Skip to main content
. 2020 Jun 24;217(9):e20190865. doi: 10.1084/jem.20190865

Figure 4.

Figure 4.

Th2 Trm cells drive the airway Th2 response to HDM via in situ proliferation but are dispensable for CD4+ T cell recruitment. (A and B) CD45.2 and CD45.1 HDM-memory mice were surgically conjoined. After 3–4 wk, both HDM-memory parabionts were left untreated or received a single dose of i.n. HDM with harvest of lungs after 72 h. (A) Schematic of parabiosis experiment. (B) Representative flow cytometry of lung CD4+ T cells from untreated CD45.2 memory parabiont demonstrating CD45.2 expression of anti-CD45 labeled CD4+ T cells. In addition, FoxP3 and GATA3 expression of anti-CD45 unlabeled CD4+ T cells as well as CD45.2 expression of FoxP3−GATA3+CD4+ T cells. (C) Quantitation of percentage of partner-derived lung Th2 cells (anti-CD45 i.v. unlabeled, FoxP3−GATA3+CD4+ T cells) assessed via flow cytometry from untreated and HDM-rechallenged memory parabionts. (D and E) CD45.2 HDM-memory mice were surgically conjoined to CD45.1 naive mice. After 3–4 wk, both parabionts received a single dose of i.n. HDM with harvest of lung after 72 h. (D) Lung naive-host–derived CD4+ T cells (anti-CD45 i.v. unlabeled, FoxP3−CD45.2−CD4+ T cells) and anti-CD45 i.v. unlabeled FoxP3−GATA3−CD4+ T cells were quantitated via flow cytometry. (E) Lung Ccl17 and Ccl22 relative expression assessed via qPCR. (F and G) Individual HDM-memory mice were left untreated or received a single dose of i.n. HDM with BAL after 72 h. BrdU was injected i.p. 2 h before BAL. (F) Representative flow cytometry of BAL Th2 cells (FoxP3−GATA3+CD4+ T cells) from HDM-memory mice without and with HDM rechallenge demonstrating Ki67 and BrdU expression. (G) Quantitation of the percentage of Ki67+ and BrdU+ BAL Th2 cells in indicated groups. Representative data show individual mice with mean ± SEM from two cumulative experiments with four to six mice per group (A–C), mean ± SEM from one of three independent experiments with three or four mice per group (D and E), or mean ± SEM from two cumulative experiments with eight mice per group (F and G). For statistical analysis, two-tailed Mann-Whitney U testing was performed for nonparametric data. One-way ANOVA analysis with Holm-Sidak’s testing was used for statistical analysis of multiple groups with paired two-tailed t tests for comparison on naive and memory parabiont groups. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.