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CD103hi Treg cells constrain lung fibrosis induced by CD103lo tissue-resident pathogenic CD4 T cells

Nature Immunology volume 20, pages 1469–1480 (2019)Cite this article

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Abstract

Tissue-resident memory T cells (TRM cells) are crucial mediators of adaptive immunity in nonlymphoid tissues. However, the functional heterogeneity and pathogenic roles of CD4+ TRM cells that reside within chronic inflammatory lesions remain unknown. We found that CD69hiCD103lo CD4+ TRM cells produced effector cytokines and promoted the inflammation and fibrotic responses induced by chronic exposure to Aspergillus fumigatus. Simultaneously, immunosuppressive CD69hiCD103hiFoxp3+ CD4+ regulatory T cells were induced and constrained the ability of pathogenic CD103lo TRM cells to cause fibrosis. Thus, lung tissue-resident CD4+ T cells play crucial roles in the pathology of chronic lung inflammation, and CD103 expression defines pathogenic effector and immunosuppressive tissue-resident cell subpopulations in the inflamed lung.

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Fig. 1: Repeated exposure to Aspergillus antigen induced tissue-resident CD4+ T cells in the inflamed lung.
Fig. 2: Tissue-resident CD69hi T cells showed unique profibrotic features.
Fig. 3: CD69hiCD103lo CD4+ T cells were the effector cytokine producers in the inflamed lung.
Fig. 4: Depletion of Foxp3+ CD4+ Treg cells resulted in exacerbation of chronic lung inflammation.
Fig. 5: CD4+ TRM cells were induced by chronic Aspergillus antigen exposure.
Fig. 6: CD44hiCD69hi CD4+ TRM regulomes show fibrotic features.
Fig. 7: CD4+ TRM cells are sufficient to induce lung inflammation accompanied by fibrotic responses.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. The RNA-Seq and ATAC-Seq data reported in this paper are available under Gene Expression Omnibus accession number GSE133399.

Code availability

The code that supports the findings of this study is available from the corresponding author upon reasonable request.

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Acknowledgements

We are grateful to Y. Kanno for critically reading and providing valuable suggestions on the manuscript. We are grateful to N. Iijima and K. J. Ishii for providing valuable suggestions on the parabiosis surgery. We are grateful to N. Takayama and K. Eto for providing valuable suggestions on the ATAC-Seq experiments. We thank T. Ito, K. Sugaya, M. Kato and T. Wada for excellent technical assistance. This work was supported by the following grants: Ministry of Education, Culture, Sports, Science and Technology Grants-in-Aid for Scientific Research (S) 26221305, JP19H05650, (C) 17K08876, 18K07164 and 19K16683; Practical Research Project for Allergic Diseases and Immunology (Research on Allergic Diseases and Immunology) from AMED (numbers JP19ek0410060 and JP19ek0410045); AMED-PRIME, AMED (number JP19gm6110005); AMED-CREST, AMED (number JP19gm1210003); the Mochida Memorial Foundation for Medical and Pharmaceutical Research, The Ichiro Kanehara Foundation for the Promotion of Medical Sciences and Medical Care; the Naito Foundation and the Takeda Science Foundation.

Author information

Author notes

  1. These authors contributed equally: Tomomi Ichikawa, Kiyoshi Hirahara, Kota Kokubo.

Authors and Affiliations

  1. Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan

    Tomomi Ichikawa, Kiyoshi Hirahara, Kota Kokubo, Masahiro Kiuchi, Ami Aoki, Yuki Morimoto, Jin Kumagai, Atsushi Onodera, Damon J. Tumes & Toshinori Nakayama

  2. First Department of Internal Medicine, University of Toyama, Toyama, Japan

    Tomomi Ichikawa & Kazuyuki Tobe

  3. AMED-PRIME, AMED, Chiba, Japan

    Kiyoshi Hirahara

  4. Institute for Global Prominent Research, Chiba University, Chiba, Japan

    Atsushi Onodera

  5. Division of Pulmonary Medicine, Department of Medicine, Jichi Medical University, Shimotsuke, Japan

    Naoko Mato & Koichi Hagiwara

  6. Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, South Australia, Australia

