FIBROSIS Copyright © 2018 T follicular helper–like cells ...Changshou Gao,4 Li Yu,5 Madhu Ramaswamy,1 Marie-Claude Gaudreau,1¶ Rob Woods,4 Ronald Herbst,1 Gianluca Carlesso1* Systemic

Taylor et al., Sci. Transl. Med. 10, eaaf5307 (2018) 7 March 2018

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F I B R O S I S

T follicular helper–like cells contribute to skin fibrosis Devon K. Taylor,1* Nanette Mittereder,1 Ellen Kuta,1† Tracy Delaney,1 Timothy Burwell,1 Karma Dacosta,2 Weiguang Zhao,2 Lily I. Cheng,2 Charles Brown,2 Anmarie Boutrin,2 Xiang Guo,3 Wendy I. White,3 Jie Zhu,4 Huifang Dong,4‡ Michael A. Bowen,4§ Jia Lin,4 Changshou Gao,4 Li Yu,5 Madhu Ramaswamy,1 Marie-Claude Gaudreau,1¶ Rob Woods,4 Ronald Herbst,1 Gianluca Carlesso1*

Systemic sclerosis (SSc) is a debilitating inflammatory and fibrotic disease that affects the skin and internal organs. Although the pathophysiology of SSc remains poorly characterized, mononuclear cells, mainly macrophages and T cells, have been implicated in inflammation and fibrosis. Inducible costimulator (ICOS), which is expressed on a subset of memory T helper (TH) and T follicular helper (TFH) cells, has been shown to be increased in SSc and associated with disease pathology. However, the identity of the relevant ICOS+ T cells and their contribution to inflammation and fibrosis in SSc are still unknown. We show that CD4+ ICOS-expressing T cells with a TFH-like phenotype infiltrate the skin of patients with SSc and are correlated with dermal fibrosis and clinical disease status. ICOS+ TFH-like cells were found to be increased in the skin of graft-versus-host disease (GVHD)–SSc mice and contributed to dermal fibrosis via an interleukin-21– and matrix metalloproteinase 12–dependent mechanism. Administration of an anti- ICOS antibody to GVHD-SSc mice prevented the expansion of ICOS+ TFH-like cells and inhibited inflammation and dermal fibrosis. Interleukin-21 neutralization in GVHD-SSc mice blocked disease pathogenesis by reducing skin fibrosis. These results identify ICOS+ TFH-like profibrotic cells as key drivers of fibrosis in a GVHD-SSc model and suggest that inhibition of these cells could offer therapeutic benefit for SSc.

INTRODUCTIONSystemic sclerosis (SSc) is an inflammatory fibroproliferative disease whose pathogenesis remains largely elusive. SSc is characterized by the excessive deposition of extracellular matrix (ECM) proteins, particularly collagen, in different organs as a result of dysregulated tissue repair (1). The deposition of ECM proteins and fibroblast replacement of paren-chymal cells occur mainly in the skin and internal organs. This condi-tion exacerbates tissue remodeling and fibrosis, which represent the main pathological manifestations associated with SSc (1). The clinical manifestations of SSc are heterogeneous and complex, and they have distinctive inflammatory and fibrotic phases, making the disease diffi-cult to treat. Although fibrosis is caused by a series of synergistic events, the dynamic interactions between myofibroblasts, macrophages, and CD4 T cells are thought to be a major component that regulates disease progression (2). Understanding the cellular and molecular mechanisms that are involved in the interactions between the innate and adaptive immune systems and stromal cells may allow the development of tai-lored medicines to replace standard of care treatments including anti- inflammatory (for example, corticosteroids) and immunosuppressant therapies (for example, methotrexate, mycophenolate mofetil, and cy-clophosphamide) that are aimed at minimizing symptoms of SSc.

Studies conducted in animal models and advances in the under-standing of the progression of SSc in patients have led to the recogni-tion that T helper (TH) cells are involved in both the early inflammatory and late fibrotic phases through interaction with differentiated fibro-blasts (3, 4). Mononuclear cells, mainly macrophages and activated effector TH cells, that produce proinflammatory cytokines [for ex-ample, interferon- (IFN-) and interleukin-6 (IL-6)] are detectable in the skin during the inflammatory phase of SSc (5–7).

Furthermore, TH cells have been implicated in the fibrotic phase of SSc (8). The fibroblasts in SSc are involved in both the synthesis and degradation of the ECM, in which matrix metalloproteinase (MMP) molecules (for example, MMP-9 and MMP-12) control the degrada-tive process and facilitate the recruitment of inflammatory cells to the site of injury (9–11).

Collagen I is one of the most abundantly expressed proteins in the collagen family in the skin, and its breakdown product, serum C-terminal telopeptide of type I collagen (s-CTX-I), has been found elevated in the serum of patients with SSc compared with healthy con-trols and correlates with modified Rodnan skin score (mRSS) (12–14). Both MMP-9 and MMP-12 are also increased in the serum and tissue of patients with SSc and correlate with the severity of skin fibrosis and vascular damage (10, 11).

The interaction between dermal fibroblasts and TH cells promotes the up-regulation of the profibrotic cytokines IL-4, IL-13, and trans-forming growth factor– (TGF-) (6, 15, 16). The TFH-associated cytokine IL-21 and its cognate, the IL-21 receptor (IL-21R), were also shown to be up-regulated in patients with SSc (17, 18). These data further support an involvement of TH effector cells and their associated cytokines in the inflammatory and fibrotic phases of SSc. However, the role of TFH-associated IL-21 during the inflammatory and fibrotic phases of the disease is still unclear.

The inducible costimulator (ICOS) protein, one of several co-stimulatory receptors of the B7/CD28 superfamily, plays an important and nonredundant role in the regulation of adaptive immune responses.

1Department of Respiratory, Inflammation and Autoimmunity, MedImmune LLC, Gaithersburg, MD 20878, USA. 2Department of Pathology, MedImmune LLC, Gaithersburg, MD 20878, USA. 3Department of Clinical Biomarkers and Computa-tional Biology, MedImmune LLC, Gaithersburg, MD 20878, USA. 4Department of Anti-body Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, MD 20878, USA. 5Clinical Pharmacology, Pharmacometrics, and DMPK (CPD), MedImmune LLC, Gaithersburg, MD 20878, USA.*Corresponding author. Email: [email protected] (D.K.T.); [email protected] (G.C.)†Present address: Preclinical Evidence Generation, GlaxoSmithKline, Rockville, MD 20850, USA.‡Present address: Cell Line Development, WuXi Biologics, Shanghai 200131, China.§Present address: Analytical Development, Juno Therapeutics Inc., Seattle, WA 98109, USA.¶Present address: Immuno-Oncology Discovery, Bristol-Myers Squibb, Princeton, NJ 08543, USA.

Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

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Unlike CD28, which is constitutively expressed on T cells, ICOS is rapidly up-regulated on activated CD4+ effector T cells, and its ex-pression is highest on T follicular helper (TFH) cells, thus positioning it as a pivotal regulator of humoral responses (19). ICOS through the binding of its cognate ICOS ligand (ICOSL; B7RP-1) elicits T cell activation, differentiation, and effector responses and controls the expansion of the effector pool size (20–22). It plays a critical role in the ontogeny and function of TFH cytokines IL-6 and IL-21 (22–26). ICOS+ TFH cells have been elevated in patients affected by systemic lupus erythematosus or rheumatoid arthritis, and these cells correlate with disease activity (27–29).

Recently, ICOS+ T cells were shown to be elevated in the skin of patients with SSc (30, 31). In addition, in a bleomycin-induced SSc mouse model, ICOS-deficient mice were protected from lung and skin fibrosis relative to wild-type animals (32). Together, these data suggest that ICOS+ T cells may play a role in skin fibrosis in SSc. However, the underlying mechanisms remain poorly defined.

We investigated the involvement of ICOS+ TH cells in patients with SSc and their relevance to inflammation and skin fibrosis. We observed that human SSc skin and lesional skin from a graft-versus-host disease (GVHD)–SSc mouse model contained a new population of TH cells demarcated as ICOS-bearing TFH-like cells that correlated with skin disease activity. Treatment with an anti-ICOS–depleting monoclonal antibody (MAb) significantly ameliorated disease by block-ing the expansion of pathogenic TFH-like cells, thereby providing support for the interplay between ICOS-expressing TH cell subsets and dysregulated fibroblasts.

