KIR2DL2 does not inhibit TCR-mediated cytotoxicity. (A) The level of KIR2DL2/2DL3/2DS2 expression on CD4⁺CD28^– T cells and CD56⁺ NK clones were examined by FACS analysis. (B) KIR⁺ CD4⁺CD28^– T-cell clones were tested for the presence of KIR2DL2, KIR2DL3, and KIR2DS2 mRNA by PCR. (C) The ability of KIR2DL2 to inhibit either T-cell–mediated (top panel) or NK cell–mediated (bottom panel) cytotoxicity was examined in a 4-hour cytotoxicity assay against anti-CD3 (•) and anti-CD3 + anti-KIR2DL2/2DL3/2DS2 (○) or anti-CD16 (▪) and anti-CD16 + anti-KIR2DL2/2DL3/2DS2 (□)–labeled P815 cells. Both anti-CD3 and anti-CD16 were tested over a wide dose range, and selected concentrations are shown. (D) The effect of KIR2DL2 on CD4⁺CD28– T cells on granule exocytosis was measured in 4-hour BLT release assay against either anti-CD3 and anti-CD3 + anti-KIR2DL2-coated P815 cells (left panel) or SEB-coated 721.221 and 721.221/HLA-Cw3 cells (right panel). Results are shown as mean ± SD of triplicate cultures.

KIR2DL2 does not affect the formation of a mature activating immune synapse. (A) Alexa488-conjugated CTXβ-labeled T cells and Alexa647-conjugated CTXβ target cells (721.221 [left panel] or 721.221/HLA-Cw3 [right panel]) were coincubated at a ratio of 1:2 in the absence or presence of the superantigen SEB (2 ng/mL) in the absence of IL-2. At the indicated time points, the percentage of T cells in conjugates was determined by FACS analysis. Results shown as means of triplicate measurements are representative of 3 experiments. (B) T cells labeled with Alexa555-conjugated CTXβ were coincubated with HLA-Cw3–positive and –negative target cells and SEB as described in panel A. Images of multiple fields were obtained by a blinded observer using a Zeiss LSM 510 confocal microscope equipped with a 63 ×/1.4 oil-immersion objective lens after 30 minutes of incubation, and conjugate formation and lipid raft clustering were analyzed by counting at least 100 T cells in different fields. Results are expressed as the percentage of T cells that were in conjugates with target cells (left panel) and the percentage of conjugates that showed lipid raft clustering at the T-cell–target-cell contact area (right panel). Results are representative of 3 experiments. (C) T cells were labeled with Alexa555-conjugated CTXβ (red; top panel) or with Alexa488-labeled CD3 mAbs (green; bottom panel). T cells and SEB-coated 721.221/HLA-Cw3 target cells were mixed at a 1:2 ratio and centrifuged for optimal cell-cell contact formation. Cells were examined at room temperature using a Zeiss LSM 510 confocal microscope equipped with a 100 ×/1.4 oil-immersion objective lens 15 minutes after initial cell contact. (D) HLA-Cw3 was visualized with Alexa488-labeled anti–HLA-ABC antibody (green), and CD3 was detected with Alexa555-labeled anti-CD3 mAb (red). Conjugates were formed and examined as described for panel C. (E) Lipid rafts (top panel) and CD3 (bottom panel) were visualized with Alexa555 (red). LFA-1 was detected by Alexa488-conjugated CD11a mAb (green). Conjugates were formed and examined as described in panel C. Taken together, the images demonstrate synapse formation with rafts and CD3 in the cSMAC; and KIR2DL2, HLA-Cw3, and LFA-1 in the pSMAC after 15 minutes. All confocal pictures are representative of at least 3 experiments with at least 5 contact areas analyzed in each experiment. Results are shown as mean ± SD of triplicate experiments.

Recruitment of KIR2DL2 to the cSMAC is delayed. (A) KIR2DL2 was visualized by Alexa555-labeled CH-L, and HLA-Cw3 was detected by Alexa488-labeled anti–MHC class I mAb. KIR2DL2⁺ CD4⁺ T cells were pelleted with 721.221/HLA-Cw3 target cells in the presence of 2 ng/mL SEB. The cells were transferred to optical dishes, and the movement of KIR2DL2 and HLA-Cw3 was observed using a confocal microscope at room temperature. Representative x-y sections for KIR2DL2 (red) and HLA-Cw3 (green) are shown for 10, 20, and 30 minutes (left 2 columns). The middle 3 columns show z-axis image reconstruction in the plane of the contact site. Line plots show a quantitative image analysis in this plane, demonstrating that the green and red fluorescence colocalize in the pSMAC after 10 minutes and in the cSMAC after 20 minutes. (B) T cells were stained with Alexa488-labeled anti-CD3 mAb and Alexa555-labeled anti-KIR2DS2/2DL2/2DL3 mAb and then coincubated with HLA-Cw3–expressing target cells as described in panel A. Representative sections at 2 time points are shown. At 10 minutes, CD3 (green) localized to the cSMAC, while KIR2DL2 (red) was found in the pSMAC. At 30 minutes, both molecules were in the cSMAC. Representative x-y section images and z-axis–reconstructed images in the plane of contact site are shown for both time points. (C) T cells were stained with Alexa555-labeled anti-CD3 mAb, and target cells (721.221/HLA-Cw3) were stained with Alexa488-labeled anti–MHC class I mAb. At 10 minutes, CD3 (red) localized to the cSMAC, while HLA-Cw3 (green) was found in the pSMAC. At 30 minutes, both molecules were in the cSMAC. Representative x-y section images and z-axis–reconstructed images in the plane of contact site are shown for both timepoints. For all panels, conjugates were formed and examined as described for Figure 2C.

