of April 13, 2018.This information is current as

T Cells+the Induction of Profibrotic CD8IL-21 Promotes Pulmonary Fibrosis through

Joseph Craft, Susan L. Swain and Ann Marshak-RothsteinTia Y. Brodeur, Tara E. Robidoux, Jason S. Weinstein,

ol.1500777http://www.jimmunol.org/content/early/2015/10/30/jimmun

published online 30 October 2015J Immunol 

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The Journal of Immunology

IL-21 Promotes Pulmonary Fibrosis through the Induction ofProfibrotic CD8+ T Cells

Tia Y. Brodeur,* Tara E. Robidoux,* Jason S. Weinstein,† Joseph Craft,†

Susan L. Swain,‡ and Ann Marshak-Rothstein*

Type 2 effector production of IL-13, a demonstrated requirement in models of fibrosis, is routinely ascribed to CD4+ Th2 cells. We

now demonstrate a major role for CD8+ T cells in a murine model of sterile lung injury. These pulmonary CD8+ T cells

differentiate into IL-13–producing Tc2 cells and play a major role in a bleomycin-induced model of fibrosis. Differentiation of

these Tc2 cells in the lung requires IL-21, and bleomycin treated IL-21– and IL-21R–deficient mice develop inflammation but not

fibrosis. Moreover, IL-21R–expressing CD8+ cells are sufficient to reconstitute the fibrotic response in IL-21R–deficient mice. We

further show that the combination of IL-4 and IL-21 skews naive CD8+ T cells to produce IL-21, which, in turn, acts in an

autocrine manner to support robust IL-13 production. Our data reveal a novel pathway involved in the onset and regulation of

pulmonary fibrosis and identify Tc2 cells as key mediators of fibrogenesis. The Journal of Immunology, 2015, 195: 000–000.

Fibrosis, defined as the excess production and accumulationof extracellular matrix components, is the final commonpathway of many chronic inflammatory conditions (1, 2).

Despite the prevalence of fibrosis as both a primary disease and aconsequence of common chronic diseases, such as asthma, sar-coidosis, and heart failure, the U.S. Food and Drug Administra-tion–approved treatments that specifically target fibrogenesis areextremely limited (3). Idiopathic pulmonary fibrosis (IPF) can be arapidly progressive disease with a poor prognosis and minimaltherapeutic options. Fibrotic interstitial lung disease also occurs inautoimmune diseases, such as systemic sclerosis (SSc) and rheu-matoid arthritis (1–5). In these diseases, cells that are beneficialduring normal tissue repair, including macrophages, T cells, andfibroblasts, drive an excessive tissue repair response, leading tofibrosis and eventual organ dysfunction (1, 2, 4).Activation of fibroblasts by TGF-b and consequent collagen

production are crucial for both wound healing and fibrosis. Ad-ditionally, Th2 cell–derived IL-4 and IL-13 are integral compo-nents of the wound-healing response because of the ability ofthese cytokines to activate fibroblasts (1, 4). IL-13 is also a potentstimulus for macrophage production of TGF-b and can drive lung

fibrosis in the absence of other lung injury (6). Th2-associatedcytokines become aberrantly upregulated in fibrosis, and CD4+ Tcells have been implicated in murine models of SSc (7, 8) andpulmonary fibrosis (9, 10).Development of murine pulmonary fibrosis following bleomycin

treatment, a sterile lung injury model, requires IL-13 (11–13),especially late in the response (10). Asthma, which is often ac-companied by subepithelial fibrosis, has largely been considered aTh2-driven IL-13–dependent disease (14, 15); however, a role forCD8+ T cells was described (14, 16, 17). Fibrosis associated withschistosomiasis also requires IL-13 production, and CD4+ T cellswere identified as the main producers of IL-13 in these systems(1). Therefore, it has been presumed that CD4+ T cells are themajor source of IL-13 in sterile fibrosis.Elevated numbers of IL-17–producing Th17 cells also were im-

plicated in models of fibrosis (10, 18). Th17 cells secrete IL-21, ahighly pleiotropic cytokine, which, in combination with other cy-tokines, amplifies the function of CD4+ and CD8+ T cells (19, 20).Interestingly, IL-21 was implicated in the induction of both Th17 (21)and Th2 cells (9, 22) and, as such, could provide a bridge for theprogression from a Th17 response to a Th2 response in the fibrosinglung. Furthermore, in human fibrotic lung samples, expression of IL-21R was detected in lymphocytic infiltrates, suggesting that IL-21–responsive lymphocytes may be involved in lung fibrosis (23, 24). Inmice, IL-21R deficiency attenuated collagen deposition in the livercaused by parasitic infection (9). Taken together, these data implicateIL-21 as potentially deleterious in pulmonary fibrosis. We now showthat IL-21 plays a critical role in the fibrotic response associated witha bleomycin model of lung injury. Unexpectedly, we also found thatIL-21 drives the activation and expansion of a novel population of IL-13–producing Tc2 cells, which serve as a critical link between in-flammation and fibrosis.

