Treg Phenotype Research Articles

Th1-like Treg in vitiligo: An incompetent regulator in immune tolerance

Vitiligo is an autoimmune skin disease resulting from epidermal melanocyte destruction mediated by CD8+T cells that breach the self-tolerance. Regulatory T cells (Tregs) are critical for keeping the CD8+T cells in check, but the deficiency of Tregs leading to the immune disequilibrium in vitiligo remains undefined. In the present study, we used RNA-sequencing (RNA-seq) to acquire the transcriptome data of Tregs from vitiligo patients and healthy controls, respectively. Further flow cytometry analysis and immunofluorescence assays substantiated the phenotype of Th1-like Tregs in vitiligo. CD8+T cell-/vitiligo serum-Treg co-culture assays and chemotaxis assays were used to functionally examine this subset of Tregs. As a result, RNA-seq, flow cytometry, and immunofluorescence all indicated the transition of bona fide Treg to the Th1-like T-bet+IFN-γ+Treg in vitiligo patients. Besides, these Th1-like Tregs exhibited significantly dampened suppression on the proliferation and activation of CD8+T cells and a markedly higher tendency to be chemoattracted by CXCL10 and CXCL16. More interestingly, vitiligo serum could even elicit bona fide Tregs of healthy controls to adopt the Th1-like phenotype and manifest impaired suppression. To conclude, Tregs from vitiligo patients are functionally disturbed and the Th1-skewed inflammatory microenvironment in the serum of vitiligo patients is responsible for the generation of Th1-like Tregs. We provide a clinical exploitable strategy that in addition to simply replenishing the bona fide Treg or promoting the homing of Treg to the skin, the normalization of the Th1-skewed inflammatory environment in vitiligo patients and targeting the incompetent Th1-like Tregs might be critical in the future treatment of vitiligo.

Lymph node fibroblastic reticular cells preserve a tolerogenic niche in allograft transplantation through laminin α4.

Lymph node (LN) fibroblastic reticular cells (FRCs) define LN niches and regulate lymphocyte homeostasis through producing diverse extracellular matrix (ECM) components. We examined the role of ECM laminin α4 (Lama4) using FRC-Lama4 conditional KO Pdgfrb-Cre–/– × Lama4fl/fl mice. Single-cell RNA-sequencing (scRNA-Seq) data showed the promoter gene Pdgfrb was exclusively expressed in FRCs. Depleting FRC-Lama4 reduced Tregs and dendritic cells, decreased high endothelial venules, impaired the conduit system, and downregulated T cell survival factors in LNs. FRC-Lama4 depletion impaired the homing of lymphocytes to LNs in homeostasis and after allografting. Alloantigen-specific T cells proliferated, were activated to greater degrees in LNs lacking FRC-Lama4, and were more prone to differentiate into effector phenotypes relative to the Treg phenotype. In murine cardiac transplantation, tolerogenic immunosuppression was not effective in FRC-Lama4 recipients, which produced more alloantibodies than WT. After lung transplantation, FRC-Lama4–KO mice had more severe graft rejection with fewer Tregs in their LNs. Overall, FRC-Lama4 critically contributes to a tolerogenic LN niche by supporting T cell migration, constraining T cell activation and proliferation, and promoting Treg differentiation. Hence, it serves as a therapeutic target for immunoengineering.

P1270: IMMUNE REGULATORY CELLS IN B-CELL LYMPHOMAS: SIGNIFICANT HETEROGENEITY ACROSS HISTOLOGIC TYPES.

