peanut or inhalants). In vitro studies of enriched human B cells revealed significantly greater IgE on SWM B cells with high CCR6 and CXCR4 expression 10 days after cells were treated with IL-4 and CD40 to stimulate class switch recombination. Both CCL20 (CCR6 ligand) and CXCL12 (ligand for CXCR4) increased the expression of IgE on SWM B cells expressing their receptors. However, they appeared to have unique pathways mediating this effect as only CCL20 increased activation-induced cytidine deaminase (AID), while CXCL12 drove proliferation of CXCR4+ SWM B cells. Lastly, correlation analysis indicated an association between CAD severity and the frequency of both CCR6+ SWM and CXCR4+ SWM B cells.Conclusions: CCR6+ SWM B cells were identified as potential producers of IgE to α-gal in CAD patients. Additionally, our findings highlighted non-chemotaxis roles of CCL20/CCR6 and CXCL12/CXCR4 signaling in mediating IgE class switching and cell proliferation of SWM B cells respectively. Results may have important implications for a better understanding and better therapeutic approaches for subjects with IgE sensitization to α-gal.

Tumor-B-cell Interactions Promote Isotype Switching to an Immunosuppressive IgG4 Antibody Response Through Upregulation of IL-10 in Triple Negative Breast Cancers

BackgroundTriple negative breast cancer (TNBC) is an aggressive breast cancer for which there is currently no targeted therapy. Tumor-infiltrating B-cells (TIB) have been observed in tumor tissues of TNBC patients, but their functional role is unclear. IgG4 is one of four antibody subclasses of IgG expressed and secreted by B cells. Unlike other IgG isotypes, IgG4 has an immunosuppressive function and is induced by Th2-type cytokines. In cancers such as melanoma, IgG4 has been linked with advanced disease and poor patient survival. Therefore, we sought to determine the role of IgG4 in TNBC. MethodsWe performed co-culture assays to examine expression of Th2 cytokines by TNBC cells with and without the presence of B cells. We also performed in vitro class switching experiments with peripheral B cells with and without co-culture with TNBC cells in the presence or absence of an IL-10 blocking antibody. We examined expression of CD20 + TIB, IgG4 and Th2 cytokines by immunohistochemistry in 152 TNBC samples. Statistical analysis was done using Log-Rank and Cox-proportional hazards tests. ResultsOur findings indicate that B cells interact with TNBC to drive chronic inflammatory responses through increased expression of inflammatory cytokines including the TH2 cytokines IL-4 and IL-10. In vitro class switching studies show that interactions between TNBC cell lines and B cells drive isotype switching to the IgG4 isotype in an IL-10 dependent manner. In patient tissues, expression of IgG4 correlates with CD20 and tumor expression of IL-10. Both IgG4 and tumor IL-10 are associated to shorter recurrence free survival (RFS) and overall survival (OS) in TNBC. In a multi-variant analysis, IL-10 was associated with poor outcomes indicating that tumor IL-10 may drive immune escape. ConclusionsThese findings indicate that interactions between TIB and TNBC results in activation of chronic inflammatory signals that suppress antibody driven immune responses

Multiple DSB Resection Activities Redundantly Promote Alternative End Joining-Mediated Class Switch Recombination

Alternative end joining (A-EJ) catalyzes substantial level of antibody class switch recombination (CSR) in B cells deficient for classical non-homologous end joining, featuring increased switch (S) region DSB resection and junctional microhomology (MH). While resection has been suggested to initiate A-EJ in model DSB repair systems using engineered endonucleases, the contribution of resection factors to A-EJ-mediated CSR remains unclear. In this study, we systematically dissected the requirement for individual DSB resection factors in A-EJ-mediated class switching with a cell-based assay system and high-throughput sequencing. We show that while CtIP and Mre11 both are mildly required for CSR in WT cells, they play more critical roles in mediating A-EJ CSR, which depend on the exonuclease activity of Mre11. While DNA2 and the helicase/HRDC domain of BLM are required for A-EJ by mediating long S region DSB resection, in contrast, Exo1’s resection-related function does not play any obvious roles for class switching in either c-NHEJ or A-EJ cells, or mediated in an AID-independent manner by joining of Cas9 breaks. Furthermore, ATM and its kinase activity functions at least in part independent of CtIP/Mre11 to mediate A-EJ switching in Lig4-deficient cells. In stark contrast to Lig4 deficiency, 53BP1-deficient cells do not depend on ATM/Mre11/CtIP for residual joining. We discuss the roles for each resection factor in A-EJ-mediated CSR and suggest that the extent of requirements for resection is context dependent.

