Expansion of tumor-associated Treg cells upon disruption of a CTLA-4-dependent feedback loop.

Foxp3+ T regulatory (Treg) cells promote immunological tumor tolerance, but how their immune-suppressive function is regulated in the tumor microenvironment (TME) remains unknown. Here, we used intravital microscopy to characterize the cellular interactions that provide tumor-infiltrating Treg cells with critical activation signals. We found that the polyclonal Treg cell repertoire is pre-enriched to recognize antigens presented by tumor-associated conventional dendritic cells (cDCs). Unstable cDC contacts sufficed to sustain Treg cell function, whereas T helper cells were activated during stable interactions. Contact instability resulted from CTLA-4-dependent downregulation of co-stimulatory B7-family proteins on cDCs, mediated by Treg cells themselves. CTLA-4-blockade triggered CD28-dependent Treg cell hyper-proliferation in the TME, and concomitant Treg cell inactivation was required to achieve tumor rejection. Therefore, Treg cells self-regulate through a CTLA-4- and CD28-dependent feedback loop that adjusts their population size to the amount of local co-stimulation. Its disruption through CTLA-4-blockade may off-set therapeutic benefits in cancer patients.

CD80 on skin stem cells promotes local expansion of regulatory T cells upon injury to orchestrate repair within an inflammatory environment.

Following tissue damage, epithelial stem cells (SCs) are mobilized to enter the wound, where they confront harsh inflammatory environments that can impede their ability to repair the injury. Here, we investigated the mechanisms that protect skin SCs within this inflammatory environment. Characterization of gene expression profiles of hair follicle SCs (HFSCs) that migrated into the wound site revealed activation of an immune-modulatory program, including expression of CD80, major histocompatibility complex class II (MHCII), and CXC motif chemokine ligand 5 (CXCL5). Deletion of CD80 in HFSCs impaired re-epithelialization, reduced accumulation of peripherally generated Treg (pTreg) cells, and increased infiltration of neutrophils in wounded skin. Importantly, similar wound healing defects were also observed in mice lacking pTreg cells. Our findings suggest that upon skin injury, HFSCs establish a temporary protective network by promoting local expansion of Treg cells, thereby enabling re-epithelialization while still kindling inflammation outside this niche until the barrier is restored.

Antibodies to Costimulatory Receptor 4-1BB Enhance Anti-tumor Immunity via T Regulatory Cell Depletion and Promotion of CD8 T Cell Effector Function.

The costimulatory receptor 4-1BB is expressed on activated immune cells, including activated T cells. Antibodies targeting 4-1BB enhance the proliferation and survival of antigen-stimulated T cells in vitro and promote CD8 T cell-dependent anti-tumor immunity in pre-clinical cancer models. We found that T regulatory (Treg) cells infiltrating human or murine tumors expressed high amounts of 4-1BB. Intra-tumoral Treg cells were preferentially depleted by anti-4-1BB mAbs in vivo. Anti-4-1BB mAbs also promoted effector T cell agonism to promote tumor rejection. These distinct mechanisms were competitive and dependent on antibody isotype and FcγR availability. Administration of anti-4-1BB IgG2a, which preferentially depletes Treg cells, followed by either agonistic anti-4-1BB IgG1 or anti-PD-1 mAb augmented anti-tumor responses in multiple solid tumor models. An antibody engineered to optimize both FcγR-dependent Treg cell depleting capacity and FcγR-independent agonism delivered enhanced anti-tumor therapy. These insights into the effector mechanisms of anti-4-1BB mAbs lay the groundwork for translation into the clinic.

A lncRNA Dleu2-encoded peptide relieves autoimmunity by facilitating Smad3-mediated Treg induction.

Micropeptides encoded by short open reading frames (sORFs) within long noncoding RNAs (lncRNAs) are beginning to be discovered and characterized as regulators of biological and pathological processes. Here, we find that lncRNA Dleu2 encodes a 17-amino-acid micropeptide, which we name Dleu2-17aa, that is abundantly expressed in T cells. Dleu2-17aa promotes inducible regulatory T (iTreg) cell generation by interacting with SMAD Family Member 3 (Smad3) and enhancing its binding to the Foxp3 conserved non-coding DNA sequence 1 (CNS1) region. Importantly, the genetic deletion of Dleu2-17aa in mice by start codon mutation impairs iTreg generation and worsens experimental autoimmune encephalomyelitis (EAE). Conversely, the exogenous supplementation of Dleu2-17aa relieves EAE. Our findings demonstrate an indispensable role of Dleu2-17aa in maintaining immune homeostasis and suggest therapeutic applications for this peptide in treating autoimmune diseases.

