Regulation of CD8+ regulatory T cells: Interruption of the NKG2A-Qa-1 interaction allows robust suppressive activity and resolution of autoimmune disease - PubMed (original) (raw)

Regulation of CD8+ regulatory T cells: Interruption of the NKG2A-Qa-1 interaction allows robust suppressive activity and resolution of autoimmune disease

Linrong Lu et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Regulation of autoreactive CD4 T cells is essential to maintain self-tolerance and prevent autoimmune disease. Although CD8 T regulatory (Treg) cells that recognize self-peptides restricted by Qa-1 (HLA-E in humans) inhibit autoreactive CD4 cells and attenuate experimental autoimmune encephalomyelitis (EAE), the mechanism of this interaction is unclear. We generated Qa-1 mutant knock-in mice that impair Qa-1 binding to the T cell receptor (TCR) and CD94/NKG2A receptors. Analysis of these mice showed that TCR-dependent recognition of Qa-1-peptide complexes on target CD4 cells is essential for suppression by CD8 Treg cells. Further analysis revealed that genetic disruption of the Qa-1-CD94/NKG2A interaction unleashes robust CD8 Treg cell activity that completely abolishes development of EAE.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Generation and analysis of Qa-1 D227K mutant knock-in mice. (A) Qa-1 genomic locus and targeting strategy. Boxes represent exons; exon 4 (gray box) indicates the mutation site. The loxP sites are represented by black triangles. TK, thymidine kinase gene; _neo_r, neomycin-resistance gene. (B) Southern blot analysis of ES cell genomic DNA. The upper band (12.5 kb) corresponds to the WT allele; the lower band (7.2 kb) represents the knock-in allele. Right: PCR genotyping of knock-in mice. WT (280 bp) and knock-in (430 bp) products represent the addition of base pairs from the remaining loxP site and surrounding sequence (WT/WT, Qa-1 WT; DK/DK, homozygous mutant knock-in mice; WT/DK, heterozygous). (C) ConA-induced Qa-1 expression of WT and D227K mutant. Splenocytes from littermates (Qa-1-deficient [KO], Qa-1 WT [WT], and Qa-1 D227K knock-in [D227K]) were individually stimulated with ConA for 40 h and analyzed for surface Qa-1 expression by FACS analysis using anti-Qa-1 Ab (BD Bioscience). (D) L cells transfected with GFP, WT Qa-1 or Qa-1 D227K, and ConA blasts from WT or Qa-1 D227K mice were used as targets in a CTL assay by CD8 cells from B6.Tla mice immunized with Qa-1 ConA blasts. Percent lysis is shown at the indicated E:T ratios. (E) Left: NK cell susceptibility of Qa-1 R72A mutant CD4 cells. CD4+ T cells from Qa-1 WT, Qa-1-deficient, Qa-1 D227K, and Qa-1-R72A mutant mice were activated by ConA for 48 h; labeled with 51Cr; and used as targets for IL-2-activated NKG2A+ NK cells in a standard 4-h killing assay. Percent lysis at an E:T ratio is shown. Data shown represents mean ± SD. (n = 3). Right: Homeostatic expansion of Qa-1 mutant CD4 cells. A total of 106 CD4+ T cells isolated from Qa-1 WT, Qa-1-deficient, Qa-1 D227K, and Qa-1 R72A mice were transferred into syngeneic _Rag2_−/− hosts. Fourteen days later, CD4+ T cells from spleen and lymph nodes were enumerated. Data, shown as mean ± SD, represent one of two independent experiments (n = 3). (F) CD4 T cells from OT-2 TCR-transgenic B6 mice activated with ConA were used to vaccinate B6 mice. Fourteen days later, purified CD8 T cells (1.5 ×106) from OT-2-immunized mice were transferred with OT-2 CD4 T cells (from Qa-1 WT, Qa-1−/−, Qa-1 D227K, and Qa-1 R72A; 1 × 106) into _Rag2_−/−_Prf1_−/− mice before challenge with OVA peptide (50 μg) in CFA. Expansion of OT-2 T cells was quantitated by enumerating OT-2 cells in both lymph nodes and spleen 2 weeks after transfer. Data are representative of two independent experiments (n = 3).

Fig. 2.

Fig. 2.

MOG-induced EAE in Qa-1 D227K mice. (A) EAE was induced into WT (n = 5) and D227K (n = 5) mice by injecting 100 μg of MOG in CFA (50 ng of MTb) with pertussis toxin on days 0 and 2. The mice were reimmunized with 100 μg of MOG in CFA on day 7. EAE disease score was determined as described in Methods. (B) Recall response. At the end of the EAE experiments, spleen and lymph node cells were pooled from three mice in each group, and the cells were restimulated with the indicated doses of MOG peptides in vitro. IFN-γ and IL-17 production was measured by ELISA using 48 h culture supernatants. Data shown are representative of two independent experiments. (C and D) Effects of preimmunization in PLP-induced EAE development. B6 Qa-1 WT and B6 Qa-1 D227K mice were preimmunized with PLP peptide (20 μg) in CFA and boosted 10 days later with PLP peptide in CFA plus pertussis toxin. Clinical score (C, Left) and incidence (C, Right) were determined after the boost (time, horizontal axes; 5–8 mice per group). (D) Lymph nodes and spleen were harvested from mice at the end of the EAE experiments, and single-cell suspensions were prepared; 4 × 105 pooled draining lymph node and splenic cells were restimulated in vitro with the indicated concentrations of PLP peptide. Production of IFN-γ was measured 48 h after culture. Data shown represent two independent experiments.

