Modulating IL-21-driven B cell responses in idiopathic inflammatory myopathies via inhibition of the JAK/STAT pathway

Abstract

Background: Idiopathic inflammatory myopathies (IIM) are autoimmune disorders characterized by muscle inflammation and autoreactive B cell responses. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway is essential for B cell functions, making it a promising therapeutic target. This study explores the potential of tofacitinib, a JAK1/JAK3 inhibitor, to modulate B cell activity in IIM.

Methods: Peripheral B cell populations from dermatomyositis (DM), anti-synthetase syndrome (ASyS) and overlap myositis (OM) patients were analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMC) or sorted memory B cells were cultured with tofacitinib and stimulated with combinations of CD40, IL-21, IL-2, BAFF and CpG. B cell proliferation, differentiation and (auto)antibody, cytokine/chemokine production were assessed by flow cytometry, Luminex, and ELISA/ELiA assays.

Results: The IIM peripheral B cell compartment had elevated transitional and naive B cells, with reduced Bmem frequencies compared to healthy donors. Tofacitinib significantly inhibited CD40/IL-21-induced B cell proliferation, plasmablast formation and function in PBMC and B cell-only cultures across all IIM subgroups, predominantly affecting the IL-21-induced differentiation and antibody production. Remarkably, tofacitinib reduced the levels of anti-Jo1 autoantibodies, as well as of CXCL10 and CXCL13 in ASyS memory B cell cultures.

Conclusions: These findings highlight the B cell involvement in IIM, evidenced by altered peripheral B cell composition in active disease and the effective inhibition of essential B cell responses, including proliferation, differentiation, and (auto)antibody production, by tofacitinib in vitro. This positions the JAK/STAT pathway as a promising new therapeutic target to modulate B cell activity in IIM.

Keywords: B cell; JAK; Myositis; Plasma cell; Tofacitinib.

Fig. 1

Peripheral B cell composition of IIM patients with subgroups DM, ASyS and OM. (A) Dot plots showing the gating strategy used to identify peripheral B cell populations in one representative patient per phenotype, including CD27⁺IgD^- switched memory cells (SWM), CD27⁺IgD⁺ non-switched memory (NSM) cells, CD27^-IgD^- double negative (DN) cells and CD27^-IgD⁺ naive B cells. (B) CD24⁺CD38⁺ transitional B cells were gated within total CD19⁺ B cells, with subtypes 1 (IgM⁺IgD^-), 2 (IgM⁺IgD⁺) and 3 (IgM^-IgD⁺) identified as shown. The percentages of the identified subtypes from the dot plots are included. (C) Within the SWM cells, CD27⁺CD38⁺ plasmablasts were identified as shown in the dot plots, and graph shows the percentage within total B cells. Dotted line in A-C represents HD reference values. DM: dermatomyositis, ASyS: anti-synthetase syndrome, OM: overlap myositis, HD: healthy donor. Graphs display the mean, SD and individual values (*p < 0.05, **p < 0.01, n = 4/IIM subgroup; n = 6 HD)

Fig. 2

Dose-dependent effects of tofacitinib on B cell responses in a 6-day HD-PBMC assay. (A) Representative histograms showing CFSE staining of PBMC to assess proliferation of CD19⁺CD20⁺ cells for conditions with tofacitinib (Tofa) or control under anti-CD40/IL-21 (left) and CpG/IL-2 stimulation (right). Gates were set using unstimulated cells as control, and percentages of proliferated CD19⁺CD20⁺ cells are shown. (B) Representative dot plots showing the percentage of gated plasmablasts (CD27⁺CD38⁺) within CD19⁺CD20⁺ B cells. Quantification of plasmablast proportions is shown for anti-CD40/IL-21 (left) and CpG/IL-2 stimulation (right). (C) Concentrations of IgG, IgM and IgA in the culture supernatants collected under anti-CD40/IL-21 (left) and CpG/IL-2 (right) stimulation. Graphs display the mean, SD and individual values (*p < 0.05, n = 6 HD)

