A reservoir of rituximab-resistant splenic memory B cells contributes to relapses after B-cell depletion therapy

Abstract

Immune thrombocytopenia (ITP) is an autoimmune disease mediated by pathogenic antibodies directed against platelet antigens, including GPIIbIIIa. Taking advantage of spleen samples obtained from ITP patients, we characterized by multiples approaches the onset of disease relapses occurring after an initial complete response to rituximab. Analysis of splenic B cell immunoglobulin heavy chain gene repertoire at bulk level and from single anti-GPIIbIIIa B cells revealed that germinal centers were fueled by B cells originating from the ongoing lymphopoiesis, but also by rituximab-resistant memory B cells, both giving rise to anti-GPIIbIIIa plasma cells. We identified a population of splenic memory B cells that resisted rituximab through acquisition of a unique phenotype and contributed to relapses, providing a new target in B cell mediated autoimmune diseases.

Maturation and persistence of the anti-SARS-CoV-2 memory B cell response

“…Single cell culture was performed as previously described (Crickx et al, 2019). Single B cells were sorted in 96-well plates containing MS40 cells expressing CD40L (kind gift from G. Kelsoe).…”

Maturation and persistence of the anti-SARS-CoV-2 memory B cell response

“…Single cell culture was performed as previously described (Crickx et al, 2019): single B cells were sorted in 96-well plates containing MS40 cells expressing CD40L (kind gift from G. Kelsoe). Cells were co-cultured at 37 C with 5% CO2 during 21 or 25 days in RPMI-1640 (Invitrogen) supplemented with 10% HyClone FBS (Thermo Scientific), 55 mM 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, 100 units/mL penicillin, 100 mg/mL streptomycin, and MEM non-essential amino acids (all Invitrogen), with the addition of recombinant human BAFF (10 ng/mL), IL2 (50 ng/mL), IL4 (10 ng/mL), and IL21 (10 ng/mL; all Peprotech).…”

Analysis of mRNA vaccination-elicited RBD-specific memory B cells reveals strong but incomplete immune escape of the SARS-CoV-2 Omicron variant

“…All culture supernatants from single-cell cultured memory B cells used in this study were generated and reported as part as a previous study (see Sokal et al, 2021b). Briefly, single B cells were sorted in 96-well plates containing MS40L lo cells expressing CD40L (kind gift from G. Kelsoe, Crickx et al, 2021;Luo et al, 2009). Cells were co-cultured at 37 C with 5% CO2 during 21 or 25 days in RPMI-1640 (Invitrogen) supplemented with 10% HyClone FBS (Thermo Scientific), 55 mM 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, 100 units/mL penicillin, 100 mg/mL streptomycin, and MEM non-essential amino acids (all Invitrogen), with the addition of recombinant human BAFF (10 ng/ml), IL2 (50 ng/ml), IL4 (10 ng/ml), and IL21 (10 ng/ml; all Peprotech).…”