Background: Immune regulatory cell populations play a fundamental role in tumor development and tumor immune evasion. Myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) considered as dominant suppressor cells which are significantly enriched in blood, bone marrow (BM), or tumor sites of patients with hematologic malignancies and often associate with high tumor burden, adverse clinical features, and outcome. Studies in lymphoma patients however, have generated conflicting results, regarding the function and phenotype of MDSC and Tregs mainly due to large heterogeneity of the disease as well as due to the lack of a unanimous nomenclature of these cell subsets in human samples. Furthermore, the molecular mechanisms via which MDSCs and Tregs contribute to clinical outcomes of disease and how they interfere with response to therapy remain elusive. Aims: To investigate the prevalence of immune suppressive population in B-cell lymphomas according to histologic, subtype providing the basis for further analysis of their biologic properties and association with clinical features and outcome of specific lymphoma subtypes. Methods: Peripheral blood (PB) and BM samples were collected from healthy individuals (n= 22) and lymphoma patients (n=50). Specifically, lymphoma subtypes were as follows:Marginal Zone (MZL, N=19), Diffuse Large B-cell Lymphoma (DLBCL, N=18) and Follicular Lymphoma (FL, N=13). Peripheral blood mononuclear cells (PBMCs) were isolated with ficoll gradient and subjected to flow cytometry analysis. MDSC characterization was based on HLADR-/lowCD11b+CD33- expression with CD14+CD15- for M-MDSCs and CD14-CD15+ for PMN-MDSCs. In addition, Treg cells were denoted as CD4+CD25highCD127-Foxp3+. Expression of PDL1 and PD1 on MDSCs and Tregs respectively, was also determined by flow cytometry. M-MDSCs and PMN-MDSCs were sorted from BM samples, RNA was extracted in order to subjected to transcriptomic analysis (RNAseq). Informed consent was obtained. Results: Frequencies of MDSCs were significantly increased in PB of all lymphoma patients compared to healthy individuals, with MZL subtype exhibiting the most prominent increase of MDSCs (p<.0001). Notably numbers of M-MDSCs were increased in FL and MZL patients and to a lesser extend in DLBCL patients while PMN-MDSCs were elevated in all subtypes with MZL displaying the higher frequencies. Regarding the Treg cells, although all lymphoma patients exhibited increased absolute numbers of CD4+CD25highCD127- Treg cells, Foxp3-expressing Tregs were significantly increased in the PB of DLBCL and MZL patients, but not in FL patients. Of interest, CD4+ and CD8+ T cells in the PB of DLBCL patients exhibited increase expression of PD1 compared to respective cell subsets from healthy individuals. Summary/Conclusion: Our findings comprehensively demonstrate enhanced numbers of MDSC and Treg immune suppressive cells in the PB of B-cell lymphoma patients while an increased heterogeneity was observed among the different histological lymphoma types. Functional characterization of MDSCs and Tregs by transcriptomic (RNAseq) and proteomic analysis are ongoing.

Abstract 1362: Metabolic engineering of CAR-T cells overcomes suppressive adenosine signaling and enhances functionality

Abstract Chimeric Antigen Receptor (CAR) T cell therapy has resulted in remarkable clinical outcomes in the context of acute and chronic lymphoblastic leukemia, but remains unsuccessful in the treatment of solid tumors. One reason for this failure is thought to be T cell dysfunction or exhaustion promoted by suppressive soluble factors within the tumor microenvironment (TME). High extracellular levels of the immunosuppressive factor adenosine (Ado) are generated in the TME via breakdown of ATP by ecto-enzymes CD39 and CD73 expressed on tumor-infiltrating immune cells. Binding of extracellular Ado to its receptor A2a on T cells results in inhibition of proliferation and effector function. Interestingly, CD39 has recently been described as a surrogate marker of exhaustion on human CAR-T cells and non-engineered T cells. Therefore, we hypothesized that CD39 expression on exhausted CAR-T cells promotes dysfunction through generation of extracellular adenosine. Using an in vitro model of T cell exhaustion, whereby human T cells express a CAR that tonically signals in an antigen-independent manner (HA CAR), we demonstrate that exhausted HA CAR T cells actively hydrolyze extracellular ATP via their elevated expression of CD39 and CD73. Moreover, exhausted CD39+ CAR T cells upregulate several genes associated with a Treg phenotype at the mRNA and protein levels, suggesting that this cell population might be suppressive. To assess whether CD39+/CD73+ CAR T cells exhibit suppressive functions, we co-cultured them with non-exhausted CD19-CAR T cells. Indeed, proliferation and secretion of IL-2 by CD19 CAR T cells were diminished when they were co-cultured with exhausted CD39+ CAR T cells, and that this suppression is dependent on the A2a receptor. Using this knowledge, we used gene-editing and overexpression approaches to engineer CAR-T cells with resistance to suppressive adenosine signaling. In contrast to genetic deletion of CD39 or CD73, which did not alleviate CAR T cell dysfunction, genetic deletion of adenosine receptor A2aR in exhausted CAR T cells resulted in phenotypic changes and a modest improvement in tumor-specific killing. Further, ectopic overexpression of adenosine deaminase (ADA) in CAR T cells led to decreased exhaustion marker expression and significantly enhanced effector function. These data indicate that ADA overexpression is an innovative approach to increase the functionality of CAR T cells through avoidance of suppressive adenosine signaling, and provides proof-of-concept that metabolic engineering of CAR-T cells can pave the way for responses in patients with solid tumors. Citation Format: Dorota Klysz, Meena Malipatlolla, Katherine Freitas, Malek Bashti, Louai Labanieh, Peng Xu, Cecilia Ramello, Amaury Lerust, Hyatt Balke Want, Kaithlen Zen Pacheco, Evan W. Weber, Shabnum Patel, Steven Feldman, Elena Sotillo, Crystal L. Mackall. Metabolic engineering of CAR-T cells overcomes suppressive adenosine signaling and enhances functionality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1362.

