Natural killer cell activation enhances immune pathology and promotes chronic infection by limiting CD8+ T-cell immunity

Abstract

Infections with HIV, hepatitis B virus, and hepatitis C virus can turn into chronic infections, which currently affect more than 500 million patients worldwide. It is generally thought that virus-mediated T-cell exhaustion limits T-cell function, thus promoting chronic disease. Here we demonstrate that natural killer (NK) cells have a negative impact on the development of T-cell immunity by using the murine lymphocytic choriomeningitis virus. NK cell-deficient (Nfil3(-/-), E4BP4(-/-)) mice exhibited a higher virus-specific T-cell response. In addition, NK cell depletion caused enhanced T-cell immunity in WT mice, which led to rapid virus control and prevented chronic infection in lymphocytic choriomeningitis virus clone 13- and reduced viral load in DOCILE-infected animals. Further experiments showed that NKG2D triggered regulatory NK cell functions, which were mediated by perforin, and limited T-cell responses. Therefore, we identified an important role of regulatory NK cells in limiting T-cell immunity during virus infection.

Fig. 1.

NK cells are activated after LCMV infection. (A) Splenocytes from naïve (Left), LCMV-Armstrong–infected (Center), and LCMV clone 13-infected (Right) mice were incubated with YAC-1 cells at the indicated effector/target ratios. ⁵¹Cr release at different effector/target ratios is shown (n = 3, mean ± SEM of duplicates). (B and C) NK1.1⁺CD3e⁻ splenocytes were analyzed by flow cytometry for surface expression of NKG2D (B) and 2B4 (C) at 2 d after infection with 2 × 10⁶ pfu of LCMV WE (n = 6; Student's t test). NKG2D expression (mean fluorescence intensity) (B) and 2B4 negative (%) (C) NK1.1⁺CD3⁻ cells are shown (n = 6; Student's t test).

Fig. 2.

NK cells limit CD8⁺ T-cell response in vivo and in vitro. WT and NK cell-depleted mice were infected with 2 × 10⁶ pfu of LCMV WE. (A) gp33-specific (Left) and np396-specific (Right) tetramer response in the spleen (% of CD8⁺ T cells) at the indicated time (days) after infection are shown (P < 0.01 for day 6 and P < 0.001 for day 8 gp33 tetramers; P < 0.001 for day 6 and 8 for np396 tetramers; two-way ANOVA, n = 6 of two independent experiments). (B) IFN-γ⁺CD8⁺ of all CD8⁺ splenocytes are shown after restimulation with gp33 (Left) or np396 (Right) peptide (P < 0.001 for days 6 and 8 postinfection, two-way ANOVA, n = 6). (C) Control or NK cell-depleted mice were infected with 200 pfu of LCMV WE. At 8 d postinfection, splenocytes were restimulated with the LCMV-specific epitope gp33, and IFN-γ production was determined (n = 7–8; two-way ANOVA). (D) A total of 2 × 10⁶ negatively sorted, CD45.1⁺ P14 T cells were transferred at 2 d postinfection with 200 pfu of LCMV WE into control and NK cell-depleted mice. CD45.1⁺CD8⁺ population was analyzed (Left; n = 6; P < 0.05 for aNK1.1 from naïve and control group using one-way ANOVA), and NKG2D–hIgG expression was measured (one representative of n = 3–6 is shown). (E) Negatively sorted T cells were activated with anti-CD3/CD28 antibodies and coincubated with purified NK cells from naïve or LCMV-infected mice (day 2, 2 × 10⁶ pfu) or naïve NK cells stimulated with IFN-α4. CD8⁺ T-cell number was compared after 72 h (n = 3–4; *P < 0.05, one-way ANOVA). (F) Nfil3^−/− and nfil3^+/− mice were infected with 2 × 10⁵ pfu of LCMV WE. At 6 d after infection, splenocytes were restimulated in vitro with the virus-specific peptides gp33 and np396, and IFN-γ production was determined by intracellular cytokine staining and FCM analysis (n = 5–6 of two independent experiments; P < 0.01 for gp33 and np396, two-way ANOVA). (G) WT or NK cell-depleted mice were infected with 2 × 10⁶ pfu of LCMV clone 13. Splenocytes from control or NK cell-depleted mice were restimulated ex vivo with the virus-derived peptides gp33 and np396 at 10 d after infection. Intracellular production of IFN-γ by CD8⁺ T cells was measured by FCM analysis (n = 6 of two independent experiments; P < 0.001 for gp33 and np396, two-way ANOVA).

