Single cell analysis of spondyloarthritis regulatory T cells identifies distinct synovial gene expression patterns and clonal fates

Regulatory T cells (Tregs) play an important role in controlling inflammation and limiting autoimmunity, but their phenotypes at inflammatory sites in human disease are poorly understood. We here analyze the single-cell transcriptome of >16,000 Tregs obtained from peripheral blood and synovial fluid of two patients with HLA-B27+ ankylosing spondylitis and three patients with psoriatic arthritis, closely related forms of inflammatory spondyloarthritis. We identify multiple Treg clusters with distinct transcriptomic profiles, including, among others, a regulatory CD8+ subset expressing cytotoxic markers/genes, and a Th17-like RORC+ Treg subset characterized by IL-10 and LAG-3 expression. Synovial Tregs show upregulation of interferon signature and TNF receptor superfamily genes, and marked clonal expansion, consistent with tissue adaptation and antigen contact respectively. Individual synovial Treg clones map to different clusters indicating cell fate divergence. Finally, we demonstrate that LAG-3 directly inhibits IL-12/23 and TNF secretion by patient-derived monocytes, a mechanism with translational potential in SpA. Our detailed characterization of Tregs at an important inflammatory site illustrates the marked specialization of Treg subpopulations.

IRF4 is Correlated With the Conversion to a Th17-like Phenotype in Regulatory T Cells From the Malignant Pleural Effusion

Background: RORγt+Foxp3+ (Th17-like) Tregs are a plastic Treg subset implicated in immune-related diseases; however, the mechanism of Treg phenotypic transformation in malignant pleural effusion (MPE) has not been elucidated.Methods: The percentage of CD4+CD25+Foxp3+Helios+ and RORγt+Foxp3+ Tregs from peripheral blood and pleural effusion mononuclear cells were measured. The level of interferon regulatory factor 4 (IRF4) mRNA expression was detected by quantitative real-time reverse transcription polymerase chain reaction. The effects of IRF4 on the induction of Tregs from patients with non-small cell lung cancer (NSCLC) were evaluated in vitro. Correlation assays between IRF4 expression and the frequency of RORγt+Foxp3+ Tregs were performed. Results: The frequency of CD4+CD25+Foxp3+Helios+ Tregs and CD4+RORγt+ Th17 cells were both increased in the MPE of NSCLC patients. The group of double-positive Foxp3+RORγt+ Treg phenotype were identified in the pleural effusion. A significant increase in the frequency of Foxp3+RORγt+ Tregs was found in MPE compared with the non-malignant pleural effusion (NPE). Compared to NPE, the relative level of IRF4 expression was increased in the MPE. IRF4 expression was positively associated with the frequency of Foxp3+RORγt+ Tregs in the PE. In vitro, the level of Helios mRNA and protein expression was reduced in induced Tregs following IRF4 over-expression. Additionally, the level of RORγt protein expression was substantially increased. However, ectopic Helios expression in induced Tregs reversed the effects induced by enhanced IRF4 expression.Conclusion: IRF4 may serve as a potential molecule that promotes the conversion of regulatory T cells from MPE to a Th17-like phenotype by modulating Helios.

Single cell analysis of spondyloarthritis regulatory T cells identifies distinct synovial gene expression patterns and clonal fates

Regulatory T cells (Tregs) play an important role in controlling inflammation and limiting autoimmunity, but their phenotypes at inflammatory sites in human disease are poorly understood. We here analyze the single-cell transcriptome of >16,000 Tregs obtained from peripheral blood and synovial fluid of two patients with HLA-B27+ ankylosing spondylitis and three patients with psoriatic arthritis, closely related forms of inflammatory spondyloarthritis. We identify multiple Treg clusters with distinct transcriptomic profiles, including, among others, a regulatory CD8+ subset expressing cytotoxic markers/genes, and a Th17-like RORC+ Treg subset characterized by IL-10 and LAG-3 expression. Synovial Tregs show upregulation of interferon signature and TNF receptor superfamily genes, and marked clonal expansion, consistent with tissue adaptation and antigen contact respectively. Individual synovial Treg clones map to different clusters indicating cell fate divergence. Finally, we demonstrate that LAG-3 directly inhibits IL-12/23 and TNF secretion by patient-derived monocytes, a mechanism with translational potential in SpA. Our detailed characterization of Tregs at an important inflammatory site illustrates the marked specialization of Treg subpopulations.