RESULTSPatients with SSc have increased frequency of TFH-like cellsOne of the main characteristic histopathological findings in SSc skin is dermal fibrosis, which is driven in part by the infiltration of macrophages and activated T lymphocytes (6). Although it is known that T cells play a key role in SSc, the characterization of these spe-cialized skin T cell subsets remains undefined. To decipher the dermal T cell profiling, we enrolled 28 patients with SSc, who underwent full clinical examination. Lesional skin biopsies were collected for im-munoprofiling. Nine of 28 skin biopsies did not pass the quality con-trol check because of poor tissue quality. The baseline demographics and clinical characteristics of patients with SSc are summarized in Tables 1 and 2. Most of these patients had diffuse SSc with multiple organ system involvement.

A representative photomicrograph of the histological hematoxylin and eosin (H&E) analysis of this patient cohort demonstrated a pro-found increase in fibrosis and lymphocyte infiltration compared with healthy controls (Fig. 1A). The expression of transcripts for cd3, cd4, and icos was confirmed to be significantly increased (P < 0.005, P < 0.05, and P < 0.05, respectively) in biopsied lesional skin from pa-tients with SSc (Fig. 1, B and C) (30, 31).

Because the expression of ICOS is associated with the activation and differentiation of TH cells into different lineages, we determined the phenotype and expression pattern of ICOS-associated infiltrating TH cells in the skin of patients with SSc. Gene expression analysis of Bcl6, Tbet, gata3, and rorc demonstrated that skin T cells displayed atypical transcriptional factors profiling (fig. S1A). Gene profiling of membrane-expressing TH lineage–specific transcripts was compared between patients with SSc and healthy controls. The expression of chemokine (c-x-c motif), receptor 5 (cxcr5), and programmed cell death 1

Table 1. Baseline demographics and clinical characteristics of all analyzed patients. Demographics and clinical characteristics are defined according to American College of Rheumatology criteria for systemic sclerosis. mRSS, modified Rodnan skin score; HAQ, health assessment questionnaire; EDAI, European disease activity index; CRP, complement reactive protein; ESR, erythrocyte sedimentation rate; FVC, forced vital capacity; anti-Scl 70, anti-topoisomerase 1 antibody.

Baseline characteristics (n = 28)

Mean age (years) ± SEM

47.3 ± 9.74

n %

Sex (n = 28)

Female 19 68

Ethnicity (n = 28)

Asian 1 3.5

Black 3 10.7

White 24 85.7

Scleroderma subtype (n = 28)

Diffuse 24 85.7

Limited 4 14.3

Median disease duration in years (range)

4.7 (0.3–16)

Sclerosis activity score (n = 28)

mRSS 23.0 ± 8.45

HAQ 1.09 ± 0.83

EDAI 4.59 ± 2.07

Disease manifestation

Raynaud’s phenomenon (n = 28)

28 100

Vascular digital ulcers (n = 28)

15 53.6

Esophageal (dysphagia and reflux) (n = 28)

24 85.7

Elevated acute-phase reactants (ESR and CRP) (n = 24)

14 58.3

Cardiopulmonary (fibrosis) plain x-ray (n = 28)

8 28.6

Restrictive defect (FVC <60% predicted) (n = 28)

4 14.3

Positive autoantibodies

Anti–Scl-70 (n = 25) 12 48.0

Anti-nuclear (n = 24) 21 87.5

Anti-cardiolipin (n = 11) 1 9.09

Medication (n = 15)

Immunosuppressant (prednisone, azathioprine, mycophenolate mofetil, or methotrexate)

15 100


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(pdcd1, also known as PD-1) was significantly elevated (P < 0.001 and P < 0.005) in SSc skin (Fig. 1C).

The association of icos with cd3, cd4, cxcr5, and pdcd1 provides a valuable understanding of the putative immune phenotype properties of TH cells in fibrotic SSc skin that are normally associated with TFH cells or their circulating TFH-like relatives. Patients with SSc also ex-pressed significantly elevated il21r mRNA expression (P < 0.0005) in the skin (Fig. 1C). Correlational analysis of the mRNA expression of pdcd1 and icos, pdcd1 and cxcr5, and icos and cxcr5 showed sig-nificant coexpression (P < 0.008, P < 0.05, and P < 0.5 × 10−5) of these genes in lesional skin (Table 3).

The phenotype of these newly identified TFH-like cells was further investigated by immunohistochemistry (IHC) and multicolor immu-nofluorescence (IF) staining, which demonstrated the presence of CD4, ICOS, and PD-1 single-positive cells localized throughout the dermis of SSc skin (Fig. 1, E and F). SSc skin sections demonstrated perivascular staining for CD4, ICOS, and PD-1 by IHC (Fig. 1E). IF analysis of CD4, ICOS, and PD-1 single-positive cells in SSc skin revealed statistically significant increases in expression (P < 0.005, P < 0.5 × 10−4, and P < 0.0005, respectively) compared with healthy control skin (Fig. 1G). Activated CD4+ICOS+ cells were also elevated in SSc skin (Fig. 1G).

Analysis of the number of PD-1+ cells versus ICOS+ cells, PD-1+ cells versus CD4+ cells, and ICOS+ cells versus CD4+ cells also demon-strated significant correlations (P < 0.5 × 10−6, P < 0.5 × 10−5, and P < 0.5 × 10−5, respectively) (Table 4). The coexpression with CD4, ICOS, and PD-1 identified the presence of infiltrated cells in SSc skin that had a cell surface phenotype reminiscent of TFH-like cells (Fig. 1H). The frequency of TFH-like cells identified by the concomi-tant expression of ICOS, PD-1, and CD4 was also significantly higher in SSc skin samples (P < 0.001; Fig. 1H) and represented about 95% of the total activated CD4+ICOS+ T cell population (Fig. 1G).

The involvement of other TH cell subsets was also explored. Quan-titative reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated the presence of T cell–associated transcripts that are characteristic of both TH1 (Fig. 1D) and TH2 cell phenotypes (fig. S1B). The mRNA expression of the TH1 transcripts cxcr3 and ifng (Fig. 1D) and TH2 genes ptgdr2 (crth2) and cxcr4 (fig. S1B) was elevated in the

skin from patients with SSc. Notably, crth2 and cxcr4 significantly correlated with each other (P < 0.5 × 10−7), whereas both crth2 and cxcr4 negatively correlated with CD4 (Table 5). Although mRNA expression of ifng correlated with cd4, no significant correla tion was observed between cxcr3 and ifng (P > 0.2) (Table 5). Comparison of crth2, cxcr4, cxcr3, and ifng with TFH-like markers cd4, cxcr5, icos, and pdcd1 also did not reveal any statistical correlation, except for ifng, suggesting that these cells are of a different lineage (table S1).

ICOS+PD-1+CXCR5+ TFH-like cells are associated with fibrosis and elicit myofibroblast differentiationTranscript analysis of molecules that are known to be elevated during active disease in the skin of patients with SSc was assessed. Although mRNAs for inflammatory chemokine (C-C motif) ligand 2 (ccl2) and ligand 5 (ccl5) were modestly increased, vascular cell adhesion molecule 1 (vcam-1) and von Willebrand factor (vwf), which are as-sociated with fibrosis and vasculopathy, were significantly elevated in SSc skin (P < 0.5 × 10−7) (Fig. 2A).

