Background/Purpose:  T follicular helper (Tfh) cells represent a CD4⁺T cell subset specialized to provide help to B cells and to induce memory B cell and plasma cell differentiation. Proportion of blood Tfh cells are increased in patients with systemic lupus erythematosus, Sjogren’s syndrome and rheumatoid arthritis (RA)¹. The study of STAT3-deficient patients showed that JAK/STAT pathway play a crucial role in human Tfh development and function². Tofacitinib is a pan JAK inhibitor with potent inhibition of JAK3 and 1 and to a minor degree JAK2. The main reported mechanism of action of this drug, already approved for RA in many countries is the inhibition of numerous proinflammatory cytokines. JAK/STAT inhibitors are known to induce a decrease in CD4⁺T cells but their effect on T-cell subpopulations is not known. We therefore investigated whether therapeutic targeting of JAK/STAT pathway could also result in the inhibition of Tfh differentiation and function.

Methods:  Mononuclear cells were isolated from peripheral blood of healthy human donors. After selection, naive CD4⁺T cells were cultured in the presence of cytokines known to induce Tfh differentiation. Tofacitinib, was added at 2 different times: first concomitantly to IL12 and anti-human CD3/CD28 beads required to promote Tfh differentiation in order to evaluate the effect of tofacitinib (at concentrations from 0 to 3 µM) on Tfh differentiation; second after 3 days of in vitro Tfh cell-differentiation to assess the effect of tofacitinib on Tfh function after 14, 24, 48 and 72 hours of tofacitinib exposure. The differentiation of naive CD4⁺T cells into Tfh was analyzed using flow cytometry (positive staining for CXCR5, ICOS, PD1). Differentiation into Th1 and Th17 cells was analyzed using flow cytometry. The function of Tfh was assessed by intracellular staining of IL21 using flow cytometry. Cell viability was assessed using flow cytometry.

Results: Induction of Tfh differentiation in vitro. We first confirmed that the protocol used to differentiate naive CD4⁺T cells into Tfh was efficacious: IL12 and anti-CD3/CD28 allowed to induce the differentiation of 33% (from 6 to 53%, n=6) of Tfh. Expression of Bcl-6, the specific transcription factor of Tfh, was significantly higher in Tfh than in non Tfh cells. Inhibition of Tfh differentiation. First, adding tofacitinib concomitantly to IL12 and beads dramatically decreased Tfh differentiation in a dose dependent manner (52% without tofacitinib vs 5% with tofacitinib, maximal inhibition of 90%). JAK/STAT inhibition also demonstrated an inhibition of Th1 and Th17. Tofactinib did not induce T-cell mortality, as demonstrated by viability test. Inhibition of Tfh function. Second, the addition of tofacitinib after Tfh-cell commitment induced a reduction of IL21. Maximal reduction was obtained after an exposure to tofacitinib during 48hours.

Conclusion: We demonstrate that JAK/STAT pathway inhibition impairs Tfh differentiation and function in vitro in healthy controls. We are currently extending our analyses to blood samples from patients with RA and other autoimmune diseases. These results highlight an additional mechanism of action of JAK/STAT inhibitors in autoimmune diseases. ¹ Ueno. J Clin Immunol, 2016; ² Ma et al. Blood, 2012

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/therapeutic-targeting-of-jakstat-pathway-inhibits-follicular-helper-t-cell-maturation-and-function/