The study was approved by the local medical ethical committee of the Amsterdam University Medical Centers, location AMC (MEC_AMC_NL65076.018.15), and location VUmc (MEC_VUmc_ NL17200.029.07). All patients and healthy controls provided informed consent for study participation and publication of the related results. The study was performed according to the principles of the Declaration of Helsinki.

For this cross-sectional study, we recruited 14 SLE patients, 12 RA patients, and 13 healthy controls who visited the Amsterdam UMC in Amsterdam, the Netherlands. All SLE patients fulfilled the American College of Rheumatology (ACR) 1997 classification criteria and all RA patients met the ACR 2010 classification criteria [17, 19]. Healthy controls were recruited at the Amsterdam UMC or obtained from Sanquin Blood Supply, Amsterdam, the Netherlands. Exclusion criteria covered patients with concomitant malignancies, other inflammatory diseases, or use of a biological or targeted synthetic disease-modifying anti-rheumatic drugs (DMARD) within the previous year (including anti-CD20 therapy). Sex, age, disease duration, medication use, disease activity, and clinical and laboratory parameters were annotated. As disease activity parameters, we used the SLE Disease Activity Index 2000 (SLEDAI-2k) [20] or Disease Activity Score 28-ESR (DAS28-ESR) [21]. Active SLE required a SLEDAI-2k score ≥ 6 with a clinical SLEDAI-2k (excluding laboratory results) score ≥ 4. Active RA required a DAS28-ESR score ≥ 2.6. Characteristics of the study participants are reported in Tables 1 and 2.

Peripheral blood mononuclear cells (PBMCs) were isolated from total blood using Ficoll/Lymphoprep separation (GE Healthcare) and cryopreserved in liquid nitrogen until further use.

PBMCs were analyzed either directly ex vivo (=unstimulated), or stimulated using 1 µg/ml ODN 2006 (CpG) for B cell stimulation, or CD3 (clone 1XE) and CD28 (clone 15E8) soluble antibodies for T cell stimulation for 6 days. Cell proliferation was measured using a CellTrace violet cell proliferation kit (ThermoFisher Scientific, Waltham, Massachusetts, USA). Proliferation and division index were calculated using the proliferation platform in FlowJo v10 software (BD Biosciences, San Jose, CA, USA). PBMCs were incubated with different Bcl-2 family inhibitors for 24 h. Cell viability was measured by flow cytometry using DiOC6 and TO-PRO-3 viability dyes.

ODN2006 CpG oligonucleotide type B was purchased from InvivoGen (San Diego, CA, USA), venetoclax was purchased from Active Biochem (Bonn, Germany), S-63845 was purchased from Chemgood (Glen Allen, VA, USA), and AZD4320 was provided by Acerta Pharma (Oss, The Netherlands).

PBMCs were stained with the antibodies specific for CD4 (Biolegend, San Diego, CA, USA), CD8 (Biolegend, San Diego, CA, USA), CD19 (BD Biosciences, San Jose, CA, USA), CD27 (BD Biosciences, San Jose, CA, USA), CD38 (BD Biosciences, San Jose, USA), CD45RA (BD Biosciences, San Jose, CA, USA), and IgD (Biolegend, San Diego, CA, USA). Cells were subsequently permeabilized and stained with antibodies targeting Bcl-2 (Biolegend San Diego, CA, USA), Bcl-XL (Cell Signaling Technology, Danvers, Massachusetts, USA), and Mcl-1 (Cell Signaling Technology, Danvers, Massachusetts, USA). PBMCs were measured on an LSR Fortessa (BD Biosciences, San Jose, USA) and analyzed using FlowJo v10.8. Gating strategy of flow cytometry analyses of B and T cell subsets are represented in Supplementary Fig. 1.

Statistical analyses were performed using Graphpad Prism 9 (Graph Pad, San Diego, CA, USA). T-tests and two-way analysis of variance (ANOVA) were used to analyze differences between groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.