The correlation of the TFH-like subset-defining molecules with clinical skin score and fibrosis biomarkers was analyzed to determine their association with disease. This analysis showed a statistically sig-nificant association between mean mRSS and CD4 (r = 0.61, P < 0.01), ICOS (r = 0.46, P < 0.05), and PD-1 (r = 0.5, P < 0.05; Fig. 2B). As-sessment of patient-reported skin worsening score, a component of the European disease activity index, also established a significant and positive correlation with CD4 infiltrating T cells (r = 0.47, P < 0.05) and a correlative trend with ICOS+ cells (r = 0.42, P = 0.06; fig. S1C). In addition, individually assessed infiltrating CD4, ICOS, and PD-1 TH cells demonstrated positive correlative trends with both s-CTX-I and VCAM-1 (fig. S1, D to I). Consistent with this finding was the obser-vation of a positive association between mRSS and ICOS/PD-1/CD4 triple-positive TFH-like cells in the skin of patients with SSc (r = 0.4, P = 0.06; Fig. 2B).

The presence of ECM remodeling fibrotic markers in the serum of patients with SSc was also evaluated to assess ICOS/PD-1/CD4 triple-positive cells with fibrosis biomarkers. A significant correla-tion was observed between infiltrating ICOS/PD-1/CD4 triple-positive TFH-like cells and s-CTX-I, which represents the degradative by- product of collagen I (r = 0.54, P < 0.05; Fig. 2B). Furthermore, VCAM-1 was also significantly up-regulated in patients with SSc and correlated with ICOS/PD-1/CD4 triple-positive TFH-like cells (r = 0.5, P < 0.05; Fig. 2B). Overall, these data support the implication of TFH-like cells in the fibrotic pathology of SSc.

Myofibroblasts are not commonly found in healthy skin and only arise in response to injury and repair. To evaluate the direct func-tional impact of the TFH-like cells described above in skin fibrosis, we cultured normal human dermal fibroblasts (NHDFs) with in vitro– differentiated CD4+ICOS+PD-1+CXCR5+ TFH-like cells using a mod-ified TFH polarization protocol (33). Under polarizing conditions, CD4+ T cells up-regulated CXCR5, ICOS, and PD-1, and a substantial subset coexpressed ICOS and PD-1 (Fig. 2C). These cells were co-cultured with NHDFs at a 1:4 ratio in the presence or absence of sub-optimal TGF- and anti-CD3/anti-CD28 beads. After 3 to 4 days in coculture, NHDFs up-regulated the expression of -smooth muscle actin (SMA) within the vimentin-positive cells (Fig. 2D). The re-sults indicate that ICOS+PD-1+CXCR5+ TFH-like cells could drive the differentiation of myofibroblasts (Vimhi SMAhi) compared with myofibroblast frequency observed in TFH-like null cultures (Fig. 2E). The CD4+ICOS+PD-1+CXCR5+ TFH-like cell–driven myofibroblast

Table 2. Baseline demographics for healthy control participants. N/A, not applicable.

Baseline characteristics (n = 20)

Mean age (years) ± SEM

41.0 ± 11.6

n %

Sex (n = 20)

Female 20 100

Ethnicity (n = 20)

Asian N/A N/A

Black N/A N/A

White N/A N/A

Disease manifestation

N/A (weight reduction surgery) 20 100


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differentiation of NHDFs was independent of TGF-, which suggests that TFH-like cells can promote SMA up-regulation and myofibro-blast differentiation in vitro.

ICOS-expressing TH cells correlate with the clinical manifestation of GVHD-SScBecause of the observed increase in TFH-like cells in human dermal SSc cells, we investigated the potential involvement of these ICOS+ TH cells in skin inflammation and fibrosis. We used the scleroderma-

tous GVHD (GVHD-SSc) mouse model developed by Ruzek et al. that is characterized by dermal infiltration of activated T cells and macrophages, resulting in skin remodeling and dermal fibrosis (fig. S2, A and B) (34). We confirmed the accumulation and expansion of CD3+ T cells and F4/80+ macrophages in the thickened dermis that coincided with the peak manifestation of skin pathology (fig. S2, C, D, and F). These data support previous findings regarding the correlation between skin T lymphocytes and the initiation and progression of GVHD- SSc cutaneous fibrosis.

Fig. 1. Phenotype of ICOS-expressing inflammatory and TH cells in the skin of patients with SSc. (A) Representative hematoxylin and eosin (H&E) staining of systemic sclerosis (SSc) and control (HC) skin samples (magnification, ×20). (B to D) SSc and normal control (HC) skin samples were assessed by TaqMan gene expression assays for the expression of T helper (TH) cell subset characteristic molecules (n = 20 healthy controls and n = 19 patients with SSc). (E) Representative immunohistochemistry (IHC) staining of SSc skin samples with CD4, in ducible costimulator (ICOS), and programmed cell death 1 (PD-1) (magnification, ×40). (F) Representative immunofluorescence staining of SSc skin with CD4 (turquoise), ICOS (green), and PD-1 (red; magnification, ×40). Yellow arrows indicate positive cells. (G) Quantitation of CD4, ICOS, PD-1, and CD4+ICOS+ infiltrating T cells in the skin of a patient with SSc by immunofluorescence. (H) Representative immunofluorescence staining of CD4+ICOS+PD-1+ T follicular helper (TFH)–like infiltrating T cells in SSc skin (magnification, ×40). Yellow arrows indicate positive cells. (I) Quantitation of CD4+ICOS+PD-1+ TFH-like T cells in the skin of a patient with SSc (n = 19 patients with SSc). Data are from a cohort of patients with SSc, as described in Tables 1 and 2. P value was determined by unpaired t test (*P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.00005).


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On the basis of the finding that T lymphocytes were associated with the onset of GVHD-SSc dermal fibrosis, we further characterized their phenotype, including expression of costimulatory molecules. mRNA assessment on lesional skin from GVHD-SSc mice was con-ducted at days 7, 14, 21, and 28. A 20-fold increase in the expression of cd3e was observed in GVHD-SSc mice compared with syngeneic mice starting on day 7 and lasting through day 28 (Fig. 3A). This finding was striking when compared with the modest twofold increase in macrophage-associated EGF module–containing, mucin-like, hormone receptor–like 1 (emr1 or f4/80) expression (Fig. 3A). The expression of f4/80 increased fourfold by day 14 before dropping to less than twofold by day 28. This observation suggests that CD3+ T cells are the major lymphocyte population involved during the onset and main-tenance of cutaneous GVHD-SSc disease.

We further assessed the mRNA expression of CD4, along with T cell costimulatory and inhibitory molecules icos, cd28, and pdcd1. The expression of these genes was substantially elevated more than five-fold as early as day 7 and remained elevated throughout the disease duration in GVHD-SSc mice (Fig. 3A). Other immune cells, including cd19 B cells, nk1-2/nkp46 natural killer cells, and cd1d natural killer T cells, with the exception of cd11c dendritic cells (DCs), were not con-siderably elevated as the CD3+CD4+ T cell compartment (fig. S2E). Overall, the data indicate that CD4+ T cells and their associated co-stimulatory and inhibitory molecules may play a central role during the initial inflammation and disease progression of cutaneous fibrosis of GVHD-SSc mice. Multiparameter fluorescence-activated cell sort-ing (FACS) analysis showed that about 60% of the lymphocytes in the lesional skin of GVHD-SSc mice were CD3+ T cells. Of these, more than 30% were CD4+ T cells, most of which (~70%) displayed a CD44+ CD62L− effector phenotype (Fig. 3B). About 60% of the effector CD4+ T cell also expressed ICOS, confirming the gene ex-pression data and underlining their potential role in the progression of GVHD-SSc (Fig. 3B). Assessment of the coexpression of CXCR5, PD-1, and ICOS demonstrated that about 40% of the effector CD4+ T cells were CXCR5+, and more than 70% of these cells had a TFH-like phenotype (ICOS+PD-1+CXCR5+; Fig. 3, B and C).

The total number of CD4+ T cells in GVHD-SSc mice was 30-fold greater than in the control group by day 8 and 40-fold greater by day 28 (Fig. 3C). Among the effector CD4+ cells (CD44hiCD62Llo/neg), ICOS+ T cells were substantially elevated (about 80-fold) (Fig. 3C). The frequency of skin F4/80 macrophages increased threefold from day 8 until the end of the study (fig. S2F).