Type III interferon drives thymic B cell activation and regulatory T cell generation.

The activation of thymic B cells is critical for their licensing as antigen presenting cells and resulting ability to mediate T cell central tolerance. The processes leading to licensing are still not fully understood. By comparing thymic B cells to activated Peyer's patch B cells at steady state, we found that thymic B cell activation starts during the neonatal period and is characterized by TCR/CD40-dependent activation, followed by immunoglobulin class switch recombination (CSR) without forming germinal centers. Transcriptional analysis also demonstrated a strong interferon signature, which was not apparent in the periphery. Thymic B cell activation and CSR were primarily dependent on type III IFN signaling, and loss of type III IFN receptor in thymic B cells resulted in reduced thymocyte regulatory T cell (Treg) development. Finally, from TCR deep sequencing, we estimate that licensed B cells induce development of a substantial fraction of the Treg cell repertoire. Together, these findings reveal the importance of steady-state type III IFN in generating licensed thymic B cells that induce T cell tolerance to activated B cells.

CD200R activation on naïve T cells by B cells induces suppressive activity of T cells via IL-24.

CD200 is an anti-inflammatory protein that facilitates signal transduction through its receptor, CD200R, in cells, resulting in immune response suppression. This includes reducing M1-like macrophages, enhancing M2-like macrophages, inhibiting NK cell cytotoxicity, and downregulating CTL responses. Activation of CD200R has been found to modulate dendritic cells, leading to the induction or enhancement of Treg cells expressing Foxp3. However, the precise mechanisms behind this process are still unclear. Our previous study demonstrated that B cells in Peyer's patches can induce Treg cells, so-called Treg-of-B (P) cells, through STAT6 phosphorylation. This study aimed to investigate the role of CD200 in Treg-of-B (P) cell generation. To clarify the mechanisms, we used wild-type, STAT6 deficient, and IL-24 deficient T cells to generate Treg-of-B (P) cells, and antagonist antibodies (anti-CD200 and anti-IL-20RB), an agonist anti-CD200R antibody, CD39 inhibitors (ARL67156 and POM-1), a STAT6 inhibitor (AS1517499), and soluble IL-20RB were also applied. Our findings revealed that Peyer's patch B cells expressed CD200 to activate the CD200R on T cells and initiate the process of Treg-of-B (P) cells generation. CD200 and CD200R interaction triggers the phosphorylation of STAT6, which regulated the expression of CD200R, CD39, and IL-24 in T cells. CD39 regulated the expression of IL-24, which sustained the expression of CD223 and IL-10 and maintained the cell viability. In summary, the generation of Treg-of-B (P) cells by Peyer's patch B cells was through the CD200R-STAT6-CD39-IL-24 axis pathway.

Zfp362 potentiates murine colonic inflammation by constraining Treg cell function rather than promoting Th17 cell differentiation.

Mucosal barrier integrity and pathogen clearance is a complex process influenced by both Th17 and Treg cells. Previously, we had described the DNA methylation profile of Th17 cells and identified Zinc finger protein (Zfp)362 to be uniquely demethylated. Here, we generated Zfp362-/- mice to unravel the role of Zfp362 for Th17 cell biology. Zfp362-/- mice appeared clinically normal, showed no phenotypic alterations in the T-cell compartment, and upon colonization with segmented filamentous bacteria, no effect of Zfp362 deficiency on Th17 cell differentiation was observed. By contrast, Zfp362 deletion resulted in increased frequencies of colonic Foxp3+ Treg cells and IL-10+ and RORγt+ Treg cell subsets in mesenteric lymph nodes. Adoptive transfer of naïve CD4+ T cells from Zfp362-/- mice into Rag2-/- mice resulted in a significantly lower weight loss when compared with controls receiving cells from Zfp362+/+ littermates. However, this attenuated weight loss did not correlate with alterations of Th17 cells but instead was associated with an increase of effector Treg cells in mesenteric lymph nodes. Together, these results suggest that Zfp362 plays an important role in promoting colonic inflammation; however, this function is derived from constraining the effector function of Treg cells rather than directly promoting Th17 cell differentiation.

