Distinct functions of tissue-resident and circulating memory Th2 cells in allergic airway disease

Abstract

Memory CD4+ T helper type 2 (Th2) cells drive allergic asthma, yet the mechanisms whereby tissue-resident memory Th2 (Th2 Trm) cells and circulating memory Th2 cells collaborate in vivo remain unclear. Using a house dust mite (HDM) model of allergic asthma and parabiosis, we demonstrate that Th2 Trm cells and circulating memory Th2 cells perform nonredundant functions. Upon HDM rechallenge, circulating memory Th2 cells trafficked into the lung parenchyma and ignited perivascular inflammation to promote eosinophil and CD4+ T cell recruitment. In contrast, Th2 Trm cells proliferated near airways and induced mucus metaplasia, airway hyperresponsiveness, and airway eosinophil activation. Transcriptional analysis revealed that Th2 Trm cells and circulating memory Th2 cells share a core Th2 gene signature but also exhibit distinct transcriptional profiles. Th2 Trm cells express a tissue-adaptation signature, including genes involved in regulating and interacting with extracellular matrix. Our findings demonstrate that Th2 Trm cells and circulating memory Th2 cells are functionally and transcriptionally distinct subsets with unique roles in promoting allergic airway disease.

Graphical abstract

Figure S1.

Allergen rechallenge promotes type 2 chemokine expression, lung inflammation, and mucus metaplasia. C57BL/6 mice were sensitized with 10 µg i.n. HDM followed by 10 µg daily challenges of i.n. HDM on days 7–11. After 6–12 wk of rest, HDM-memory mice were left untreated or rechallenged with a single dose of i.n. HDM followed by tissue harvest 72 h later. (A) Schematic of i.n. HDM treatment. (B) Lung Ccl11 and Ccl24 relative RNA levels assessed via qPCR. (C) Lung Ccl17 and Ccl22 relative RNA levels assessed via qPCR. (D) Representative images of lung H&E and PAS staining in indicated groups. White arrows indicate blood vessels, and black arrows indicate airways. Scale bars represent 100 µm. Representative data show individual mice with mean ± SEM from one of two independent experiments with three or four mice per group. For statistical analysis, one-way ANOVA analysis with Holm-Sidak’s testing was used for statistical analysis of multiple groups. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Figure 1.

Memory Th2 cells orchestrate the recall response to HDM in an allergen-specific manner. (A–H) C57BL/6 mice were sensitized and challenged with i.n. HDM, rested for 6–12 wk, and left untreated or rechallenged with a single dose of i.n. HDM followed by tissue harvest 72 h later. (A) Lung Il5 and Il13 relative RNA levels assessed via qPCR. (B) Lung parenchymal (anti-CD45 i.v. unlabeled) eosinophils were quantitated via flow cytometry. (C) Lung histology scores. (D) Mucus scores. (E) Airway resistance was measured in indicated groups after increasing doses of methacholine. (F and G) Th2 cells (FoxP3⁻GATA3⁺CD4⁺ T cells) were quantitated by flow cytometry in the (F) lung parenchyma (anti-CD45 i.v. unlabeled) and (G) mLNs in indicated groups. (H) Quantitation of lung parenchymal ILC2 (anti-CD45 i.v. unlabeled, lineage⁻CD4⁻Thy1.2⁺CD127⁺ST2⁺ cells). (I and J) C57BL/6 and Rag2^−/− mice were sensitized and challenged with i.n. HDM, rested for 6–12 wk, and left untreated or rechallenged with a single dose of i.n. HDM followed by tissue harvest 72 h later. (I) Lung Il5 and Il13 relative RNA levels assessed via qPCR. (J) Lung parenchymal (anti-CD45 i.v. unlabeled) eosinophils were quantitated via flow cytometry. (K and L) C57BL/6 mice were sensitized and challenged with i.n. HDM, rested for 6–12 wk, and left untreated or rechallenged with a single dose of i.n. HDM or i.n. A. alternata followed by tissue harvest 72 h later. (K) Lung Il5 and Il13 relative RNA levels assessed via qPCR. (L) Th2 cells (FoxP3⁻GATA3⁺CD4⁺ T cells) were quantitated by flow cytometry in the lung parenchyma (anti-CD45 i.v. unlabeled). Representative data show individual mice with mean ± SEM from one of three independent experiments with four mice per group (A–H) or mean ± SEM from one of two independent experiments with three or four mice per group (I–L). One-way ANOVA analysis with Holm-Sidak’s testing was used for statistical analysis of multiple groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. ns, not significant.

