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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.

The circulating precursor cells that give rise to human resident memory T cells (T RM ) are poorly characterized. We used an in vitro differentiation system and human skin–grafted mice to study T RM generation from circulating human memory T cell subsets. In vitro T RM 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 (T EM ) had the highest conversion rate to T RM in vitro and in vivo, but central memory T cells (T CM ) persisted longer in the circulation, entered the skin in larger numbers, and generated increased numbers of T RM . In summary, T CM are highly efficient precursors of human skin T RM , a feature that may underlie their known association with effective long-term immunity.

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.

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.

Vitiligo is a chronic inflammatory skin disorder characterized by the loss of melanocytes. While a T helper cell (Th)1/cytotoxic T cell (Tc)1-skewed immune response is now well demonstrated in vitiligo, recent data suggest that the T-cell component could be more complex, involving different combinatorial T-cell subsets.To analyse the phenotype and function of circulating CD4This is a monocentric, prospective, descriptive and exploratory study. Multiparametric flow cytometry analyses were performed to evaluate the surface expression of homing and T-cell-subset markers together with intracellular cytokine production in peripheral blood mononuclear cells from 60 patients with vitiligo, 25 patients with psoriasis and 28 healthy donors.Vitiligo peripheral blood circulating effector and central memory T cells expressed similar proportions of skin-homing markers. Decrease in the frequencies of circulating CD4The decreased frequencies of circulating Th1/Tc1, Th17/Tc17 and Th1/Th17-Tc1/Tc17 cells suggest a possible migration of these T-cell subsets into the skin of patients with vitiligo. These could be targeted to prevent flares of the disease. What is already known about this topic? Vitiligo is a chronic inflammatory skin disorder associated with the loss of melanocytes. Vitiligo is characterized by a T helper cell (Th)1/cytotoxic T cell (Tc)1-skewed immune response in the skin. What does this study add? A thorough analysis of the phenotype and function of circulating memory T cells suggests the migration of Th1/Tc1, Th17/Tc17 and Th1/Th17-Tc1/Tc17 cell subsets in the skin. What is the translational message? A better understanding of the different immune T-cell subsets involved in vitiligo could lead to better therapeutic options. Linked Comment: Matos. Br J Dermatol 2020; 183:803.

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

Copyright © 2021 Matos, Hirakawa, Alho, Neleman, Graca and Ritz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., 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., This work was supported by NIH grant P01CA229092, EADV Research Fellowship, Rene-Touraine Fellowship and a generous contribution from the Fundação para a Ciência e a Tecnologia (FCT), FCT SFRH/BD/98980/2013

Emerging data show that tissue-resident memory T (T

Psoriasis is a chronic and common disease mediated by resident memory T cells that negatively affects a broad range of people worldwide. One of the oldest and most commonly used treatments is phototherapy. We reviewed the existing literature on the four main ultraviolet B (UVB) modalities of phototherapy in the management of psoriasis: heliotherapy, broadband UVB, narrowband UVB, and excimer laser and lamp. Despite the many studies done on these therapies, there is significant variation in their prescription and use. Phototherapy remains one of the most effective and safest treatments for psoriasis. We provide an updated comprehensive overview of UVB phototherapy for psoriasis to help physicians optimize their choice of modality and dosing regimen to ensure optimal outcomes for psoriasis patients. © 2016 Elsevier Inc. All rights reserved.

© 2018 by The American Society of Hematology, 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., This work was supported by National Institutes of Health (NIH), National Institute of Arthritis and Musculoskeletal and Skin Diseases grants R01 AR063962 (R.A.C.), R01 AR056720 (R.A.C.), P30 AR069625 (R.A.C.), T32 AR-07098-36 (T.S.K.; supplied salary for J.T.O.), NIH, National Institute of Allergy and Infectious Diseases grant R01 AI097128 (T.S.K. and R.A.C.), NIH, National Cancer Institute grant R01 CA203721 (R.A.C. and T.S.K.), and the AstraZeneca Foundation/Faculty of Medicine of the University of Lisbon Research Grant (T.R.M.).

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 Vβ and Vα subfamilies. We identified 15 TCRβ 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 αβ 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 αβ 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 αβ T cells. In summary, IL-17-producing αβ 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.

High-throughput sequencing (HTS) of the T-cell receptor (TCR) has advanced rapidly in recent years, giving the unique opportunity to identify and quantify every distinct T-cell clone present within any biological sample. This sensitive and accurate technique provides the relative frequency of each individual clone within the full T-cell repertoire, allowing to study particular single T-cell clones of interest as well as the concomitant analysis of all different clones of the whole population. HTS is essential to expand our knowledge on the diversity of the TCR repertoire in homeostasis or pathologic conditions, as well as to understand the development and kinetics of antigen-specific T-cell responses that critically determine protective immunity (i.e. vaccination) and immune-related disorders (i.e. auto-immunity and cancer). Interestingly, HTS can be tailored for personalized medicine, having the potential to monitor therapeutic interventions and reveal prognostic and diagnostic biomarkers. In this article, we briefly review the methodology, advances and limitations of HTS, and describe emerging applications of this novel technique in the field of investigative dermatology, including studying the pathogenesis of T cells in allergic dermatitis and its application in diagnosis, early recurrence detection and therapy monitoring of cutaneous T-cell lymphoma.

