Inducible down-regulation of MHC class I results in natural killer cell tolerance

Abstract

Natural killer (NK) cells are innate lymphocytes that are thought to kill cells that down-regulate MHC class I (MHC-I) through "missing-self" recognition. NK cells from B2m^-/- mice that lack surface MHC-I, however, are not autoreactive as predicted by the missing-self hypothesis. As a result, it is unclear if MHC-I down-regulation in vivo induces NK cell reactivity or tolerance to missing-self. Here, we generated a floxed B2m mouse to acutely down-regulate MHC-I in vivo in a host that normally expresses MHC-I. Global down-regulation of MHC-I induced NK cell hyporesponsiveness and tolerance to missing-self without overt missing-self reactivity. In contrast, down-regulation of MHC-I on a small fraction of hematopoietic cells triggered missing-self reactivity. Surprisingly, down-regulation of MHC-I only on CD4⁺ T cells predominately induced tolerance to missing-self without resetting NK cell responsiveness. In this setting, inflammation triggered substantial missing-self reactivity. These results show that MHC-I down-regulation can induce either NK cell tolerance or killing in vivo and that inflammation promotes missing-self reactivity.

Figure 1.

Conditional deletion of B2m leads to loss of surface MHC-I. (A) The targeted B2m^tm1a allele is depicted before (top) and after (middle) removal of the LacZ and neomycin resistance (neo^r) genes by FLP recombinase. Germline-expressed Cre recombinase was used to generate the B2m^Δ allele (bottom). (B) Representative histograms of H-2K^b and H-2D^b expression on total lymphocytes from spleens of WT, B2m^fl/fl, B2m^−/−, and B2m^Δ/Δ mice. (C) Representative dot plots showing the percentage of T cells (CD19⁻ CD3⁺) that express CD4 or CD8. Data in B and C are representative of two independent experiments with three mice per group. (D) Total splenic CD8⁺ T cell number in WT, B2m^fl/fl, B2m^−/−, and B2m^Δ/Δ mice (n = 6 mice per group). Data in D are combined from two independent experiments. (E and F) Splenocytes from WT, B2m^fl/fl, B2m^−/−, and B2m^Δ/Δ mice were labeled with CFSE and differentially labeled with CT violet and CT far red as indicated. Labeled cells were injected i.v. into WT recipient mice, and donor cells were recovered from spleens of recipients after 2 d. (E) Representative dot plots showing the relative percentages of transferred cells (CFSE⁺) recovered from the spleens of WT recipient mice that were depleted of NK cells with anti-NK1.1 (right) or undepleted (middle). (F) Percentage of NK cell–specific rejection of donor cells by WT recipient mice (n = 4–5 recipient mice per group). Data in F are representative of two independent experiments with four to five recipient mice per group that received the same mix of donor cells in each experiment. Statistical significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test. Each symbol in D and F represents an individual mouse. Error bars indicate mean ± SEM; ****, P < 0.0001; ns, not significant.

Figure 2.

Global down-regulation of MHC-I does not induce overt NK cell missing-self reactivity. (A) Representative histograms showing H-2K^b expression on CD45⁺ lymphocytes from peripheral blood of B2m^fl/fl R26-Cre-ER^T2 mice treated with tamoxifen starting on day 0. Mice were injected i.p. with anti-NK1.1 antibody to deplete NK cells or with PBS control as indicated. (B) The percentage of H-2K^b–deficient CD45⁺ cells that accumulate in the blood of tamoxifen-treated B2m^fl/fl R26-Cre-ER^T2 mice over time (n = 4 mice per group). Similar results to those in A and B were observed in a second experiment with three mice per group. (C and D) B2m^fl/fl, B2m^fl/fl R26-Cre-ER^T2, and B2m^Δ/Δ mice were treated with tamoxifen or vehicle control on days 0–4, and splenocytes were harvested and labeled with CFSE plus CT violet and CT far red as indicated on day 16. Labeled splenocytes were i.v. injected into WT recipients, and recovery of donor cells was analyzed after 2 d. (C) Representative dot plots showing the relative percentages of transferred cells (CFSE⁺) recovered from the spleens of WT recipient mice that were depleted of NK cells with anti-NK1.1 (right) or undepleted (left). (D) NK cell–specific rejection of the indicated donor cells combined from three independent experiments in which donor cells were injected on day 14 or 16 (each symbol represents an average of four to five recipient mice injected with the same mix of donor cells; two-way ANOVA with Bonferroni’s multiple comparisons test). CT far red labeling was inverted in one of the replicates in D. Error bars indicate mean ± SEM; ****, P < 0.0001; ns, not significant. Tam, tamoxifen; Veh, vehicle.

