Regulatory T cells in skin mediate immune privilege of the hair follicle stem cell niche.

Immune tolerance is maintained in lymphoid organs (LOs). Despite the presence of complex immune cell networks in non-LOs, it is unknown whether self-tolerance is maintained in these tissues. We developed a technique to restrict genetic recombination to regulatory T cells (Tregs) only in skin. Selective depletion of skin Tregs resulted in T cell-mediated inflammation of hair follicles (HFs). Suppression did not rely on CTLA-4, but instead on high-affinity interleukin-2 (IL-2) receptor expression by skin Tregs, functioning exclusively in a cell-extrinsic manner. In a novel model of HF stem cell (HFSC)-driven autoimmunity, we reveal that skin Tregs immunologically protect the HFSC niche. Finally, we used spatial transcriptomics to identify aberrant IL-2 signaling at stromal-HF interfaces in a rare form of human alopecia characterized by HFSC destruction and alopecia areata. Collectively, these results reveal the fundamental biology of Tregs in skin uncoupled from the systemic pool and elucidate a mechanism of self-tolerance.

Early-life inflammation primes a T helper 2 cell-fibroblast niche in skin.

Inflammation early in life can prime the local immune milieu of peripheral tissues, which can cause lasting changes in immunological tone that confer disease protection or susceptibility1. The cellular and molecular mechanisms that prompt changes in immune tone in many nonlymphoid tissues remain largely unknown. Here we find that time-limited neonatal inflammation induced by a transient reduction in neonatal regulatory T cells causes a dysregulation of subcutaneous tissue in mouse skin. This is accompanied by the selective accumulation of type 2 helper T (TH2) cells within a distinct microanatomical niche. TH2 cells are maintained into adulthood through interactions with a fibroblast population in skin fascia that we refer to as TH2-interacting fascial fibroblasts (TIFFs), which expand in response to TH2 cytokines to form subcutaneous fibrous bands. Activation of the TH2-TIFF niche due to neonatal inflammation primes the skin for altered reparative responses to wounding. Furthermore, we identify fibroblasts in healthy human skin that express the TIFF transcriptional signature and detect these cells at high levels in eosinophilic fasciitis, an orphan disease characterized by inflammation and fibrosis of the skin fascia. Taken together, these data define a previously unidentified TH2 cell niche in skin and functionally characterize a disease-associated fibroblast population. The results also suggest a mechanism of immunological priming whereby inflammation early in life creates networks between adaptive immune cells and stromal cells to establish an immunological set-point in tissues that is maintained throughout life.

Layilin Anchors Regulatory T Cells in Skin.

Regulatory T cells (Tregs) reside in nonlymphoid tissues where they carry out unique functions. The molecular mechanisms responsible for Treg accumulation and maintenance in these tissues are relatively unknown. Using an unbiased discovery approach, we identified LAYN (layilin), a C-type lectin-like receptor, to be preferentially and highly expressed on a subset of activated Tregs in healthy and diseased human skin. Expression of layilin on Tregs was induced by TCR-mediated activation in the presence of IL-2 or TGF-ß. Mice with a conditional deletion of layilin in Tregs had reduced accumulation of these cells in tumors. However, these animals somewhat paradoxically had enhanced immune regulation in the tumor microenvironment, resulting in increased tumor growth. Mechanistically, layilin expression on Tregs had a minimal effect on their activation and suppressive capacity in vitro. However, expression of this molecule resulted in a cumulative anchoring effect on Treg dynamic motility in vivo. Taken together, our results suggest a model whereby layilin facilitates Treg adhesion in skin and, in doing so, limits their suppressive capacity. These findings uncover a unique mechanism whereby reduced Treg motility acts to limit immune regulation in nonlymphoid organs and may help guide strategies to exploit this phenomenon for therapeutic benefit.

Regulatory T cells promote innate inflammation after skin barrier breach via TGF-ß activation.

Regulatory T cells (Tregs) use multiple mechanisms to attenuate inflammation and prevent autoimmunity. Tregs residing in peripheral (i.e., nonlymphoid) tissues have specialized functions; specifically, skin Tregs promote wound healing, suppress dermal fibrosis, facilitate epidermal regeneration, and augment hair follicle cycling. Here, we demonstrated that skin Tregs were transcriptionally attuned to interact with their tissue environment through increased expression of integrin and TGF-ß pathway genes that influence epithelial cell biology. We identified a molecular pathway where skin Tregs license keratinocytes to promote innate inflammation after skin barrier breach. Using a single-cell discovery approach, we identified preferential expression of the integrin αvß8 on skin Tregs Upon skin injury, Tregs used this integrin to activate latent TGF-ß, which acted directly on epithelial cells to promote CXCL5 production and neutrophil recruitment. Induction of this circuit delayed epidermal regeneration but provided protection from Staphylococcus aureus infection across a compromised barrier. Thus, αvß8-expressing Tregs in the skin, somewhat paradoxical to their canonical immunosuppressive functions, facilitated inflammation acutely after loss of barrier integrity to promote host defense against infection.