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.

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.

The health benefits of natural sunlight have been noted since the rise of civilization, even without the knowledge of its mechanisms of action. Currently, phototherapy remains an effective and widely used treatment for a variety of skin diseases. Ultraviolet radiation, from either the sun or artificial light sources, has a profound immunomodulatory effect that is responsible for its beneficial clinical outcomes. Ultraviolet radiation mostly induces the innate while suppressing the adaptive immune system, leading to both local and systemic effects. It is antigen specific, acts on both effector and regulatory T cells, alters antigen-presenting cell function, and induces the secretion of cytokines and soluble mediators. This review provides an overview of the immunologic mechanisms by which ultraviolet radiation is responsible for the therapeutic effects of phototherapy.

Idelalisib is a small-molecule inhibitor of PI3Kd with demonstrated efficacy for the treatment of relapsed/refractory chronic lymphocytic leukemia (CLL). To evaluate idelalisib as front-line therapy, we enrolled 24 subjects in a phase 2 study consisting of 2 months of idelalisib monotherapy followed by 6 months of combination therapy with idelalisib and the anti-CD20 antibody ofatumumab. After a median follow-up period of 14.7 months, hepatotoxicity was found to be a frequent and often severe adverse event. A total of 19 subjects (79%) experienced either grade >= 1 ALT or AST elevation during the study, and 13 subjects (54%) experienced grade >= 3 transaminitis. The median time to development of transaminitis was 28 days, occurring before ofatumumab introduction. Younger age and mutated immunoglobulin heavy chain status were significant risk factors for the development of hepatotoxicity. Multiple lines of evidence suggest that this hepatotoxicity was immune mediated. A lymphocytic infiltrate was seen on liver biopsy specimens taken from 2 subjects with transaminitis, and levels of the proinflammatory cytokines CCL-3 and CCL-4 were higher in subjects experiencing hepatotoxicity. All cases of transaminitis resolved either by holding the drug, initiating immunosuppressants, or both, and rates of recurrent toxicity were lower in patients taking steroids when idelalisib was reinitiated. A decrease in peripheral blood regulatory T cells was seen in patients experiencing toxicity on therapy, which is consistent with an immune-mediated mechanism. These results suggest that caution should be taken as drugs within this class are developed for CLL, particularly in younger patients who have not received prior disease-specific therapy. This study was registered at www.clinicaltrials.gov as #NCT02135133

Introduction Phosphatidylinositol-3-kinase (PI3K) inhibition has emerged as a novel targeted mechanism for achieving durable remission in CLL in both the untreated and relapsed/refractory setting. Despite this profound efficacy, PI3K inhibition with the delta isoform inhibitor, idelalisib, has been linked with significant autoimmune toxicity including hepatitis and colitis. Treatment naïve patients receiving idelalisib with ofatumumab in a phase 2 trial had high rates of autoimmune hepatitis within the first month of treatment (Lampson, Blood, 2016). We have previously shown that a reduction in CD4+ regulatory T cells (Tregs) likely predisposes to this toxicity. Here we report additional details of T cell populations observed in these idelalisib-treated patients, with and without severe toxicity. Methods To better characterize the immunologic mechanism of this toxicity, we evaluated samples from study participants using single cell mass cytometry (CyTOF) with a panel of 26 surface membrane and 9 intracellular markers focused on T cell populations (Lampson et al). CyTOF was performed on 26 peripheral blood mononuclear cell (PBMC) samples from 14 patients, at baseline and one month on therapy, the timepoint of autoimmune hepatitis. Flow cytometry standard (fcs) files were processed using FlowJo to gate on viable, singlet cells. The CD3+ events from each sample were exported as individual fcs files to Cytobank.org for analysis. Patients were analyzed in two groups: absent or mild hepatitis (grades 0,1, and 2) vs severe hepatitis (grades 3 and 4) as defined by CTCAEv4.0. viSNE was used to visualize high-dimensional CyTOF data and identify islands for validation (Amir, Nature Biotechnology, 2013). We confirmed these findings by isolating cell populations as percentages of total CD4+ T cells. Results Our analysis of four individual groups demonstrated that CD4+ T cells as a percentage of CD3+ T cells were greater in high toxicity patients than in low toxicity, at baseline and one month, but without significant change during therapy (62.5 vs. 48.2 at baseline; n=9 and n=4, respectively). Evaluation of Tregs showed significant decrease following idelalisib exposure. This was seen when defining Treg cells as CD4+CD25+CD127- as well as when adding Foxp3 expression. viSNE analysis further identified discrete populations. CD4+CD39+Foxp3+ Tregs This is a Treg subset that expresses CD39 and Helios (Figure 1). Across timepoint and toxicity, it is more than two-fold higher in patients with high toxicity, representing 9.6% of CD4+ cells in high toxicity compared with 4.1% in low toxicity. After one cycle of treatment, these percentages declined to 7.5% and 3.2%, respectively. Further, CD39 is expressed by 67% of all Treg cells in this cohort and has been previously suggested to associate with autoimmune response (Zhao, Frontiers in Immunology, 2017). A relative decrease is observed in all patients after exposure, but the reduction of CD39 expressing Treg cells is more robust in low toxicity patients. Naïve CD4+ T Cell: CD62L+CCR7+CD45RA+HLA-DR- The population of naïve T cells is present at baseline in low and high toxicity patients, 5.4% and 10.5% of CD4+ cells, respectively (Figure 2). After one month of exposure to idelalisib, this population in high toxicity patients increased to 14% but remained the same in low toxicity. Conclusion This CyTOF analysis among patients with and without severe autoimmune hepatic toxicity on idelalisib demonstrates relative expansion of CD4 populations in patients who developed toxicity. We identify two CD4 T cell subsets in patients experiencing severe hepatotoxicity. The CD4+CD39+ Treg population emerges as a leading candidate for further evaluation. In leukemic cells, CD39 has been reported to promote differentiation of specific Tregs (Vaisitti, International Journal of Molecular Sciences, 2018). Furthermore, CD39 expression on T-cells has been shown to confer worse outcomes in CLL (Perry, Annals of Hematology, 2012). The mechanism for this remains unknown.The second population is a subset of naïve T cells that grows following treatment only in high toxicity patients. An expanded cohort from this trial is currently being analyzed with an updated T cell focused CyTOF panel to further evaluate these findings. If validated, CD39 expressing T cells may help predict susceptibility to severe autoimmune mediated hepatotoxicity prior to PI3K-based treatment of CLL. Disclosures Brown: TG Therapeutics: Consultancy; Verastem: Consultancy, Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; Loxo: Consultancy; Sunesis: Consultancy; Sun Pharmaceutical Industries: Research Funding; Roche/Genentech: Consultancy; Genentech: Consultancy; Boehringer: Consultancy; Pharmacyclics: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Invectys: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Research Funding; Abbvie: Consultancy.

