Articles
https://doi.org/10.1038/s41590-019-0494-y

CD103hi Treg cells constrain lung fibrosis induced
by CD103lo tissue-resident pathogenic CD4 T cells
Tomomi Ichikawa1,2,12, Kiyoshi Hirahara1,3,12, Kota Kokubo1,12, Masahiro Kiuchi1, Ami Aoki1,
Yuki Morimoto1, Jin Kumagai1, Atsushi Onodera1,4, Naoko Mato5, Damon J. Tumes 1,6,
Yoshiyuki Goto7,8, Koichi Hagiwara5, Yutaka Inagaki 9, Tim Sparwasser10, Kazuyuki Tobe2 andToshinori Nakayama 1,11*
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, lungtissue-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.

I

mmunological memory is central to acquired immunity1.
Memory T cells direct rapid and efficient protection against previously encountered pathogens2. Several subsets of memory T cell
have been identified (that is, effector memory T cells (TEM cells)
and central memory T cells (TCM cells))3. TEM cells are abundantin nonlymphoid tissues and express low levels of the chemokine
receptor CCR7 and CD62L, whereas TCM cells express both CCR7
and CD62L3. Another subset of memory T cells—tissue-resident
memory T cells (TRM cells)—have been defined and are characterized by expression of the C-type lectin CD69 and the integrin
CD103 (refs. 4–7). TRM cells preferentially localize in epithelial barrier
tissues and are required for protection at mucosal sites, such as the
respiratory tract, gastrointestinal tract, reproductive tract and skin,in both mice and humans8–10.
Heterogeneity of effector T cell populations, including circulating memory T cells, has been well defined11–14. In the case of CD8+
TRM cells, distinct populations of CD8+ TRM cells play importantroles in the pathology of T cell-mediated inflammatory diseases15,16.
CD49a+CD8+ TRM cells are enriched in the skin of patients with vitiligo, whereas interleukin-17 (IL-17)-producing CD49a−CD8+ TRM
cells are frequently detected in patients with psoriasis15.
CD4+ T cells in the lung aid the development of CD103hiCD8+
TRM cells after influenza virus infection17. In a mouse model of vaginal herpes simplex virus infection, CD4+ TRM cells in the genital
mucosa are required for full protection from herpes simplex virus18.
Tissue regulatory T cells (Treg cells) reside in nonlymphoid tissue andcontribute to the maintenance of host homeostasis19–21. Pathogenicroles for memory CD4+ T cells, which particularly memory-type
pathogenic TH2 cells that produce high levels of IL-5, have been
identified13,22. Similar pathogenic TH2 cells producing high levels of

IL-5 have been reported in the skin of patients with atopic dermatitis23, as well as the peripheral blood of patients with eosinophilic
gastrointestinal and allergic diseases24–26. Amphiregulin-producing
fibrosis-inducing pathogenic TH2 cells have been identified ineosinophilic airway inflammation27. However, the functional heterogeneity and pathogenic roles of CD4+ TRM cells that reside withinchronic inflammatory lesions remain unknown. Moreover, themolecular mechanisms through which CD4+ TRM cells are generated are largely undefined.
Fungi are the most widespread microbes encountered by mammalian hosts28. Sensitization to fungi plays a major role in thedevelopment, persistence and severity of lower airway diseases inpatients with asthma29. Sensitivity to Aspergillus fumigatus, which
is one of the most common environmental fungi, is associated withan increased risk of adult-onset asthma30. The underlying cellularand molecular mechanisms that control the chronic inflammationinduced by fungal infection are poorly understood.
Herein, we investigate the function of CD4+ TRM cells involved in
the pathogenesis of fibrotic responses during chronic lung inflammation induced by repeated exposure to A. fumigatus. We foundthat tissue-resident CD44hiCD69hi CD4+ T cells had high expression of fibrosis-related genes, relative to circulating CD44lo or
CD44hiCD69lo CD4+ T cells. Moreover, low expression of CD103
defined pathogenic effector CD4+ TRM cells, and high expressionof CD103 defined tissue-resident immunosuppressive Treg cells thatpossessed distinct transcriptional and epigenetic programs in the
inflamed lung. Among CD44hiCD69hi CD4+ T cells, CD103lo CD4+
TRM cells promoted the pathology of fibrotic responses in the lungthrough the enhanced production of effector cytokines such as
IL-5 and IL-13. In contrast, CD103hi tissue-resident Treg cells that

Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan. 2First Department of Internal Medicine, University of Toyama,
Toyama, Japan. 3AMED-PRIME, AMED, Chiba, Japan. 4Institute for Global Prominent Research, Chiba University, Chiba, Japan. 5Division of Pulmonary
Medicine, Department of Medicine, Jichi Medical University, Shimotsuke, Japan. 6Centre for Cancer Biology, University of South Australia and SA
Pathology, Adelaide, South Australia, Australia. 7Division of Molecular Immunology, Medical Mycology Research Center, Chiba University, Chiba, Japan.
8
Division of Mucosal Symbiosis, International Research and Development Center for Mucosal Vaccines, Institute of Medical Science, The University ofTokyo, Tokyo, Japan. 9Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Isehara, Japan. 10Department of Medical
Microbiology and Hygiene, University Medical Center, Johannes Gutenberg University Mainz, Mainz, Germany. 11AMED-CREST, AMED, Chiba, Japan.
12
These authors contributed equally: Tomomi Ichikawa, Kiyoshi Hirahara, Kota Kokubo. *e-mail: tnakayama@faculty.chiba-u.jp
1

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Articles
expressed Foxp3 ameliorated fibrotic responses. Thus, functionallydistinct tissue-resident CD4+ T cell populations regulate the pathology of chronic inflammatory responses, including fungal-induced
fibrotic responses in the lung.

Results

Repeated exposure to Aspergillus antigen resulted in chronic lunginflammation and lung fibrotic responses. Dysregulated inflammation promotes fibrotic changes in various organs, including thelung31. To investigate the immunological events that occur during
fibrotic changes in the lung, we developed an experimental model inwhich mice were repeatedly exposed intranasally to Aspergillus antigen for 7 weeks (Supplementary Fig. 1a). A histological analysis of
the lungs revealed massive infiltration of inflammatory cells aroundthe bronchi (Fig. 1a). Repeated antigen exposure also induced
fibrotic responses in the peri-bronchial region, as evidenced byincreased staining by Sirius Red, as well as increased collagen levelsin the lung compared with those in controls (Fig. 1b,c). The expression of the fibrotic signature gene collagen type 1 alpha 1 (Col1a1)
was enhanced in the lungs of mice with repeated exposure to fungal antigen (Supplementary Fig. 1b). In addition, transgenic micecontaining the enhancer and promoter sequences of the Col1a2
gene linked to green fluorescent protein (collagen 1a2-GFP mice)
showed increased GFP-positive cells that produced collagen in thelung (Supplementary Fig. 1c). Effector cytokines—particularly IL-5
and IL-13—were substantially increased in the bronchoalveolar
lavage (BAL) fluid of mice repeatedly exposed to Aspergillus antigen(Supplementary Fig. 1d), and were associated with the infiltration of
inflammatory cells, including eosinophils, neutrophils and lymphocytes, and enhanced airway hyper-responsiveness (Supplementary
Fig. 1e,f). Thus, repeated exposure to Aspergillus antigen resulted
in chronic lung inflammation accompanied by increased lungfibrotic responses.
Increased numbers of CD4+ T cells were detected in the lungs,
but not the spleen, of Aspergillus antigen-exposed mice, andthe lung CD4+ T cells had increased proportions of cells with a
CD44hiCD62Llo phenotype, suggesting that memory-type CD4+
T cells may be involved in the induction of fibrotic responses
in this model of chronic lung inflammation (Supplementary
Fig. 1g–i). Moreover, in T cell-deficient Foxn1nu mice, the lunginflammation and fibrotic responses were significantly ameliorated,indicating that T cells play an important role in the lung pathology(Supplementary Fig. 1j,k).
Repeated exposure to Aspergillus antigen induced CD69hi CD4+
tissue-resident T cells in the inflamed lung. Repeated exposure
to the fungus antigen induced a subpopulation of CD44hiCD62Llo
CD4+ T cells in the lungs that expressed high levels of CD69
(Fig. 1d). CD69 expression on CD44hiCD62Llo CD4+ T cells
in the spleen was unchanged by fungal exposure in the lungs(Supplementary Fig. 2a). To characterize the CD44hiCD62LloCD69hi
CD4+ T cells in the lungs, we performed an unbiased flow cytometry
screening analysis. We identified 12 upregulated cell surface markers
and one downregulated cell surface marker (Supplementary Fig. 2b).
Among these molecules, CD103 (a ligand for E-cadherin) exhibiteda bimodal expression pattern, while the other cell surface molecules
did not (Fig. 1e and Supplementary Fig. 2c). CD44hiCD62LloCD69hi
CD4+ T cells from the inflamed lung, but not the spleen, exhibited increased numbers of CD103hi T cells compared with controls(Fig. 1f and Supplementary Fig. 2d–f). Since both CD69 and CD103
have recently been defined as markers for CD8+ tissue TRM cells5,
we analyzed whether the CD44hiCD62LloCD69hi CD4+ T cells
were tissue resident. After repeated exposure to the Aspergillus
antigen for 7 weeks, the mice were intravascularly injected with
CD4-PE antibody and sacrificed 3 min later to distinguish between
tissue-localized cells and blood-borne cells32 (Supplementary

