Treg Expansion Research Articles

Effects of ruxolitinib on murine regulatory T cells are immune-context dependent

Aplastic anemia is a bone marrow failure (BMF) disorder characterized by pancytopenia and hypocellular marrow from an immune-mediated etiology. Regulatory T cells (Tregs) prevent autoimmunity by suppressing autoreactive T cells. We recently demonstrated the efficacy of ruxolitinib (RUX), a JAK 1/2 inhibitor, in attenuating murine BMF. Herein, we investigated the changes of Tregs in the context of RUX treatment for murine BMF. Tregs are conventionally identified by surface expression of CD4 and CD25, in addition to intracellular transcription factor FoxP3. RUX promoted the expansion of Tregs in BMF mice defined by increased expression of FoxP3 in CD4 T cells but suppressed expression of activation marker CD25 in CD4 and CD8 T cells. In this context, CD25 is no longer a reliable surface marker for Tregs. We observed strong co-expression of FoxP3 with surface marker GITR instead of CD25 in RUX-treated BMF mice. Fluorescence-activated cell sorting (FACS)-sorted CD4+GITRhi cells showed high FoxP3 expression and intact suppressive function in vitro, suggesting GITR to be a surrogate marker for Tregs. In contrast to its expansive effect on Tregs in BMF, RUX suppressed Tregs in normal and sublethal irradiation conditions, indicating that the effects of RUX on Tregs are immune-context dependent.

Cancer-associated fibroblasts drive CXCL13 production in activated T cells via TGF-beta.

Tumour-reactive T cells producing the B-cell attractant chemokine CXCL13, in solid tumours, promote development of tertiary lymphoid structures (TLS) and are associated with improved prognosis and responsiveness to checkpoint immunotherapy. Cancer associated fibroblasts are the dominant stromal cell type in non-small cell lung cancer (NSCLC) where they co-localise with T cells and can influence T cell activation and exhaustion. We questioned whether CAF directly promote CXCL13-production during T cell activation. We characterised surface markers, cytokine production and transcription factor expression in CXCL13-producing T cells in NSCLC tumours and paired non-cancerous lung samples using flow cytometry. We then assessed the influence of human NSCLC-derived primary CAF lines on T cells from healthy donors and NSCLC patients during activation in vitro measuring CXCL13 production and expression of cell-surface markers and transcription factors by flow cytometry. CAFs significantly increased the production of CXCL13 by both CD4+ and CD8+ T cells. CAF-induced CXCL13-producing cells lacked expression of CXCR5 and BCL6 and displayed a T peripheral helper cell phenotype. Furthermore, we demonstrate CXCL13 production by T cells is induced by TGF-β and limited by IL-2. CAF provide TGF-β during T cell activation and reduce availability of IL-2 both directly (by reducing the capacity for IL-2 production) and indirectly, by expanding a population of activated Treg. Inhibition of TGF-β signalling prevented both CAF-driven upregulation of CXCL13 and Treg expansion. Promoting CXCL13 production represents a newly described immune-regulatory function of CAF with the potential to shape the immune infiltrate of the tumour microenvironment both by altering the effector-function of tumour infiltrating T-cells and their capacity to attract B cells and promote TLS formation.

Brain-specific regulatory T cell expansion limits cognitive decline.

Recent work from Lemaitre and colleagues leveraged a central nervous system (CNS)-specific gene delivery approach to expand regulatory T cells (Treg) in aged mice. CNS-restricted Treg expansion reversed age-related glial cell transcriptomic changes and prevented aspects of cognitive decline, unveiling immune modulation as a potential approach to protect cognitive function with age.

Stimulation of Immune Checkpoint Molecule B and T-Lymphocyte Attenuator Alleviates Experimental Crescentic Glomerulonephritis.