Fig. 3.

NKG2D triggers NK_reg functions. (A) Mice were treated with NKG2D-blocking antibody or isotype control (day −1, day 2 postinfection) and infected with 200 pfu of LCMV WE (day 0). On day 2, 2 × 10⁶ negatively sorted, CFSE-labeled, and congenically marked P14 T cells were transferred into animals of both groups. On day 4 after infection, CFSE expression and expansion of CD45.1⁺CD8⁺ cells were determined (n = 3–4; Student's t test). (B and C) Mice were treated with NKG2D-blocking antibody or an isotype control (day −1, 12 h postinfection) and infected with 2 × 10⁶ pfu of LCMV WE. gp33-tetramer⁺ was measured at 8 d after infection (n = 3; Student's t test) (B), and IFN-γ production was determined after restimulation with the virus-specific epitopes gp33 and np396 (C) (n = 3; two-way ANOVA).

Fig. 4.

NK cell-mediated perforin production limits T-cell immunity. (A and B) Splenocytes (50,000) from P14⁺CD45.1⁺ mice were transferred into perforin^−/− and WT mice and infected with 2 × 10⁶ pfu of LCMV WE. (A) At 8 d postinfection, IFN-γ production of CD8⁺ T cells after restimulation with gp33 was determined in single-cell suspensions of spleen and liver tissue (n = 6; P < 0.01, Student's t test). (B) One group was treated with an NK cell-depleting antibody, and IFN-γ production was monitored (n = 5–6; *P < 0.05 for aNK1.1, perforin^−/−, and aNK1.1 perforin^−/− vs. control, Newman–Keuls one-way ANOVA). (C) A total of 2 × 10⁶ negatively sorted, CFSE-labeled T cells from P14⁺CD45.1⁺ mice were transferred into WT and perforin^−/− mice on day 2 postinfection with 200 pfu of LCMV WE. (Left) After 48 h, CFSE expression of the CD45.1⁺CD8⁺ population was analyzed. (Right) Relative amount (%) of CD45.1⁺CD8⁺ cells in perforin^−/− mice compared with WT mice (n = 4; P < 0.01, Student's t test).

Fig. 5.

NK cells promote development of chronic infection and immunopathology. (A) WT or NK cell-depleted mice were infected with 2 × 10⁶ pfu of LCMV clone 13. Virus titers were determined from spleen, liver, kidney, and lung tissue at 10 d after infection [n = 8 of two independent experiments; P < 0.001 except liver (P < 0.05) of ₁₀log(Titer), Student's t test]. (B) Control and NK cell-depleted mice were infected with 10⁵ pfu of LCMV DOCILE. Virus titers were measured after 33 d (n = 5, Student's t test). (C–E) WT or NK cell-depleted mice were infected with 2 × 10⁶ pfu of LCMV WE. (C) At 6 and 8 d postinfection, virus titers were analyzed in spleen tissue. (n = 6). *P < 0.05, significant difference for ₁₀log(Titer), Student's t test. (D) Liver from control and NK cell-depleted mice were analyzed for LCMV nucleoprotein expression by immunohistology. One representative picture is shown (n = 5). (E) WT mice or NK cell-depleted mice were infected with 2 × 10⁶ pfu of LCMV. Hepatitis induction was assessed by measuring ALT activity in the sera at different time points (n = 2–11; P < 0.05 for day 10 and P < 0.01 for day 12 postinfection, two-way ANOVA). (F) Nfil3^−/− and nfil3^+/− mice were infected with 2 × 10⁵ pfu of LCMV WE. ALT activity in the serum of nfil3^−/− and nfil3^+/− mice was determined at 11 d postinfection (n = 7–8; P < 0.01 using Student's t test).