Materials and MethodsMouse strains

BALB/c and C57BL/6 (B6) mice were purchased from The Jackson Lab-oratory. IL-21R–knockout mice originally created by Warren Leonard (25)were provided by Oliver Dienz (University of Vermont), and IL-21–knockout mice were obtained from Lexicon-Mutant Mouse RegionalResource Centers. Both IL-21R–knockout and IL-21–knockout micewere backcrossed to C57BL/6J mice for .10 generations. The IL-21

*Division of Rheumatology, Department of Medicine, University of MassachusettsMedical School, Worcester, MA 01605; †Section of Rheumatology, Departmentof Internal Medicine, Yale University School of Medicine, New Haven, CT 06520;and ‡Department of Pathology, University of Massachusetts Medical School,Worcester, MA 01605

ORCID: 0000-0002-3643-8018 (S.L.S.).

Received for publication April 2, 2015. Accepted for publication October 2, 2015.

This work was supported by National Institutes of Health Grants AR055634 (to A.M.-R.),T32 AI095213 and T32 GM107000 (to T.Y.B.), P01AI46530 (to S.L.S.), T32AR07107(to J.S.W.), and R01AR040072, R21AR062842, and P30AR053495 (to J.C.).

Address correspondence and reprint requests to Dr. Ann Marshak-Rothstein, Divisionof Rheumatology, Department of Medicine, University of Massachusetts MedicalSchool, 364 Plantation Street, LRB 309, Worcester, MA 01605. E-mail address:ann.rothstein@umassmed.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: B6, C57BL/6; BALF, bronchoalveolar lavage fluid;ICS, intracellular staining; IPF, idiopathic pulmonary fibrosis; i.t., intratracheal(ly);LDLN, lung-draining lymph node; a-smAc, a-smooth muscle actin; SSc, systemicsclerosis; Treg, regulatory T cell.

Copyright� 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00

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reporter line IL-21–mKat also was described (26). All mouse experi-ments were approved by the Institutional Animal Care and Use Com-mittee at the University of Massachusetts Medical School.

Lung treatments

Twelve- to sixteen-week-old mice were anesthetized by isoflurane in-halation. A total of 50 ml sterile bleomycin sulfate (Sigma), 0.05 U/mouse(low dose) or 0.15 U/mouse (high dose), recombinant mouse IL-21(R&D Systems) 1 mg/mouse, or PBS was instilled into the lungs ofmice by oropharyngeal aspiration (27). For experiments in which miceof different genotypes were compared, the mice were matched by age(6 2 wk).

Lung and lymph node tissue harvesting

Mice were sacrificed by cervical dislocation and immediate removal of thediaphragm. Lungs were perfused with PBS through the right ventricle toremove blood. Bronchoalveolar lavage fluid (BALF) was collected withthree flushes of 0.5 ml PBS. Lung-draining lymph nodes (LDLNs) weredissected and dissociated in HBSS using frosted glass slides. The rightbronchus was sutured, and the right and postcaval lobes were removed andplaced in HBSS, minced, and forced through a 100-mm nylon mesh filter to

generate a single-cell suspension. The left lobe was inflated with 10%neutral buffered formalin, the left bronchus was sutured, and the left lobewas removed and submerged in 10% buffered formalin at room tempera-ture until processed for paraffin embedding (.24 h).

Collagen measurement

The entire left lobe was minced into fine pieces and placed in 2 ml of asolution of acetic acid (Sigma; 0.5 M) and pepsin. After 24–48 h, the solutionwas spun at 4˚C for 20 min, and the lung extracts were frozen until thecollagen-concentration assay was performed. Sircol (Biocolor, Belfast, U.K.)or a Sirius Red Total Collagen Detection kit (Chondrex) was used, accordingto the manufacturer’s instructions, with similar results.

Histopathology and immunohistochemistry

Formalin-fixed, paraffin-embedded lung tissue was sectioned into 5-mmsections and stained with H&E or Masson’s trichrome stain. Immuno-histochemistry for a-smooth muscle actin (a-smAc; Santa Cruz Bio-technology; clone 1A4, 1:500 dilution) was performed without Agretrieval; goat anti-mouse IgG2a-HRP (Santa Cruz Biotechnology;1:500 dilution) was used as a secondary Ab, with diaminobenzene(Vector Laboratories) as a substrate. Mayer’s hematoxylin was used as a

FIGURE 1. IL-21 promotes pulmonary fibrosis

following lung injury. B6 and Il21r2/2 mice were

treated i.t. with bleomycin or PBS and sacrificed

at day 14. (A) Formalin-fixed, paraffin-embedded

lung sections were stained with Masson’s tri-

chrome (original magnification320). (B) Collagen

concentrations in lung extracts were quantitated

by a colorimetric assay. (C) Lung sections were

stained by immunohistochemistry for a-smAc

to detect activated fibroblasts. Data in (A)–(C)

are representative of three independent experi-

ments (PBS→B6, n = 6; PBS→Il21r2/2, n = 4;

bleomycin→B6, n = 11; bleomycin→Il21r-/-,

n = 11; bleomycin→Il212/2, n = 5). Original

magnification 320. (D) BALB/c mice were

treated with a single instillation of recombinant

mouse IL-21 i.t. or PBS; at day 10, lungs were

harvested, fixed in formalin, embedded in par-

affin, and stained with H&E and Masson’s tri-

chrome (original magnification310). (E) Collagen

concentrations in lung extracts from PBS-, bleo-

mycin-, IL-17A–, and IL-21–treated mice were

measured by colorimetric assay. Results shown

are compiled from four independent experi-

ments with three to four mice per group. Data

are mean 6 SEM. **p # 0.01, ***p # 0.001,

Student t test.