Treatment of acute, crescentic glomerulonephritis (GN) consists of unspecific and potentially toxic immunosuppression. T cells are central in the pathogenesis of GN, and various checkpoint molecules control their activation. The immune checkpoint molecule B and T-lymphocyte attenuator (BTLA) has shown potential for restraining inflammation in other T-cell-mediated disease models. To investigate its role in GN in a murine model of crescentic nephritis, the authors induced nephrotoxic nephritis in BTLA-deficient mice and wild-type mice. They found that BTLA has a renoprotective role through suppression of local Th1-driven inflammation and expansion of T regulatory cells and that administration of an agonistic anti-BTLA antibody attenuated experimental GN. These findings suggest that antibody-based modulation of BTLA may represent a treatment strategy in human glomerular disease. Modulating T-lymphocytes represents a promising targeted therapeutic option for glomerulonephritis (GN) because these cells mediate damage in various experimental and human GN types. The immune checkpoint molecule B and T-lymphocyte attenuator (BTLA) has shown its potential to restrain inflammation in other T-cell-mediated disease models. Its role in GN, however, has not been investigated. We induced nephrotoxic nephritis (NTN), a mouse model of crescentic GN, in Btla -deficient ( BtlaKO ) mice and wild-type littermate controls and assessed disease severity using functional and histologic parameters at different time points after disease induction. Immunologic changes were comprehensively evaluated by flow cytometry, RNA sequencing, and in vitro assays for dendritic cell and T-cell function. Transfer experiments into Rag1KO mice confirmed the observed in vitro findings. In addition, we evaluated the potential of an agonistic anti-BTLA antibody to treat NTN in vivo . The BtlaKO mice developed aggravated NTN, driven by an increase of infiltrating renal Th1 cells. Single-cell RNA sequencing showed increased renal T-cell activation and positive regulation of the immune response. Although BTLA-deficient regulatory T cells (Tregs) exhibited preserved suppressive function in vitro and in vivo , BtlaKO T effector cells evaded Treg suppression. Administration of an agonistic anti-BTLA antibody robustly attenuated NTN by suppressing nephritogenic T effector cells and promoting Treg expansion. In a model of crescentic GN, BTLA signaling effectively restrained nephritogenic Th1 cells and promoted regulatory T cells. Suppression of T-cell-mediated inflammation by BTLA stimulation may prove relevant for a broad range of conditions involving acute GN.

Cancer cell plasticity and MHC-II-mediated immune tolerance promote breast cancer metastasis to lymph nodes.

Tumor-draining lymph nodes (TDLNs) are important for tumor antigen-specific T cell generation and effective anticancer immune responses. However, TDLNs are often the primary site of metastasis, causing immune suppression and worse outcomes. Through cross-species single-cell RNA-Seq analysis, we identified features defining cancer cell heterogeneity, plasticity, and immune evasion during breast cancer progression and lymph node metastasis (LNM). A subset of cancer cells in the lymph nodes exhibited elevated MHC class II (MHC-II) gene expression in both mice and humans. MHC-II+ cancer cells lacked costimulatory molecule expression, leading to regulatory T cell (Treg) expansion and fewer CD4+ effector T cells in TDLNs. Genetic knockout of MHC-II reduced LNM and Treg expansion, while overexpression of the MHC-II transactivator, Ciita, worsened LNM and caused excessive Treg expansion. These findings demonstrate that cancer cell MHC-II expression promotes metastasis and immune evasion in TDLNs.

Bioengineered particles expand myelin-specific regulatory T cells and reverse autoreactivity in a mouse model of multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease characterized by autoreactive immune cells damaging myelinated nerves, impairing brain function. Treatments aim for tolerance induction to reeducate the immune system to recognize myelin as "self" rather than "foreign." As peripheral immune tolerance is primarily mediated by regulatory T cells (Tregs), we developed a therapy to support Treg expansion and activity in vivo. To target, engage, and activate myelin-specific Tregs, we designed a biodegradable microparticle (MP) loaded with rapamycin and functionalized with a biased interleukin-2 (IL-2) fusion protein and a major histocompatibility complex (MHC) class II loaded with a myelin peptide. These tolerogenic MPs (Tol-MPs) were validated in vitro and then evaluated in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Tol-MPs promoted sustained disease reversal in 100% of mice and full recovery in 38% of mice with symptomatic EAE. Tol-MPs are a promising platform for treatment of autoimmune diseases.