Ultraviolet radiation (UVR) can have a beneficial biologic impact on skin, but it is also the most significant environmental risk factor for skin cancer development. Photocarcinogenesis comprises a complex interplay between the carcinogenic UVR, skin, and the immune system. UVB is absorbed by the superficial skin layers and is mainly responsible for direct DNA damage, which, if unrepaired, can lead to mutations in key cancer genes. UVA is less carcinogenic, penetrates deeper in the dermis, and mainly causes indirect oxidative damage to cellular DNA, proteins, and lipids, via photosensitized reactions. UVR not only induces mutagenesis, altering proliferation and differentiation of skin cells, but also has several immunosuppressive effects that compromise tumor immunosurveillance by impairing antigen presentation, inducing suppressive cells, and modulating the cytokine environment. This review focuses upon molecular and cellular effects of UVR, regarding its role in skin cancer development. info:eu-repo/semantics/publishedVersion

© 2016 by The American Society of Hematology, The development and maintenance of immune tolerance after allogeneic hematopoietic stem cell transplantation (HSCT) requires the balanced reconstitution of donor-derived CD4 regulatory T cells (CD4Tregs) as well as effector CD4 (conventional CD4 T cells [CD4Tcons]) and CD8 T cells. To characterize the complex mechanisms that lead to unbalanced recovery of these distinct T-cell populations, we studied 107 adult patients who received T-replete stem cell grafts after reduced-intensity conditioning. Immune reconstitution of CD4Treg, CD4Tcon, and CD8 T cells was monitored for a 2-year period. CD3 T-cell counts gradually recovered to normal levels during this period but CD8 T cells recovered more rapidly than either CD4Tregs or CD4Tcons. Reconstituting CD4Tregs and CD4Tcons were predominantly central memory (CM) and effector memory (EM) cells and CD8 T cells were predominantly terminal EM cells. Thymic generation of naive CD4Tcon and CD8 T cells was maintained but thymic production of CD4Tregs was markedly decreased with little recovery during the 2-year study. T-cell proliferation was skewed in favor of CM and EM CD4Tcon and CD8 T cells, especially 6 to 12 months after HSCT. Intracellular expression of BCL2 was increased in CD4Tcon and CD8 T cells in the first 3 to 6 months after HSCT. Early recovery of naive and CM fractions within each T-cell population 3 months after transplant was also strongly correlated with the subsequent development of chronic graft-versus-host disease (GVHD). These dynamic imbalances favor the production, expansion, and persistence of effector T cells over CD4Tregs and were associated with the development of chronic GVHD., This work was supported by a Collaborative Research Grant from the Harvard Medical School-Portugal Program in Translational Research HMSP-ICT/0001/201, and National Institutes of Health, National Cancer Institute grants CA183559, CA183560, and CA142106.

