Central memory T cells are the most effective precursors of resident memory T cells in human skin.

The circulating precursor cells that give rise to human resident memory T cells (TRM) are poorly characterized. We used an in vitro differentiation system and human skin-grafted mice to study TRM generation from circulating human memory T cell subsets. In vitro TRM differentiation was associated with functional changes, including enhanced IL-17A production and FOXP3 expression in CD4+ T cells and granzyme B production in CD8+ T cells, changes that mirrored the phenotype of T cells in healthy human skin. Effector memory T cells (TEM) had the highest conversion rate to TRM in vitro and in vivo, but central memory T cells (TCM) persisted longer in the circulation, entered the skin in larger numbers, and generated increased numbers of TRM. In summary, TCM are highly efficient precursors of human skin TRM, a feature that may underlie their known association with effective long-term immunity.

Presence of Skin Tissue-Resident Memory T Cells in Human Nonmalignant and Premalignant Melanocytic Skin Lesions and in Melanoma.

ABSTRACT: The infiltration of tissue-resident memory (TRM) cells in melanoma correlates with improved survival, suggesting an important role for TRM cells in immunity against melanoma. However, little is known about the presence of TRM cells in nonmalignant and premalignant melanocytic lesions. This study aimed to evaluate the presence of TRM cells in human skin melanocytic lesions, representing the spectrum from healthy skin to metastatic melanoma. FFPE sections from healthy skin, sun-exposed skin, benign nevi, lentigo maligna (LM), primary LM melanoma, and primary cutaneous and metastatic melanoma were analyzed by immunohistochemistry. The number of infiltrating cells expressing TRM-associated markers, CD3, CD4, CD8, CD69, CD103, and CD49a, was quantified by digital analyses. Multiplex immunofluorescence was performed to analyze coexpression of TRM cell markers. More T cells and CD69+ cells were found in melanoma lesions, as compared with healthy skin and nevi. CD103+ and CD49a+ cell numbers did not significantly differ. More importantly, no differences were seen in expression of all markers between healthy skin and benign nevi. Similar results, except for CD69, were observed in LM melanoma, as compared with LM and sun-exposed skin. Interestingly, multiplex immunofluorescence showed that nevi tissues have comparable CD103+ T cell numbers with healthy skin but comprise more CD103+ CD8+ cells. Expression of TRM cell markers is significantly increased in melanoma, as compared with nonmalignant skin. Our data also show that TRM cells are not abundantly present already in premalignant tissues. Further studies on the specificity of TRM cells for melanocyte/melanoma antigens may reveal their significance in cancer immunosurveillance.

Labeling and tracking of immune cells in ex vivo human skin.

Human skin harbors various immune cells that are crucial for the control of injury and infection. However, the current understanding of immune cell function within viable human skin tissue is limited. We developed an ex vivo imaging approach in which fresh skin biopsies are mounted and then labeled with nanobodies or antibodies against cell surface markers on tissue-resident memory CD8+ T cells, other immune cells of interest, or extracellular tissue components. Subsequent longitudinal imaging allows one to describe the dynamic behavior of human skin-resident cells in situ. In addition, this strategy can be used to study immune cell function in murine skin. The ability to follow the spatiotemporal behavior of CD8+ T cells and other immune cells in skin, including their response to immune stimuli, provides a platform to investigate physiological immune cell behavior and immune cell behavior in skin diseases. The mounting, staining and imaging of skin samples requires ~1.5 d, and subsequent tracking analysis requires a minimum of 1 d. The optional production of fluorescently labeled nanobodies takes ~5 d.

