Background/Purpose . Response Gene to Complement (RGC)-32 was cloned and characterized by our group as one of the genes induced by complement activation. It encodes an intracytoplasmatic protein that promotes cell cycle activation via cyclin B1-CDC2 activation and Akt phosphorylation in vascular smooth muscle and endothelial cells. RGC-32 is also a downstream target of TGF-β in fibroblasts and renal proximal tubular cells and plays a role in renal fibrogenesis. The expression and function of RGC-32 in immune cells has not been studied.  As Th17 and induced T regulatory cells (iTreg) differentiate under the control of TGF-β, we determined whether RGC-32 plays a role in Th17/Treg balance using RGC-32 KO mice generated in our lab. In addition, using two established models of chronic graft versus host disease (cGVHD), we assessed whether absence of RGC-32 on either donor CD4 cells or host B cells alters autoimmune parameters of disease.

Methods .  Naïve CD4⁺ cells purified from WT or RGC-32 KO splenocytes were cultured under Th1, Th2, Th17 and Treg conditions. The expression of IFN-γ, IL-4, IL-17A, FoxP3, T-bet, RORγt, and GATA-3 were determined by flow cytometry, ELISA and RT-PCR. Lymphocytes  isolated from the large intestine lamina propria of WT and RGC-32^-/-mice were stained for IL-17A. Phosphorylation of STAT3, Smad2 and 3 were assessed by Western blot.

Parent-into-F1 cGVHD was induced with 12×10⁶ WT or RGC-32^-/- CD4 cells injected into B6D2F1 recipients. Bm12-into-B6 cGVHD was induced by injecting 100×10⁶ Bm12 splenocytes into WT or RGC-32^-/-hosts. In both models, parameters of cGVHD including donor CD4, host B cell number and activation, anti-ssDNA autoAb production were assessed at two weeks after disease induction.

Results . RGC-32^-/- CD4⁺ T cells failed to differentiate normally to Th17 lineage as demonstrated by a significant reduction in IL-17 and RORγt mRNA and protein expression in vitro and a decreased proportion of IL-17⁺ cells in lamina propria lymphocytes in vivo. Decreased phosphorylation of Smad2 but not of STAT3 and Smad3 was noted in RGC-32^-/- CD4⁺ cells. A trend for increased iTreg differentiation was also noted while Th1 and Th2 differentiation did not differ between WT and RGC-32^-/-CD4 cells.

In P-into-F1 cGHVD mice, RGC-32^-/- donor CD4 cells displayed decreased expansion and proliferation compared to WT donor cells. Consistent with the in vitro data, RGC-32^-/- donor CD4 cells displayed lower proportion of IL-17⁺ cells.  Bm12-into- B6 cGVHD induced in RGC-32^-/- mice was characterized by decreased anti-dsDNA titers versus WT B6 recipients.

Conclusion . RGC-32 is a novel mediator of Th17 differentiation. In cGVHD expression of RGC-32 on CD4 cells is critical for their proliferation, expansion and IL-17 differentiation while expression of RGC-32 on B cells is critical for optimal autoantibody production. These data support the idea that RGC-32 blockade has the potential to attenuate autoimmune parameters of cGVHD and possibly reverse abnormalities in the Th17 and Treg cell pathways that contribute to lupus pathogenesis. These observations provide a compelling rationale for further investigating the therapeutic potential of RGC-32 blockade in murine and human lupus.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/lack-of-response-gene-to-complement-32-impairs-th17-differentiation-and-attenuates-lupus-like-chronic-graft-versus-host-disease/