Allergen exposure functionally alters influenza-specific CD4+ Th1 memory cells in the lung

Abstract

CD4+ lung-resident memory T cells (TRM) generated in response to influenza infection confer effective protection against subsequent viral exposures. Whether these cells can be altered by environmental antigens and cytokines released during heterologous, antigen-independent immune responses is currently unclear. We therefore investigated how influenza-specific CD4+ Th1 TRM in the lung are impacted by a subsequent Th2-inducing respiratory house dust mite (HDM) exposure. Although naïve influenza-specific CD4+ T cells in the lymph nodes do not respond to HDM, influenza-specific CD4+ TRM in the lungs do respond to a subsequent allergen exposure by decreasing expression of the transcription factor T-bet. This functional alteration is associated with decreased IFN-γ production upon restimulation and improved disease outcomes following heterosubtypic influenza challenge. Further investigation revealed that ST2 signaling in CD4+ T cells during allergic challenge is necessary to induce these changes in lung-resident influenza-specific CD4+ TRM. Thus, heterologous antigen exposure or ST2-signaling can drive persistent changes in CD4+ Th1 TRM populations and impact protection upon reinfection.

Figure 1.

NP_311–325:I-A^b–specific and Der p 1_117–127:I-A^b–specific CD4⁺ T cells are not crossreactive or activated by heterologous responses. (A) In mice administered respiratory HDM (top) or PR8 (bottom) as illustrated in the experimental timeline shown, lung-draining mediastinal lymph nodes and lung tissues were isolated and stained with Der p 1_117–127:I-A^b and NP_311–325:I-A^b tetramers and analyzed by flow cytometry. Representative flow plots are shown. Lung samples were pregated on cells that are negative for the intravenous Thy1.2 label to demarcate cells located in the lung parenchyma. (B) Representative flow cytometry plots of CD4⁺ T cells isolated from the mediastinal lymph node or lung 43 d after infection with PR8 (top) or following PR8 and respiratory HDM (bottom) as illustrated in the experimental timeline shown. Lung samples were pregated on cells negative for the intravenous Thy1.2 label to demarcate cells located in the lung parenchyma. (C) Summary data of the total number of NP-specific or Der p 1–specific tetramer-positive cells in the mediastinal lymph node or lungs of mice from each condition as represented in B. Data are pooled from three to six mice per group from three independent experiments and were analyzed by unpaired t test. Graphs show mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. IAV, influenza A virus. o.p., oropharyngeal. pfu, plaque-forming units.

Figure 2.

HDM-induced airway inflammation following influenza A virus infection decreases the frequency of T-bet–expressing NP-specific CD4⁺ T_RM in the lung. (A) Schematic representing the experimental timeline. (B) Representative flow cytometry plots of CD4⁺ T cells isolated from the lung at the time point indicated in A. (C) Summary data of the percentage of T-bet⁺ NP-specific cells, geometric mean fluorescence intensity of T-bet in NP-specific cells, and the percentage of ST2⁺ NP-specific cells as represented in B. Data are pooled from eight mice per group from two independent experiments. (D) Representative flow cytometry plots of CD4⁺ T cells isolated from the lung at the time point indicated in A. (E) Summary data of the percentage of GATA-3⁺ NP-specific cells and geometric mean fluorescence intensity of GATA-3 in NP-specific cells as represented in D. Data are pooled from six mice per group from two independent experiments. (F) Representative flow cytometry plots of CD4⁺ T cells isolated from the lung at the time point indicated in A. (G) Summary data of the percentage of Foxp3⁺ NP-specific cells and geometric mean fluorescence intensity of Foxp3 in NP-specific cells as represented in F. Data are pooled from five to seven mice per group from two independent experiments. (H) Schematic of the experimental timeline for data in I and J. (I) Representative flow cytometry plots of CD4⁺ T cells isolated from the lung at experimental day 64. (J) Summary data of the percentage of T-bet⁺ NP-specific cells, the geometric mean fluorescence intensity of T-bet in NP-specific cells, and the percentage of ST2⁺ NP-specific cells as represented in I. Data are pooled from seven mice per group from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Graphs show mean ± SD and data were analyzed by unpaired t test. pfu, plaque-forming units. IAV, influenza A virus. o.p., oropharyngeal. gMFI, geometric mean fluorescence intensity.

Figure 3.

NP-specific CD4⁺ T_RM in the lung exhibit a decreased potential to produce IFN-γ and IL-10 following allergic sensitization and challenge. (A) Schematic of the experimental timeline for data in B and C. (B) Representative flow cytometry plots of CD4⁺ T cells isolated from the lung at experimental day 10 or day 40. (C) Summary data of the percentage of YFP⁺ or tdTomato⁺ NP-specific cells and the geometric mean fluorescence intensity of YFP in NP-specific cells as represented in B. Data are pooled from two to five mice per group from two independent experiments. (D) Schematic of the experimental timeline for data in E–H. (E) Representative flow cytometry plots of CD4⁺ T cells isolated from the lung at experimental day 43 and either incubated in media (unstimulated control) or stimulated with PMA/ionomycin for 4.5 h to assess cytokine production potential. (F) Summary data of the percentage of IFN-γ⁺ NP-specific cells and the geometric mean fluorescence intensity of IFN-γ in NP-specific cells as represented in E. Data are pooled from 11–13 mice per group from three independent experiments. (G) Representative flow cytometry plots of CD4⁺ T cells isolated from the lung at experimental day 43 and either incubated in media (unstimulated control) or stimulated with PMA/ionomycin for 4.5 h to assess cytokine production potential. (H) Summary data of the percentage of IL-10⁺ or IL-13⁺ NP-specific cells as represented in G. Data are pooled from 11–13 mice per group from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Graphs show mean ± SD and data were analyzed by unpaired t test. pfu, plaque-forming units. IAV, influenza A virus. o.p., oropharyngeal. gMFI, geometric mean fluorescence intensity.