Nature Immunology
Fig. 2g,h). Exposure to the fungal antigen induced substantial
numbers of tissue-resident CD44hi CD4+ T cells in the inflamedlung (Supplementary Fig. 2i), but not the spleen (Supplementary
Fig. 2j). In the control group, approximately 40% of the CD44hiCD69hi
CD4+ T cells were protected from in vivo labeling of by the CD4antibody, and more than 95% were protected in the Aspergillus antigen-treated group (Fig. 1g,h and Supplementary Fig. 2k). In boththe control and Aspergillus antigen-treated groups, the majority ofCD44loCD69lo CD4+ T cells were circulating cells. Next, we analyzed
the flow cytometry data using t-distributed stochastic linear embedding (t-SNE) plots—a dimensionality reduction method33. t-SNE
plots confirmed that almost all of the CD44hiCD69hi CD4+ T cells
in the Aspergillus antigen-treated group were within the population protected from intravascular labeling of CD4 (Supplementary
Fig. 2l). Consistent with these results, histological analysis of theinflamed lung exhibited massive infiltration of CD69hi CD4+ T cells,
as well as collagen deposition (Fig. 1i,j and Supplementary Fig. 2m).
Thus, repeated exposure to fungal antigen induced CD69hi CD4+
tissue-resident T cells in the inflamed lung.
Tissue-resident CD69hi CD4+ T cells expressed high levels offibrosis-related genes. To determine the characteristics of CD4+
T cells induced by fungal antigen stimulation, we isolated four subpopulations of CD4+ T cells (CD44lo, CD44hiCD69lo, CD44hiCD69hi
CD103lo and CD44hiCD69hiCD103hi T cells) from the inflamedlung and examined the global gene expression profile by RNA
sequencing (RNA-Seq; Fig. 2a and Supplementary Fig. 3a,b).
Principal component analysis revealed that each subpopulation of
CD4+ T cells exhibited a distinct transcriptional profile (Fig. 2a,bottom). In total, 27 and 56 genes were specifically expressed in
CD44hiCD69hiCD103lo and CD44hiCD69hiCD103hi T cells, respectively (Supplementary Fig. 3c). Effector cytokines such as Il17a, Ifng,
Il4, Il13 and Il5 were upregulated in CD69hiCD103lo T cells but not
in CD69hiCD103hi T cells (Fig. 2b, left). The specifically upregulated
genes in CD69hiCD103hi T cells included Foxp3, Klrg1, Prdm1 andRorc, which indicated that CD69hiCD103hi T cells express genes characteristic of Treg cells19,21,34 (Fig. 2b, right). Pdcd1, Rora and a seriesof chemokine receptors including Cxcr6, Ccr4, Ccr2, Ccr5 and Ccr8
were upregulated in both CD69hiCD103lo and CD69hiCD103hi T cells
compared with CD44lo and CD44hiCD69lo T cells (Supplementary
Fig. 3d). Both CD44hiCD69hiCD103lo and CD44hiCD69hiCD103hi
T cells expressed significantly less S1pr1 than CD44lo and CD44hi
CD69lo T cells, and the low expression of S1pr1 is required for theestablishment of CD8+ TRM cells35 (Supplementary Fig. 3e,f). Both
CD69hiCD103lo and CD69hiCD103hi T cells also exhibited downregulation of the chemokine receptor Ccr7, which is required for
T cell exit from peripheral tissues36 (Supplementary Fig. 3f). To
validate the differential transcriptomes between CD69hiCD103lo
and CD69hiCD103hi T cells, we performed a co-expression network
analysis using CD44lo, CD69hiCD103lo and CD69hiCD103hi T cells
using a previously published method37 (Fig. 2c). Overlaying theexpression differences of the included genes revealed that one
cluster was defined by CD69hiCD103lo T cells, and the other was
defined by CD69hiCD103hi T cell-associated genes (Supplementary
Fig. 3g). Fibrosis-related genes were upregulated in both tissue-resident CD44hiCD69hiCD103lo and CD44hiCD69hiCD103hi
T cells compared with CD44lo or CD44hiCD69lo T cells (Fig. 2d–f,
Supplementary Fig. 3h and Supplementary Table 1) . Distinct sets of
upregulated genes were observed in the CD44hiCD69hiCD103lo andCD44hiCD69hiCD103hi T cells. These results indicated that tissueresident CD69hi CD4+ T cells were distinct in the transcriptome
profile from circulating CD4+ T cells.
Tissue-resident CD69hiCD103lo CD4+ T cells exhibit enhanced
cytokine production. Approximately 12% of CD69hiCD103lo
T cells expressed GATA3 (the master regulator of TH2 cells),
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Controlb

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1.50Control
Asp-AgFig. 1 | Repeated exposure to Aspergillus antigen induced tissue-resident CD4+ T cells in the inflamed lung. a, Representative histological sections of thelungs from control BALB/c mice (left) and Aspergillus antigen-exposed BALB/c mice (Asp-Ag; right). Sections were stained with hematoxylin and eosin.
Ten mice per group were analyzed. b, Representative histological sections of the lungs from control and Asp-Ag mice. Sections were stained with Sirius
Red. Ten mice per group were analyzed. c, Quantification of collagen protein in the lungs from control (n = 6) and Asp-Ag mice (n = 11). Pooled findings
from two independent experiments are shown. d, Cell surface markers of CD44hiCD62Llo CD4+ T cells in the lungs of control (blue lines) and Asp-Ag mice(red), along with the isotype-matched control antibody (gray). Representative findings from two independent experiments are shown. e, Cell surface
markers on CD44hiCD62LloCD69hi CD4+ T cells in the lungs of control (blue lines) and Asp-Ag mice (red), along with the isotype-matched control
antibody (gray). f, Pooled data showing the proportion of CD103hi T cells in CD44hiCD62LloCD69hi CD4+ T cells from control (n = 5) and Asp-Ag mice(n = 6). Representative findings from two independent experiments are shown. g, Representative plots of intravenous staining of anti-CD4 and anti-CD103
staining in CD44hiCD69hi CD4+ T cells (top) and CD44loCD69lo CD4+ T cells (bottom) in the lungs of control and Asp-Ag mice. iv, intravenous injection.
h, Percentage of tissue-resident CD4+ T cells in the CD44hiCD69hi CD4+ T cell population (red) and CD44loCD69lo CD4+ T cell population (blue) fromcontrol (open symbols; n = 9) and Asp-Ag mice (filled symbols; n = 8). i, Representative confocal micrograph images of the lung stained with anti-CD4(red), anti-CD69 (green) and anti-type l collagen (blue) from Asp-Ag mice. Representative findings of three independent experiments are shown.
j, Absolute numbers of CD69hiCD4+ T cells in the lungs of control and Asp-Ag mice (n = 8 mice per group). Data are presented as means ± s.d. inc, f, h and j. P values were calculated by two-sided Mann–Whitney U-test in c, f, h and j.
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d

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a Fibroblastb Tissue remodelingc Epithelial cell differentiaion
d Growth factor
e Chronic inflammatory response
f Agingg epithelial–mesenchymal transition
h TGF-β signalingi Extracellular marix/collagen/fibronection
j Angiogenesisk Myoblast

1.0

0

–1.0

Relative gene expression(z score)

S100a10
Esm1
Itgb1
Anxa2
Hist1h1b
Il2rb
Lgals1
Mki67
Rora
S100a4
S100a6Adam8Areg
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Calca
Capg
Ccr2
Cd44
Csda
Ehd1
Id2
Il10
Il1rl1
Il2ra
Lmna
Maf
Penk
Podnl1
Tiam1
Vim
Ern1
Furin
Ifng
Igfbp7
Il13
Il4
Il5
Nrp1
Ptprs
Rbpj
Spry1
Tnfsf11
Vdr
Xcl1
Bmpr2
Csf1
Dst
Foxp3
Itgav
Itgb8
Lamc1
Myo1e
Pmaip1
Prdm1
Ptpn5
Ramp1
Sdc4

Ehd1

interacts wit
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h

Ern1

hi

e

Foxp3

Foxp3

hi

CD44 CD69 CD103
T cell-specific moduleloCorrelation (n = 5,464)hihiloCD44 CD69 CD103 T cell-specific gene (n = 25)hihihi
CD44 CD69 CD103 T cell-specific gene (n = 54)
Correlated gene (n = 404)