Figure 3.

Global down-regulation of MHC-I induces loss of NK cell licensing. (A) B2m^fl/fl and B2m^fl/fl R26-Cre-ER^T2 mice were treated with tamoxifen or vehicle control followed by i.v. injection of labeled donor splenocytes as indicated. (B) Representative histograms showing the relative percentages of transferred B2m^fl/fl and B2m^Δ/Δ cells (CFSE⁺ CT far red^low) recovered from the spleens of recipient mice after 2 d. B2m^fl/fl and B2m^Δ/Δ cells were differentially labeled with CT violet as indicated. (C) Summary of NK cell–specific rejection of B2m^Δ/Δ donor cells (n = 4–5 mice per group; two-way ANOVA with Bonferroni’s multiple comparisons test). Data in C are combined from two independent experiments. Similar results were seen with WT and B2m^−/− donor splenocytes (CFSE⁺ CT far red^high) that were cotransferred in these experiments. (D–G) B2m^fl/fl, B2m^fl/fl R26-Cre-ER^T2, and B2m^Δ/Δ mice were treated with tamoxifen or vehicle control on days 0–4, and splenocytes were stimulated on day 14 with plate-bound anti-NK1.1 antibody or PMA and ionomycin. (D) Representative dot plots showing percentage of NK cells (CD3⁻ CD19⁻ NKp46⁺) that express Ly49C and IFN-γ after stimulation with plate-bound anti-NK1.1. (E) Percentage of NK cells that express IFN-γ after stimulation with plate-bound anti-NK1.1 (n = 3–4 mice per group; one-way ANOVA with Bonferroni’s multiple comparisons test). (F) Relative production of IFN-γ by Ly49C⁺ and Ly49C⁻ NK cells after stimulation with anti-NK1.1 is quantified by an Ly49C licensing ratio (see Materials and methods; n = 3–4 mice per group; one-way ANOVA with Bonferroni’s multiple comparisons test). (G) Percentage of NK cells that express IFN-γ after stimulation with PMA and ionomycin (n = 3–4 mice per group). Data in D–G are representative of two independent experiments with three to four mice per group. Each symbol in C and E–G represents an individual mouse. Error bars indicate mean ± SEM; ***, P < 0.001; ****, P < 0.0001; ns, not significant. Tam, tamoxifen; Veh, vehicle.

Figure 4.

Down-regulation of MHC-I is sufficient for missing-self recognition of a limited number of cells. (A) Mixed BM chimeras were generated by reconstituting irradiated WT (CD45.1) mice with different ratios of BM from WT (CD45.1) and B2m^fl/fl R26-Cre-ER^T2 (CD45.2) mice. After 18–21 wk, chimeras were treated with tamoxifen or vehicle control starting on day 1, and peripheral blood was analyzed weekly from day 0 to 28. Chimeras were injected with anti-NK1.1 to deplete NK cells or with PBS on day −2 and every 7 d after. (B) Percentage of B2m^fl/fl R26-Cre-ERT2 cells remaining in the blood on day 28 relative to day 0. Percentage of CD45.2⁺ cells was assessed within all CD45.1 and/or CD45.2⁺ lymphocytes. (C) Splenocytes from B2m^fl/fl and B2m^fl/fl R26-Cre-ER^T2 mice were labeled with CT far red and differentially labeled with CT violet and i.v. injected into WT recipient mice (20 × 10⁶ of each donor). Recipient mice were subsequently treated with tamoxifen or vehicle for 5 d. Recipient mice were injected i.p. with anti-NK1.1 to deplete NK cells or with PBS control. (D) Representative histograms showing H-2K^b expression on transferred B2m^fl/fl R26-Cre-ER^T2 cells (CT far red⁺ CT violet^high) from blood of recipient mice over time. (E) Percentage of transferred B2m^fl/fl R26-Cre-ER^T2 cells that are H-2K^b–deficient in the blood of recipient mice over time (n = 6 mice per group; two-way repeated measure ANOVA with Bonferroni’s multiple comparisons test). Asterisks in E indicate statistical significance between the tamoxifen-treated groups treated with anti-NK1.1 or PBS. (F) Representative histograms showing the relative percentages of transferred cells (CT far red⁺) recovered from the spleens of recipient mice on day 14. B2m^fl/fl (Cre⁻), and B2m^fl/fl R26-Cre-ERT2 (Cre⁺) donor cells were distinguished by CT violet labeling as indicated. (G) Percentage of NK cell–specific rejection of B2m^fl/fl R26-Cre-ER^T2 donor cells by WT recipient mice treated as indicated (n = 6 mice per group; one-way ANOVA with Bonferroni’s multiple comparisons test). Data in B, E, and G are combined from two independent experiments in which all recipients within an experiment received the same mix of donor BM or splenocytes. Each symbol in B and G represents an individual recipient mouse. Error bars indicate mean ± SEM; *, P < 0.05; ****, P < 0.0001; ns, not significant. Tam, tamoxifen; Veh, vehicle.

