Fig 2: The immunisation regimen and virus challenge studies in mice. The immunisation schedule and challenge study in mice. 6-8-week-old specific pathogen-free (SPF) female C57BL/6 mice were divided into three groups (n = 78 per group), including the negative control groups (pVAX and pVAX-LC3B groups), and the experimental group (MEV group). On weeks 0 and 2, mice were intramuscularly vaccinated two times with 50 μg of the corresponding DNA plasmid (pVAX, pVAX-LC3B, and MEV-DNA) in 100 μL of PBS into the gastrocnemius muscle of the hind legs. On week 4, mice were intramuscularly vaccinated with 100 μL of PBS or 50 μg of the corresponding protein (LC3B and MEV-Pro) in 100 μL of PBS. On week 7, mice were sacrificed, and samples were collected (n = 6 per group). The rest of the mice of each group were intranasally challenged with influenza viruses and monitored until week 9 (n = 72 per group).

Fig 3: RAPL inhibits the phosphorylation of LC3 at Thr50 by Mst1.a Direct binding between RAPL and ATG8 family proteins. Pull-down assays were performed using purified GST-fused RAPL and 293T lysates expressing CLIP-LC3 or flag-GABARAP. Three independent experiments were performed. b Interaction of RAPL with purified LC3. Immunoprecipitation of myc-tagged RAPL in 293T cell lysate was followed by the addition of recombinant LC3b. Myc-RAPL and LC3 were detected with anti-Myc and anti-LC3 antibodies, respectively. Two independent experiments were performed. c FRET assay between RAPL and LC3. FRET between mCherry-RAPL and YPet-LC3 in adhered lymphocytes was measured (number of cells measured: n = 20). Lymphocytes expressing YPet-FL-mCherry24 (positive control, PC, n = 17), or mCherry-RAPL with YPet alone (n = 20) were used as positive and negative controls, respectively. d Role of LC3 phosphorylation in the canonical autophagy pathway. In the presence of nutrient starvation, Mst1 phosphorylates LC3b at Thr50 to inhibit binding of LC3b to FYCO1, an adaptor protein for kinesin, thereby promoting dynein-dependent transport of autophagosomes to the perinuclear region42. e Specific inhibition of Mst1-mediated LC3 phosphorylation at Thr50 by RAPL in 293T cells. Indicated tagged proteins were overexpressed in 293T cells and detected by western blotting with lysates and tag-specific antibodies. For Nore1A, α-tubulin and phosphorylated LC3 (p-LC3), antigen specific antibodies were used. N = 7. f Increased inhibition of LC3 phosphorylation caused by Rap1 activation in 293T cells. N = 7. g Effect of RAPL knockdown on LFA1 accumulation at the contact surface. BAF/LFA1 cells infected with virus harboring control shRNA (shCont): n = 40 or shRNA against RAPL (shRAPL): n = 44. h Effect of RAPL knockdown on LFA1(β2) and LC3 clustering. shCont: n = 38; shRAPL: n = 39. The error bars represent the SEM. Statistical analyses were performed using unpaired (g, h) nonparametric two-sided Student’s t tests, or one-way ANOVA with Dunnett correction (e), or Turkey correction (c, f) for multiple comparisons. The error bars represent the SEM. Source data are provided as a Source Data file.

Fig 4: 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 5: Dephosphorylation of LC3 promotes LFA1-mediated lymphocyte adhesion.a Effect of LC3b phosphorylation at T50 on lymphocyte adhesion to ICAM1-coated dishes under PMA stimulation. LC3b with mutation of T50 to Ala (LC3b-T50A, dephosphorylated form, n = 49) or to Glu (LC3b-T50E, constitutively phosphorylated form, n = 60) or without mutation (LC3b-WT, n = 45) was introduced into LC3b-knockout BAF/LFA1 cells (LC3b-KO). WT: parental cells before LC3b deletion, n = 48; (-): LC3b-KO without CLIP-LC3b expression, n = 47. b Effect of LC3b phosphorylation on LFA1 levels at the contact surface of the cells shown in (a). WT: n = 40; (-): n = 44; LC3b-WT: n = 40; LC3b-T50A: n = 45; LC3b-T50E: n = 68. c Effect of ciliobrevin D (cilD), a dynein inhibitor, on lymphocyte adhesiveness. Vehicle: n = 38; cilD: n = 40. d Effect of cilD on the contact surface level of LFA1. Vehicle: n = 30; cilD: n = 31. e Effects of Rose Bengal Lactone (RBL), a kinesin inhibitor, on lymphocyte adhesiveness. Vehicle: n = 43; RBL: n = 48. f Effect of RBL on LFA1 levels at the contact surface. Vehicle: n = 36; RBL: n = 36. g A model of LC3-mediated LFA1 transport to enhance lymphocyte adhesion. Outside-in signaling activates AMPK and induces the formation of intracellular clusters to facilitate lymphocyte adhesion. Activated LFA1 forms intracellular clusters, which are partly derived from surface LFA1, with Rab7 and LC3b. Direct interaction of LC3b with RAPL inhibits LC3b phosphorylation at T50 promoted by Rap1 activation via outside-in signaling, leading to FYCO1- and kinesin-dependent transport of LFA1+LC3+ clusters to the cell contact surface and promoting the accumulation of LFA1 at the surface to increase lymphocyte adhesion. Statistical analyses were performed using unpaired nonparametric two-sided Student’s t tests (c–f), or one-way ANOVA with Dunnett correction (a, b) for multiple comparisons. Source data are provided as a Source Data file.