Listeria monocytogenes infection enhances the interaction between rat non-classical MHC-Ib molecule and Ly49 receptors

Abstract

Murine NK cell Ly49 receptors, functionally analogous to KIRs in humans recognize MHC class I molecules and play a key role in controlling NK cell function. We have previously shown that the paired activating Ly49s4 and inhibitory Ly49i4 receptors recognize undefined non-classical MHC-Ib ligands from the RT1-CE region in rats. Here, the RT1-CE16 gene of the RT1^d haplotype was stably transfected into the mouse RAW macrophage cell line, termed RAW-CE16^d cells. Combining RAW-CE16^d cells with Ly49 expressing reporter cells demonstrated Ly49i4 and Ly49s4 specificity for CE16^d. The Ly49s4/i4:CE16^d interaction was confirmed by specific MHC-I blocking monoclonal Abs. Further, we used our in vitro model to study the effect of Listeria monocytogenes (LM) on CE16^d after infection. LM infection and IFN-γ stimulation both led to enhanced CE16^d expression on the surface of transfected RAW-CE16^d cells. Interestingly, the reporter cells displayed increased response to LM-infected RAW-CE16^d cells compared with IFN-γ-treated RAW-CE16^d cells, suggesting a fundamental difference between these stimuli in supporting enhanced Ly49 recognition of CE16^d. Collectively, our data show that Ly49s4 and Ly49i4 recognize the non-classical RT1-CE16^d molecule, which in turn is up-regulated during LM infection and thereby may contribute to NK-mediated responses against infected cells.

Keywords: CE16 molecule; Listeria monocytogenes; Ly49; MHC-Ib; NK cells; RT1-CEd.

Figure 1.

Expression of rat CE16^d on CE16^d-transfected mouse RAW cells. Mouse RAW cells (upper panel), or RAW cells transfected to express rat CE16^d (RAW-CE16^d, lower panel) were surface stained with the following mAbs specific for rat (solid lines); anti-FLAG (M2, left panel), anti-MHC-I (classical MHC-Ia and non-classical MHC-Ib, OX18, middle panel), or anti-MHC-Ib (AAS5, right panel) for flow cytometric analysis. Negative secondary Ab control is depicted in stippled line. Shown are representative plots from four independent experiments.

Figure 2.

LM infection and IFN-γ stimulation enhance expression of CE16^d on RAW-CE16^d cells. (a) Representative histogram overlays of LM-infected (gray shaded), IFN-γ-stimulated (solid thick line) and untreated (solid thin line) RAW-CE16^d cells. Negative secondary Ab control is denoted (stippled line). Staining with anti-FLAG (left panel) and the anti-MHC-Ib mAbs AAS1 (middle panel) or AAS5 (right panel) are presented. (b) Summary data show geometric mean (gMFI) ± SEM of RAW-CE16^d cells treated as described above versus untreated controls, stained with AAS1 (left panel) and AAS5 (right panel). The plots and values shown are representative of four independent experiments. P < 0.05 with Student’s t-test.

Figure 3.

Ly49s4 recognizes the non-classical class I molecule CE16^d and mediates a stronger response to CE16^d upon LM infection. (a) BWZ-Ly49s4 reporter cells were incubated with untransfected RAW cells (all in beige bars) or RAW-CE16^d cells; uninfected (white bars), treated with IFN-γ (light gray bars)or infected with LM (black bars). For the blocking experiments, cell cultures were incubated with 3 µg/well of purified mAbs, or 10 µl of Ab-containing hybridoma supernatants. Stimulation of reporter cells with Ionomycin served as a positive control (dark gray bar). Responses were measured in β-galactosidase activity. All experiments were performed in triplicates and quantitative data of five independent experiments are presented. P < 0.5, P < 0.01 with Student’s t-test. (b) A representative visualization of BWZ-Ly49s4 reporter cell responses against RAW-CE16^d displaying color development in microwells is shown.

Figure 4.

Ly49s4, but not Ly49s5 recognizes CE16^d, and Ly49s4 recognition of CE16^d is enhanced with live but not heat-killed LM treatment of CE16^d expressing cells. (a) A representative experiment of RAW-CE16^d cells in triplicates co-incubated with Ly49s4 reporter cells (white bars) or Ly49s5 reporter cells (black bars). Stimulation with Ionomycin (positive control), uninfected (negative control), IFN-ɣ treatment and LM infection are indicated and identical to those in 3 a. (b) A representative experiment of BWZ-Ly49s4 reporter cells in triplicates with heat-killed HkLM treated RAW-CE16^d cells (gray bars) compared with uninfected (white bars), IFN-γ-treated (light gray bars) or live LM-infected (black bars) is shown. Data shown are representative of three independent experiments.

Figure 5.

The ectodomain of the Ly49i4 inhibitory receptor recognizes CE16^d. (a) Dot plots of the Ly49i4-CD3ζ chimera-expressing reporter cells treated with medium (negative control) or Ionomycin (positive control) (upper row), co-incubated with RAW-CE16^d cells (first panel), that were uninfected (second row), treated with IFN-γ (third row), exposed to HkLM (fourth row) or infected with live LM (fifth row). Parallel co-incubations performed with the indicated MHC-specific blocking Abs to define the ligand specificity of the response included: 10 µl of Ab-containing hybridoma supernatants OX18 (second panel column), 3 µg/well of purified AAS1 (third panel column) and 3 µg/well of purified AAS6 (fourth panel column). Responses were measured in EGFP fluorescence. The plots and values shown are representative of four independent experiments. (b) Statistical significance between Ly49i4 reporter cells in co-culture with CE16^d expressing cells uninfected (white bars), treated with IFN-γ (light gray bars), infected with heat-killed LM (dark gray bars) or live LM (black bars) was determined with a two-tailed unpaired Student’s t-test (P < 0.02). Data, summarized from four independent experiments, represent means ± SEM of standardized reporter cell EGFP expression.