Background/Purpose: Tofacitinib (tofa) inhibits cytokine signaling mediated by JAK1 JAK3 pathways leading therefore to a decrease in Th17 and an increase of Treg cells. The effect of tofa on B cell profile has been poorly explored. We aimed at studying the in vitro effect of tofa on B cell cytokine production (pro and anti-inflammatory) and plasmablast differentiation in the context of JAK-dependent and independent stimuli. The in vivo effect of tofa on B cell cytokine production was also assessed in mice.

Methods: Isolated B cells were cultured in vitro for 4 to 8 days in absence or presence of tofa and with JAK-dependent (CD40L, IL-2 and IL-21) or JAK-independent (CpG, a TLR9 ligand) stimuli. Cytokine production was assessed by flow cytometry and ELISA. Plasmablasts were defined as CD19⁺CD27⁺CD38⁺ cells. p-STAT-3 was quantified by western blot at 30,120 and 240 minutes. IL-6, IL-10 and IFN-γ neutralizing antibodies were added in some experiments. In vivo, mice were treated with tofa or placebo for 8 days and B cells were analyzed in inguinal lymph nodes and peritoneal fluid.

Results: In presence of JAK-dependent IL-2/IL-21 stimuli, tofa (1 µM) decreased the levels of IL-6 (2.7%[1.0-7.6] vs 8.9%[7.1-18.1], n=7, p=0.01 for tofa and control respectively), IFN-γ, IL-10 (0.8%[0.5-1.4] vs 2.6%[0.7-3.3], p=0.04), IL-35 (Mean Fluorescence Intensity [MFI] =89.5[56.1-99.6] vs 163[125-239], p=0.01) produced by B cells.

In presence of the JAK-independent stimulation CpG, tofa also decreased IL-6 (30.2%[20.4-48.2] vs 35.5%[27.3-59.0], n=9, p=0.05), IFN-γ, IL-10 (1.9%[0.6-2.5] vs 2.5%[0.9-5.1], p=0.02) and IL-35 (MFI: 129.4[85.2-354.6] vs 177.7[132.5-566.8], p=0.001) in B cells. Whereas IL-2/IL-21 induced a phosphorylation of STAT3 at 30 min, CpG induced a delayed activation of JAK/STAT pathway, suggesting that cytokines produced in response to CpG have an autocrine effect with a secondary activation of JAK/STAT pathway which can be blocked by tofa. To confirm this hypothesis, we cultured CpG activated B cells in the presence of cytokine-neutralizing antibodies and found that anti-IL-10 antibodies decreased the effect of CpG on IL-6 and IL-10 secretion and that tofa had then a decreased effect on those cytokines. In addition, tofa inhibited the plasmablasts differentiation in IL-2/IL-21 conditions (0.09%[0.05-0.17] vs 2.4%[1.4-3.3], n=6, p=0.03), but not with CpG (3.27%[0.57-4.97] vs 3.94%[0.73-8.41], p=0.15). In vivo, tofa decreased the frequency of IL-35 secreting B cells (0.8%[0.42-0.97] vs 1.6%[0.7-2.4, p=0.05] in peritoneal fluid but not IL-10 and TGF-β.

Conclusion: In addition to blocking cytokine pathways, tofa had a cellular impact on B cells, modifying their ability to produce pro- and anti-inflammatory cytokines both in JAK dependent and independent conditions. In JAK independent conditions, this effect of tofa was at least partly explained by the inhibition of an autocrine loop. Moreover, tofa decreased the differentiation of B cells into plasmablasts under conditions mimicking an interaction with T cells but not with TLR activation only, suggesting that tofa might have a lower impact on T-independent humoral immunity. We are currently evaluating the impact of tofa on B cells in tofa-treated rheumatoid arthritis patients.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/does-tofacitinib-impact-b-cell-functions/