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

Abstract

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.

Keywords: B cells; CITESeq; IgE class switching; alpha-gal; coronary artery disease.

Figure 1

Single cell multi-omics platform identified an association between circulating CCR6+ switched memory B cells and α-gal sensitization. (A) Representative metacluster UMAP showing 10 distinguished B cell subsets by using Louvain clustering. (B,C) Heatmap showing median expression of surface markers from metaclustering (B), table showing potential phenotypes of each B cell subset resulting from Louvain clustering (C). (D) Biaxial plots to compare frequency of each cluster as a percentage of total B cells in subjects with and without α-gal sensitization. *p < 0.05 by Mann-Whitney test.

Figure 2

CXCR4 signaling is enriched in CCR6+ SWM B cells of α-gal sensitized subjects. (A) Volcano plot of 488 gene array demonstrating DEGs of cluster 7 upregulated (blue) and downregulated (orange) in IgE α-gal + subjects. (B,C) Significant cellular processes (B) and canonical pathways (C) enriched in IgE α-gal + in cluster 7 cells. ns, non-significant.

Figure 3

CXCR4 surface expression increases in CCR6+ SWM B cells of α-gal sensitized subjects. Biaxial plots to compare geometric means (GM) of CXCR4 across 10 B cell clusters between subjects with and without α-gal sensitization. *p < 0.05 by Mann-Whitney test.

Figure 4

CCR6 and CXCR4 are important for immunoglobulin isotype switching. (A) Percentage of SWM of total B cells at day 0 before stimulation and at day 10 post with 20 ng/mL IL-4 and ± 10 ug/mL anti-CD40 stimulation. (B–D) Percentage of IgG+, IgE+, and CD138+ of CCR6- SWM or CCR6+ SWM at day 0 (B), day 3 (C), and day 10 (D) after culturing with 20 ng/mL IL-4 and 10 ug/mL anti-CD40. (E–G) Percentage of IgG+, IgE+, and CD138+ of CXCR4- SWM or CXCR4+ SWM at day 0 (E), day 3 (F), and day 10 (G) after with 20 ng/mL IL-4 and 10 ug/mL anti-CD40. *p < 0.05 by Mann-Whitney test.

Figure 5

CCL20/CCR6 intrinsic signaling augments IgE isotype switching through induction of AID expression. (A) Percentage of IgE+ of CCR6- SWM and CCR6+ SWM after 10 days culture with 20 ng/mL IL-4, 10 ug/mL anti-CD40 ± 20 ng/mL CCL20. (B) Percentage of IgG+ of CCR6- SWM and CCR6+ SWM after 10 days culture with 20 ng/mL IL-4, 10 ug/mL anti-CD40 ± 20 ng/mL CCL20. (C) Expression of AID measured by MFI on CCR6- SWM and CCR6+ SWM after 3 days culture with 20 ng/mL IL-4, 10 ug/mL anti-CD40 ± 20 ng/mL CCL20. (D) Percentage of cell proliferation measured by cell-trace violet of CCR6- SWM and CCR6+ SWM after 3 days culture with 20 ng/mL IL-4, 10 ug/mL anti-CD40 ± 20 ng/mL CCL20. *p < 0.05 by Mann-Whitney test.

Figure 6

CXCL12/CXCR4 intrinsic signaling drives proliferation of SWM B cells. (A) Percentage of IgE+ of CXCR4- SWM and CXCR4+ SWM after 10 days culture with 20 ng/mL IL-4, 10 ug/mL anti-CD40 ± 100 ng/mL CXCL12. (B) Percentage of IgG+ of CXCR4- SWM and CXCR4+ SWM after 10 days culture with 20 ng/mL IL-4, 10 ug/mL anti-CD40 ± 100 ng/mL CXCL12. (C) Expression of AID measured by MFI on CXCR4- SWM and CXCR4+ SWM after 3 days culture with 20 ng/mL IL-4, 10 ug/mL anti-CD40 ± 100 ng/mL CXCL12. (D) Percentage of cell proliferation measured by cell-trace violet of CXCR4- SWM and CXCR4+ SWM after 3 days culture with 20 ng/mL IL-4, 10 ug/mL anti-CD40 ± 100 ng/mL CXCL12. *p < 0.05 by Mann-Whitney test.

Figure 7

Chemokine receptor inhibitors reduce IgE isotype switching. (A) Percentage of IgE+ and IgG+ of CCR6+ SWM after 10 days culture with 20 ng/mL IL-4, 10 ug/mL anti-CD40 ± 20 ng/mL CCL20 ± 5uM CCR6i1. (B) Percentage of IgE+ and IgG+ of CXCR4+ SWM after 10 days culture with 20 ng/mL IL-4, 10 ug/mL anti-CD40 ± 100 ng/mL CXCL12 ± 5 uM AMD3100. *p < 0.05 by Mann-Whitney test.

Figure 8

CXCR4 and CCR6 expression on SWM B cells directly associates with CAD severity. (A,B) Linear regression between GM of CCR6 (A) and CXCR4 (B) on cluster 4 and 7 and CAD Gensini severity score. (C,D) Multivariate regression between GM of CCR6 (C) and CXCR4 (D) on cluster 4 and 7 and CAD Gensini severity score with age, sex, diabetes status, hypertension and statin use as covariates. *p < 0.05 by Mann-Whitney test.