2 IL-21 DRIVES PROFIBROTIC Tc2 CELLS IN PULMONARY FIBROSIS

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counterstain for immunohistochemistry. Before sectioning and stain-ing, tissue blocks were coded, and the pictures were taken blinded. ForH&E and trichrome staining, images were acquired randomly. Fora-smAc staining, areas of focal consolidation were chosen for imageacquisition (also blinded) to identify regions of inflammation and/orfibrosis.

Flow cytometry

Single-cell suspensions were surface stained or intracellularly stained withcombinations of the following Abs: IL-21R–biotin, CD3-PE-Cy7, -biotin(clone 145-2C11), CD4–eFluor 450, -allophycocyanin (clone RM4-5),CD8-FITC, -allophycocyanin, -PE-Cy7 (clone 53-6.7), CD25-PerCP-Cy5.5 (clone PC61.5), CD69–eFluor 450, -PerCP-Cy5.5, IL-21–allophy-cocyanin (clone FFA21), IL-13–PE (clone eBio13A), IL-17A–PerCP–Cy5.5 (clone eBio17B7), and Foxp3-PE (clone FJK-16s) (all from eBio-science). Streptavidin-PerCP was from BD Biosciences. For Foxp3staining, cells were fixed and permeabilized using the Foxp3/TranscriptionFactor Staining Buffer Set (eBioscience), according to the manufacturer’sinstructions. For intracellular cytokine staining (ICS), single-cell suspen-sions were stimulated ex vivo with plate-bound anti-CD3 (BioLegend; 5mg/ml) or PMA (100 ng/ml) plus ionomycin (1 mg/ml; both from Sigma)in complete RPMI 1640 for 2 h at 37˚C. GolgiStop (BD Biosciences;1:100) was added, and cultures were incubated for an additional 2–3 h at37˚C. Cells were stained with Fixable Viability Dye eFluor 780 (eBio-science), per the manufacturer’s instructions. Cells were surface stained,and ICS was performed using Cytofix/Cytoperm and Perm/Wash (BDBiosciences), per the manufacturer’s protocol.

Cytokine measurements

LDLN or BALF single-cell suspensions (200,000–400,000 cells/well) wereseeded onto anti-CD3 (BioLegend; 5 mg/ml)-coated plates in RPMI 1640.Supernatants were collected at 24 h and analyzed by ELISA for IL-13 (BDBiosciences), IL-21 (eBioscience), IFN-g (BD Biosciences), and IL-17A(eBioscience). ATGF-b1 Quantikine ELISA Kit (R&D Systems) was usedto perform ELISA on cell-free BALF supernatant, according to the man-ufacturer’s instructions, with the exception of the acid treatment step toactivate latent TGF-b.

In vitro T cell differentiation

CD8+ T cells were purified from spleen cell suspensions by positive se-lection (BD iMag CD8a beads). Cells were stimulated with plate-bound

anti-CD3 (2 mg/ml) and soluble anti-CD28 (BioLegend; 2 mg/ml), with or

without recombinant mouse IL-4 (BD Biosciences; 10 ng/ml), recombi-

nant human IL-2 (BD Biosciences; 50 ng/ml), recombinant mouse IL-21

(R&D Systems; 50 ng/ml), or recombinant human TGF-b (PeproTech;

5 ng/ml). Anti-CD25 Ab was purified from PC61 hybridoma super-natant (a gift from Dr. M. Shlomchik, University of Pittsburgh) andused at a concentration of 20 mg/ml. Cells were split on day 2 and thenas needed. On day 4 or 5, T cells were restimulated with anti-CD3 forICS or restimulated to harvest supernatants for ELISA after 24 h.

Statistical methods

Data analysis was performed using Prism 6 (GraphPad) software. Thep values were calculated using unpaired the two-tailed Student t test. Thep values , 0.05 were considered not significant.

ResultsIL-21R signaling drives fibrosis but is dispensable forinflammation after sterile lung injury

To determine whether IL-21 plays a role in a sterile lung injurymodel of fibrosis, we compared the effects of intratracheal (i.t.)instillation of bleomycin in wild-type B6 mice and IL-21R–deficient(Il21r2/2) B6 mice. We found a striking attenuation in peri-bronchiolar fibrosis in Il21r2/2 mice at day 14 after bleomycininjury, as detected by Masson’s trichrome staining (Fig. 1A).Quantification of collagen concentration in lung extracts at 14 dby a colorimetric collagen-binding assay further confirmed thatboth Il21r2/2 and IL-21-deficient (Il212/2) mice had signifi-cantly decreased collagen deposition in the lung compared withbleomycin-treated B6 mice (Fig. 1B). Moreover, a-smAc, aprotein expressed by collagen-producing myofibroblasts, wasreadily detected in the bleomycin-injected B6 mice but wasalmost completely absent from Il21r2/2 mice (Fig. 1C). Inaccordance with a previous report using IL-21 reporter mice(28), we found that a small population of CD4+ T cells harvestedfrom B6 mice constitutively expressed IL-21. IL-21–expressingCD4+ T cells had an ∼7-fold higher CD62L+CD44+ surface phenotyperelative to the total CD4+ population. To determine whether airwayT cells produced increased levels of IL-21 following bleomycintreatment, we cultured BALF cells with anti-CD3 and assayed su-pernatants for IL-21 by ELISA. We found that BALF from bleomycin-treated mice produced more IL-21 than PBS-treated controls(Supplemental Fig. 1). TGF-b was shown to be an integral part offibrogenesis (6, 11), although IL-13–dependent, TGF-b–independent

