IntroductionThe human FOXP3+ regulatory T (Treg) cells, a subset of CD4+ T cells with immunosuppressive function, are essential for the maintenance of immune homeostasis and tolerance. Treg cells are a heterogeneous population, subdivided into a less stable “effector” subset and a more stable “naïve” subset. Under inflammatory conditions, Treg cells can lose their immunosuppressive properties, contributing to the development of autoimmune diseases. The TGFβ signaling is pivotal for the induction of Treg cells from naïve CD4 T cells and the thymic development of natural Treg cells. However, how TGFβ signaling regulates established naïve and effector Treg cells is not fully understood.MethodsHuman naïve and effector Treg cells were isolated from healthy donors using flow cytometry. Different subsets of Treg cells were treated with a TGFβ inhibitor or genetically modified to express SKI or ARKADIA via lentiviral transduction. Treg cell phenotype, stability and signaling regulation were analyzed using flow cytometry, western blotting, transcriptomic and meta-analyses. The functionality of Treg cells was analyzed by in vitro coculture assays.ResultsWe find that the TGFβ signaling is differentially regulated in Treg subsets, with higher activity in the naïve Treg subset. Blockade of TGFβ pathway destabilizes both naïve and effector Treg cells, disrupting their immunosuppressive functions, with effector Treg cells being more susceptible. Further analysis shows that naïve Treg cells express lower levels of SKI protein, a negative regulator of TGFβ signaling suppressed by TGFβ-induced protein degradation. SKI overexpression destabilizes Treg cells and disrupts their immune suppressive function. Transcriptomic and meta-analyses reveal that TGFβ blockade and SKI overexpression commonly modulate pathways crucial for Treg to effector T cell conversion, downregulating Treg signature genes and upregulating effector T cell markers, which are validated as potential SKI targets. Importantly, overexpression of ARKADIA, an E3 ubiquitin ligase of SKI, efficiently reduces SKI levels, enhancing Treg cell stability and functionality under both TGFβ inhibition and chronic proinflammatory cytokine stimulation.ConclusionOur results identify a previously unrecognized role of the TGFβ-ARKADIA-SKI axis in regulating the stability and functionality of human Treg subsets, highlighting novel strategies for harnessing TGFβ-associated pathways to stabilize human Treg cells for clinical applications.

Regulatory T cells (Tregs) are central to immune homeostasis and controlling inflammation through multiple mechanisms, however, recent discoveries and advances in technology reveal that Tregs exert a diverse array of functions beyond mere immunosuppression, adapting uniquely to the specialized environments of tissues. This review delves into the multifaceted, tissue-specific mechanisms of Tregs, highlighting their roles in tissue repair, inflammatory modulation, and tolerance maintenance. We explore the developmental, functional, and metabolic pathways that drive Treg specialization across distinct organs, such as the central nervous system, gastrointestinal tract, joints, skin, and lungs, and examine how these insights advance the design of novel, targeted therapies for autoimmune and inflammatory disorders. This review will emphasize non-suppressive functions, discussing how Tregs can be harnessed in therapeutic applications tailored to specific tissue microenvironments, offering a promising new direction for the treatment of autoimmune diseases.

Inflammatory bowel disease (IBD) is an umbrella term for two conditions (Crohn’s Disease and Ulcerative Colitis) that is characterized by chronic inflammation of the gastrointestinal tract. The use of pre-clinical animal models has been invaluable for the understanding of potential disease mechanisms. However, despite promising results of numerous therapeutics in mouse colitis models, many of these therapies did not show clinical benefits in patients with IBD. Single cell RNA-sequencing (scRNA-seq) has recently revolutionized our understanding of complex interactions between the immune system, stromal cells, and epithelial cells by mapping novel cell subpopulations and their remodeling during disease. This technology has not been widely applied to pre-clinical models of IBD. ScRNA-seq profiling of murine models may provide an opportunity to increase the translatability into the clinic, and to choose the most appropriate model to test hypotheses and novel therapeutics. In this review, we have summarized some of the key findings at the single cell transcriptomic level in IBD, how specific signatures have been functionally validated in vivo, and highlighted the similarities and differences between scRNA-seq findings in human IBD and experimental mouse models. In each section of this review, we highlight the importance of utilizing this technology to find the most suitable or translational models of IBD based on the cellular therapeutic target.

