Regulatory T cell (Treg) therapy is an emerging platform for controlling immune overactivation. Persistence of infused Tregs is limited by insufficient IL-2, which is essential for Treg survival and function. IL-2 activates many immune cells, imposing a challenge for the selective provision of IL-2 to infused Tregs. In this study, we found that infusions of orthogonal (ortho) IL-2 failed to enhance Tregs expressing a corresponding orthoIL-2 receptor (IL-2R) in a mouse model of autoimmune diabetes. Engineering Tregs with an orthoIL-2 tethered to its receptor achieved selective autocrine signaling; increased CD25, CTLA-4, and Foxp3 expression; supported Treg persistence without exogenous IL-2 in vivo; and improved the efficacy of Treg prevention of autoimmune diabetes. Inserting the tethered orthoIL-2 construct into the Foxp3 locus enabled Treg-specific self-reinforced expression through the activation of the Foxp3 locus by increased IL-2 signaling. Together, these results illustrate a safe and effective cell-engineering solution for overcoming Tregs' dependency on exogenous IL-2, thereby achieving superior therapeutic efficacy.

Type 1 diabetes is an autoimmune condition in which the pancreatic β cells that produce insulin are destroyed by the body's immune system. For 100 years, diet and insulin injections have been the only effective treatment. Recent advances have led to significant progress in our understanding of the pathogenesis of the disease and the interplay between the environment, components of the immune system, and the β cells that are targeted. This has led to new therapies that rebalance the immune system and finally offer the promise of a cure.

In type 1 diabetes (T1D), the immune system mistakenly attacks the pancreatic islet β cells resulting in the loss of insulin secretion. Insulin-replacement therapy developed more than a century ago provided means to manage the symptoms of diabetes without addressing the root cause of the disease-the faulty immune system. A healthy immune system has built-in mechanisms to limit unwanted, excessive immune activation and prevents damages to self-tissues. These immune self-tolerance mechanisms are often impaired in autoimmune patients including those with T1Ds. Understanding how immune self-tolerance is broken in patients with T1D can inform the design of new curative therapies that correct the immune defects. In this paper, we will summarize the mechanisms of immune tolerance, review their relevance to T1Ds, and discuss novel therapeutic approaches to rebalance the immune system for the treatment of T1Ds.

Regulatory T cell therapies have shown promise for treating autoimmune diseases and aiding transplantation. We summarize a recent NIAID/HESI-sponsored workshop that addressed key issues in non-clinical and clinical development vital to advancing this immune tolerance paradigm.

In November 2022, teplizumab became the first drug approved to delay the course of any autoimmune disease and to change the course of type 1 diabetes (T1D) since the discovery of insulin. The path to its approval took more than 30 years with both successes and failures along the way that would have normally led to its abandonment in other circumstances. Development of the drug was based on studies in preclinical models and parallels efforts in transplantation. From a series of innovative adaptations in response to issues related to adverse events and immunogenicity, humanized Fc receptors (FcR) nonbinding antibodies were developed with improved clinical outcomes and safety as well as new mechanisms. Importantly, as a result of these developments, teplizumab has been able to achieve efficacy over extended periods of time without global immune suppression. The approval of teplizumab represents a significant first step toward achieving escape from T1D and, in the future, reversal of the disease.

IntroductionThe success of regulatory T cell (Treg) therapies depends on the source of Treg and the quality of the Treg manufacturing product that maintains Treg identity. Commonly used methods to identify Treg, including assessment of FOXP3 expression and demethylation of the Treg-specific demethylated region (TSDR), may not be sufficient on their own to ensure that Treg cell therapy drug products have an optimal identity and phenotype prior to infusion into patients.MethodsTo address this critical need, we developed a robust framework to molecularly characterize Treg products using next-generation sequencing. By systematically profiling Treg and effector T cells (Teff) pre- and post-expansion, we defined the molecular fingerprints for expanded Treg products. We employed a non-parametric algorithm to score Treg manufacturing products for their cell identity and expansion fingerprints.ResultsThe identity fingerprint reflects Treg cell identity by effectively distinguishing Treg from Teff cells irrespective of their activation status, with 100% sensitivity and specificity, while the expansion fingerprint discriminates expanded versus endogenous Treg or Teff cells. We also showed that the identity fingerprint predicts Treg stability in in vitro settings and can be used to illustrate differences in drug products generated using distinct strategies. We further applied fingerprinting to bulk RNA sequencing (RNA-seq) data from endogenous and expanded Treg cells in a Phase 2 clinical trial for type 1 diabetes (T1D), demonstrating its ability to capture Treg identity and expansion in an independent study.DiscussionThis Treg fingerprinting method provides a powerful tool to molecularly characterize Treg products, potentially enabling correlative analysis with the safety and efficacy outcomes of Treg-based cell therapies.