Xcl1

Epithelial celldifferentiationhi

CD44 CD69 CD103
T cell-specific genes

Itgae
Tnfrsf8
Tnfrsf9
Foxp3
Klrg1
Rln3
Itgb8
Havcr2
AA467197
Il1r2
Ptpn5
Ly6a
Lamc1
Arl5a
Ikzf2
Neb
Arnt2
Sdc4
Itgav
Gzmb
Gm2a
Lilrb4
Mmd
Pmaip1
Tigit
Glrx
Prdm1
Cst7
Serpina3g
Slc15a3
Csf1
Dhrs3
Fam110a
Gp49a
AW112010
Myo1e
Cd83
Jdp2
Nfil3
Osbpl3Cysltr2
Ccr6
Lmnb1
Dst
Rorc
Acsbg1
Lag3
Mdfic
Asb2
Top2a
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Hnrpll
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Bmpr2
Hist1h2bm
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lo

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CD44 CD69 CD103 TcellschiRelative gene expression(z score)PC

PC1 (66.4%)

hi

CD44 CD69 CD103
T cell-specific genes

Xcl1
Igfbp7
Il5
Ccl1
Ifng
Tnfsf11
Vdr
Tbx21
Plac8
Tnfsf8
Il13
Il4
Spry1
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Furin
Il17a
Cd82
Nrp1
Rbpj
Ern1
Ctla2aSerpinb6a
Phlda1
Ptprs
Ptms
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Itgb1

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0 10 10 10 10
CD44-PB

hi

CD44 CD69 CD103
T cell-specific genesloCD44 T cellshiloCD44 CD69 T cellshihiloCD44 CD69 CD103 T cellshihihi
CD44 CD69 CD103 T cells

Fig. 2 | Tissue-resident CD69hi T cells showed unique profibrotic features. a, Top: cell-sorting strategy for the four groups of CD4+ T cells in the lungs
of Asp-Ag mice. Bottom: principal component analysis plot of RNA-Seq data from four groups of CD4+ T cells (n = 8). Duplicates of pooled samples areshown. b, Heat maps of genes with a greater than eightfold change in expression unique to CD44hiCD69hiCD103lo (left; n = 27) or CD44hiCD69hiCD103hi
T cells (right; n = 56), compared with CD44lo T cells, based on the mean value of each duplicate. c, Visualization of the co-regulation network based on
the 487 genes selected using the criteria in the Methods. Red and blue nodes represent CD44hiCD69hiCD103lo CD4+ T cell-specific genes (n = 25) andCD44hiCD69hiCD103hi CD4+ T cell-specific genes (n = 54), respectively, as determined in Supplementary Fig. 2c. Red and blue ellipses reveal specificmodules of CD44hiCD69hiCD103lo and CD44hiCD69hiCD103hi CD4+ T cells, respectively. d, Network visualization of fibrosis-related Gene Ontology
terms (n = 91). Dots indicate genes that were upregulated in CD44hiCD69lo (green), CD44hiCD69hiCD103lo (red) and CD44hiCD69hiCD103hi T cells(blue), compared with CD44lo T cells. e, Heat map depicting fibrosis-related genes that were substantially upregulated among four groups of CD4+
T cells in the lungs of Asp-Ag mice. f, Numbers of genes encoding fibrosis-related factors in each pathway among four groups of CD4+ T cells in thelungs of Asp-Ag mice.
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Articles

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10

3

10
0

2

14.5

hi

7 7.6

3

4

10

10

5

10

Foxp3-AF647

2

0 10

CD103

CD103-PE

10

4

CD4-PE-Cy7

5

10

4

10

5

10

4

10

3

10

10
0

2

10

5

10

4

10

3

10
0

2

67.5 17.3
1.5

7.2

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32.5

0 10

2

10

3

10

4

10

10

5

10

4

3

10

3

10
0

2

10
0

2

10

5

10

5

10

4

10

4

10

3

10

3

10
0

2

10
0

2

5

2

c
Cells in CD69 CD103 CD4 T cells (%)

3

10

10

4

12.2 ± 8.2

54.7 7.2
15.6

21.2 4.3
32.8

5

2

10

3

10

0 10

10

4

5

10

GATA3-AF488

60

37.7 ± 9.7+

Cells in CD69 CD103 CD4 T cells (%)

4.6
11.9

Rorγt-BV421

40

+

4.4

0 10

GATA3-AF488b

29.5 34.5
8.5

Rorγt-BV421

CD103

5

10

10

Foxp3-AF647

hi

a

lo

hi

11.8 ± 5.8
2030

10.6 ± 4.9hihi

5.2 ± 2.4
0.9 ± 0.6
08.3 ± 3.6

1.6 ± 2.1

0
lo

hi

hi

Foxp3
hi
GATA3

Foxp3
hi
GATA3

hi

Foxp3
hi
RORγt

10

5

10

4

10

3

10
0

2

10

5

10

4

10

3

10
0

2

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10

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10

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10

4

10

4

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5

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4

10

3

10
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2

12.7

2

3

4

10

5

10

10

3

4

10

10

5

2

0 10

20.2 4.3
2.4

IL-13-FITC

2

0 10
10
CD4-PE-Cy7

CD103

lo

70.8

IL-5-PE

CD103-BV421

0 10

2

0 10

10

3

10

4

5

10

IL-4-APC

e

IL-4
30

P = 0.0079

4

10

5

2

10

0 10

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3.5

0 10

2

3

10

4

10

10

5

3

4

10

10

5

2

3

10

4

10

5

10

IFNγ-APC

IL-17

IL-13
30

30

10

17.2 0.3
5.3

0 10

IL-4-APC

IL-5
30

3

10

IL-17A-FITC

10

CD103

hi

d

lo

Foxp3
hi
RORγt

IFN-γ
30

Cytokine-producing cells (%)

P = 0.0079

15

15

15

P = 0.0079

15

15
P = 0.0079

P = 0.0079

0

0

0

0

0

Fig. 3 | CD69hiCD103lo CD4+ T cells were the effector cytokine producers in the inflamed lung. a, Representative intracellular staining profiles ofFoxp3, GATA3 and Rorγt in CD69hiCD103hi CD4+ Treg cells (top) and CD69hiCD103lo CD4+ T cells (bottom) in the lungs of Asp-Ag mice. More than
three independent experiments were performed, with similar results. b,c, Pooled data of intracellular staining profiles of Foxp3, GATA3 and Rorγt in
CD69hiCD103lo CD4+ T cells (b) and CD69hiCD103hi CD4+ Treg cells (c) in the lungs of Asp-Ag wild-type mice (n = 16 samples stained with Foxp3 andGATA3; n = 11 samples stained with Foxp3 and Rorγt). Data represent means ± s.d. d, Representative intracellular staining profiles of IL-5, IL-4, IL-13, IFN-γ
and IL-17 in CD69hiCD103hi CD4+ Treg cells (top) and CD69hiCD103lo CD4+ T cells (bottom) in the lungs of Asp-Ag mice. More than three independent
experiments were performed, with similar results. e, Percentages of the indicated cytokines in CD69hiCD103lo CD4+ T cells (red) and CD69hiCD103hi CD4+
Treg cells (blue) in the lungs of Asp-Ag mice. The findings of five pooled independent experiments are shown. Data represent means ± s.d. P values were
calculated by two-sided Mann–Whitney U-test.

