Detection of NKR-P1B ligands. (A) BWZ.CD3ζ/P1B cells were stimulated with plate-bound anti-NKR-P1B/C mAb (PK136) or control Abs, including isotype control (IgG2aκ) mAb (100 μg/ml), an anti-NKR-P1A/D mAb (10A7; GenBank accession no. AF342896), and purified total mouse IgG. Stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (IONO) served as a control. (B) BWZ.CD3ζ/P1B cells stimulated with various cell lines and normal cells. We cocultured 5 × 10⁴ each of BWZ.CD3ζ/P1B cells and stimulator cells overnight. (C) Upper shows staining of cell lines with NKR-P1B tetramers (shaded region) or phycoerythrin-conjugated streptavidin alone (region outlined in black). Lower shows that binding is blocked with 4A6 mAb (cells preincubated with 50 μg/ml purified 4A6 mAb for 30 min on ice). (D) Response of BWZ.CD3ζ/P1B cells is blocked with 4A6 mAb or PK136 (anti-NKR-P1B) mAb but not with control mAbs. We cocultured 5 × 10⁴ BWZ.CD3ζ/P1B cells with equal numbers of MNK-1 stimulator cells.

Ocil/Clr-b cDNA clone encoding a ligand for the NKR-P1 inhibitory receptors. (A) The 4A6 staining of 293T cells transiently transfected 48 h earlier with the pMXS expression vector containing the 1B8 cDNA. Isotype control Ab DX5 did not stain the cells, as shown in the other channel. (B) Ocil/Clr-b, but not Clr-f or Clr-g, is a ligand of NKR-P1B and NKR-P1D. We used 293T cells that had been transiently transfected with expression plasmids encoding Ocil/Clr-b, Clr-f, or Clr-g to stimulate an equal number of BWZ indicator cells expressing chimeric NKR-P1A, NKR-P1B, NKR-P1C, or NKR-P1D receptors. (C) The transiently transfected cells used as stimulator cells in B were stained 18 h after transfection with 4A6 mAb (10D7 mAb served as a control). Markers were placed according to staining of untransfected 293T cells.

Expression pattern of Ocil/Clr-b on hematopoietic cells. (A) Adult spleen cells express high levels of Ocil/Clr-b ex vivo. (B) Staining of adult thymocyte subsets reveals reduced expression on DP cells (DN, CD4^-CD8^-; DP, CD4⁺CD8⁺; CD4SP, CD4⁺CD8^-; and CD8SP, CD4^-CD8⁺). (C) High Ocil/Clr-b expression in CD45⁺ fetal liver cells but not in CD45^- fetal liver cells (enriched in erythroid lineage cells). Fetal liver cells (day 14 of gestation) were analyzed by flow cytometry without Lympholyte enrichment. (D) Ocil/Clr-b expression on spleen cells is unaltered in β₂m^-/- mice.

Ocil/Clr-b is frequently down-regulated on mouse tumor cells. The cell lines or cell populations indicated were stained with biotin-conjugated 4A6 and phycoerythrin-conjugated streptavidin secondary (shaded region) or phycoerythrin-conjugated streptavidin alone (region outlined in black) and analyzed by flow cytometry.

Ocil/Clr-b expression imparts resistance of tumor target cells to natural killing. (A) BWZ cells, selected to express endogenous (AKR strain) Ocil/Clr-b, efficiently engage NKR-P1B but not NKR-P1D. BWZ.Ocil⁺ cells (see Fig. 8C), or control BWZ cells, were tested for stimulation of BWZ reporter cells expressing different NKR-P1 fusion receptors. (B) Expression of Ocil/Clr-b (endogenous isoform) protects BWZ targets from lysis by NK cells from CD1 or NIH Swiss mice, which express NKR-P1B, but it has no effect on lysis by NK cells from B6 mice, which express NKR-P1D and not NKR-P1B. Day 4-6 LAK cells from each strain were used as effector cells with BWZ.Ocil⁺ and parental BWZ(-) target cells in ⁵¹Cr-release assays. Results are representative of three independent experiments. (C) Ab to Ocil/Clr-b reverses resistance of BWZ.Ocil cells to NK cells expressing NKR-P1B. LAK cells were used as effectors against BWZ.Ocil and parental BWZ(-) cells in ⁵¹Cr-release assays. Anti-Ocil/Clr-b 4A6 mAb (50 μg/ml) reverses resistance of BWZ.Ocil cells to lysis by CD1 LAK cells.