Foxp3+ regulatory T cells in the central nervous system and other nonlymphoid tissues.

Foxp3+ regulatory T (Treg) cells are indispensable for the maintenance of immunologic self-tolerance as well as for the confinement of autoimmune inflammation after the breach of self-tolerance. In order to fulfill these tasks, Treg cells operate in secondary lymphoid tissues and nonlymphoid tissues. The conditions for Treg cell stability and for their modes of action are different according to their compartment of residence. In addition, Treg cells initiate residency programs to inhabit niches in nonlympoid tissues (NLT) in steady state and after re-establishment of previously deflected homeostasis for extended periods of time. These NLT Treg cells are different from lymphoid tissue residing Treg cells and are functionally specialized to subserve not only immune functions but support intrinsic functions of their tissue of residence. This review will highlight current ideas about the functional specialization of NLT Treg cells in particular in the central nervous system (CNS) and discuss challenges that we are facing in an effort to exploit the power of NLT Treg cells for maintenance of tissue homeostasis and perhaps also tissue regeneration.

Human embryonic stem cell-derived mesenchymal stromal cells suppress inflammation in mouse models of rheumatoid arthritis and lung fibrosis by regulating T-cell function.

BACKGROUND AIMS: Rheumatoid arthritis (RA) is characterized by an overactive immune system, with limited treatment options beyond immunosuppressive drugs or biological response modifiers. Human embryonic stem cell-derived mesenchymal stromal cells (hESC-MSCs) represent a novel alternative, possessing diverse immunomodulatory effects. In this study, we aimed to elucidate the therapeutic effects and underlying mechanisms of hESC-MSCs in treating RA. METHODS: MSC-like cells were differentiated from hESC (hESC-MSCs) and cultured in vitro. Cell proliferation was assessed using Cell Counting Kit-8 assay and Ki-67 staining. Flow cytometry was used to analyze cell surface markers, T-cell proliferation and immune cell infiltration. The collagen-induced arthritis (CIA) mouse model and bleomycin-induced model of lung fibrosis (BLE) were established and treated with hESC-MSCs intravenously for in vivo assessment. Pathological analyses, reverse transcription-quantitative polymerase chain reaction and Western blotting were conducted to evaluate the efficacy of hESC-MSCs treatment. RESULTS: Intravenous transplantation of hESC-MSCs effectively reduced inflammation in CIA mice in this study. Furthermore, hESC-MSC administration enhanced regulatory T cell infiltration and activation. Additional findings suggest that hESC-MSCs may reduce lung fibrosis in BLE mouse models, indicating their potential to mitigate complications associated with RA progression. In vitro experiments revealed a significant inhibition of T-cell activation and proliferation during co-culture with hESC-MSCs. In addition, hESC-MSCs demonstrated enhanced proliferative capacity compared with traditional primary MSCs. CONCLUSIONS: Transplantation of hESC-MSCs represents a promising therapeutic strategy for RA, potentially regulating T-cell proliferation and differentiation.

Fragile Treg cells: Traitors in immune homeostasis?

Regulatory T (Treg) cells play a key role in maintaining immune tolerance and tissue homeostasis. However, in some disease microenvironments, Treg cells exhibit fragility, which manifests as preserved FoxP3 expression accompanied by inflammation and loss of immunosuppression. Fragile Treg cells are formatively, phenotypically and functionally diverse in various diseases, further complicating the role of Treg cells in the immunotherapeutic response and offering novel targets for disease treatment by modulating specific Treg subsets. In this review, we summarize findings on fragile Treg cells to provide a framework for characterizing the formation and role of fragile Treg cells in different diseases, and we discuss how this information may guide the development of more specific Treg-targeted immunotherapies.

PPARγ marks splenic precursors of multiple nonlymphoid-tissue Treg compartments.