Abstract 2325: Targeting kynurenine pathway using novel IDO/TDO dual inhibitor (AT0174) to modulate tumor microenvironment in platinum resistant non-small cell lung cancer cancer: An immunometabolism compliment markers

Abstract We discovered a crucial change common to all platinum resistant (CR) NSCLC cells, wherein a high level of reactive oxygen species (ROS) modulates tumor metabolism. CR cells activate the kynurenine (KYN) pathway (KP) during excessive oxidative stress to maintain high ROS for proliferation by catabolizing tryptophan (TRP) to KYN via indoleamine 2,3-dioxygenase-1 (IDO1); and by expressing (3-5x) higher levels of programmed death-ligand 1 (PD-L1) compared to platinum sensitive cells (p&amp;lt;0.02; n=8). We detected significant increases in IDO1 activity (3-4x), identified by the KYN/TRP ratio, in CR cells’ culture media (p&amp;lt;0.05; n=8). Extracellular KYN is known to program naïve T-cells to the immune suppressive regulatory T-cell (T-reg) phenotype. Using a human peripheral blood mononuclear cell (PBMC) co-culture system, we found a higher frequency of regulatory T-cells (Tregs; CD4+CD25+Foxp3+) and a lower frequency of effector T-cells (Teffs CD3+CD8+) and NK cell (CD3-CD314+CD335+) activity in co-culture with CR cells compared to cisplatin sensitive cancer cells. Importantly, inhibition of IDO1 and Tryptophan 2,3-Dioxygenase (TDO2), the enzymes involved in the rate limiting step in the KP, with the dual inhibitor AT-0174 at 20µM (48h) suppressed Tregs, but enhanced Teffs and NK cell populations in CR co-cultures. Next, we used a syngeneic mouse model of LLC (Lewis lung cancer) vs cisplatin resistant LLC-CR, to examine the effects of AT-0174 (170 mg/kg; P.O. once daily) in combination with anti-PD1 antibody (10 mg/ml; I.P. once every 3 days). Consistent with the in vitro model, analysis of mice with LLC-CR tumors found higher intratumoral T-reg and lower NK cell populations. Treatment with AT-0174 resulted in higher tumor infiltrating lymphocytes of Teffs and NK and lower Treg frequency in LLC-CR tumors than treatment with a selective IDO1 inhibitor (epacadostat). More importantly, the combination of AT-0174+antiPD1 reversed the immunosuppressive tumor microenvironments with further enhancement in Teffs and NK frequencies and diminished tumor growth in CR allografts with a significant reduction of total tumor weight compared with LLC mouse tumors (n=10, P&amp;lt;0.01). Our new findings support the potential therapeutic efficacy of the dual IDO/TDO inhibitor AT-0174 as an immuno-therapeutic treatment that enhances tumor immune surveillance and blocks tumor metabolism related survival mechanisms in platinum resistant NSCLC tumors. Support by the Dept. of Veterans Affairs. Citation Format: Sydney Spector, Chunjing Wu, Dan Nguyen, George Theodore, Ashley Garcia, Lynn Feun, Niramol Savaraj, Medhi Wangpaichitr. Targeting kynurenine pathway using novel IDO/TDO dual inhibitor (AT0174) to modulate tumor microenvironment in platinum resistant non-small cell lung cancer cancer: An immunometabolism compliment markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2325.

Phenotype, Function, and Clinical Significance of CD26+ and CD161+Tregs in Splenic Marginal Zone Lymphoma.

Regulatory T-cells (Treg) are essential to Tregs homeostasis and modulate the antitumor immune response in patients with lymphoma. However, the biology and prognostic impact of Tregs in splenic marginal zone lymphoma (SMZL) have not been studied. Biopsy specimens from 24 patients with SMZL and 12 reactive spleens (rSP) from individuals without lymphoma were analyzed by using CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), CyTOF (mass cytometry) analysis, and flow cytometry to explore the phenotype, transcriptomic profile, and clinical significance of intratumoral Tregs and their subsets. The biological characteristics and cell signaling pathways of intratumoral Treg subsets were confirmed by in vitro functional assays. We found that Tregs are more abundant in SMZL patients' spleens than rSP, and Tregs from patients with SMZL and rSP can be separated into CD161+Treg and CD26+Treg subsets. CD161+Tregs are increased in SMZL but have dysregulated immune function. We found that CD161+Treg and CD26+Tregs have unique gene expression and phenotypic profiles and are differentially correlated with patient outcomes. Specifically, increased CD161+Tregs are significantly associated with a favorable prognosis in patients with SMZL, whereas CD26+Tregs are associated with a poor prognosis. Furthermore, activation of the IL2/STAT5 pathway contributes to the induction of CD26+Tregs and can be reversed by STAT5 inhibition. IL2/STAT5-mediated expansion of CD26+Tregs contributes to a poor clinical outcome in SMZL and may represent a therapeutic opportunity in this disease.