    Damon J. Tumes

  7. Division of Molecular Immunology, Medical Mycology Research Center, Chiba University, Chiba, Japan

    Yoshiyuki Goto

  8. Division of Mucosal Symbiosis, International Research and Development Center for Mucosal Vaccines, Institute of Medical Science, The University of Tokyo, Tokyo, Japan

    Yoshiyuki Goto

  9. Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Isehara, Japan

    Yutaka Inagaki

  10. Department of Medical Microbiology and Hygiene, University Medical Center, Johannes Gutenberg University Mainz, Mainz, Germany

    Tim Sparwasser

  11. AMED-CREST, AMED, Chiba, Japan

    Toshinori Nakayama

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  1. Tomomi Ichikawa
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Contributions

T.I., K. Hirahara and T.N. conceived of and designed the experiments. T.I., K. Hirahara, K.K., M.K., A.A., Y.M., J.K., A.O. and N.M. performed the experiments. T.I., K. Hirahara, K.K., M.K., A.O., D.J.T., Y.G., K. Hagiwara, Y.I., T.S., K.T. and T.N. analyzed and interpreted the data. T.I., K. Hirahara, D.J.T. and T.N. wrote, reviewed and edited the paper.

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Correspondence to Toshinori Nakayama.

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Supplementary Figure 1 Repeated exposure to Aspergillus antigen resulted in chronic lung inflammation.

(a) A schematic illustration of 7-week repeated Aspergillus antigen exposure. (b) The quantitative RT-PCR of Col1a1 in the lung from control BALB/c mice (control) and Aspergillus antigen-exposed mice (Asp-Ag) (n = 10 mice per group). (c) Representative confocal micrographs of the lung stained with anti-GFP (green), anti-type-I collagen (purple), anti-CD3 (yellow) and DAPI (blue) from wild-type C57BL/6 mice (WT) and Collagen 1a2-GFP transgenic mice with or without Aspergillus antigen exposure. Scale bars represent 20 μm. Representative findings of five independent experiments are shown. (d) The cytokine concentrations in BAL fluid from control mice (control) (n = 6) and Aspergillus antigen-exposed mice (Asp-Ag) (n = 8). Findings pooled from two independent experiments are shown. (e) The absolute number of total cells (total), macrophages (Mac), eosinophils (Eos), neutrophils (Neut) and lymphocytes (Lymph) in BAL fluid from control mice (control) (n = 6) and Aspergillus antigen-exposed mice (Asp-Ag) (n = 11). Findings pooled from two independent experiments are shown. (f) Airway hyperresponsiveness (AHR) was assessed by increased airway resistance with increasing doses of methacholine. Findings pooled from two independent experiments are shown (n = 5 mice per group). (g) The representative gating strategy for CD4+ T cells in the lung. (h, i) The cell surface profiles of CD44 and CD62L in the lung (h) and spleen (i) from control mice and Aspergillus antigen-exposed mice (Asp-Ag) (left), and pooled data from 5 mice per group (right). Data are the mean ± SD (1b, 1d, 1e, 1h and 1i) or the mean ± SEM (1f). P-values were calculated using the two-sided Mann-Whitney U test (1b, 1d, 1e, 1h and 1i) or two-way ANOVA test (1f). (j) Representative histological sections of the lung stained with Hematoxylin and Eosin from Aspergillus antigen-exposed wild-type BALB/c mice (left) and Foxn1nu mice (right) in lower magnification (upper) and higher magnification (lower) views. Scale bars represent 1 mm or 100 μm. Representative findings of two independent experiments are shown. (k) Representative histological sections of lungs stained with Sirius Red from Aspergillus antigen-exposed wild-type mice (left) and Foxn1nu mice (right). Scale bars represent 100 μm. More than three mice per group were analyzed.

Supplementary Figure 2 Repeated exposure to Aspergillus antigen induced CD69hi CD4+ tissue-resident T cells in the lung.

