CD8+ regulatory T cells induced by T cell vaccination protect against autoimmune nephritis

Abstract

Autoreactive T cells play a pivotal role in the pathogenesis of autoimmune kidney disease. T cell vaccination (TCV) may limit autoimmune disease and induce CD8+ regulatory T cells (Tregs). We used Heymann nephritis (HN), a rat model of human membranous nephritis, to study the effects of TCV on autoimmune kidney disease. We harvested CD4+ T cells from renal tubular antigen (Fx1A) -immunized rats and activated these cells in vitro to express the MHC Class Ib molecule Qa-1. Vaccination of Lewis rats with these autoreactive Fx1A-induced T cells protected against HN, whereas control-primed T cells did not. Rats that underwent TCV had lower levels of proteinuria and serum creatinine and significantly less glomerulosclerosis, tubular damage, and interstitial infiltrates. Furthermore, these rats expressed less IFN-γ and IL-6 in splenocytes, whereas the numbers of Tregs and the expression of Foxp3 were unchanged. In vitro cytotoxicity assays showed CD8+ T cell-mediated elimination of Qa-1-expressing CD4+ T cells. In vivo, TCV abrogated the increase in Qa-1-expressing CXCR5+ TFH cells observed in HN compared with controls. Taken together, these results suggest that TCV protects against autoimmune kidney disease by targeting Qa-1-expressing autoreactive CD4+ cells.

Figures

Figure 1.

TCV through autoreactive CD4+ T cells increased the expression of Qa-1. (A) Fx1A was generated from Sprague–Dawley rat kidneys as previously described. Lewis rats were immunized with Fx1A as the experimental group and CFA as the control group. CD4+ T cells were purified from draining LNs of the two groups of rats 10 days after footpad injection of Fx1A or CFA and cultured with conA for 40 hours. The CD4+ T cells from Fx1A- and CFA-immunized rats were irradiated at 30 Gy and injected into two groups of Lewis rats through the tail vein (5×106 cells/rat). Fx1A was injected into each of their hind footpads 3 weeks after TCV. (B and C) CD4+ T cells were isolated from draining LNs from both (B) CFA- and (C) Fx1A-immunized rats and cultured on PBS or conA for 40 hours. Qa-1 expression on CD4+ T cells was increased from 13% (PBS) to 18% (conA) for CFA-immunized rats and 11% to 27% for Fx1A-immunized rats after conA stimulation (P<0.05, n=6).

Figure 2.

TCV reduced proteinuria, inflammation, and protected renal function in HN, and the protection was induced by antigen-specific TCV. HN + TCV rats had significantly reduced (A) proteinuria excretion at weeks 10 and 12 and (B) serum creatinine compared with HN rats. (C) Semiquantitative scores of morphologic changes at week 12 showed significantly less damage in glomeruli and tubules and reduced interstitial monocyte infiltration in HN + TCV rats compared with HN rats. (D) Interstitial infiltration scores of macrophage and CD8+ and CD4+ cells on immunohistochemical sections showed reduced kidney infiltration in HN + TCV rats. (E) Comparison of Fx1A IgG responses in the three groups of rats; anti-Fx1A antibody levels expressed as percentage binding of known positive serum (anti-Fx1A titer; 1:200) measured by ELISA were significantly reduced by TCV in the course of HN. (F) Proteinuria and (G) serum creatinine were significantly reduced in Fx1A-primed TCV rats compared with CFA-primed TCV (n=6 for each group, mean ± SD). *P<0.05; **P<0.01.

Figure 3.

TCV reduced renal damage in HN. (A) Histology of renal representative sections under PAS staining showed reduced renal damage and cellular infiltration in rats that received TCV. Magnification, ×200. (B) Representative sections of kidneys showed that TCV reduced subepithelial glomerular immune deposits. Magnification, ×400. (C) In the TCV (CFA) control group, the renal injury was equivalent to the standard HN rats group, whereas TCV (Fx1A) showed less injury. Magnification, ×200.

Figure 4.

Photomicrographs of immunohistochemical sections from normal, CFA, HN, and HN + TCV groups of rats showing reduction in infiltration with macrophages, CD4+ T cells, and CD8+ T cells in the HN + TCV group. Magnification, ×400.

Figure 5.

In vitro cytotoxicity analysis showed the induction of cytotoxic CD8+ T cells by antigen-specific TCV. (A) CD4+ cells isolated from TCV or nonimmunized rats were stained with high or low concentrations of CFSE, sorted depending on Qa-1 high or low by flow cytometry, and cocultured alone or with TCV CD8+ T cells. (B) Qa-1 high CD4+ cells were significantly eliminated when cocultured with TCV CD8+ cells (**P<0.01), but CD4+ Qa-1 low cells were not eliminated. Anti–Qa-1 antibody inhibited the cytotoxic effects of TCV-derived CD8+ T cells on Qa-1 high CD4 T cells. (C) In vitro cytotoxicity assays were performed using CD4 target cells and CD8 effector cells from rats that had received either Fx1A TCV or CFA TCV using the same method as in A and B. There was no effect for CFA-derived CD8+ T cells to eliminate Qa-1–expressing CD4+ T cells in vitro compared with the cytotoxicity of CD8+ T cells isolated from Fx1A-derived TCV rats that were added to Qa-1 high CD4+ T cells in coculture. These results are representative of three independent experiments.

Figure 6.

Characterization of TFH cells in HN. (A) Splenocytes were isolated from CFA and HN rats and analyzed by flow cytometry to assess the percentage of TFH cells between control (CFA injected) and HN (Fx1A treated) rats. There were significantly higher numbers of the CD4+ CXCR5+ TFH cells in HN rats than in the CFA group (*P<0.05; **P<0.01). (B) After gating on CD4+ cells, Qa-1 was highly expressed on CXCR5+ TFH cells (96% of total CD4+ cells). (C) Sorted CD4+ CXCR5+ TFH cells expressed a higher level of BCL6 mRNA than CD4+CXCR5− T cells by real-time PCR (*P<0.05). (D) Splenocytes were isolated from rats with HN and TCV, and then, sorted CD8+ T cells were analyzed by flow cytometry (purity>92%). The sorted CD8+ T cells were then stained with CD11b/c with limited CD11b/c expression. (E) Flow cytometric analysis showed TFH cells (CXCR5+ CD4+ T cells) increased to 12% after HN induction compared with normal rats (5% CXCR5+ of total CD4+ T cells); there is a significant decrease in TFH numbers in HN rats receiving TCV induced with Fx1A. However, HN rats receiving TCV induced with CFA had a similar increase in TFH cells (10%) as HN rats alone (n=6).

Figure 7.

Real-time quantitative PCR of cytokine and Foxp3 mRNA levels from splenocytes and kidney 12 weeks postimmunization with Fx1A. (A and B) HN + TCV rats had significantly lower levels of IL-6 and IFN-γ and higher levels of TGF-β and IL-10 than (A) splenocytes from HN rats (*P<0.05). (C) There was higher level of IFN-γ in kidney for HN + TCV rats compared with HN rats (*P<0.05). Splenic Foxp3 mRNA level was not different among the three groups of rats. Flow cytometric analysis showed no difference in Foxp3+ Treg numbers in circulation between (D) HN and (E) HN + TCV groups.