Fig. 3

Tofacitinib dampens IL-21-induced B cell responses in a 6-day HD-PBMC culture. (A) Representative histograms (left) showing the effect of tofacitinib (Tofa) or control on the percentage of proliferating B cells, as measured by CFSE staining, under IL-21 or anti-CD40 stimulation. Gates were set using unstimulated cells as control. Quantification (right) of the percentage of proliferated B cells. (B) Representative dot plots (left) showing the gating of CD27⁺CD38⁺ plasmablasts within CD19⁺CD20⁺ B cells. Quantification (right) of the plasmablast proportions. (C) Concentration of IgG, IgM and IgA in culture supernatants from IL-21 (top) or anti-CD40 (bottom)-stimulated cells. Graphs show the mean, SD and individual values (*p < 0.05, n = 6 HD)

Fig. 4

Tofacitinib treatment of PBMC from DM, ASyS and OM significantly reduces B cell proliferation, but mainly affects their differentiation and antibody production in a 6-day culture. (A) Histograms show the percentage of proliferation based on CFSE staining from a representative ASyS patient within CD19⁺CD20⁺ B cells. Graphs include analysis from all samples. (B) Percentage of CD27⁺CD38⁺ plasmablasts (of CD19⁺CD20⁺ B cells) and of CD138⁺ PC (of CD3^- cells). (C) Representative dot plots and analysis of the percentage of naive and memory populations (within CD19⁺CD20⁺ B cells): switched memory (SWM) (CD27⁺IgD^-), non-switched memory (NSM) (CD27⁺IgD⁺), naive (CD27^-IgD⁺) and double negative (DN) (CD27^-IgD^-). (D) Analysis of the IgG, IgM and IgA levels in the culture supernatants. DM: dermatomyositis, ASyS: anti-synthetase syndrome, OM: overlap myositis. Tofa: tofacitinib (2.5 µM). Graphs show the mean, SD and individual values (**p < 0.01; ***p < 0.001, n = 4/IIM subgroup)

Fig. 5

Effects of tofacitinib on B cell differentiation from ASyS patients in a 12-day PBMC culture. PBMC were cultured with BAFF/CpG/IL-21, with or without tofacitinib, and DMSO was used as control. (A) Representative dot plots and analysis of the percentage of naive (CD27^-IgD⁺), switched memory (SWM, CD27⁺IgD^-), double negative (CD27^-IgD^-), non-switched memory (NSM, CD27⁺IgD⁺) cells. (B) Representative dot plots and analysis of the percentage of CD27⁺CD38⁺ plasmablasts (within CD19⁺CD20⁺ B cells) and of CD138⁺ PC (within CD3^- cells). (C) Total levels of IgG, IgM and IgA detected by ELISA and (D) anti-Jo1 detected with ELiA. (E) Levels of CXCL10 and CXCL13 in supernatants measured by Luminex. ASyS: anti-synthetase syndrome. Tofa: tofacitinib (2.5 µM). Graphs show the mean, SD and individual values (*p < 0.05, **p < 0.01, n = 4 ASyS patients)

Fig. 6

Effects of JAK inhibition using tofacitinib in an 8-day memory B cell expansion and differentiation assay from ASyS patients. (A) Dotplots from one representative ASyS patient (also in B-C) showing the identification of memory and naive populations within CD19⁺CD20⁺ B cells: switched memory (SWM, CD27⁺IgD^-), non-switched memory (NSM, CD27⁺IgD⁺), CD27^-IgD⁺ and double negative (DN, CD27^-IgD^-) cells after an 8-day in vitro culture. Previously sorted memory B cells were cultured for 8 days with irradiated CD40L-expressing L-cells, and stimulated with BAFF/IL-21/IL-2 in the presence/absence of tofacitinib at days 0, 2 and 4. (B) Dotplots showing the percentage of CD27⁺CD38⁺ plasmablasts, showing the count in graphs. (C) Dotplots showing the CD138⁺ PC percentage (of B cells), representing the count in graphs. (D) IgG levels measured in the culture supernatants are shown as ng/mL. (E) Chemokine levels measured in the culture supernatants are shown as pg/mL. Tofa: tofacitinib (1.0 and 2.5 µM). Graphs show the mean, SD and individual values (*p < 0.05, n = 4 ASyS patients at day 0 and n = 5 ASyS patients at days 2–4)