CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity.

The incidence of autoimmune diseases has significantly increased over the past 20 years. Excessive host immunoreactions and disordered immunoregulation are at the core of the pathogenesis of autoimmune diseases. The traditional anti-tumor chemotherapy drug CPT-11 is associated with leukopenia. Considering that CPT-11 induces leukopenia, we believe that it is a promising drug for the control of autoimmune diseases. Here, we show that CPT-11 suppresses T cell proliferation and pro-inflammatory cytokine production in healthy C57BL/6 mice and in complete Freund's adjuvant-challenged mice. We found that CPT-11 effectively inhibited T cell proliferation and Th1 and Th17 cell differentiation by inhibiting glycolysis in T cells. We also assessed CPT-11 efficacy in treating autoimmune diseases in models of experimental autoimmune encephalomyelitis and psoriasis. Finally, we proved that treatment of autoimmune diseases with CPT-11 did not suppress long-term immune surveillance for cancer. Taken together, these results show that CPT-11 is a promising immunosuppressive drug for autoimmune disease treatment.

Sialyl Lewis X Defines an Activated and Functional Regulatory T Cell Subpopulation in Mice.

Attempts have been made to elucidate the functional markers of regulatory T cells (Tregs), CD4+Foxp3+ T cells with an immunosuppressive function. Sialyl Lewis X (sLex), a tetrasaccharide Ag, is involved in leukocyte trafficking as selectin ligands and is a marker of highly differentiated Tregs in humans. However, the importance of sLex in murine Tregs remains unknown. In this study, we report that sLex defines the activated and functional subset of murine Tregs. The contact hypersensitivity model showed that murine Tregs strongly express sLex upon activation, accompanied by functional Treg marker elevation, such as Foxp3, CD25, CD103, CD39, and granzyme B. RNA sequencing analysis revealed sLex-positive (sLex+) Tregs expressed genes involved in Treg function at a higher level than sLex-negative (sLex-) Tregs. Using an in vitro suppression assay, we found that sLex+ Tregs could more efficiently suppress naive CD4+ T cell proliferation than sLex- Tregs. In the murine contact hypersensitivity elicitation model, the topical sLex+ Treg injection into the ears suppressed ear inflammation more efficiently than that of sLex- Tregs. Our results indicate that sLex could serve as a unique surface marker of activated and functional Tregs with immunosuppressive functions in mice.

Prevention of experimental autoimmune encephalomyelitis by targeting 6-sulfo sialyl Lewis X glycans involved in lymphocyte homing.

Lymphocyte homing to peripheral lymph nodes (PLN) is critical for immune surveillance. However, autoimmune diseases such as multiple sclerosis (MS) can occur due to excessive immune responses in the PLN. Here we show that 6-sulfo sialyl Lewis X (6-sulfo sLex) glycans on high endothelial venules that function as ligands for l-selectin on lymphocytes play a critical role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2 double-knockout mice lacking the expression of 6-sulfo sLeX glycans, the EAE symptoms and the numbers of effector Th1 and Th17 cells in the draining lymph nodes (dLN) and spinal cords (SC) were significantly reduced. To determine whether 6-sulfo sLeX could serve as a target for MS, we also examined the effects of anti-glycan monoclonal antibody (mAb) SF1 against 6-sulfo sLeX in EAE. Administration of mAb SF1 significantly reduced EAE symptoms and the numbers of antigen-specific effector T cells in the dLN and SC in association with suppression of critical genes including Il17a and Il17f that are involved in the pathogenesis of EAE. Taken together, these results suggest that 6-sulfo sLeX glycan would serve as a novel target for MS.

Opposing functions of circadian protein DBP and atypical E2F family E2F8 in anti-tumor Th9 cell differentiation.

Interleukin-9 (IL-9)-producing CD4+ T helper cells (Th9) have been implicated in allergy/asthma and anti-tumor immunity, yet molecular insights on their differentiation from activated T cells, driven by IL-4 and transforming growth factor-beta (TGF-ß), is still lacking. Here we show opposing functions of two transcription factors, D-binding protein (DBP) and E2F8, in controlling Th9 differentiation. Specifically, TGF-ß and IL-4 signaling induces phosphorylation of the serine 213 site in the linker region of the Smad3 (pSmad3L-Ser213) via phosphorylated p38, which is necessary and sufficient for Il9 gene transcription. We identify DBP and E2F8 as an activator and repressor, respectively, for Il9 transcription by pSmad3L-Ser213. Notably, Th9 cells with siRNA-mediated knockdown for Dbp or E2f8 promote and suppress tumor growth, respectively, in mouse tumor models. Importantly, DBP and E2F8 also exhibit opposing functions in regulating human TH9 differentiation in vitro. Thus, our data uncover a molecular mechanism of Smad3 linker region-mediated, opposing functions of DBP and E2F8 in Th9 differentiation.