We next quantified the TH cell subsets in the lesional skin of GVHD-SSc mice and observed that TFH- and TH1-like cell subsets were greatly expanded compared with the control group (Fig. 3, C to F). This also included a 30-fold expansion of ICOS+ cells within the

TH1 subset (Fig. 3C). The presence of the TH2-like (CCR6− CXCR3−) cell subset (fig. S2G) in the skin was also confirmed, but the expan-sion of this pool was less than that of the TH1- and TFH-like cells.

In addition, T lymphocytes isolated from the skin were FACS- sorted for CD3+CD4+CD44hiCD62Llo/neg and intracellularly stained for TH-associated transcription factors. About 90% of T cells expressed Tbet+, 40% expressed GATA-3+, 9% expressed Foxp3, and about 3% were Bcl-6+ (fig. S2H). The transcription factor profiling demonstrates an atypical heterogeneous TH cell environment. It should be noted that the Foxp3+ regulatory T cell population was expanded through-out the onset and progression of the GVHD murine SSc, although the expression of ICOS remained unaltered within this subset (fig. S2I). These data further confirm the heterogeneity and potential plasticity of the inflammatory CD4+ T cells in the skin of GVHD-SSc mice.

ICOS+ T cells drive GVHD-induced fibrosisThe prominence of CD4+ICOS+ T cells in SSc and in the skin of GVHD-SSc mice may suggest involvement of these cells in inflamma-tion, fibrosis, or both. To test this hypothesis, we treated GVHD-SSc mice from day 12 to allow cell engraftment and disease initiation with a glycoengineered immunoglobulin G1 (IgG1) anti-ICOS MAb with enhanced antibody-dependent cellular cytotoxicity (ADCC) activity (anti–ICOS-aFuc MAb; table S2). Anti–ICOS-aFuc MAb elicited ef-fective depletion of ICOS-expressing T cells, as demonstrated in vitro by the ADCC assay and in vivo after injection into GVHD-SSc mice (fig. S3, A and B). The administration of the anti–ICOS-aFuc MAb in GVHD mice resulted in inhibition of disease manifestation, as indicated by the in-life clinical scoring as early as day 17 and for the entire dura-tion of the study (Fig. 4A). Disease severity, along with incidence of dermal lesions and alopecia, was reduced compared with the con-trol groups. Unlike the depleting anti–ICOS-aFuc MAb, a blocking anti–ICOS MAb (anti–ICOS-TM) that share the Fab region directed against the ligand-binding domain of ICOS failed to effectively block disease progression (Fig. 4A). This occurred although the anti–ICOS-TM blockade MAb suppressed germinal center responses in the spleen (fig. S4, A and B). Therefore, the in-life mean clinical score data indicated that depletion of ICOS-expressing T cells can control GVHD-SSc to a much greater degree than simply blocking ICOS-ICOSL interactions.

To assess the impact of the anti–ICOS-aFuc MAb on lymphocyte infiltration, tissue remodeling, and fibrosis, we assessed dorsal skin from the different treatment animal groups by H&E and Masson’s trichrome (MT) staining. Treatment with anti–ICOS-aFuc MAb re-sulted in substantially reduced mononuclear cells infiltration, collagen deposition, and thickening of the skin (Fig. 4, B and C). IHC analysis also confirmed the reduction of CD3+ T cells and F4/80+ macrophages in the skin of anti–ICOS-aFuc MAb–treated mice (fig. S4, C and D).

Table 3. Correlation of mRNA transcript signature for TFH-like phenotypic molecules in fibrotic skin of patients with systemic sclerosis.

mRNA transcript combination

Correlation analysis

P r

PDCD-1 versus ICOS 0.0082 0.54

PDCD-1 versus CXCR5 0.025 0.45

ICOS versus CXCR5 6.25 × 10−5 0.77

Table 4. Association profile of the frequency of TFH-like phenotypic marker expression in fibrotic skin-infiltrating cells determined by immunofluorescence.

Immunofluorescence stain combination


P r

PD-1 versus ICOS 2.30 × 10−6 0.89

PD-1 versus CD4 3.05 × 10−5 0.83

ICOS versus CD4 6.27× 10−5 0.81


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Despite controlling early disease manifestation, the blocking anti–ICOS-TM MAb failed to control the histological characteristics of the disease, including infiltrating leukocytes, dermal thickening, and fibrosis, when compared with the depleting anti–ICOS-aFuc MAb (Fig. 4, B and C).

To further explore the effects of depletion of ICOS+ T cells, we performed FACS analysis of T cell subsets isolated from lesional skin. Treatment with anti–ICOS-aFuc MAb inhibited the expansion of total CD4+ and effector CD4+ICOS+ T cells threefold or greater when com-pared with the isotype MAb–treated group (Fig. 4D). Furthermore, anti–ICOS-aFuc MAb treatment depleted the ICOS-expressing TFH-like cells and the TH1-like cells (45- and 12-fold reduction, respectively) (Fig. 4D). These results collectively confirmed the key contribution of ICOS-bearing TH cell subsets to the initiation and progression of skin inflammation and dermal fibrosis.

ICOS+ TH cells contribute to the proinflammatory and profibrotic conditions in GVHD-SScOn the basis of the considerable improvement of GVHD-SSc after treatment with anti–ICOS-aFuc MAb, we examined the correlation between ICOS+ TH cell depletion and the expression of profibrotic cytokines and proinflammatory factors that contribute to inflamma-tion and cutaneous fibrosis. Enzyme-linked immunospot (ELISpot) assessment demonstrated a significant increase (P < 0.05) in the number of IFN-–, IL-4–, and IL-21–producing TH and TFH-like cells in GVHD- SSc mice (Fig. 4E). However, the frequency of IL-13–associated TH2 cytokine–producing cells remained unchanged in GVHD-SSc mice compared with syngeneic animals.

The presence of these proinflammatory and profibrotic cytokines in TH and TFH-like subsets was also confirmed by intracellular flow cytometry assays using sorted CD4+ effector skin T cells (fig. S5). The data demonstrated that treatment with the anti–ICOS-aFuc MAb in-hibited the proinflammatory IFN-, as well as IL-4 and IL-21 ex-pression, whereas the production of the profibrotic cytokine IL-13 in the skin of GVHD- SSc mice was not impacted by ICOS-aFuc MAb treatment (Fig. 4E).

IL-21, one of the prominent TFH-associated cytokines, has been linked to fibrosis (17, 18, 35). The expression of IL-21 transcript was significantly increased (P < 0.005) in the skin from GVHD mice and

almost completely inhibited by anti–ICOS-aFuc MAb treatment (Fig. 5A). This finding is in alignment with the observed reduction in the numbers of skin IL-21–producing lymphocytes (Fig. 4E). These data confirmed that treatment with ICOS-depleting MAb was re-sponsible for the prominent inhibition of TH1- and TFH-like cytokines, including IL-21, which could be one of the key drivers of cutaneous GVHD-SSc.

To further assess the effects of depletion of ICOS+ TH cells, we measured the expression of a broader array of mediators involved in inflammation and fibrosis by quantitative RT-PCR and multianalyte profiling of soluble serum proteins. Molecules involved in inflamma-tion and lymphocyte recruitment, such as monocyte chemotactic protein-1 (mcp-1) and regulated on activation normal T cell expressed and secreted (rantes), were significantly induced (P < 0.05) under GVHD conditions as early as day 12 (fig. S6A) and subsequently reduced 5- to 20-fold upon the depletion of ICOS-expressing T cells (Fig. 4, F and G). Similarly, the serum expression of other inflammatory mediators, such as IL-6 and MCP-5, was up-regulated in GVHD-SSc mice and profoundly reduced upon the removal of ICOS-expressing T cells (Fig. 4G and fig. S6, B and C).

Vascular alterations are a major component of disease manifesta-tion in SSc (34, 36). Serum expression of vasculopathy mediators, such as endothelin-1 and vWF, was increased in GVHD-SSc mice (Fig. 4G and fig. S6D) and significantly reduced (P < 0.0005) in mice treated with anti–ICOS-aFuc MAb. Finally, fibrotic mediators, such as VCAM-1 and MMP-9, were markedly elevated in GVHD-SSc mice (Fig. 4G and fig. S6E), and their expression subsequently declined (P < 0.0005) after the depletion of ICOS-expressing T cells.

