Background/Purpose: Follicular T helper (Tfh) cells have been correlated with germinal center (GC) formation, autoantibody production and disease severity in human systemic lupus erythematosus (SLE). Follicular regulatory T cells (Tfr) are a regulatory CD4 T (Treg) subset that counteract Tfh cells and suppress GC B cell differentiation.  We recently identified that interleukin 21 (IL-21) promotes Tfh differentiation in autoimmune BXD2 mice that develop spontaneous GCs. However, the role of IL-21 in regulating Tfr is unknown. The aim of this study is to determine the modulatory effects of IL-21 on Tfr/Tfh balance in BXD2 mice.

Methods: Real-time PCR analysis was used to determine the expression of Treg-related genes in sorted Tfr cells after in vitro cultured with IL-21 (50ng/ml) stimulation. Exogenous IL-21 was provided to BXD2-Il21^-/- mice via adenovirus (Ad)-IL21 administration to investigate the effect of IL-21 on Tfr in vivo. Sorted Tfr cells from BXD2-Il21^-/- mice were co-cultured with CD4 T cells and B cells in vitro as well as transferred into young BXD2 mice in vivoto determine the function of Tfr. FACS staining was used to determine the percent and phenotype of Tfh and Tfr. Confocal imaging analysis was used to visualize the distribution of Tfr cells and GCs. ELISA was used to determine serum levels of IL-21 and TGF-β1. ELISPOT was used to determine the generation of antibody producing B cells.

Results: GL-7⁺Fas⁺ GC B cells and CXCR5⁺ICOS⁺ Tfh cells were significantly reduced, but the frequency of FoxP3⁺CXCR5⁺ICOS⁺ Tfr cells was 2-fold higher in BXD2-Il21^-/- mice than in wild-type BXD2 mice, although the frequencies of conventional Treg cells were equivalent. The Tfr cells in BXD2-Il21^{– /-} mice expressed high level of other Treg markers including CTLA4, TGF-β1 and GITR. FoxP3⁺Tfr cells were localized in spleen follicular areas of BXD2-Il21^-/- mice. Transfer of Tfrs from BXD2-Il21^-/- mice into BXD2 mice although decreased GC size and reduced IgM, it did not suppress IgG autoantibody producing B cells in the spleen. AdIL-21 administration to BXD2-Il21^-/- mice increased GC B cells but decreased the serum TGF-β1, membrane TGF-β1 expression in Tfrs and the ratio of Tfr/Tfh in the spleen. In vitro, IL-21 decreased FoxP3⁺ and significantly reduced Tgfb, Gitr and Il2 expression in Tfrs. IL-21 also counteracted Tfr-induced B cells death and inhibition of Tfhs.

Conclusion: The present study suggests that IL-21 positively promotes autoreactive GC reactions in BXD2 mice at least partially by decreasing Tfrs and compromising the suppressive function of Tfrs on B cells and Tfhs. The results further suggest that IL-21 can skew the balance between Tfr and Tfhs to favor Tfh differentiation. Thus, IL-21 blockage is necessary when developing a Tfr transferring therapeutic strategy to inhibit autoimmune GC response.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/il-21-inhibited-follicular-regulatory-t-cells-to-promote-autoreactive-germinal-center-development-in-autoimmune-bxd2-mice/