Copyright © 2015, American Association for the Advancement of Science., The skin of an adult human contains about 20 billion memory T cells. Epithelial barrier tissues are infiltrated by a combination of resident and recirculating T cells in mice, but the relative proportions and functional activities of resident versus recirculating T cells have not been evaluated in human skin. We discriminated resident from recirculating T cells in human-engrafted mice and lymphoma patients using alemtuzumab, a medication that depletes recirculating T cells from skin, and then analyzed these T cell populations in healthy human skin. All nonrecirculating resident memory T cells (TRM) expressed CD69, but most were CD4(+), CD103(-), and located in the dermis, in contrast to studies in mice. Both CD4(+) and CD8(+) CD103(+) TRM were enriched in the epidermis, had potent effector functions, and had a limited proliferative capacity compared to CD103(-) TRM. TRM of both types had more potent effector functions than recirculating T cells. We observed two distinct populations of recirculating T cells, CCR7(+)/L-selectin(+) central memory T cells (TCM) and CCR7(+)/L-selectin(-) T cells, which we term migratory memory T cells (TMM). Circulating skin-tropic TMM were intermediate in cytokine production between TCM and effector memory T cells. In patients with cutaneous T cell lymphoma, malignant TCM and TMM induced distinct inflammatory skin lesions, and TMM were depleted more slowly from skin after alemtuzumab, suggesting that TMM may recirculate more slowly. In summary, human skin is protected by four functionally distinct populations of T cells, two resident and two recirculating, with differing territories of migration and distinct functional activities., This work was supported by a generous charitable contribution from Edward P. Lawrence, Esq., R01 AR063962 NIH/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) (to R.A.C.), R01 AR056720 NIH/NIAMS (to R.A.C.), a Damon Runyon Clinical Investigator Award (to R.A.C.), the SPORE in Skin Cancer P50 CA9368305 NIH/National Cancer Institute (to T.S.K.), and R01 AI097128 National Institute of Allergy and Infectious Diseases (to T.S.K. and R.A.C.).

Introduction: CD4+ FoxP3+ CD25+ regulatory T cells (Treg) are required for maintenance of immune tolerance and immune homeostasis. Quantitative or functional Treg deficiency has been correlated with autoimmune disease, allergy, allograft rejection and graft versus host disease. Conversely, increased Treg can suppress tumor immunity resulting in tumor progression. Treg express a large number of cellular markers that reflect their level of maturation, functionality, activation and migratory capacity. Nevertheless, it has not previously been possible to integrate the expression of these various markers and correlate their expression with human Treg differentiation in vivo. Methods: We used single cell mass cytometry (CyTOF) to simultaneously study 36 phenotypic and functional markers of human Treg in samples obtained from umbilical cord blood (CB) (n=4) and healthy adult donors (n=10). Wanderlust trajectory detection algorithm was used to analyze temporal positioning of Treg across development. To quantify Treg heterogeneity we used ACCENSE; an analysis tool that combines a nonlinear dimensionality reduction algorithm (t-SNE) with spectral clustering algorithm and automated cell classification into subpopulations. Results: Using Wanderlust to characterize Treg maturation, the majority of CB Treg were naive (CD45RA+) and CB memory Treg (CD45RA-) were poorly differentiated with minimal expression of activation (HLA-DR) and pro-apoptotic (CD95) markers (Figure 1A). Adult Treg contained few naive cells and mature Treg effector cells expressed high levels of activation and pro-apoptotic markers. (Figure 1B). Using Wanderlust together with spearman correlation, 5 discrete stages of Treg maturation were identified; 1) recent thymic emigrants (RTE), 2) naive, 3) effector, 4) activated and 5) terminal effector. RTE Treg defined by expression of CD45RA, CD31 and CCR7, also expressed markers of proliferation (KI67) and functionality (Tbet, PDL-1, Helios) at low levels but lacked functional CTLA4 and TIM-3. Naive Treg lacked expression of CD31 but expressed other markers characteristic of RTE. Effector Treg expressed increased levels of CD95, CTLA-4, CCR7, GITR, Helios and FoxP3 but lacked HLA-DR. Activated effector Treg expressed the highest levels of FoxP3, Helios and Ki67, along with functional markers (CD28, CXCR3, ICOS, GITR, CD39, CTLA-4, TIM-3) and homing molecules (vascular endothelial CCR5, gut addressin ACT-1, skin addressins CCR4, CLA). Activated Treg express the highest levels and diversity of functional markers along with the ability to migrate to different tissues. Lastly, terminal effector Treg express a more restricted set of functional and homing markers (CD28, CTLA-4, ICOS and CCR5) with less diversity. Markers of exhaustion (PD-1 and TIM-3) are also expressed by effector, activated and terminal effector Treg. Pro-apoptotic markers (CD95high BCL2low) are primarily expressed by activated and terminal effector Treg. Using ACCENSE to evaluate Treg diversity allowed further identification of discrete Treg sub-populations within each maturation state. RTE and naive Treg appear very homogeneous and appear as a single cluster in both CB and adults. In contrast, effector, activated and terminal effector Treg are more heterogeneous and are visualized as 9 distinct clusters (Figure 1C, D). This clustering reflects distinct subsets of memory Treg that co-express various combinations of functional markers in our panel. All 9 Treg effector populations are present in CB, but at much lower levels. Treg effector cell diversity is maintained but does not increase as Treg mature and expand in adults and RTE/naive Treg become less prevalent. Conclusion: Our study is the first to quantify human Treg heterogeneity based on expression of a large set of activation, proliferation, tissue homing and functional markers in conjunction with stages of Treg maturation and differentiation. These studies define 5 stages of Treg maturation and 10 clusters of Treg diversity based on differential expression of phenotypic and functional markers. Similar approaches can be applied to describe maturation and diversity of other cell populations. Further application of this CyTOF panel can be used to study Treg maturation and diversity after hematopoietic stem cell transplantation and in immune and inflammatory diseases, to identify specific defects that may contribute to immune dysfunction. Disclosures No relevant conflicts of interest to declare.