CD30+OX40+ Treg is associated with improved overall survival in colorectal cancer

Regulatory T cells (Tregs) are often enriched in tumors, where their immunosuppressive function has a key role in tumor persistence and progression. In colorectal cancer (CRC), however, Tregs are frequently associated with an improved clinical outcome. Tumor-infiltrating Tregs have been shown to exhibit a distinct signature comprising the co-stimulatory molecules (OX40, 4-1BB), cytokine receptors (IL1R2, IL21R, CCR8, CD30), and co-inhibitory molecules (PD-L1, TIGIT). Here, we showed by flow cytometry that circulating CD45RO+ Tregs from patients with CRC (n = 25) have elevated CD30 and OX40 expression compared to healthy subjects (n = 14). We identified co-expression of CD30 and OX40 on circulating CD45RO+ Tregs using single-cell images captured by the DEPArray™ system. The frequency of CD30+OX40+CD45RO+ Tregs was significantly higher in CRC patients than in healthy subjects (P < 0.001). Importantly, receiver operating characteristic analysis confirmed that this CD30+OX40+ Treg subset could strongly discriminate between CRC patients and healthy subjects with the highest accuracy of 92.3%, an AUC of 0.92, a sensitivity of 88%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 82.35%, and a trade-off value of 3.44%, compared to other Treg subsets. Consistently, multiplex-IHC/IF of tumor-infiltrating Tregs revealed a significant association between high densities of CD30+OX40+ Tregs and improved overall survival; no such association was found for other subsets. These data suggest a potential role for CD30+OX40+ Tregs as a diagnostic or prognostic biomarker in CRC.

Monosexual Cercariae of Schistosoma japonicum Infection Protects Against DSS-Induced Colitis by Shifting the Th1/Th2 Balance and Modulating the Gut Microbiota

Inflammatory bowel disease (IBD)-related inflammation is closely associated with the initiation and progression of colorectal cancer. IBD is generally treated with 5-aminosalicylic acid and immune-modulating medication, but side effects and limitations of these therapies are emerging. Thus, the development of novel preventative or therapeutic approaches is imperative. Here, we constructed a dextran sodium sulphate (DSS)-induced IBD mouse model that was infected with monosexual Schistosoma japonicum cercariae (mSjci) at day 1 or administered dexamethasone (DXM) from days 3 to 5 as a positive control. The protective effect of mSjci on IBD mice was evaluated through their assessments of their clinical signs, histopathological lesions and intestinal permeability. To uncover the underlying mechanism, the Th1/Th2 balance and Treg cell population were also examined. Additionally, the alterations in the gut microbiota were assessed to investigate the interaction between the mSjci-modulated immune response and pathogenic microbiome. Mice treated with DSS and mSjci showed fewer IBD clinical signs and less impaired intestinal permeability than DSS-treated mice. Mechanistically, mSjci modulated the Th1/Th2 balance by repressing IFN-γ production, promoting IL-10 expression and enhancing the Treg subset population. Moreover, mSjci notably reshaped the structure, diversity and richness of the gut microbiota community and subsequently exerted immune-modulating effects. Our findings provide evidence showing that mSjci might serve as a novel and effective protective strategy and that the gut microbiota might be a new therapeutic target in IBD.

The role of Treg subtypes in glomerulonephritis

While Th1 and Th17 T effector cells are pathogenic drivers of glomerulonephritis (GN), regulatory T cells (Tregs) potently protect from renal tissue injury. Recently, it has become evident that different Treg subtypes exist. Among these are lineage specific Treg1 and Treg17 cells, which are specialized to down regulate either Th1 or Th17 T effector cell responses. Interestingly, programming of specialized Tregs and the corresponding T helper effector cells depend on the same lineage specific master transcription factors Tbet (Th1/Treg1) and STAT3 (Th17/Treg17). Furthermore, early control of T effector cell priming in secondary lymphoid organs by specialized Tregs was described. One central mechanism of T effector cell control by the corresponding Treg subtype seems to be expression of the same chemokine receptor repertoire, which facilitates their co-localization. More recently, another intriguing Treg subset was identified, which expresses Foxp3 together with the Th17 characteristic transcription factor RORγt. While these Foxp3+RORγt+ Tregs were shown to be highly immunosuppressive, studies in GN also identified pro-inflammatory potential via secretion of IL-17. Many questions regarding this unusual Treg subset remain, including their origin, stability, and mechanisms of action. Further characterization of the renal Treg landscape during GN will help to identify novel immunosuppressive mechanisms and develop successful Treg-directed therapies. In this review, we summarize the currently available data about specialized Treg subsets and discuss their role in GN.