Fig. 3.

Fig. 3.

MOG-induced EAE development in Qa-1 R72A mice. Left: CD4 cells were purified from MOG-immunized EAE mice [WT (A) and R72A (B)] and transferred into _Rag2_−/−_Prf1_−/− hosts with or without CD8 cells from the EAE mice. Mice were then immunized with MOG peptide with pertussis toxin, and the development of EAE was monitored daily. Data shown represent two independent experiments (n = 5). Right: Draining lymph nodes and spleens were collected 35 days after EAE induction. Single-cell suspensions were pooled and stimulated with the indicated concentrations of MOG peptide. IFN secretion was measured by ELISA after 48 h of culture. To ensure complete removal of NK cells from donor T cell populations, NK cells were depleted as described in Methods. (C–E) 2D2-induced EAE. (C) 2D2 CD4+ T cells (106) enriched from Qa-1 WT and R72A 2D2 TCR-transgenic mice were transferred into syngeneic _Rag2_−/−_Prf1_−/− hosts (n = 5) with or without CD8 Treg cells generated as described in Methods. Hosts were then immunized with 10 μg of MOG/CFA and 200 μg of pertussis toxin (on days 0 and 2). Development of EAE was scored daily as described in Methods. (D) In vivo suppression of R72A 2D2 expansion by CD8 regulatory cells. 2D2 CD4+ T cells (106) from Qa-1 WT or R72A mice were transferred into syngeneic _Rag2_−/−_Prf1_−/− hosts (n = 3) with or without CD8 Treg cells generated as described in Methods. Hosts were then immunized with 10 μg of MOG/CFA. 2D2 CD4 T cells in the draining lymph nodes and spleens were enumerated after 14 days. Data are shown as mean ± SD (n = 3). (E) Single-cell suspensions were prepared from draining lymph nodes collected as described above and pooled according to each group. The lymph node cells were stimulated with the indicated concentrations of MOG peptide in the presence of irradiated splenocytes as antigen-presenting cells. IL-2 secretion was measured by ELISA 48 h after culture.

Fig. 4.

Fig. 4.

Susceptibility of Qa-1 R72A 2D2 cells to CD8+ Treg cells. CD8 cells were purified from the draining lymph nodes of R72A 2D2 and CD8 suppressor cell cotransferred mice. 2D2 CD4 T cells were isolated from Qa-1 WT, D227K, and R72A mice and stimulated in vitro with different doses of MOG peptide in the presence of irradiated splenocytes as antigen-presenting cells with or without purified CD8 suppressor cells. (A) Proliferation of CD4 T cells was measured 60 h later by 3H-thymidine incorporation. Different CD4:CD8 ratios were used in similar experiments, and proliferation of CD4 T cells on stimulation with 30 μg of MOG peptide was measured (Lower). (B) IL-2 secretion was measured by ELISA 48 h after culture; relative IL-2 secretion is shown (normalized to no CD8 control cultures). To confirm specificity of Qa-1-restricted suppression, anti-Qa-1 Ab was included in one group at a concentration of 10 μg/ml. The data shown represent three independent experiments.

Fig. 5.

Fig. 5.

(A) Suppression of Qa-1 R72A 2D2 cell expansion by CD8 Treg cells requires perforin. First, 2D2 CD4+ T cells (106) from Qa-1 R72A mice were transferred into syngeneic _Rag2_−/−_Prf1_−/− hosts (n = 3) with CD8 suppressor cells generated in B6 mice or mice deficient in perforin and Fas ligand. Hosts were then immunized with 10 μg of MOG/CFA. 2D2 CD4 T cells in draining lymph nodes and spleen were enumerated after 14 days. Data are shown as mean ± SD (n = 3). (B) In vitro suppression of Qa-1 R72A 2D2 CD4 cell response by CD8 Treg cells. CD8 cells were purified from the draining lymph nodes of Qa-1 R72A 2D2 and CD8 suppressor cells cotransferred into mice. 2D2 CD4 T cells were isolated from Qa-1 R72A mice and stimulated in vitro with different doses of MOG peptide in the presence of irradiated splenocytes as antigen-presenting cells with or without different purified CD8 suppressor cells, as indicated. Proliferation of CD4 T cells was measured 60 h later by 3H-thymidine incorporation. Data shown represent two independent experiments. (C) In vitro lysis of Qa-1 R72A CD4 targets by CD8 Treg cells from 2D2-immunized mice. Qa-1 WT and Qa-1 R72A 2D2 CD4 T cells were activated by MOG peptide for 48 h and used as target cells in a lysis assay by CD8 T cells purified from OT2-immunized mice. CD8 cells from naïve B6 mice were used as controls. Percent lysis is shown at the indicated E:T ratios.

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