CD4+CD25highFoxp3+ regulatory T-cells (Tregs) are functionally characterized for their ability to suppress the activation of multiple immune cell types and are indispensable for maintaining immune homeostasis and tolerance. Disruption of this intrinsic brake system assessed by loss of suppressive capacity, cell numbers, and Foxp3 expression, leads to uncontrolled immune responses and tissue damage. The conversion of Tregs to a pathogenic pro-inflammatory phenotype is widely observed in immune mediated diseases. However, the molecular mechanisms that underpin the control of Treg stability and suppressive capacity are incompletely understood. This review summarizes the concepts of Treg cell stability and Treg cell plasticity highlighting underlying mechanisms including translational and epigenetic regulators that may enable translation to new therapeutic strategies. Our enhanced understanding of molecular mechanism controlling Tregs will have important implications into immune homeostasis and therapeutic potential for the treatment of immune-mediated diseases.

Wepreviously showed that treating NOD mice with an agonistic monoclonalanti-TLR4/MD2 antibody (TLR4-Ab) reversed acute type one diabetes (T1D). Here,we show that TLR4-Ab reverses T1D by induction of myeloid derived suppressorcells (MDSC). Unbiased gene expression analysis after TLR4-Ab treatmentdemonstrated upregulation of genes associated with CD11b+Ly6G+ myeloid cells,and downregulation of T cell genes. Further RNAseq of purified, TLR4-Ab treatedCD11b+ cells showed significant upregulation of genes associated with bonemarrow-derived CD11b+ cells and innate immune system genes. TLR4-Ab significantlyincreased percentages and numbers of CD11b+ cells. TLR4-Ab-induced CD11b+cells, derived ex vivo from TLR4-Ab treated mice, suppress T cells, andTLR4-Ab-conditioned bone marrow cells suppress acute T1D when transferred intoacutely diabetic mice. Thus, the TLR4-Ab-induced CD11b+ cells, by currentlyaccepted definition, are MDSCs able to reverse T1D. To understand the TLR4-Ab mechanismwe compared TLR4-Ab to TLR4 agonist LPS (which cannot reverse T1D). TLR4-Abremains sequestered at least 48 times longer than LPS within early endosomes,alters TLR4 signaling and downregulates inflammatory genes and proteinsincluding NFkB. TLR4-Ab in the endosome therefore induces a sustained,attenuated inflammatory response providing an ideal “second signal” for theactivation/maturation of MDSCs that can reverse acute T1D.

Primary biliary cholangitis (PBC) is a prototypical organ-specific autoimmune disease that is mediated by autoreactive T-cell attack and destruction of cholangiocytes. Despite the clear role of autoimmunity in PBC, immune-directed therapies have failed to halt PBC, including biologic therapies effective in other autoimmune diseases. MicroRNA (miRNA) dysregulation is implicated in the pathogenesis (PBC). In the dominant-negative TGF-β receptor type II (dnTGFβRII) mouse model of PBC, autoreactive CD8 T cells play a major pathogenic role and demonstrate a striking pattern of miRNA down-regulation. Enoxacin is a small molecule fluoroquinolone that enhances miRNA biogenesis, partly by stabilizing the interaction of transactivation response RNA-binding protein with Argonaute (Ago) 2.We hypothesized that correcting aberrant T-cell miRNA expression with enoxacin in dnTGFβRII mice could modulate autoreactive T-cell function and prevent PBC. Here, we show that liver-infiltrating dnTGFβRII CD8 T cells have significantly decreased levels of the miRNA biogenesis molecules prolyl 4-hydroxylase subunit alpha 1 (P4HA1) and Ago2 along with significantly increased levels of granzyme B and perforin. Enoxacin treatment significantly up-regulated miRNAs in dnTGFβRII CD8 T cells and effectively treated autoimmune cholangitis in dnTGFβRII mice. Enoxacin treatment directly altered T cells both ex vivo and in vitro, resulting in altered memory subset numbers, decreased proliferation, and decreased interferon-γ production. Enoxacin significantly decreased CD8 T-cell expression of the transcription factor, Runx3, and significantly decreased perforin expression at both the mRNA and protein levels.Enoxacin increases miRNA expression in dnTGFβRII CD8 T cells, reduces CD8 T-cell pathogenicity, and effectively halted progression of autoimmune biliary disease. Targeting the miRNA pathway is a therapeutic approach to autoimmunity that corrects pathological miRNA abnormalities in autoreactive T cells.