Figure 5.

Minimal NK cell reactivity is observed after down-regulation of MHC-I on CD4⁺ T cells. B2m^fl/fl and B2m^fl/fl CD4-Cre-ER^T2 mice were treated with tamoxifen or vehicle control on days 0–4, and spleens were harvested for flow cytometry on day 14. (A) Percentage of NK cells (CD45⁺ CD19⁻ CD3⁻ NK1.1⁺ NKp46⁺), B cells (CD45⁺ CD3⁻ CD19⁺), CD8⁺ T cells (CD45⁺ CD19⁻ CD3⁺ CD4⁻ CD8⁺), and CD4⁺ T cells (CD45⁺ CD19⁻ CD3⁺ CD8⁻ CD4⁺) that express H-2K^b after treatment with tamoxifen (closed bars) or vehicle (open bars; n = 10 mice per group). (B) Representative histograms showing H-2K^b expression on the indicated cell types. (C) Representative dot plots showing the percentage of splenic T cells (CD45⁺ CD19⁻ CD3⁺) that express CD4 or CD8. (D) Percentage of splenic T cells that express CD4 (n = 10 mice per group). (E) Number of CD4⁺ splenic T cells (n = 10 mice per group). Data in A, D, and E are combined from three independent experiments. (F and G) B2m^fl/fl and B2m^fl/fl CD4-Cre-ER^T2 mice were treated with tamoxifen or vehicle control on days 0–4 followed by i.v. injection of labeled donor splenocytes on day 14. Recipient spleens were harvested after 2 d to analyze donor cell recovery. (F) Representative histograms showing the relative percentages of transferred cells (CFSE⁺) recovered from the spleens of recipient mice. (G) Summary of NK cell–specific rejection of B2m^Δ/Δ donor cells (n = 4–6 mice per group). Data in G is combined from two independent experiments in which all recipients within an experiment received the same mix of donor cells. Statistical significance was calculated by two-way ANOVA with Bonferroni’s multiple comparisons test. Each symbol in A, D, E, and G represents an individual mouse. Error bars indicate mean ± SEM; **, P < 0.01; ****, P < 0.0001; ns, not significant. Tam, tamoxifen; Veh, vehicle.

Figure 6.

NK cell responsiveness and receptor repertoire are not affected by MHC-I down-regulation on CD4⁺ T cells. B2m^fl/fl, B2m^fl/fl CD4-Cre-ER^T2, and B2m^Δ/Δ mice were treated with tamoxifen or vehicle control on days 0–4, and splenocytes were harvested on day 14 for anti-NK1.1 stimulation and receptor repertoire analysis. (A) Representative dot plots showing the percentage of total NK cells (CD45⁺ CD3⁻ CD19⁻ NKp46⁺) that express Ly49C and IFN-γ after stimulation with plate-bound anti-NK1.1. (B) Summary of the percentage of total NK cells that express IFN-γ after stimulation with plate-bound anti-NK1.1 (n = 3 mice per group; one-way ANOVA with Bonferroni’s multiple comparisons test). (C) The Ly49C licensing ratio for total NK cells after stimulation with anti-NK1.1 (n = 3 mice per group; one-way ANOVA with Bonferroni’s multiple comparisons test). Data in B and C are representative of three independent experiments with three to four mice per group. (D) The percentage of NK cells (CD3⁻ CD19⁻ NK1.1⁺ NKp46⁺) that express the indicated receptor (n = 10 mice per group). Data in D are combined from three independent experiments. (E) Representative histograms showing expression of the indicated receptor on NK cells from B2m^fl/fl and B2m^fl/fl CD4-Cre-ER^T2 mice treated with tamoxifen. Each symbol in B–D represents an individual mouse. Error bars indicate mean ± SEM; ** P < 0.01; *** P < 0.001; ****, P < 0.0001; ns, not significant. Tam, tamoxifen; Veh, vehicle.