IgM-MM is predominantly a pre–germinal center disorder and has a distinct genomic and transcriptomic signature from WM

Immunoglobulin M (IgM) multiple myeloma (MM) is a rare disease subgroup. Its differentiation from other IgM-producing gammopathies such as Waldenström macroglobulinemia (WM) has not been well characterized but is essential for proper risk assessment and treatment. In this study, we investigated genomic and transcriptomic characteristics of IgM-MM samples using whole-genome and transcriptome sequencing to identify differentiating characteristics from non–IgM-MM and WM. Our results suggest that IgM-MM shares most of its defining structural variants and gene-expression profiling with MM, but has some key characteristics, including t(11;14) translocation, chromosome 6 and 13 deletion as well as distinct molecular and transcription-factor signatures. Furthermore, IgM-MM translocations were predominantly characterized by VHDHJH recombination-induced breakpoints, as opposed to the usual class-switching region breakpoints; coupled with its lack of class switching, these data favor a pre–germinal center origin. Finally, we found elevated expression of clinically relevant targets, including CD20 and Bruton tyrosine kinase, as well as high BCL2/BCL2L1 ratio in IgM-MM, providing potential for targeted therapeutics.

Longitudinal Dynamics of Human B-Cell Response at the Single-Cell Level in Response to Tdap Vaccination

To mount an adequate immune response against pathogens, stepwise mutation and selection processes are crucial functions of the adaptive immune system. To better characterize a successful vaccination response, we performed longitudinal (days 0, 5, 7, 10, and 14 after Boostrix vaccination) analysis of the single-cell transcriptome as well as the B-cell receptor (BCR) repertoire (scBCR-rep) in plasma cells of an immunized donor and compared it with baseline B-cell characteristics as well as flow cytometry findings. Based on the flow cytometry knowledge and literature findings, we discriminated individual B-cell subsets in the transcriptomics data and traced over-time maturation of plasmablasts/plasma cells (PB/PCs) and identified the pathways associated with the plasma cell maturation. We observed that the repertoire in PB/PCs differed from the baseline B-cell repertoire e.g., regarding expansion of unique clones in post-vaccination visits, high usage of IGHG1 in expanded clones, increased class-switching events post-vaccination represented by clonotypes spanning multiple IGHC classes and positive selection of CDR3 sequences over time. Importantly, the Variable gene family-based clustering of BCRs represented a similar measure as the gene-based clustering, but certainly improved the clustering of BCRs, as BCRs from duplicated Variable gene families could be clustered together. Finally, we developed a query tool to dissect the immune response to the components of the Boostrix vaccine. Using this tool, we could identify the BCRs related to anti-tetanus and anti-pertussis toxoid BCRs. Collectively, we developed a bioinformatic workflow which allows description of the key features of an ongoing (longitudinal) immune response, such as activation of PB/PCs, Ig class switching, somatic hypermutation, and clonal expansion, all of which are hallmarks of antigen exposure, followed by mutation & selection processes.

Class-Switching of B Lymphocytes by DNA and Cell Immunization for Stereospecific Monoclonal Antibodies against Native GPCR

To develop efficient applications of monoclonal antibodies for therapeutic purposes, stereospecific recognition of the target antigens is needed. DNA immunization is one of the best methods for sensitizing B lymphocytes that can produce conformation-specific antibodies. Here we verified the class-switching of monoclonal antibodies by DNA immunization followed by cell immunization for the generation of stereospecific monoclonal antibodies against native G protein-coupled receptor (GPCR) using the optimized stereospecific targeting (SST) technique. This technology facilitates the efficient selection of sensitized B lymphocytes through specific interaction with the intact antigen via B-cell receptors (BCRs). We demonstrate that multiple DNA immunizations followed by a single cell immunization in combination with a longer sensitization period (three to four months) are an appropriate sensitizing strategy for the generation of IgG-type stereospecific monoclonal antibodies by class-switching, and the characteristics of antibody production could be transferred to hybridoma cells provided by the optimized SST technique.