Introduction: Idelalisib (idela) is a highly selective oral inhibitor of PI3Kδ that is currently FDA-approved in conjunction with rituximab for the treatment of relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL). In clinical trials performed in the R/R setting, the overall response rate was 70-80%, while the frequency of significant toxicity was easily manageable (grade ≥3 transaminitis 14%, pneumonitis 3%, grade ≥3 diarrhea 14%) (Coutre EHA 2015). Given the high efficacy of idela, we are evaluating the combination of idela plus ofatumumab (ofa) as first-line therapy for CLL in a phase II study. Surprisingly, we noted much higher rates of grade 3-4 transaminitis in particular, as well as more pneumonitis and colitis, than previously reported. Preclinical data and clinical response to corticosteroids suggest that these toxicities may be autoimmune in origin. Methods: These results describe the clinical characteristics and toxicities of the first 21 subjects enrolled in a single-arm phase II study of idela plus ofa in previously untreated CLL patients in need of therapy. Subjects received idela 150mg twice daily during a 2 month lead-in period prior to the addition of weekly ofa infusions x 8 followed by monthly infusions x 4. For the first 2 months, subjects were monitored for toxicities with weekly clinic visits and biweekly serum chemistries. Single cell mass cytometry (CyTOF) permits the simultaneous evaluation of up to 36 markers without marker emission overlap, allowing for a comprehensive phenotypic and functional analysis of T cell subsets. We used CyTOF to compare T cell subset number and function between subjects who experienced no toxicity (n=2) and a portion of subjects (n=5) who experienced grade ≥3 toxicity while on idela. Results: After a median follow-up of 8.1 months (range 0.7-10.8 months), sixteen subjects (76%) had experienced a grade 3 or higher toxicity. The most frequent grade ≥3 adverse events were transaminitis (n=12, 57%), enterocolitis (n=3, 14%), and pneumonitis (n=2, 10%). The subjects who experienced grade ≥3 toxicities, or who experienced multiple toxicities of at least grade 2 (n =13), were younger (median age 65 vs. 75 years, p=0.047) and had higher absolute lymphocyte counts (median 71466 vs. 19250 cells/µL, p =0.017) compared to subjects who experience no or low grade toxicity (n=7). The median time to onset of transaminase elevation was 28 days (range 14-274 days), with most occurring between days 20-30. Two subjects with ongoing elevation of grade 4 transaminitis after holding idela underwent liver biopsy. These biopsies showed increased activated cytotoxic T cells within the liver parenchyma compared to normal controls with CLL. In all cases, the organ toxicities have abated with the initiation of immunosuppressive therapy. Sixteen subjects (76%) required steroids and one subject (5%) required mycophenolate mofetil. Preclinical data suggest that PI3Kδ is critical to the function of regulatory T cells (Tregs), and inhibition of PI3Kδ leads to autoimmunity. Indeed, CyTOF analysis demonstrated that five out of six tested subjects (83%) had a decrease in the percentage of Tregs after one cycle of idela therapy. Tregs from subjects who experienced grade ≥3 toxicity had lower baseline expression level of functional markers (GITR, T-bet, TIM-3) and higher expression level of apoptotic markers (CD95) compared to subjects who experienced no toxicity. After one cycle of idela therapy, expression levels of the Treg effector markers granzyme β, HLA-DR, and PD-1 decreased in subjects who experienced toxicity, but increased in those subjects who did not. Conclusions: The use of idela as first-line therapy in CLL results in more frequent and severe toxicities than its use in the R/R setting. Multiple lines of evidence suggest that this toxicity is immune-mediated: the delayed time to onset, an immune cell infiltrate in biopsies of affected organs, and abatement of toxicity with immunosuppressants. Affected patients had depressed Treg functionality at baseline and lost markers of Treg activation after idela therapy, suggesting that they may be particularly sensitive to PI3Kδ blockade and Treg inhibition. In addition to elucidating the mechanisms of idelalisib, these studies will hopefully allow us to better screen and select patients in whom idela therapy will be well tolerated. Disclosures No relevant conflicts of interest to declare.