Collectively, these observations demonstrated that the deletion of ICOS+ TH cells can effectively block GVHD disease manifestations and provide protection from inflammation and fibrosis. Blocking the interaction of ICOS with its ligand was not sufficient to prevent skin pathology, and this finding further emphasizes the direct involvement of ICOS+ T effector cells in skin inflammation and fibrosis.

Anti-ICOS depleting MAb ameliorates GVHD-SSc by perturbing the interaction between fibroblasts and TH cellsMicroarray gene expression analysis conducted on GVHD-SSc skin showed that the expression of several MMPs were significantly in-creased between day 21 and day 28 compared with the syngeneic control animals (37). Quantitative RT-PCR analysis confirmed a sig-nificant increase (P < 0.05) in the expression of mmp-12 in GVHD-SSc mice and its subsequent significant inhibition (P < 0.05) in response to anti-ICOS–depleting MAb (Fig. 5A). Likewise, the expression of il21, described previously, and il21r transcript was both elevated in GVHD-SSc mice and significantly inhibited (P < 0.0005) by treatment with anti-ICOS–depleting MAb (Fig. 5A). In addition, the expression of IL-21R on fibroblasts (CD3−, CD19−, CD11c−, and F4/80−) isolated from fibrotic skin was increased compared with expression in syn-geneic control mice (P < 0.05; Fig. 5B).

To investigate the relevance of the IL-21/IL-21R axis in the cross- talk between ICOS+ TFH-like cells and stromal cells involved in skin fibrosis, we isolated dorsal dermal fibroblasts from GVHD-SSc and syngeneic mice. Analysis of the supernatant collected from GVHD fibroblast cultures showed that MMP-12 expression increased almost twofold compared to the control graft and was undetectable in the ICOS-aFuc MAb treatment group (P < 0.05; Fig. 5C). To assess the impact of IL-21 as a profibrotic cytokine on MMP-12 biology, we stimulated GVHD-derived skin fibroblasts with recombinant IL-21.

Table 5. Correlation of mRNA transcript signature for TH2 and TH1 phenotypic molecules in fibrotic skin of patients with systemic sclerosis.

TH2 mRNA transcript combination


P r

CRTH2 versus CXCR4 7.34 × 10−7 0.88

CRTH2 versus CD4 0.0013 −0.68

CXCR4 versus CD4 0.00059 −0.71

TH1 mRNA transcript combination


P r

CXCR3 versus IFN- 0.2 −0.3

CXCR3 versus CD4 0.014 −0.55

IFN- versus CD4 0.0045 0.62


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TaqMan RT-PCR analysis demonstrated that IL-21 induced signif-icant mRNA expression of mmp-12 and il21r (P < 0.05) (Fig. 5, D and E). These data suggest that IL-21–producing ICOS+ TFH-like cells could cooperate with dermal fibroblasts in a profibrotic loop that in-volves the expression of IL-21R and MMP-12 production via IL-21.

IL-21 neutralization protects from skin fibrosis in GVHD-SSc miceTo explore a direct link between IL-21 and disease pathogenesis, we conducted IL-21 neutralization studies in GVHD-SSc mouse model. The neutralization of IL-21 (IL-21R–Fc was administered starting

Fig. 2. Disease correlation and functional impact of ICOS-expressing TFH-like cells on myofibroblast differentiation. (A) TaqMan gene expression profile of indicated molecules involved in fibrosis in SSc versus HC. (B) Cor-relations of the mRSS with cell numbers and serum markers for extracel-lular matrix turnover and vascular damage in fibrotic skin of patients with SSc. (C) CD4+ICOS+PD-1+CXCR5+ TFH-like cells were differentiated in vitro on plate- bound anti-CD3 with soluble anti-CD28 monoclonal antibodies (MAbs) in the presence of ICOSL-Fc, interleukin-1 (IL-1), IL-12, and IL-6 for 4 days. (D and E) SMA+Vim+-differentiated myofibroblasts were determined by flow cytometry after direct coculture assay of in vitro–differentiated TFH-like cells with normal human dermal fibroblasts (NHDFs) at a T cell–to–fibroblast ratio of 1:4 in the presence or absence of suboptimal transforming growth

factor– (TGF-) and anti-CD3/anti-CD28 antibodies (FMO, fluorescence minus one). All correlations were carried out using Spearman’s correlation analyses (n = 19 pa-tients with SSc). Data represent two independent experiments. P value was determined by unpaired t test (*P < 0.05, ***P < 0.0005, and ****P < 0.00005).


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at day −1, three times per week) resulted in significant reductions (P < 0.05) in skin inflammation and fibrosis, as determined by disease clinical and histological scores (Fig. 6, A and B). H&E and MT staining

of dorsal skin demonstrated that IL-21 neutralization resulted in the reduction of infiltrating mononuclear cells, skin remodeling, and dermal fibrosis compared with control mice (Fig. 6C). These results support the involvement of IL-21 in skin fibrosis and disease patho-genesis mediated by ICOS-expressing TFH-like cells. Notably, IL-21 neutralization also functionally affected germinal center B cells in the spleen, as measured by a reduction in GL-7+CD95hi–expressing B cells (Fig. 6D). TaqMan RT-PCR analysis of genes associated with TFH-like markers (cd4, icos, pdcd1, and cxcr5), fibrotic markers [mmp-12, mmp-13, mmp-1a, tgf-1, fibroblast growth factor 15 (fg f15), and vcam-1], and inflammatory mediators (ccl5, ccl2, and il6) demon-strated that IL-21 modulates molecules involved with the pathogenesis of GVHD-SSc (Fig. 6E and table S3). IL-21 neutralization resulted in statistically significant reductions (P < 0.005) in the TFH-associated markers cd4, icos, and pd-1 while demonstrating a trend toward reduced cxcr5 (Fig. 6E). The fibrotic genes mmp-12, mmp-13, mmp-1a, tgf-1, fgf15, and vcam-1 were all profoundly reduced in the IL-21R–Fc–treated cohort (Fig. 6E), supporting the histological finding showing reduced MT staining and less fibrosis (Fig. 6C). Consistent with our previous findings, inflammatory mediators ccl5, ccl2, and il6 were also affected by IL-21 neutralization, resulting in their significant reduc-tion (P < 0.005; Fig. 6E). Collectively, this result demonstrates that IL-21 neutralization can affect immune cells and their associated fac-tors that are involved in driving disease pathogenesis and skin fibro-sis in the GVHD-SSc mouse model.

DISCUSSIONHuman SSc is an autoimmune disease that is mediated by the dys-regulation of the innate and adaptive immune systems, leading to inflammation, fibrosis, and vascular damage. Treatment of SSc re-mains very challenging because the mechanisms responsible for the initiation and maintenance of the disease are largely uncharacterized. Here, we demonstrated that the lesional skin of patients with SSc con-tains a subset of infiltrating CD4+ICOS+PD-1+ TFH-like cells that correlate with disease activity. The identification of infiltrating TFH-like cells in the skin of patients with SSc unravels a potential new contri-bution of this cell population to the proinflammatory and fibrotic mechanisms in this disease.