FIGURE 2. IL-21R deficiency

does not prevent lung inflamma-

tion after bleomycin injury. B6 and

Il21r2/2 mice were treated i.t. with

bleomycin or PBS and sacrificed at

day 14. (A) Formalin-fixed, paraffin-

embedded lung sections were stained

with H&E (original magnification

320). (B) BALF and LDLN cells

were collected at day 14, and cell

numbers per mouse were deter-

mined. Data are compiled from three

or four independent experiments. *p#

0.05, **p # 0.01, ***p # 0.001,

****p # 0.0001. ns, p . 0.05.

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fibrosis was described (29). We found that TGF-b increased in B6 andIl21r2/2 mice in response to bleomycin (Supplemental Fig. 2).Because IL-21R deficiency led to such dramatic effects on

bleomycin-induced fibrosis, we next asked whether IL-21 by itself

could elicit fibrosis. Strikingly, IL-21–treated mice showed evi-

dence of inflammation and fibrosis 10 d later, as detected histo-

logically by H&E and trichrome stains (Fig. 1D), as well as by the

quantitative collagen assay (Fig. 1E). Collagen deposition elicited

by rIL-21 instillation was comparable to bleomycin instillation

and rIL-17A, which was shown to drive fibrosis (10). Thus, IL-21

is indispensible for bleomycin-induced lung fibrosis, and transient

local increases in IL-21 can drive lung fibrosis.To determine whether IL-21 deficiency led to decreased inflam-

mation and, therefore, attenuated fibrosis, we assessed the extent of

inflammation in bleomycin-treated mice by histology and by enu-

merating lung-infiltrating cells. Unexpectedly, H&E staining indi-

cated a similar extent of mononuclear cell infiltration in B6 and

Il21r2/2 mice (Fig. 2A). In addition, comparably increased num-

bers of mononuclear cells were recovered from the BALF of

bleomycin-treated B6 and Il21r2/2 mice compared with PBS-

treated control mice, and the total number of LDLNs tended to

be slightly lower in bleomycin-treated Il21r2/2 mice compared

with bleomycin-treated B6 mice, but the difference was not

statistically significant (Fig. 2B). Together, these results identify

IL-21 signaling as a critical event in the transition between

inflammation and fibrosis.

CD8+ T cell recruitment is impaired in Il21r2/2 mice aftersterile lung injury

The pathogenicity of T cells in bleomycin-induced fibrosis has beendisputed (30); however, T cell depletion was shown to attenuatefibrosis (31), and T cells produce pathogenic cytokines in thissystem (10, 18). Thus, we wanted to determine whether decreasedT cell recruitment or activation was responsible for the attenuatedpulmonary fibrosis in Il21r2/2 mice. At day 14 postbleomycininjury, we stained single-cell suspensions of lymphocytes har-vested from LDLNs for T cell surface markers and markers ofactivation. Il21r2/2 mice had roughly half (an average of 48%less) the number of CD8+ T cells in the LDLNs compared with B6mice after bleomycin injury (Fig. 3A). The total average numberof CD4+ T cells was decreased by only 30% in Il21r2/2 micecompared with B6 controls after bleomycin treatment. Further-more, surface staining of the activation marker CD69 on CD4+

and CD8+ T cells was comparable between bleomycin-treated B6and Il21r2/2 mice (Fig. 3B). CD4+CD25+Foxp3+ regulatoryT cells (Tregs) can dampen inflammation, including T cell re-sponses (32, 33). However, the frequency of Tregs in LDLNs ofIl21r2/2 mice was not increased compared with B6 mice andactually tended to be lower than in B6 mice, suggesting that Tregsare not likely to be responsible for decreased CD8+ proliferationor recruitment (Fig. 3C). These data show that IL-21 is a keyfactor in maximizing the CD8+ T cell response during lungfibrogenesis, and it has a lesser effect on the CD4+ T cell response.

FIGURE 3. IL-21/IL-21R interactions are required for optimal CD8+ T cell recruitment to the lung following bleomycin injury. B6 and Il21r2/2 mice

were treated with bleomycin or PBS i.t. and sacrificed at day 14. (A) Total number of CD4+ and CD8+ T cells in LDLNs at day 14. (B) LDLN single-cell

suspensions were analyzed by flow cytometry for surface expression of CD69. (C) Surface and intranuclear staining of LDLN single-cell suspensions was

performed to determine Treg frequency by coexpression of CD25 and Foxp3, as determined by flow cytometry. Data are compiled from two independent

experiments with similar results (PBS→B6, n = 6; PBS→Il21r2/2, n = 4; bleomycin→B6, n = 8; bleomycin→Il21r2/2, n = 8). Data are mean with an error

bar representing SEM. *p , 0.05, **p # 0.01, Student t test.

