'Unlicensed' natural killer cells dominate the response to cytomegalovirus infection

Abstract

Natural killer (NK) cells expressing inhibitory receptors that bind to self major histocompatibility complex (MHC) class I are 'licensed', or rendered functionally more responsive to stimulation, whereas 'unlicensed' NK cells lacking receptors for self MHC class I are hyporesponsive. Here we show that contrary to the licensing hypothesis, unlicensed NK cells were the main mediators of NK cell-mediated control of mouse cytomegalovirus infection in vivo. Depletion of unlicensed NK cells impaired control of viral titers, but depletion of licensed NK cells did not. The transfer of unlicensed NK cells was more protective than was the transfer of licensed NK cells. Signaling by the tyrosine phosphatase SHP-1 limited the proliferation of licensed NK cells but not that of unlicensed NK cells during infection. Thus, unlicensed NK cells are critical for protection against viral infection.

Figure 1. MHC class I inhibition overrides NK cell licensing

(a) IFN-γproduction and degranulation by naïve Ly49H⁺ B6 NK cells stimulated ex vivo with RMA, RMA-S, RMA-S-m157, or RMA-m157 targets in the presence or absence of blocking antibody to H-2K^b. Plots are gated on Ly49H⁺ NK1.1⁺ cells. (b) Average IFN-γproduction and degranulation by Ly49H⁺ B6 NK cells from 4 mice stimulated with RMA-S-m157 or RMA-m157 targets in the presence or absence of blocking antibody to H-2K^b. Error bars indicate the standard error of the mean. (c) Average IFN-γproduction and degranulation by Ly49D⁺ B6 NK cells from 4 mice stimulated with RMA-Hm1-C4 targets in the presence or absence of blocking antibody to H-2K^b. Error bars indicate the standard error of the mean. Data are representative of five experiments with 3–4 mice per experiment

Figure 2. Licensed NK cells become under-represented during MCMV infection

(a) Unlicensed Ly49C/I⁻ (black line)and licensed Ly49C/I⁺ (grey fill) Ly49H⁺ NK cells from naïve (top row) or 36 hour MCMV-infected (bottom row). B6 mice were analyzed for CD69 upregulation and ex vivo IFN-γand granzyme B expression. Ly49C/Iexpression was analyzed on Ly49H⁺ NK cells from (b) B6 wild-type mice and (c) H2K^bD^b−/− animals prior to infection and 5 days after MCMV infection. Data are representative of three experiments with 3–4 mice per group.

Figure 3. Ly49C/I is stably expressed and limits proliferation of NK cells during MCMV infection

(a) CFSE dilution by Ly49C/I⁺Ly49H⁺ (gray fill) and Ly49C/I⁻ Ly49H⁺ (black line) NK cells in naïve recipients and 5 days after MCMV infection. Data are representative of five experiments with 3–4 animals each. (b) Ly49C/I expression on NK cells sorted as either Ly49G2⁺Ly49C/I⁻ (grey fill) or Ly49G2⁻ Ly49C/I⁺ (black line), transferred into naïve recipients, and analyzed for Ly49C/I expression six days after MCMV infection. Data are representative of two experiments.

Figure 4. Ly49C/I-mediated inhibition requires functional SHP-1

IFN-γ and degranulation by mixed bone marrow chimeric (a) Ly49H⁺ wild-type NK cells and (b) Ly49H⁺ SHP-1^Me-v NK cells stimulated with RMA-S-m157 or RMA-m157 targets in the presence or absence of blocking antibody to H-2K^b. Data are representative of three experiments with 3–4 mice per experiment. (c) Ly49C/Iexpression was analyzed on mixed bone marrow chimeric Ly49H⁺ wild-type and SHP-1^Me-v NK cells prior to infection and 5 days after MCMV infection. Data are representative of three experiments with 3–4 mice per group. (d) CFSE dilution by Ly49C/I⁺ (gray fill) and Ly49C/I⁻ (black line) mixed bone marrow chimeric Ly49H⁺ wild-type and SHP-1^Me-v NK cells 5 days after MCMV infection. Data are representative of three experiments with 2–3 animals each.

Figure 5. Unlicensed NK cells control MCMV infection

MCMV titers in the (a) salivary glands and (b) liver one-week post infection in B6 wild-type mice either untreated or depleted of Ly49C/I⁺ NK cells, Ly49G2⁺ NK cells, or all NK cells. Data are representative of two experiments with five mice per group. MCMV titers in the (c) salivary glands and (d) liver one-week post infection of B6 wild-type mice and B2m^−/− mice, both depleted of CD8⁺ T cells. Data are representative of two experiments with five mice per group.

Figure 6. Licensed NK cells do not protect neonates from MCMV infection

Ly49h^−/− neonates received (a) 1 × 10⁵ Ly49H⁺ NK cells sorted as either Ly49C/I⁺ or Ly49G2⁺Ly49C/I⁻ or PBS or (b) 7.5 × 10⁴ Ly49H⁺ NK cells from donors pre-depleted of Ly49C/I⁺ or Ly49G2⁺ cells in vivo or PBS and challenged with 2 × 10³ pfu of MCMV. Animals were monitored daily for morbidity.