The CD4+ICOS+PD-1+ TFH-like cells, together with ICOS express-ing TH1 cells, were also increased in the skin of the GVHD-SSc mouse model that recapitulates some of the key features of human SSc making this a viable preclinical model to investigate their roles in fibrosis. The prominent expression of ICOS on T cells in the dermis suggests that this costimulatory molecule may represents a critical factor in guiding the fate and frequency of TFH-like cells. The skin-infiltrating TFH-, TH1-, and TH2-like effector cells identified in GVHD-SSc mice along with their associated cytokines IL-21, IFN-, and IL-4 represent an expanded list of players in fibrotic driven disease pathology. Human skin biopsies from patients with SSc showed cells of a similar profile to their murine counterpart, with a profound up-regulation of cd3, cd4, cxcr5, pdcd1, icos, and il21r, which demarcated TFH-like cells, thereby linking them to a profibrotic pathology. Notably, the tran-scription factor profiling in human and murine SSc skin for Bcl-6, Tbet, gata3, and rorc demonstrated an atypical expression pattern that may represent cells in a state poised for quick response to injury in an inflamed tissue such as SSc skin. TFH-like markers transcripts con-firmed the correlation between the expression of cxcr5 and pdcd1, cxcr5 and icos, and icos and pdcd1, demonstrating their possible expression on

Fig. 3. GVHD-SSc skin-infiltrating TFH-like and TH1 cells express ICOS. (A) Kinetics of infiltrating T cell– and macrophage-associated genes in graft-versus-host disease (GVHD)–SSc mice (n = 7). Mice were sacrificed on days 7, 14, 21, and 28 after adoptive transfer of splenocytes. The mean gene expression signals were obtained from the skin, and fold differences were calculated in comparison with the syngeneic con-trol group (n = 7). (B) Cells were isolated from murine GVHD-SSc skin and analyzed by flow cytometry to identify T cell subsets. (C) The frequency of specific T cell sub-sets was examined by flow cytometry. All TH cell lineages were assessed from a parent effector T cell gate (CD44hiCD62Lhi/lo). Subsets were compared with syngeneic con-trols. Data represents three independent experiments. P value was determined by unpaired t test (**P < 0.005 and ***P < 0.0005).


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Fig. 4. Anti-ICOS–depleting MAb reduced pathogenic TH subsets and their associated proinflam-matory and profibrotic mediators involved in tissue remodeling in GVHD-SSc. (A) Evaluation of the mean clinical disease score during biweekly treatment starting on day 8 with anti-ICOS–depleting MAb (n = 5), anti-ICOS–blocking MAb (n = 5), or isotype control MAb and phosphate-buffered saline (PBS) (n = 10). Black asterisks indicate statistical significance for anti–ICOS-aFuc MAb versus PBS group, red asterisks indicate statistical significance for anti–ICOS-aFuc MAb versus isotype MAb group, and blue asterisks indicate

statistical significance for anti–ICOS-aFuc MAb versus anti–ICOS-Tm MAb. (B) H&E assessment of dorsal skin collected from treatment groups at 4 weeks (magnification, ×10). (C) Histological assessment by Masson’s trichrome (MT) staining (magnification, ×10). (D) Quantification of T cell subset frequency after treatment with anti-ICOS MAbs was carried out by flow cytometry. (E) Single-cell suspensions from GVHD-SSc mouse skin of different treatment groups were restimulated in vitro with soluble anti-CD3 MAb for 48 hours on plates coated with IFN-, IL-21, anti–IL-4, and anti–IL-13 antibodies. The cytokine-producing T cells were quantitated in an enzyme-linked immunospot assay. (F) Total RNA samples isolated from skins of all treatment groups were subjected to TaqMan gene expression analysis against a selected gene set. Data were expressed as fold expression changes compared with syngeneic group. P value for gene expression data was calculated using one-way analysis of variance (ANOVA). (G) Serum samples from each treatment group were assessed for selected molecules using the multiplexed immunoassay profiling platform. Data are graphed on a log2 scale to illustrate the fold change. Data represent three independent experiments. P value was determined by unpaired t test, and the ANOVA model was used to estimate the mean differences in the log2 scale of the protein concentrations (*P < 0.05, **P < 0.005, and ***P < 0.0005). ns, not significant; vWF, von Willebrand factor.


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the same cells. This was further supported by the coexpression of CD4, ICOS, and PD-1 in lesional skin using confocal microscopy. However, this associative trend was not consistent among of the evaluated TH1 and TH2 transcripts, indicating a possible predominance of the TFH-like cells and identifying them as a distinctive lineage in-volved in fibrosis.

Although the expression of ICOS has been recently demonstrated in SSc skin (30, 31), our results associated its expres-sion with TFH-like and IFN-–producing cells and demonstrate that this popula-tion is present in human SSc skin and in murine GVHD-SSc. TFH-like cells of a similar phenotype were also recently de-scribed in renal biopsies of patients with lupus nephritis and were inferred to con-tribute in situ to inflammation (38). Like-wise, these TFH-like cells were observed in the circulation of patients with systemic lupus erythematosus and correlated with elevated autoantibody and disease ac-tivity, which is indicative of their rela-tionship to disease pathogenesis (27). These TFH-like cells may also be linked to TH1- driven pathology, as evidenced by recent data that support the development of TH1 cells via a pathway that includes cellular transition by a TFH route (39). Such plas-ticity may create a phenotypic hetero-geneous environment between TH1 and TFH, where Tbet repressed conventional TFH functionality (39). In addition, IL-21+IFN-+ double-secreting cells that were Bcl-6–negative and coexpressed ICOS were observed in a Plasmodium chabaudi in-fection model and found to be involved in parasite clearance (40). It would be in-teresting to explore this finding in future SSc studies.

The TFH-like cells identified in the skin of patients with SSc may serve a different and intriguing fibrosis function. This is supported by their correlation with mRSS and CTX-I, as well as their direct impact on myofibroblast differentiation, which is supportive of their abilities to drive a fibrotic mechanism. Furthermore, SSc skin TFH-like cells correlated with serum VCAM-1, a biomarker for vascular dam-age that is a key event in fibrosis (41). Despite the limitation of our small sample size used in this study, the correlation seen between TFH-like cells and these pa-rameters that reflect active disease was striking and identifies a causal relationship linking them to a role in disease patho-genesis. Most recently, T cell activation pathways and collagen accumulation that have been associated with a range of pro-

fibrotic molecules, such as CTX-I, N-terminal type III procollagen peptide, and MMP-derived degradative neoepitopes of type III col-lagen, were used to monitor the downstream effect of type I IFN receptor blockade in patients with SSc (14). Our finding further as-sociates a subset of skin-infiltrating T cells with fibrotic involve-ment in SSc. Moreover, the impact of their depletion on molecules

Fig. 5. Impact of ICOS+ T cell deletion on profibrotic molecules involved in TH cell–fibroblast cross-talk in GVHD-SSc mice. (A) Total skin RNA samples from syngeneic (n = 5), PBS and isotype MAb (n = 10), or anti–ICOS-aFuc MAb (n = 5)–treated GVHD-SSc mice were profiled for MMP-12, IL-21, and IL-21R using TaqMan gene expression. (B) Mean fluorescence intensity (MFI) of IL-21R on CD3−CD19−CD11c−F4/ 80−– gated cells isolated from lesional skin assessed by flow cytometry. (C) MMP-12 expression in supernatants from syngeneic or GVHD-SSc skin fibroblasts cultured for 48 hours was determined by enzyme- linked immunosorbent assay. (D and E) Single-cell suspension was prepared from digested skin of syngeneic and GVHD-SSc mice. Cells

were cultured for 12 days with two passages to yield confluent fibroblasts. Fibroblasts isolated from either syngeneic or GVHD-SSc animals were stimulated with recombinant IL-21 for 18 hours, followed by RNA isolation and real-time reverse transcription polymerase chain reaction analysis for IL-21R and MMP-12. Data are representative of two to three independent experiments. P value was determined by unpaired t test (*P < 0.05, **P < 0.005, and ***P < 0.0005).


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associated with inflammation (42), vasculopathy (9), and fibrosis (11) in SSc suggests that ICOS-bearing TH1 and TFH-like cells may be in-volved during the early inflammatory and subsequent fibrotic phases of the disease.

