Tim-4 Effector B Cells Express a Th17-Like Proinflammatory Cytokine Module That Is Restrained by Nur77

Introduction/Rationale: B cells expressing various proinflammatory cytokines potentiate immune responses in infection, autoimmunity and transplantation. However, phenotypic markers for such effector B cells (Beffs) are lacking and regulation of their proinflammatory potential is uncharacterized. Tim-4+ B cells express IFNγ and promote islet allograft rejection. We now examine expression and regulation of other proinflammatory cytokines by Tim-4+ B cells.

Methods: RNAseq/ATACseq: +/- 24h stimulation with anti-IgM+IL-23. Cytokines: intracellular flow cytometry (IFC) of splenocytes stimulated 5h with PMA, ionomycin, BrefeldinA. Transplants: BALB/c islets under the kidney capsule of chemically diabetic B6 mice; heterotopic Bm12 hearts into B6 mice.

Results: Tim-4+ Beffs uniquely express RORgt, which along with IL-23, drive expression of a pathogenic Th17-like proinflammatory module comprising Il17a, Il17f, Il22, Csf2, Il6, and Il1b. RNAseq/ATACseq identified Nr4a1 (Nur77) as a potential TF regulating Tim-4+ B cells. Nur77floxCd19CRE+/- (Nur77 BKO) mice exhibited a 1.5X increase in Tim-4+ Beffs, and these had increased RORgt (2.5x), IL17(1.7x), IL22(2x), GM-CSF(1.5x), IL6(2x), & IL1b(2x) expression vs. Cre-controls (IFC). Notably, Tim1+ Breg frequency and IL-10 expression were decreased (30% & 60% respectively). Compared to Cre controls, Nur77 BKO mice exhibited shorter islet allograft survival (MST 14d vs 20d; p< 0.05) and accelerated rejection of BM12 hearts (MST 30d vs 65d; p< 0.01). In vitro data reveal tamoxifen-induced KO of B cell Nur77 acutely increases IL-17 expression, now enabling studies to determine how Nur77 regulates the Tim-4+ Beff transcriptome.

Conclusion: Tim-4 is a broad marker for Beffs that promote allograft rejection via expression of a pro-inflammatory module driven by IL-23 and RORgt. Nur77 constrains Tim-4+ Beffs and maintains a normal balance between Beffs and Bregs. This enhances our understanding of B cell biology and provides insight into immune regulation and tolerance.