and 12% expressed Rorγt (the master regulator of TH17 cells)(Fig. 3a,b and Supplementary Fig. 4a), with few cells co-expressing GATA3 or Rorγt together with Foxp3 (the master regulator
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of Treg cells) (0.9 and 5.2%, respectively). In contrast, between 60
and 80% of CD69hiCD103hi T cells expressed Foxp3, with approximately 50% of these co-expressing Rorγt, and 10% co-expressingArticles
GATA3 (Fig. 3a,c and Supplementary Fig. 4b). On stimulation
with phorbol 12-myristate 13-acetate (PMA) and ionomycin for
4 h, CD69hiCD103lo T cells exhibited significantly higher production of effector cytokines, such as IL-4, IL-5, IL-13, IL-17A
and interferon-γ (IFN-γ), compared with CD69hiCD103hi T cells(Fig. 3d,e and Supplementary Fig. 4c). These results indicate thattissue-resident CD69hiCD103lo CD4+ T cells produce effector cytokines in the Aspergillus antigen-induced chronically inflamed lung.
Foxp3+ Treg cells suppress the pathology of chronic lung inflammation. Next, we investigated the immunological function of
Foxp3+ CD4+ T cells during chronic lung inflammation. Foxp3DTR
mice, which express the diphtheria toxin receptor (DTR) under
the control of the Foxp3 gene locus, were repeatedly exposed
to Aspergillus antigen followed by intranasal administration ofDiphtheria toxin to deplete Foxp3hi CD4+ T cells (Supplementary
Fig. 5a). The intranasal administration of Diphtheria toxin resulted
in a significant decrease of Foxp3hi CD4+ T cells in the lung, but notin the spleen (Supplementary Fig. 5b,c). As expected, the percentage
of CD69hiCD103hi T cells was significantly decreased as a result of
the deletion of Foxp3+ CD4+ T cells (Supplementary Fig. 5d). A coexpression network analysis showed alterations in the gene expression of the remaining CD69hiCD103hi T cells from the lungs ofAspergillus antigen-exposed Foxp3DTR mice after the administrationof Diphtheria toxin (Fig. 4a, right). The remaining CD69hiCD103hi
T cells showed increased expression of effector cytokine genes, such
as Il4, Il5, Il13 and Ifng (Fig. 4a, boxes in right panel), accompanied
by an increase in the Foxp3loGATA3hi cell subpopulation (Fig. 4b
and Supplementary Fig. 5e) The administration of Diphtheria toxin
also resulted in a significant increase in CD103loFoxp3loGATA3hi
T cells and a significant decrease in CD103loFoxp3loRorγthi T cells(Supplementary Fig. 5f,g). CD69hiCD103hi T cells in Foxp3DTR
mice showed enhanced expression of IL-5 and IL-13 (Fig. 4c andSupplementary Fig. 5h). A histological analysis of the lungs of
Foxp3DTR mice revealed a further increase in the infiltration of
inflammatory cells around the bronchi (Fig. 4d). The absolute
numbers of infiltrating inflammatory cells in BAL fluid were significantly increased in Foxp3DTR mice compared with wild-typemice (Fig. 4e). Foxp3DTR mice showed substantially higher levels offibrosis compared with those in control groups, as assessed by Sirius
Red staining and collagen levels (Fig. 4f,g). These results indicatethat Foxp3+ Treg cells suppress the pathology of Aspergillus antigeninduced chronic inflammation in the lung. Moreover, we investigated whether Foxp3+Rorγt+ Treg cells regulated the Aspergillus
antigen-induced lung inflammation in vivo (Supplementary
Fig. 5i,j). Rorcfl/flFoxp3-cre mice showed similar numbers of infiltrating inflammatory cells in BAL fluid compared with those in Rorcfl/
flmice after the administration of Aspergillus antigen for 7 weeks(Fig. 4h). These data indicate that Foxp3+Rorγt+ Treg cells do notcontribute to the suppression of Aspergillus antigen-induced chronic
inflammation in the lung.
Global views of the chromatin landscapes in CD103lo TRM andCD103hi tissue-resident Treg cells reveal their differential regulomes. To assess whether the tissue-resident CD44hiCD69hi CD4+
T cells induced by chronic Aspergillus antigen exposure are CD4+
TRM cells, we performed parabiosis experiments using Thy1.1
and Thy1.2 mice that were subjected to a 7-week challenge withAspergillus antigen followed by a 7-week rest period to allow
memory T cell formation (Fig. 5a). Pairs of naive Thy1.1 mice andallergen-challenged Thy1.2 mice with a 7-week rest period were
surgically joined, and the pairs of mice were analyzed 2 weeks later.
After intravenous staining with anti-CD4 antibody, we found thatcongenically marked CD4+ T cells were mixed, almost equivalently,
in the circulating CD4+ T cells of both the naive and challenged
parabionts (Fig. 5b,c). In contrast, in the tissue-resident CD4+

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T cells, the population of Thy1.2+ cells in the lungs of the challenged
Thy1.2 parabionts was significantly larger than that in the naive
Thy1.1 parabionts. These parabiosis experiments using mice witha 7-week rest period indicate that chronic Aspergillus antigen exposure induced CD4+ TRM cells (Fig. 5b,c).
Analysis of chromatin accessibility can give detailed and highly
informative data about gene regulation and the functional stateof cells. We performed an assay to detect transposase-accessible
chromatin using sequencing (ATAC-Seq), using lung CD4+ T cells
recovered from mice subjected to a 7-week Aspergillus antigen
challenge with a 7-week rest period (Fig. 6a and Supplementary
Fig. 6a). A gating strategy based on the expression of CD44, CD69
and CD103 was again used (see Fig. 2a, top). Principal component
analysis revealed that each subpopulation of CD4+ T cells exhibiteda distinct regulome profile (Fig. 6b). Among a total of 26,449 accessible regulatory elements from four subpopulations of CD4+ T cells
in the lung, approximately 37% were common to all subpopulations,whereas the majority (63%) of the regions were either unique to asingle subpopulation (CD69hiCD103lo T cell-specific region: 7.93%;
CD69hiCD103hi T cell-specific region: 11.00%) or shared by two
subpopulations of CD4+ T cells (common region: 11.39%) (Fig. 6c).
The enrichment of consensus transcription factor motifs showedthat specific transcription factors were differentially enrichedin each subpopulation of CD4+ T cells (Supplementary Fig. 6b).
For example, Tcf7 and Lef1 were enriched in CD44lo T cells, andStat6 and GATA3 were enriched in CD44hiCD69hiCD103lo T cells,
whereas Foxp3 was enriched in CD44hiCD69hiCD103hi T cells.
More importantly, we found that the signature genes in each subpopulation were epigenetically regulated (Fig. 6d–f). For example,
the regulatory elements of effector cytokines such as II4, Il13 andIl17a, which were ranked in the top 10% (Fig. 6d, left), were preferentially accessible in CD44hiCD69hiCD103lo T cells (Supplementary
Fig. 6c,d). In contrast, regulatory elements of Foxp3 and Itgae, whichwere ranked in top 10% (Fig. 6e, left), were selectively accessible inCD44hiCD69hiCD103hi T cells (Supplementary Fig. 6e,f). The specific regulatory elements of fibrosis-related genes were highly accessible in both CD44hiCD69hiCD103lo and CD44hiCD69hiCD103hi
CD4+ T cells compared with CD44lo or CD44hiCD69lo CD4+
T cells (Fig. 6g and Supplementary Fig. 6g,h). More importantly,
the enriched pathways of the fibrosis-related genes revealed by
ATAC-Seq were similar to those identified by RNA-Seq analysis.
Thus, high-resolution profiling of chromatin landscapes by ATACSeq identified subpopulation-specific regulatory elements neargenes that may specify the function of CD44hiCD69hiCD103lo andCD44hiCD69hiCD103hi T cells.
Tissue-resident CD4+ T cells in the lungs shape the inflammation accompanied by fibrotic responses. Next, we investigated thepathophysiological roles of CD103lo TRM cells and CD103hi tissueresident Treg cells in vivo. Mice subjected to the 7-week Aspergillus
antigen challenge followed by a 7-week rest period were rechallenged with a single dose of antigen, and the infiltration of inflammatory cells in BAL fluid was found to be increased compared withthat in mice without the antigen rechallenge in vivo (Fig. 7a andSupplementary Fig. 7a). Increased infiltration of inflammatory cells
in the lung parenchyma, and enhanced collagen deposition aroundthe bronchi, were also observed 24 and 48 h after rechallenge of
Aspergillus antigen (Supplementary Fig. 7b,c).
To prevent T cells from repositioning from secondary lymphoidorgans to the tissue, we administered FTY720 (ref. 38) (Supplementary
Fig. 7d). As expected, FTY720 treatment resulted in a dramatic
decrease in CD4+ T cells in the peripheral blood mononuclear cells(Fig. 7b). FTY720 treatment followed by a single Aspergillus antigen
challenge induced a significant decrease of circulating CD4+ T cells,
but not in tissue-resident CD4+ T cells in the lungs (Fig. 7c). At thesame time, comparable numbers of inflammatory cells infiltrated in
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a

lo

hi

CD44

hi

CD44 CD69 CD103

lo

hi

hi

hi

CD44 CD69 CD103
Il4

Wild type

Il13
IFNγ
Ccl1
Il5

Correlation(5,464 edges)

Foxp3-DTR

Il4
Il13
IFNγ
Ccl1

–1
0
1
Relative gene expression(z score; 483 genes)

Il5

b

40

c

P < 0.0001

Wild type

2.3

104

P < 0.0001
P = 0.0006

IL-5-PE(%)

Foxp3-DTR

20

P > 0.999

Wild typeFoxp3hihi
RORγt

Foxp3-DTR

e

IL-13-PE

Foxp3lohi
RORγt

P = 0.0083

12.0

103
102
0
0 102 103 104 105

0 102 103 104 105

IFN-γ-APC

105

1.1 0.1
2.0

14.4 1.8
2.2

104

103

103

102
0

102
0
0 102 103 104 105

0 102 103 104 105

IL-4-APCf

Wild typeFoxp3-DTR

5

Absolute cell number (×10 )d

Foxp3hihi
GATA3

11.1 0.6
4.3

104

102
0

104

Foxp3lo
hi
GATA3

105

0.3
12.2

103

105

0

Foxp3-DTR

Wild type105

6.0

100 µm

100 µm0
1 mm

g

Foxp3-DTRh
P = 0.0467

Absolute cell number (×105)