Peripheral host T cells survive hematopoietic stem cell transplantation and promote graft-versus-host disease.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in hematopoietic stem cell transplantation (HSCT). Donor T cells are key mediators in pathogenesis, but a contribution from host T cells has not been explored, as conditioning regimens are believed to deplete host T cells. To evaluate a potential role for host T cells in GVHD, the origin of skin and blood T cells was assessed prospectively in patients after HSCT in the absence of GVHD. While blood contained primarily donor-derived T cells, most T cells in the skin were host derived. We next examined patient skin, colon, and blood during acute GVHD. Host T cells were present in all skin and colon acute GVHD specimens studied, yet were largely absent in blood. We observed acute skin GVHD in the presence of 100% host T cells. Analysis demonstrated that a subset of host T cells in peripheral tissues were proliferating (Ki67+) and producing the proinflammatory cytokines IFN-γ and IL-17 in situ. Comparatively, the majority of antigen-presenting cells (APCs) in tissue in acute GVHD were donor derived, and donor-derived APCs were observed directly adjacent to host T cells. A humanized mouse model demonstrated that host skin-resident T cells could be activated by donor monocytes to generate a GVHD-like dermatitis. Thus, host tissue-resident T cells may play a previously unappreciated pathogenic role in acute GVHD.

Author Correction: Tissue patrol by resident memory CD8+ T cells in human skin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Maturation and Phenotypic Heterogeneity of Human CD4+ Regulatory T Cells From Birth to Adulthood and After Allogeneic Stem Cell Transplantation.

CD4+ Regulatory T cells (Treg) play a critical role in maintaining immune homeostasis. Various Treg subsets have been identified, however the heterogeneity of Treg subpopulations during development remains uncharacterized. Using mass cytometry we obtained single cell data on expression of 35 functional markers to examine the heterogeneity of Treg cells at birth and in adults. Unsupervised clustering algorithms FlowSOM and ACCENSE were used to quantify Treg heterogeneity. As expected, Treg in umbilical cord blood were predominately naïve while Treg in adult blood were predominately central memory and effector memory cells. Although umbilical cord blood Treg are mostly naïve cells, we observed multiple phenotypic Treg subsets in cord blood. Nevertheless, peripheral blood in adults contained higher percentages of Treg and the heterogeneity of Treg was significantly increased in adults. We also studied Treg heterogeneity throughout a 2-year period after allogeneic hematopoietic stem cell transplantation (alloHSCT) and in patients with chronic graft-versus-host disease (cGVHD). Treg heterogeneity recovered rapidly after alloHSCT and gradually increased in the first two years post-transplant. However, patients with cGVHD had significantly fewer distinct Treg subpopulations, proposing a correlation between a disrupted Treg heterogeneity and cGVHD. Our study is the first to compare human Treg heterogeneity at birth, in healthy adults and in patients after alloHSCT with and without cGVHD. This approach to characterize Treg heterogeneity based on expression of a large panel of functional markers may enable future studies to identify specific Treg defects that contribute to immune dysfunction.

Skin-resident memory T cells as a potential new therapeutic target in vitiligo and melanoma.

Tissue-resident memory T (TRM ) cells are abundant in the memory T cell pool and remain resident in peripheral tissues, such as the skin, where they act as alarm sensors or cytotoxic killers. TRM cells persist long after the pathogen is eliminated and can respond rapidly upon reinfection with the same antigen. When aberrantly activated, skin-located TRM cells have a profound role in various skin disorders, including vitiligo and melanoma. Autoreactive TRM cells are present in human lesional vitiligo skin and mouse models of vitiligo, which suggests that targeting these cells could be effective as a durable treatment strategy for vitiligo. Furthermore, emerging evidence indicates that induction of melanoma-reactive TRM cells is needed to achieve effective protection against tumor growth. This review highlights seminal reports about skin-resident T cells, focusing mainly on their role in the context of vitiligo and melanoma, as well as their potential as therapeutic targets in both diseases.

Tissue patrol by resident memory CD8+ T cells in human skin.

Emerging data show that tissue-resident memory T (TRM) cells play an important protective role at murine and human barrier sites. TRM cells in the epidermis of mouse skin patrol their surroundings and rapidly respond when antigens are encountered. However, whether a similar migratory behavior is performed by human TRM cells is unclear, as technology to longitudinally follow them in situ has been lacking. To address this issue, we developed an ex vivo culture system to label and track T cells in fresh skin samples. We validated this system by comparing in vivo and ex vivo properties of murine TRM cells. Using nanobody labeling, we subsequently demonstrated in human ex vivo skin that CD8+ TRM cells migrated through the papillary dermis and the epidermis, below sessile Langerhans cells. Collectively, this work allows the dynamic study of resident immune cells in human skin and provides evidence of tissue patrol by human CD8+ TRM cells.

