Fig 1: Expression of pQa-1 bound with Qdm/Qdm-like peptide inhibits NKG2A+ NK cell activation and prevents tumor rejection in vivo.C57BL/6 splenocytes were co-cultured with CHO cells expressing the indicated constructs and the NK cells were subsequently analyzed by flow cytometry (A–F). The ratio of IFN? production between NKG2A+ and NKG2A- NK cells is shown in the bar graphs. (A) Representative dot plots of NK cell IFN? production upon stimulation with CHO cells expressing vector, pQa-1, or Qa-1 in the presence of Qdm-k peptide (AMVPRTLLL). (B) Splenocytes were co-cultured with indicated CHO cells in the presence of Qdm-k or control peptide. (C) Co-cultures were performed as in (B) in the presence of isotype or 20D5 (anti-NKG2A/C/E) antibody. (D) IFN? and (E) CD107a expression by NK cells in response to CHO cells expressing pQa-1 single chain trimer (pQa-1-SCT) was performed as in (C). (F) Same experiment as in (D) and (E) was conducted after CHO-pQa-1-SCT and CHO-V cells were treated with or without 0.5 U/ml PI-PLC. (A–F) Representative experiments are shown from two to three independent experiments per panel. Bars in the figures represent mean ±SEM of duplicates. (G) 5000 Qa-1-restricted Ln12 T cells were co-cultured overnight with titrating amounts of human T2 cells (TAP-deficient cells) expressing vector, Qa-1, or pQa-1. The amount of IFN? in the supernatants was determined by ELISA. EC7.1-Qa-1, a mouse TAP- and MHC-Ia-deficient lymphoma cell line transduced to express Qa-1 served as a positive control. Mean ±SD of triplicates is shown. (H) Lung metastasis formation 14 days after intravenous injection B16F10 melanoma cells expressing pQa-dtSCT or vector control (B16-pQa-1 or B16-V). Dots over each bar represent individual mice, cumulative data from two independent experiments. Two-tailed unpaired t test was used (*=p < 0.05; **=p < 0.01).
Fig 2: Downregulation of MHC-I by pK3 leads to NK killing susceptibility, while pQa-1 co-expression provides protection in an NKG2A-dependent manner.(A) The GFP+ target cells (RMA cells expressing pK3 or pK3 RM by IRES-GFP vector or co-expressing pK3 and pQa-1) were mixed with the effectors (NK cells isolated from wildtype C57BL/6 or NKG2A-/- mice and activated by IL-2 for 6 days) at indicated E:T ratio and cultured at 37°C for 4 hr before stained with propidium iodide (PI) and analyzed by flow cytometry. NK killing%=[PI+ target %/(PI+ target%+PI- target%)*100]. Bars represent mean ±SD of four replicates. Dunnett's multiple comparisons test is used. (B) Working model for how RHVP-encoded pK3 and pQa-1 work in concert to evade CTL and NK killing. In normal circumstances, cytotoxic lymphocytes can survey foreign antigen through TCR recognition of MHC-I/peptide complexes at the cell surface, and NK cells can sense defects in antigen presentation and processing via engagement of HLA-E/Qa-1 by the inhibitory receptor CD94/NKG2A (left panel). In RHVP-infected cells, pK3 induces rapid degradation of MHC-I in the ER thus preventing CTL activation and clearance of infected cells; On the other hand, pQa-1 lacks an MHC-I like TM region and is thus resistant to pK3, which allows its cell surface expression and CD94/NKG2A engagement thus undermining ‘missing self’ recognition by NK cells (right panel).
Fig 3: The Qdm-ß2m-pQa-1 single chain trimer (SCT) specifically recognizes the CD94/NKG2A receptor.(A) Spleen lymphocytes isolated from wildtype C57BL/6 or NKG2A-/- knockout mice were stained with PE-labeled pQa-1-dtSCT tetramer (PE-pQa-1 Tet), or PE-labeled streptavidin (PE-SA) as negative control, at room temperature for 1 hr followed by staining with a mixture of fluorochrome labeled antibodies containing either anti-NKG2A/C/E (20D5) or isotype control for 30 min. The stained cells were acquired by BD Canto II and data were analyzed with FlowJo software. Representative data of four (wildtype) and two (NKG2A-/-) independent experiments are shown. NK cells were defined as the live NK1.1+CD3-CD19- population, while CD8 T cells were gated on the live CD19- CD3+CD8+ population.
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