Cutting edge: impaired MHC class I expression in mice deficient for Nlrc5/class I transactivator

Abstract

MHC class I and class II are crucial for the adaptive immune system. Although regulation of MHC class II expression by CIITA has long been recognized, the mechanism of MHC class I transactivation has been largely unknown until the recent discovery of NLRC5/class I transactivator. In this study, we show using Nlrc5-deficient mice that NLRC5 is required for both constitutive and inducible MHC class I expression. Loss of Nlrc5 resulted in severe reduction in the expression of MHC class I and related genes such as β(2)-microglobulin, Tap1, or Lmp2, but did not affect MHC class II levels. IFN-γ stimulation could not overcome the impaired MHC class I expression in Nlrc5-deficient cells. Upon infection with Listeria monocyogenes, Nlrc5-deficient mice displayed impaired CD8(+) T cell activation, accompanied with increased bacterial loads. These findings illustrate critical roles of NLRC5/class I transactivator in MHC class I gene regulation and host defense by CD8(+) T cell responses.

FIGURE 1

Nlrc5 is required for the constitutive expression of MHC class I in various organs. (A) Expression of MHC class I (H2-K^b) in the indicated organs of wild-type mice (+/+, n=3), or mice heterozygous (+/−, n=4) or homozygous (−/−, n=3) for the targeted disruption of the Nlrc5 gene, as determined by qPCR analysis using gene-specific primers. Error bars represent ± SEM. *p < 0.05. (B) Expression of β2m, Tap1, Lmp2 and H2-M3 in the spleen and thymus of Nlrc5+/+ (n = 3) and Nlrc5−/− mice (n = 3) were analyzed by qPCR using the indicated gene-specific primers. Error bars represent ± SEM. *p < 0.05; **p < 0.01; ns, not significant. (C) Analysis of Nlrc5, MHC class II (H2-Aa), and CIITA gene expression in splenocytes, as determined by qPCR. n = 3. Error bars represent ± SEM. ns: not significant. (D) Western blot analysis of MHC class I (H2-K^b) and β2m protein expression in the spleen and thymus from mice with the indicated genotype. The expression of Hsp90 is shown as a loading control.

FIGURE 2

Reduced MHC class I expression in various cell types of Nlrc5-deficient mice. Single cell suspension of spleen (A, left panel & D), thymus (B, left panel) and lymph node (C, left panel) from Nlrc5 +/− (grey line) and Nlrc5−/− (black line) mice were analyzed by flow cytometry for the expression of MHC class I (H2-K/D/L, in A, B & C, left panel) or MHC class II (D) molecules (IA^b/IA^d), gated on CD8+, CD4+, B220+, F4/80+ or CD11c+ cells. Shaded region represent the isotype control. Data are representative of two independent experiments. Bar graphs (A, B & C, right panel) present the mean and SEM of the corresponding MFI (n = 3 for each genotype). *p < 0.05; **p < 0.01.

FIGURE 3

IFN-γ stimulation does not rescue the reduced MHC class I expression observed in Nlrc5-deficient mice. (A) Splenocytes from Nlrc5+/+, Nlrc5+/− and Nlrc5−/−mice (n = 3 for each genotype) were stimulated for 18 hrs with IFN-γ (100 U/ml) and transcript levels were analyzed by qPCR using the indicated gene-specific primers. Error bars represent ± SEM. **p < 0.01; ns, not significant. (B) MHC class I (H2-K^b) and β2m protein expression in splenocytes and thymocytes from mice with the indicated genotype. Cell extracts were prepared 18 hrs post stimulation with IFN-γ and analyzed by Western blotting. Hsp90 levels are depicted as a loading control. (C) Splenocytes from Nlrc5+/+, Nlrc5+/− and Nlrc5−/− mice (n = 3 for each genotype) were cultured overnight in the presence of IFN-γ and were analyzed by flow cytometry for the expression of MHC class I molecules (H2-K/D/L), gated on CD8+, CD4+, B220+, F4/80+, and CD11c+ cells. Bar graphs represent the mean and SEM of the corresponding MFI.

FIGURE 4

Nlrc5 is required for MHC class I mediated immune responses. (A) Nlrc5-deficient B cells display impaired antigen-specific stimulation of CD8+ T cells. CFSE-labeled OT-1 (CD45.1) CD8+ T cells were co-cultured with SIINFEKL (1μM) pulsed B220+ splenocytes from Nlrc5+/− or Nlrc5−/− mice at a ratio of 1:25 (B:T) for 72 hrs. Proliferation of CD45.1 gated cells was determined by flow cytometric analysis of CFSE dilution. Data are representative of two independent experiments (n=3). (B) Nlrc5+/+, Nlrc5+/− or Nlrc5−/− mice were infected with L. monocytogenes (LM) (1 × 10⁴ CFU). Splenocytes and hepatic leucocytes isolated from Nlrc5+/+ and Nlrc5−/− mice at day 6 after infection were cultured with heat-killed LM for 16 hours and analyzed by flow cytometry for the expression of IFN-γ gated on CD8⁺ cells. Numbers indicate percentage of cells. Data are representative of two independent experiments (n=3). Bar graphs (B, right panel) present the mean and SEM of the percentage of CD8+ IFN-γ+ cells (n = 3 for each genotype). *p < 0.05. (C) Bacterial load in the spleen and liver of LM infected Nlrc5+/+, Nlrc5+/− or Nlrc5−/− mice were determined. Data are representative of two independent experiments *p < 0.05; **p < 0.01.