NLRC5 knockdown in CD4+CD25+ Treg cells attenuates cardiac allograft rejection through enhanced immunosuppressive function

Purpose: CD4⁺CD25⁺ regulatory T cells (Tregs) are critical mediators of immune tolerance, but their therapeutic efficacy in preventing alloimmune rejection in organ transplantation is constrained by functional instability. NLRC5, a transcriptional regulator of major histocompatibility complex class I, has emerging roles in immune modulation. This study investigated the immunomodulatory role of NLRC5 in CD4⁺CD25⁺ Tregs during cardiac allograft rejection.

Methods: In vitro, shRNA-mediated NLRC5 knockdown (sh-NLRC5) was performed in Tregs, followed by cytokine profiling using enzyme-linked immunosorbent assay, effector T cell (Teff) proliferation suppression assay using flow cytometry, and transwell migration assay. For in vivo validation, heart transplant recipients received adoptive transfers of Tregs transfected with sh-NLRC5. Graft survival was monitored, the cardiac pathology was assessed by hematoxylin-eosin and Masson staining. The balance between T-helper type 1 (Th1) and T-helper type 2 (Th2) was confirmed by flow cytometry, and Foxp3 expression was examined using Reverse transcription PCR and Western blot assays.

Results: In the mouse model, the NLRC5 expression in splenic Tregs was lower in the tolerance group compared to the rejection group. Knockdown of NLRC5 in Tregs led to inhibition of inflammatory cytokines and Teff cell proliferation, while enhanced Treg migration. In vivo, injection of sh-NLRC5 Tregs extended the survival time of heart-transplanted mice, improved cardiac pathology, decreased the Th1/Th2 cell ratio and increased Foxp3 expression.

Conclusions: These results indicated that NLRC5-modified Tregs hold great promise for preventing alloimmune rejection in heart transplantation, providing a potential novel therapeutic approach.

CD4(+)CD25(+) Treg; NLRC5; Th1/Th2; cardiac allograft; immune tolerance.