P179 CD19hi B cells express T-bet and display activated metabolism and function in rheumatoid arthritis and systemic lupus erythematosus patient samples

Abstract

Abstract Background/Aims CD19, a co-receptor of the B cell receptor, fine-tunes the threshold for B-cell activation with implications for cell metabolism and function. Activating receptors such as CD40, IFNγR and TLRs also influence B cell activation. B cell subpopulation, double negative (DN, IgD-CD27-) T-bet+ B cells, is considered pathogenic in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Here, we explored whether CD19hi B cells differed from CD19lo B cells with regards to proliferation, metabolism, and function. Methods Peripheral blood samples from nine healthy participants, 16 RA and 20 SLE patients were analysed. Subgroup analysis of rituximab-treated (RTX-T), n = 6 versus rituximab naïve (RTX-N), n = 6, was performed. B cells or peripheral blood mononuclear cells were stimulated with anti-IgG, anti-IgM and anti-human CD40 antibodies and a TLR9 agonist for 20 hours prior to flow cytometric analysis of B cell expression of phosphorylated AMPK (pAMPK, the master regulator of metabolism), intracellular expression of Ki67 (proliferation marker), and cytokines IL-6 and IL-10. Statistical analysis was performed using GraphPad Prism v10.0.1. Results Compared to CD19+CD20+ B cells, CD19+CD20- B cells: 1) were predominantly composed of DN (55% versus 19%, p < 0.01) and IgD-CD27+ memory subpopulations (37% versus 25%, p = 0.1094); 2) were detectable at a hierarchical frequency, RTX-T>RTX-N>HC cohorts; 3) had a greater frequency of Ki67+ cells (38% versus 5%, p < 0.01); 4) displayed higher CD38 MFI (41478.0 versus 10589.0, p < 0.01). Approximately 50% of CD19hi B cells were T-bet+, compared to 28% of CD19lo B cells (n = 33, p < 0.0001). Compared to CD19lo, CD19hi B cells 1) expressed (MFI) significantly greater TLR7, TLR9, IFNγR and pAMPK (n = 7 for all except pAMPK, n = 4), and 2) had greater frequency of IL6+ and IL10+ B cells (n = 7, p < 0.05) (Table). Conclusion CD20- B cell subpopulations with potent proliferative capacity express CD19. Further, CD19hi B cells: 1) upregulate the TLR-IFNγ-T-bet pathway; 2) have significantly greater expression of pAMPK; 3) have higher cytokine expression compared to CD19lo B cells. These data suggest the activation pathway, metabolic activity, and functional profile of CD19hi cells. Therefore, the potential of CD19hi cells to promote inflammation provides strong rationale for targeting CD19 in autoimmune disease with emerging therapies such as CAR-T cells and T cell engagers. Disclosure K. Shah: Grants/research support; KS has received funding for research from Roche. M.J. Leandro: Consultancies; MJL acted as a consultant for Roche and Genentech. Other; MJL received funds less than $10000 and support to attend educational conferences. M.S. Cragg: Consultancies; Consultant for BioInvent International, Boehringer Ingelheim, GSK, Radiant, iteos, Surrozen, Hanall and Mestag. Honoraria; He has received honoraria/speakers fees from Boehringer Ingelheim, Amgen, Roche and Symphogen. Grants/research support; BioInvent International, GSK and UCB. F. Kollert: Corporate appointments; FK is employed by Roche. Shareholder/stock ownership; FK has stock ownership at Roche. F. Schuler: Corporate appointments; FS is employed by Roche. Shareholder/stock ownership; FS has patents and stock ownership at Roche. D.A. Isenberg: None. A. Akbar: None. C. Klein: Corporate appointments; CK is employed by Roche. Shareholder/stock ownership; CK has patents and stock ownership at Roche. V. Reddy: Grants/research support; VR has received a research grant from Roche.