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Blockade of IL-7 signaling suppresses inflammatory responses and reverses alopecia areata in C3H/HeJ mice

Blocking the overexpression of IL-7 in lesional alopecis skin can reverse alopecia areata in C3H/HeJ mice. The interleukin-7 (IL-7) signaling pathway plays an important role in regulation of T cell function and survival. We detected overexpression of IL-7 in lesional skin from both humans and C3H/HeJ mice with alopecia areata (AA), a T cell–mediated autoimmune disease of the hair follicle. We found that exogenous IL-7 accelerated the onset of AA by augmenting the expansion of alopecic T cells. Conversely, blockade of IL-7 stopped the progression of AA and reversed early AA in C3H/HeJ mice. Mechanistically, we observed that IL-7Rα blockade substantially reduced the total number of most T cell subsets, but relative sparing of regulatory T cells (Tregs). We postulated that short-term anti–IL-7Rα treatment in combination with a low dose of Treg-tropic cytokines might improve therapeutic efficacy in AA. We demonstrated that short-term IL-7Rα blockade in combination with low doses of Treg-tropic cytokines enhanced therapeutic effects in the treatment of AA, and invite further clinical investigation.


INTRODUCTION
Alopecia areata (AA) is a chronic inflammatory disease that attacks the hair follicle (HF) (1)(2)(3)(4); however, the etiology and pathogenesis of AA remain incompletely understood. AA is a T cell-mediated autoimmune disease involving the collapse of HF immune privilege, which develops because of genetic and environmental factors leading to cytotoxic CD8 + T cell activity, cytokine release, and apoptosis of the HF (4-6). We recently identified CD8 + NKG2D + T cells as the key pathogenic cells in AA, which guided our investigation of the cytokines that drive their activation and function (5,6). Our transcriptional profiling analysis revealed that the c cytokines interleukin-2 (IL-2), IL-7, and IL-15 were up-regulated in lesional skin from both humans and C3H/HeJ mice with AA (6). Blockade of either of IL-2 or IL-15 using antibodies (Abs) prevented the development of AA in the C3H/HeJ skin-grafted model (6); however, the role of IL-7 in AA remains undefined. IL-7 plays an important role in immune system development and homeostasis by promoting lymphoid cell growth and survival (7)(8)(9). The effect of IL-7 on T cells is controlled by the expression of the IL-7 receptor (IL-7R) complex (composed of the  chain and IL-7R), as well as by the state of differentiation of the T cell and the availability of the cytokine IL-7 itself (7)(8)(9). In addition to the critical role of IL-7 in naïve and memory T cell growth and survival, recent research also revealed that IL-7 enhances the function and expansion of interferon- (IFN-)-producing T helper type 1 (T H 1) cells (10). IL-7 plays a key role in the pathogeneses of multiple T celldependent autoimmune diseases, including multiple sclerosis (MS), type 1 diabetes (T1D), rheumatoid arthritis (RA), and Sjögren's syndrome (SS) (8). Therefore, a therapeutic strategy aimed at blocking the IL-7 signaling pathway may prevent or reverse the autoimmune disorders in mouse models of these diseases (10)(11)(12).
We and others previously showed that AA exhibits a notable IFN gene expression signature in lesional skin from both humans and C3H/HeJ mice with AA (3,6). IFN- may contribute to the collapse of HF immune privilege by up-regulating major histocompatibility complex (MHC) class I expression in the HF and inducing apoptosis of the HF (13). Here, we demonstrated that IL-7 is up-regulated in lesional skin from both humans and C3H/HeJ mice with AA. We investigated the role of IL-7 in the development and onset of AA in the C3H/HeJ mouse model and found that IL-7 enhances the cytotoxic T cell type 1 (Tc1) immune response in C3H/HeJ mice. We demonstrated that blockade of IL-7R reduces the number of alopecic T cells yet relatively spares regulatory T cells (T regs ), leading to the reversal of AA. Furthermore, we found that anti-IL-7R treatment in combination with low dose of T reg -tropic cytokines (such as IL-2 and IL-33) enhanced its therapeutic benefit in the treatment of AA without severe T cell depletion. Together, we defined a previously unidentified role of IL-7 in the development of AA and revealed critical underlying disease mechanisms for disease resolution. These findings provide evidence that therapeutic approaches blocking IL-7 signaling alone or in combination with T reg -tropic cytokines may represent a promising new combination therapy in the treatment of human AA.
AA scalp biopsies, IL-7R was also detected in T cells infiltrating the HFs in which the expression of IL-7 was up-regulated (6), compared to HF from normal control scalp skin (Fig. 1, E and F).
We investigated the effects of IL-7-mediated signaling in skininfiltrating T cells. Signal transducer and activator of transcription 5 (STAT5) is a key transcription factor downstream of IL-7R, and IL-7 requires STAT5 to induce Bcl-2 expression and to maintain effector T cells (9). Phosphorylation of STAT5 (pSTAT5) levels was determined in T cells following stimulation with IL-7 by using a pSTAT5-specific monoclonal Ab (mAb) and intracellular flow cytometry. We found that IL-7 drove pSTAT5 almost equally in CD8 + T cells within both skin and lymphoid organs (Fig. 1, G and H), indicating that the IL-7 signaling pathway was functional in skininfiltrating CD8 + T cells. Because IL-7R expression is lower on T regs than non-T regs (Fig. 1I), IL-7 drove less pSTAT5 in T regs compared to CD4 + non-T regs (Fig. 1, J and K) (9). Accordingly, B cells that lack the expression of IL-7R showed no detectable response to IL-7 ( fig. S1B). Together, these results indicate that overexpression of IL-7 in lesional skin may enable IL-7-dependent T cells to better survive in skin.