STAP-2 Is a Novel Positive Regulator of TCR-Proximal Signals.

TCR ligation with an Ag presented on MHC molecules promotes T cell activation, leading to the selection, differentiation, and proliferation of T cells and cytokine production. These immunological events are optimally arranged to provide appropriate responses against a variety of pathogens. We here propose signal-transducing adaptor protein-2 (STAP-2) as a new positive regulator of TCR signaling. STAP-2-deficient T cells showed reduced, whereas STAP-2-overexpressing T cells showed enhanced, TCR-mediated signaling and downstream IL-2 production. For the mechanisms, STAP-2 associated with TCR-proximal CD3ζ immunoreceptor tyrosine activation motifs and phosphorylated LCK, resulting in enhancement of their binding after TCR stimulation. In parallel, STAP-2 expression is required for full activation of downstream TCR signaling. Importantly, STAP-2-deficient mice exhibited slight phenotypes of CD4+ T-cell-mediated inflammatory diseases, such as experimental autoimmune encephalomyelitis, whereas STAP-2-overexpressing transgenic mice showed severe phenotypes of these diseases. Together, STAP-2 is an adaptor protein to enhance TCR signaling; therefore, manipulating STAP-2 will have an ability to improve the treatment of patients with autoimmune diseases as well as the chimeric Ag receptor T cell therapy.

Single-Cell Analysis Revealed the Role of CD8+ Effector T Cells in Preventing Cardioprotective Macrophage Differentiation in the Early Phase of Heart Failure.

Heart failure is a complex clinical syndrome characterized by insufficient cardiac function. Heart-resident and infiltrated macrophages have been shown to play important roles in the cardiac remodeling that occurs in response to cardiac pressure overload. However, the possible roles of T cells in this process, have not been well characterized. Here we show that T cell depletion conferred late-stage heart protection but induced cardioprotective hypertrophy at an early stage of heart failure caused by cardiac pressure overload. Single-cell RNA sequencing analysis revealed that CD8+T cell depletion induced cardioprotective hypertrophy characterized with the expression of mitochondrial genes and growth factor receptor genes. CD8+T cells regulated the conversion of both cardiac-resident macrophages and infiltrated macrophages into cardioprotective macrophages expressing growth factor genes such as Areg, Osm, and Igf1, which have been shown to be essential for the myocardial adaptive response after cardiac pressure overload. Our results demonstrate a dynamic interplay between cardiac CD8+T cells and macrophages that is necessary for adaptation to cardiac stress, highlighting the homeostatic functions of resident and infiltrated macrophages in the heart.

The Curcumin Analog GO-Y030 Controls the Generation and Stability of Regulatory T Cells.

Regulatory T cells (Tregs) play a crucial role in preventing antitumor immune responses in cancer tissues. Cancer tissues produce large amounts of transforming growth factor beta (TGF-ß), which promotes the generation of Foxp3+ Tregs from naïve CD4+ T cells in the local tumor microenvironment. TGF-ß activates nuclear factor kappa B (NF-κB)/p300 and SMAD signaling, which increases the number of acetylated histones at the Foxp3 locus and induces Foxp3 gene expression. TGF-ß also helps stabilize Foxp3 expression. The curcumin analog and antitumor agent, GO-Y030, prevented the TGF-ß-induced generation of Tregs by preventing p300 from accelerating NF-κB-induced Foxp3 expression. Moreover, the addition of GO-Y030 resulted in a significant reduction in the number of acetylated histones at the Foxp3 promoter and at the conserved noncoding sequence 1 regions that are generated in response to TGF-ß. In vivo tumor models demonstrated that GO-Y030-treatment prevented tumor growth and reduced the Foxp3+ Tregs population in tumor-infiltrating lymphocytes. Therefore, GO-Y030 exerts a potent anticancer effect by controlling Treg generation and stability.

Combination of apoptotic T cell induction and self-peptide administration for therapy of experimental autoimmune encephalomyelitis.