Introduction: Idelalisib, a specific small molecule inhibitor of the delta isoform of PI3 kinase (PI3Kd), has recently been FDA approved for the treatment of relapsed CLL. Expression of PI3Kd is predominantly restricted to leukocytes, and inhibition of PI3Kd promotes apoptosis of neoplastic CLL cells. Patients who receive idelalisib often experience toxicities that appear to be immune mediated. In an ongoing phase II study of idelalisib-ofatumumab at DFCI, 16 of 21 patients experienced grade 3 or greater autoimmune toxicities (14 transaminitis, 2 enteritis/colitis and 3 presumed drug-induced pneumonitis). Methods: To identify potential causes of autoimmunity in patients receiving idelalisib, we used single cell mass cytometry (CyTOF) with a panel of 26 surface membrane and 9 intracellular markers to provide a comprehensive phenotypic and functional analysis of all peripheral blood lymphocytes. Blood samples were obtained at baseline prior to therapy and at onset of autoimmune symptoms, which occurred at a median of 28 days from start of therapy. Results in 5 severely affected patients were compared with 3 patients who did not develop autoimmune toxicities and 15 healthy donors. The second sample from unaffected patients was matched in time to the affected patient samples. Results: Within CD3 T cells, idelalisib therapy led to an increased percentage of CD4 T cells and decreased percentage of CD8 T cells. Within CD4 T cells, CD25high Foxp3+ regulatory T cells (Treg) were reduced, resulting in a marked increase of the ratio of conventional CD4 T (Tcon)/Treg and increased CD8/Treg ratio. These findings were similar in patients who developed autoimmune toxicities and those who did not. Analysis of effector memory (EM; 45RA- CCR7-), central memory (CM; 45RA- CCR7+), terminal effector (TEMRA; 45RA+ CCR7-) and naive (45RA+ CCR7+) subsets within Treg, Tcon and CD8 T cells was comparable before and after idelalisib therapy and with healthy individual controls. To evaluate the heterogeneity of each population we utilized viSNE, which allows visualization of complex high-dimensional cytometry data on a two-dimensional plot. Treg in patients who developed autoimmune phenomena were compared with Treg from patients who did not. In patients who developed autoimmunity, baseline Treg had higher expression of PD-1 and lower expression of functional markers, such as GITR, Tbet, CXCR3, PDL-1, granzyme-β and TIM-3. In some cases, expression of these functional markers declined further with idelalisib therapy (Figure 1). Cytotoxic T lymphocyte antigen 4 (CTLA4) is constitutively expressed by Treg, and its deficiency has been shown to limit suppressive function of Treg. Inducible co-stimulator (ICOS) also mediates the suppressive functions of CD4 Treg by regulating cell surface expression of CTLA-4. Interestingly, expression of CTLA-4 and ICOS did not change during idelalisib therapy (Figure 1). Susceptibility to apoptosis was monitored by expression of pro-survival Bcl-2 and pro-apoptotic CD95 (FAS). Treg from patients developing autoimmunity expressed lower levels of Bcl-2 and higher levels of CD95 compared to those who did not. Conversely, Treg of non-affected patients retained higher expression of functional markers and became more active, assessed by increased expression of HLA-DR. The use of CyTOF combined with our comprehensive panel allowed us to delineate up to 13 distinct Treg sub-populations among healthy controls. In contrast, CLL patients often lacked such broad Treg heterogeneity. Expression of functional markers by Tcon and CD8 T cells from patients who would develop side effects, were also slightly lower, suggesting that autoimmune toxicities were not due to expansion or activation of CD8 or Tcon effector cells but due to the defect in both number and function of Tregs, which became insufficient to maintain the immune tolerance. Conclusion: These studies allowed us to identify defects in both Treg number and function, which may be responsible for the autoimmune toxicities in patients receiving idelalisib therapy. Further studies may allow the development of a reliable predictor of these toxicities by evaluation of the Treg markers prior to drug exposure which could be used to guide therapy. Moreover, the identification of idelalisib as a Treg inhibitor suggests that it could be used as an immunomodulatory agent to target the Treg pathway, in patients with solid tumors. Disclosures No relevant conflicts of interest to declare.