Foxp3+CD4+ regulatory T cells (Tregs) regulate most types of immune response as well as several processes important for tissue homeostasis, for example, metabolism and repair. Dedicated Treg compartments-with distinct transcriptomes, T cell receptor repertoires, and growth/survival factor dependencies-have been identified in several nonlymphoid tissues. These Tregs are specifically adapted to function and operate in their home tissue-When, where, and how do they take on their specialized characteristics? We recently reported that a splenic Treg population expressing low levels of the transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) contains precursors of Tregs residing in visceral adipose tissue. This finding made sense given that PPARγ, the "master regulator" of adipocyte differentiation, is required for the accumulation and function of Tregs in visceral adipose tissue but not in lymphoid tissues. Here we use single-cell RNA sequencing, single-cell Tcra and Tcrb sequencing, and adoptive-transfer experiments to show that, unexpectedly, the splenic PPARγlo Treg population is transcriptionally heterogeneous and engenders Tregs in multiple nonlymphoid tissues beyond visceral adipose tissue, such as skin and liver. The existence of a general pool of splenic precursors for nonlymphoid-tissue Tregs opens possibilities for regulating their emergence experimentally or therapeutically.

Mucosal IgA immune complex induces immunomodulatory responses in allergic airway and intestinal TH2 disease.

BACKGROUND: IgA is the most abundant immunoglobulin at the mucosal surface and although its role in regulating mucosal immunity is not fully understood, its presence is associated with protection from developing allergic disease. OBJECTIVE: We sought to determine the role of IgA immune complexes for therapeutic application to mucosal allergic responses. METHODS: Trinitrophenol (TNP)-specific IgA immune complexes were applied, using TNP-coupled ovalbumin (OVA), to airway and gut mucosal surfaces in systemically sensitized allergic animals to regulate allergen challenge responses. Animals were assessed for both pathologic and immune-mediated responses in the lung and gut, respectively, using established mouse models. RESULTS: The mucosal application of IgA immune complexes in the lung and gut with TNP-OVA regulated TH2-driven allergic response in the lung and gut, reducing TH2 cytokines and mucus (lung) as well as diarrhea and temperature loss (gut), but increasing IL-10 and the number of regulatory T cells. The IgA-OVA immune complex did not alter peanut-induced anaphylaxis, indicating antigen specificity. Using OVA-specific DO.11-green fluorescent protein IL-4 reporter mouse-derived TH2-skewed cells in a transfer model demonstrated that mucosal IgA immune complex treatment reduced TH2-cell expansion and increased the number of regulatory T cells. To address a potential mechanism of action, TGF-ß and IL-10 were induced in bone marrow-derived dendritic cells when they were exposed to IgA immune complex, suggesting a regulatory phenotype induced in dendritic cells that also led to an altered primary T-cell-mediated response in in vitro OVA-specific assays. CONCLUSIONS: These studies highlight one possible mechanism of how allergen-specific IgA may provide a regulatory signal to reduce the development of allergic responses in the lung and gut.

mTOR signaling at the crossroads of environmental signals and T-cell fate decisions.

The evolutionarily conserved serine/threonine kinase mTOR (mechanistic target of rapamycin) forms the distinct protein complexes mTORC1 and mTORC2 and integrates signals from the environment to coordinate downstream signaling events and various cellular processes. T cells rely on mTOR activity for their development and to establish their homeostasis and functional fitness. Here, we review recent progress in our understanding of the upstream signaling and downstream targets of mTOR. We also provide an updated overview of the roles of mTOR in T-cell development, homeostasis, activation, and effector-cell fate decisions, as well as its important impacts on the suppressive activity of regulatory T cells. Moreover, we summarize the emerging roles of mTOR in T-cell exhaustion and transdifferentiation. A better understanding of the contribution of mTOR to T-cell fate decisions will ultimately aid in the therapeutic targeting of mTOR in human disease.

Noninvasive ultrasound stimulation to treat myocarditis through splenic neuro-immune regulation.