Phase I/II Trial of a Combination of Anti-CD3/CD7 Immunotoxins for Steroid-Refractory Acute Graft-versus-Host Disease.

Effective therapies for treating patients with steroid-refractory acute graft-versus-host-disease (SR-aGVHD), particularly strategies that reduce the duration of immunosuppression following remission, are urgently needed. The investigated immunotoxin combination consists of a mixture of anti-CD3 and anti-CD7 antibodies separately conjugated to recombinant ricin A (CD3/CD7-IT), which induces in vivo depletion of T cells and natural killer (NK) cells and suppresses T cell receptor activation. We conducted a phase I/II trial to examine the safety and efficacy of CD3/CD7-IT in 20 patients with SR-aGVHD; 17 of these patients (85%) had severe SR-aGVHD, and all 20 patients had visceral organ involvement, including 18 (90%) with gastrointestinal (GI) involvement and 5 (25%) with liver involvement. A validated 2-biomarker algorithm classified the majority of patients (11 of 20) as high risk. On day 28 after the start of CD3/CD7-IT therapy, the overall response rate was 60% (12 of 20), with 10 patients (50%) achieving a complete response. The 6-month overall survival rate was 60% (12 of 20), including 64% (7 of 11) classified as high risk by biomarkers. The 1-week course of treatment with CD3/CD7-IT caused profound but transient depletion of T cells and NK cells, followed by rapid recovery of the immune system with a diverse TCR Vß repertoire, and preservation of Epstein-Barr virus- and cytomegalovirus-specific T cell clones. Furthermore, our results indicate that CD3/CD7-IT appeared to be safe and well tolerated, with a relatively low prevalence of manageable and reversible adverse events, primarily worsening of hypoalbuminemia, microangiopathy, and thrombocytopenia. These encouraging results suggest that CD3/CD7-IT may improve patient outcomes in patients with SR-aGVHD.

A primary role for human central memory cells in tissue immunosurveillance.

Central memory T cells (TCM) patrol lymph nodes, providing central immunosurveillance against known pathogens, but have not been described as conducting primary tissue immunosurveillance. We analyzed the expression of tissue-homing addressins in human TCM vs effector memory T cells (TEM) from the same donors. In humans, the majority of human TCM were tropic for either skin or gut, and the overall tissue tropism of TCM was comparable to that of TEM TCM were present in healthy, noninflamed human skin, lung, colon, and cervix, suggesting a role for TCM in the primary immunosurveillance of peripheral tissues. TCM also had potent effector functions; 80% of CD8+ TCM produced TC1/TC2/TC17/TC22 cytokines. TCM injected into human skin-grafted mice migrated into skin and induced inflammatory eruptions comparable to TEM-injected mice. In summary, human TCM express peripheral tissue-homing receptors at levels similar to their effector memory counterparts, are found in healthy human tissues, have impressive effector functions, and can act alone to induce skin inflammation in human engrafted mice. Our studies support a novel role for human TCM in primary immunosurveillance of peripheral tissues and highlight the important role of this long-lived cell type in tissue-based immune responses.

Clinically resolved psoriatic lesions contain psoriasis-specific IL-17-producing αß T cell clones.

In psoriasis, an IL-17-mediated inflammatory skin disease, skin lesions resolve with therapy, but often recur in the same locations when therapy is discontinued. We propose that residual T cell populations in resolved psoriatic lesions represent the pathogenic T cells of origin in this disease. Utilizing high-throughput screening (HTS) of the T cell receptor (TCR) and immunostaining, we found that clinically resolved psoriatic lesions contained oligoclonal populations of T cells that produced IL-17A in both resolved and active psoriatic lesions. Putative pathogenic clones preferentially utilized particular Vß and Vα subfamilies. We identified 15 TCRß and 4 TCRα antigen receptor sequences shared between psoriasis patients and not observed in healthy controls or other inflammatory skin conditions. To address the relative roles of αß versus γδ T cells in psoriasis, we carried out TCR/δ HTS. These studies demonstrated that the majority of T cells in psoriasis and healthy skin are αß T cells. γδ T cells made up 1% of T cells in active psoriasis, less than 1% in resolved psoriatic lesions, and less than 2% in healthy skin. All of the 70 most frequent putative pathogenic T cell clones were αß T cells. In summary, IL-17-producing αß T cell clones with psoriasis-specific antigen receptors exist in clinically resolved psoriatic skin lesions. These cells likely represent the disease-initiating pathogenic T cells in psoriasis, suggesting that lasting control of this disease will require suppression of these resident T cell populations.