BACKGROUND: Clinical trials on multiple sclerosis with repeated injections of monoclonal antibodies depleting CD4+ T cells have not resulted in much success as a disease therapy. Here, we developed an immunotherapy for EAE in mice by combining a transient depletion of T cells together with the administration of neuron derived peptides. METHODS: EAE was induced in SJL and C57BL/6 mice, by proteolipid protein peptide PLP139-151 (pPLP) and myelin-oligodendrocyte glycoprotein MOG35-55 (pMOG) peptides, respectively. Anti-CD4 and anti-CD8 antibody were injected intraperitoneally before or after peptide immunization. EAE scores were evaluated and histology data from brain and spinal cord were analyzed. Splenocytes were isolated and CD4+, CD4+CD25- and CD4+CD25+ T cells were purified and cultured in the presence of either specific peptides or anti-CD3 antibody and proliferation of T cells as well as cytokines in supernatant were assessed. FINDINGS: This experimental treatment exhibited therapeutic effects on mice with established EAE in pPLP-susceptible SJL mice and pMOG-susceptible C57BL/6 mice. Mechanistically, we revealed that antibody-induced apoptotic T cells triggered macrophages to produce TGFß, and together with administered auto-antigenic peptides, generated antigen-specific Foxp3+ regulatory T cells (Treg cells) in vivo. INTERPRETATION: We successfully developed a specific immunotherapy to EAE by generating autoantigen-specific Treg cells. These findings have overcome the drawbacks of long and repeated depletion of CD4+ T cells, but also obtained long-term immune tolerance, which should have clinical implications for the development of a new effective therapy for multiple sclerosis. FUND: This research was supported by the Intramural Research Program of the NIH, NIDCR.

Loss of TET proteins in regulatory T cells promotes abnormal proliferation, Foxp3 destabilization and IL-17 expression.

Ten-eleven translocation (TET) proteins regulate DNA methylation and gene expression by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Although Tet2/Tet3 deficiency has been reported to lead to myeloid cell, B-cell and invariant natural killer T (iNKT) cell malignancy, the effect of TET on regulatory T cells (Tregs) has not been elucidated. We found that Tet2/Tet3 deficiency in Tregs led to lethal hyperproliferation of CD4+Foxp3+ T cells in the spleen and mesenteric lymph nodes after 5 months of age. Additionally, in aged Treg-specific Tet2/Tet3-deficient mice, serum IgG1, IgG3, IgM and IgE levels were markedly elevated. High IL-17 expression was observed in both Foxp3+ and Fopx3- CD4+ T cells, and adoptive transfer of Tet2/Tet3-deficient Tregs into lymphopenic mice inhibited Foxp3 expression and caused conversion into IL-17-producing cells. However, the conserved non-coding DNA sequence-2 (CNS2) region of the Foxp3 gene locus, which has been shown to be particularly important for stable Foxp3 expression, was only partly methylated. We identified novel TET-dependent demethylation sites in the Foxp3 upstream enhancer, which may contribute to stable Foxp3 expression. Together, these data indicate that Tet2 and Tet3 are involved in Treg stability and immune homeostasis in mice.

Brain regulatory T cells suppress astrogliosis and potentiate neurological recovery.

In addition to maintaining immune tolerance, FOXP3+ regulatory T (Treg) cells perform specialized functions in tissue homeostasis and remodelling1,2. However, the characteristics and functions of brain Treg cells are not well understood because there is a low number of Treg cells in the brain under normal conditions. Here we show that there is massive accumulation of Treg cells in the mouse brain after ischaemic stroke, and this potentiates neurological recovery during the chronic phase of ischaemic brain injury. Although brain Treg cells are similar to Treg cells in other tissues such as visceral adipose tissue and muscle3-5, they are apparently distinct and express unique genes related to the nervous system including Htr7, which encodes the serotonin receptor 5-HT7. The amplification of brain Treg cells is dependent on interleukin (IL)-2, IL-33, serotonin and T cell receptor recognition, and infiltration into the brain is driven by the chemokines CCL1 and CCL20. Brain Treg cells suppress neurotoxic astrogliosis by producing amphiregulin, a low-affinity epidermal growth factor receptor (EGFR) ligand. Stroke is a leading cause of neurological disability, and there are currently few effective recovery methods other than rehabilitation during the chronic phase. Our findings suggest that Treg cells and their products may provide therapeutic opportunities for neuronal protection against stroke and neuroinflammatory diseases.

IL-6, IL-17 and Stat3 are required for auto-inflammatory syndrome development in mouse.