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Protection from fibrosis in Il21r2/2 mice is associated with adecrease in IL-13, but not IL-17A, production by T cells

IL-21 is a reported amplifier of both Th17 and Th2 responses(21, 22). To investigate the role of IL-21 within a microenviron-ment that favors the differentiation of both in profibrotic Th2 andTh17 responses, we analyzed LDLN T cells for cytokine pro-duction. At day 14 following bleomycin injury, LDLN cells ob-tained from PBS- and bleomycin-treated mice were stimulatedin vitro with plate-bound anti-CD3 for 24–48 h. Supernatants wereanalyzed by ELISA for IL-17A, as a readout of Th17 cells, andIL-13, as a readout for Th2 cells. T cells from the bleomycin-treated B6 and Il21r2/2 mice produced comparable amounts ofIL-17A (Fig. 4A). However, T cells from bleomycin-treatedIl21r2/2 mice secreted 3-fold less IL-13 than did those frombleomycin-treated B6 mice (Fig. 4B). To determine whetherIL-13 was being produced by airway lymphocytes, we treatedB6 and Il212/2 (cytokine-deficient, receptor-sufficient) micewith PBS or bleomycin and harvested BALF cells at day 14.ELISA of supernatants from BALF cells cultured with plate-boundanti-CD3 showed that IL-13 production by T cells was increased inbleomycin-treated B6 mice compared with controls, albeit at lowerlevels than LDLN cultures (Fig. 4C). Cells that were culturedwithout anti-CD3 did not produce IL-13, indicating that T cellswere producing the IL-13 in these cultures (data not shown). Sim-ilar to IL-21R–deficient LDLNs, IL-21–deficient BALF cells pro-duced less IL-13 in response to bleomycin (Fig. 4C).In other models of fibrosis, CD4+ Th2 cells were found to be the

major source of IL-13 (1, 4, 9), but because we found that CD8+

T cell numbers were most affected in IL-21R–deficient mice, wedecided to assess the subset distribution of the IL-13–producingcells in the bleomycin-treated mice. Surprisingly, as enumerated by

ICS of CD4+ and CD8+ T cells from LDLNs, many of the IL-13–producing cells were CD8+ cells. In addition, Il21r2/2 mice had asignificantly lower frequency of these cells (Fig. 4D, 4E). The de-creased frequency of IL-13–producing CD8+ T cells in Il212/2

mice, taken together with the decreased total number of CD8+

T cells (Fig. 3A), resulted in strikingly fewer IL-13–producingCD8+ T cells overall. These data indicate that bleomycin-inducedCD8+ T cell differentiation into profibrotic, type 2 effectors ishighly dependent on IL-21.

The combination of IL-4 and IL-21 efficiently promotes thein vitro differentiation of CD8+ T cells into Tc2 cells

To better understand how IL-21 promotes IL-13 production byCD8+ T cells in the injured lung, we decided to evaluate the im-portance of IL-21 and other cytokines in the in vitro generation ofTc2 cells. It was reported that Tc2 cells require IL-2 and IL-4 fordifferentiation (34, 35). However, IL-2 is not strongly upregulatedin the lungs in response to bleomycin injury (36). Therefore, wesought to determine whether IL-21 was sufficient for Tc2 differ-entiation in vitro, as determined by IL-13 production. We activatedpurified CD8+ T cells and assessed the ability of IL-21 to supporttheir capacity to produce IL-13 in conjunction with IL-4, with orwithout IL-2. We found that IL-21 could supplant IL-2 in Tc2differentiation (Fig. 5A, 5B). Remarkably, blockade of the high-affinity IL-2R, CD25, enhanced IL-13 production almost 2-fold atthe expense of IFN-g production (Fig. 5B, 5C), suggesting that,upon activation, high-affinity IL-2 signaling does not drive CD8+

T cells into Tc1- and Tc2-differentiation pathways equally, butrather inhibits Tc2 differentiation.TGF-b has both potent profibrotic and anti-inflammatory func-

tions. To understand how these roles affected Tc2 differentiation,

FIGURE 4. Lack of IL-21R significantly impairs IL-13 production in response to sterile lung injury. (A and B) B6 and Il21r2/2 mice were treated i.t.

with PBS or bleomycin. At day 14, the mice were sacrificed, and LDLNs were harvested. LDLN cell suspensions collected from PBS- and bleomycin-

treated mice were stimulated with anti-CD3; culture supernatants were harvested 24 h later and assayed for IL-17A and IL-13 by ELISA. Data are compiled

from two independent experiments with similar results. (PBS→B6, n = 6; bleomycin→B6, n = 9; bleomycin→Il21r2/2, n = 7). (C) B6 and Il212/2 mice

were treated i.t. with PBS or bleomycin. At day 14, the mice were sacrificed, and BALF cells were harvested. BALF cells were stimulated with anti-CD3,

and culture supernatants were harvested 24 h later and assayed for IL-13 by ELISA (PBS→B6, n = 3; PBS→Il212/2, n = 2; bleomycin→B6, n = 6;

bleomycin→Il212/2, n = 4). (D and E) At day 14, LDLN T cells from bleomycin-treated mice were restimulated, and cytoplasmic staining was performed

for IL-13. Data are compiled from three independent experiments (bleomycin→B6, n = 12; bleomycin→Il21r2/2, n = 10). Data in (E) are representative of

three independent experiments that yielded similar results with more than three mice per group. Data are mean with an error bar representing SEM.