IFN- increased IL-7 production in skin
Type I IFNs and IFN- induced IL-7 production in keratinocytes, stroma, and epithelial cells in previous studies (16,17). Because lesional skin from both patients with AA and C3H AA mice exhibited a notable IFN gene signature (6), we postulated that IFN- produced by skin-infiltrating mononuclear cells, specifically CD8 + T cells, might promote local production of IL-7 in the skin. To investigate the role of IFN- signaling in IL-7 production, we injected recombinant mouse IFN- intradermally in areas of the skin of C3H NC mice (18). We detected up-regulation of IL-7 in the skin following IFN- treatment compared to phosphate-buffered saline (PBS) ( S2A). Moreover, using a mAb that targets IFN- receptor  (IFN-R) to block IFN- signaling, we observed a significant decrease in the expression of IL-7 in the skin from anti-IFN-R-treated mice compared to controls ( fig. S2B). These results indicate that IFN- signaling may promote IL-7 production in the skin under homeostasis or in the context of inflammatory conditions such as AA, which may, in turn, promote the survival of IL-7-dependent lymphocytes (16).
IL-7 promoted the development of Tc1 and accelerates the development of AA IL-7 promotes T H 1 cell responses, as well as priming and inducing activation of autoreactive T cells in mouse models, such as experimental autoimmune encephalomyelitis (EAE) and nonobese diabetic (NOD) (10,11). Because AA is a T cell-mediated autoimmune disease of the HF, next, we assessed whether IL-7 influenced the development of T H 1 or Type 1 CD8 + T cells (Tc1) in C3H/HeJ mice. Freshly isolated naïve CD4 + T cells and naïve CD8 + T cells were stimulated with anti-CD3 in the presence of IL-7. Consistent with previous reports in other models (10,11), significantly greater percentages of IFN- + CD4 + T cells and IFN- + CD8 + T cells were induced by IL-7 than PBS (Fig. 2, A and B). Cytotoxic CD8 + T cells led to HF destruction through the release of cytotoxic mediators, including granzymes (GZMs) and perforin-1 (PRF1), comprising the cytotoxic T lymphocyte (CTL) gene signature that we previously defined in AA lesional skin from both human AA and C3H/HeJ AA mice (6). c cytokines including IL-2, IL-15, and IL-21 are known to augment the cytotoxic function of CD8 + T cells by inducing the production of those cytotoxic mediators (19). We observed that IL-7 also enhanced the production of GZMA, GZMB, and PRF-1 by CD8 + T cells (Fig. 2, C and D). Given the role of IL-7 in promoting T H 1 and Tc1 responses in vitro, we next evaluated the function of IL-7 in vivo by exogenous administration of IL-7 using the C3H/HeJ skin-grafted model. To extend the in vivo life span and activity of IL-7, we took advantage of a highly bioactive form of IL-7 complexed to anti-IL-7 Ab (IL-7c) with some modifications (20,21). C3H/HeJ mice were administered with IL-7c 2 weeks after skin grafting, a time point at which effector T cells begin to proliferate and accumulate in the SDLNs (6). We treated the mice with a lower dose of IL-7c (0.5-g IL-7 with 5-g mAb M25) than previous reports (1.5-g IL-7 with 15-g mAb M25) (20,21), for a total of two times at 7-day intervals. IL-7c-treated mice displayed faster and more severe hair loss than PBS-treated mice (Fig. 2, E and F). The role of IL-7 in accelerating AA development was further confirmed by comparing the frequencies of skin-infiltrating CD8 + T cells and IFN--producing CD8 + T cells from IL-7c-or PBS-treated mice (Fig. 2, G and H).
We also examined the effects of IL-7 on immune cells from lymphoid organs 1 week after the last dose of IL-7c administration. In agreement with previous reports in other mouse models (20,21), IL-7c increased the total number of lymphocytes in lymphoid organs ( fig.  S3A). We also found that IL-7c robustly increased the frequency of CXCR3 + CD8 + T cells, T-bet + CD8 + T cells, and IFN--producing CD8 + T cells in SDLNs ( Fig. 2I and fig. S3B) but only slightly increased the frequency of CD4 + T cell counterparts ( fig. S3C). The frequency of CD4 + T regs in IL-7c-treated mice may have been decreased because of the low response to IL-7 stimulation ( Fig. 1K and  fig. S3C). Collectively, these results demonstrate that IL-7 plays a key role in the development of AA by enhancing Tc1 responses in C3H/HeJ mice. IL-7R blockade prevented disease onset in in C3H/HeJ skin-grafted model of AA We next investigated role of endogenous IL-7 in AA pathogenesis by IL-7 signaling blockade. The anti-mouse IL-7R (clone A7R34) Ab has been used to block IL-7R signaling when administered in vivo (11,12,22). C3H/HeJ skin-grafted mice were treated with A7R34 or isotype control starting on the day of alopecic skin grafting to induce AA. In the control group, all mice developed hair loss by 7 weeks after skin grafting. In contrast, none of anti-IL-7R-treated mice displayed any signs of hair loss (Fig. 3, A and B). The thymic stromal lymphopoietin (TSLP) receptor shares the IL-7R chain but pairs with the cytokine receptor like factor 2, rather than c, as the second chain comprising the TSLP receptor complex (23). The role of the anti-IL-7R mAb could potentially inhibit signaling from both IL-7 and TSLP in vivo. We ruled out a potential role of TSLP in AA development by treating the C3H/HeJ skin-grafted mice with anti-TSLP neutralizing mAbs (Fig. 3, C and D) (24). These results indicated that endogenous IL-7 plays a pivotal role in AA development in C3H/HeJ skin-grafted mice.