Modulation of the Response to Mycobacterium leprae and Pathogenesis of Leprosy.

The initial infection by the obligate intracellular bacillus Mycobacterium leprae evolves to leprosy in a small subset of the infected individuals. Transmission is believed to occur mainly by exposure to bacilli present in aerosols expelled by infected individuals with high bacillary load. Mycobacterium leprae-specific DNA has been detected in the blood of asymptomatic household contacts of leprosy patients years before active disease onset, suggesting that, following infection, the bacterium reaches the lymphatic drainage and the blood of at least some individuals. The lower temperature and availability of protected microenvironments may provide the initial conditions for the survival of the bacillus in the airways and skin. A subset of skin-resident macrophages and the Schwann cells of peripheral nerves, two M. leprae permissive cells, may protect M. leprae from effector cells in the initial phase of the infection. The interaction of M. leprae with these cells induces metabolic changes, including the formation of lipid droplets, that are associated with macrophage M2 phenotype and the production of mediators that facilitate the differentiation of specific T cells for M. leprae-expressed antigens to a memory regulatory phenotype. Here, we discuss the possible initials steps of M. leprae infection that may lead to active disease onset, mainly focusing on events prior to the manifestation of the established clinical forms of leprosy. We hypothesize that the progressive differentiation of T cells to the Tregs phenotype inhibits effector function against the bacillus, allowing an increase in the bacillary load and evolution of the infection to active disease. Epigenetic and metabolic mechanisms described in other chronic inflammatory diseases are evaluated for potential application to the understanding of leprosy pathogenesis. A potential role for post-exposure prophylaxis of leprosy in reducing M. leprae-induced anti-inflammatory mediators and, in consequence, Treg/T effector ratios is proposed.

Low Memory T Cells Blood Counts and High Naïve Regulatory T Cells Percentage at Relapsing Remitting Multiple Sclerosis Diagnosis.

ObjectiveThe aim of this study is to assess the peripheral immune system of newly diagnosed patients with relapsing remitting multiple sclerosis (RRMS) and compare it to healthy controls (HC).MethodsThis cross-sectional study involves 30 treatment-naïve newly diagnosed patients with RRMS and 33 sex- and age-matched HC. Peripheral blood mononuclear cells were analyzed regarding: i) thymic function surrogates [T cell receptor excision circles (TRECs) and recent thymic emigrants (RTEs)]; ii) naïve and memory CD4+ and CD8+ T cells subsets; iii) T helper (Th) phenotype and chemokine receptors expression on CD8+ T cells subsets; iv) regulatory T cell (Tregs) phenotype; and exclude expression of activating/inhibitory receptors by natural killer (NK) and NKT cells. Analyses were controlled for age, sex, and human cytomegalovirus (HCMV) IgG seroprevalence.ResultsNewly diagnosed patients with RRMS and HC have equivalent thymic function as determined by similar numbers of RTEs and levels of sjTRECs, DJβTRECs, and sj/DJβTREC ratio. In the CD8+ T cells compartment, patients with RRMS have a higher naive to memory ratio and lower memory cell counts in blood, specifically of effector memory and TemRA CD8+ T cells. Interestingly, higher numbers and percentages of central memory CD8+ T cells are associated with increasing time from the relapse. Among CD4+ T cells, lower blood counts of effector memory cells are found in patients upon controlling for sex, age, and anti-HCMV IgG seroprevalence. Higher numbers of CD4+ T cells (both naïve and memory) and of Th2 cells are associated with increasing time from the relapse; lower numbers of Th17 cells are associated with higher MS severity scores (MSSS). Patients with RRMS have a higher percentage of naïve Tregs compared with HC, and lower percentages of these cells are associated with higher MSSS. Percentages of immature CD56bright NK cells expressing the inhibitory receptor KLRG1 and of mature CD56dimCD57+ NK cells expressing NKp30 are higher in patients. No major alterations are observed on NKT cells.ConclusionCharacterization of the peripheral immune system of treatment-naïve newly diagnosed patients with RRMS unveiled immune features present at clinical onset including lower memory T cells blood counts, particularly among CD8+ T cells, higher percentage of naïve Tregs and altered percentages of NK cells subsets expressing inhibitory or activating receptors. These findings might set the basis to better understand disease pathogenesis.