IL-27-containing exosomes secreted by innate B-1a cells suppress and ameliorate uveitis.

IL-27 is a heterodimeric cytokine composed of Ebi3 and IL-27p28 and can exert proinflammatory or immune suppressive effects depending on the physiological context. Ebi3 does not contain membrane-anchoring motifs, suggesting that it is a secreted protein while IL-27p28 is poorly secreted. How IL-27p28 and Ebi3 dimerize in-vivo to form biologically active IL-27 is unknown. Major impediment to clinical use of IL-27 derives from difficulty of determining exact amount of bioavailable heterodimeric IL-27 needed for therapy. To understand how IL-27 mediates immune suppression, we characterized an innate IL-27-producing B-1a regulatory B cell population (i27-Breg) and mechanisms i27-Bregs utilize to suppress neuroinflammation in mouse model of uveitis. We also investigated biosynthesis of IL-27 and i27-Breg immunobiology by FACS, immunohistochemical and confocal microscopy. Contrary to prevailing view that IL-27 is a soluble cytokine, we show that i27-Bregs express membrane-bound IL-27. Immunohistochemical and confocal analyses co-localized expression of IL-27p28 at the plasma membrane in association with CD81 tetraspanin, a BCR-coreceptor protein and revealed that IL-27p28 is a transmembrane protein in B cells. Most surprising, we found that i27-Bregs secrete IL-27-containing exosomes (i27-exosomes) and adoptive transfer of i27-exosomes suppressed uveitis by antagonizing Th1/Th17 cells, up-regulating inhibitory-receptors associated with T-cell exhaustion while inducing Treg expansion. Use of i27-exosomes thus obviates the IL-27 dosing problem, making it possible to determine bioavailable heterodimeric IL-27 needed for therapy. Moreover, as exosomes readily cross the blood-retina-barrier and no adverse effects were observed in mice treated with i27-exosome, results of this study suggest that i27-exosomes might be a promising therapeutic approach for CNS autoimmune diseases.

Development of a CCR8 monoclonal antibody to block CCR8 signaling and to abolish the immunosuppressive function of Treg for the treatment of cancers.

e14506 Background: The tumor microenvironment (TME) contains regulatory T (Treg) cells, which play a critical role in immunosuppression and are thus an attractive target for cancer therapy. The ideal approach would be to inhibit Tregs within tumors while sparing those in peripheral and normal tissues. Tumor-associated Tregs express the chemokine receptor CCR8, which is involved in the induction, expansion, migration, and immunosuppression of Tregs, making targeted inhibition of CCR8 signaling a potential therapeutic strategy against cancer. Methods: We have developed IPG0521, a monoclonal antibody antagonist of CCR8, and characterized its properties, including signaling blocking, chemotaxis, species cross-reactivity, anti-tumor efficacy, and synergistic antitumor effect with PD1 antibody. Single-cell RNA sequencing with CD45+ cells isolated from tumors treated with or without IPG0521 was also employed for profiling the mechanism of the antitumor effect of IPG0521 in the murine H22 cancer model. Results: IPG0521 inhibits receptor-mediated signaling and chemotaxis in Treg cells with a single-digit nanomolar IC50. It is cross-reactive in five species: mouse, rat, dog, monkey, and human. IPG0521 has shown excellent anti-tumor efficacy in several murine syngeneic cancer models, and its anti-tumor effect is enhanced when combined with a PD-1 antibody. Its sustained anti-tumor efficacy has been demonstrated in the murine H22 cancer model via even withdrawing the antibody during the study. The results of deep single-cell RNA sequencing on tumor-infiltrating lymphocytes (TILs) showed that the transcriptional profiles of these Treg cells could be distinguished into three distinct subsets based on molecular, functional, and immunosuppressive properties. These subsets were defined as the Treg_Foxp3 high subset, which exhibited low immunosuppression, the Treg_Foxp3 mild subset, which demonstrated mild immunosuppression, and the Treg_Foxp3 low subset, which had high proliferation. IPG0521 treatment resulted in a significant increase in the Treg_ Foxp3 high subset and a significant decrease in the Treg _ Foxp3 mild subset while no change in the Treg_ Foxp3 low subset, indicating a reduction in Treg immunosuppression. This change in Treg cells also altered the composition of other immune cells, including a significant increase in neutrophils and natural killer (NK) cells, which ultimately inhibited tumor growth. Conclusions: IPG0521 has potent anti-tumor effects in multiple tumor types, including lung, liver, and breast cancers, in both syngeneic and immune-humanized mouse models. It inhibits Tregs immunosuppression and activates CD8+ T cells to achieve its anti-tumor effects. These results support the development of IPG0521, which is currently under IND filing, as a novel therapeutic agent for the treatment of cancers.