5.0
Collagen in left lung (mg)Wild type

1 mm

2.5

0

Wild typeFoxp3-DTR

6.0

fl/fl

Rorc

fl/fl

Rorc Foxp3-cre
P = 0.773
P = 0.658
3.0
P = 0.982

P = 0.983
P = 0.842
0
Mac

Lymph

Neut

Eos

Total

Fig. 4 | Depletion of Foxp3+ CD4+ Treg cells resulted in exacerbation of chronic lung inflammation. a, Relative gene expression profiles in the co-regulation
network in naive CD44loCD69lo (left), CD69hiCD103lo CD4+ (middle) and CD69hiCD103hi CD4+ T cells (right) from the lungs of Asp-Ag wild-type mice
(BALB/c background) (top) and Foxp3-DTR transgenic mice (bottom). b, Intracellular staining profiles of Foxp3, GATA3 and Rorγt in CD69hiCD103hi
CD4+ T cells in the lungs of Asp-Ag wild-type mice (n = 9) and Foxp3-DTR transgenic mice (n = 9). c, Intracellular staining profiles of IFN-γ and IL-5 (top)and IL-13 and IL-4 (bottom) in CD69hiCD103hi CD4+ T cells in the lungs of Asp-Ag wild-type mice (left) and Foxp3-DTR transgenic mice (right). Three
independent experiments were performed, with similar results. d, Representative histological sections of the lungs from Asp-Ag wild-type mice and Foxp3DTR transgenic mice. Sections were stained with hematoxylin and eosin. More than nine mice per group were analyzed. e, Total numbers of cells in the
BAL fluid from Asp-Ag wild-type (n = 9) and Foxp3-DTR transgenic mice (n = 9). f, Representative histological sections of lungs from Asp-Ag wild-type(left) and Foxp3-DTR transgenic mice (right). Sections were stained with Sirius Red. More than nine mice per group were analyzed. g, Quantificationof collagen protein in the lungs from Asp-Ag wild-type (n = 15) and Foxp3-DTR transgenic mice (n = 18). h, Absolute numbers of total cells (Total),
macrophages (Mac), eosinophils (Eos), neutrophils (Neut) and lymphocytes (Lymph) in BAL fluid from Asp-Ag Rorcfl/fl (C57BL/6J background; n = 13)
and Rorcfl/flFoxp3-cre mice (n = 15). Data are represented as means ± s.d. P values were calculated by two-way ANOVA (b) or two-sided Mann–Whitney
U-test (e, g and h).
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Articles
Intranasal administrationof 50 µg mouse−1
once per week1
Thy1.2

3

4

1

2

5

6

7

8

3

4

5

6

7

8
Weeks

9

10

11

85.2 0.1
3.5 11.2

103

103

2

102
0
–102

10
0
–102
103 104 105

1
2
Weeks
Assay
12

1.7

105

22.9

3.6 0
3.5 92.9

4

10

103

103

102
0
–102

102
0
–102

Thy1.2-APC

100

Challenged
parabiont
lung

Naive
parabiontlung

103 104 105

0Circulating CD4+ T cells

P = 0.0006

1.5

10

0
103 104 105
Thy1.1-FITC

3.0
Absolute number ofThy1.2+CD4+ T cells (×107)

3.0

49.9 0.1
5.8 44.2

4

3

Tissue-resident CD4+ T cells

103 104 105

105

10

0
103 104 105
CD4 (iv )-PE

14

53.1 0.1
6.1 40.7

0

4

102
0
–102

13

104

103 104 105

0

14Parabiosis

105

103

105

CD4-PE-Cy7

105

20.6

104

0

13

Circulating
CD4+ T cells

4

10

Thy1.2

1.2

102
0
–102

Thy1.1

12

11

Tissue-resident
CD4+ T cells
105

Absolute number ofThy1.2+CD4+ T cells (×106)

10

Restingphase(7 weeks)

Lung

c

9

Challengephase(4 weeks)

Sensitization
phase(3 weeks)

Thy1.1b

2

Intranasal administrationof 20 µg mouse−1
twice per week

3.0

P = 0.3176
1.5

0Challenged
parabiont
lung

Naive
parabiont
lung

Absolute number ofThy1.1+CD4+ T cells (×107)a

Nature Immunology

P = 0.9015
1.5

0Challenged
parabiont
lung

Naive
parabiontlungFig. 5 | CD4+ TRM cells were induced by chronic Aspergillus antigen exposure. a, Schematic of the experimental protocol. Mice were repeatedly exposed
to Aspergillus antigen for 7 weeks, with a 7-week rest period followed by the parabiosis experiment. b, Representative plots of cell-surface staining ofanti-Thy1.1 and anti-Thy1.2 in tissue-resident CD4+ T cells (left) and circulating CD4+ T cells (right) in the lungs from Asp-Ag Thy1.2 mice (top) and Thy1.1
mice (bottom). Three independent experiments were performed, with similar results. c, Absolute numbers of Thy1.2+ CD4+ T cells in the tissue-resident
CD4+ T cell population (left), Thy1.2+ CD4+ T cells in the circulating CD4+ T cell population (middle) and Thy1.1+ CD4+ T cells in the circulating CD4+
T cell population (right) from the lungs of Asp-Ag Thy1.2 (challenged parabiont lung) and Thy1.1 mice (naive parabiont lung) (n = 7 mice per group).
Data represent means ± s.d. P values in were calculated by two-sided Mann–Whitney U-test.

the BAL fluid, accompanied by fibrotic responses in the lungs of themice with FTY720 treatment (Fig. 7d,e). These results indicate thatlung inflammation induced by rechallenge of the Aspergillus antigen
was induced by CD4+ TRM cells.
Finally, we performed a set of experiments using the administration of FTY720 and a single challenge of Aspergillus antigen,with or without intraperitoneal injection of anti-CD103 antibodies(Supplementary Fig. 7e). The administration of anti-CD103 antibodies resulted in a substantial depletion of CD44hiCD69hiCD103hi
circulating CD4+ T cells (Supplementary Fig. 7f,g). The administration of a single dose of Aspergillus antigen to mice that received

anti-CD103 antibody treatment resulted in exacerbation of lunginflammation, accompanied by increased inflammatory cell infiltration and fibrotic responses compared with mice treated with
control antibodies (Fig. 7f,g). Thus, the CD103hi subpopulation of
CD44hiCD69hi CD4+ T cells constrains lung inflammation and theextent of fibrotic responses.

Discussion

We have demonstrated that repeated fungal exposure induced lunginflammation and fibrotic responses, and the presence of two functionally distinct CD4 TRM populations in the lung. CD69hiCD103lo
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a
105

104

104

103
0
–103 0 103

103
0
–103

104 105–103 0 103

50

PC2 (13.0%)

CD103-FITC

CD69-PEb
105

104 1050CD44-PB
CD44lo T cells
CD44hiCD69lo T cells

CD44hiCD69lo T cells–100

CD44hi CD69hi CD103hi T cellscCD44lo T cells–50

CD44hiCD69hi CD103lo T cells

–50

0

50

100

CD44hiCD69hi CD103lo T cells

150

CD44hi CD69hi CD103hi T cells

PC1 (64.2%)

Variable (63%)

Common (37%)

Cell type
Rep 1
Rep 2

CD44loCD44hiCD69lo
CD44hiCD69hi CD103lo
CD44hi CD69hi CD103hi

Rep 1
Rep 2
Rep 1
Rep 2
Rep 1
Rep 2

5

0

10

log2[RPM]

d

g
200

Normalized tag countCD44lo T cellsaIgfbp7
Il4
Tnfsf8

100
50
Ifng

Igfbp7
Il13
Tnfsf8
Igfbp7
Tnfsf8
Itgb1
Il17a
Itgb1
Tnfsf8Tnfsf11
Xcl1 Tnfsf8
Ccl1
Tnfsf11
Rbpj
Rbpj
Il17a

200

400

g

Normalized tag count

20

Arnt2

0

Gzmb
Ikzf2
Crip1
Glrx
Tigit
Pmaip1
Itgae
Klrg1
Lmnb1
Itgav
Ccr6
Mmd
Jdp2
Tigit
Itgav
AW112010
Prdm1 Pmaip1
Dhrs3Itgae
Mdfic
Ikzf2
Havcr2
Ptpn5 Nfil3Lamc1
Arnt2

200

400

600

23%

f

Fibrosis-related genes

Fibrosis-related genes

Fibrosis-related genes (ATAC)

Fibrosis-related genes (ATAC)

CD44hiCD69hiCD103hi T cells
a
15
b
k
12

9

j

c

6

9

j

3
1

i

i

c

6

d

d

i

Genes with peaks (top 10%)
Genes with peaks
Genes without peaks

800

e

h
g

f

e

h
g

f

Fibrosis-related genes

Fibrosis-related genes

Fibrosis-related genes (ATAC)

Fibrosis-related genes (ATAC)

700
600

Normalized tag countg3
1

Ranked open chromatins

f

e

h

12

42%

58%

d

f

CD44hiCD69hiCD103lo T cells
a
15
b
k

Itgb8

c

6

i

e

h

Genes with peaks

Foxp3

40

d

Genes with peaks (top 10%)800 1,000 1,200

100

9

3
1

Genes without peaks

600

120

60

6

j

3
1

Ranked open chromatins

80

c

i

Xcl1

Tnfsf11

0

12

9

43%

57%

CD44hiCD69lo T cells
a
15
b
k

b

12

16%
j

150

e

15

k

Il10
Tox2
Il1rl1
Ctla4
Il1rl1Cish Stx11
Cd44
Cish
Fgl2
Fgl2

500
400
300
200
100

Ctla4
Rora
Il1rl1
Ccr4 Capg
Dusp4
Cxcr6 Vim
Ccr5
Atp2b4
Ahnak
Ccr4
Calca
Samsn1
Pdcd1
S100a11
Ccr2
Mafg
Rora
Ttc39c
S100a4
Lmna
Maf
Il2ra
Ahr Ccr8
Anxa2 Ahrr
Il18rap S100a6
Podnl1
Fam129a Ttc39c Adam8