Rho-GTPase dependent leukocyte interaction generates pro-inflammatory thymic Tregs and causes arthritis

Conditional mutation of protein geranylgeranyltransferase type I (GGTase-I) in macrophages (GLC) activates Rho-GTPases and causes arthritis in mice. Knocking out Rag1 in GLC mice alleviates arthritis which indicates that lymphocytes are required for arthritis development in those mice. To study GLC dependent changes in the adaptive immunity, we isolated CD4+ T cells from GLC mice (CD4+GLCs). Spleen and joint draining lymph nodes (dLN) CD4+GLCs exhibited high expression of Cdc42 and Rac1, which repressed the caudal HOXA proteins and activated the mechanosensory complex to facilitate migration. These CDC42/RAC1 rich CD4+GLCs presented a complete signature of GARP+NRP1+IKZF2+FOXP3+ regulatory T cells (Tregs) of thymic origin. Activation of the β-catenin/Lef1 axis promoted a pro-inflammatory Th1 phenotype of Tregs, which was strongly associated with arthritis severity. Knockout of Cdc42 in macrophages of GLC mice affected CD4+ cell biology and triggered development of non-thymic Tregs. Knockout of Rac1 and RhoA had no such effects on CD4+ cells although it alleviated arthritis in GLC mice. Disrupting macrophage and T cell interaction with CTLA4 fusion protein reduced the Th1-driven inflammation and enrichment of thymic Tregs into dLNs. Antigen challenge reinforced the CD4+GLC phenotype in non-arthritic heterozygote GLC mice and increased accumulation of Rho-GTPase expressing thymic Tregs in dLNs. Our study demonstrates an unexpected role of macrophages in stimulating the development of pro-inflammatory thymic Tregs and reveal activation of Rho-GTPases behind their arthritogenic phenotype.

Identification of EPZ004777 and FG2216 as inhibitors of TGF-β1 induced Treg cells by screening a library of epigenetic compounds

AimsRegulatory T cells play an essential role in immune tolerance and homeostasis. Their long-term stability depends on epigenetic modifications and identification of small molecule modulators of Treg differentiation therefore has many applications. In this study, we performed a novel functional screen of an epigenetic compound library to identify compounds that can modulate generation of TGF-β1 induced T regulatory cells. Main methodsA screening strategy based on IFN-γ ELISA was designed to screen epigenetic compound library. Effect of hit compounds on Treg phenotype and function was assessed by RT-PCR, flow cytometry and suppression assays. TGF-β signalling proteins were assayed by western blotting. Chromatin Immunoprecipitation (ChIP) assay was used to assess epigenetic modifications at Foxp3 gene locus. Key findingsWe screened 160 compounds to identify hits capable of reversing TGF-β induced inhibition of IFN-γ production in activated spleen cells and CD4+ T cells. Two compounds EPZ004777 and FG-2216 consistently reversed TGF-β1 iTregs in terms of (a) differentiation of naïve T cells into CD4+CD25+Foxp3+Treg cells, (b) Foxp3 target gene expression and (c) Treg suppressive function without affecting TGF-β downstream signalling. ChIP assay revealed that the compounds were able to reverse – TGF- β mediated decrease in epigenetic marks H3K27me3 and 5-mC and an increase in epigenetic marks H3K4me3 and H3K27Ac in the promoter and conserved non coding sequence (CNS1) regions of the Foxp3 gene. SignificanceEPZ004777 and FG-2216 have been identified as potent epigenetic modulators that can reverse TGF-β1 induced T regulatory cells and may be used to treat diverse immune disorders.

Role of type l IFN in lacrimal gland-protective Treg dysfunction in nonobese diabetic mouse model of Sjögren’s disease

Abstract Sjogren’s disease is a chronic autoimmune disease of the lacrimal and salivary glands characterized by foci of inflammation in the tissue composed of aggregations of lymphocytes. Male non-obese diabetic (NOD) mice spontaneously develop autoimmunity of the lacrimal glands. Tregs from these mice are dysfunctional. Disruption of the type I interferon (IFN) signaling pathway protects NOD mice from developing lacrimal gland disease. Whether type l IFN signaling drives lacrimal gland protective Treg dysfunction is not known. Here we investigated the role of type l IFN in modulating Treg phenotype and function. Through adoptive transfer studies comparing cells from wild-type or IFNAR1-deficient donor mice, we found that recipients of IFNAR1-deficient cells had fewer foci of inflammation. IFNAR1 expression was correlated with a population of CD44loCD62Lhi Tregs. Co-adoptive transfer of both WT and IFNAR1-deficient cells determined that this altered Treg phenotype was due to intrinsic type l IFN signaling. In vitro studies suggest that Treg phenotype can be shaped by IFNα in a dose dependent manner. Ongoing in vivo studies will interrogate the impact of IFNAR1 expression on Treg function. Type l IFN signaling plays a role in shaping Treg phenotype and may antagonize Treg function. These studies will help define the role of type l IFN signaling in early pathogenesis of Sjögren’s disease and may provide insight into other systemic autoimmune diseases. Supported by grants from NIH (R01EY027731-01A1)

