Abstract Alopecia areata (AA) is an autoimmune disease with a 2% lifetime incidence. Commonly presenting as defined patches of hair loss on the scalp, AA is characterized by the presence of T cells surrounding the hair bulb and is thought to result as a consequence of the breakdown of immune privilege of the hair follicle. IL-27 is a cytokine with context-dependent pro- and anti-inflammatory properties that has been used as a potential therapy in models of autoimmune diseases and cancer. The objective of this study was to determine if IL-27 has therapeutic potential in AA. To address this, we used an adeno-associated virus that drives the overexpression of IL-27 (AAV-IL27) in our skin-graft induction model of AA. We found that mice that received AAV-IL27 were fully protected from disease development and had minimal CD8 T cell infiltration into the hair follicles. Further, we saw an increase in IL-10 producing CD4 T cells, supporting a potential mechanism by which IL-27 prevents disease development. Interestingly, we found that mice treated with AAV-IL27 generated a robust population of NKG2D expressing CD8 T cells, which has previously been associated with the presence of disease. These results suggest that IL-27 may act in an immunoregulatory manner in AA, by promoting immunosuppressive T cells in the SDLNs. Further studies are needed to further define the specific mechanisms by which IL-27 contributes to the prevention of AA and its utility as a treatment for AA.

Abstract Alopecia Areata (AA) is a prevalent autoimmune disease that presents as nonscarring hair loss. AA is associated with the loss of immune tolerance to the hair follicle and marked by a robust immune cell infiltration and production of pro-inflammatory cytokines. In AA, CD8 T cells accumulate around and within the hair follicle and have been identified as pathogenic effectors of disease. The close spatial relationship between cytotoxic CD8 T cells and the hair follicle end-organ target invites further study of the interactions between them. Our transcriptional analysis has revealed CD8 T cells infiltrating AA skin exhibit increased gene expression of perforin and granzymes, which are key mediators of cytolysis. However, the contributions of these cytolytic molecules in AA are currently unknown. Our objective was to investigate the role of perforin in the onset of AA by using C3H/HeJ mice globally deficient in Prf1 (PKO). Interestingly, PKO mice were capable of developing spontaneous AA. Using a skin-graft induction model, we observed that wild type (WT) and PKO recipient mice showed equivalent onset and progression of AA. However, when a CD8-induction model was used, we found CD8 T cells from PKO AA mice were inferior at inducing disease when compared to CD8 T cells from WT AA mice. Overall, our findings suggest that perforin-mediated cytolysis is not required for the autoimmune attack of the hair follicle, and that other effector mechanisms of CD8 T cells are drivers of AA.

Alopecia areata (AA) is an autoimmune disease with a 2% lifetime incidence. AA presents as hair loss and is characterized histologically by the presence of CD4 and CD8 T cells surrounding the hair bulb. Although CD4 T cells comprise a majority of the cellular infiltrate, little is known about the contribution of this population to disease pathogenesis. An emerging animal model of AA involves adoptive transfer of in vitro activated and expanded bulk skin draining lymph node (SDLN) cells from AA mice, resulting in the induction of disease in recipient mice. The objective of this study was to investigate the mechanisms by which those cells transfer disease. Expanded bulk SDLN cells overwhelmingly consisted of CD3+ T cells, including both CD8 and CD4 T cells. To address whether these T cell populations could separately induce AA, we first sorted CD8 T cells from the SDLNs of AA mice and found these cells potently induced disease in recipient mice. Surprisingly, isolated CD4 T cells from AA mice also had a robust capacity to induce disease, and they did so more efficiently than CD4 T cells from unaffected (UA) mice. High dimensional flow cytometric analysis of the SDLNs indicated that an enhanced number of CD4 T cells in AA mice exhibited an effector-like phenotype. Development of AA following transfer of expanded CD4 T cells relied on the presence of endogenous CD8 T cells and host responsiveness to IFN-γ, but, in contrast, loss of IL-17 signaling appeared to have no effect. Together, these data suggest that activated CD4 T-helper type 1 effector cells contribute to the activation of CD8 T cells and the subsequent attack of the hair follicle. Further studies are needed to further define the specific mechanisms by which CD4 T cells contribute to the development of AA. Dept of VA (I01BX004907), NIH/NIAMS (R01AR077194, K08AR069111, T32AI007485)