13%
47%
53%

Areg

0

500

Genes with peaks (top 10%)
Genes with peaks
Genes without peaks

1,000 1,500 2,000 2,500

a Fibroblastb Tissue remodelingc Epithelial cell differentiaion
d Growth factor
e Chronic inflammatory resposef Agingg epithelial–mesenchymal transition
h TGF-β signalingi Extracellular marix/collagen/fibronection
j Angiogenesisk Myoblast

Ranked open chromatins

Fig. 6 | CD44hiCD69hi CD4+ TRM regulomes show fibrotic features. a, Cell-sorting strategy for the four groups of CD4+ T cells in the lungs of Asp-Ag micesubjected to a 7-week Aspergillus antigen challenge followed by a 7-week rest period (Supplementary Fig. 5a). b, Principal component analysis plots of
ATAC-Seq data from the four groups of CD4+ T cells (n = 8). Duplicates of pooled samples are shown. c, Comparison of global ATAC peaks in the fourgroups of CD4+ T cells in the lungs of mice subjected to a 7-week Aspergillus antigen challenge followed by a 7-week rest period. A heat map (right) shows
the signal intensity (log2[reads per million mapped reads (RPM)]) of each ATAC peak. Rep, replication. d–f, Left: cumulative normalized tag counts of
ATAC peaks in the indicated regions (CD44hiCD69hiCD103lo T-cell-specific region (d), CD44hiCD69hiCD103hi T-cell-specific region (e) and the common
region (f)), plotted against ranked open chromatins. Right: pie charts depict specific genes in the indicated regions with or without ATAC peaks (n = 27
in d, n = 56 in e and n = 60 in c). g, Numbers of genes encoding fibrosis-related factors with ATAC peaks in each pathway among the four groups of CD4+
T cells in the lungs of Asp-Ag mice. The results are overwritten on the profiles shown in Fig. 2f.

CD4+ TRM cells produced effector cytokines such as IL-5 and IL-13,
and were directly involved in the pathology of fungus-induced
fibrotic responses in the lung. In contrast with the reported functions
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of CD103hi CD8+ TRM cells15, CD103hi tissue-resident CD4+ T cells
frequently expressed Foxp3, and depletion of Foxp3+ CD4+ Treg cells
or CD103hi CD4+ T cells resulted in enhanced fibrotic responses inArticles

Nature Immunology
P = 0.0286b40

Sensitization (–); rechallenge (–)

P = 0.2222 P = 0.0079

cSensitization (+); rechallenge (+)
15.0

P = 0.0286

P = 0.0286

20

P = 0.3429
P = 0.0286

0

Lymph

Neut

Eos

–10

–1

Days before
Asp-Ag exposure

0
Mac

10.0

8.0
Absolute number (×105)

Sensitization (+); rechallenge (–)

Total

Circulating
CD4+ T cells
P = 0.0002

Tissue-resident
CD4+ T cells

Sensitization (–); rechallenge (+)
CD4+ T cells (%)

Absolute cell number (×104)

30.0

P = 0.16
4.0

0

PBS

Absolute number (×105)

a

5.0

0

FTY720PBS

FTY720PBS
FTY720deAbsolute cell number (×104)

30.0

Control
PBS
FTY720Control

PBS

FTY720

P = 0.574

P = 0.879
15.0

P = 0.442

100 µm

100 µm

100 µm

100 µm

100 µm

100 µmP = 0.574
P = 0.0207
0
Mac

Lymph

Neut

Eos

Total

g

P = 0.0002
25

30

100 µmP = 0.274 P < 0.0001

P = 0.315
0
Lymph

Neut

Eos

Total

Anti-CD103 antibody;rechallenge (+)

Mac

100 µmRatio of stained bronchus
area to basement
membrane area

Control antibody; rechallenge (–)
Anti-CD103 antibody; rechallenge (–)
Control antibody; rechallenge (+)
Anti-CD103 antibody; rechallenge (+)

P = 0.0002

Control antibody;rechallenge (+)

Absolute cell number (×104)

50

Control antibody;rechallenge (–)

f

P = 0.0001
P = 0.0414
P = 0.0429

15

0Control antibody; rechallenge (–)
Control antibody; rchallenge (+)
Anti-CD103 antibody; rechallenge (+)

100 µmFig. 7 | CD4+ TRM cells are sufficient to induce lung inflammation accompanied by fibrotic responses. a, Absolute numbers of total cells, macrophages,
eosinophils, neutrophils and lymphocytes in BAL fluid from four groups of mice (no sensitization and no rechallenge; no sensitization but rechallenge;
sensitization but no rechallenge; and sensitization and rechallenge; n = 4 per group). b, Proportion of CD4+ T cells in peripheral blood mononuclear cells
from PBS- (n = 5) and FTY720-treated mice (n = 5). c, Absolute numbers of tissue-resident T cells (left) and circulating T cells (right) in the lungs from
PBS- and FTY720-treated Asp-Ag mice (n = 8 mice per group). d, Absolute numbers of total cells, macrophages, eosinophils, neutrophils and lymphocytes
in BAL fluid from control mice (n = 5), PBS-treated mice (n = 8) and FTY720-treated mice (n = 8). e, Representative histological sections of lungs fromcontrol mice, PBS-treated mice and FTY720-treated mice. Sections were stained with hematoxylin and eosin (top) or Sirius Red (bottom). More than
three mice per group were analyzed. f, Absolute numbers of total cells, macrophages, eosinophils, neutrophils and lymphocytes in BAL fluid from fourgroups of mice (control antibody with no rechallenge (n = 5); control antibody with rechallenge (n = 8); anti-CD103 antibody with no rechallenge (n = 5);
and anti-CD103 antibody with rechallenge (n = 10)). g, Left: representative histology of mouse lung specimens stained with Sirius Red (n = 2 independent
experiments). Right: ratios of areas of randomly selected regions stained with Sirius Red surrounding the bronchi versus areas of the associated basement
membranes (control antibody with no rechallenge: n = 6 samples from n = 3 mice; control antibody with rechallenge: n = 9 samples from n = 3 mice;anti-CD103 antibody with rechallenge: n = 11 samples from n = 4 mice). Data represent means ± s.d. P values were calculated by two-sided Mann–Whitney
U-test (a–d and f) or one-way ANOVA (g).
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the lung. Moreover, we found that CD44hiCD69hiCD103lo pathogenic
effector CD4+ TRM cells and CD44hiCD69hiCD103hi tissue-resident
CD4+ Treg cells are transcriptionally, epigenetically and functionally distinct. Thus, this study highlights novel tissue-resident CD4+
T cell populations in the chronically inflamed lung that play crucial
roles in the establishment of fibrotic responses in the lung.
In the current study, we identified that the lung tissue-resident CD44hiCD69hi CD4+ T cells induced by repeated exposure
to fungal antigens consist of CD69hiCD103lo CD4+ TRM cells andCD69hiCD103hi Treg cells, both of which showed enhanced expression of fibrosis-related genes compared with circulating CD44hi
naive or CD44hiCD69lo CD4+ T cells. The fibrosis-related genesignature appeared to be acquired during the differentiation into
tissue-resident CD4+ T cells. Among the fibrosis-related genes
upregulated in tissue-resident CD44hiCD69hi CD4+ T cells, significant differences were observed between CD103lo CD4+ TRM cells
and CD103hi Treg cells. The profibrotic cytokine genes Il5 and Il13
were selectively expressed in CD69hiCD103lo CD4+ TRM cells. In
contrast, CD69hiCD103hiFoxp3+ Treg cells expressed high levels of
Itgae and Foxp3 and constrained the fungal antigen-induced fibrotic
responses in the inflamed lung. Interestingly, both Itgae and Foxp3
are induced by the fibrosis-associated cytokine transforming growth
factor-β (TGF-β), indicating that they may act as a rheostat limiting
fibrotic responses. We identified CD69hiCD103lo CD4+ TRM cells asa pathogenic cell population promoting massive lung inflammation
with fibrotic responses after repeated exposure to fungal antigens.
The CD69hiCD103lo CD4+ TRM cells exhibited unique genome-wide
transcriptomes and regulomes, with enhanced production of effector cytokines, including IL-4, IL-5, IL-13, IL-17A and IFN-γ. It has
been reported that amphiregulin (encoded by Areg, and produced
from pathogenic TH2 cells) plays a crucial role in fibrotic responses
in eosinophilic inflammation in the lung via the production of
osteopontin by eosinophils30. Amphiregulin produced from Treg
cells is also reported to be important for tissue repair processes20,27,39.
In the current study, we found that both CD69hiCD103lo CD4+ TRM
and CD69hiCD103hi Treg cells expressed high levels of Areg. Although
it is not known whether tissue repair processes occur in the current
experimental model of fungus-induced inflammation, a profibrotic
function of amphiregulin may play a distinct role. Importantly, thefibrosis-related genes upregulated in CD69hiCD103hi Treg cells weredistinct from those upregulated in CD69hiCD103lo TRM cells. Blimp1(encoded by Prdm1) is known to be crucial for both the appropriate function of Treg cells and stabilization of Foxp3 expression in
Treg cells40,41. We found high expression of both Foxp3 and Prdm1
in CD69hiCD103hi Treg cells, which indicates an immunosuppressiverole, rather than a profibrotic role, in the inflammatory tissue.
We demonstrated that repeated fungal antigen stimulationinduced CD69hiCD103hi CD4+ T cells, a substantial proportion ofwhich were Foxp3hiRorγthi cells. Surprisingly, Rorc deletion in Foxp3positive cells resulted in little impact on the extent of lung inflammation, which indicates that Foxp3hiRorγthi Treg cells might not have an
inhibitory function in the lung inflammation induced by repetitive
exposure to the Aspergillus antigen. In contrast, gut microbiotainduced Foxp3hiRorγthi Treg cells were reported to regulate type 2
immune responses in the intestine19,42. Although microbiota-induced
Foxp3hiRorγthi CD4+ T cells are maintained in the intestine undernormal steady-state conditions, lung CD69hiCD103hiFoxp3hiRorγthi
Treg cells were only detected after repeated exposure to fungal antigens. Thus, these two populations appeared to be induced by distinct
mechanisms, and might have different functions in vivo, althoughboth populations express Rorγt and Foxp3.
An important finding from the present study is that CD103
is a key molecule that can distinguish the function of lung CD4+
TRM cells. CD103 is well appreciated as a key molecule for the epithelial localization of CD8+ TRM cells6,43. The binding of CD103 toE-cadherin increases T cell adherence to epithelial cells44, suggesting
Nature Immunology | www.nature.com/natureimmunologythat CD103 may compartmentalize CD69hiCD103hi CD4+ T cells
from CD69hiCD103lo CD4+ TRM cells within the mucosal tissue in thelung. In addition, the localization of CD69hiCD103hi CD4+ T cells
adjacent to epithelial cells may confer the suppressive function of
CD69hiCD103hi CD4+ T cells, because epithelial cells are known tobe the main source of TGF-β in the lung45. At the same time, TGF-β
is known to be important for upregulation of CD103 expression andrepression of Klf2 expression35. Indeed, CD69hiCD103hi CD4+ T cells
downregulated expression of Klf2. Interestingly, CD69hiCD103hi
CD4+ T cells also had high expression of Klrg1, which encodes
KLRG1, another binding partner of E-cadherin46. Thus, tissue-localization in vivo may be closely related to the unique characteristics oftissue-resident CD4+ T cell subpopulations.
Patients with severe persistent asthma and allergic bronchopulmonary aspergillosis often show airway remodeling, structural
alterations of the airway wall (such as subepithelial thickening),
vascular changes and fibrosis47–49. High sensitivity to fungi—especially to Aspergillus—increases the risk of adult-onset asthma30.
Our findings indicate that CD4+ TRM cells could be potential therapeutic targets for patients with chronic fungal inflammation andfibrotic responses.
In summary, we identified pathogenic and immunosuppressive
tissue-resident CD4+ T cell subpopulations (CD69hiCD103lo CD4+
TRM cells and CD69hiCD103hiFoxp3+ CD4+ Treg cells, respectively)in the chronically inflamed lung with fibrotic responses. We havehighlighted the epigenetically regulated functional dichotomyof the tissue-resident CD4+ T cell subpopulations that govern
the pathophysiology of the fungus-induced fibrotic responses.
Understanding cellular and molecular mechanisms through which
distinct tissue-resident CD4+ T cells are generated will be essential
for the development of new therapeutic strategies for intractableinflammation-associated fibrotic diseases.