IL-7R blockade prevented disease onset in T cell receptor retrogenic mouse model AA
Retrogenic T cell receptor (TCR) mouse models consist of bone marrow cells from a recombination activating gene 1 (RAG-1)-deficient C57BL/6 mouse transduced with a retroviral vector that carries specific TCR  and  chains, grafted into RAG-1-deficient C57BL/6 mice (25,26). Here, we adapted the previously described 1MOG244 TCR retrogenic system to induce spontaneous AA in C57BL/6 mice ( fig. S4, A to C). Analysis of the T cell compartment of these mice revealed that nearly 40% of CD8 + T cells coexpressed NKG2D, consistent with the central role for these cells in the C3H/HeJ mouse model of AA ( fig. S4D) (6).
Previous studies showed that neither IFN-, tumor necrosis factor- (TNF-), nor IL-17 played a significant role in the pathogenesis of AA in the 1MOG244 TCR retrogenic mouse model (26). To test whether IL-7 signaling affected alopecic T cells in this mouse model, we labeled the 1MOG244 CD8 + T cells from donor AA mice with CellTrace Violet before adoptive transfer into C57BL/6 RAG-1deficient recipients, followed by anti-IL-7R treatment. As shown by CellTrace Violet dilution, anti-IL-7R markedly inhibited 1MOG244 CD8 + T cell proliferation (Fig. 3E). Furthermore, anti-IL-7R mAb administration prevented hair loss in all treated mice, compared to control mice that all developed AA after 1MOG244 CD8 + T cell adoptive transfer (Fig. 3, F and G) (26). Together, we demonstrated an essential role of IL-7/IL-7R signaling in promoting the proliferation and function of AA effector T cells in both the C3H/HeJ skin-grafted mouse model and the C57BL/6 1MOG244 TCR retrogenic model.

IL-7R blockade reversed early AA in C3H/HeJ mice
To evaluate the efficacy of anti-IL-7R in a clinically relevant context, we asked whether anti-IL-7R could reverse newly established disease by initiating therapy 5 to 7 weeks after skin grafting, a time point at which grafted C3H/HeJ mice begin losing hair (6). Notably, after 8 weeks of anti-IL-7R mAb administration, more than 60% of treated mice displayed hair regrowth in areas of previous hair loss, whereas all control mice developed progressive and extensive hair loss (Fig. 4, A and B). Consistent with hair regrowth in treated mice, anti-IL-7R substantially reduced skin inflammation shown by staining of histological markers of the disease (CD8, MHC class I, and MHC class II) (Fig. 4C). Flow cytometry analysis of skin single cell suspensions further revealed that the frequency of CD45 + immune infiltrates was markedly reduced in anti-IL-7R-treated mice (Fig. 4D). Total CD8 + T cell frequency and IFN--producing CD8 + T cell frequency among skin CD45 + immune infiltrates were both significantly reduced in anti-IL-7R-treated mice (Fig. 4D). Concordantly, global analysis of skin gene expression revealed that both the IFN and CTL components of the ALADIN (Alopecia Areata Disease Severity Index) score strongly discriminated between anti-IL-7R-treated mice and control animals (Fig. 4, E and F). Collectively, IL-7R blockade reduced the number of alopecic effector T cells present and inhibited their function in affected skin, leading to disease reversal.