Rheumatoid arthritis (RA) is an autoimmune disease that primarily affects the synovial joints and can lead to bone erosion and cartilage damage. One hallmark of RA is anticitrullinated protein autoantibodies (ACPA) and memory citrulline-specific B-cells, which have been implicated in RA pathogenesis. While depletion of B-cells with Rituximab improves clinical responses in RA patients, this treatment strategy leaves patients susceptible to infections. Here we use of Siglec-engaging Tolerance-inducing Antigenic Liposomes (STALs) to selectively target the citrulline-specific B-cells. ACPA production from purified human RA patients' B-cells in vitro was achieved through a set of stimulation conditions, which includes the following: BAFF, anti-CD40, IL-21, and LPS. In vivo generation of citrulline specific B-cells and ACPA production was accomplished by antigenic liposomes consisting of monophosphoryl lipid A (MPLA) and a cyclic citrullinated peptide (CCP) administered to SJL/J mice. We show that STALs that codisplay a high affinity CD22 glycan ligand and synthetic citrullinated antigen (CCP STALs) can prevent ACPA production from RA patients' memory B-cells in vitro. These CCP STALs were also effective in inducing tolerance to citrullinated antigens in SJL/J mice. The results demonstrate that tolerization of the B-cells responsible for ACPA can be achieved by exploiting the inhibitory receptor CD22 with high-affinity glycan ligands. Such a treatment strategy could be beneficial in the treatment of RA.

We show here that soluble CD137 (sCD137), the alternately spliced gene product of Tnfsfr9, effectively treats acute type 1 diabetes (T1D) in nonobese diabetic (NOD) mice. sCD137 significantly delayed development of end-stage disease, preserved insulin+ islet beta cells, and prevented progression to end-stage T1D in some mice. We demonstrate that sCD137 induces CD4+ T cell anergy, suppressing antigen-specific T cell proliferation and IL-2/IFN-gamma secretion. Exogenous IL-2 reversed the sCD137 anergy effect. sCD137 greatly reduces inflammatory cytokine production by CD8 effector memory T cells, critical mediators of beta cell damage. We demonstrate that human T1D patients have decreased serum sCD137 compared to age-matched controls (as do NOD mice compared to NOD congenic mice expressing a protective Tnfsfr9 allele), that human sCD137 is secreted by regulatory T cells (Tregs; as in mice), and that human sCD137 induces T cell suppression in human T cells. These findings provide a rationale for further investigation of sCD137 as a treatment for T1D and other T cell-mediated autoimmune diseases.

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a naïve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

We previously reported that CD137 (encoded by Tnfrsf9) deficiency suppressed type 1 diabetes (T1D) progression in NOD mice. We also demonstrated that soluble CD137 produced by regulatory T cells contributed to their autoimmune-suppressive function in this model. These results suggest that CD137 can either promote or suppress T1D development in NOD mice depending on where it is expressed. In this study, we show that NOD.Tnfrsf9-/- CD8 T cells had significantly reduced diabetogenic capacity, whereas absence of CD137 in non-T and non-B cells had a limited impact on T1D progression. In contrast, NOD.Tnfrsf9-/- CD4 T cells highly promoted T1D development. We further demonstrated that CD137 was important for the accumulation of β cell-autoreactive CD8 T cells but was dispensable for their activation in pancreatic lymph nodes. The frequency of islet-infiltrating CD8 T cells was reduced in NOD.Tnfrsf9-/- mice in part because of their decreased proliferation. Furthermore, CD137 deficiency did not suppress T1D development in NOD mice expressing the transgenic NY8.3 CD8 TCR. This suggests that increased precursor frequency of β cell-autoreactive CD8 T cells in NY8.3 mice obviated a role for CD137 in diabetogenesis. Finally, blocking CD137-CD137 ligand interaction significantly delayed T1D onset in NOD mice. Collectively, our results indicate that one important diabetogenic function of CD137 is to promote the expansion and accumulation of β cell-autoreactive CD8 T cells, and in the absence of CD137 or its interaction with CD137 ligand, T1D progression is suppressed.