Recurrence Rates Over 20 Years in the Treatment of Malignant Melanoma: Immediate Versus Delayed Reconstruction.

Wide local excision (WLE) with a safety margin is the standard of treatment for primary head and neck cutaneous malignant melanoma (HNCMM). Studies have demonstrated inconsistency in recurrence rates following immediate versus delayed reconstruction. The objectives of this study were to assess and compare recurrence rates after WLE of HNCMM followed by immediate or delayed reconstruction in determining recurrence-free survival estimates. METHODS: A consecutive, retrospective analysis of 451 patients undergoing WLE of primary HNCMM followed by reconstruction over a period of 20 years was performed. Patients were divided into 2 groups based on timing of reconstruction (immediate versus delayed). Univariate analyses were performed to assess distributions. Kaplan-Meier survival analysis and multivariate Cox proportional hazard analyses were performed to estimate recurrence-free survival. RESULTS: Tumor specimen positive margins were comparable between immediate and delayed reconstruction groups (P = 0.129). Univariate analysis demonstrated comparable local melanoma recurrence after immediate or delayed reconstruction (41.4% versus 53.3%; P = 0.399). After adjusting for prognostic factors, multivariate analysis also failed to demonstrate an association between reconstruction timing and local recurrence-free survival (P = 0.167). CONCLUSIONS: In this long-term study, we were not able to demonstrate an association between reconstruction timing and local recurrence-free survival after excision WLE of HNCMM, rendering immediate reconstruction a reliable approach. In addition, the presence of ulceration and a positive sentinel lymph node were positively associated with the risk of recurrence.

Research Techniques Made Simple: High-Throughput Sequencing of the T-Cell Receptor.

High-throughput sequencing (HTS) of the T-cell receptor (TCR) is a rapidly advancing technique that allows sensitive and accurate identification and quantification of every distinct T-cell clone present within any biological sample. The relative frequency of each individual clone within the full T-cell repertoire can also be studied. HTS is essential to expand our knowledge on the diversity of the TCR repertoire in homeostasis or under pathologic conditions, as well as to understand the kinetics of antigen-specific T-cell responses that lead to protective immunity (i.e., vaccination) or immune-related disorders (i.e., autoimmunity and cancer). HTS can be tailored for personalized medicine, having the potential to monitor individual responses to therapeutic interventions and show prognostic and diagnostic biomarkers. In this article, we briefly review the methodology, advances, and limitations of HTS of the TCR and describe emerging applications of this technique in the field of investigative dermatology. We highlight studying the pathogenesis of T cells in allergic dermatitis and the application of HTS of the TCR in diagnosing, detecting recurrence early, and monitoring responses to therapy in cutaneous T-cell lymphoma.

Research Techniques Made Simple: Mass Cytometry Analysis Tools for Decrypting the Complexity of Biological Systems.

Mass cytometry by time-of-flight experiments allow analysis of over 40 functional and phenotypic cellular markers simultaneously at the single-cell level. The data dimensionality escalation accentuates limitations, inherent to manual analysis, as being subjective, labor-intensive, slow, and often incapable of showing the detailed features of each unique cell within populations. The subsequent challenge of examining, visualizing, and presenting mass cytometry data has motivated continuous development of dimensionality reduction methods. As a result, an increasing recognition of the inherent diversity and complexity of cellular networks is emerging, with the discovery of unexpected cell subpopulations, hierarchies, and developmental pathways, such as those existing within the immune system. Here, we briefly review some frequently used and accessible mass cytometry data analysis tools, including principal component analysis (PCA); spanning-tree progression analysis of density-normalized events (SPADE); t-distributed stochastic neighbor embedding (t-SNE)-based visualization (viSNE); automatic classification of cellular expression by nonlinear stochastic embedding (ACCENSE); and cluster identification, characterization, and regression (CITRUS). Mass cytometry, used together with these innovative analytic tools, has the power to lead to key discoveries in investigative dermatology, including but not limited to identifying signaling phenotypes with predictive value for early diagnosis, prognosis, or relapse and a thorough characterization of intratumor heterogeneity and disease-resistant cell populations, that may ultimately unveil novel therapeutic approaches.