Regulatory T Cells Expressing Tumor Necrosis Factor Receptor Type 2 Play a Major Role in CD4+ T-Cell Impairment During Sepsis

Sepsis causes inflammation-induced immunosuppression with lymphopenia and alterations of CD4+ T-cell functions that renders the host prone to secondary infections. Whether and how regulatory T cells (Treg) are involved in this postseptic immunosuppression is unknown. We observed in vivo that early activation of Treg during Staphylococcus aureus sepsis induces CD4+ T-cell impairment and increases susceptibility to secondary pneumonia. The tumor necrosis factor receptor 2 positive (TNFR2pos) Treg subset endorsed the majority of effector immunosuppressive functions, and TNRF2 was particularly associated with activation of genes involved in cell cycle and replication in Treg, probably explaining their maintenance. Blocking or deleting TNFR2 during sepsis decreased the susceptibility to secondary infection. In humans, our data paralleled those in mice; the expression of CTLA-4 was dramatically increased in TNFR2pos Treg after culture in vitro with S. aureus. Our findings describe in vivo mechanisms underlying sepsis-induced immunosuppression and identify TNFR2pos Treg as targets for therapeutic intervention.

CTLA-4 blockade reverses the Foxp3+ T-regulatory-cell suppression of anti-tuberculosis T-cell effector responses

BackgroundsIt has been well described that Foxp3+ T regulatory (Treg) cells suppress immune responses and that murine cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) can control the function of Foxp3+Treg cells. However, it remains unknown about the role of CTLA-4 pathway in Treg suppression of T cell responses in tuberculosis (TB).MethodsWe assessed TB-driven changes in CTLA-4-expressing Foxp3+ Treg and conducted CTLA-4 blocking mechanistic studies ex vivo in 126 subjects with active TB, latent TB or uninfected statuses.ResultsFrequencies of CTLA-4-expressing Treg cells were increased in the circulation of pulmonary TB patients and in the pleural compartment of TB pleuritis. Six-month anti-TB treatment significantly reduced CTLA-4+ Treg subset. Notably, antibody blocking of CTLA-4 pathway (CTLA-4 blockade) reversed the ability of Treg cells to suppress anti-TB Th1 responses and abrogated the Treg-mediated suppression of TB antigen-stimulated proliferative response. The CTLA-4 blockade reversed the Treg suppression of the ability of T cells to restrict intracellular BCG and M. tuberculosis growth in macrophages.InterpretationThe study uncovered previously-unreported observations implicating that the CTLA-4 blockade abrogates the capability of Treg cells to suppress anti-TB immune responses or immunity. Findings support the rationale for exploring the CTLA-4 blockade as potential host-directed therapy against TB.FundThis work is supported in part by the National Natural Science Foundation of China (30901277, 81671553, 81501359), and the Key Technologies Research and Development Program for Infectious Diseases of China (2017ZX10201302-004).

Propranolol Suppresses the T-Helper Cell Depletion-Related Immune Dysfunction in Cirrhotic Mice

Bacterial translocation (BT) and splenomegaly contribute to cirrhosis-associated immune dysfunction (CAID) including T cell depletion, infection, and chronic inflammation. β-blockers have been reported to decrease BT and improve splenomegaly. This study explores the modulation of β1 and β2 adrenergic receptors (ADRB1/ADRB2) by propranolol treatment on the peripheral and splenic immune dysfunction of cirrhotic mice. In vivo experiments were performed in bile duct ligation (BDL)- and thioacetamide (TAA)-cirrhotic mice receiving two weeks of propranolol treatment. Acute effects of propranolol were evaluated in T-helper (Th) cells isolated from spleen of cirrhotic mice. Over-expression of β1 and β2 adrenergic receptors (ADRB1/ADRB2) in spleen and T lymphocytes was associated with high peripheral/splenic lipopolysaccharide binding protein levels. Moreover, a decrease in Th cells percentage, increase in Treg subset, and cytokines were accompanied by increased apoptosis, proliferation, and reduced white pulp hyperplasia in cirrhotic mice, which were counteracted by propranolol treatment. The Th-cell depletion, systemic inflammation, BT, and infection were improved by chronic propranolol treatment. Acute propranolol treatment inhibited apoptosis, Treg-conditioned differentiation, and promoted Th2-conditioned differentiation through ADRB-cyclic adenosine monophosphate (cAMP) signals in cirrhotic mice. In conclusion, suppression of ADRB1 and ADRB2 expressions in spleen and splenic T lymphocytes by acute and chronic propranolol treatment ameliorate systemic and splenic immune dysfunction in cirrhosis.