Fig 1: Integrin αL (CD11a) mutant alleles are LOF in vitro.(A) Schematic representation of the LFA-1 integrin heterodimer. (B) WT integrin αL and predicted impact of the patients’ variants. (C) Protein immunoblots of αL and β2 in HEK293T cells transfected with αL variants and β2. MWs (in kilodalton) are shown on the right. GAPDH, glyceraldehyde phosphate dehydrogenase. (D) Surface expression of LFA-1 subunits in HeLa cells transfected with αL variants and β2-YFP, assessed by flow cytometry. See also fig. S2E for quantification. (E) Surface (S) and intracellular (I) αL/β2 levels in HEK293T cells transfected with αL variants and β2 (mean; N = 3). See also fig. S2D. MFI, median fluorescence intensity. (F) Confocal images of HeLa cells transfected with αL-CFP (blue) and β2-YFP (yellow), with ER (red) and membrane (white) staining. Scale bars, 10 μm. (G) Protein immunoblots of αL and β2 after IP with an anti–LFA-1 (TS2/4) antibody in HEK293T cells transfected with αL and β2. (H) Protein immunoblots of αL and β2 after IP with an anti-HA antibody in HEK293T cells transfected with αL and β2-HA. (I) Homotypic aggregation of αL-KO Jurkat cells transduced with αL variants, induced by Mg2+/EGTA or activating anti-β2 Ab (CBR LFA1/2) (mean ± SEM; N = 4). Right: Representative microscopy images. Scale bars, 100 μm. NS, nonstimulated. (J) ICAM-1 binding to αL-KO Jurkat cells expressing αL variants using ICAM-1–pseudotyped VLPs containing mNEON (means ±SEM; N ≥ 2). (K) Adhesion of αL-KO Jurkat cells expressing αL variants to immobilized integrin ligands after Mg2+/EGTA stimulation (mean ± SEM; N = 4). (L) Migration of αL-KO Jurkat cells expressing αL variants through Transwell inserts coated with integrin ligands, with CXCL12 in the lower chamber (mean ± SEM; N = 3). (M and N) Expression and function of all homozygous αL variants found in public databases (see table S7) or in patients (red) in HEK293T cells transfected with αL and β2. (M) Heatmap showing LFA-1 expression and adhesion to ICAM-1 and ICAM-3, relative to WT. (N) Variants plotted by activity (ICAM-1 binding) versus AF. See also fig. S2H. Two-way ANOVA tests with Dunnett’s correction were used in (I), (K), and (L) (variants versus WT). ***P < 0.001; ns, not significant. Representative results from N ≥ 3 are shown in (C), (D), (F), (G), and (H).