Auto-inflammatory syndrome, a condition clinically distinct from rheumatoid arthritis, is characterized by systemic inflammation in tissues such as major joints, skin, and internal organs. Autonomous innate-immune activation is thought to promote this inflammation, but underlying pathological mechanisms have not been clarified nor are treatment strategies established. Here, we newly established a mouse model in which IL-1 signaling is conditionally activated in adult mice (hIL-1 cTg) and observed phenotypes similar to those seen in auto-inflammatory syndrome patients. In serum of hIL-1 cTg mice, IL-6 and IL-17 levels significantly increased, and signal transducer and activator of transcription 3 (Stat3) was activated in joints. When we crossed hIL-1 cTg with either IL-6- or IL-17-deficient mice or with Stat3 conditional knockout mice, phenotypes seen in hIL-1 cTg mice were significantly ameliorated. Thus, IL-6, IL-17 and Stat3 all represent potential therapeutic targets for this syndrome.

Reprogramming of Th1 cells into regulatory T cells through rewiring of the metabolic status.

T helper type 1 (Th1) cells form one of the most stable CD4 T-cell subsets, and direct conversion of fully differentiated Th1 to regulatory T (Treg) cells has been poorly investigated. Here, we established a culture method for inducing Foxp3 from Th1 cells of mice and humans. This is achieved simply by resting Th1 cells without T-cell receptor ligation before stimulation in the presence of transforming growth factor-beta (TGF-ß). We named the resulting Th1-derived Foxp3+ cells Th1reg cells. Mouse Th1reg cells showed an inducible Treg-like phenotype and suppressive ability both in vitro and in vivo. Th1reg cells could also be induced from in vivo-developed mouse Th1 cells. Unexpectedly, the resting process enabled Foxp3 expression not through epigenetic changes at the locus, but through metabolic change resulting from reduced mammalian target of rapamycin complex 1 (mTORC1) activity. mTORC1 suppressed TGF-ß-induced phosphorylation of Smad2/3 in Th1 cells, which was restored in rested cells. Our study warrants future research aiming at development of immunotherapy with Th1reg cells.

Inhibition of Nr4a Receptors Enhances Antitumor Immunity by Breaking Treg-Mediated Immune Tolerance.

Enhanced infiltration of regulatory T cells (Treg) into tumor tissue is detrimental to patients with cancer and is closely associated with poor prognosis as they create an immunosuppressive state that suppresses antitumor immune responses. Therefore, breaking Treg-mediated immune tolerance is important when considering cancer immunotherapy. Here, we show that the Nr4a nuclear receptors, key transcription factors maintaining Treg genetic programs, contribute to Treg-mediated suppression of antitumor immunity in the tumor microenvironment. Mice lacking Nr4a1 and Nr4a2 genes specifically in Tregs showed resistance to tumor growth in transplantation models without exhibiting any severe systemic autoimmunity. The chemotherapeutic agent camptothecin and a common cyclooxygenase-2 inhibitor were found to inhibit transcriptional activity and induction of Nr4a factors, and they synergistically exerted antitumor effects. Genetic inactivation or pharmacologic inhibition of Nr4a factors unleashed effector activities of CD8+ cytotoxic T cells and evoked potent antitumor immune responses. These findings demonstrate that inactivation of Nr4a in Tregs breaks immune tolerance toward cancer, and pharmacologic modulation of Nr4a activity may be a novel cancer treatment strategy targeting the immunosuppressive tumor microenvironment.Significance: This study reveals the role of Nr4a transcription factors in Treg-mediated tolerance to antitumor immunity, with possible therapeutic implications for developing effective anticancer therapies. Cancer Res; 78(11); 3027-40. ©2018 AACR.

Negative Regulation of Cytokine Signaling in Immunity.

Cytokines are key modulators of immunity. Most cytokines use the Janus kinase and signal transducers and activators of transcription (JAK-STAT) pathway to promote gene transcriptional regulation, but their signals must be attenuated by multiple mechanisms. These include the suppressors of cytokine signaling (SOCS) family of proteins, which represent a main negative regulation mechanism for the JAK-STAT pathway. Cytokine-inducible Src homology 2 (SH2)-containing protein (CIS), SOCS1, and SOCS3 proteins regulate cytokine signals that control the polarization of CD4+ T cells and the maturation of CD8+ T cells. SOCS proteins also regulate innate immune cells and are involved in tumorigenesis. This review summarizes recent progress on CIS, SOCS1, and SOCS3 in T cells and tumor immunity.

Generation of allo-antigen-specific induced Treg stabilized by vitamin C treatment and its application for prevention of acute graft versus host disease model.

