Expression of dnDAP12 and/or dnDAP10 in CD8⁺ T cells from LGLL patients reduces adaptor signaling, cytotoxicity toward CRL-2598 cells, and granule redistribution. (A) Expression of dnDAP12 blocks Syk phosphorylation. AD293 cells were cotransfected with plasmids encoding Syk and 1 of the following FLAG-tagged DAP12 constructs: wild type (WT, lanes 2), DAP12^Y75A (lane 3), or DAP12^Y64A;Y75A (lane 4). Single transfections with Syk and WT DAP12 are included as negative controls (lanes 1, 5, and 6). After cross-linking with anti-Flag antibody, DAP12 was immunoprecipitated before Western blot analyses with an antiphosphotyrosine antibody (4G10). The blot was subsequently stripped and reprobed with an anti-Syk antibody confirming that the phosphorylated protein is Syk (data not shown). (B) Expression of dnDAP10 and dnDAP12 reduce the cytotoxic potential of CD8⁺ T cells from LGLL patients. The cytotoxic properties of genetically modified CD8⁺ T cells from LGLL patients were evaluated in 5-hour ⁵¹Cr release assays. CD8⁺ cells were infected with recombinant vaccinia virus encoding CD56 (negative control), dnDAP10, dnDAP12, or a combination of dnDAP10 and dnDAP12(Y75A), as well as dnMEK (positive control) and mixed at an E/T ratio of 50:1 with CRL-2598 target cells. Mock-infected CD8⁺ T cells from LGLL patients and healthy controls are included for comparison. Representative results from 1 LGLL patient are shown: cells from 5 other patients yielded similar results. The inhibitory effects of dnDAP10 and dnDAP12 occurred at E/T cell ratios ranging from 50:1 to 6:1 (data not shown). The mean and SEM of triplicate wells are shown. (C) Expression of dnDAP12 and/or dnDAP10 in CD8⁺ T cells from LGLL patients reduces cytotoxicity toward synovial cells (HTB293). Direct cytotoxicity was performed using 5-hour ⁵¹Cr release assays as described in Figure 7B. Results from 1 representative patient and healthy control are shown: similar results were obtained from 5 additional patients. The mean and SEM of triplicate wells are shown. (D) Target cell–induced granule redistribution is blocked in CD8⁺ T LGLL expressing dnDAP10 and/or dnDAP12. A proportion of the cells described in panel B were mixed at 1:1 E/T ratio and examined for granule redistribution. Cells were cytospun onto slides and lytic granules visualized with (i) a FITC-conjugated anti–granzyme B antibody (green). (ii) CRL-2598 target cells were prestained with Cell-Tracker Orange (red: Molecular Probes). DAPI was used for nuclear staining (blue) of both cell types. Results using untreated CD8⁺ T LGLL cells are shown at (iii) 0 and (iv) 10 minutes after mixing. Representative results using CD8⁺ T LGLL cells expressing (v) CD56 (negative control), (vi) dnDAP10, (vii) dnDAP12(Y75A), (viii) dnDAP10, and dnDAP12(Y75A) and (ix) dnMEK are shown. Results from 1 representative experiment are shown: results from 4 other patients yielded similar results (data not shown).