(a) Cell surface markers of CD44hiCD62Llo CD4+ T cells in the spleen from control (blue lines) and Aspergillus antigen-exposed mice (red) with isotype-matched control antibody (gray). Representative findings from two independent experiments are shown. (b) The ratio of cells positive for the indicated cell surface marker in CD44hiCD62LloCD69hi CD4+ T cells in comparison to CD44hiCD62LloCD69lo CD4+ T cells from control mice (horizontal axis) and Aspergillus antigen-exposed mice (Asp-Ag, vertical axis). Upper and lower diagonal lines indicate the expression ratio in Aspergillus antigen-exposed mice in comparison to control mice is >2 (red) or <2 (blue), respectively. (c) The cell surface markers on CD44hiCD62LloCD69hi CD4+ T cells in the lung from control (blue lines) and Aspergillus antigen-exposed mice (red) with isotype-matched control antibody (gray). Representative findings from two independent experiments are shown. (d) Pooled data showing the absolute number of CD103hi T cells in CD44hiCD62LloCD69hi CD4+ T cells from control mice (n = 5) and Aspergillus antigen-exposed mice (n = 6). (e, f) Pooled data showing the proportion (e) and absolute number (f) of CD44hiCD62LloCD69hiCD103hi CD4+ T cells in the spleen from control mice (n = 5) and Aspergillus antigen-exposed mice (n = 6). (g) A schematic illustration of repeated Aspergillus antigen exposure followed by intravenous staining for CD4+ T cells. (h) Intravenous staining of anti-CD4 (CD4 (iv)) and anti-CD4 staining in CD4+ T cells in the peripheral blood from control mice (control) and Aspergillus antigen-exposed mice (Asp-Ag). Representative findings from two independent experiments are shown. Data are the mean ± SD. P-values were calculated using the two-sided Mann-Whitney U test (2d, 2e, and 2f). (i) Intravenous staining of anti-CD4 (CD4 (iv)) and anti-CD44 staining in CD4+ T cells in the lung from control mice (control) and Aspergillus antigen-exposed mice (Asp-Ag) (left), and the percentage of tissue-resident T cells in CD4+ T cell population from control mice (control) and Aspergillus antigen-exposed mice (Asp-Ag) (n = 9 mice per group). (j) Intravenous staining of anti-CD4 (CD4 (iv)) and anti-CD44 staining in CD4+ T cells in the spleen from control mice (control) and Aspergillus antigen-exposed mice (Asp-Ag) (left), and the percentage of tissue-resident T cells in CD4+ T cell population from control mice (control) and Aspergillus antigen-exposed mice (Asp-Ag) (n = 9 mice per group). (k) The absolute number of tissue-resident T cells in the CD44hiCD69hi CD4+ T cell population (red) and the CD44loCD69lo CD4+ T cell population (blue) from control mice (control; n = 9) and Aspergillus antigen-exposed mice (Asp-Ag; n = 8), which were investigated in Fig. 1h. (l) The t-distributed stochastic linear embedding (t-SNE) plot of CD4+ T cells in the lung from control (left) and Aspergillus antigen-exposed mice (right). CD4(iv) (-) CD4+ T cells and CD44hiCD69hi CD4+ T cells in the t-SNE space of the CD4+ T cells (green) are shown in red and blue, respectively. Representative findings from two independent experiments are shown. (m) Representative confocal micrograph of the lung in a control mouse (control) stained with anti-CD4 (red), anti-CD69 (green), and anti-type l collagen (blue). Scale bars represent 50 μm. Representative findings of three independent experiments are shown. Data are the mean ± SD. P-values were calculated using the two-sided Mann-Whitney U test (2i, 2j, and 2k).

Supplementary Figure 3 Tissue-resident CD69hi CD4+ T cells expressed high levels of fibrosis-related genes.