IL-7R blockade inhibited T effector cell function and favored immune regulation
We next investigated the immunological consequences of anti-IL-7R treatment on different T cell populations. CD8 + T cells within SDLNs from anti-IL-7R-treated C3H/HeJ mice produced significantly less IFN- and IL-2 compared to controls (Fig. 5A). We next evaluated the function of effector T cells. We and others previously showed that CD8 + T cells isolated from SDLNs of C3H/HeJ AA mice can induce AA in normal-haired young C3H/HeJ recipients (6,27). As expected, all recipient mice developed AA by 10 weeks after adoptive transfer of SDLN T cells from isotype-treated mice. In contrast, same number of SDLN CD8 + T cells from anti-IL-7R-treated C3H/HeJ mice failed to induce AA during the time of observation (15 weeks after cell transfer) (Fig. 5, B and C), indicating that anti-IL-7R treatment suppressed alopecic effector T cells in treated mice.
Because IL-7 is a critical factor for lymphocyte survival, we next analyzed the changes in lymphocytes in SDLN and spleen after treatment. Anti-IL-7R treatment significantly reduced the total numbers of SDLN cells, CD3 + T cells, CD4 + T cells, CD8 + T cells, and B cells compared to controls (Fig. 5D). Among CD8 + T cells, three populations, including CD8 + NKG2D + T cells, CD8 + CD44 + CD62L − T cells, and IFN--producing CD8 + T cells, were markedly reduced in number and frequency in anti-IL-7R-treated C3H/HeJ mice (fig. S5, A and B) (6). In agreement with previous reports in other mouse models (8,11), C3H/HeJ mice treated with anti-IL-7R showed a significant increase in the frequency of PD-1 + CD44 + T cells and T regs within SDLNs compared to controls ( Representative flow cytometric plots and summary graphs of GZMA, GZMB, or PRF1-producing CD8 + T cells. Data are represented as the means ± SD of three independent experiments. *P < 0.05 and ***P < 0.001 (unpaired Student's t test). (E and F) C3H skin-grafted mice were given IL-7c (0.5-g IL-7 mixed with 5-g anti-IL-7 mAb M25, 37°C for 20 min) once a week for 2 weeks after 2 weeks after skin grafting. Representative images (E) and the incidence of AA onset (F) in grafted C3H/HeJ mice treated with IL-7c (n = 6) or PBS (n = 6). *P < 0.05, log-rank test. (G and H) Representative fluorescence-activated cell sorting plots and summary graphs of the percentages of T cell profiles in the skin of grafted mice treated with IL-7c or PBS. Data are represented as the means ± SD of two independent experiments. **P < 0.01 (unpaired Student's t test). (I) Summary graphs of the percentages of CD8 + T cell subsets within SDLNs. Data are represented as the means ± SD of two independent experiments. **P < 0.01 and ***P < 0.001 (unpaired Student's t test). Photo credit: Mice pictures taken by Zhenpeng Dai, Columbia University. decrease in their absolute numbers, the T reg /T eff cell (T reg /CD8) ratios were increased in the SDLNs from anti-IL-7R-treated C3H/ HeJ mice (Fig. 5I). We also observed that the suppressive function of T regs from anti-IL-7R-treated C3H/HeJ mice was not significantly changed when compared to T regs from control Ab-treated mice ( fig. S6A), indicating that the effect of anti-IL-7R treatment spares T regs , consistent with previous reports (11). We observed that the expression of PD-L1 (programmed cell death 1 ligand 1) and PD-L2 were markedly up-regulated in lesional skin from C3H/ HeJ AA mice compared to controls ( fig. S7A), consistent with our previously published gene expression microarray data (6). Skininfiltrating T cells from anti-IL-7R-treated C3H/HeJ mice contained a higher percentage of PD-1 + CD8 + T cells than controls (fig. S7B). Moreover, the PD-1 + CD8 + T cells from anti-IL-7R-treated C3H/HeJ mice produced less IFN- and TNF- compared to controls ( fig. S7C). Given the critical roles of T regs and programmed cell death protein 1 (PD-1)/PD-L pathway in peripheral tolerance (28,29), we postulated that IL-7R blockade might favor immune tolerance in the context of AA (8,11).
We next investigated whether T regs or the PD-1/PD-L pathway played a role in AA reversal after anti-IL-7R treatment. First, C3H/HeJ AA mice were treated with anti-IL-7R for 4 weeks. Then, the mice were administered either anti-PD-1 mAb (to block the PD-1 signaling pathway), or anti-FR4 (folate receptor 4) mAb (to deplete T regs ) (30,31). Previous reports showed that PD-1/PD-L1 blockade abrogates the protective effects of anti-IL-7R treatment in the NOD mouse model (11). However, we found that PD-1 blockade neither accelerated nor delayed disease in treated AA mice compared to controls (Fig. 5J). In contrast, following T reg depletion, we observed an increase in hair loss in AA mice (Fig. 5J), indicating that an increased frequency of T regs might be one of the underlying mechanisms during AA resolution following IL-7R blockade. Moreover, we found a protective role of T regs in AA development after T reg depletion in C3H/HeJ skin-grafted mice (fig .  S6B). Together, these results demonstrate that IL-7R blockade reduced alopecic effector T cell frequency and, simultaneously, spared T regs during AA resolution.

A combination of anti-IL-7R and low-dose T reg -tropic cytokine promotes resolution of AA
Given the role of T regs in controlling pathogenic autoimmune response in AA (Fig. 5O), we next compared the frequency of T regs in lymphoid organs and skin in C3H/HeJ mice, with or without AA. We found that the frequency of T regs increased within the SDLNs yet significantly decreased in the skin over the course of the disease (Fig. 6, A and B), suggesting that disruption of the T reg /T eff cell balance in the skin could be a contributing factor in AA (32). Manipulating T regs is a promising strategy to treat autoimmunity (33). The T reg -tropic cytokines IL-2 and IL-33 are crucial for the survival and stability of T regs in vivo (34,35). Low-dose IL-2 has shown therapeutic value in the treatment of several autoimmune diseases, including a small pilot study in AA (36,37). Likewise, the cytokine IL-33 has recently been shown to expand tissue T regs and to enhance their suppressive capacity in vivo (38). We found that neither low doses of IL-2 nor of IL-33 used in our study were able to prevent AA development in C3H/HeJ skin-grafted mice (Fig. 6, C and D), possibly because of strong inflammatory responses in AA that overcome the suppressive function of T regs in alopecic skin.
Although long-term anti-IL-7R treatment showed efficacy in AA resolution (Fig. 4), it also had the undesirable effect of significantly reducing the total numbers of lymphocytes (Fig. 5E). Because anti-IL-7R treatment reduced T effectors, but relatively spared the T regs (Fig. 5), we postulated that a short-term anti-IL-7R treatment to suppress strong inflammatory responses transiently without inducing lymphopenia, in combination with low-dose IL-2 or IL-33 to increase the frequency of T regs , might promote AA resolution. We added either low-dose IL-2 or low-dose IL-33, as described in previous reports, after anti-IL-7R mAb administration (total of doses) (Fig. 6E) (39). We observed that IL-2 and IL-33 combinatorial immunotherapy did not completely reverse the AA in most treated mice (Fig. 6F). Nevertheless, the combinatorial immunotherapy increased the frequency of T regs in mice skin and showed effects on slowing AA progression compared to PBS (Fig. 6, G to I), suggesting a role for T regs in AA development. Our results demonstrated that short-term IL-7R blockade in combination with T reg -trophic cytokines may have therapeutic benefit in the treatment of AA.

