ANA-Reactive IgG Memory B Cells Differentiate and Proliferate Abnormally Via T-Dependent Stimulation in Patients with Systemic Lupus Erythematosus

Objectives Abnormal B cell activation and differentiation and production of pathogenic autoreactive antibodies play foundational roles in the pathogenesis of systemic lupus erythematosus (SLE).[1,2] We aimed to better understand whether IgG memory B cells in patients with SLE are “pre-activated” compared to those from healthy individuals using a T-dependent B cell activation cocktail and whether a difference exists between ANA+ antigen-experienced cells compared to their ANA− counterparts. Methods We isolated peripheral blood mononuclear cells from whole blood obtained from healthy individuals (n=6) and patients with SLE (n=7). We selected patients with SLE with clinically inactive disease, defined as a clinical SLEDAI of 0. Using a previously developed assay to identify autoreactive cells, we isolated ANA+ and ANA− IgG memory B cells using fluorescence-activated cell sorting and cultured them in vitro with CD40L and IL-21.[3] Plasmablast differentiation and immunoglobulin production were subsequently assessed using flow cytometry and IgG ELISA, respectively. Statistical analyses were performed using paired t-test with p<0.05 being significant. Results We observed statistically significant group differences in response to T-dependent stimulation with CD40L and IL-21 between ANA+ and ANA− memory B cells in healthy individuals and patients with SLE. ANA+ cells from healthy individuals had significantly less plasma cell differentiation compared to ANA− cells, but this difference was not seen in patients with SLE (p=0.0046 and p=0.7371, respectively) (Figure 1a). Comparable findings were seen in IgG immunoglobulin production between healthy individuals and patients with SLE (p=0.0231 and p=0.9961, respectively) (Figure 1b). Mean age of patients with SLE was 41.0 ± 14.6 years and mean disease duration was 13.1 ± 5.8 years. Mean SLEDAI was 1.86 ± 2.04, mean SDI was 0.29 ± 0.48, and mean PGA was 0.37 ± 0.46. 85.7% (6/7) of patients with SLE were female and 57.1% (4/7) of patients with SLE had serological activity. All patients with SLE were on hydroxychloroquine and 28.6% (2/7) of patients required either immunosuppressive medications or corticosteroids. All healthy controls were ANA negative by Hep-2 IFA testing. Conclusion We demonstrate a regulatory checkpoint in healthy individuals which inhibits differentiation of ANA+ IgG memory B cells into plasmablasts using a model of T-dependent stimulation. This checkpoint is not seen in patients with SLE. This abnormality may partially explain the production of deleterious antibodies in patients with SLE. References [1.] Nie Y. Clin Rev Allergy Immunol 2022;62:301-23. [2.] Tsokos GC. N Engl J Med 2011;365:2110-21. [3.] Malkiel S. Arthritis Rheumatol 2016;68:2210-20.