Contribution of Thy1+ NK cells to protective IFN-γ production during Salmonella typhimurium infections

Abstract

IFN-γ is critical for immunity against infections with intracellular pathogens, such as Salmonella enterica. However, which of the many cell types capable of producing IFN-γ controls Salmonella infections remains unclear. Using a mouse model of systemic Salmonella infection, we observed that only a lack of all lymphocytes or CD90 (Thy1)(+) cells, but not the absence of T cells, Retinoic acid-related orphan receptor (ROR)-γt-dependent lymphocytes, (NK)1.1(+) cells, natural killer T (NKT), and/or B cells alone, replicated the highly susceptible phenotype of IFN-γ-deficient mice to Salmonella infection. A combination of antibody depletions and adoptive transfer experiments revealed that early protective IFN-γ was provided by Thy1-expressing natural killer (NK) cells and that these cells improved antibacterial immunity through the provision of IFN-γ. Further analysis of NK cells producing IFN-γ in response to Salmonella indicated that less mature NK cells were more efficient at mediating antibacterial effector function than terminally differentiated NK cells. Inspired by recent reports of Thy1(+) NK cells contributing to immune memory, we analyzed their role in secondary protection against otherwise lethal WT Salmonella infections. Notably, we observed that a newly generated Salmonella vaccine strain not only conferred superior protection compared with conventional regimens but that this enhanced efficiency of recall immunity was afforded by incorporating CD4(-)CD8(-)Thy1(+) cells into the secondary response. Taken together, these findings demonstrate that Thy1-expressing NK cells play an important role in antibacterial immunity.

Fig. 1.

Thy1-expressing CD3⁻CD4⁻CD8⁻ cells are required for early control of S. Typhimurium infection. (A and B) C57BL/6, Rag1/Je^−/−, Rag2^−/−γc^−/−, IFN-γ^−/−, CD1d^−/−, µMT, ROR-γt^−/−, GK1.5Tg, GK1.5/2.43Tg mice were infected i.v. with 200 cfu S. Typhimurium BRD509. Survival was assessed (A) and/or bacterial numbers in the spleen were determined on day 21 after infection (B). (C–F) GK1.5Tg (C and D) and GK1.5/2.43Tg (E and F) mice were injected i.p. with PBS or antibodies against mouse CD8 (2.43), Thy1 (30-H12), or IFN-γ (HB-170-15). Forty-eight hours later mice were infected i.v. with 200 cfu S. Typhimurium BRD509. Injection of depleting antibodies was continued twice weekly for 3 wk. Mice were culled on day 21 after infection, and bacterial numbers (C and E) and serum IFN-γ (D and F) were assessed. Data are representative of at least two pooled independent experiments. Individual data points (C–F) and mean ± SEM of at least six mice per group (B) and at least seven mice per group (A) are shown. Statistical analyses: paired Student t test (E and F) and one-way ANOVA followed by Bonferroni multiple comparison test (B–D). ***P < 0.001, **P < 0.01.

Fig. 2.

NK cell precursors and immature NK cells express Thy1 and are depleted by anti-Thy1 antibodies. (A) GK1.5Tg and (B) GK1.5/2.43Tg mice were injected with antibodies and infected with S. Typhimurium BRD509 as in Fig. 1. Splenic NK cell (CD3⁻DX5⁺NK1.1⁺) numbers were enumerated at day 21 after infection. (C–E) B6 mice were treated i.p. with PBS or anti-Thy1 (30H-12) twice weekly for 3 wk. CD27^hi/lo and CD11b^hi/lo splenic NK cells (CD3⁻DX5⁺NK1.1⁺) were analyzed for the expression of Thy1 (black histograms) 48 h after the last antibody treatment (C). DX5^+/− and NK1.1^+/− splenic NK cell precursor subsets (CD19⁻CD3⁻CD4⁻β-TCR⁻CD122⁺) were analyzed for the expression of Thy1 (black histograms) and enumerated at 48 h (D) or at different time points over 3 wk (E) after injection of anti-Thy1. Representative FACS plots (C and D) and mean cell numbers ± SEM of at least five mice (A–E) are shown. Data are representative of at least two independent experiments. Cells gated on viable single lymphocytes. Statistical analyses: paired Student t test for individual cell populations (B–D), one-way ANOVA (A), or two-way ANOVA followed by Bonferroni multiple comparison test (E). ***P < 0.001, **P < 0.01, *P < 0.05.