IL-7R polymorphisms and AA risk
Recently, polymorphisms in IL-7R have been shown to contribute to the susceptibilitour geneomic datay of developing autoimmune disorders in human complex diseases (40). In genome-wide association studies (GWAS), several IL-7R single-nucleotide polymorphisms were associated with autoimmune disorders, including MS, T1D, ulcerative colitis (UC), sarcoidosis, and primary biliary cirrhosis (PBC) (40). However, the mechanisms by which these variations alter risk of disease remain to be elucidated. To ask whether the IL-7 pathway has relevance in human AA, we reanalyzed our previous GWAS data in AA (5), we found that one singlenucleotide polymorphism (rs6891096) in the IL-7R region showed a nominal AA association (P = 0.000717) ( fig. S8A). Moreover, we found that the production of IFN- by T cells stimulated by IL-7 was higher from patients compared to control subjects ( fig. S8B). This suggests that IL-7 may reduce the threshold for activation of alopecic T cells, under a predisposed genetic background, leading to AA progression s.

DISCUSSION
Previous studies proposed that disequilibrium between the production of proinflammatory cytokines and anti-inflammatory cytokines could be involved in the persistence of AA (1, 2). The lack of targeted therapies for AA can be attributed in part to an incomplete understanding of the cytokine pathways that underlie AA pathogenesis (42,43). In RA and psoriasis, for example, the identification of proinflammatory cytokines critically involved in pathogenesis led to effective therapies using blocking Abs to interrupt these signaling pathways necessary for sustained inflammatory responses in tissue, such as TNF- and IL-17A, respectively (44,45). In this study, we demonstrated the critical role of IL-7 in AA by showing that (i) IL-7 is highly up-regulated in AA lesional skin; (ii) administration of exogenous IL-7 accelerated the onset of AA; and (iii) blockade of IL-7 signaling by administrating anti-IL-7R mAb ameliorated ongoing AA and reversed established AA.
In agreement with our previous gene expression studies, we showed that both humans and C3H/HeJ mice with AA show up-regulated expression of IL-7 in lesional skin. IFNs drive several proinflammatory pathways that facilitate pathogenic recognition and activation of adaptive and innate responses, including the (B and C) Normal C3H/ HeJ mice (n = 6) were transferred with 10 × 10 6 activated CD8 + T cells (either from anti-IL-7R or control mAb-treated mice). Representative images (B) and the incidence of AA (C). ***P < 0.001 (log-rank test). (D to I) The animals were treated as in Fig. 4. (D) The total numbers of immune cell subsets within SDLNs of the anti-IL-7R or control mAb-treated mice. (E to F) Representative flow cytometric plots and summary graphs of the percentages of PD-1 + T cells within indicated T cell populations. (G to H) Representative flow cytometric plots and summary graphs of the percentages of T regs within total CD4 + T populations. (I) Bar diagram depicts the ratio of T regs :CD8 + T effectors within SDLNs from the two groups. Data are represented as the means ± SD of two independent experiments. **P < 0.01 (unpaired Student's t test). (J) The incidence of AA onset in C3H/HeJ mice were treated with anti-IL-7R mAb following treatment with anti-PD-1 mAb (n = 10), T reg depletion (n = 10), or control mAb (n = 10). ns, not significant; *P < 0.05, log-rank test. Photo credit: Mice pictures taken by Zhenpeng Dai, Columbia University. recruitment of immune effectors through the induction of chemokines (46). The role of IFN- in AA was previously established using knockout studies and pharmacological administration of cytokines that precipitated disease (47). IL-7 promotes T H 1 responses, as well as priming activation of autoreactive T cells in several mouse models of autoimmune diseases, including EAE, T1D, and SS (8,(10)(11)(12)22). We asked whether IL-7 plays a role in the enhanced T H 1 responses and IFN- production in AA. We showed that IL-7 accelerated the development of AA and increased the number of IFN--producing T cells infiltrating the HFs. IL-7 is mainly produced by stromal cells in lymphoid tissues and bone marrow, and other tissues including skin are also known sites of IL-7 production (15). It was previously reported that IFN- induces expression of IL-7 in keratinocytes (48). To this end, we showed that treatment with IFN- increased the expression of IL-7 in the skin, and likewise, blockade of IFN- signaling pathway decreased the expression of IL-7 in the skin. This result revealed a positive feedback regulation between the IFN- and IL-7 signaling cascades in the skin (16). IL-7 is an important factor in the regulation of T cell survival and function, and IL-7 as well as its receptor are involved in T celldependent autoimmune processes (8). The role of the IL-7/IL-7R pathway in autoimmune diseases is underscored by several recent studies demonstrating that IL-7R blockade in the EAE and NOD mouse models had therapeutic benefit (10,11). To define the role of IL-7 in the pathogenesis of AA, we found that therapeutic anti-IL-7R treatment in C3H/HeJ AA mice induced hair regrowth and reversed early disease. Furthermore, anti-IL-7R treatment reduced inflammation and markedly reduced skin infiltration by T cells. T cells from mice treated with anti-IL-7R produced less IFN- and IL-2 and did not induce AA in C3H/HeJ recipients as effectively as T cells from isotype control-treated mice, indicating that IL-7 is critical for the functioning of T effector cells in AA. Our results showed that the efficacy of anti-IL-7R mAbs is associated with the induction of lymphodepletion. However, the IL-7 biology in C3H/ HeJ mouse model and other rodent models shows differences from that in primates and humans, because administration of high doses of anti-IL-7R antagonistic mAb has not been reported to induce lymphopenia in primates and in human phase 1 trial (49,50).