KIR2DL2 affects early and late TCR-mediated signaling events differently. (A) KIR2DL2⁺ T cell clones were stimulated with SEB-coated 721.221 or 721.221/HLA-Cw3 target cells for the indicated time points. Cell lysates were tested for phosphorylated ZAP70, phosphorylated PLC-γ1, and phosphorylated Vav1. (B) KIR2DL2⁺ T-cell clones were stimulated with 1 μg/mL control IgG, 1 μg/mL, anti-CD3 + 1 μg/mL IgG, or 1 μg/mL anti-CD3 + 1 μg/mL anti-KIR2DL2 for the indicated timepoints. The amount of total and phosphorylated ERK1/2 was determined by ELISA. The amount of phosphorylated ERK1/2 relative to the amount of total ERK1/2 is shown as mean of triplicate measurements. Results are representative of 3 experiments. (C) KIR2DL2⁺ T-cell clones were stimulated with SEB-coated 721.221 or 721.221/HLA-Cw3 target cells for 60, 90, and 120 minutes. Cell lysates were tested for phosphorylated PLC-γ1 and phosphorylated Vav1. (D) KIR2DL2⁺ T-cell clones were stimulated as in panel B and the level of ERK1/2 phosphorylation was established as in panel B. Results are shown as mean ± SD of triplicate measurements.

KIR2DL2 inhibits TCR-mediated IFN-γ production and cell proliferation. (A) KIR2DL2⁺ T cells were stimulated with SEB-coated 721.221 or 721.221/HLA-Cw3 target cells. Gene expression was profiled at 0, 4, and 24 hours. Only those genes that showed at least a 3-fold increase in expression after activation were selected, and the expression levels of these genes under the 2 culture conditions were compared. KIR2DL2 engagement by HLA-Cw3 reduced the stimulation-induced up-regulation of the majority of genes. (B) Induction of IFN-γ was found to be strongly inhibited by gene array. To confirm this result, KIR2DL2⁺ T cells were incubated with anti-CD3 + IgG (150 ng/mL) or anti-CD3 + anti-KIR2DL2 (150 ng/mL) coated P815 cells (top panel); or with SEB-coated 721.221 or 721.221/HLA-Cw3 target cells (bottom panel) for 48 hours. IFN-γ in the supernatant was quantified by ELISA. Results are shown as mean ± SD of triplicate cultures representative of 5 experiments. (C) CFSE-labeled KIR2DL2⁺ T cells were incubated with SEB-coated 721.221 or 721.221/HLA-Cw3 target cells for 5 days. The percentage of T-cell proliferating was determined by FACS analysis. Results are shown as mean and SD of triplicates representative of 3 experiments.

KIR2DL2 decreases immune synapse stability. (A) Lipid rafts were visualized with Alexa555-conjugated CTXβ (red) in KIR2DL2⁺ T cells pelleted with SEB-coated 721.221 or 721.221/HLA-Cw3 target cells. The conjugates were transferred to optical dishes, and the kinetics of rafts (red) disbanding in contact areas was monitored by confocal microscopy. A representative picture after 90 minutes is shown. At that time, the raft persisted in conjugates with MHC class I–negative cells (top panel) but had resolved in conjugates with HLA-Cw3–positive cells (bottom panel). (B) Cells were prepared and treated as described in panel A, and images of 10 fields were visualized with a 63 ×/1.4 oil-immersion objective lens. The percentage of T cells in conjugates with target cells (top panel) and the percentage of conjugates with lipid rafts at the contact zone (bottom panel) were determined. Results are shown as mean ± SD of 3 independent experiments.

Sustained TCR-mediated signaling is required for the transcriptional activation of the IFN-γ gene. KIR2DL2⁺ T cells were incubated with SEB-coated 721.221 or 721.221/HLA-Cw3 target cells. Anti–MHC class II monoclonal antibodies (L243) were added to the cultures at the specified time points, and the cultures were continued for a total of 48 hours. Supernatants were harvested, and IFN-γ was quantified by ELISA. Results are shown as mean ± SD of triplicate cultures. TCR synapse dissociation within the first 90 minutes mimics the inhibitory effect of KIR2DL2.