Acquisition of external major histocompatibility complex class I molecules by natural killer cells expressing inhibitory Ly49 receptors

Abstract

Murine natural killer (NK) cells express inhibitory Ly49 receptors specific for major histocompatibility complex (MHC) class I molecules. We report that during interactions with cells in the environment, NK cells acquired MHC class I ligands from surrounding cells in a Ly49-specific fashion and displayed them at the cell surface. Ligand acquisition sometimes reached 20% of the MHC class I expression on surrounding cells, involved transfer of the entire MHC class I protein to the NK cell, and was independent of whether or not the NK cell expressed the MHC class I ligand itself. We also present indirect evidence for spontaneous MHC class I acquisition in vivo, as well as describe an in vitro coculture system with transfected cells in which the same phenomenon occurred. Functional studies in the latter model showed that uptake of H-2D(d) by Ly49A+ NK cells was accompanied by a partial inactivation of cytotoxic activity in the NK cell, as tested against H-2D(d)-negative target cells. In addition, ligand acquisition did not abrogate the ability of Ly49A+ NK cells to receive inhibitory signals from external H-2D(d) molecules. This study is the first to describe ligand acquisition by NK cells, which parallels recently described phenomena in T and B cells.

Figure 1.

(A) Ly49A⁺ cells acquire H-2D^d after transfer into BALB/c mice. 20–50 × 10⁶ NWNA splenocytes were inoculated into BALB.B and BALB/c, respectively. After 16–18 h, spleens were recovered and NK1.1⁺ cells were analyzed for their expression of Ly49A and H-2D^d. The density plots show cells gated on the NK1.1⁺ population. The figure shows one representative experiment out of 45. (B) H-2D^d is acquired at high levels 20 min after transfer of B6 cells into BALB/c. Spleens were recovered after transfer at indicated time points and H-2D^d expression on NK1.1⁺Ly49A⁺ B6 cells after transfer to BALB/c and BALB.B is shown. (C) Ly49A⁺ NK1.1⁺ cells lose H-2D^d expression after one night in culture. B6 cells were transferred into BALB/c mice and spleens were recovered after 18 h. Cells were cultured in rIL-2 and analyzed for H-2D^d expression at indicated time points.

Figure 1.

(A) Ly49A⁺ cells acquire H-2D^d after transfer into BALB/c mice. 20–50 × 10⁶ NWNA splenocytes were inoculated into BALB.B and BALB/c, respectively. After 16–18 h, spleens were recovered and NK1.1⁺ cells were analyzed for their expression of Ly49A and H-2D^d. The density plots show cells gated on the NK1.1⁺ population. The figure shows one representative experiment out of 45. (B) H-2D^d is acquired at high levels 20 min after transfer of B6 cells into BALB/c. Spleens were recovered after transfer at indicated time points and H-2D^d expression on NK1.1⁺Ly49A⁺ B6 cells after transfer to BALB/c and BALB.B is shown. (C) Ly49A⁺ NK1.1⁺ cells lose H-2D^d expression after one night in culture. B6 cells were transferred into BALB/c mice and spleens were recovered after 18 h. Cells were cultured in rIL-2 and analyzed for H-2D^d expression at indicated time points.

Figure 1.

(A) Ly49A⁺ cells acquire H-2D^d after transfer into BALB/c mice. 20–50 × 10⁶ NWNA splenocytes were inoculated into BALB.B and BALB/c, respectively. After 16–18 h, spleens were recovered and NK1.1⁺ cells were analyzed for their expression of Ly49A and H-2D^d. The density plots show cells gated on the NK1.1⁺ population. The figure shows one representative experiment out of 45. (B) H-2D^d is acquired at high levels 20 min after transfer of B6 cells into BALB/c. Spleens were recovered after transfer at indicated time points and H-2D^d expression on NK1.1⁺Ly49A⁺ B6 cells after transfer to BALB/c and BALB.B is shown. (C) Ly49A⁺ NK1.1⁺ cells lose H-2D^d expression after one night in culture. B6 cells were transferred into BALB/c mice and spleens were recovered after 18 h. Cells were cultured in rIL-2 and analyzed for H-2D^d expression at indicated time points.

Figure 2.