CD22, a sialic acid-binding Ig-type lectin (Siglec) family member, is an inhibitory coreceptor of the BCR with established roles in health and disease. The restricted expression pattern of CD22 on B cells and most B cell lymphomas has made CD22 a therapeutic target for B cell-mediated diseases. Models to better understand how in vivo targeting of CD22 translates to human disease are needed. In this article, we report the development of a transgenic mouse expressing human CD22 (hCD22) in B cells and assess its ability to functionally substitute for murine CD22 (mCD22) for regulation of BCR signaling, Ab responses, homing, and tolerance. Expression of hCD22 on transgenic murine B cells is comparable to expression on human primary B cells, and it colocalizes with mCD22 on the cell surface. Murine B cells expressing only hCD22 have identical calcium (Ca2+) flux responses to anti-IgM as mCD22-expressing wild-type B cells. Furthermore, hCD22 transgenic mice on an mCD22-/- background have restored levels of marginal zone B cells and Ab responses compared with deficiencies observed in CD22-/- mice. Consistent with these observations, hCD22 transgenic mice develop normal humoral responses in a peanut allergy oral sensitization model. Homing of B cells to Peyer's patches was partially rescued by expression of hCD22 compared with CD22-/- B cells, although not to wild-type levels. Notably, Siglec-engaging antigenic liposomes formulated with an hCD22 ligand were shown to prevent B cell activation, increase cell death, and induce tolerance in vivo. This hCD22 transgenic mouse will be a valuable model for investigating the function of hCD22 and preclinical studies targeting hCD22.

Type 1 diabetes (T1D) is currently an incurable disease, characterized by a silent prodromal phase followed by an acute clinical phase, reflecting progressive autoimmune destruction of insulin-producing pancreatic β-cells. Autoreactive T cells play a major role in β-cell destruction, but innate immune cell cytokines and costimulatory molecules critically affect T-cell functional status. We show that an agonistic monoclonal antibody to TLR4/MD-2 (TLR4-Ab) reverses new-onset diabetes in a high percentage of NOD mice. TLR4-Ab induces antigen-presenting cell (APC) tolerance in vitro and in vivo, resulting in an altered cytokine profile, decreased costimulatory molecule expression, and decreased T-cell proliferation in APC:T-cell assays. TLR4-Ab treatment increases T-regulatory cell (Treg) numbers in both the periphery and the pancreatic islet, predominantly expanding the Helios(+)Nrp-1(+)Foxp3(+) Treg subset. TLR4-Ab treatment in the absence of B cells in NOD.scid mice prevents subsequent T cell-mediated disease, further suggesting a major role for APC tolerization in disease protection. Specific stimulation of the innate immune system through TLR4/MD-2, therefore, can restore tolerance in the aberrant adaptive immune system and reverse new-onset T1D, suggesting a novel immunological approach to treatment of T1D in humans.

Abstract Viral, parasitic or bacterial infections can prevent T1D in NOD mice. The “hygiene hypothesis” proposes that the incidence of autoimmunity is increasing due to decreased immune stimulation in childhood, offering a rationale for stimulation of innate immunity as a treatment for T1D. We demonstrate that a TLR4/MD-2 specific agonistic monoclonal antibody, UT18, can not only prevent T1D, but also reverse new onset T1D (diagnosed by polyuria, weight loss, and elevated blood glucose). UT15, a control TLR4/MD-2 non-agonistic monoclonal antibody, neither prevented nor reversed T1D. In the prevention studies, UT18 treatment resulted in decreased insulitis vs. UT15 treatment. UT18 treatment permanently reversed new onset T1D in 9/13 mice, while UT15 treated mice rapidly progressed to end-stage diabetes (>600 mg/dl). Mice successfully treated with UT18 had less severe islet infiltration and increased insulin positive beta-cell area compared to UT15 controls. Transfer of CD4+ and CD8+ T-cells (from mice successfully treated with UT18) did not cause diabetes for up to 75 days post transfer, whereas cells transferred from UT15 and non-diabetic NOD mice rapidly caused disease. The lack of T cell mediated diabetes transfer suggests that T cell tolerance was restored in new onset diabetic mice treated with UT18. Restoration of T cell tolerance via TLR-4/MD-2 mediated innate immune stimulation is a promising new pathway for treatment of T1D and other T cell mediated autoimmune diseases.