Alopecia Areata (AA) is a prevalent autoimmune disease that results in nonscarring hair loss. Disease is associated with the collapse of an immune privileged state of the hair follicle, characterized by increased MHC expression, immune cell infiltration and the presence of pro-inflammatory cytokines in the microenvironment. CD8 T cells have been identified as the main contributors to disease and the activating receptor NKG2D can identify this pathogenic population. There is currently one FDA-approved drug for AA, a JAK1/2 inhibitor, which is thought to act by dampening inflammatory mediators during disease. However, the potential of adverse effects and refractory patients necessitates the development of alternative therapeutics. Emerging data from our lab suggests that the observed expansion of CD4 T regulatory cells (Tregs), relative to effector CD8 T cells during AA, is inadequate to prevent disease onset. We hypothesized that pharmacologic enhancement of Treg numbers may restrain development of AA. To address this, we investigated the protective potential of Treg expansion using a murine model of AA. In vivo expansion of CD4 +CD25 +FoxP3 +Tregs was achieved using an IL-2 cytokine/antibody complex (IL-2C), which is known to selectively expand and enhance Tregs. We found that 6 weeks of treatment with IL-2C halted the progression of hair loss, while control mice exhibited robust hair loss. Additionally, IL-2C treatment led to a reduction in NKG2D +CD8 T cells and IFNγ-producing CD8 T cells found infiltrating the skin, suggesting that increasing Tregs in the system is an effective strategy for restraining disease. Our findings provide translational insight for the use of Treg enrichment as a therapeutic strategy for patients with AA. Supported by grants from NIH (R01 AR077194) and Dept. of VA (I01 BX004907)

Alopecia Areata (AA) is a common autoimmune disease characterized by non-scarring hair loss ranging from patches on the scalp to complete hair loss involving the entire body. Disease onset is hypothesized to follow the collapse of immune privilege of the hair follicle, which results in an increase in self-peptide/MHC expression along the follicular epithelium. Hair loss is associated with infiltration of the hair follicle with putatively self-reactive T cells. This process is thought to skew the hair follicle microenvironment away from a typically homeostatic immune state towards one of active inflammation. This imbalance is mediated in part by the dominating presence of specific cytokines. While interferon-γ (IFNγ) has been identified as the key player in AA pathogenesis, many other cytokines have also been shown to play pivotal roles. Mechanistic studies in animal models have highlighted the contribution of common gamma chain (γc) cytokines such as IL-2, IL-7, and IL-15 in augmenting disease. IFNγ and γc cytokines signal through pathways involving receptor activation of Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). Based on these findings, JAK/STAT pathways have been targeted for the purposes of therapeutic intervention in the clinical setting. Case reports and series have described use of small molecule JAK inhibitors leading to hair regrowth among AA patients. Furthermore, emerging clinical trial results show great promise and position JAK inhibitors as a treatment strategy for patients with severe or recalcitrant disease. Demonstrated efficacy from large-scale clinical trials of the JAK inhibitor baricitinib led to the first-in-disease FDA-approved treatment for AA in June of 2022. This review aims to highlight the JAK/STAT signaling pathways of various cytokines involved in AA and how targeting those pathways may impact disease outcomes in both laboratory and clinical settings.

Alopecia Areata (AA) is a common autoimmune disease characterized by the loss of immune privilege of the hair follicle. CD8+ T cells have been identified as the main effectors in AA, however, little is known about the role of regulatory T cells (Tregs) and why they fail to control disease. The imbalance of Tregs: effectors is thought to influence the development of many autoimmune diseases. Enrichment of the regulatory compartment has been gaining momentum in the field and currently there are many ongoing clinical trials involving Treg therapy as a treatment for autoimmune diseases. We were interested in exploring the effect of enhancing Treg numbers on the onset and development of AA in a murine C3H skin graft-induction model. In vivo treatment with recombinant IL-2 complexed to anti-IL-2 monoclonal antibody (IL-2C) resulted in selective expansion of CD4+ CD25+ FoxP3+ Tregs and enhanced expression of functional Treg markers compared to IgG control. Both continuous treatment and transient treatment with IL-2C resulted in the complete prevention of AA. Continuous IL-2C treatment resulted in an increased frequency of Tregs in the skin (2,652 vs. 594 Tregs/g skin), suggesting their protective role in hair follicle tolerance. Treg levels remained elevated in skin at 3 months after stopping treatment, indicating how IL-2C treatment may deliver long-lasting tolerogenic benefits. Ongoing studies may elucidate the potential for IL-2C to inhibit hair loss during active disease. Findings from these studies may provide translational insight into the use of in vivo Treg enrichment as a clinical therapeutic option for AA patients.