Introduction: CD4+ FoxP3+ CD25+ regulatory T cells (Treg) play a central role in the maintenance of immune tolerance and prevention of chronic graft-versus-host disease (cGVHD) after allogeneic stem cell transplantation (SCT). Treg constitutively express high-affinity interleukin-2 (IL-2) receptors and murine models have established that IL-2 is a critical homeostatic regulator of Treg in vivo. We previously reported that daily administration of low-dose IL-2 in patients with cGVHD induces selective expansion of Treg and NK cells and results in clinical improvement in approximately 50% of patients. However, the mechanisms responsible for these selective effects and the influence of IL-2 therapy on other lymphocytes have not been established due to the limited resolution of traditional cell analytic methods such as flow cytometry. Methods: Single cell mass cytometry (CyTOF) with a panel of 33 markers was used to simultaneously examine the phenotypic and functional effects of low-dose IL-2 on lymphocyte populations in vitro and in vivo. The analytic panel included 22 cell surface markers to identify distinct T, B and NK cell subsets and 11 intracellular markers to measure functional status and activation of specific signaling pathways. viSNE, a cytometry analysis tool, was used to visualize high-dimensional cytometry data on a two-dimensional map. Results: In unstimulated lymphocytes from healthy donors, constitutive expression of CD25 (IL-2Ra) at high levels was restricted to Treg and CD56bright NK cells. Central memory (CM) and effector memory (EM) subsets of conventional CD4 T cells (Tcon) and CM CD8 T cells expressed low levels of CD25. Within the Treg population, the highest expression of CD25 was closely associated with expression of Helios transcription factor. Helios+ Treg also express higher levels of FoxP3, HLA-DR and CD95 and lower levels of BCL2 compared to Helios- Treg. To examine responses to IL-2, we stimulated peripheral blood mononuclear cells (PBMC) from healthy donors with IL-2 for 15 min in vitro (Figure 1). At low IL-2 concentrations (1 to 10 IU/ml), pSTAT5 was preferentially activated in Treg. Notably, pSTAT5 activation was more robust in memory Treg than naïve Treg and in Helios+ Treg than Helios- Treg. In addition, we observed activation of pSTAT5 in CD56bright NK cells at low concentrations of IL-2 (10 IU/ml). Higher IL-2 concentrations (100-1000 IU/ml) were required to activate pSTAT5 in Tcon, CD8 T cells and CD56dim NK cells. At high IL-2 concentrations, pSTAT5 was activated in all Treg, NK, Tcon and CD8 subsets. To examine the response to IL-2 in vivo, we examined PBMC from 14 patients with chronic GVHD receiving daily low-dose IL-2 using the same CyTOF panel of markers. Without additional in vitro stimulation, pSTAT5 expression was increased preferentially in Helios+ Treg. Peak pSTAT5 expression occurred 1 week after starting IL-2 and decreased with continued IL-2 therapy. Similarly, increased expression of FoxP3, CD25, HLA-DR and Ki67 occurred primarily in Helios+ Treg with peak expression at 1 week. At later time points during IL-2 therapy, changes in Treg included increased expression of CD95, CTLA4, PD-1, BIM and BCL2. Although there was no activation of pSTAT5 in CD4 Tcon and CD8 T cells, expression of PD-1 increased in effector memory subsets of Tcon and CD8 T cells 1 week after starting IL-2 therapy. Selective expansion of CD56bright NK cells was also noted, with peak activation at 1 week. No other changes were noted in Tcon, CD8 T cells and B cells. All changes observed during IL-2 therapy returned to baseline levels 4 weeks after treatment was stopped. However, examination of PBMC from 8 patients who received continuous daily low-dose IL-2 therapy for approximately 1 year showed that all of the changes noted above persisted during extended therapy. Conclusion: Comprehensive analysis of T, B and NK cells from healthy donors revealed that low concentrations of IL-2 result in selective activation of Helios+ Treg and CD56bright NK cells. Higher concentrations of IL-2 are required for activation of CD4 Tcon, CD8 T cells and CD56dim NK cells. Identical populations are activated in patients with cGVHD receiving daily low-dose IL-2 and these functional effects persist during extended IL-2 therapy. Although the function of Helios transcription factor is not well defined, Helios expression identifies those Treg most primed to respond to low concentrations of IL-2 in vitro and in vivo. Disclosures Armand: Infinity Pharmaceuticals: Consultancy; Merck: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding. Antin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees. Soiffer:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Introduction: Cytokines play important roles in the activation, proliferation, differentiation and survival of T cells. Previous studies have revealed that individual cytokines selectively activate different T cell populations and also function at specific stages of T cell differentiation. For example, IL-2 supports the development of CD4 regulatory T cells. IL-7 is required for naive conventional CD4 T cell (Tcon) homeostasis, whereas naive CD8 T cell homeostasis requires both IL-7 and IL-15. In contrast, IL-6 promotes Th17 T cell differentiation. The functions of each cytokine are partly defined by the differential expression of specific multi-unit receptors but the selective homeostatic effects of individual cytokines are still incompletely understood. Methods: We stimulated peripheral blood mononuclear cells from healthy donors with varying concentrations of IL-2, IL-7, IL-15 and IL-6 for 15 min in vitro. Single cell mass cytometry (CyTOF) with a panel of 33 markers was used to simultaneously examine signaling pathways activated by each cytokine in distinct T cell subsets. viSNE, a cytometry analysis tool, was used to visualize high-dimensional cytometry data on a two-dimensional map. Expression of pSTAT5 was used to monitor activation induced by IL-2, IL-7 and IL-15; pSTAT3 was used to monitor activation by IL-6. Results: In CD4 Tcon, relatively high concentrations of IL-2 (100-1000 IU/ml) are required to induce pSTAT5 (Figure 1). However even at high concentrations, IL-2 activation was selective for memory Tcon subsets. In contrast, IL-7 induced pSTAT5 at very low concentrations (1-10 IU/ml). Although all Tcon were affected, activation was more robust in memory than naive Tcon subsets at all IL-7 concentrations. IL-15 activation of pSTAT5 required at least 10 IU/ml and only memory Tcon subsets were activated even at high IL-15 concentrations. Whereas IL-2, IL-7 and IL-15 preferentially activated memory Tcon subsets, IL-6 selectively activated pSTAT3 in naive and central memory (CM) Tcon subsets at low concentrations (10 IU/ml). At high IL-6 concentrations (100-1000 IU/ml) effector memory (EM) Tcon were also activated. CD8 T cells (Figure 2) are relatively insensitive to IL-2, and only CM CD8 T cells are activated at high IL-2 concentrations (100 IU/ml). Although all CD8 T cell subsets were activated at very high IL-2 concentrations (1000 IU/ml), pSTAT5 activation remained most evident in CM CD8 T cells. Similar to Tcon, IL-7 induced pSTAT5 in CD8 T cells at very low IL-7 concentrations (1-10 IU/ml). However unlike Tcon, pSTAT5 activation was most prominent in naive and CM CD8 T cells. EM CD8 T cells were activated at higher IL-7 concentrations but TEMRA CD8 T cells were resistant to IL-7 stimulation. IL-15 induced pSTAT5 equally in all CD8 T cell subsets but relatively high concentrations (100-1000 IU/ml) were required. Similar to CD4 Tcon, IL-6 induced selective pSTAT3 activation in naive CD8 T cells. Activation of naive CD8 T cells was observed at low concentrations of IL-6 and both EM and TEMRA were resistant to very high IL-6 concentrations (100-1000 IU/ml). Conclusion: This detailed analysis of cytokine signaling has identified differential effects of IL-2, IL-7, IL-15 and IL-6 on different subsets of CD4 Tcon and CD8 T cells. Whereas CD4 Treg are activated at low IL-2 concentrations, CD4 Tcon and CD8 T cells are relatively resistant to IL-2. At high IL-2 concentrations, activation was most prominent for memory CD4 Tcon and CM CD8 T cells. In contrast, low concentrations of IL-7 are sufficient to activate both CD4 Tcon and CD8 T cells. Within these populations, memory Tcon and naive CD8 cells were preferentially activated at low IL-7 concentrations. Within the CD8 T cell population, IL-15 activated all subsets equally. Within CD4 Tcon, IL-15 preferentially activates memory subsets. IL-6 acts at low concentrations and primarily on naive cells in both CD4 Tcon and CD8 T cells. In all experiments, these effects do not require TCR antigen activation and therefore reflect the potency and differential activity of homeostatic signals supported by these cytokines. Importantly, high concentrations used for in vitro experiments are not likely achieved in vivo but may reflect toxicities of high dose exogenous cytokine therapies or cytokine release syndromes. In contrast, differential effects observed at low concentrations more likely reflect physiologic homeostatic effects of these cytokines in vivo. Disclosures No relevant conflicts of interest to declare.