Online content

Any methods, additional references, Nature Research reporting
summaries, source data, statements of code and data availability andassociated accession codes are available at https://doi.org/10.1038/
s41590-019-0494-y.
Received: 30 May 2019; Accepted: 12 August 2019;
Published: xx xx xxxx

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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 valuablesuggestions on the parabiosis surgery. We are grateful to N. Takayama and K. Eto forproviding 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 thefollowing grants: Ministry of Education, Culture, Sports, Science and Technology Grantsin-Aid for Scientific Research (S) 26221305, JP19H05650, (C) 17K08876, 18K07164 and
19K16683; Practical Research Project for Allergic Diseases and Immunology (Researchon Allergic Diseases and Immunology) from AMED (numbers JP19ek0410060 andJP19ek0410045); AMED-PRIME, AMED (number JP19gm6110005); AMED-CREST,
AMED (number JP19gm1210003); the Mochida Memorial Foundation for Medical andPharmaceutical Research, The Ichiro Kanehara Foundation for the Promotion of Medical
Sciences and Medical Care; the Naito Foundation and the Takeda Science Foundation.

Author 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.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information is available for this paper at https://doi.org/10.1038/
s41590-019-0494-y.
Correspondence and requests for materials should be addressed to T.N.
Peer review information Zoltan Fehervari was the primary editor on this article andmanaged its editorial process and peer review in collaboration with the rest of theeditorial team.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.
© The Author(s), under exclusive licence to Springer Nature America, Inc. 2019

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Nature Immunology
Methods

Mice. The animals used in this study were backcrossed to C57BL/6J or BALB/
cAJcl mice more than ten times. Collagen 1a2-GFP transgenic mice (C57BL/6J
background) have been reported previously50. ‘Depletion of regulatory T cell’(DEREG) mice (BALB/cAJcl background) were provided by T. Sparwasser(Johannes Gutenberg University). Foxp3-YFP-IRES-Cre mice (C57BL/6J
background) were provided by A. Y. Rudensky (Memorial Sloan Kettering Cancer
Center)51. All mice were used at 6–8 weeks of age and were maintained under
specific-pathogen-free conditions. BALB/cAJcl, BALB/c Foxn1nu and C57BL/6J
mice were purchased from CLEA Japan. Rorcfl/fl mice (C57BL/6J background)
were purchased from The Jackson Laboratory. All of the Rorcfl/fl and Foxp3YFP-IRES-Cre mice used in this study were male. The DREG mice used in thisstudy were both male and female. All other mice used in this study were female.
Same-sex littermates were randomly assigned to experimental groups. The research
proposals were reviewed by the ethics committee for animals at Chiba University(registration numbers 29–98 and 29–99).
Induction of chronic lung inflammation by repeated intranasal administrationof Aspergillus antigen. A. fumigatus antigen (Greer) was administered intranasallyfor 7 weeks in a modified protocol based on the protocol described by Kerzerho
et al.52. For the first 3 weeks, 50 μg of antigen was administered once a week. For
the following 4 weeks, 20 μg of antigen was administered twice a week. In someexperiments, mice were analyzed 7 weeks after the last antigen administration.
Assays were performed 24 h after the last antigen administration.
Histology and immunofluorescence. Pathological changes were evaluated byhematoxylin and eosin, periodic acid–Schiff, Sirius Red and immunofluorescencestaining, as previously described53,54. Cryostat sections of the lungs were stained
and mounted with Fluorescent Mounting Medium (DakoCytomation). All
immunofluorescence and histological analyses were carried out with a confocal
laser microscope (LSM710; Carl Zeiss). Anti-CD4 (RM4-5; 552775; BD), antiCD3e (2C11; 12-0031-82; eBioscience), anti-CD69 (AF2386; R&D Systems)
and anti-type l collagen (LB-1102; LSL-LB-1102; LSL) were used for staining
lung samples.
Isolation of CD4+ T cells from the lung. After euthanasia, mice were perfusedwith 5 ml cold phosphate buffered saline (PBS) solution through the left and rightventricle, respectively. Lungs were dissociated in a C tube (Miltenyi Biotec) indigestion buffer (RPMI 1640 supplemented with collagenase type III (200 U ml−1;
Worthington) and DNase I (200 μg ml−1; Sigma–Aldrich)) using a gentleMACS
tissue dissociator (Miltenyi Biotec). After 30 min at 37 °C, samples were treatedwith final gentleMACS dissociation. Cell suspensions were passed through a
70-μm cell strainer (Greiner Bio-One). Lung mononuclear cells were separatedusing by Percoll solution (GE Healthcare).
Ex vivo analysis of CD4+ T cells from the inflamed lung. Anti-CD3ε (145-2C11;553061; BD), anti-CD4 (RM4-5; 552775; BD), anti-CD8a (53-6.7; 100725;
BioLegend), anti-CD44 (IM7; 103020; Biolegend), anti-CD62L (MEL-14;
553150, 553152 and 560514; BD), anti-TCRβ (H57-597; 553174; BD), anti-CD69(H1.2F3; 561932 and 560689; BD), anti-CD103 (2E7; 121421; BioLegend; andM290; 557494 and 557495; BD), anti-CD25 (PC61; 553866 and 561048; BD),
anti-CD122 (IL-2Rβ; TM-Beta 1; 553362 and 564762; BD), anti-CD127 (IL7Rα;
SB/199; 552543 and 564175; BD), anti-CD30L (RM153; 559232; BD), anti-I-COS(7E.17G9; 552146; BD), anti-CD200 (OX-90; 565543; BD), anti-PSGL-1 (2PH1;555306; BD), anti-CD11a (2D7; 553121; BD), anti-CD180 (RP/14; 117706;
BioLegend), anti-LAG3 (C9B7W; 552380; BD), anti-IL10Ra (2B11/CXCR4;551966; BD), anti-CCR7 (4B12; 560682; BD), anti-RANK-L (IK22-5; 560295;
BD), anti-CD309 (Avas12; 136403; BioLegend), anti-NKG2D (CX5; 558403;
BD), anti-Thy1.2 (30-H12; 105312; BioLegend), anti-Thy1.1 (OX-7; 202504;
BioLegend), anti-GATA3 (L50-823; 560074 and 560163; BD), anti-Foxp3 (MF23;
560408 and 560401; BD) and anti-Rorγt (Q31-378; 562607 and 562894; BD) wereused to stain the samples.
For unbiased fluorescence-activated cell sorting (FACS) screening, CD4+
T cells isolated from the lung were stained with antibodies against markers (Mouse
Cell Surface Marker Screening Panel; BD) or corresponding isotype-matchedantibodies. A FACS Verse or FACS Canto ll was used for the multi-parameter
analysis, and data were analyzed using the FlowJo software program (Tree Star).
Intravenous staining of CD4+ T cells localized in the vasculature of the lung.
Intravenous staining of CD4+ T cells was carried out as previously described55. For
intravascular cell staining of CD4+ cells, mice were intravenously injected with
0.5 μg anti-CD4 antibody (RM4-4; 116006 and 116004; BioLegend) diluted in
100 μl PBS, 3 min before sampling.
Depletion of Foxp3+ T cells by intranasal administration of Diphtheria toxin.
For the depletion of Foxp3+ cells, Foxp3-DTR transgenic or wild-type mice weretreated with the intranasal administration of 100 ng Diphtheria toxin (Sigma–
Aldrich) in 30 μl PBS, 1 h before the administration of Aspergillus antigen during
the last 3 weeks of antigen administration.
Nature Immunology | www.nature.com/natureimmunology