Type 1 diabetes (T1D) is an auto immune disease perpetuated by intricate inter­ actions between innate and adaptive immune responses, which collaborate to cause destruction of insulin producing islet β cells. Antigen­specific CD8 and CD4 T cells ultimately destroy pancreatic islet β cells; however, clinical trials directed at dominant T­cell epitopes in T1D (e.g., glu­ tamic acid decarboxylase or insulin) have failed to reverse disease [1]. This does not necessarily mean that antigen­specific ther­ apies will not work, but highlights the need to uncover new therapeutic approaches and/ or combination therapies. Why have T­cell directed therapies failed? There may be fun­ damental innate immune defects that estab­ lish and maintain persistent auto reactive T­cell responses. These innate immune defects have been highlighted both in the mouse model of T1D, nonobese diabetic (NOD) mice, as well as in T1D patients reviewed elsewhere [2]. It is thus possible that correcting or ‘re­tuning’ the innate immune response will have beneficial effects in controlling autoreactive adaptive immune responses. The role of innate immunity in T1D may explain the rapidly rising incidence of T1D since the mid­20th century [3,4]. The ‘hygiene hypothesis’ explains the rising inci­ dence of T1D, as well as other autoimmune diseases, by positing that the lack of innate immune stimulation early in life primes the adaptive immune system toward auto­ immunity. The NOD mouse model sup­ ports this hypothesis, since mice housed in colonies with a greater bacterial burden have lower incidence of T1D compared with mice housed in ‘clean’ cages [5]. This suggests that innate immune stimulation may be beneficial in the prevention of T1D. There is indeed a large literature on effi­ cacy of therapeutic agents which stimulate or modulate the innate immune system to prevent T1D in NOD mice. However, although predictive biomarkers for T1D are being investigated, preventative therapeutics are currently inutile until an accurate marker is found. The unsolved clinical problem remains reversal of new onset disease. New onset T1D presents with elevated blood glucose levels, polyuria, failed glucose toler­ ance test and weight loss; these symptoms all arise after destruction of pancreatic islet β cells have already progressed. These clini­ cal features are present in the NOD mouse model and our lab (and others) has changed our focus from T1D prevention to reversal, which is a much more difficult task. Some adaptive immune modulatory agents have reversed T1D and induced hypertrophy of islet β cells in the NOD mouse model [6], but with limited effect in humans (gener­ Targeting innate immunity for treatment of Type 1 diabetes

Nonobese diabetic (NOD) mice are genetically programmed to spontaneously develop type one diabetes (T1D). Multiple Insulin dependent diabetes (Idd) genetic loci have been identified but their functional effects are mostly poorly understood. TnfsfR9, expressing the protein product CD137, is a strong candidate gene in the Idd9.3 locus, and NOD.B10 Idd9.3 mice are significantly protected from type one diabetes (T1D). We previously showed that nonobese diabetic (NOD) mice have a deficiency in the numbers of CD137(pos) T regulatory cells, that CD137(pos) Tregs are the source of soluble CD137 (sCD137), and that NOD mice have low serum levels of sCD137. To test the hypothesis that correcting low levels of sCD137 could affect the disease, we constructed a lentiviral vector producing recombinant sCD137; this physiologic sCD137 is glycosylated and exists primarily as a dimer. NOD mice treated with the recombinant sCD137 are protected from developing T1D. Insulitis is significantly decreased, but not eliminated in the sCD137 treated mice, however insulin producing pancreatic beta cells are preserved despite residual insulitis. To begin to understand the protective immune mechanisms of sCD137, we tested sCD137 in vitro. It was previously suggested that sCD137 simply blocked the interaction between CD137 (on T cells) and CD137 ligand (on antigen presenting cells (APCs)). Here however, we use an APC independent assay and demonstrate that sCD137 can actively suppress highly purified CD4 T cells in a CD137L dependent fashion. These results support the hypothesis that sCD137 acts in a negative feedback loop to actively suppress over-zealous immune responses, and that it can be used clinically to suppress autoimmunity. sCD137 is an important Treg derived natural immunosuppressive molecule that regulates effector T cells to avert diabetes in vivo.