Antigen-specific regulatory T cells (Tregs) possess the potential to reduce excess immune responses in autoimmune diseases, allergy, rejection after organ transplantation and graft-versus-host disease (GVHD) following hematopoietic stem cell transplantation. Although in vitro-expanded antigen-specific induced Tregs (iTregs) have been considered to be a promising therapeutic agent against such excessive immune reactions, the instability of iTregs after transfer is a fundamental problem in their clinical application. In this study, we searched for the optimal way to generate stable iTregs for the prevention of the murine GVHD model, in which conventional iTregs are reported to be inefficient. Allo-antigen-specific iTregs were generated by co-culturing naive T cells with allogenic dendritic cells in the presence of TGF-ß and retinoic acid. By examining various agents and genes, we found that vitamin C stabilized Foxp3 expression most effectively in adoptively transferred iTregs under a GVHD environment. Vitamin C treatment caused active DNA demethylation specifically on the conserved non-coding sequence 2 (CNS2) enhancer of the Foxp3 gene locus in allo-antigen-specific iTregs and reduced iTreg conversion into pathogenic exFoxp3 cells. Vitamin C-treated iTregs suppressed GVHD symptoms more efficiently than untreated iTregs. Vitamin C also facilitated induction of a FOXP3high iTreg population from human naive T cells, which was very stable even in the presence of IL-6 in vitro. The treatment of vitamin C for iTreg promises innovative clinical application for adoptive Treg immunotherapy.

Improvement of Foxp3 stability through CNS2 demethylation by TET enzyme induction and activation.

Since induced regulatory T cells (iTregs) can be produced in a large quantity in vitro, these cells are expected to be clinically useful to induce immunological tolerance in various immunological diseases. Foxp3 (Forkhead box P3) expression in iTregs is, however, unstable due to the lack of demethylation of the CpG island in the conserved non-coding sequence 2 (CNS2) of the Foxp3 locus. To facilitate the demethylation of CNS2, we over-expressed the catalytic domain (CD) of the ten-eleven translocation (TET) protein, which catalyzes the steps of the iterative demethylation of 5-methylcytosine. TET-CD over-expression in iTregs resulted in partial demethylation of CNS2 and stable Foxp3 expression. We also discovered that TET expression was enhanced under low oxygen (5%) culture conditions, which facilitated CNS2 DNA demethylation and stabilization of Foxp3 expression in a TET2- and TET3-dependent manner. In combination with vitamin C treatment, which has been reported to enhance TET catalytic activity, iTregs generated under low oxygen conditions retained more stable Foxp3 expression in vitro and in vivo and exhibited stronger suppression activity in a colitis model compared with untreated iTregs. Our data indicate that the induction and activation of TET enzymes in iTregs would be an effective method for Treg-mediated adoptive immunotherapy.

D-mannose induces regulatory T cells and suppresses immunopathology.

D-mannose, a C-2 epimer of glucose, exists naturally in many plants and fruits, and is found in human blood at concentrations less than one-fiftieth of that of glucose. However, although the roles of glucose in T cell metabolism, diabetes and obesity are well characterized, the function of D-mannose in T cell immune responses remains unknown. Here we show that supraphysiological levels of D-mannose safely achievable by drinking-water supplementation suppressed immunopathology in mouse models of autoimmune diabetes and airway inflammation, and increased the proportion of Foxp3+ regulatory T cells (Treg cells) in mice. In vitro, D-mannose stimulated Treg cell differentiation in human and mouse cells by promoting TGF-ß activation, which in turn was mediated by upregulation of integrin αvß8 and reactive oxygen species generated by increased fatty acid oxidation. This previously unrecognized immunoregulatory function of D-mannose may have clinical applications for immunopathology.

Induction and maintenance of regulatory T cells by transcription factors and epigenetic modifications.

Regulatory T cells (Tregs) are an essential cell subset for the maintenance of immune homeostasis. Foxp3 (Forkhead box P3) is the Treg master gene which is essential for immune suppressing activity. In addition, Tregs are characterized by a distinct pattern of gene expression, including upregulation of immune-suppressive genes and silencing of inflammatory genes. The molecular mechanisms of Treg development and maintenance have been intensively investigated. Tregs are characterized by expression of the transcription factor Foxp3. Several intronic enhancers and a promoter at the Foxp3 gene locus were shown to play important roles in Treg differentiation. The enhancers have been designated as conserved non-coding sequences (CNSs) 0, 1, 2, and 3. We showed that the transcription factors Nr4a and Smad2/3 are essential for the development of thymic Tregs and induced Tregs, respectively. Recently, Treg-specific DNA demethylation has been shown to play an important role in Treg stability. DNA demethylation of CNS2 has been implicated in Treg stability, and recent reports have revealed that the ten-eleven translocation (Tet) family of demethylation factor plays an important role in CpG demethylation at CNS2. This article reviews the recent progress on the roles of transcription factors and epigenetic modifications in the differentiation, maintenance, and function of Tregs.