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The Role of Dendritic Cell Subsets in the Maintenance of Effector and Regulatory T-cells in the Skin

Final Report Summary - DCMERT (The Role of Dendritic Cell Subsets in the Maintenance of Effector and Regulatory T-cells in the Skin)

Based on the data generated in this proposal, our manuscript is currently under the 2nd round of revision to be considered for publication in the journal Nature. Our manuscript is titled “Regulatory T cells in skin facilitate epithelial stem cell differentiation”. A draft of the manuscript, including the most prominent data are attached in the list of final publications of this report.

Elucidating the local regulation of stem cell function in tissues is an area of active investigation. One specific component of the cutaneous immune system, Regulatory T-cells (Tregs), localize to hair follicles. Hair follicles house the major stem cell compartment in skin and are critical for hair follicle function. Hair follicles in mammalian skin undergo bouts of regeneration, cycling between highly synchronized phases of quiescence and growth (anagen). Interestingly, polymorphisms in genes that control Treg function are strongly associated with the most common autoimmune disorder of man - Alopecia Areata (characterized by defects in hair follicle cycling). Further, augmenting Tregs is proving to be clinically efficacious in this disease. In the work conducted as part of this proposal, we functionally dissect the role of Tregs in hair follicle-associated stem cell biology and make the following novel observations:

1) The abundance, proliferative index and activation state of skin Tregs (as evidenced by flow cytometric expression of Ki67, CD25, ICOS, GITR, and CTLA-4) correlated with stage of the HF cycle.

2) To determine whether these cells play an active role in the process of HF cycling, we employed a well-characterized model of depilation-induced HF regeneration. In this model, mice with dorsal skin HFs in telogen are depilated to remove hair shafts (anagen induction), and thus begins the process of hair regeneration. To determine if Tregs play a role in this process, we utilized mice transgenic for the diphtheria toxin receptor under the control of the Foxp3 promoter (Foxp3DTR). These mice allow for robust depletion of Tregs following administration of Diphtheria toxin (DT). Ablation of Tregs markedly reduced hair regrowth when compared to wild type (WT) control mice treated with or without DT.

3) We next sought to elucidate the cellular mechanisms responsible for Treg mediated induction of hair regeneration. The HF bulge region is the best characterized niche for adult skin epithelial SC residence. Activation of bulge HFSCs is required anagen onset. Induction of HFSC proliferation was significantly reduced in mice depleted of Tregs early after depilation. These data suggest that Tregs directly stimulate HFSC function during hair follicle regeneration.

4) To elucidate potential molecular mechanisms by which Tregs directly influence HFSC function, we performed high throughput sequencing to identify genes and pathways of interest. We hypothesized that pathways involved in this process would be preferentially expressed in skin Tregs when compared to Tregs found in SDLNs. Thus, we performed whole transcriptome RNA sequencing on Tregs purified from skin and compared expression profiles to Tregs isolated from skin draining lymph nodes (SDLNs). Most notably, Jag1, a ligand of the Notch signaling pathway, was among the highest differentially expressed genes between skin and SDLN Tregs. To functionally determine if Notch signaling plays a role in Treg-mediated enhancement of HFSC activation, we attempted to rescue the HFSCs proliferative defect observed in Treg-depleted mice by the exogenous addition of Jag1. Microbeads coated with Jag1-Fc or control Fc were subcutaneously administered to DT-treated Foxp3DTR mice and activation of HFSCs was quantified by flow cytometry 4 days post-depilation. In these experiments, exogenous Jag1 was able to partially rescue HFSC activation.

5) To definitively test whether Jag1 expression on Tregs is required for anagen induction, mice expressing a Jag1 conditional allele (Jag1fl/fl) were crossed to Foxp3-cre mice to specifically ablate Jag1 expression in Tregs. Early after anagen induction, Foxp3Cre/CreJag1fl/fl mice had significantly attenuated proliferative capacity of bulge HFSCs when compared to age- and gender-matched littermate controls. To determine the hair regeneration capacity in the absence and presence of Jag1 on Tregs, Foxp3Cre/CreJag1fl/fl and control mice were depilated and dorsal skin hair regrowth was quantified. Consistent with the diminished proliferative capacity of HFSCs, hair regrowth in Foxp3Cre/CreJag1fl/fl mice was significantly decreased relative to controls revealing an essential function for Jag1 expression on Tregs for hair follicle activation. Collectively, these findings suggest that Tregs in skin utilize the Notch pathway to directly augment HFSC proliferation.

The non-immunologic functions of cutaneous Tregs, a major skin-resident immune cell population, have yet to be defined. To the best of our knowledge, this is the first report of Tregs influencing hair follicle biology. We identify a functional requirement of skin Tregs for the regeneration of HFs through enhancing bulge HFSC activation and differentiation. Treg-mediated induction of SC activation provides a novel pathway by which an immune cell population influences SC behavior in tissues, with a marked impact on regeneration, and ultimately tissue function. These results also suggest that Treg impairment may contribute to dysregulated HF cycling observed in patients with AA, reported to be the most common autoimmune disease of man. Our work establishes a foundation for the development of new therapeutic strategies targeting the Treg-Notch axis in this disease and potentially other tissue regenerative disorders.