Fig 2: CCL21 activated the CD11a-ICAM1 axis. (A) Five major cell subsets (CD45+ cells, CD31+ cells, EpCAM+ cells, NG2+ cells, and SPARC+ cells) were sorted and cultured from fresh LUAD tissues using flow cytometry. (B) Determination of CCL21 secretion by CD45+, CD31+, EpCAM+, NG2+, and SPARC+ cells cultured in CSE medium. The supernatant from cultured cells was collected and assayed using ELISA. (C) The t-SNE plots showed epithelial cell subpopulations in subcluster analysis, according to current smoker (above) and non-smoker (below) single-cell RNA sequencing. (D) Correlation between CCL21 and CD11a in the TCGA-lung database. (E) CD4+ T cells were extracted by magnetic beads and co-cultured with A549 cells treated either with or without CCL21 for 48 hours. The expression of CD11a in peripheral blood lymphocytes was assayed by flow cytometry. (F) CD4+ T cells were extracted by magnetic beads and co-cultured with A549 cells treated with or without CSE or CSE+ αCCL21 for 48 hours. The expression of CD11a in peripheral blood lymphocytes was assayed by flow cytometry. (G) CD4+ T cells were extracted by magnetic beads and co-cultured with A549 cells treated either with or without CCL21 for 48 hours. B cell and T cell interactions were detected by the T cells static adhesion assay (scale bar=100 µm). (H) CD4+ T cells were extracted by magnetic beads and co-cultured with A549 cells treated with or without CSE or CSE+ αCCL21 for 48 hours. B cell and T cell interactions were detected by the T cells static adhesion assay (scale bar=100 µm). (I) B cells co-cultured with CD4+ T cells treated with CCL21 recombinant protein schematic. (J) Immunofluorescence staining of B cells co-cultured with CD4+ T cells using anti-CD4 anti-B, anti-CD11a, and anti-ICAM-1 antibodies (scale bar=100 µm). (K) The co-immunoprecipitation assay demonstrated the interaction between CD11a and ICAM1. (L) Representative immunofluorescence images of stained lung tissue derived from smoking and non-smoking patients using an anti-CD20, anti-CD4, anti-CD11a, and anti-ICAM-1 antibodies (scale bar=50 µm). Data are presented as the mean ± SD. ns, not significant. *p<0.05, ***p<0.001, two-tailed Student’s t-test. CSE, cigarette smoke extract; LUAD, lung adenocarcinoma; TCGA, The Cancer Genome Atlas; t-SNE, t-distributed stochastic neighbor embedding.