Robust IL-2-dependent antitumor immunotherapy requires targeting the high-affinity IL-2R on tumor-specific CD8+ T cells

BackgroundDevelopment of interleukin (IL)-2-dependent antitumor responses focus on targeting the intermediate affinity IL-2R to stimulate memory-phenotypic CD8+ T and natural killer (NK) cells while minimizing regulatory T cell (Treg) expansion....

Th17/Treg balance is regulated by myeloid-derived suppressor cells in experimental autoimmune myocarditis.

Autoimmune myocarditis is caused by both innate and adaptive immune responses. Many studies have found that myeloid-derived suppressor cells (MDSCs) suppress T-cell responses and reduce immune tolerance, while MDSCs may serve as a key player in inflammatory responses and pathogenesis in variety of autoimmune diseases. However, research into the role of MDSCs in experimental autoimmune myocarditis (EAM) remains lacking. We discovered that the expansion of MDSCs in EAM was closely related to the severity of myocardial inflammation. At an early stage of EAM, both adoptive transfer (AT) and selective depletion of MDSCs could inhibit the expression of IL-17 in CD4+ cells and downregulate the Th17/Treg ratio, alleviating excessive inflammation of EAM myocarditis. In another experiment, in addition, MDSCs transferred after selective depletion could increase IL-17 and Foxp3 expressions in CD4+ cells, as well as the Th17/Treg ratio, contributing to the aggravation of myocardial inflammation. MDSCs promoted the Th17 cell induction under Th17-polarizing conditions in vitro but suppressed Treg expansion. These findings suggest that MDSCs play a plastic role in sustaining mild inflammation in EAM by shifting Th17/Treg balance.

Negative Feedback Expansion of Tregs Caused by Endogenous IL-2 Limits the Activity of IL-2-based Therapies

Stimulating effector T-cells (Teffs) without inducing regulatory T-cells (Tregs) has been the primary goal of IL-2-based therapies for cancer. Recently, modified IL-2 designed for differential T-cell expansion for the treatment of cancer has failed in the clinic. We propose that treatments based on exogenous administrations of modified IL-2 are inherently undermined by a negative feedback loop, caused by IL-2 secreted endogenously from activated effector T-cells. This endogenous IL-2 secretion subsequentially induces Treg expansion and inhibits the immune response that is essential for cancer clearance. Here, we demonstrate that treatments utilizing exogenous modified IL-2 indeed induce Treg expansion. To circumvent this negative feedback, we computationally designed a novel monoclonal humanized antibody (AU-007) that binds human IL-2 with pM affinity at a predefined epitope and completely blocks IL-2 binding to CD25 that is highly expressed on Tregs, without hindering IL-2 binding to CD122/CD132 dimer receptor expressed over effector cells. This epitope-specific, high-affinity antibody controls endogenous IL-2 and prevents it from expanding Tregs while allowing it to expand Teffs. We show that controlling endogenous IL-2 using AU-007 abrogates the negative feedback loop and replaces it with a positive feedback loop that enhances the expansion of NK cells and Teffs, an effect considered favorable for cancer immunotherapy.