Impact of constitutive FLT3 signaling on dendritic cell development and function in a genetically-engineered mouse model of Acute Myeloid Leukemia

Abstract Acute myeloid leukemia (AML) is a hematological cancer with a very poor prognosis. FLT3-ITD mutations that cause constitutive FLT3 signaling are commonly seen in patients. Our lab and others have shown that AML patient samples exhibit hallmarks of immune exhaustion such as T cell dysfunction, increased MDSCs, and increased Tregs that associate with worse survival. In solid tumor models DCs have been shown to be important for anti-tumor T cell activity and we aim to further describe the role of DCs in the immune response to leukemia. FLT3 signaling is critical for DC development but the effects of FLT3-ITD on DCs and how it contributes to leukemogenesis remains unclear. In a novel FLT3-ITD driven AML mouse model we found that DCs from these mice have elevated pFLT3, indicating constitutive FLT3 signaling. Moreover, significantly expanded DC progenitors and DCs in the bone marrow and spleen are observed. Analysis of serum cytokines identified increased levels of IL-27, IL-10, and IL-17a in leukemia mice compared to healthy controls, suggesting that the expansion of DCs in these leukemia mice may be driving pathogenic expansion of T helper subsets. Furthermore, we measured higher levels of circulating Th2, Th17, and Treg phenotypes in the blood compared to healthy controls. Thus, we hypothesize that FLT3-ITD DCs are a significant contributor to T cell skewing in AML resulting in poor anti-tumor responses. We are using scRNA-seq methods to identify transcriptional changes in FLT3-ITD DCs that lead to the phenotypes we identified. Understanding how FLT3-ITD DCs contribute to AML immune suppression is critical to interpreting leukemia etiology and the development of targeted therapies such as immune checkpoint blockade or small molecule inhibitors.

Demonstration of regulatory CD8 T cell prevalence, phenotype, and functions in autoimmune patients treated with a tolerizing peptide vaccine.

Abstract INTRODUCTION Others have described a subset of CD8 T cells with immunosuppressive characteristics in inflammatory disease settings. CD8 Treg activation, through a canonical T cell receptor, results in oligoclonal expansion and perforin dependent elimination of pathogenic CD4 T cells. We have demonstrated the CD8 Treg network in Celiac patients, and that CD8 Tregs can be modulated to enhance their cytolytic elimination of pathogenic CD4 T cells. Here, we evaluate culture conditions to enhance CD8 Treg functions, and describe the impact of a gluten tolerizing peptide vaccine on CD8 Treg in Celiac disease patients. METHODS We examined the phenotype and function of Celiac patient-derived peripheral blood CD8 Tregs using flow cytometry, multicolor immunohistochemical staining, supernatant analysis, and TCR sequencing. We then evaluated the impact of a gluten tolerizing peptide vaccine on the CD8 Treg network in Celiac patient derived peripheral blood and tissues. RESULTS Using our previously reported phenotypic and functional readouts to define CD8 Treg activity, we describe culture conditions that support oligoclonal CD8 Treg cell expansion, plasticity, and cytolytic function. In patients treated with a gluten tolerizing vaccine, we found evidence for CD8 Treg expansion and preferential recruitment to duodenal tissues; however, pathogenic CD4 T cells were still detectable, suggesting insufficient CD8 Treg functional activation. CONCLUSION We describe parameters that support cytolytic CD8 Treg function, as well as the impact of a gluten tolerizing vaccine on the mobilization of CD8 Treg. Collectively, our data support the potential for CD8 Treg directed therapies to eliminate pathogenic CD4 T cells in autoimmune diseases.

Trafficking and persistence of alloantigen-specific chimeric antigen receptor regulatory T cells in Cynomolgus macaque

SummaryAdoptive transfer of chimeric antigen receptor regulatory T cells (CAR Tregs) is a promising way to prevent allograft loss without the morbidity associated with current therapies. Non-human primates (NHPs) are a clinically relevant model to develop transplant regimens, but manufacturing and engraftment of NHP CAR Tregs have not been demonstrated yet. Here, we describe a culture system that massively expands CAR Tregs specific for the Bw6 alloantigen. In vitro, these Tregs suppress in an antigen-specific manner without pro-inflammatory cytokine secretion or cytotoxicity. In vivo, Bw6-specific CAR Tregs preferentially traffic to and persist in bone marrow for at least 1 month. Following transplant of allogeneic Bw6+ islets and autologous CAR Tregs into the bone marrow of diabetic recipients, CAR Tregs traffic to the site of islet transplantation and maintain a phenotype of suppressive Tregs. Our results establish a framework for the optimization of CAR Treg therapy in NHP disease models.