***p # 0.001, Student t test. ns, p . 0.05.

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we also skewed Tc2 cells in the presence of TGF-b. As expected,TGF-b limited the proliferation of CD8+ T cell cultures (data notshown). However, the TGF-b–treated CD8+ T cells produced IFN-gbut not IL-13 (Fig. 5B, 5C). Additionally, we found that TGF-b, inconjunction with IL-4, decreased the level of IL-21R expression(Fig. 5D), thereby downregulating CD8+ T cell responsiveness toIL-21. This effect was not induced by TGF-b or IL-4 alone. Thisfinding suggests that, although TGF-b has indispensable profibroticeffects on stromal cells, such as fibroblasts, it negatively regulatesprofibrotic CD8+ T cells. Taken together, these data indicate that IL-21 can supplant IL-2 to promote Tc2 differentiation during fibro-genesis and that high-affinity IL-2R signaling and TGF-b coun-terregulate the effects of IL-21 signaling.

Tc2 cells self-prime with IL-21

Because Th17 cells can be amplified by autocrine and/or paracrineIL-21 during differentiation (37), we hypothesized that autocrineIL-21 would amplify Tc2 differentiation. Using an in vitro acti-vation system, we skewed wild-type and IL-21–deficient purifiedCD8+ T cells using IL-2 or IL-21, with or without IL-4. After thecells had reverted to a resting phenotype and been washed free ofany residual cytokine, they were restimulated with plate-boundanti-CD3. Cytokine production was initially quantified by ELISA.Intriguingly, only IL-21/IL-4, and not IL-2/IL-4 or IL-21 alone,generated CD8+ effectors capable of copious IL-21 secretion uponrestimulation (without the addition of exogenous cytokines), asmeasured by ELISA. The IL-21 detected was highly unlikely to havebeen “carried over” from the primary stimulation, because IL-21 was

detected only in IL-21+IL-14 and not in cultures that had been sup-plemented with IL-21 alone (Fig. 6A). Activation of CD8+ T cellsobtained from an IL-21 reporter mouse line confirmed the results ofthe ELISA (Fig. 6B). Moreover, in accordance with our hypothesis,there was a striking difference in IL-13 production by IL-21–deficientand IL-21–sufficient Tc2 cultures. IL-21–deficient CD8+ T cells didnot secrete IL-13 when treated with IL-21 and IL-4, but wild-type B6CD8+ T cells secreted high levels of IL-13 (Fig. 6C). Therefore,paracrine IL-21 drives CD8+ T cell secretion of IL-21, which, in turn,promotes IL-13 production through an autocrine pathway.

IL-21R–sufficient CD8+ T cells restore fibrotic phenotype ofIL-21R–deficient mice

The impact of IL-21 deficiency on CD8+ T cell number andfunction pointed to the possibility that IL-21–responsive CD8+

T cells are required for bleomycin-induced lung fibrosis. To testthis hypothesis, purified splenic B6 CD4+ and CD8+ T cells fromuntreated mice were adoptively transferred into B6 and Il21r2/2

mice, and the recipients were treated with bleomycin. Remark-ably, injection of B6 CD8+ T cells, but not CD4+ T cells, “res-cued” IL-13 production by LDLN T cells from bleomycin-treatedIl21r2/2 recipients but had little effect on the IL-13 response ofB6 recipients (Fig. 7A). Moreover, adoptive transfer of IL-21R–sufficient CD8+ T cells into Il21r2/2 mice, but not B6 mice, led to asignificant increase in collagen concentration in lung extracts afterbleomycin treatment, whereas adoptive transfer of IL-21R–sufficientCD4+ T cells had a negligible effect (Fig. 7B). Restoration of fibro-sis and fibroblast activation by IL-21R–sufficient CD8+ T cells was

FIGURE 5. IL-21 drives Tc2 differentiation in vitro. Purified splenic CD8+ T cells from naive B6 mice were cultured in the presence of the indicated

cytokines and Abs and activated with plate-bound anti-CD3 and soluble anti-CD28 for 4–5 d. (A–C) CD8+ T cell cultures were restimulated using anti-CD3

in the presence of GolgiStop, and cytoplasmic staining for IL-13 and IFN-g was performed. A representative flow cytometry plot is shown in (A), and a

summary is shown in (B) and (C). Data are mean6 SEM and are representative of five independent experiments. (D) Surface IL-21R expression on cultured

CD8+ T cells was analyzed by flow cytometry at day 5 postprimary stimulation. Shaded line graphs show Il21r2/2 control subjected to the same stain. Data

are representative of three independent experiments. *p , 0.05, **p # 0.01, unpaired Student t test.

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confirmed histologically by immunohistochemical staining for col-lagen and a-smAc (Fig. 7C). Thus, IL-21–responsive CD8+ T cellsare necessary and sufficient for reconstituting the fibrotic phenotypefollowing lung injury in Il21r2/2 mice.