In the NOD mouse model, IL-7R blockade not only reduced the number of activated effector T cells but also involved the upregulation of PD-1 and signaling via its ligand PD-L1, leading to long-term tolerance as shown (11). PD-1/PD-L has been implicated as a critical pathway for peripheral tolerance (51). PD-L expression is often elevated in response to tissue insult and inflammation, and PD-1 is overexpressed on effector T cells (51). This may represent the "last hope" of the tissue or organ as it attempts to regulate selfdestructive T cells. Although PD-1-ligand expression is increased in the HF of C3H/HeJ mice with AA, the disease still develops, suggesting a defect in this signaling pathway in stopping disease progression. We showed that IL-7R blockade reverses the onset of AA with a concomitant increase in the expression of PD-1 on T cells in C3H/HeJ mice. PD-1 blockade did not significantly accelerate AA. However, we cannot exclude a role for the PD-1/PD-L signaling pathway in AA, because this pathway is significantly up-regulated in AA lesional skin. Our data are consistent with previous reports in other mouse models of disease in which the role of the PD-1 signaling pathway may become less prominent because IL-7R blockade induces multiple mechanisms of action for disease resolution (52).
T regs are central to maintain immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses (32,33), and T reg defects contribute to the loss of tolerance in human autoimmune diseases and mouse models (53). We observed that the ratio of T reg /T eff cells significantly decreased in the skin of C3H/HeJ mice over the course of the AA. Enhancing T reg is one strategy to treat autoimmune diseases. IL-2 is a key survival factor for T regs , and low-dose IL-2 therapy has been used in clinical trials for several autoimmune diseases (37). However, the efficacy of lowdose IL-2 in human AA treatment was not clear. IL-33 is an IL-1 family cytokine that signals via ST2, and IL-33/ST2 signaling is known to expand tissue-resident T regs and increase their immunomodulatory function in several mouse model of inflammatory disorders (54). Low-dose IL-33 has not been reported in the treatment of AA. Our results suggest that anti-IL-7R treatment depleted most effector T cells and increased the relative frequency of T regs , which then play a role in AA recovery. However, we found that low-dose IL-2 or low-dose IL-33 alone used in our study was not effective in the treatment of C3H AA, perhaps because of the strong inflammatory responses in AA. Because long-term anti-IL-7R mAb treatment induces broad lymphodepletion, we postulated that a short-term anti-IL-7R treatment without inducing lymphopenia, in combination with low-dose IL-2 or IL-33, might have greater efficacy in disease reversal. We found that low-dose IL-2 or IL-33 combined with IL-7R blockade augmented the frequency of T regs with skin, and this combination treatment showed some efficacy in halting disease progression, but it did not completely reverse AA in most treated mice. We observed that a short-term anti-IL-7R treatment suppresses the action of effector T cells only transiently, possibly because of the ability of IL-15 and/or other compensatory gamma chain cytokines that provide "back-up" support for the alopecic CD8 T cells (6). Previous reports in other disease models showed that disease protection by IL-2 is highly dose dependent (34). Our results suggest the need for careful evaluation of the dose of trophic cytokines used to expand T regs avoiding unintended exacerbation of the alopecic T cell response within the skin. T reg instability has been reported in NZW mice (35). T regs in peripheral lymphoid organs in C3H mice did not show wide differences in proportions with other inbred strains of mice [except for New Zealand white mice (NZW)], when compared with the published data ( Fig. 6B) (35). We also showed that C3H mouse T regs displayed strong suppression in vitro, as well as sensitivity to trophic cytokines in our study. These results suggest T reg functioning normally in C3H/HeJ mice.
In summary, we demonstrated that IL-7 plays an important role in the pathogenies of AA and that IL-7R blockade, in combination with a low dose of IL-2 or IL-33, may represent a promising new therapeutic candidate in the treatment of AA. Further investigation needed to determine whether anti-IL-7R leads to active tolerance as a mechanism of AA reversal.