New Flow Cytometric Methods for Monitoring STAT5 Signaling Reveal Responses to SARS-CoV-2 Antigen-Specific Stimulation in FOXP3+ Regulatory T Cells also in Patients with Advanced Chronic Lymphocytic Leukemia

Increased frequency of CD4+CD25+ regulatory T-cells (Treg) has been associated with disease progression in chronic lymphocytic leukemia (CLL). Flow cytometric methods, which allow for the simultaneous analysis of their specific transcription factor Foxp3 and activated STAT proteins, together with proliferation can help to elucidate the signaling mechanisms driving Treg expansion and suppression of FOXP3- conventional CD4+T-cells (Tcon). Herein, we first report a novel approach in which STAT5 phosphorylation (pSTAT5) and proliferation (BrdU-FITC incorporation) could be analyzed specifically in FOXP3+ and FOXP3- responding cells after CD3/CD28 stimulation. The addition of magnetically purified CD4+CD25+ T-cells from healthy donors to cocultured autologous CD4+CD25- T-cells resulted in suppression of Tcon cell cycle progression accompanied by a decrease in pSTAT5. Next, a method using imaging flow cytometry is presented for the detection of cytokine-dependent pSTAT5 nuclear translocation in FOXP3-expressing cells. Finally, we discuss our experimental data obtained by combining Treg pSTAT5 analysis and antigen-specific stimulation with SARS-CoV-2 antigens. Applying these methods on samples from patients revealed Treg responses to antigen-specific stimulation and significantly higher basal pSTAT5 in CLL patients treated with immunochemotherapy. Thus, we speculate that through the use of this pharmacodynamic tool, the efficacy of immunosuppressive drugs and their possible off-target effects can be assessed.

Immunosuppressive Polymeric Nanoparticles Targeting Dendritic Cells Alleviate Lupus Disease in Fcgr2b-/- Mice by Mediating Antigen-Specific Immune Tolerance.

Dendritic cells (DCs) are the most potent antigen-presenting cells that have multifaceted functions in the control of immune activation and tolerance. Hyperresponsiveness and altered tolerogenicity of DCs contribute to the development and pathogenesis of system lupus erythematosus (SLE); therefore, DC-targeted therapies aimed at inducing specific immune tolerance have become of great importance for the treatment of SLE. This study developed a new nanoparticle (NP) containing a biodegradable PDMAEMA-PLGA copolymer for target-oriented delivery to DCs in situ. PDMAEMA-PLGA NPs provided sustained drug release and exhibited immunosuppressive activity in FLT3L and GM-CSF-derived bone marrow in conventional DCs (BM-cDCs). PDMAEMA-PLGA NPs improved dexamethasone capability to convert wild-type and Fcgr2b-/- BM-cDCs from an immunogenic to tolerogenic state, and BM-cDCs treated with dexamethasone-incorporated PDMAEMA-PLGA NPs (Dex-NPs) efficiently mediated regulatory T cell (Treg) expansion in vitro. Dex-NP therapy potentially alleviated lupus disease in Fcgr2b-/- mice by mediating Foxp3+ Treg expansion in an antigen-specific manner. Our findings substantiate the superior efficacy of DC-targeted therapy using the PDMAEMA-PLGA NP delivery system and provide further support for clinical development as a potential therapy for SLE. Furthermore, PDMAEMA-PLGA NP may be a versatile platform for DC-targeted therapy to induce antigen-specific immune tolerance to unwanted immune responses that occur in autoimmune disease, allergy, and transplant rejection.

OX40L-expressing recombinant modified vaccinia virus Ankara induces potent antitumor immunity via reprogramming Tregs.