Fig. 3.

Thy1⁺ NK cells produce IFN-γ and contribute to Salmonella control. (A and B) Rag1/Je^−/− mice were injected i.v. with 1 × 10⁸ cfu heat-killed S. Typhimurium (HKST), and IFN-γ secretion by CD3⁻NK1.1⁺ cells was assessed 2 h later in the spleen (A). IFN-γ⁺ (black) and IFN-γ⁻ (gray) NK 1.1⁺ cells were assessed for expression of Thy1 (B). (C–F) Rag1/Je^−/− mice were treated i.p. with PBS or anti-Thy1 (30H-12) and infected i.v. with 200 cfu S. Typhimurium BRD509 48 h later. Injection of depleting antibodies was continued twice weekly for 3 wk. Bacterial numbers in spleen (E) and liver (F), serum IFN-γ levels (D), and NK cell numbers (C) were assessed on day 21 after infection. Data are representative of two independent experiments. Representative FACS plot (A) and histogram (B) or individual data points (C–F) are shown. Statistical analysis: paired Student t test. ***P < 0.001.

Fig. 4.

Transfer of IFN-γ–competent NK cells improves Salmonella control in Rag2^−/−γc^−/− mice: (A–D) 1 × 10⁶ purified in vitro–activated NK cells or PBS were injected i.v. into naïve Rag2^−/−γc^−/− mice on day 5 and 6 after in vitro culture. Mice were i.v. infected with 200 cfu BRD509 24 h later. Some mice were injected twice weekly i.p. with antibodies against mouse Thy1 (30-H12). Bacterial numbers in spleen (A) and liver (B) were assessed, and serum samples were analyzed for IFN-γ by cytometric bead array (C) at 23 d after infection. In separate experiments, survival (D) was assessed. Data are representative of at least two independent experiments. Survival plots of 10 mice (D) or individual pooled data points (A–C) are shown. Statistical analyzes: one-way ANOVA followed by Bonferroni multiple comparison test (A–C); log-rank (Mantel-Cox) test (D). ***P < 0.001, **P < 0.01.

Fig. 5.

NK cells are not essential for control of primary infection when other cell types are present. (A) B6 and CD1d^−/− mice were injected i.p. with PBS or antibodies against mouse NK1.1 (PK136) and infected i.v. with 200 cfu S. Typhimurium BRD509 48 h later. Injection of depleting antibodies or PBS was continued twice weekly for 3 wk. Splenic NK cell numbers (A) and bacterial counts in spleen (B) and liver (C) were determined on day 21 after infection. Data are representative of two pooled independent experiments with seven to eight mice per group. Statistical analyses: paired Student t test (A). ***P < 0.001, **P < 0.01.

Fig. 6.

Thy1⁺ NK cells are required for optimal vaccine-mediated protection. (A) B6 mice were orally infected with 5 × 10⁹ cfu BRD509, Δedd ΔpfkA ΔpfkB Salmonella, or left uninfected. Twelve weeks later, mice were orally infected with 1 × 10⁷ cfu WT Salmonella SL1344. Survival was assessed over 120 d. (B–D) Mice were vaccinated or left uninfected as in A. Two days before challenge with WT Salmonella SL1344, mice were treated i.p. with PBS or antibodies against mouse CD8 (2.43) and CD4 (GK1.5), NK1.1 (PK136), or Thy1 (30-H12). Antibody treatment was maintained twice weekly for the duration of the experiment. Survival was assessed over 15 d. Data are representative of two independent experiments with 9–16 mice per group. Statistical analyses: log-rank (Mantel-Cox) test. *P < 0.05.