Fig. 5.

CD4 + IL-17-expressing cells (Th17) are significantly increased in the lung by exposure to CS+PIC. WT C57B/6 mice were exposed to CS+PIC and the lungs harvested on day 21 after the initiation of smoke exposure. Lung immune cells were analyzed by multicolor flow cytometry. Cells were stained in a single panel for Gr1, NK1.1, B220, TCRβ, TCRγδ, CD8, CD4, IL17A secretion, and INFγ secretion (A–I) or for CD3ε, CD90, CD127, Rorγt, IL17A, and INFγ (J–L). A: cells expressing Gr1, Nk1.1, or neither were gated and percent positive staining with IL-17A shown as total positive cell number in M. In B, Gr1, Nk1.1 double-negative cells were gated into Tcrγδ (D–F)- and Tcrβ (C)-positive populations, which were then gated into IFNγ-negative and IL-17A-positive populations in room air (E) compared with CS+PIC (F)-treated mice. IL-17A-positive cells were defined using pooled cells stained without surface capture for IL-17A (D). In C, Tcrβ-positive cells were gated into CD8- and CD4-positive populations. Shown in in G–J, Tcrβ, CD4+ or CD8 (not shown) cells were gated into IL-17A-positive and IL-17-negative populations, which were enumerated as shown in M. J: lung cells were gated into CD3ε and CD90-positive and -negative populations. Innate lymphoid cells (CD3ε neg, CD90 positive) were gated into CD127 positive/IL-17-negative (ILC1) and CD127-negative/IL-17A-positive (ILC3) populations in room air (K) and CS+PIC-exposed mice (L) and enumerated in M. IL-17A-positive cells were defined by pooled cells stained without IL-17A surface capture. IL-17A-positive ILC3 cells were also positive for Rorγt (not shown). In M, total numbers of IL-17A-expressing cell types (PMNs, NK, CD4, CD8, γδ, and ILC3) in room air (open bars) and CS+PIC-exposed (solid bars) mice (n = 3 in each group) are shown. *P < 0.05, **P < 0.01 as determined by Student’s t-test.