(a) A scatter plot showing the FKPM of a single gene in two individual sample sets in CD44lo T cells, CD44hiCD69lo T cells, CD44hiCD69hiCD103lo T cells, and CD44hiCD69hiCD103hi T cells. (b) A heatmap showing the similarity and clustering of CD44lo T cells, CD44hiCD69lo T cells, CD44hiCD69hiCD103lo T cells and CD44hiCD69hiCD103hi T cells using the gene expression profiles. (c) Venn diagram depicts the number of genes with expression levels that were increased >8-fold in CD44hiCD69hiCD103lo T cells (red circle) or CD44hiCD69hiCD103hi T cells (blue circle) in comparison to CD44lo T cells. (d) Genes for which a >8-fold expression change was observed in both CD44hiCD69hiCD103lo T cells and CD44hiCD69hiCD103hi T cells (n= 60) in comparison to CD44lo T cells are depicted by the heatmap. (e) The Venn diagram depicts the number of genes with expression levels that were decreased <0.25-fold in CD44hiCD69hiCD103lo T cells (red circle) or CD44hiCD69hiCD103hi T cells (blue circle) in comparison to CD44lo T cells. (f) Genes for which a <0.25-fold expression change was uniquely observed in CD44hiCD69hiCD103lo T cells (n= 7), CD44hiCD69hiCD103hi T cells (n= 23), or both CD44hiCD69hiCD103lo T cells and CD44hiCD69hiCD103hi T cells (n= 18) in comparison to CD44lo T cells are depicted by heatmap. (g) The relative gene expression profiles in the co-regulation network in naive (CD44lo) CD4+ T cells (left), CD69hiCD103lo CD4+ T cells (middle), and CD69hiCD103hi CD4+ T cells (right). (h) The list of GO terms in the indicated categories that are involved in fibrotic responses. Genes that are included in each GO term are listed on Supplementary Table 1.

Figure Supplementary 4 Tissue-resident CD69hiCD103lo CD4+ T cells exhibit high levels of cytokine production.

(a, b) The absolute numbers of Foxp3-, GATA3- and Rorγt-positive cells in CD44hiCD69hiCD103lo CD4+ T cells (a) and CD44hiCD69hiCD103hi CD4+ Treg cells (b) in the lung from Aspergillus antigen-exposed wild-type mice, which were investigated in Fig. 3b, c (samples stained with Foxp3 and GATA3; n=16, samples stained with Foxp3 and RORγt; n=11). (c) The absolute numbers of the indicated cytokines in CD44hiCD69hiCD103lo CD4+ T cells (red) and CD44hiCD69hiCD103hi CD4+ Treg cells (blue) in the lung from Aspergillus antigen-exposed mice (Asp-Ag), which were investigated in Fig. 3e. Findings of five pooled independent experiments are shown. Data are the mean ± SD. P-values were calculated using the two-sided Mann-Whitney U test.

Supplementary Figure 5 Foxp3+ Treg cells suppress the pathology of chronic lung inflammation.

(a) A schematic illustration of the repeated exposure to Aspergillus antigen accompanied by the intranasal administration of diphtheria toxin. (b, c) Intracellular-staining profiles of Foxp3 (right) in the lung (b) and spleen (c) from Aspergillus antigen-exposed (Asp-Ag) wild-type mice (n = 9) and Foxp3-DTR transgenic mice (n = 9) from three independent experiments (right). Findings of two pooled independent experiments are shown. (d) Percentages of CD69hiCD103hi CD4+ T cells (left) and CD69hiCD103lo CD4+ T cells (right) in the lung from Aspergillus antigen-exposed wild-type (n = 9) and Foxp3-DTR transgenic mice (n = 9). Findings of two pooled independent experiments are shown. (e) The absolute numbers of Foxp3-, GATA3- and Rorγt-positive cells in CD69hiCD103hi CD4+ T cells in the lung from Aspergillus antigen-exposed wild-type mice (n = 9) and Foxp3-DTR transgenic mice (n = 9), which were investigated in Fig. 4b. Findings of two pooled independent experiments are shown. (f) Intracellular-staining profiles of Foxp3, GATA3, and Rorγt in CD69hiCD103lo CD4+ T cells in the lung from Aspergillus antigen-exposed wild-type mice (n = 9) and Foxp3-DTR transgenic mice (n = 9). Findings of two pooled independent experiments are shown. (g) The absolute number of Foxp3-, GATA3- and Rorγt-positive cells in CD69hiCD103lo CD4+ T cells in the lung from Aspergillus antigen-exposed wild-type mice (n = 9) and Foxp3-DTR transgenic mice (n = 9), which were investigated in Supplementary Fig. 4f. Findings of two pooled independent experiments are shown. (h) The percentages of the indicated cytokines in CD69hiCD103hi CD4+ T cells in the lung from Aspergillus antigen-exposed mice (Asp-Ag). The findings of three pooled independent experiments are shown. Data are the mean ± SD. P-values were calculated using the two-sided Mann-Whitney U test (5b, 5c, and 5d), a two-way ANOVA (5e, 5f, and 5g) or an unpaired two-sided Student’s t-test (5h). (i) A schematic illustration of the repeated exposure of Rorcfl/fl mice and Rorcfl/flFoxp3-cre mice to Aspergillus antigen for 7 weeks. (j) The absolute numbers of CD69hiCD103hiFoxp3hiRorγthi CD4+ T cells in the lung from Rorcfl/fl mice and Rorcfl/flFoxp3-cre mice subjected to repeated exposure to Aspergillus antigen (n = 5 mice per group). Data are the mean ± SD. P-values were calculated using the two-sided Mann-Whitney U test in 5j.

