Ly49P recognition of cytomegalovirus-infected cells expressing H2-Dk and CMV-encoded m04 correlates with the NK cell antiviral response

Abstract

Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-D(k) and the MCMV m04 protein. Using H2 chimeras between H2-D(b) and -D(k), we demonstrate that the H2-D(k) peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04-deletion mutant (Deltam04) virus infection. MA/My mice, which express Ly49P and H2-D(k), are resistant to MCMV; however, infection with Deltam04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Deltam04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatibility complex class I-restricted recognition of virally infected cells by an activating NK cell receptor.

Figure 1.

Activation via Ly49P requires H2-D^k. (A) H2^k proteins were detected on MCMV-infected cells. BALB.K MEFs were infected with GFP-MCMV (MOI 0.5 and 0.1) and stained with mAbs against: H2-D^k, H2-K^k, or an isotype-matched control antibody. GFP-DIM MCMV-infected cells expressing H2-D^K and H2-K^K molecules are shown in circles. (B) NIH3T3 cells were transduced with H2^k-encoding retroviruses and their surface expression was analyzed. BALB.K MEFs were a positive control. (C) NKL-Ly49P cells were co-cultured with uninfected (white bars) or MCMV-infected (black bars) cells. MCMV-infected or uninfected MEF.K, NIH3T3 cells (Ø), or NIH3T3 cells transduced with H2-D^k and/or -K^k were used as stimulators. IFN-γ was detected by ELISA. (D) NKL-Ly49H cells were a positive control. Data are representative of four independent experiments. Error bars represent the SD.

Figure 2.

α1α2 of H2-D^k are necessary for Ly49P recognition. (A) Schematic representation. H2-D^k is represented in gray, H2-D^b in white, and β₂m as white dotted circle. (B) Surface expression of the native H2 and chimeras on NIH3T3 cells was assayed using antibodies against the α1α2 domains of H2-D^k and/or -D^b. (C) NKL-Ly49P cells were co-cultured with uninfected (white bars) or MCMV-infected (black bars) cells. MCMV-infected or uninfected NIH3T3 cells expressing native H2 or chimeras were stimulators. Significant (P < 0.01) activation of NKL-Ly49P cells depended on the presence of α1α2 domains of H2-D^k on MCMV-infected cells. (D) NKL-Ly49H cells were a control. Data are representative of three independent experiments.

Figure 3.

Ly49P-mediated recognition requires m04. Ly49P or Ly49H NFAT-GFP reporter cells were co-cultured with MEF.K infected with MCMV-deletion mutants and GFP expression was assessed (shaded histograms, infected; open histograms, uninfected). Data are representative of four independent experiments.

Figure 4.

m04 complements Δm04 MCMV infection in Ly49P reporter cell assays. (A) Transient m04 transfection complements the Δm04 viral infection in the Ly49P reporter assays. Ly49P reporter cells were co-cultured with MEF.K either untransfected or transfected with m04 (+m04), m90 (+m90), or m157 (+m157). Stimulators were uninfected or infected with WT or Δm04 MCMV. (B) Stable m04 expression complements the Δm04 viral infection in the Ly49P reporter assays. Ly49P reporter cells were co-cultured with mRFP (+mRFP) or V5-m04 (+V5-m04) positive MEF.K cells. Stimulators were treated as in A. The histogram shows mRFP expression in mRFP-MEF.K cells (black) and untransduced cells (gray). (C) H2^k and V5-m04 are expressed on the surface of MCMV-infected cells. Uninfected and WT or Δm04 MCMV-infected V5-m04 MEF.K cells used in the reporter assay shown in (B) were costained with anti-H2-D^k, anti-V5, and anti-m06. Plots represent live-gated cells. (D) Top histograms display V5-m04 expression (black) in uninfected V5-m04 MEF.K (left) and V5-m04 NIH3T3-H2-D^k cells (right), compared with untransfected cells (gray). Middle and bottom histograms display WT or Δm04 MCMV-infected cells gated on the top right (D^k+m06⁺) and bottom right (D^k+m06⁻) quadrants of untransfected MEF.K (gray) and V5-m04 MEF.K (black) cells. The percentage of V5-m04⁺ cells in each gated population is indicated. Data are representative of three independent experiments.

Figure 5.

m04 deletion abrogates NK-cell mediated MA/My resistance. (A) MA/My mice were infected with 5 × 10⁵ PFU of WT or Δm04 MCMV. 4 d a.i., viral loads in the spleen and liver were assessed. (B) MA/My and B6 mice were either untreated or depleted of NK cells 48 h before infection. 4 d a.i., viral titers in the spleen were assessed. Three to four mice per group were used. Data are representative of three independent experiments.