The role of tumor necrosis factor receptor 2 in experimental autoimmune encephalomyelitis

Abstract Multiple sclerosis (MS) is an autoimmune neuroinflammatory disease that affects over 2 million people globally. HLA-DR2b is a major histocompatibility complex (MHC) locus in humans responsible for antigen-specific immune responses. HLA-DR2b (DRB1*15:01) is a major genetic risk factor for MS. Experimental autoimmune encephalomyelitis (EAE) is the animal model for MS and can provide insights into the mechanisms behind MS pathogenicity. Tumor necrosis factor receptor 2 (TNFR2) was suspected to control EAE severity by altering regulatory T cell (Treg) function and impairing remyelination. In the absence of TNFR2, mice expressing the human DR2b allele and lacking mouse MHC II develop progressive EAE upon adoptive transfer of myelin oligodendrocyte glycoprotein (MOG)-reactive CD4 T cells. These mice show altered Treg phenotype, including decreased expression of Helios, a marker of thymic Tregs, by splenic Tregs from DR2bΔR2 mice compared with DR2b mice. However, it remains unclear how the presence or absence of TNFR2 alters the expression of specific protective genes in both astrocytes and Tregs in the context of EAE. Using the DR2bΔR2 and DR2b mouse models, we investigated the impact of TNFR2 on protective astrocyte subsets and regulatory T cells by single-cell RNA sequencing (scRNA-seq) in the context of both active and adoptive transfer EAE. Our results elucidate mechanisms underlying the protective function TNFR2 via modulating astrocyte and regulatory T cell function in EAE. Our results may aid in the development of new treatments for MS. Supported by grant 1RO1NS117742 from the NIH/NINDS (T.G.F.) and by NIH/NIGMS RISE grant GM60655 (BB).

Alpha-synuclein peptides presented on chimeric MHC class Ib molecules prevent loss of substantia nigra neurons in an animal model for Parkinson’s disease

Abstract Aim An accumulating body of evidence suggests the involvement of alpha-synuclein (aSyn) specific autoreactive T cells in Parkinson’s disease (PD). Here, we investigate whether novel antigen-specific tolerance-inducing biomolecules, that present peptide antigens on MHC class Ib-related molecules, can inhibit neurodegeneration and prevent PD in vivo. Methods The alpha3 domain of the human MHC class Ib molecule HLA-G inhibits T cells via the human ILT-2 or the murine PIRB receptor. We fused this HLA-G alpha3 domain to MHC class I alpha1-alpha2 antigen presenting domains, antigenic peptides and beta2-microglobulin. These single-chain AutoImmunity Modifying Biologicals (AIM Bios) induce antigen-specific tolerogenic CD8+ Treg cells in human cells in vitro and in mice in vivo. We used this platform to prevent autoimmune disease symptoms in mouse models for autoimmune diseases, including an aSynA53T-AAV driven model for PD. Results Single-chain model AIM Bios with MHC-derived antigen-presenting domains and antigenic peptides polarize cognate CD8+ T cells towards a CD8+CD122+ IL-10 secreting Treg phenotype. Such antigen-specific Treg can also be induced in mice in vivo. Disease-associated aSyn CD8+ epitopes were identified in aSynA53T-AAV PD mice. Corresponding AIM Bios prevented mobility impairments. Post mortem histopathological assessment confirmed the induction a favorable in-situ immune cell composition and the rescue of substantia nigra neurons. The translational potential of this approach deserves further exploration. Supported by grants from the IZKF Wuerzburg A421 and by Aeterna Zentaris Inc.

CD6 – a costimulatory receptor at the nexus of Teff / Treg development

Abstract The balance of proinflammatory effector T cells (Teff) and anti-inflammatory regulatory T cells (Treg) is associated with autoimmune and inflammatory diseases. CD6 is a costimulatory receptor expressed on T cells with its greatest expression on pathogenic Teff cells, while Tregs are CD6low. Notably T cells from CD6knockout mice fail to differentiate to Th17 cells but were seen to skew to a Treg phenotype more rapidly. Although the role of CD6 in Teff development is well characterized, here we sought to investigate the role of CD6 in the development of Treg cells. Itolizumab, a humanized anti-CD6 monoclonal antibody that modulates cell surface levels of CD6, was used to generate CD6low naïve T cells, after which itolizumab was removed and Tregs were differentiated using a standard protocol. Tregs derived from CD6low cells had greater co-expression of FOXP3 and HELIOS (~2-fold) vs. Tregs derived from isotype-treated CD6high. These Tregs possessed greater suppressive function, with increased inhibition of proliferation and cytokine release by stimulated T responder cells by at least 50% more than Tregs derived from CD6high cells; this was verified in three different donors. Similar observations were made when measuring levels of cytokines. CD6low Tregs were able to suppress T responder’s production of proinflammatory cytokines by 60–90% compared to CD6high Tregs. This is the first study to directly characterize the role of CD6 in the development and activity of Treg cells. These data suggest that reduced levels of cell surface CD6 facilitate the development of Treg cells with greater stability and suppressive activity and that modulating the levels of CD6 may decrease the Teff/Treg ratio in patients with autoimmune and inflammatory diseases.

