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B cells initiate and perpetuate autoimmune disease processes. Interleukin (IL)-4 and IL-21 produced by follicular helper T cells are required for B cell activation, germinal centre formation, immunoglobulin class switching and plasma cell differentiation.1 ,2 The JAK inhibitor tofacitinib is approved for treatment of rheumatoid arthritis. We recently reported that tofacitinib can suppress IL-17 and interferon-γ production by CD4+ T cells3 and inhibit the T cell stimulatory capacity of human monocyte-derived dendritic cells.4 However, whether this action involves B cell activation remains unclear.
Here we investigated the in vitro effects of tofacitinib on the gene regulatory network that controls B cell class switching and differentiation. Purified CD19+ B cells were stimulated with B cell antigen receptor (BCR), soluble CD40 ligand (sCD40L) and cytokines with/without tofacitinib. Culture medium was replenished on day 3. Cell viability tests revealed that, although B cell survival decreased considerably over time, the possibility of pharmacological toxicity by tofacitinib could be excluded (data not shown).
The expression of AICDA was slightly induced by cytokines or BCR/sCD40L alone, while costimulation with BCR, sCD40L and cytokines, especially IL-4, caused robust gene expression (figure 1A). BCL6 and XBP-1 exhibited similar expression patterns, at day 2 and day 5, respectively (figure 1A). BCR/sCD40L costimulated expression of AICDA and XBP-1was inhibited by tofacitinib in a dose-dependent manner (figure 1B, C). Tofacitinib did not inhibit BCL6 gene expression on day 2. In contrast with tofacitinib, inhibitors of the Src-family kinase (PP1, PP2) and a JAK2 inhibitor did not affect the expression of AICDA (figure 1B). Methotrexate exhibited a modest suppressive effect on AICDA expression compared with tofacitinib (figure 1D). In addition, AICDA gene expression levels after CpG stimulation were comparable in the presence/absence of tofacitinib (figure 1E). Finally, tofacitinib markedly and dose-dependently abrogated IgG production by B cells stimulated with BCR, sCD40L and IL-4 (figure 1F). These data describe tofacitinib as an effective inhibitor of B cell development.
Next, the effect of tofacitinib on activated B cells was examined. Tofacitinib was added on day 2 of B cell stimulation, and its effects on gene expression and immunoglobulin G (IgG) production were assessed on day 5. Tofacitinib abrogated AICDA, BCL6, XBP-1 expression and IgG production to similar levels seen with BCR/sCD40L alone (figure 2A, B). BCL6 protein levels were also reduced by tofacitinib (data not shown). This suggested that tofacitinib may inhibit the cytokine-mediated maintenance of BCL6 at the later time point. Previous studies indicated that Bach2 impairs germinal centre formation and class switching,5 its downregulation enhances plasma cell differentiation from IgG1 memory B cells6 and that Bach2 also represses effector programmes that stabilise regulatory T cell function.7 Uniquely, Bach2 was induced by BCR and sCD40L, but expression was significantly inhibited by IL-4. Interestingly, the addition of tofacitinib resulted in the recovery of Bach2 expression in a dose-dependent manner (figure 2A).
Finally, we investigated the effect of tofacitinib on the production of proinflammatory and anti-inflammatory cytokines by activated B cells. The strong induction of IL-6 gene expression and protein production by BCR, sCD40L and IL-4 stimulation was significantly abrogated by tofacitinib in a dose-dependent manner. Meanwhile, IL-10, which is produced by regulatory B cells,8 was not affected by tofacitinib even at the highest dose (figure 2C). These results suggest that tofacitinib suppresses B cell activation, differentiation and class switching, while maintaining B cell regulatory function.
In conclusion, our results provide new insights into the mechanisms of action of tofacitinib in the treatment of autoimmune diseases.
Contributors All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. YT had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Funding This work was supported in part by a Research Grant-In-Aid for Scientific Research from the Ministry of Health, Labour and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the University of Occupational and Environmental Health, Japan.
Competing interests YT has received consulting fees, speaking fees and/or honoraria from Chugai Pharma, Mitsubishi-Tanabe Pharma, Eisai Pharma, Pfizer, Abbott Immunology Pharma, Janssen Pharma, Takeda Industrial Pharma, Astra-Zeneca, Astellas Pharma, Asahi-kasei Pharma and GlaxoSmithKline and has received research grant support from Bristol-Myers Squibb, Mitsubishi-Tanabe Pharma, MSD, Takeda Industrial Pharma, Astellas Pharma, Eisai Pharma, Chugai Pharma, Pfizer and Daiichi-Sankyo. KS is employee of the Mitsubishi Tanabe Pharma Corporation.
Provenance and peer review Not commissioned; externally peer reviewed.
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