BACKGROUND: The cholinergic anti-inflammatory pathway (CAP) has been widely studied to modulate the immune response. Current stimulating strategies are invasive or imprecise. Noninvasive low-intensity pulsed ultrasound (LIPUS) has become increasingly appreciated for targeted neuronal modulation. However, its mechanisms and physiological role on myocarditis remain poorly defined. METHODS: The mouse model of experimental autoimmune myocarditis was established. Low-intensity pulsed ultrasound was targeted at the spleen to stimulate the spleen nerve. Under different ultrasound parameters, histological tests and molecular biology were performed to observe inflammatory lesions and changes in immune cell subsets in the spleen and heart. In addition, we evaluated the dependence of the spleen nerve and cholinergic anti-inflammatory pathway of low-intensity pulsed ultrasound in treating autoimmune myocarditis in mice through different control groups. RESULTS: The echocardiography and flow cytometry of splenic or heart infiltrating immune cells revealed that splenic ultrasound could alleviate the immune response, regulate the proportion and function of CD4+ Treg and macrophages by activating cholinergic anti-inflammatory pathway, and finally reduce heart inflammatory injury and improve cardiac remodeling, which is as effective as an acetylcholine receptor agonists GTS-21. Transcriptome sequencing showed significant differential expressed genes due to ultrasound modulation. CONCLUSIONS: It is worth noting that the ultrasound therapeutic efficacy depends greatly on acoustic pressure and exposure duration, and the effective targeting organ was the spleen but not the heart. This study provides novel insight into the therapeutic potentials of LIPUS, which are essential for its future application.

Clinical and experimental treatment of allergic asthma with an emphasis on allergen immunotherapy and its mechanisms.

Allergen immunotherapy (AIT) is currently the only form of treatment that modifies allergic asthma. Pharmacotherapy alone seeks to control the symptoms of allergic asthma, allergic rhinitis, and other atopic conditions. In contrast, AIT can induce long-term physiological modifications through the immune system. AIT enables individuals to live improved lives many years after treatment ends, where they are desensitized to the allergen(s) used or no longer have significant allergic reactions upon allergen provocation. The leading forms of treatment with AIT involve injections of allergen extracts with increasing doses via the subcutaneous route or drops/tablets via the sublingual route for several years. Since the initial attempts at this treatment as early as 1911 by Leonard Noon, the mechanisms by which AIT operates remain unclear. This literature-based review provides the primary care practitioner with a current understanding of the mechanisms of AIT, including its treatment safety, protocols, and long-term efficacy. The primary mechanisms underlying AIT include changes in immunoglobulin classes (IgA, IgE, and IgG), immunosuppressive regulatory T-cell induction, helper T cell type 2 to helper T cell type 1 cell/cytokine profile shifts, decreased early-phase reaction activity and mediators, and increased production of IL-10, IL-35, TGF-ß, and IFN-γ. Using the databases PubMed and Embase, a selective literature search was conducted searching for English, full-text, reviews published between 2015 and 2022 using the keywords (with wildcards) "allerg*," "immunotherap*," "mechanis*," and "asthma." Among the cited references, additional references were identified using a manual search.

Significance of regulatory T cells in cancer immunology and immunotherapy.

Immunosuppression in the tumour microenvironment (TME) attenuates antitumor immunity, consequently hindering protective immunosurveillance and preventing effective antitumor immunity induced by cancer immunotherapy. Multiple mechanisms including immune checkpoint molecules, such as CTLA-4, PD-1, and LAG-3, and immunosuppressive cells are involved in the immunosuppression in the TME. Regulatory T (Treg) cells, a population of immunosuppressive cells, play an important role in inhibiting antitumor immunity. Therefore, Treg cells in the TME correlate with an unfavourable prognosis in various cancer types. Thus, Treg cell is considered to become a promising target for cancer immunotherapy. Elucidating Treg cell functions in cancer patients is therefore crucial for developing optimal Treg cell-targeted immunotherapy. Here, we describe Treg cell functions and phenotypes in the TME from the perspective of Treg cell-targeted immunotherapy.

Adherent-invasive Escherichia coli LF82 aggravated intestinal inflammation in colitis mice by affecting the gut microbiota and Th17/Treg cell differentiation balance.