Alopecia Areata (AA) is a common autoimmune disease characterized by T cell infiltration of the hair follicle, resulting in nonscarring hair loss. Although CD4 T cells comprise the majority of the infiltrate, little is known about their contribution to disease pathogenesis. Previously we have identified an increased number of IFN-γ producing CD4+ T cells in the skin-draining lymph nodes (SDLN) as compared to unaffected (UA) controls. The objective for this study was to determine the role that CD4 T cells play in the pathogenesis of AA. Utilizing spectral flow cytometry, we found CD4 T cells exhibiting an effector phenotype were more numerous in the SDLN of AA mice as compared to that from UA mice. To address the role CD4 T cells play in AA, we adapted a model of AA whereby in vitro expanded bulk lymph node (LN) cells from previously affected AA mice were adoptively transferred into previously unaffected mice. When CD4 T cells were specifically sorted from SDLNs of AA mice using this protocol, these cells robustly transferred AA, contrasting with minimal induction by CD4 T cells from UA mouse LNs or spleens from AA mice. Additionally, this induction was found to be dose-dependent, with increased numbers of CD4 T cells inducing disease in recipient mice at a higher rate. In addition, we found that the endogenous CD8 T cell population is critically required for the transfer of CD4 T cell-induced disease, and that CD4 T cells-mediated disease relies on IFN-γ signaling for the emergence of AA. Our data suggest that activated CD4 T-helper type 1 effector cells contribute to the activation of CD8 T cells, enabling the attack of the hair follicle. Further studies are needed to elucidate the specific mechanisms by which CD4 T cells and IFN-γ contribute to the development of AA.

Alopecia areata (AA) is an autoimmune disease characterized by T cell infiltration of the hair follicle. The breakdown of tolerance to the hair follicle is thought to be the driving force in the nonscarring hair loss seen in patients. There are currently no FDA approved treatments for AA. IL-27 plays a pleotropic role across many other autoimmune diseases; however, whether its immunoregulatory effects can be leveraged for therapeutic applications in AA is unknown. To address this, we used an adeno-associated virus (AAV) that drives overexpression of IL-27 in our C3H/HeJ skin graft-induction model of AA. Briefly, AAV-IL-27 administration was followed by disease induction using skin grafts from an AA-affected donor. Mice were observed weekly for disease progression. We found that exogenous IL-27 protected mice from disease development. Additionally, we saw an increased presence of FoxP3+ CD4 T cells and IL-10-producing CD8 T cells in the skin draining lymph nodes of mice treated with AAV-IL-27, suggesting roles for these immunoregulatory populations in the prevention of autoimmunity. Interestingly, NKG2D-expressing CD8 T cells, previously associated to correlate with the presence of disease, were present in mice treated with AAV-IL-27, raising the possibility that IL-27 may be useful in late stages of disease. Our findings support further studies examining use of IL-27 in the treatment of AA patients.

GPCRs are highly desirable drug targets for human disease. Although GPCR dysfunction drives development and progression of many tumors, including breast cancer (BC), targeting individual GPCRs has limited efficacy as a cancer therapy because numerous GPCRs are activated. Here, we sought a new way of blocking GPCR activation in HER2+ BC by targeting a subgroup of GPCRs that couple to Gi/o proteins (Gi/o-GPCRs). In mammary epithelial cells of transgenic mouse models, and BC cell lines, HER2 hyperactivation altered GPCR expression, particularly, Gi/o-GPCR expression. Gi/o-GPCR stimulation transactivated EGFR and HER2 and activated the PI3K/AKT and Src pathways. If we uncoupled Gi/o-GPCRs from their cognate Gi/o proteins by pertussis toxin (PTx), then BC cell proliferation and migration was inhibited in vitro and HER2-driven tumor formation and metastasis were suppressed in vivo. Moreover, targeting Gi/o-GPCR signaling via PTx, PI3K, or Src inhibitors enhanced HER2-targeted therapy. These results indicate that, in BC cells, HER2 hyperactivation drives aberrant Gi/o-GPCR signaling and Gi/o-GPCR signals converge on the PI3K/AKT and Src signaling pathways to promote cancer progression and resistance to HER2-targeted therapy. Our findings point to a way to pharmacologically deactivate GPCR signaling to block tumor growth and enhance therapeutic efficacy.

Cancer metastasis is the major cause of tumor mortality and has been attributed in part to the presence of a minority subpopulation of cancer stem cells (CSCs) in the bulk of tumor cells. We showed previously that WDR26, a scaffolding/adaptor protein that is highly upregulated in breast cancer, promotes breast cancer growth and metastasis. Here we show WDR26 was required for maintaining the CSC populations in breast cancer cells and the formation of lung metastases. Downregulation of WDR26 in breast cancer cells impaired the CSC-like activities and reduced the CSC population. Mammary gland-specific deletion of WDR26 in the MMTV-PyMT mouse model of breast cancer had a little effect on primary tumor formation but largely abolished spontaneous lung metastasis. WDR26 promoted β-catenin activation via AKT and GSK3, and the activity of AKT, GSK3 and β-catenin was required for maintaining the CSC population in breast cancer cells. Our results have identified a novel, WDR26-dependent pathway that links breast CSC activities to tumor metastatic potential. Citation Format: Dharmendra K. Bhargava, Wei Wang, Maddison Lensing, Songhai Chen. WDR26 regulates an AKT-Gsk3-Wnt/b-catenin signaling cascade to maintain the breast cancer stem cell population and controls cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4672.