Effective depletion of immune suppressive regulatory T cells (Tregs) in the tumor microenvironment without triggering systemic autoimmunity is an important strategy for cancer immunotherapy. Modified vaccinia virus Ankara (MVA) is a highly attenuated, non-replicative vaccinia virus with a long history of human use. Here, we report rational engineering of an immune-activating recombinant MVA (rMVA, MVA∆E5R-Flt3L-OX40L) with deletion of the vaccinia E5R gene (encoding an inhibitor of the DNA sensor cyclic GMP-AMP synthase, cGAS) and expression of two membrane-anchored transgenes, Flt3L and OX40L. Intratumoral (IT) delivery of rMVA (MVA∆E5R-Flt3L-OX40L) generates potent antitumor immunity, dependent on CD8+ T cells, the cGAS/STING-mediated cytosolic DNA-sensing pathway, and type I IFN signaling. Remarkably, IT rMVA (MVA∆E5R-Flt3L-OX40L) depletes OX40hi regulatory T cells via OX40L/OX40 interaction and IFNAR signaling. Single-cell RNA-seq analyses of tumors treated with rMVA showed the depletion of OX40hiCCR8hi Tregs and expansion of IFN-responsive Tregs. Taken together, our study provides a proof-of-concept for depleting and reprogramming intratumoral Tregs via an immune-activating rMVA.

CD4 +Treg expansion arrests disease progression in a pre-clinical model of alopecia areata

Abstract Alopecia Areata (AA) is a prevalent autoimmune disease that results in nonscarring hair loss. Disease is associated with the collapse of an immune privileged state of the hair follicle, characterized by increased MHC expression, immune cell infiltration and the presence of pro-inflammatory cytokines in the microenvironment. CD8 T cells have been identified as the main contributors to disease and the activating receptor NKG2D can identify this pathogenic population. There is currently one FDA-approved drug for AA, a JAK1/2 inhibitor, which is thought to act by dampening inflammatory mediators during disease. However, the potential of adverse effects and refractory patients necessitates the development of alternative therapeutics. Emerging data from our lab suggests that the observed expansion of CD4 T regulatory cells (Tregs), relative to effector CD8 T cells during AA, is inadequate to prevent disease onset. We hypothesized that pharmacologic enhancement of Treg numbers may restrain development of AA. To address this, we investigated the protective potential of Treg expansion using a murine model of AA. In vivo expansion of CD4 +CD25 +FoxP3 +Tregs was achieved using an IL-2 cytokine/antibody complex (IL-2C), which is known to selectively expand and enhance Tregs. We found that 6 weeks of treatment with IL-2C halted the progression of hair loss, while control mice exhibited robust hair loss. Additionally, IL-2C treatment led to a reduction in NKG2D +CD8 T cells and IFNγ-producing CD8 T cells found infiltrating the skin, suggesting that increasing Tregs in the system is an effective strategy for restraining disease. Our findings provide translational insight for the use of Treg enrichment as a therapeutic strategy for patients with AA. Supported by grants from NIH (R01 AR077194) and Dept. of VA (I01 BX004907)

Abstract 305: Dietary β-carotene Accelerates Atherosclerotic Resolution By Promoting Treg Expansion In The Atherosclerosis Lesion

Forkhead box P3 ( FoxP3 ) + regulatory T cells (Tregs) mediate atherosclerosis resolution and regression by promoting an anti-inflammatory environment in the lesion. FoxP3 is upregulated by retinoic acid, the active form of vitamin A, but whether vitamin A status influences atherosclerosis resolution remains unexplored. We hypothesized that β-carotene, the main vitamin A precursor in human diet, accelerates atherosclerosis resolution by upregulating FoxP3 expression to promote Treg expansion. We induced atherosclerosis in FoxP3 EGFP mice fed Western diet control (WD-Control) and injected with an anti-sense oligonucleotide targeting the LDLR (ASO-LDLR) for 16 weeks. After this period, we harvested a subset of mice as Baseline, while the remaining mice underwent atherosclerosis resolution upon the interruption of ASO-LDLR infusions to recover LDLR expression and normalize plasma lipids for a period of three weeks. During this time, mice either continued on the same WD-Control or were switched to a Western diet supplemented with 50 mg of β-carotene/kg of diet (WD- β-carotene). To examine the implication of Tregs, we injected a subset of WD-β-carotene with anti-CD25 (WD-β-carotene + anti-CD25), while WD-Control and a second subset of WD- β-carotene mice were injected with IgG control. We estimated atherosclerosis resolution by quantifying macrophage and collagen contents in the lesions present at the level of the aortic root. WD-β-carotene-fed mice injected with IgG showed a reduction in macrophage content accompanied by an increase in collagen in comparison to all the remaining groups, while WD-β-carotene + anti-CD25 lesions resembled those present in WD-Control mice. We examined Treg the relative number of CD25 + FoxP3 + and CD25 - FoxP3 + cells in the spleen, blood, and the lesion. CD25 + FoxP3 + numbers decreased in response to anti-CD25 infusions in all the tissues examined. Dietary β-carotene favored an increase of 269% and 653% in CD25 - FoxP3 + Tregs in comparison to the Baseline of WD-Control groups, respectively. These effects occurred independently of changes in plasma lipids or changes in body weight. Our data show that β-carotene supplementation results in Treg expansion and acceleration of atherosclerosis resolution by increasing Treg number.