Figure S2.

Identification of Th2 Trm cells via flow cytometry. C57BL/6 mice were sensitized and challenged with i.n. HDM and then rested for 6–12 wk followed by lung tissue harvest. (A) Representative flow cytometry on lung cells in HDM-memory mice to identify Th2 cells (anti-CD45 i.v. unlabeled, FoxP3⁻GATA3⁺CD4⁺ T cells). (B) Quantitation of lung Th2 cells (anti-CD45 i.v. unlabeled, FoxP3⁻GATA3⁺CD4⁺ T cells) in naive and HDM-memory mice via flow cytometry. (C) Representative histogram demonstrating CD69 staining (and fluorescence minus one control staining) of lung Th2 cells from HDM-memory mice (left panel) with quantitation of percent CD69⁺ cells from lung Th2 cells and FoxP3⁻GATA3⁻CD4⁺ T cell populations (right panel). (D) C57BL/6 mice were sensitized and challenged with i.n. A. alternata, rested for 6–8 wk, and left untreated or rechallenged with a single dose of i.n. A. alternata. Quantitation of lung Th2 cells (anti-CD45 i.v. unlabeled, FoxP3⁻GATA3⁺CD4⁺ T cells) in indicated groups. Representative data show individual mice with mean ± SEM from one of two independent experiments with three or four mice per group. For statistical analysis, a two-tailed t test was performed for parametric data, and a two-tailed Mann-Whitney U test was performed for nonparametric data. *, P < 0.05; ****, P < 0.0001. FSC-A, forward scatter area; FSC-H, forward scatter height; SSC-A, side scatter area.

Figure S3.

Function and trafficking of tissue-resident and circulating memory Th2 cells in HDM-memory mice. (A and B) C57BL/6 mice were sensitized and challenged with i.n. HDM and rested for 6–12 wk. (A) Lung cells from HDM-memory mice underwent CD4⁺ T cell negative selection followed by treatment with anti-CD3 and anti-CD28 for 12–16 h. Representative flow cytometry of anti-CD45 i.v. unlabeled (intraparenchymal) and labeled (intravascular) Th2 cells after intracellular cytokine staining for IL-5 and IL-13. (B) Geometric mean fluorescence intensity (gMFI) for IL-5 and IL-13 expression in indicated groups. (C and D) 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 mLN and lung after 72 h. (C) Representative flow cytometry of lung parenchymal eosinophils (anti-CD45 i.v. unlabeled, CD11c^loSiglec-F⁺ cells) from indicated groups. (D) Quantitation of BAL eosinophils from indicated groups. (E) CD45.2 and CD45.1 HDM-memory mice were surgically conjoined. After 3–4 wk, lungs from both parabionts were harvested. Quantitation of percent partner-derived lung memory CD4⁺ T cells (anti-CD45 i.v. unlabeled, FoxP3⁻GATA3⁻CD4⁺ T cells and FoxP3⁻GATA3⁺CD4⁺ T cells) assessed via flow cytometry. (F) 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 lungs after 72 h. Lung FoxP3⁺CD4⁺ T cells (T regs) were quantitated via flow cytometry. (G) Individual HDM-memory mice were left untreated or received a single dose of i.n. HDM with BAL after 72 h. BAL Th2 cells were quantitated via flow cytometry. Representative data show individual mice with mean ± SEM from one of two independent experiments with three or four mice per group (A and B), or one of three independent experiments with three or four mice per group (C, D, and F) or cumulative data from two independent experiments (E and G). For statistical analysis, a two-tailed t test was performed for parametric data (B and G). 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 of parabionts (D and F). A two-tailed Mann-Whitney U test was performed for nonparametric data (E). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 2.