Study design
The objectives of the present study were to examine the role of IL-7 in the development of AA and to determine whether blocking IL-7 signaling could prevent or reverse disease in mouse model of AA. For our study, AA was established by either skin grafting, adoptive T cell transfer, or TCR retrogenic T cell transfer. Experiments were designed to validate cytokines (including IL-2, IL-7, and IL-33) or mAbs (including anti-IL-7R, anti-TSLP, anti-PD-1, anti-FR4, and isotype mAbs) in disease prevention or acceleration. On the basis of preliminary experiments and a type I error (α) = 0.05, we calculated that a minimum of five mice per group were required for statistical significance for our prevention and treatment studies for 80% power to detect an effect in at least 50% of animals included in each experiment. The primary outcome variable between two groups was hair loss. Calculations were performed using G*Power software. Mouse hair status was recorded after each treatment.

Mice
C3H/HeJ and B6.129S7-Rag1 tm1Mom /J (Rag1 −/− B6) mice were obtained from the Jackson laboratory and maintained under specific pathogen-free conditions. Experiments were performed in compliance with institutional guidelines as approved by the Institutional Animal Care and Use Committee of Columbia University.

Generation of C3H/HeJ AA mice
We used two different methods to induce AA in C3H/HeJ mice, including skin grafting and adoptive T cell transfer from SDLN, both of which were described previously (6). For the skin graft model, a piece of skin (1-cm diameter) from a C3H/HeJ donor mouse with extensive AA was grafted onto the dorsal back skin of 8-to 10-week-old C3H/HeJ recipients and covered with a bandage for 10 days. For adoptive T cell transfer, 5 million magnetic bead (MACS, Miltenyi Biotec)-purified CD8 + T cells isolated from SDLN were transferred intradermally into normal-haired young C3H/HeJ recipients (6). Hair status was examined twice weekly.

Antibodies
We performed flow cytometric staining with fluorophore-conjugated Abs that were obtained from BioLegend, BD Biosciences, and Thermo

Flow cytometry and fluorescence activated cell sorting
Single-cell suspensions were stained with fixable, viability live/dead Fixable Blue (Thermo Fisher Scientific) in PBS for 15 min at room temperature, after which they were washed twice with RPMI 1640 with 10% fetal bovine serum (FBS). Nonspecific Ab binding was blocked using mouse or human TruStain FcX (BioLegend). For surface marker staining, Abs were diluted at 1:200 and incubated with cells in BD brilliant stain buffer (BD Biosciences) on ice for 30 min. For intracellular staining, the cells then were fixed and permeabilized using a FOXP3 staining kit (Thermo Fisher Scientific) for detection of FoxP3, T-bet, GZMA, GZMB, PRF-1, IL-13, IL-17A, TNF-, or IFN-, according to the FoxP3 staining protocol from the manufacturer. Viable cell populations were gated on the basis of forward and side scatters and by Fixable Blue staining and acquired with a BD LSR II flow cytometer (BD Biosciences). Analysis was carried out using FlowJo software (TreeStar).
For cell sorting, cells were incubated with fluorochromeconjugated Abs in staining buffer (PBS with 2% FBS), on ice for 20 min. The samples were run on an Influx cell sorter (BD Biosciences). Viable cell populations were gated on the basis of forward and side scatters and 4′,6-diamidino-2-phenylindole (DAPI) staining (BioLegend). The viability and purity of sorted cells were greater than 95%.

Preparation of single-cell suspensions
SDLNs or spleens were homogenized and filtered through a 70-m cell strainer. Splenocytes were depleted of erythrocytes by RBC Lysis Buffer (Thermo Fisher Scientific). After the mice were euthanized, hair was removed using hair clippers. Skin was then defatted and digested in 0.25% trypsin for 20 min at 37°C. Epidermis was separated from the dermis using forceps and scalpel blades. The dermis was finely minced and digested for 45 min at 37°C with collagenase type IV (1 mg/ml; Worthington) and deoxyribonuclease I (0.05 mg/ ml; Sigma-Aldrich) in RPMI 1640 with 5% FBS in a shaker. Digested skin was homogenized, filtered through a 70-m cell strainer, and washed before staining or stimulation.

Cell culture and stimulation
All cells were cultured in RPMI 1640 supplemented with 10% FCS, 4 mM GlutaMAX, 20 mM Hepes, 0.05 mM 2-mercaptoethanol, penicillin (1000 U/ml), and streptomycin (1 mg/ml) (all from Thermo Fisher Scientific). Purified CD4 + T cells or CD8 + T cells were stimulated with Dynabeads mouse T-activator CD3/CD28 (Thermo Fisher Scientific) at a bead-to-cell ratio of 2:1 for 4 days in the presence of recombinant murine IL-7 (10 ng/ml) (PeproTech) for T H 1 or Tc1 cell induction in 24-well plates (Corning). To detect intracellular cytokines, cells were restimulated with eBioscience Cell Stimulation Cocktail (Thermo Fisher Scientific) for 5 hours plus eBioscience Protein Transport Inhibitor Cocktail (Thermo Fisher Scientific). To detect IFN- and IL-2 production, T cells were stimulated with Dynabeads mouse T-activator CD3/CD28 for 2 days. The production of IFN- or IL-2 was measured by an enzyme-linked immunosorbent assay kit (BioLegend).
T reg suppression assays CD25 high CD4 + T regs and CD8 + T cells were isolated from mice SDLNs on an Influx cell sorter (30). The CD8 + T cells were labeled with 2.5 M CellTrace Violet (Thermo Fisher Scientific), cultured with the T regs at various ratios, and stimulated with platedbound anti-CD3 (1 g/ml) (145-2C11; Bio X Cell) and anti-CD28 (2 g/ml) (37.51; Bio X Cell) in a flat-bottom 96-well plate (Thermo Fisher Scientific) for 4 days. The effector T cells were gated on CD8, and the percent suppression was calculated according to previous reports (55).