Regulatory T cells aid Leishmania majorimmune evasion by suppressing effector responses against the parasite in an antigen-specific manner

Abstract Despite the generation of a strong T cell response, clearance of Leishmania major is incomplete and leaves a pool of chronically infected cells. Understanding of the persistence is lacking, but Leishmania major driven induction of the immunosuppressive microenvironment through recruitment of regulatory T cells at the site of infection has been proposed to prevent parasite clearance in vivo. Here, we used a novel TCR transgenic mouse model, where CD4 +T cells recognize an immunodominant peptide derived from Leishmania-glycosomal phosphoenolpyruvate carboxykinase (PEPCK), to visualize the dynamics of anti-L. major CD4 +T cell responses and characterize mechanisms which restrain their effector function. We show that PEPCK-specific Tregs are able to suppress nearly 80% of IFNg produced by PEPCK-specific Th1 cells in vitro though the production of IL-10, while decreasing IL-12, TNF, and IL-2. Intravital microscopy studies characterizing PEPCK-specific CD4 +T cell migration dynamics in live mice show a significant recruitment of adoptively transferred cells to the lesion site in vivo, displaying cellular behaviors consistent with antigen recognition, yet augmented at the chronic stage, indicating a fundamentally altered environment. Injection of heat-killed L. major into mice which have already healed a L. major infection, distal to the original lesion, induced a rapid expansion of PEPCK-specific Tregs at the original site of infection, which suppressed endogenous anti-Leishmania response through IL-10 and led to parasite expansion and lesion reactivation. Our results show that antigen recognition by Tregs plays an important role in suppressing effector responses against L. major and thus maintaining concomitant immunity. Research funded by a grant from the GSK-CIHR Partnered Chair Program

Chimeric cytokine receptor provides IL-2 signaling for engineered regulatory T cell therapy

Abstract Regulatory T cells (Tregs) are potent immune suppressors and critical to the maintenance of immunological tolerance. Interleukin 2 (IL-2) is essential for Treg survival, expansion, phenotypic stability, and suppressive function. However, Tregs do not produce IL-2 and are dependent on exogenous sources of IL-2. This poses a significant challenge for Treg cell therapies in tissues with no or limited IL-2 availability in the microenvironment. Therefore, we have developed engineered Treg cells containing a chimeric cytokine receptor that provides an IL-2 signal. The chimeric cytokine receptor, termed IL-9 tethered switch receptor (IL-9TSR), consists of interleukin 9 (IL-9) tethered to the extracellular domain of IL9RA paired with the intracellular domain of IL2RB. We have shown that Tregs expressing IL-9TSR can survive and expand in the absence of exogenous IL-2 in vitro and in vivo but require TCR stimulation for prolonged persistency. IL-9TSR Tregs have a stable FOXP3 +HELIOS +phenotype and superior suppressive function when compared to control Tregs. In conclusion, we have generated Treg cells engineered to survive and function in a low IL-2 environment. Treg cell therapies equipped with IL-2 signaling enable increased persistency and allow for potential use in more clinical indications than previously possible.