The presence of TFH-like cells in the lesional skin of GVHD-SSc is profound and raises important questions about their infiltration and expansion. Although the maintenance of TFH cells in germinal centers requires B cells, interactions between DCs and naive TH cells are critical to initiate the TFH cell program (43, 44). This was supported in studies that have shown that in the context of abundant and prolonged antigen

presentation, DCs are the favored drivers of TFH cell differentiation, whereas B cells function in a more antigen-stringent environment (43). It is possible that DC is the major player in the generation of TFH-like cells in GVHD-SSc mice. In addition to their role in costimulatory pathways involving CD28/B7, ICOS/ICOSL, and OX40/OX40L, DC- derived IL-6 and IL-21 are required for the differentiation of TFH cells (26, 45, 46). Elevated IL-6 in patients with SSc correlated with in-creased skin thickness, fibrosis, and differentiation of IL-4–producing TH2 cells (47–49). In addition to IL-6 and IL-21, IFN- mRNA and protein were also elevated in SSc and GVHD-SSc environments and

Fig. 6. IL-21 neutralization reduces skin fibrosis in GVHD-SSc mice. (A) Live mean clinical score of syngeneic (n = 5) and GVHD-SSc mice treated with either IL-21R–Fc (n = 7) or control protein-Fc (n = 7) three times per week starting at day −1. (B) Histological skin scoring was measured using a rubric assessing tissue inflamma-tion and fibrosis. (C) Histological assessment by H&E and MT staining of dorsal skin (magnification, ×20). (D) Splenic germinal B cells were enumerated by flow cytometry using a CD19+GL-7hiCD95hi gate. (E) TaqMan gene expression assay was conducted on RNA samples isolated from the skins of all treatment groups. Data were expressed as fold expression change relative to the syngeneic group. Data represent two independent experiments. P value was determined by unpaired t test (*P < 0.05, **P < 0.005, and ***P < 0.0005).


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may also contribute to the development and expansion of dermal TFH-like cells. It has been shown that heightened IFN- signaling con-tributed to the initial expansion of TFH cells by up-regulating the expression of Bcl-6 (50). Together, these data provide a likely mech-anism for the generation and expansion of TFH-like cells.

In addition to the requirement for the formation of TFH cells, IL-21 is a major cytokine produced by TFH cells that drives the activation of several lymphocyte populations and various nonimmune cells. Recent reports have implicated IL-21 under several autoimmune con-ditions, including skin inflammation (psoriasis) and SSc dermal fi-brosis (17, 51). Most recently, IL-21–producing CD8 cytolytic-2 T cells have been shown to elicit lung fibrosis in a bleomycin sterile lung injury model, further supporting its functionality beyond reg-ulation of B cell responses (52). In a related study, synovial fluid–sorted CD4+IL-21+ cells could induce the secretion of MMP-1 and MMP-3 by a fibroblast-like synoviocyte cell that promoted inflammation and joint pathology in rheumatoid arthritis patients, further supporting other nonclassic roles for cells with this phenotype (53). Signaling of IL-21 requires the interaction with IL-21R whose expression was elevated in the patient cohort investigated in this study and in an-other published population (51). Moreover, our study suggests that the IL-21 signaling axis was active at the stromal level to influence dermal remodeling by modulating MMPs and fibroblast growth fac-tor in a profibrotic loop that involves the expression of IL-21R. The elimination of IL-21–producing ICOS+ TFH-like cells or neutralization of IL-21 similarly inhibited molecules, such as IFN-, IL-6, TGF-, FGF15, VCAM, and MMPs, that are implicated in both the inflam-matory and profibrotic phases characterizing disease pathogenesis seen in GVHD-SSc. These data provide a link between the IL-21/IL-21R axis and fibrosis and further implicate IL-21–producing TFH-like cells in disease pathology. Several cytokines, including IL-1, tumor ne-crosis factor–, and MCP-1, have been correlated with increased ex-pression of IL-21R in the skin from patients with SSc (17). However, none of these cytokines was directly involved in the regulation of IL-21 and IL-21R expression by fibroblasts.

The blockade of the ICOS/ICOSL axis was not sufficient to control the expansion of skin-infiltrating TH1- and TFH-like cells and the production of proinflammatory and profibrotic cytokines. Such be-havior could not have been attributed to a lack of pharmacological activity by the MAb because both the depleting and blocking ICOS MAbs showed similar inhibition of IgG expression and TFH cell– dependent B cell activation of and germinal center B cell formation in the secondary lymphoid tissues of GVHD-SSc mice. This inability may arise, in part, from the activation of alternative pathways, such as the CD28-B7, CD40-CD40L, or 4-1BB-4-1BBL axes, which are expected to be activated in such a highly inflamed environment (54).

IL-13 has been shown to be associated with human SSc patho-genesis, correlated with the inflammatory subtype of SSc and most recently implicated as a driver of fibrosis in a similar GVHD-SSc pathological environment (55, 56). Although the GVHD-SSc model we used focused on the functional consequences of ICOS-expressing T cells at the peak fibrotic window (day 28), Greenblatt et al. char-acterized elevated IL-13 expression at around day 14, which is more reflective of an inflammatory phase that may be regulated by a dif-ferent cytokine profile (56). The depletion of ICOS+ TH cells affected the number of macrophages but not the number of IL-13–producing T cells. This may suggest that other IL-13–producing cells are at play in our model (18, 57). Although we did not evaluate the expression of IL-13 at day 14, serum analysis at day 12 revealed the presence of

a diverse array of proinflammatory factors. Despite these differences, both studies are in agreement that CCL2 is up-regulated and that CD4+ T cells are key sources of profibrotic cytokines.

There are some limitations to our study that should be highlighted. First, the size of our SSc patient cohort is small and did not allow for stratification based on medication. The size may have also affected the statistical power of the study that could have supported alternative interpretation of the data. Despite this, our conclusion and hypothesis on the involvement of TFH-like cells in SSc were well supported by statistically significant associations of these cells with molecules im-plicated in disease pathology. Second, the size of the patient’s biopsies was limited and did not allow for the investigation of T cells eluted from patient’s skin. Future work would be required to incorporate this approach and compare the immune infiltrates in the lesional and nonlesional skin from patients with SSc. Third, there may be some limitation in the GVHD-SSc mouse model that may not have com-pletely mimics the human disease. Finally, the fact that the use of an anti-ICOS–depleting reagent does not specifically target TFH-like cells is well noted; however, no reagents are currently available to selectively target TFH-like or other TH subsets that would allow for a robust phar-macodynamic assessment of their direct contribution to disease pa-thology in mouse models. To that end, the anti-ICOS MAb used in our study is neither TFH-like cell-specific nor a global T cell–depleting agent but allowed us to effectively assess ICOS+ TH cells.

Collectively, the data identify an important interplay between infiltrating ICOS+ T cell subsets and dysregulated fibroblasts, demon-strated by profoundly reduced inflammation and fibrosis in GVHD-SSc after the removal of ICOS+ T cells. The increased frequency of ICOS-expressing TFH-like cells have been described in several auto-immune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and inclusion body myositis (27–29, 58–61). Here, we ex-panded the role of ICOS+ T cells, including TFH-like cells, to SSc- associated fibrosis as a result of their positive correlation with disease score and ECM remodeling molecules that are related to disease pathogenesis. Although the etiology of most autoimmune diseases is still largely unknown, the results presented here further support the notion that ICOS may be an attractive therapeutic target for the treatment of fibroproliferative disorders.

MATERIALS AND METHODSStudy designGVHD-SScThe study was initiated to evaluate the contribution of ICOS+ TH cells in patients with SSc and in a murine model that recapitulate key characteristic features of the human disease. The significance of ICOS+ TH cells to the pathological manifestation of inflammation and skin fibrosis was incorporated in this study to support the va-lidity of testing specific therapeutics. The GVHD-SSc model gener-ated by the injection of 8- to 10-week-old female spleen cells from B10.D2 mice into 8- to 10-week-old Rag2-deficient BALB/c female mice shared characteristic feature with the human disease inclusive of immune inflammation, dermal fibrosis, and vasculopathy. GVHD-SSc animals were randomly assigned to control [phosphate- buffered sa-line (PBS), isotype control MAb, or control Fc) or treatment groups (ICOS-TM or ICOS-aFuc MAb and IL-21R–Fc). Sample size and dis-ease end time points were selected on the basis of previous studies. In-life clinical score was performed in a blinded fashion. Image analy-sis processing on skin sections were blind-graded by a board-certified