CD4+CD25+Foxp3+ regulatory T cells (Treg) play a critical role in establishment of immune tolerance and prevention of graft versus host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). The recovery and maintenance of Treg after HSCT is dependent on homeostatic factors including the generation of naïve Treg from hematopoietic precursor cells, the proliferation and expansion of mature Treg and the survival of Treg in vivo. We previously reported that Treg in healthy donors are more susceptible to mitochondrial apoptotic priming than conventional T cells (Tcon). Mitochondrial priming is increased after hematopoietic stem cell transplantation in all T cell subsets and particularly in patients with chronic GVHD. Interleukin-2 (IL-2) is a critical homeostatic cytokine that is required for Treg development, expansion, activity and survival. We previously demonstrated that daily administration of low-dose IL-2 resulted in selective expansion of Treg in vivo, clinical improvement of chronic GVHD (Koreth et al. NEJM 2011), increased Treg Bcl2 expression and increased Treg resistance to apoptosis in vitro (Matsuoka et al. SciTM, 2013). These observations led us to undertake a more detailed analysis of apoptotic pathways in functionally and phenotypically distinct Treg subsets including naïve Treg, recent thymic emigrants (RTE) Treg and memory Treg. To examine the mechanisms whereby IL-2 affects susceptibility to apoptosis in each Treg subset, we used a functional flow cytometry-based assay (apoptotic profiling) to measure mitochondrial membrane depolarization in response to a panel of pro-apoptotic BH3 peptides (BIM, BID, BAD, NOXA, PUMA, BMF, HRK). This assay allowed us to compare "priming", which we define as susceptibility to BH3 peptide-induced mitochondrial membrane depolarization, in naïve, RTE and memory subsets. We also examined cytoplasmic expression of Bcl2 as an additional measure of susceptibility to apoptosis in each subset. In resting blood obtained from healthy donors (n=25), memory Treg were more "primed" than either naïve or RTE subsets when exposed to several BH3 peptides (BID, NOXA, PUMA, BMF and the combination of BAD+NOXA) (Figure 1). Memory Treg were also found to have decreased expression of Bcl2 compared to naïve or RTE subsets. Thus, memory Treg in healthy individuals are more susceptible to apoptosis than other Treg subsets through the mitochondrial pathway (Figure 1). To examine the functional effects of IL-2 on T cell homeostasis, Treg and Tcon were purified by cell sorting and cultured for 5 days with different concentrations of IL-2 (0, 10, 100, 1000 U/ml). Low-concentration IL-2 (10 U/ml) lowered apoptotic priming and increased Bcl2 expression in all Treg subsets. Similar effects were observed in all Tcon subsets, but higher concentrations of IL-2 (≥100 U/ml) were required to decrease apoptotic priming and increase Bcl2 expression in Tcon subsets. The peak effect of IL-2 on Treg priming occurred on day 3 but these effects appeared more rapidly in naïve and RTE Treg subsets than in memory Treg. However, the effect of IL-2 on apoptotic priming persisted for a longer period in memory Treg than in naïve and RTE Treg subsets. The peak effect of IL-2 on Bcl2 expression occurred on day 3 in all Treg subsets. To compare the effect of IL-2 on Treg and Tcon subsets, we examined apoptotic priming and Bcl2 expression after 3 day incubation with different IL-2 concentrations (0.001 - 1000 U/ml). Very low-concentration IL-2 (1 U/ml) lowered priming and increased Bcl2 expression in memory Treg, but higher concentrations of IL-2 (≥10 U/ml) were required to increase Bcl2 expression and decrease priming in naïve and RTE Treg. All Tcon subsets showed a similar pattern of response but higher concentrations of IL-2 (≥100 U/ml) were required. Memory Treg are the predominant regulatory population in patients with cGVHD and the high level of priming in this subset contributes to the inability to maintain adequate numbers of CD4 Treg after HSCT. In healthy donors, low concentrations of IL-2 are sufficient to reduce mitochondrial priming and increase Bcl2 expression in memory Treg. IL-2 has similar effects on Tcon but 10-100 fold higher concentrations of IL-2 are required. These concentration-dependent effects of IL-2 likely contribute to the selective expansion and persistence of Treg in patients with cGVHD receiving daily low-dose IL-2 therapy after HSCT. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.