![The effect of the L135P mutation on STX11 function. (A) Clinical data from the patient at first presentation (16 mo old) and second presentation (24 mo old). (B–C) A 51Cr-release assay performed on days 4 (B) and 7 (C) using activated Stx11-KO murine CD8+ T cells show a rescue of cytotoxic function when WT STX11 is expressed, but reduced cytotoxicity when STX11-L135P is expressed. The assay was done using CD8+ T cells from Bl/6.OT1 mice following their incubation with SIINFEKL-treated syngeneic EL4 target cells. Data represent mean ± SEM from n = 3 independent experiments. NT, non-targeting gRNA-transfected cells (as per [3]). (D) Degranulation was measured by the externalization of CD107a on CD8+ T cells from BL/6.OT1 mice following their incubation with SIINFEKL-treated syngeneic EL4 targets. Reduced levels of degranulation were observed in Stx11-KO CD8+ T cells expressing STX11-L135P on both days 4 and 7, compared to cells expressing WT STX11 (STX11 WT). Shown is a representative result of three independent experiments. NT, non-targeting gRNA-transfected cells. (E–H) Western immunoblot analysis of recombinant human STX11 in various cell types. β-actin was used as a loading control. NT, non-targeting gRNA-transfected cells. (E) Expression of recombinant STX11-WT and STX11-L135P in Stx11-KOmurine primary CD8+ T cells. Shown is a representative result of three independent experiments. NT, non-targeting gRNA-transfected cells. (F) Expression of recombinant STX11-WT and STX11-L135P in STX11-KO human primary CD8+ T cells. Shown is a representative result of two independent experiments. NT, non-targeting gRNA-transfected cells. (G) Expression of recombinant STX11-WT and STX11-L135P in human fibroblast cell line BJ3 with or without transduction of recombinant STXBP2-WT. Shown is a representative result of three independent experiments. (H) Quantification of three independent western blots, one of which is shown in Fig. 1 G. The left plot shows the fold increase in expression of the STX11-L135P mutant relative to STX11-WT in BJ3 cells, either without STXBP2 (7.0 ± 1.1, mean ± SEM, N = 3) or with STXBP2 overexpression (6.9 ± 0.4, mean ± SEM, N = 3). The right plot shows actin-normalized expression levels of STX11-WT and STX11-L135P, with expression differences of 17.9 ± 1.5 (without STXBP2 overexpression) and 17.7 ± 1.4 (with STXBP2 overexpression) (mean ± SEM, N = 3), respectively. E:T, Effector:target. Source data are available for this figure: SourceData F1.](https://cdn.rupress.org/rup/content_public/journal/jhi/1/4/10.70962_jhi.20250100/1/jhi_20250100_fig1.png?Expires=1768235340&Signature=BWGRoor7DIWYgdeRdngnOUZkPbCz9zbuP~1a3CrkYaIpuguxPkS~IRw47RnJerW-p6LseL5NVoZXDj12xliD1BFzPPAN3UdE6XQ6K6msZDBBNCUe-yi3opBkdmqkdk3r6Ra0-M9RBaphhujwNvB9n0YRyFH24QwbcRuFDPUuA3T1J5IivIebpySH8i8JF2TpVzXDHZzUaS6bMQL0SJrqt3B7YmDmIzphvHNf8kiU635EfUAVOznhbikFzYltAuBfgZByStcpxw9CAV8NnyWdj2tiJKa3ofEk3PEdEQn5PtCtpIjiAVY8pyDsH537LpBuXV7Z5flzOxOmaWO1xmh1rQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The effect of the L135P mutation on STX11 function. (A) Clinical data from the patient at first presentation (16 mo old) and second presentation (24 mo old). (B–C) A 51Cr-release assay performed on days 4 (B) and 7 (C) using activated Stx11-KO murine CD8+ T cells show a rescue of cytotoxic function when WT STX11 is expressed, but reduced cytotoxicity when STX11-L135P is expressed. The assay was done using CD8+ T cells from Bl/6.OT1 mice following their incubation with SIINFEKL-treated syngeneic EL4 target cells. Data represent mean ± SEM from n = 3 independent experiments. NT, non-targeting gRNA-transfected cells (as per [3]). (D) Degranulation was measured by the externalization of CD107a on CD8+ T cells from BL/6.OT1 mice following their incubation with SIINFEKL-treated syngeneic EL4 targets. Reduced levels of degranulation were observed in Stx11-KO CD8+ T cells expressing STX11-L135P on both days 4 and 7, compared to cells expressing WT STX11 (STX11 WT). Shown is a representative result of three independent experiments. NT, non-targeting gRNA-transfected cells. (E–H) Western immunoblot analysis of recombinant human STX11 in various cell types. β-actin was used as a loading control. NT, non-targeting gRNA-transfected cells. (E) Expression of recombinant STX11-WT and STX11-L135P in Stx11-KOmurine primary CD8+ T cells. Shown is a representative result of three independent experiments. NT, non-targeting gRNA-transfected cells. (F) Expression of recombinant STX11-WT and STX11-L135P in STX11-KO human primary CD8+ T cells. Shown is a representative result of two independent experiments. NT, non-targeting gRNA-transfected cells. (G) Expression of recombinant STX11-WT and STX11-L135P in human fibroblast cell line BJ3 with or without transduction of recombinant STXBP2-WT. Shown is a representative result of three independent experiments. (H) Quantification of three independent western blots, one of which is shown in Fig. 1 G. The left plot shows the fold increase in expression of the STX11-L135P mutant relative to STX11-WT in BJ3 cells, either without STXBP2 (7.0 ± 1.1, mean ± SEM, N = 3) or with STXBP2 overexpression (6.9 ± 0.4, mean ± SEM, N = 3). The right plot shows actin-normalized expression levels of STX11-WT and STX11-L135P, with expression differences of 17.9 ± 1.5 (without STXBP2 overexpression) and 17.7 ± 1.4 (with STXBP2 overexpression) (mean ± SEM, N = 3), respectively. E:T, Effector:target. Source data are available for this figure: SourceData F1.