(A) Expression of H-2D^d, H-2K^d, and H-2L^d on NK1.1⁺ B6 cells after transfer to BALB/c mice. MHC class I expression is indicated as percentage of the respective MHC class I expression in BALB/c (100%). Each bar represent the mean value from four individual mice from two experiments and SDs are indicated as bars. Paired Student's t tests were used to compare the relative fluorescence intensities between Ly49A⁺ and Ly49A⁻ cell populations after they had been normalized against BALB/c. P < 0.01 for H-2D^d, P < 0.05 for H-2K^d, and nonsignificant results for H-2L^d. (B) Comparison of H-2D^d acquisition among three different H-2D^d–binding Ly49 receptors. Density plots showing H-2D^d expression on Ly49A, G2, and D populations after B6->BALB/c transfer. All cells are gated on NK1.1.

Figure 2.

(A) Expression of H-2D^d, H-2K^d, and H-2L^d on NK1.1⁺ B6 cells after transfer to BALB/c mice. MHC class I expression is indicated as percentage of the respective MHC class I expression in BALB/c (100%). Each bar represent the mean value from four individual mice from two experiments and SDs are indicated as bars. Paired Student's t tests were used to compare the relative fluorescence intensities between Ly49A⁺ and Ly49A⁻ cell populations after they had been normalized against BALB/c. P < 0.01 for H-2D^d, P < 0.05 for H-2K^d, and nonsignificant results for H-2L^d. (B) Comparison of H-2D^d acquisition among three different H-2D^d–binding Ly49 receptors. Density plots showing H-2D^d expression on Ly49A, G2, and D populations after B6->BALB/c transfer. All cells are gated on NK1.1.

Figure 3.

Western blot analysis showing the H-2D^d protein from sorted B6VA49A cells after transfer to BALB/c. CFSE-labeled B6VA49A cells were transferred into BALB/c and sorted on a FACSVantage™. Immunoprecipitation with an anti–H-2D^d mAb was performed followed by Western blot analysis. 1, 1.5 × 10⁶ untransferred B6VA49A cells; 2, 1.5 × 10⁶ sorted B6VA49A cells after transfer to BALB/c; 3, 1.5 × 10⁶ BALB/c cells; 4, 5.0 × 10⁶ BALB/c cells; 5, mixture of 98% B6VA49A cells and 2% BALB/c cells (1.5 × 10⁶ cells in total). The figure shows one experiment out of four.

Figure 4.

(A). Downregulation of Ly49A and Ly49C on TAP1⁻/β₂m⁻ NK cells transferred to BALB/c, BALB.B, and (BALB/c × BALB.B)F₁ recipients. Black bars show median fluorescence intensity before transfer, white bars after transfer to BALB.b, gray bars after transfer to BALB/c and hatched bars after transfer to (BALB/c × BALB.B)F₁. (B) TAP1⁻/β₂m⁻/Ly49A⁺ cells acquire H-2D^d but not H-2K^b after transfer of NK cells to (BALB/c × BALB.B)F₁ mice. In the same transfer, Ly49C⁺ NK cells acquire H-2K^b and some H-2D^d. Histograms show H-2D^d and H-2K^b expression on Ly49A⁺ (bold lines) and Ly49A⁻ (thin lines) cells, and on Ly49C⁺ (bold lines) and Ly49C⁻ (thin lines). All cells are gated on NK1.1. The figure shows one experiment out of four.

Figure 4.

(A). Downregulation of Ly49A and Ly49C on TAP1⁻/β₂m⁻ NK cells transferred to BALB/c, BALB.B, and (BALB/c × BALB.B)F₁ recipients. Black bars show median fluorescence intensity before transfer, white bars after transfer to BALB.b, gray bars after transfer to BALB/c and hatched bars after transfer to (BALB/c × BALB.B)F₁. (B) TAP1⁻/β₂m⁻/Ly49A⁺ cells acquire H-2D^d but not H-2K^b after transfer of NK cells to (BALB/c × BALB.B)F₁ mice. In the same transfer, Ly49C⁺ NK cells acquire H-2K^b and some H-2D^d. Histograms show H-2D^d and H-2K^b expression on Ly49A⁺ (bold lines) and Ly49A⁻ (thin lines) cells, and on Ly49C⁺ (bold lines) and Ly49C⁻ (thin lines). All cells are gated on NK1.1. The figure shows one experiment out of four.

Figure 5.

(A) Staining of mosaic DL6 mice with NK1.1 and H-2D^d reveals three populations with different H-2D^d levels; a low (R1), intermediate (R2), and a high (R3) population. (B) Percentage of Ly49A⁺ cells among NK1.1⁺ cells in the three populations. The graph shows a summary of 16 consecutive experiments including 16 individual mice. Error bars show SD. Paired Student's t tests were used to compare the amount of Ly49A-positive cells in the intermediate population R2 compared with the negative population R1 (P < 0.001) and between the intermediate population R2 compared with the positive population R3 (P < 0.001).