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pathologist in a blinded fashion. Flow cytometry on the lesional skin of GVHD-SSc mice, ELISpot assay, and TaqMan gene assessment were incorporated to quantify and define skin-infiltrating immune cells. The data analysis on treatment groups was performed by a blinded statistician. Animal experiments were performed in accordance with animal protocols approved by MedImmune Animal Care and Use Committee in compliance with Association for Assessment and Ac-creditation of Laboratory Animal Care guidelines.Scleroderma patientsThe human study was conducted in accordance with the Declaration of Helsinki, International Council for Harmonization Guidance for Good Clinical Practice, and all local laws. The ethics committee from each participating hospital approved the study protocol and all amend-ments. All subjects provided written informed consent before study participation. Subjects (males and females, ≥18 years of age) that ful-fill the American Rheumatism Association (American College of Rheumatology) preliminary classification criteria for SSc with at least moderate skin thickening (score of at least 2 by mRSS) in at least one area suitable for biopsy (that is, arms, legs, or trunk) were in-cluded. Patients who received any investigational drug therapy in-cluding depleting and nondepleting T and B biologic therapies before the study (or within five half-lives of ingestion, whichever is longer) and patients with any opportunistic infection or serious infection or with history of severe viral infection and positive tests for hepatitis A, B, and C were also excluded. Quantitative confocal IF microscopy and TaqMan gene profile were incorporated to correlate findings with fibrosis markers in the disease cohort.Patients and clinical assessmentLesional skin biopsies were obtained from 19 of 28 enrolled adult patients with SSc who had at least moderate skin thickening and fulfilled the American College of Rheumatology criteria for SSc (Table 1). The study protocol was reviewed and approved by the Institutional Review Board or independent ethics committee of each participating center before study initiation. Written informed consent was ob-tained from each participant before study entry.

Eligible patients were not treated with any MAb therapy at any time and did not receive more than 20 mg/day of leflunomide within the 6 months before the study. Doses of certain concomitant medica tions, such as cyclophosphamide, prednisone, methotrexate, and mycophe-nolate mofetil, were also reduced within the 21 days before the study.

No stratification of patient outcomes with medication was reported because of the size of the clinical cohort. In addition, the patients’ medical histories demonstrated the involvement of other organ systems, including cardiovascular, gastrointestinal, respiratory, musculo-skeletal, endocrine, immunological, neurological, and urogenital sys-tems. However, no correlation was made with skin manifestation or disease duration because of the small sample size and the limited scope of this work. The dermatological features captured in patients’ medical histories included alopecia areata, chronic dry skin, proximal skin thickening, and recurrent rashes. The antinuclear antibody test data, summarized in Table 1, were not segregated into individual tests to differentiate anti-RNA polymerase III antibodies from anticentromere antibodies for prognostic determination because this was outside the scope of our study. Biopsies from 20 healthy adult volunteers were used as controls (Table 2). Primary data are reported in table S4.

Statistical analysisThe analysis of variance (ANOVA) model was used to estimate the mean differences in the log2 scale of the protein concentrations among

animal groups; a P value of less than 0.05 was considered statistically significant. Statistics software (SAS version 9.1, SAS Institute Inc.) was used for statistical analysis. Additional statistical analysis was per-formed using GraphPad Prism 6 for flow cytometric, clinical scoring, ELISpot, and gene expression data using one-way ANOVA or un-paired t test (two-tailed). Correlation analyses were carried out using Pearson’s or Spearman correlation coefficients, and P values of less than 0.05 were considered statistically significant.

SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/10/431/eaaf5307/DC1Materials and MethodsFig. S1. Skin transcription factor profiling and correlational analysis of infiltrating T cells and molecules related to disease activity.Fig. S2. Evaluation of cellular subsets in the fibrotic skin of mice with GVHD-induced skin fibrosis.Fig. S3. Anti–ICOS-aFuc MAb elicits efficient depletion of ICOS-expressing T cells.Fig. S4. Anti-ICOS treatment blocks B cell functional response.Fig. S5. Skin-infiltrating T cells express both proinflammatory and profibrotic cytokines.Fig. S6. Anti-ICOS treatment reduced the systemic levels of molecules involved in lymphocyte function, migration, and fibrosis.Table S1. Correlative comparison of mRNA transcript signature for TH1, TH2, and TFH phenotypic molecules in fibrotic skin of patients with SSc.Table S2. Summary of binding affinity for parental and therapeutic antibody constructs.Table S3. Designated probes used for screening human SSc and murine GVHD-SSc genes.Table S4. Primary data.References (62–64)

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Acknowledgments: We thank T. O’Day and J. Bakken for the technical support. We thank G. Sims for critically reading the manuscript. Funding: The research in this manuscript

was fully funded by MedImmune/AstraZeneca. Author contributions: D.K.T. and G.C. designed the experiments, supervised the project, helped with the flow cytometry assay, analyzed the data, and wrote the manuscript. N.M. performed the mRNA extraction, quantitative PCR analysis, and ELISpot assays. E.K. performed flow cytometry, mRNA extraction, quantitative PCR analysis, and enzyme-linked immunosorbent assay. T.D. and T.B. helped set up the in vivo models and performed the in vivo experiments. K.D. prepared tissue for the histochemical analysis and helped with image acquisition. W.Z. and L.I.C. graded the coded skin sections, performed the analysis and quantitation of cellular infiltrating subsets on IHC and IF stained tissues, and analyzed the skin fibrosis data. C.B. and A.B. performed IHC and IF staining of samples. X.G. and W.I.W. performed the assays for ECM turnover and vascular damage serum biomarkers in patients with SSc and analyzed the data. J.Z., H.D., M.A.B., J.L., and C.G. engineered the isotype control, TM, fucosylated, and glycoengineered anti-ICOS MAbs. M.R. and M.-C.G. performed the TFH-like differentiation experiments. L.Y. performed the statistical analysis. M.R. and M.-C.G. performed the TFH-like differentiation experiments for T cell fibroblast coculture assays. R.W. conducted the binding affinity measurement of fucosylated and glycoengineered antibodies. R.H. provided the clinical samples and data and helped with manuscript editing. Competing interests: All authors of this manuscript are MedImmune employees and own AstraZeneca stock and/or stock interests.

Submitted 25 February 2016Resubmitted 3 October 2017Accepted 13 February 2018Published 7 March 201810.1126/scitranslmed.aaf5307

Citation: D. K. Taylor, N. Mittereder, E. Kuta, T. Delaney, T. Burwell, K. Dacosta, W. Zhao, L. I. Cheng, C. Brown, A. Boutrin, X. Guo, W. I. White, J. Zhu, H. Dong, M. A. Bowen, J. Lin, C. Gao, L. Yu, M. Ramaswamy, M.-C. Gaudreau, R. Woods, R. Herbst, G. Carlesso, T follicular helper–like cells contribute to skin fibrosis. Sci. Transl. Med. 10, eaaf5307 (2018).


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like cells contribute to skin fibrosis−T follicular helper

CarlessoLin, Changshou Gao, Li Yu, Madhu Ramaswamy, Marie-Claude Gaudreau, Rob Woods, Ronald Herbst and GianlucaI. Cheng, Charles Brown, Anmarie Boutrin, Xiang Guo, Wendy I. White, Jie Zhu, Huifang Dong, Michael A. Bowen, Jia Devon K. Taylor, Nanette Mittereder, Ellen Kuta, Tracy Delaney, Timothy Burwell, Karma Dacosta, Weiguang Zhao, Lily

DOI: 10.1126/scitranslmed.aaf5307, eaaf5307.10Sci Transl Med

not-so-helpful T cells could dampen dermal fibrosis and bring relief to scleroderma patients.IL-21, an important T follicular helper cell cytokine, ameliorated disease in the mouse model. Targeting these−anti

expressing inducible costimulator (ICOS) correlated with disease scores. Blocking these cells with anti-ICOS or like cells−model to elucidate the key players in this disease. They observed that a subset of T follicular helper based mouse− examined immune cells from patients and in the skin of a graft-versus-host diseaseet al.Taylor

Scleroderma, also known as systemic sclerosis, is a devastating disease involving multi-organ fibrosis.Scleroderma's little helpers

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FIBROSIS Copyright © 2018 T follicular helper–like cells ...Changshou Gao,4 Li Yu,5 Madhu Ramaswamy,1 Marie-Claude Gaudreau,1¶ Rob Woods,4 Ronald Herbst,1 Gianluca Carlesso1* Systemic