DiscussionFibrosis is a frequent outcome of chronic inflammation, butdespite extensive investigation, the pathways responsible for thetransition from inflammation to fibrosis remain incompletelyunderstood. Overexuberant type 2 responses characterized byhigh levels of IL-4 and IL-13 are often linked to fibrogenesis;however, the initiating signals for such responses are not clear.Through the analysis of IL-21R–deficient mice, we showed thatlung fibrosis and optimal IL-13 production in response to bleo-mycin lung injury are dependent on IL-21R signaling, thusidentifying IL-21 as a potential therapeutic target for fibroticdiseases. Previous work showed that bleomycin-induced lungfibrosis is ameliorated by neutralization of IL-13 or depletion ofIL-13–responsive cells (12, 13, 38). In accordance with thesestudies, we found that IL-21R deficiency and protection fromfibrosis were associated with a striking reduction in IL-13 pro-duction by lung T cells ex vivo. Intriguingly, protection fromfibrosis in Il21r2/2 mice was not associated with a significantreduction in inflammation or ex vivo IL-17 production. Il21r2/2

mice also were shown to develop less severe fibrosis in aschistosomiasis-associated liver fibrosis model; however, thiseffect was attributed to IL-21 induction of Th2 cells and thepromotion of increased macrophage sensitivity to Th2 cytokines,such as IL-4 and IL-13 (9).We further showed that, in the bleomycin model, IL-13–se-

creting CD8+ T cells, or Tc2 cells, and not Th2 cells, weremost profoundly impacted by IL-21R deficiency. This findingwas unexpected because CD4+ T cells have been charged with

driving fibrosis through the production of IL-17A (10, 18) andIL-13 (11, 12).The physiological significance of Tc2 cells in vivo had been

controversial because most functional studies of Tc2 cells used invitro–skewed cells for adoptive transfer. In vitro–differentiated Tc2cells are less potent effectors in pulmonary viral clearance and tumorrejection compared with Tc1 cells (39, 40). However, in murineasthma, Tc2 cells develop and increase airway inflammation andhyperresponsiveness (14). We now show that IL-21–dependentTc2 cells are required for lung fibrosis after sterile lung injury,because IL-13 production and fibrosis were restored by trans-ferring IL-21R+/+ CD8+ T cells (not activated in vitro) intoIl21r2/2 mice treated with bleomycin. Adoptive transfer of IL-21R+/+ CD4+ T cells did not “rescue” fibrosis in Il21r2/2 mice,suggesting that CD8+ T cell cytotoxicity, in addition to IL-13production, could be crucial for fibrosis. These data are consis-tent with previous reports in which depletion of CD8+ T cells orperforin deficiency almost completely prevented bleomycin-induced pulmonary fibrosis (31, 41).In addition to identifying a novel role for Tc2 cells during fibrosis,

our results points to a unique role for IL-21 in Tc2 differentiation.Studies by other investigators showed that Tc2 differentiation can beachieved in vitro using a combination of IL-2 and IL-4 (34, 35). Inthis study, we showed that IL-21, in conjunction with IL-4, canreplace the need for exogenous IL-2 in Tc2 differentiation. Coun-terintuitively, CD25 blockade enhanced Tc2 differentiation in re-sponse to IL-21/IL-4, which again pointed to a specific role for IL-21 during fibrogenesis that cannot be replaced by IL-2. Most im-portantly, we also found that only IL-4 + IL-21–differentiated cellsacquired the capacity to produce high levels of their own self-sustaining IL-21.We further found that CD8+ T cells produced IL-21 in vivo

in bleomycin-treated mice. To the best of our knowledge, this

FIGURE 6. Autocrine IL-21 promotes Tc2 phenotype. (A) Purified splenic CD8+ T cells from naive B6 mice were cultured in the presence of the

indicated cytokines and Abs for 4 d, washed, and restimulated using anti-CD3. Cell supernatants were harvested at 24 h and assayed for IL-21. Data shown

are compiled three independent experiments. (B) Purified CD8+ T cells from wild-type and IL-21–mKat reporter mice were stimulated as indicated, and

mKate expression was analyzed by flow cytometry at day 4 without restimulation or addition of exogenous cytokines. Data are representative of two

independent experiments. (C) Purified CD8+ T cells from wild-type and Il212/2 mice were stimulated as indicated, and the concentration of IL-13 in the

culture supernatants was determined. Data in (C) are mean 6 SEM and are representative of three independent experiments.

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is the first report of IL-21 production by CD8+ T cells with relevanceto a disease model. IL-21 message was detected in T-bet andeomesodermin double-knockout Tc17 cells skewed in vitro bya combination of IL-6 and TGF-b; however, IL-21 was not de-tected in Tc17 cultures by ELISA (42). The finding that CD8+

T cells produce IL-21 in vivo could reflect a phenotype that isspecific to a sterile type II environment and not mounted duringviral or parasitic infections.Interestingly, fibroblasts in the gut express IL-21R and upreg-

ulate matrix metalloproteinases in response to IL-21 (43), raisingthe possibility that CD8+ T cell–derived IL-21 could act directlyon fibroblasts. Moreover, IL-21 production by Tc2 cells couldfurther reinforce a type II immune response due to the robusteffects of IL-21 on CD4+ T cells and innate immune effectors(44, 45). Strikingly, IL-21 instillation into the lungs of mice in theabsence of other injurious stimuli led to fibrosis in addition to thedevelopment of both IL-13– and IL-21–producing CD8+ T cells.Thus, the foregoing data show that IL-21 is both necessary andsufficient for pulmonary fibrosis.In studying the differentiation of Tc2 cells, we found that TGF-