TregGF-ß: Deletion of TGF-ß in Tregs revisited.

Treg-specific ablation of TGF-ß clarifies the complicated role of Treg-derived TGF-ß in immunosuppression in vivo.

Evaluation of SARS-CoV-2 serology assays reveals a range of test performance.

Appropriate use and interpretation of serological tests for assessments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, infection and potential immunity require accurate data on assay performance. We conducted a head-to-head evaluation of ten point-of-care-style lateral flow assays (LFAs) and two laboratory-based enzyme-linked immunosorbent assays to detect anti-SARS-CoV-2 IgM and IgG antibodies in 5-d time intervals from symptom onset and studied the specificity of each assay in pre-coronavirus disease 2019 specimens. The percent of seropositive individuals increased with time, peaking in the latest time interval tested (>20 d after symptom onset). Test specificity ranged from 84.3% to 100.0% and was predominantly affected by variability in IgM results. LFA specificity could be increased by considering weak bands as negative, but this decreased detection of antibodies (sensitivity) in a subset of SARS-CoV-2 real-time PCR-positive cases. Our results underline the importance of seropositivity threshold determination and reader training for reliable LFA deployment. Although there was no standout serological assay, four tests achieved more than 80% positivity at later time points tested and more than 95% specificity.

Test performance evaluation of SARS-CoV-2 serological assays.

BACKGROUND: Serological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data. METHOD: We conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 128 plasma or serum samples from 79 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system. RESULTS: Among specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8-100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.7-94.8%. No consistent cross-reactivity was observed. CONCLUSION: Our evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies.

Regulatory T cells in skin injury: At the crossroads of tolerance and tissue repair.

Skin injury is a highly inflammatory process that is carefully regulated to mitigate tissue damage and allow for proper barrier repair. Regulatory T cells (Tregs) are crucial coordinators of the immune response to injury in several organs. Here, we review the emerging role of Tregs in facilitating skin repair after injury. We focus on recently discovered interactions between lymphocytes and nonhematopoietic cells during wound healing and discuss how these interactions are regulated both by "classical" suppressive mechanisms of Tregs and by "nonclassical" reparative Treg functions.

Regulatory T cells use arginase 2 to enhance their metabolic fitness in tissues.

Distinct subsets of Tregs reside in nonlymphoid tissues where they mediate unique functions. To interrogate the biology of tissue Tregs in human health and disease, we phenotypically and functionally compared healthy skin Tregs with those in peripheral blood, inflamed psoriatic skin, and metastatic melanoma. The mitochondrial enzyme, arginase 2 (ARG2), was preferentially expressed in Tregs in healthy skin, increased in Tregs in metastatic melanoma, and reduced in Tregs from psoriatic skin. ARG2 enhanced Treg suppressive capacity in vitro and conferred a selective advantage for accumulation in inflamed tissues in vivo. CRISPR-mediated deletion of this gene in primary human Tregs was sufficient to skew away from a tissue Treg transcriptional signature. Notably, the inhibition of ARG2 increased mTOR signaling, whereas the overexpression of this enzyme suppressed it. Taken together, our results suggest that Tregs express ARG2 in human tissues to both regulate inflammation and enhance their metabolic fitness.

Treg-Cell Control of a CXCL5-IL-17 Inflammatory Axis Promotes Hair-Follicle-Stem-Cell Differentiation During Skin-Barrier Repair.

Restoration of barrier-tissue integrity after injury is dependent on the function of immune cells and stem cells (SCs) residing in the tissue. In response to skin injury, hair-follicle stem cells (HFSCs), normally poised for hair generation, are recruited to the site of injury and differentiate into cells that repair damaged epithelium. We used a SC fate-mapping approach to examine the contribution of regulatory T (Treg) cells to epidermal-barrier repair after injury. Depletion of Treg cells impaired skin-barrier regeneration and was associated with a Th17 inflammatory response and failed HFSC differentiation. In this setting, damaged epithelial cells preferentially expressed the neutrophil chemoattractant CXCL5, and blockade of CXCL5 or neutrophil depletion restored barrier function and SC differentiation after epidermal injury. Thus, Treg-cell regulation of localized inflammation enables HFSC differentiation and, thereby, skin-barrier regeneration, with implications for the maintenance and repair of other barrier tissues.