Phosphorylation assays
Single-cell suspensions were incubated with recombinant murine IL-7 (20 ng/ml; PeproTech) at 37°C for 20 min. The cells were subsequently fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Fixed cells were permeabilized with 90% icecold methanol for 30 min on ice, washed once, and stained with cell surface markers (CD45, CD3, CD4, CD8, and CD25) and antiphospho-STAT5 (C71E5, Cell Signaling Technology) for 2 hours at room temperature. The cells were acquired with a BD LSR II flow cytometer (BD Biosciences), and analysis was carried out using FlowJo software (Treestar).

In vivo treatment
For IL-7 induction studies, C3H/HeJ mice (without AA) received a single intradermal injection of 400 ng recombinant murine IFN- (PeproTech) in the areas of the back skin, and PBS was used as a control. After 48 hours, skin biopsies were taken. C3H/HeJ mice (without AA) received an injection of 200-g anti-IFN-Ra (GR-20; Bio X Cell) or isotype control immunoglobulin G (IgG) (Bio X Cell) by intraperitoneal injection two times weekly for 4 weeks. For IL-7 stimulation in vivo, C3H/HeJ grafted mice were given IL-7c [0.5-g recombinant murine IL-7 mixed with 5-g anti-IL-7 mAb (M25; Bio X Cell), 37°C for 20 min], once a week for 2 weeks after 2 weeks after skin grafting. The preparation of IL-7/M25 complex was described as previously (20).
For AA prevention studies, treatment was started at the day of grafting (using the C3H/HeJ grafted model) or adoptive T cell transfer (using the retrogenic TCR C57BL/6 model). Anti-IL-7R mAb (A7R34; Bio X Cell), anti-TSLP (28F12; BioLegend), or isotype control IgG (Bio X Cell) was administrated by intraperitoneal injection (250 g) two times weekly for 6 to 8 weeks. In separate experiment recombinant human IL-2 (PeproTech) was administered at 25,000 U per dose ip daily for 5 days (39); or recombinant murine IL-33 (BioLegend) was administered at 2 g per dose ip daily for 3 days (39).
For AA reversal studies, anti-IL-7R mAb (A7R34; Bio X Cell) or isotype control IgG was administered into early-onset AA mice (5 to 7 weeks after skin grafting) by intraperitoneal injection (500 g) two times weekly for 8 weeks. For Ab combination treatment, earlyonset AA mice were treated anti-IL-7R mAb (500 g) two times weekly for two to 4 weeks, after which anti-PD-1 (RMP1-14; Bio X Cell) was administered at 200 g per dose intraperitoneally (ip) twice per week for 3 weeks or anti-FR4 (TH4; BioLegend) was administered at 200 g per dose ip twice per week for 2 weeks. For cytokine combination treatment, AA mice were treated anti-IL-7R mAb (500 g) two times for 1 week, after which recombinant human IL-2 (Pepro-Tech) was administered at 25,000 U per dose ip daily for 5 days; recombinant murine IL-33 (BioLegend) was administered at 2 g per dose ip daily for 3 days.

RNA sequencing and bioinformatic analysis
Total cellular RNA was extracted using the RNeasy Kit (Qiagen) from skin homogenates. RNA quality and quantity were determined using an Agilent BioAnalyzer (Agilent Technologies). mRNA was converted to complementary DNA (cDNA) using random primers and reverse transcriptase. cDNA library construction and RNA sequencing were performed at GENEWIZ. RNA sequencing reads and read quality were performed using STAR Aligner with the corresponding Ensembl 84 reference genomes and FastQC v0.11.2. To visualize the raw similarity or divergence of the gene expression profile of each sample, we used the normalized hit count from each sample to perform unsupervised analyses. The hit counts of each sample were normalized by the DESeq2 package in Bioconductor, which can estimate variance-mean dependence in count data from high-throughput sequencing assays and test for differential expression based on a model using the negative binomial distribution (56). Principal components analysis (PCA) was performed with the plotPCA function in DESeq2 to display the overall difference of the gene expression profile in each sample and how similar or dissimilar they are to each other in an unsupervised manner. To visualize the difference in expression in the ALADIN genes (6), the normalized hit counts of the ALADIN genes in each sample were plotted as a heatmap using the pheatmap package in Bioconductor. Similarly, the unsupervised hierarchical clustering of the samples was based on their similarity or difference in expression in ALADIN genes, naïve to their treatment.

Statistics
Statistical analyses were performed using the GraphPad Prism 7.0 software. Groups of data were compared using a two-tailed Student's t test. Log-rank tests were used to analyze the hair loss or regrowth curves. One-way analysis of variance (ANOVA) was used for mean differences comparison from multiple groups. Data in bar and dot graphs are means ± SD. Significance is indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.