Th2 Trm cells and circulating memory Th2 cells collaborate to promote Th2 cell expansion and type 2 cytokine production within the lung. (A–H) 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 mLN and lung after 72 h. (A) Schematic of the parabiosis experiment. (B) Representative flow cytometry of mLN CD4⁺ T cells isolated from indicated groups demonstrating FoxP3 and GATA3 expression. (C) Percentage of CD45.2⁺ cells (from memory parabiont) of FoxP3⁻GATA3⁻ and FoxP3⁻GATA3⁺ CD4⁺ T cells from the mLN of naive and memory parabionts. (D) Quantitation of Th2 cells from mLNs from indicated groups. (E) Total mLN cells were restimulated ex vivo with 25 µg/ml HDM for 72 h and levels of IL-5 and IL-13 protein in the supernatant was measured via ELISA. (F) Representative flow cytometry of lung CD4⁺ T cells from indicated groups demonstrating FoxP3 and GATA3 expression. (G) Lung parenchymal Th2 cells (anti-CD45 i.v. unlabeled, FoxP3⁻GATA3⁺CD4⁺ T cells) were quantitated via flow cytometry. (H) Lung Il5 and Il13 relative RNA levels assessed via qPCR. Representative data show individual mice with mean ± SEM from one of three independent experiments with three or four mice per group. For statistical analysis, 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 of naive and memory parabiont groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 3.

Th2 Trm cells and circulating memory Th2 cells perform nonredundant functions upon HDM rechallenge. (A–I) 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 or BAL after 72 h. (A) Quantitation of eosinophils (anti-CD45 i.v. unlabeled, CD11c^loSiglec-F⁺ cells) from the lung parenchyma of indicated groups. (B) Lung Ccl11 and Ccl24 relative RNA levels assessed via qPCR. (C) Representative histograms demonstrating BAL eosinophil cell surface expression of CD11b and CD62L (top panel) and geometric mean fluorescence intensity (gMFI; bottom panel) from naive parabionts (purple circles) and memory parabionts (blue circles) as determined by flow cytometry. (D) H&E-stained (top panel) and PAS-stained (bottom panel) lung sections from indicated groups. White arrows indicate blood vessels, and black arrows indicate airways. Scale bars represent 100 µm. (E) Lung histology scores. (F) Mucus scores. (G) Airway resistance was measured in indicated group after increasing doses of methacholine. (H) Immunohistochemistry for lung CD4⁺ T cells in naive and memory parabionts 72 h after HDM challenged. White arrows indicate blood vessels, and black arrows indicate airways. Scale bars represent 100 µm. (I) BAL Th2 cells (FoxP3⁻GATA3⁺CD4⁺ T cells) were quantitated in indicated groups via flow cytometry. Representative data show individual mice with mean ± SEM from one of three independent experiments with three to four mice per group (A–F and H–I) or mean ± SEM from two cumulative experiments with four mice per group (G). For statistical analysis, 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 < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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.

Figure 5.

Shared and distinct transcriptional profiles of Th2 Trm and circulating memory Th2 cells. (A–E) FoxP3^YFPCre mice were sensitized and challenged with intranasal HDM and rested for 6 wk followed by lung and LN/spleen (SLO) harvest. (A) Representative flow cytometry of lung and SLO CD4⁺ T cells indicating YFP and ST2 expression as well as sorting gates for YFP⁻ST2⁻ and YFP⁻ST2⁺ populations. (B) Principal component analysis (PCA) plot based on differentially expressed genes from RNA-seq data of indicated memory CD4⁺ T cell populations. (C) Heatmap of expression values of T cell subset genes, including Th1, Th2, Th17, Tfh, and Trm up-regulated and down-regulated genes. (D) Bar graph of statistical significance (false discovery rate [FDR] by the DAVID tool) of pathway enrichment among genes overexpressed in lung YFP⁻ST2⁺CD4⁺ T cells relative to SLO YFP⁻ST2⁺CD4⁺ T cells. (E) Heatmap of a selected subset of genes known to be involved in ECM biology and cell adhesion. Data are from three independent experiments with five HDM-memory mice pooled for each experiment (cell sort and RNA isolation). (F) Model of memory Th2 cell subsets during allergen rechallenge. Transcriptionally distinct memory Th2 cell subsets include Th2 Trm cells persisting in the lung parenchyma (indicated in blue) and circulating memory Th2 cells in the blood (Th2 Circ; indicated in red). After allergen rechallenge, circulating memory Th2 cells traffic into the lung parenchyma and produce type 2 cytokines, promoting perivascular inflammation, CCL11 and CCL24 production, and eosinophil recruitment. Activation of Th2 Trm cells leads to in situ proliferation and production of type 2 cytokines near the airways, which promotes mucus metaplasia (green epithelial cells), CCL11 and CCL24 production, and eosinophil activation. CCL11 and CCL24 are likely produced from multiple structural and immune cell types following allergen rechallenge. Created with BioRender.com.