Figure 4.

HDM-exposed NP-specific CD4⁺ T_RM maintain decreased T-bet expression after heterosubtypic infection and are associated with improved disease severity. (A) Schematic of the experimental timeline for data in B–H. (B) The total number of CD4⁺ cells per 20 μl peripheral blood of mice at experimental day 48 prior to FTY720 administration and on day 55 prior to sacrifice. Data are pooled from 17–23 mice per time point from three independent experiments. (C) Representative flow cytometry plots of cells isolated from the lung at experimental day 55. Box gate denotes the Thy1.2⁻ NP-specific CD4⁺ cells in E. (D) Summary data of the number of NP-specific CD4⁺ cells in the lung at experimental day 55 as represented in C. Data are pooled from 6–12 mice per group from three independent experiments. (E) Representative flow cytometry plots of NP-specific CD4⁺ T cells isolated from the lung at experimental day 55. (F) Summary data of the percentage of T-bet⁺ NP-specific cells, the geometric mean fluorescence intensity of T-bet in NP-specific cells, and the percentage of ST2⁺ NP-specific cells as represented in E. Data are pooled from 10–12 mice per group from three independent experiments. (G) Graph summarizing morbidity following X31 infection as a percent of starting (experimental day 50) weight. Data are pooled from 2–12 mice per group from five independent experiments. (H) Quantification of viral load in whole lung tissue at experimental day 55 as measured by qRT-PCR. The dotted line indicates limit of detection. Data are pooled from 2–12 mice per group from five independent experiments. *, P < 0.05; ^## or **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Graphs show mean ± SD. Data in B, D, F, and G were analyzed by unpaired t test. Data in H were analyzed by Mann–Whitney test. pfu, plaque-forming units. IAV, influenza A virus. o.p., oropharyngeal. gMFI, geometric mean fluorescence intensity.

Figure S1.

Proinflammatory cytokine levels in the lung are reduced after heterosubtypic challenge in mice exposed to HDM following PR8 infection and positively correlate with increased viral load. (A) Schematic of the experimental timeline for data in B. (B) Quantification of Ifng, Il6, and Tnf transcript expression in whole lung tissue on experimental day 55 as measured by qRT-PCR. Data are pooled from 1–12 mice per group from five independent experiments. (C) Correlation of viral transcript expression and Ifng expression in whole lung tissue, Il6 expression in whole lung tissue, and level of T-bet expression in NP-specific CD4⁺ T cells isolated from the lung on experimental day 55. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Graphs in B show mean ± SD. Data in B were analyzed by Mann–Whitney test, data in C were analyzed by Spearman’s rank correlation. pfu, plaque-forming units. IAV, influenza A virus; o.p., oropharyngeal. gMFI, geometric mean fluorescence intensity.

Figure S2.

NP-specific CD4⁺ T_RM in the lung express IL-18Ra. (A) Schematic of the experimental timeline for data in B and C. (B) Representative flow cytometry plots of CD4⁺ T cells isolated from the lung at experimental day 27. (C) Summary data of the percentage of IL-18Ra⁺ NP-specific cells as represented in B. Data are pooled from six mice per group from two independent experiments. *, P < 0.05. Graphs show mean ± SD and data were analyzed by unpaired t test. pfu, plaque-forming units. IAV, influenza A virus. o.p., oropharyngeal.

Figure 5.

Diminished T-bet expression in NP-specific CD4⁺ T_RM in the lung can be driven by IL-33 and is dependent on ST2 signaling in CD4⁺ T cells. (A) Schematic of the experimental timeline for data in B–C. (B) Representative flow cytometry plots of CD4⁺ T cells isolated from the lung at experimental day 27. (C) Summary data of the percentage of T-bet⁺ NP-specific cells, the geometric mean fluorescence intensity of T-bet in NP-specific cells, and the percentage of ST2⁺ NP-specific cells as represented in B. Data are pooled from six mice per group from two independent experiments. (D) Schematic of the experimental timeline for data in E and F. (E) Representative flow cytometry plots of CD4⁺ T cells isolated from the lung on experimental day 27. Red box gate and numbers define the T-bet⁺ population and associated frequency, respectively. (F) Summary data of the percentage of T-bet⁺ NP-specific cells, the geometric mean fluorescence intensity of T-bet in NP-specific cells, and the percentage of ST2⁺ NP-specific cells as represented in E. Data are pooled from eight to nine mice per group from three independent experiments. (G) Schematic of the experimental timeline for data in H–J. (H) Representative flow cytometry plots and summary data of ST2 expression by CD4⁺ and CD4⁻ cells isolated from the lung at experimental day 48. Plots are pregated on CD45.2⁺, B220⁻, CD8⁻, CD19⁻, F4/80⁻, CD11b⁻, CD11c⁻, TCR γΔ⁻, and NKG2d⁻ cells. Data are pooled from 10–11 mice per group from three independent experiments. (I) Representative flow cytometry plots of CD4⁺ T cells isolated from the lung at experimental day 48. (J) Summary data of the percentage of T-bet⁺ NP-specific cells as represented in I. Data are pooled from 10–11 mice per group from three independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Graphs show mean ± SD and data were analyzed by unpaired t test. pfu, plaque-forming units. IAV, influenza A virus. o.p., oropharyngeal. gMFI, geometric mean fluorescence intensity.