Research Techniques Made Simple: Experimental Methodology for Single-Cell Mass Cytometry.

Growing recognition of the complexity of interactions within cellular systems has fueled the development of mass cytometry. The precision of time-of-flight mass spectrometry combined with the labeling of specific ligands with mass tags enables detection and quantification of more than 40 markers at a single-cell resolution. The 135 available detection channels allow simultaneous study of additional characteristics of complex biological systems across millions of cells. Cutting-edge mass cytometry by time-of-flight (CyTOF) can profoundly affect our knowledge of cell population heterogeneity and hierarchy, cellular state, multiplexed signaling pathways, proteolysis products, and mRNA transcripts. Although CyTOF is currently scarcely used within the field of investigative dermatology, we aim to highlight CyTOF's utility and demystify the technique. CyTOF may, for example, uncover the immunological heterogeneity and differentiation of Langerhans cells, delineate the signaling pathways responsible for each phase of the hair cycle, or elucidate which proteolysis products from keratinocytes promote skin inflammation. However, the success of mass cytometry experiments depends on fully understanding the methods and how to control for variations when making comparisons between samples. Here, we review key experimental methods for CyTOF that enable accurate data acquisition by optimizing signal detection and minimizing background noise and sample-to-sample variation.

Discovery of skin lymphocytes was a game changer in experimental dermatology.

A substantial part of ongoing research in experimental dermatology focuses on skin T cells-for that reason, we find important to highlight the pioneering work of Jan D. Bos et al. from 1987 (The skin immune system (SIS): Distribution and immunophenotype of lymphocyte subpopulations in normal skin) https://www.ncbi.nlm.nih.gov/pubmed/3494791. This key article sets the record straight, once and for all, about the presence of lymphocytes in healthy skin, characterized the immunophenotypes of subpopulations, quantified these cells and studied their location. It was perhaps the critical discoveries made by Bos et al. that fuelled the scientific community's interest in skin lymphocytes, contributing to a new generation of cutaneous immunology research. We briefly describe additional scientific breakthroughs made since 1987. Nonetheless, the study of cutaneous lymphocytes remains essential to understand the relationship of these cells to human diseases and to develop therapies that can be leveraged to selectively mobilize, enhance or deplete these cells.

Low-dose IL-2 selectively activates subsets of CD4+ Tregs and NK cells.

CD4+ regulatory T cells (CD4Tregs) play a critical role in the maintenance of immune tolerance and prevention of chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. IL-2 supports the proliferation and survival of CD4Tregs and previous studies have demonstrated that IL-2 induces selective expansion of CD4Tregs and improves clinical manifestations of chronic GVHD. However, mechanisms for selective activation of CD4Tregs and the effects of low-dose IL-2 on other immune cells are not well understood. Using mass cytometry, we demonstrate that low concentrations of IL-2 selectively induce STAT5 phosphorylation in Helios+ CD4Tregs and CD56brightCD16- NK cells in vitro. Preferential activation and expansion of Helios+ CD4Tregs and CD56brightCD16- NK cells was also demonstrated in patients with chronic GVHD receiving low-dose IL-2. With prolonged IL-2 treatment for 48 weeks, phenotypic changes were also observed in Helios- CD4Tregs. The effects of low-dose IL-2 therapy on conventional CD4+ T cells and CD8+ T cells were limited to increased expression of PD-1 on effector memory T cells. These studies reveal the selective effects of low-dose IL-2 therapy on Helios+ CD4Tregs and CD56bright NK cells that constitutively express high-affinity IL-2 receptors as well as the indirect effects of prolonged exposure to low concentrations of IL-2 in vivo.