Figure 2.
The CD14+ classical monocyte subset activates high levels of TGFβ via integrin αvβ8 and MMP14. (A and B) PBMCs were gated on single, live HLA-DR+ cells, then as CD14+CD16− classical monocytes (green gate), CD14+CD16+ intermediate monocytes (red gate), and CD14−CD16+ nonclassical monocytes (blue gate). Levels of integrin β8 were measured on each subset (colored histogram, integrin β8 antibody; gray histogram, isotype control). Representative flow cytometry plots (A) and quantification of integrin β8 levels (B); n = 19 healthy donors. (C and D) Monocyte subsets were isolated by flow cytometry sorting and co-cultured overnight with a TGFβ reporter cell line (C; n = 10–13 donors) in the presence of an isotype or anti–integrin β8 antibody (D; n = 3–6 donors). Bars show mean and SD. (E and F) MMP14 expression was measured by real-time PCR, normalizing to the housekeeping gene B2M (E; n ≥ 5 for each subset) and flow cytometry (F; n = 8 for each subset) in monocyte subsets. Bars show mean and SD. (G) Active TGFβ production by CD14+ monocytes in the presence of an isotype control, integrin β8 blocking, or MMP14 blocking antibody (n = 6 donors). Bars show mean and SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant; P values were determined by Friedman test with Dunn’s multiple comparisons post-test (B, F, and G), Wilcoxon matched-pairs signed rank test (D), and Kruskal–Wallis with Dunn’s multiple comparisons post-test (C and E).