Inhibit NLRC5 regulate CD4+ T cellsfunctionandimmunological protection onallograftinmice

Abstract

Objective To study the effect of inhibiting NLRC5 on CD4+ T cells function and allografts immune protective effects in mice. Methods Mice spleen-derived CD4+ T cells were cultured, purified andtransfected with short hairpin RNA (shRNA) lentiviral vector NLRC5-RNA interference (RNAi)-green fluorescent protein (GFP). The mice allogenic islet transplantation model andskintransplantation model were established, and 24 h prior to transplantation each recipient mice was given CD4+ T cells, NLRC5-RNAi-T cells or equal amount of phosphate buffer (PBS), named T cell group, NLRC5-RNAi group and control group respectively. The survival of islet grafts andskingraftsof transplanted recipients was observed, and on the day 7, the islet graft glucose tolerance, the percentage of T cell subsets in the spleenaswellas related cytokines interleukin (IL)-10 and interferon (IFN)-γwere observed. Results The experimental data demonstratedthat NLRC5-RNAi significantly prolonged the survival timewith a median survival time of (19.0±3.4) days inislet allograftand (15.4±3.8) days inskin allograft, which is significantly longer than control [(11.6±1.9) d, t=5.156, P<0.01] and T cell group [(10.0±1.4) d, t=4.151, P<0.01] inislet allograft, aswellascontrol [(8.0±0.9) d, t=3.375, P<0.05] and T cell group [(7.8±0.8) d, t=3.375, P<0.05] inskin allograft. The results of enzyme linked immunosorbent assay (ELISA) showed that the expression of IL-10 (344.0±4.1) ng/L inisletallograft and (275.9±12.5) ng/L in skinallograftin NLRC5-RNAi group were significantly higher than control group (t=124.141, 20.121, P<0.01) and T cell group (t=32.605, 17.900, P<0.01); the expression of IFN-γ (85.1±6.6) ng/L inisletallograftand (96.6±2.5) ng/L inskinallograftin NLRC5-RNAi group were lower than control group (t=7.633, 7.490, P<0.05) and T cell group (t=10.972, 13.286, P<0.01). Flow cytometric analysis revealed that compared to control group and T cell group, the NLRC5-RNAi group dramatically increased the population of Th2 [(0.190±0.053)%, t=5.220, 5.278, P<0.05] inislet allograft and [(0.130±0.012)%, t=21.060, 9.470, P<0.05] in skin allograft, as well asreduced the population of Th1 [(0.810±0.036)%, t=6.219, 5.276, P<0.05] inislet allograft and [(0.180±0.026)%, t=9.248, 25.324, P<0.05] in skin allograft. Conclusion After inhibiting the expression of NLRC5 gene, the expression of Th2 type cytokines in CD4+ T cells is increased, which prolongs the survival time of grafts and has positive significance for the induction of transplantation tolerance. Key words: NLRC5; Lentivirus transduction; CD4+ T cells; Islet transplantation; Skin transplantation; Immune tolerance