Figure 7.

MCMV infection and poly(I:C) induce missing-self reactivity toward MHC-I–deficient CD4⁺ T cells. (A–C) B2m^fl/fl and B2m^fl/fl CD4-Cre-ER^T2 mice were treated with tamoxifen on days 0–4, injected with PBS or anti-NK1.1 antibody on day 9, and infected with 5 × 10³ PFU of Δm157 MCMV on day 11. Splenocytes were harvested 4 d after infection for flow cytometry as depicted in A. Tam, tamoxifen; Veh, vehicle. (B) The percentage of CD4⁺ T cells (CD45⁺ CD19⁻ CD3⁺ CD8⁻ CD4⁺) that express H-2K^b in the spleens of mice 4 d after infection (n = 5–8 mice per group; two-way ANOVA with Bonferroni’s multiple comparisons test). (C) The number of splenic CD4⁺ T cells 4 d after infection (n = 5–8 mice per group; two-way ANOVA with Bonferroni’s multiple comparisons test). (D–H) B2m^fl/fl and B2m^fl/fl CD4-Cre-ER^T2 mice were treated with tamoxifen on days 0–4, followed by i.p. injection of poly(I:C) or PBS on days 6, 9, and 12. Mice were injected with anti-NK1.1 to deplete NK cells or with PBS control on day −2 and every 7 d after. (D) Representative dot plots showing CD4 and CD8 expression on peripheral blood T cells (CD45⁺ CD19⁻ CD3⁺) on days 0 and 14. (E) Percentage of blood T cells that express CD4 over time (n = 4–7 mice per group). (F) Representative histogram showing H-2K^b expression on day 14 splenic CD4⁺ T cells from anti-NK1.1– or PBS-treated B2m^fl/fl CD4-Cre-ER^T2 mice treated with tamoxifen and poly(I:C). (G) Percentage of splenic CD4⁺ T cells that express H-2K^b on day 14 (n = 4–9 mice per group; one-way ANOVA with Bonferroni’s multiple comparisons test). (H) Number of splenic CD4⁺ T cells on day 14 (n = 4–9 mice per group; one-way ANOVA with Bonferroni’s multiple comparisons test). Data in B, C, E, G, and H are combined from two independent experiments. Each symbol in B, C, G, and H represents an individual mouse. Error bars indicate mean ± SEM; *, P < 0.05; ****, P < 0.0001.

Figure 8.

IL-15 agonist induces missing-self reactivity toward MHC-I–deficient CD4⁺ T cells. B2m^fl/fl CD4-Cre-ER^T2 mice were treated with tamoxifen on days 0–4 and i.p. injected with IL-15 + IL-15Rα-Fc (0.6 µg + 3 µg) on days 6, 8, and 10. Mice were injected with anti-NK1.1 or PBS on days 4, 5, and 12. (A) Representative dot plots showing CD4 and CD8 expression on CD45⁺ blood lymphocytes. (B) Percentage of CD45⁺ blood lymphocytes that express CD4 over time. (C) Percentage of blood CD4⁺ T cells (CD45⁺ CD8⁻ CD4⁺) that express H-2K^b over time. (D) Representative dot plot showing CD4 and CD8 expression by day 14 spleen T cells (CD45⁺ CD19⁻ NK1.1⁻ CD1d-Tet⁻ CD3⁺). (E) Number of splenic CD4⁺ T cells on day 14. (F) Representative histogram showing H-2K^b expression on day 14 splenic CD4⁺ T cells. (G) Percentage of splenic CD4⁺ T cells that express H-2K^b on day 14. Data in B, C, E, and G are combined from two independent experiments (n = 6 mice per group; unpaired t test). Each symbol in E and G represents an individual mouse. Error bars indicate mean ± SEM; ****, P < 0.0001.