Fig 2: Identification of factors colocalizing with LFA1 intracellular clusters.a LFA1 (αLβ2) intracellular cluster (ICC) formation concomitant with lymphocyte adhesion. Adhesion of lymphocytes (BAF/LFA1) to the ICAM1-displaying surface via LFA1 results in the formation of LFA1 ICCs inside the cell. LFA1 was visualized by fusing a SNAP tag to the C-terminus of β222. b Colocalization of β2-SNAP (shown in magenta) with factors involved in the regulation of vesicular trafficking (shown in green). Colocalized spots are indicated by arrows. Colocalization of LFA1 (β2) with TGN38 (TGN, n = 15), Rab7 (n = 9) or LC3 (n = 12) was quantified using Mander’s coefficient22. c Co-trafficking of β2-SNAP and CLIP-LC3 in adhered lymphocytes. Yellow arrows and cyan arrowheads indicate the ICCs containing both β2 and LC3 (see also Supplementary Movie 1). d Trajectories of co-trafficked ICCs indicated in (c). The area of the cell containing the visualized ICC is indicated by a dotted line. e Colocalization analysis of β2-SNAP with the ATG8 family proteins, LC3 (n = 14) and GABARAP (n = 14). f FRET assay between β2 and LC3. FRET between β2-mCherry and YPet-LC3 in adhered lymphocytes was measured (number of cells measured: n = 17). Lymphocytes expressing YPet-FL-mCherry24 (positive control, PC, n = 16), and β2-mCherry with YPet alone (n = 19) were used as positive and negative controls, respectively. The error bars represent the SEM (standard error of the mean). Statistical analyses were performed using unpaired nonparametric two-sided Student’s t test (e), one-way ANOVA with Dunnett correction (b) or Turkey correction (f) for multiple comparisons. Source data are provided as a Source Data file.

Fig 3: Importance of ATG8 family proteins for LFA1-dependent lymphocyte adhesion.a Western blotting analysis of LC3b and GABARAP in BAF/LFA1. LC3b-KO: BAF/LFA1 with deletion of LC3b; GABARAP-KO: BAF/LFA1 with deletion of GABARAP. Two independent experiments were performed. b FACS analysis of LFA1 expression in BAF/LFA1 cells. Surface expression of LFA1 was monitored with monoclonal antibodies (Mean Fluorescence Intensity, MFI: αL [No staining, WT, LC3b-KO, GABARAP-KO] = [83.3, 22400, 22300, 26500], β2 [WT, LC3b-KO, GABARAP-KO] = [23300, 16000, 23900]). Total expression of LFA1 was monitored using the SNAP tag protein fused to β2 (MFI: SNAP [No staining, WT, LC3b-KO, GABARAP-KO] = [77.1, 33700, 30300, 39900]). c Adhesion assay. Areas of cell adhesion were monitored by interference reflection microscopy. WT: n = 45; LC3b-KO: n = 45; GABARAP-KO: n = 48. d Accumulation of LFA1 at the contact surface. Surface LFA1 was stained with a dye-conjugated non-blocking monoclonal antibody and adhered to ICAM1-coated dishes in the presence of PMA. The fluorescence intensity of LFA1 at the contact surface was measured by TIRFM. WT: n = 36; LC3b-KO: n = 35; GABARAP-KO: n = 42. e LFA1 clustering inside the cell. Intracellular clusters were visualized using a SNAP tag fused to β2 by SRRF microscopy. The size of clusters was measured using Image J (clusters were defined as particles >0.05 µm2). WT: n = 36; LC3b-KO: n = 27; GABARAP-KO: n = 31. f Working flow describing the preparation of T-cell blasts with deletion of the gene of interest by the CRISPR/Cas9 system. g Western blotting analysis of LC3b in primary T-cell blasts. NC: negative control. Two independent experiments were performed. h FACS analysis of LFA1 surface expression in T-cell blasts. i Adhesion assay using T-cell blasts. Cells were stimulated with an anti-CD3 monoclonal antibody and applied onto ICAM1-coated dishes. NC: n = 60; LC3b: n = 54. j Accumulation of LFA1 at the contact surface of adhered T-cell blasts. NC: n = 51; LC3b: n = 63. Statistical analyses were performed using unpaired nonparametric two-sided Student’s t test (i, j), or one-way ANOVA with Dunnett correction (c–e) for multiple comparisons. Source data are provided as a Source Data file.