Microparticle-delivered stimulator of interferon genes (STING) agonist suppresses experimental autoimmune encephalomyelitis (EAE) via expansion of FoxP3 +CD4 +Tregs

Abstract Background: The cGAS-cyclic GMP-AMP (cGAMP)-stimulator of intracellular STING receptors induces a powerful IFN-I response. Microparticles (MPs), synthesized biodegradable polymer, passively target the APCs, resulting in intracellular delivery of immuno-regulatory treatment with dose sparing and better tolerability. cGAMP MP treatment of EAE induced both IFN-I-dependent and -independent immunosuppressive responses through induction of IL-27 and IL-10. Results: We show that cGAMP MP treatment reduced clinical disease scores of EAE after 3 doses. By flow cytometry, cGAMP MPs decreased absolute number of mononuclear cells in the CNS infiltrates, and the percentages of CD3 −CD45 highCD11 +b Ly6C +monocytes, CD11c +and CD11c +CD11b +DCs, as well as their expression of CD80 and CD86, suggestive of tolerogenic phenotype. Moreover, we detected an increased percentage of FOXP3 +CD4 +Tregs in CNS infiltrates, with increased expression of IL-10, IL-27R, and Granzyme B, and decreased percentage of IL-17A +CD4 +cells in cGAMP MP-treated mice. Furthermore, the supernatants of the in vitroco-cultured cGAMP MP-treated monocytes with CD3 +CD4 +, isolated from EAE spleenocytes, show increased IL-10 and IL-27 levels. Additionally, the in vitroD3 +CD4 +cells show increased FOXP3 +expression with higher CTLA-4, and GITR in cGAMP treated group. Moreover, FOXP3cre+/+ IL-27RA fl/fl mice showed more aggressive disease compared to wild-type mice in attempt to test whether IL-27 induced effect of cGAMP MPs is mediated via Tregs. Conclusion: cGAMP MPs suppress RREAE through induction of tolerogenic APCs and increase secretion of IL-10 and IL-27, which induce Treg expansion indicating its potential use as a tolerogenic therapy in patients with RRMS.

A KIR x CD8 targeting bispecific modulator enhances regulatory CD8 T cell functions, and reduces inflammation in models of autoimmune disease

Abstract INTRODUCTION: We have characterized a subset of CD8 T cells (CD8 Treg) with immunosuppressive characteristics in inflammatory disease settings. CD8 Treg activation results in their oligoclonal expansion and elimination of pathogenic CD4 T cells. Here we describe a bispecific CD8 Treg modulator that activates autoimmune patient derived CD8 Treg resulting in the cytolytic elimination of pathogenic CD4 T cell in vitro, ex vivo, and in vivo. METHODS: We tested target and cell binding of a novel bispecific CD8 Treg modulator by Octet and flow cytometry. The functional impact was evaluated using cytotoxicity assays and supernatant analysis in vitro, using Celiac patient derived biopsies ex vivo, and in acute GVHD studies using human PBMCs. RESULTS: We evaluated the efficacy and mechanism of action of a novel bispecific CD8 Treg modulator directed toward CD8 and inhibitory KIR2DL1/2/3. The modulator enhanced cytolytic CD8 Treg functions in vitro using autoimmune patient PBMCs, increased activated CD4 T cell death, and decreased pro-inflammatory cytokines. In Celiac patient derived duodenal tissues, the modulator increased CD8 Treg prevalence and reduced epithelial cell death after gliadin peptide stimulation. In acute GVHD studies, CD8 Treg modulator treated mice had CD8 Treg expansion in a dose dependent fashion and reduced inflammation. CONCLUSIONS: A novel KIR x CD8 targeting bispecific modulator restores cytolytic function of CD8 Treg, resulting in a decrease of pathogenic CD4 T cells. We postulate that the bispecific CD8 T cell modulator can reduce autoreactive CD4 T cell prevalence to reestablish immune balance and may represent a selective and broadly applicable therapeutic approach for the treatment of autoimmune disease.