ArticlesFlow cytometry for the analysis of cytokine production. CD4+ T cells isolated
from the lung were stimulated with phorbol 12-myristate 13-acetate (50 ng ml−1)
and ionomycin (500 ng ml−1) for 4 h in the presence of monensin (2 μM). Anti-IL-4(11B11; 554436 and 562915; BD), anti-IL-5 (TRFK5; 554396; BD; and 504304;
BioLegend), anti-IL-13 (eBio13A; 12-7133-41 and 53-7133-82; eBioscience), antiIL-17 (TC11-18H10.1; 559502 and 563354; BD; and 506910; BioLegend) and antiIFNγ (XMG1.2; 554411 and 554412; BD; and 17-7311-81; eBioscience) were used
for intracellular staining of mouse samples.
t-SNE analysis. t-SNE analysis was performed using the FlowJo software program(version 10.2; FlowJo).
Quantitative real-time PCR. Total RNA isolation, complementary DNA synthesisand quantitative real-time PCR were described previously56. Primers and Roche
Universal probes were purchased from Sigma–Aldrich and Roche, respectively. The
gene expression was normalized with the β-actin messenger RNA signals.
RNA-Seq. RNA-Seq was carried out as previously described57. Total cellular RNA
was extracted with TRIzol reagent (Invitrogen). For complementary DNA library
construction, we used a SMARTer Stranded Total RNA-Seq Kit v2-Pico Input
Mammalian (Clontech) according to the manufacturer’s protocol. Sequencing oflibrary fragments was performed on the HiSeq 1500 System. For the data analysis,
read sequences (50 base pairs) were aligned to the mm10 mouse reference genome(University of California, Santa Cruz; December 2011) using Bowtie 2 (version
2.0.0) and TopHat (version 1.3.2). The fragments per kilobase of exon per million
mapped reads for each gene were calculated using Cufflinks (version 2.0.2).
Analysis and graphic display of RNA-Seq data. A total of 9,085 genes for which
the maximum fragments per kilobase of exon per million mapped reads value in all
samples was ≥1.0 were subjected to further analyses. A principal component analysis
was performed using R (https://www.r-project.org/). Clustering was performed using
APCluster (an R Package for Affinity Propagation Clustering). The transcriptionalsignatures of CD44hiCD69lo, CD44hiCD69hiCD103lo and CD44hiCD69hiCD103hi CD4+
T cells were defined with genes for which the expression was eightfold higher orfourfold lower than that in CD44loCD69lo CD4+ T cells.
For the visualization of the co-regulation network, the 500 genes in the CD44hi
CD4+ T cell groups that showed the greatest variation compared with the naive(CD44loCD69lo) CD4+ T cell group were subjected to further analyses. The firstneighbor genes were determined using the following two criteria: (1) a correlationof >0.8; and (2) a ratio of norm of 0.8–1.25. The network graph of 483 genes was
visualized using Cytoscape (http://www.cytoscape.org/).
The genes specifically upregulated in CD44hiCD69lo, CD44hiCD69hiCD103lo or
CD44hiCD69hiCD103hi CD4+ T cells were determined as fibrosis-related genes when
they were included in the gene lists of the indicated Gene Ontology terms. The firstneighbor Gene Ontology terms were determined using the following criterion: a
Simpson coefficient of ≥0.2. Node connections were determined based on the overlapof related to elemental gene products among the Gene Ontology terms. The network
graph of 483 genes and Gene Ontology terms was visualized using Cytoscape.
ATAC-Seq. ATAC-Seq was carried out as previously described, with minor
modification58,59. Some 50,000 cells were pelleted and washed with 50 μl 1× PBS,
followed by treatment with 50 μl lysis buffer. The nuclei were re-suspended in a
40-μl transposition reaction with 2 μl Tn5 transposase (15027865 and 15027866;
Illumina) to tag and fragmentize the accessible chromatin. The reaction was
incubated at 37 °C with shaking at 300 r.p.m. for 30 min. The fragmentized DNA
was purified using a QIAGEN MinElute kit and amplified with 11 or 12 PCR
cycles, based on the amplification curve. The libraries were sequenced for 50 cycles(paired-end reads) on a HiSeq 1500.
Analysis of ATAC-Seq data. The ATAC-Seq data were mapped to the mouse
genome build NCBI37/mm9 using the Bowtie (version 2.4.2.6; http://bowtie-bio.
sourceforge.net/index.shtml) and HOMER tag directories, which were createdby the HOMER platform60 (makeTagDirectory). Reproducible ATAC peaks wereannotated to the closest gene or transcriptional start site with proximity-based
annotation using HOMER annotatePeaks.pl (mm9 genome build)60. BigWig files
were generated from the aligned SAM or BED file formats using SAMtools61,
BEDTools62 and the University of California, Santa Cruz genomeGoverageBed
and bedGraphToBigWig, and normalized to 1 million reads. Only regions called
in both replicates were used in the downstream analysis. Peak intensities (‘tags’
column) were normalized as tags per 10 million reads in the original library.
A downstream analysis and heat map generation were performed. The Integrative
Genomics Viewer software program (https://software.broadinstitute.org/software/igv/) was used for the visualization of BiGWig files. A motif enrichment analysis
was performed with the findMotifsGenome.pl command in the HOMER package
using a 200-base pair window.
Measurement of the collagen level in the lung tissue. Quantification of
the collagen level of the left lung was performed using a Sircol collagenassay (Biocolor).

Articles
Airway hyper-responsiveness. The airway function was assessed by measuring thechanges in lung resistance in response to increasing doses of inhaled methacholine,
as described previously53.
Parabiosis. Mice were anesthetized, and the corresponding lateral aspects wereshaved. These aspects were then incised from the hip to behind the ear, andsutured together with a surgical needle (91–2124; Sansyo) and suture thread(DEBB0404; Akiyama-Seisakusyo).
FTY720 treatment. From 10 d to 1 d before the assay, 25 µg FTY720 (10006292;
Cayman) dissolved in 200 µl saline was administered intraperitoneally to eachmouse once a day.
Anti-CD103 antibody treatment. At 8, 6, 4 and 2 d before the assay, 100 µg
InVivoMAb anti-mouse CD103 (M290; BE0026; Bio X Cell) was administeredintraperitoneally to each mouse once a day.
Statistical analysis. Data were analyzed with the GraphPad Prism software
program (version 6). Comparisons were performed using the Mann–Whitney
U-test (two tailed), an unpaired t-test, one-way analysis of variance (ANOVA) with
Tukey’s multiple comparisons test, or two-way ANOVA with Bonferroni’s multiple
comparisons test. P < 0.05 was considered to indicate statistical significance.
Reporting Summary. Further information on research design is available in the
Nature Research Reporting Summary linked to this article.

Data availability

The data that support the findings of this study are available from thecorresponding author upon reasonable request. The RNA-Seq and ATAC-Seq datareported 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 thecorresponding author upon reasonable request.

Nature Immunology
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