Figure 2
Figure 2. Patient's perforin mutations, D49N and K285del, are detrimental to NK cell cytotoxicity. (A) KHYG1 cells were virally transduced with nonsilencing (ns) shRNA or shPRF1 constructs to down-regulate perforin expression. Perforin expression was then restored in KHYG1 shPRF1 cells by virally transducing cells with WT-perforin construct (KHGY1 shPRF1+ WT). KHYG1 shPRF1 cells were also transduced with the empty vector for control (KHYG1 shPRF1+ GFP). (i) Western immunoblot shows the relative amounts of perforin expression in each of the stable KHYG1 cell populations. (ii) Four-hour 51Cr release assays, against target K562 cells at the effector/target (E:T) ratios indicated, show a 90% reduction in function in KHYG1 shPRF1 cells and full restoration in KHYG1 shPRF1+ WT cells. Data are mean ± SE of 8 independent experiments. (Bi) Table shows patients 1 to 9 identified in the literature who inherited deletion mutations in the intensely basic (282KKKKHK) region of PRF1. (ii-iv) KHYG1 shPRF1 cells were virally transduced with K285del-perforin, sorted on the basis of identical mean GFP fluorescence (compared with KHYG1 shPRF1+ GFP and KHYG1 shPRF1+ WT cells), and then analyzed for perforin expression and cytotoxicity. For clarity, background levels seen for KHYG1 shPRF1+ GFP cells were subtracted from total 51Cr release levels (to reflect the activity of reintroduced recombinant perforin) and standardized against WT-perforin at a 10:1 E/T ratio. Data are mean ± SE of 3 independent experiments. (Ci-iii) KHYG1 shPRF1 cells were virally transduced with D49N- and D49NT51D-perforin, sorted on the basis of identical mean GFP fluorescence (compared with KHYG1 shPRF1+ GFP and KHYG1 shPRF1+ WT cells), and then analyzed for perforin expression and cytotoxicity. The values plotted represent standardized 51Cr release levels, as described in subpanels ii through iv. The data shown are mean ± SE of 7 independent experiments. (iii inset) Perforin-expressing KHYG1 shPRF1 cells were sorted to achieve identical protein expression and analyzed for perforin cytotoxicity. The values plotted represent standardized 51Cr release levels (as described in subpanels ii-iv). Data are mean ± SE of 3 to 5 independent experiments for each cell line. Corresponding Western blots are shown in supplemental Figure 4. (iv) Table lists FHL patients who inherited putative gain of glycosylation mutations in perforin.

Patient's perforin mutations, D49N and K285del, are detrimental to NK cell cytotoxicity. (A) KHYG1 cells were virally transduced with nonsilencing (ns) shRNA or shPRF1 constructs to down-regulate perforin expression. Perforin expression was then restored in KHYG1 shPRF1 cells by virally transducing cells with WT-perforin construct (KHGY1 shPRF1+ WT). KHYG1 shPRF1 cells were also transduced with the empty vector for control (KHYG1 shPRF1+ GFP). (i) Western immunoblot shows the relative amounts of perforin expression in each of the stable KHYG1 cell populations. (ii) Four-hour 51Cr release assays, against target K562 cells at the effector/target (E:T) ratios indicated, show a 90% reduction in function in KHYG1 shPRF1 cells and full restoration in KHYG1 shPRF1+ WT cells. Data are mean ± SE of 8 independent experiments. (Bi) Table shows patients 1 to 9 identified in the literature who inherited deletion mutations in the intensely basic (282KKKKHK) region of PRF1. (ii-iv) KHYG1 shPRF1 cells were virally transduced with K285del-perforin, sorted on the basis of identical mean GFP fluorescence (compared with KHYG1 shPRF1+ GFP and KHYG1 shPRF1+ WT cells), and then analyzed for perforin expression and cytotoxicity. For clarity, background levels seen for KHYG1 shPRF1+ GFP cells were subtracted from total 51Cr release levels (to reflect the activity of reintroduced recombinant perforin) and standardized against WT-perforin at a 10:1 E/T ratio. Data are mean ± SE of 3 independent experiments. (Ci-iii) KHYG1 shPRF1 cells were virally transduced with D49N- and D49NT51D-perforin, sorted on the basis of identical mean GFP fluorescence (compared with KHYG1 shPRF1+ GFP and KHYG1 shPRF1+ WT cells), and then analyzed for perforin expression and cytotoxicity. The values plotted represent standardized 51Cr release levels, as described in subpanels ii through iv. The data shown are mean ± SE of 7 independent experiments. (iii inset) Perforin-expressing KHYG1 shPRF1 cells were sorted to achieve identical protein expression and analyzed for perforin cytotoxicity. The values plotted represent standardized 51Cr release levels (as described in subpanels ii-iv). Data are mean ± SE of 3 to 5 independent experiments for each cell line. Corresponding Western blots are shown in supplemental Figure 4. (iv) Table lists FHL patients who inherited putative gain of glycosylation mutations in perforin.

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