b, a cytokine abundantly expressed in fibrotic tissues, specificallyinhibited Tc2 differentiation. The finding that TGF-b decreasedIL-21R expression on CD8+ T cells when combined with IL-4 wassurprising because of the profibrotic roles of both cytokines.However, this could reflect a “brake” on fibrogenesis by de-creasing positive feedback through IL-21R. It should be noted thatbleomycin-induced lung fibrosis is a self-limiting disease aftersingle i.t. instillations, and a TGF-b–dependent feedback mecha-nism could contribute to the spontaneous resolution of fibrosis.TGF-b is also produced by macrophages (46), including alterna-

tively activated macrophages, which are known to accumulate inthe fibrotic lung (47, 48). The capacity of macrophage-derivedTGF-b to suppress Tc2 differentiation might explain why mac-rophage depletion after fibrosis is established was found to exac-erbate, and not ameliorate, collagen deposition (49). Additionally,the decrease in the expression of IL-21R in response to TGF-bcould be a mechanism by which Treg suppression of Tc2 cells ispromoted. Because IL-21 impairs Treg function (50–52), Treg-derived TGF-b could limit an IL-21R/IL-21 positive-feedbackloop, thus ensuring the potency of nearby Tregs by dampening localIL-21 production.In addition to a role for IL-21 in Tc2 skewing, we found that IL-

21R signaling was required for optimal CD8+ T cell proliferation/recruitment. Both the frequency and total number of CD8+ T cellswere reduced in Il21r2/2 mice during fibrogenesis compared withwild-type controls. These data are consistent with a significant bodyof work characterizing the mitogenic effects of IL-21 on CD8+

T cells. In the NOD model of diabetes, lack of IL-21R signalingalso decreases CD8+ T cells and attenuates disease; however, in theNOD system, it was reported that IL-21R+ CD4+ T cells and den-dritic cells recruit cytotoxic CD8+ T cells into pancreatic islets (53).In contrast, we found that direct engagement of IL-21R on CD8+

T cells was required for fibrosis and optimal IL-13 productionduring lung fibrogenesis. Because fibrotic disease is a Th2/Tc2-driven response, as opposed to Th1/Tc1-driven NOD disease, ourresults point to a requirement for IL-21R for a robust CD8+ T celleffector activity that is specific to the type 2 environment caused bysterile fibrotic lung injury. Whether our findings can be extended toother sterile injury models of fibrosis (e.g., silica) or an infectionmodel (e.g., hypersensitivity pneumonitis) remains to be determined.

FIGURE 7. IL-21R on CD8+ T cells is required for optimal IL-13 production and collagen deposition. (A) A total of 106 purified splenic CD4+ or CD8+

T cells from naive B6 mice was injected i.v. into B6 and Il21r2/2 mice at day 21. At day 0, mice were treated i.t. with PBS or bleomycin. LDLN cell

suspensions from PBS- and bleomycin-treated mice were collected at day 14 and stimulated with anti-CD3. Cell supernatants were harvested at 24 h and

assayed for IL-13. (B) Lungs were harvested at day 14, and lung extracts were quantitated for collagen concentration by Sircol assay. Data in (A) and (B) are

compiled from two independent experiments (PBS→B6, n = 5; PBS→Il21r2/2, n = 4; bleomycin→B6, n = 9; bleomycin→Il21r2/2, n = 8; bleomycin→B6 +

wild-type CD4, n = 5; bleomycin→B6 + wild-type CD8, n = 8; bleomycin→Il21r2/2 + wild-type CD4, n = 5; bleomycin→Il21r2/2 + wild-type

CD8, n = 9). Data are mean6 SEM. (C) Lung sections obtained at day 14 were stained for a-smAc to detect fibroblast activation (original magnification320).

***p # 0.001, unpaired Student t test. ns, p $ 0.05.

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CD8+ T cells were positively correlated with disease severity inpatients with lung fibrosis (54) and CD8+ T cell activation is in-creased in early, diffuse SSc (55). We now establish a role forCD8+ T cells among the repair/fibrosis class of effectors duringsterile inflammation. Importantly, a number of studies linked Tc2cells to fibrosis in human patients. CD8+ T cells from SSc patientssecrete copious IL-13 and can activate fibroblasts in an IL-13– andSTAT-6–dependent manner (56). Additionally, in the peripheralblood of SSc patients, a higher frequency of CD8+ T cells pro-duces IL-13 than CD4+ T cells (56, 57). It also was reported thatthe frequency of Tc2 cells in the lungs of IPF patients is cor-related with disease severity and shortness of breath (54), andCD8+ T cells are as, if not more, abundant than CD4+ T cells inthe lungs of IPF patients (58, 59). These studies, taken togetherwith the current report, challenge the predominant CD4+ T cell–centric view of fibrosis. Our data clearly point to an IL-21/IL-13axis during fibrogenesis, requiring CD8+ T cells as effectors onboth sides of this axis. Overall, these results have importantimplications for the rational design of IL-21–targeted therapiesthat could be used to treat fibrosis resulting from conditions suchas SSc, IPF, or chronic inflammation.

DisclosuresThe authors have no financial conflicts of interest.

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10 IL-21 DRIVES PROFIBROTIC Tc2 CELLS IN PULMONARY FIBROSIS

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