Figure 5.

(A) Staining of mosaic DL6 mice with NK1.1 and H-2D^d reveals three populations with different H-2D^d levels; a low (R1), intermediate (R2), and a high (R3) population. (B) Percentage of Ly49A⁺ cells among NK1.1⁺ cells in the three populations. The graph shows a summary of 16 consecutive experiments including 16 individual mice. Error bars show SD. Paired Student's t tests were used to compare the amount of Ly49A-positive cells in the intermediate population R2 compared with the negative population R1 (P < 0.001) and between the intermediate population R2 compared with the positive population R3 (P < 0.001).

Figure 6.

(A) Acquisition of H-2D^d by DL1 NK cells after transfer to BALB/c mice. Histogram overlays show H-2D^d expression on Ly49A⁺ (bold line) and Ly49A⁻ (thin line) DL1 cells after transfer to BALB/c. H-2D^d expression on DL1 cells is measured with a mAb recognizing α₃ of H-2D^d in order to distinguish H-2D^d derived from BALB/c since DL1 cells express a chimeric H-2D^d molecule containing α₃ from H-2L^d. (B) H-2D^d expression on Ly49A⁺ and Ly49A⁻ NK cells from H-2D^d transgenic D8 mice. The figure shows six individual mice. In both A and B, cells gated on NK1.1⁺ cells.

Figure 6.

(A) Acquisition of H-2D^d by DL1 NK cells after transfer to BALB/c mice. Histogram overlays show H-2D^d expression on Ly49A⁺ (bold line) and Ly49A⁻ (thin line) DL1 cells after transfer to BALB/c. H-2D^d expression on DL1 cells is measured with a mAb recognizing α₃ of H-2D^d in order to distinguish H-2D^d derived from BALB/c since DL1 cells express a chimeric H-2D^d molecule containing α₃ from H-2L^d. (B) H-2D^d expression on Ly49A⁺ and Ly49A⁻ NK cells from H-2D^d transgenic D8 mice. The figure shows six individual mice. In both A and B, cells gated on NK1.1⁺ cells.

Figure 7.

(A) Acquisition of H-2D^d on RNK.Ly49A cells. EL-4D^d cells were labeled with CFSE and cocultured overnight with RNK.Ly49A or RNK-16 effector cells and analyzed by flow cytometry using anti–H-2D^d mAb plus goat anti–mouse biotin antiserum plus streptavidin-PE, as described previously. Panels show H-2D^d stainings of cell mixtures containing EL-4D^d cells cocultured with RNK-16 cells (left) or RNK.Ly49A (right), respectively. One experiment of five. (B) H-2D^d staining of RNK.Ly49A cells cultured overnight alone (dotted line), together with EL-4 (thin solid line) or together with EL-4D^d (thick solid line). (C) Relative acquisition of H-2D^d by RNK.Ly49A and RNK-16 cells after cocultures with EL-4D^d in the presence of antibodies against Ly49A or either one of the classical MHC class I molecules expressed on EL4-D^d: H-2K^b, H-2D^b, and H-2D^d. Acquisition of H-2D^d by RNK.Ly49A was only prevented when antibodies against either Ly49A or H-2D^d was added to the culture. H-2D^d staining is plotted relative (fold-increase) to the staining of RNK.Ly49A or RNK-16 cells cultured alone in the same experiment. (D) Kinetics of H-2D^d uptake. Median values of H-2D^d staining on gated RNK-16 or RNK.Ly49A cells after coculture with EL-4D^d cells for various time periods. One experiment of four with similar results.

Figure 8.

RNK.Ly49A cells acquire H-2D^d-molecules in a patch-like pattern on the cell surface. CFSE (green) labeled RNK.Ly49A (a-i) and RNK-16 (j-r) cells were cocultured with unlabeled EL-4D^d cells. All cells were stained with α-H-2D^d (red) mAb. The cells were analyzed on a LSCM and displayed as a series of sequential cross-sections. Each image represents one RNK and one EL-4D^d cell. Acquired H-2D^d-molecules are observed as red dots on the green RNK.Ly49A cells, as indicated by the arrow in c. Scale bar is 5 μm.

Figure 9.

Reduced cytotoxic activity by sorted RNK.Ly49A cells after acquisition of H-2D^d. Lysis of ⁵¹Cr-labeled YB2/0 cells and YB2/0-D^d cells by sorted RNK.Ly49A cells cocultured overnight with EL-4 (white squares), EL-4D^d (black squares), and no cells (white circles). The figure shows one representative experiment of four.