Figure Supplementary 6 CD103lo TRM and CD103hi tissue-resident Treg cells have distinct regulomes.

(a) A schematic illustration of repeated exposure of mice to Aspergillus antigen for 7 weeks with a 7-week rest period followed by an ATAC-Seq assay. (b) A heatmap showing the relative enrichment of TF motifs in the indicated CD4+ T cell subpopulation. (c, d, e, f, g) Comparison of ATAC-seq signals across tissue-resident CD4+ T cell signature genes, including (c) Il4, Il5 and Il13 loci, (d) Il17a, and Il17f loci, (e) Foxp3 locus, (f) Itgae locus, and (g) Areg locus. (h) The fibrosis-related genes expressed in CD44hiCD69hiCD103lo T cells unique (left, n= 15), CD44hiCD69hiCD103hi T cells unique (middle, n= 13), and both CD44hiCD69hiCD103lo T cells and CD44hiCD69hiCD103hi T cells (right, n= 27)are categorized into the indicated groups.

Figure Supplementary 7 CD4+ TRM cells in the lung induce the inflammation accompanied by fibrotic responses.

(a) A schematic illustration of the repeated exposure of mice to Aspergillus antigen for 7 weeks with a 7-week rest period followed by challenge with a single dose of Aspergillus antigen. (b, c) Representative histological sections of the lungs stained with Hematoxylin and Eosin (upper panel), PAS (middle panel), and Sirius red (lower panel) from four groups of mice (Sensitization (-) Rechallenge (-), Sensitization (-) Rechallenge (+), Sensitization (+) Rechallenge (-), Sensitization (+) Rechallenge (+) 24 hours (b) or 48 hours (c) after rechallenge of Aspergillus antigen are shown. Scale bars represent 100 μm. More than three mice per group were analyzed. (d) A schematic illustration of the repeated exposure of mice to Aspergillus antigen for 7 weeks with a 7-week rest period with FTY720 treatment followed by challenge with a single dose of Aspergillus antigen. (e) A schematic illustration of the repeated exposure of mice to Aspergillus antigen for 7 weeks with a 7-week rest period with FTY720 treatment and anti-CD103 treatment followed by challenge with a single dose of Aspergillus antigen. (f) A representative plot of intravenous staining of anti-CD4 (CD4 (iv)) and anti-CD103 staining in CD44loCD69lo CD4+ T cells (left), and CD44hiCD69hi CD4+ T cells (right) in the lung from control antibody-treated mice (control ab.) and anti-CD103 antibody-treated mice (anti-CD103 ab.). Representative findings from two independent experiments are shown. (g) The absolute numbers of CD44hiCD69hiCD103hi CD4+ T cells in the lung from anti-control antibody-treated mice (control ab.) and anti-CD103 antibody-treated mice (anti-CD103 ab.) (n = 3 mice per group). Data are the mean ± SD. P-values were calculated using an unpaired two-sided Student’s t-test (g).

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Ichikawa, T., Hirahara, K., Kokubo, K. et al. CD103hi Treg cells constrain lung fibrosis induced by CD103lo tissue-resident pathogenic CD4 T cells. Nat Immunol 20, 1469–1480 (2019). https://doi.org/10.1038/s41590-019-0494-y

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