Different subpopulations of regulatory T cells in human autoimmune disease, transplantation, and tumor immunity.

CD4+CD25+ regulatory T cells (Tregs), a subpopulation of naturally CD4+ T cells that characteristically express transcription factor Forkhead box P3 (FOXP3), play a pivotal role in the maintenance of immune homeostasis and the prevention of autoimmunity. With the development of biological technology, the understanding of plasticity and stability of Tregs has been further developed. Recent studies have suggested that human Tregs are functionally and phenotypically diverse. The functions and mechanisms of different phenotypes of Tregs in different disease settings, such as tumor microenvironment, autoimmune diseases, and transplantation, have gradually become hot spots of immunology research that arouse extensive attention. Among the complex functions, CD4+CD25+FOXP3+ Tregs possess a potent immunosuppressive capacity and can produce various cytokines, such as IL‐2, IL‐10, and TGF‐β, to regulate immune homeostasis. They can alleviate the progression of diseases by resisting inflammatory immune responses, whereas promoting the poor prognosis of diseases by helping cells evade immune surveillance or suppressing effector T cells activity. Therefore, methods for targeting Tregs to regulate their functions in the immune microenvironment, such as depleting them to strengthen tumor immunity or expanding them to treat immunological diseases, need to be developed. Here, we discuss that different subpopulations of Tregs are essential for the development of immunotherapeutic strategies involving Tregs in human diseases.

IL-21 plays an important role in modulating "Th17-Treg" cell axis in leprosy Type 1 reactions.

Leprosy type 1 reaction (T1R) is a cell-mediated inflammatory reaction which involves skin and peripheral nerves in leprosy. Lesions with T1R have higher production of IL-17 cytokine from CD4+ T cells along with lower TGF-β producing FOXP3+ CD4+ Tregs. IL-21 is an important cytokine that promotes the development and stability of Th17 cells in an autocrine manner. It can play an important role in the pathogenesis of T1R in leprosy. However, the mechanism by which IL-21 influences the pathogenic progress of leprosy T1R remains poorly understood. In the present study, we evaluated the expression of IL-21 cytokine in skin lesions of both non-reactional (NR) and T1R via immuno-histochemistry and quantitative PCR (qPCR). Further, expression of various genes (IL-17A, IL-17F, TGF-β, FOXP3, RORC and IL-21) was also measured by qPCR in cultured cells. We also analyzed the secretion of various cytokines such as of IL-21, IL-17A/F and TGF-β in the culture supernatants by ELISA. In addition, differentiation of Th17 and Treg cells were studied in PBMC cultures after stimulation with Mycobacterium leprae sonicated antigens and rIL-21 for 48hrs and the phenotypes of Th17 and Tregs were determined by flowcytometric analysis. Our results clearly indicate that IL-21+T cells were significantly higher in both peripheral blood and skin lesions of T1R as compared to NR patients. Moreover, we observed that recombinant IL-21 cytokine inhibited TGF-β producing Treg cells differentiation along with up-regulating Th17 cells under in-vitro conditions. The gene expression of IL-21 was significantly negatively correlated with Treg and positively correlated with Th17 cell markers in T1R patients. Our results suggested that IL-21 promotes T1R mediated inflammation via modulating the balance of Th17 and Treg cell populations.

INTERLEUKIN-6 NEUTRALIZATION AND REGULATORY T CELLS ARE ADDITIVE IN CHONDROPROTECTION FROM IL-1BETA-INDUCED INFLAMMATION

Purpose: Inflammation is increasingly recognized as a key driver of inflammation in osteoarthritis (OA) and is present in most patients suffering from OA, yet the immunoregulation of OA remains relatively understudied. Immunosuppressive Regulatory T cells (Tregs) are present within the joint and actively secrete anti-inflammatory factors but are unable to restore homeostasis within the joint. Persistent inflammation within the joint, driven by specific inflammatory cytokines including IL-1β and IL-6, can lead to destabilization of Treg phenotype and function.