The imbalance of Th17 and Treg cell differentiation, intestinal flora imbalance, and intestinal mucosal barrier damage may be important links in the occurrence and development of inflammatory bowel disease (IBD) since Th17 and Treg differentiation are affected by the intestinal flora. This study aimed to explore the effect of Escherichia coli (E. coli) LF82 on the differentiation of Th17 and Treg cells and the role of the intestinal flora in mouse colitis. The effects of E. coli LF82 infection on intestinal inflammation were evaluated by analyzing the disease activity index, histology, myeloperoxidase activity, FITC-D fluorescence value, and claudin-1 and ZO-1 expression. The effects of E. coli LF82 on the Th17/Treg balance and intestinal flora were analyzed by flow cytometry and 16S rDNA sequencing. Inflammatory markers, changes in the intestinal flora, and Th17/Treg cells were then detected after transplanting fecal bacteria from normal mice into colitis mice infected by E. coli LF82. We found that E. coli LF82 infection can aggravate the intestinal inflammation of mice colitis, destroy their intestinal mucosal barrier, increase intestinal mucosal permeability, and aggravate the imbalance of Th17/Treg differentiation and the disorder of intestinal flora. After improving the intestinal flora imbalance by fecal bacteria transplantation, intestinal inflammation and intestinal mucosal barrier damage were reduced, and the differentiation balance of Th17 and Treg cells was restored. This study showed that E. coli LF82 infection aggravates intestinal inflammation and intestinal mucosal barrier damage in colitis by affecting the intestinal flora composition and indirectly regulating the Th17 and Treg cell differentiation balance.

Insulin Receptor Substrate 1 Signaling Inhibits Foxp3 Expression and Suppressive Functions in Treg Cells through the mTORC1 Pathway.

Regulatory T (Treg) cells play an important role in immune homeostasis by inhibiting cells within the innate and adaptive immune systems; therefore, the stability and immunosuppressive function of Treg cells need to be maintained. In this study, we found that the expression of insulin receptor substrate 1 (IRS1) by Treg cells was lower than that by conventional CD4 T cells. IRS1-overexpressing Treg cells showed the downregulated expression of FOXP3, as well as Treg signature markers CD25 and CTLA4. IRS1-overexpressing Treg cells also showed diminished immunosuppressive functions in an in vitro suppression assay. Moreover, IRS1-overexpressing Treg cells were unable to suppress the pathogenic effects of conventional T cells in a transfer-induced colitis model. IRS1 activated the mTORC1 signaling pathway, a negative regulator of Treg cells. Moreover, IRS1 destabilized Treg cells by upregulating the expression of IFN-γ and Glut1. Thus, IRS1 acts as a negative regulator of Treg cells by downregulating the expression of FOXP3 and disrupting stability.

Enhancing Regulatory T Cells to Treat Inflammatory and Autoimmune Diseases.

Regulatory T cells (Tregs) control immune responses and are essential to maintain immune homeostasis and self-tolerance. Hence, it is no coincidence that autoimmune and chronic inflammatory disorders are associated with defects in Tregs. These diseases have currently no cure and are treated with palliative drugs such as immunosuppressant and immunomodulatory agents. Thereby, there is a great interest in developing medical interventions against these diseases based on enhancing Treg cell function and numbers. Here, we give an overview of Treg cell ontogeny and function, paying particular attention to mucosal Tregs. We review some notable approaches to enhance immunomodulation by Tregs with therapeutic purposes including adoptive Treg cell transfer therapy and discuss relevant clinical trials for inflammatory bowel disease. We next introduce ways to expand mucosal Tregs in vivo using microbiota and dietary products that have been the focus of clinical trials in various autoimmune and chronic-inflammatory diseases.

Epigenetics: An opportunity to shape innate and adaptive immune responses.

Epigenetics connects genetic and environmental factors: it includes DNA methylation, histone post-translational modifications and the regulation of chromatin accessibility by non-coding RNAs, all of which control constitutive or inducible gene transcription. This plays a key role in harnessing the transcriptional programs of both innate and adaptive immune cells due to its plasticity and environmental-driven nature, piloting myeloid and lymphoid cell fate decisions with no change in their genomic sequence. In particular, epigenetic marks at the site of lineage-specific transcription factors and maintenance of cell type-specific epigenetic modifications, referred to as 'epigenetic memory', dictate cell differentiation, cytokine production and functional capacity following repeated antigenic exposure in memory T cells. Moreover, metabolic and epigenetic reprogramming occurring during a primary innate immune response leads to enhanced responses to secondary challenges, a phenomenon known as 'trained immunity'. Here, we discuss how stable and dynamic epigenetic states control immune cell identity and plasticity in physiological and pathological conditions. Dissecting the regulatory circuits of cell fate determination and maintenance is of paramount importance for understanding the delicate balance between immune cell activation and tolerance, in healthy conditions and in autoimmune diseases.