2B4 inhibits NK-cell fratricide

Abstract

2B4 (CD244) and its ligand, CD48, are expressed on all natural killer (NK) cells. In studies using 2B4-deficient, CD48-deficient, or wild-type NK cells with blocking antibodies, we found that in the absence of 2B4-CD48 interactions, activated murine NK cells kill each other. We also show that NK-NK fratricide in the absence of 2B4-CD48 interaction is dependent on perforin both in vitro and in vivo. 2B4 has been reported to have activating, costimulatory, and inhibitory functions on murine NK cells. 2B4-mediated inhibition of NK-cell fratricide explains some of the paradoxes of 2B4 function reported in studies of murine NK cells. We show that in the absence of 2B4 signaling, activated NK cells have defective cytotoxicity and proliferation because of fratricide and not due to the absence of a 2B4-dependent activation signal.

Figure 1

In the absence of 2B4-CD48 interactions, activated NK cells undergo perforin-dependent fratricide. LAK cells activated with IL-2 in vitro were used as effectors and targets in cytotoxicity assays. (A) WT and 2B4 KO LAK cells were used as effectors against WT LAK cells. (B) WT LAK cells were used as effectors against WT and CD48 KO LAK cells. (C) WT LAK cells were used as effectors against untreated, or anti-CD48 antibody–coated WT, or β2m KO LAK cells. To coat LAK targets, cells were incubated for 15 minutes in 10 μg/mL anti-CD48 antibody at room temperature. Coated cells were then washed and used in killing assays. (D) WT and perforin KO LAK cells were loaded with chromium and incubated in the presence of anti-CD16/CD32 blocking antibody plus anti-2B4, anti-CD48, anti-NK1.1, or anti-H-2K^b antibody. Chromium release, which indicates lysis due to fratricide, was measured after 6 hours of incubation. (E) WT and perforin KO LAK cells were incubated in the presence of anti-CD16/CD32 blocking antibody plus anti-2B4, anti-CD48, or anti-NK1.1 antibody. Annexin V and PI staining was measured after 6 to 7 hours of incubation with blocking antibody. The percents of Annexin V⁺, PI⁺ apoptotic cells are noted in the upper right quadrants of the FACS dot plots. Results are representative of 4 independent experiments. Error bars are standard deviation (SD).

Figure 2

Perforin-dependent fratricide causes defective NK function in the absence of 2B4-CD48 interactions. (A) WT and perforin KO LAK cells were stimulated with anti-NK1.1 mAb–coated plates in the presence of anti-2B4 or anti-CD48 blocking antibodies. After 6 hours of stimulation, culture supernatants were measured for granzyme secretion via the BLT esterase assay. *P < .05. Results are representative of 3 independent experiments. (B) WT and perforin KO NK cells were cultured in complete media supplemented with IL-2 in the presence of anti-2B4 or anti-CD48 blocking antibodies. Thymidine incorporation was measured at different times during culture. Results are representative of 3 independent experiments. (C) Five days after injection with CpG, NK cells from WT, 2B4 KO, perf KO, and perf/2B4 DKO mice were counted. Fold expansions were calculated by dividing NK numbers of mice injected with CpG by NK numbers in noninjected control mice; n=3 mice per group, and data are representative of 2 independent experiments. Error bars are SD.