In autoimmunity, an imbalance of effector (Teff) and regulatory (Treg)T cells contributes to inflammation and tissue destruction. CD2, highly expressed on Teff and at lower levels on Treg and naive T cells (Tn), is an attractive target for depleting Teff at sites of inflammation. SBT115301 is a second generation CD2-targeting fusion protein containing the cognate receptor of CD2, lymphocyte function associated antigen-3 (LFA-3; CD58). In in vitro and in vivo studies, SBT115301 preferentially decreased CD2hi-expressing Teff cells compared to Treg and Tn. In a phase 1 clinical trial, SBT115301 selectively reduced memory T cells. SBT115301 was well tolerated aside from decreases of CD4+ T cells in some participants in the highest dose IM and IV cohorts. Anti-drug antibodies decreased exposure of SBT115301 in some participants without affecting the pharmacodynamics. These data support further study of SBT115301 as a monotherapy or in combination with other drugs in autoimmune indications.

In autoimmune Type 1 diabetes (T1D), immune cells infiltrate and destroy the islets of Langerhans - islands of endocrine tissue dispersed throughout the pancreas. However, the contribution of cellular programs outside islets to insulitis is unclear. Here, using CO-Detection by indEXing (CODEX) tissue imaging and cadaveric pancreas samples, we simultaneously examine islet and extra-islet inflammation in human T1D. We identify four sub-states of inflamed islets characterized by the activation profiles of CD8 + T cells enriched in islets relative to the surrounding tissue. We further find that the extra-islet space of lobules with extensive islet-infiltration differs from the extra-islet space of less infiltrated areas within the same tissue section. Finally, we identify lymphoid structures away from islets enriched in CD45RA + T cells - a population also enriched in one of the inflamed islet sub-states. Together, these data help define the coordination between islets and the extra-islet pancreas in the pathogenesis of human T1D.

Adoptive regulatory T cell (Treg) therapy is an emergent treatment paradigm for restoring immune tolerance in patients with autoimmune and inflammatory diseases. While preclinical models have demonstrated the remarkable efficacy of Treg cell therapy, lineage-tracing studies have also revealed that murine Tregs can lose lineage identity and adopt a proinflammatory state. Whether human Tregs exhibit a similar susceptibility to inflammatory cytokine-mediated lineage decommitment remains inconclusive. Here, we present an in vitro model of human Treg destabilization characterized by silencing of the Treg lineage-defining transcription factor, FOXP3, loss of suppressive activity, and gain of proinflammatory functions. Analysis of single-cell ATAC and transcriptomic profiles of Tregs during destabilization revealed a switch in chromatin accessibility between two elements at the IRF4 locus. Excision of the distal IRF4 element enabled Tregs to resist inflammatory cytokine-induced reprogramming, presenting a cell-engineering strategy to design more effective Treg therapies.

Adoptive regulatory T-cell (Treg) therapy is an emerging therapeutic paradigm for promoting immune tolerance in transplant and autoimmune disease settings. Prior investigations demonstrate that murine Tregs can undergo epigenetic reprogramming within chronically inflamed tissue environments, resulting in acquisition of proinflammatory functions and the capacity to exacerbate tissue damage. Despite the ramifications of Treg lineage decommitment for cell therapy applications, inflammation-induced human Treg cell fate decisions remain poorly understood. Here, we present a robust in vitromodel of IL6, IL1β, and IL23-driven Treg instability characterized by progressive FOXP3 and HELIOS downregulation, FOXP3conserved non-coding sequence (CNS)2 enhancer re-methylation, diminished in vitrosuppressive function, and elevated proinflammatory cytokine expression. To gain insight into the gene regulatory networks enabling the loss of Treg identity, we generated single-cell transcriptomic and chromatin accessibility profiles of primary human Tregs maintained in the presence or absence of IL6, IL1β, and IL23. Unsupervised clustering revealed a dysfunctional Treg population with an epigenetic signature consistent with murine Treg to “exTreg” conversion, including altered chromatin accessibility at the IFNγ, IL17A, and FOXP3CNS2 loci. Inference of transcription factor (TF)-associated changes in chromatin accessibility indicated a key role for E26 transformation-specific (ETS) family members. Ongoing experiments aim to identify specific TF modules that can be targeted to better safeguard the function and stability of Treg therapeutics.

Engineered regulatory T (T reg ) cells have emerged as precision therapeutics aimed at inducing immune tolerance while reducing the risks associated with generalized immunosuppression. This Viewpoint highlights the opportunities and challenges for engineered T reg cell therapies in treating autoimmune and other inflammatory diseases.

Insulin is a central autoantigen in the pathogenesis of T1D, and thymic epithelial cell expression of insulin under the control of the Autoimmune Regulator ( Aire ) is thought to be a key component of maintaining tolerance to insulin. In spite of this general working model, direct detection of this thymic selection on insulin-specific T cells has been somewhat elusive. Here, we used a combination of highly sensitive T cell receptor transgenic models for detecting thymic selection and sorting and sequencing of Insulin-specific CD4+ T cells from Aire-deficient mice as a strategy to further define their selection. This analysis revealed a number of unique t cell receptor (TCR) clones in Aire-deficient hosts with high affinity for insulin/major histocompatibility complex (MHC) ligands. We then modeled the thymic selection of one of these clones in Aire-deficient versus wild-type hosts and found that this model clone could escape thymic negative selection in the absence of thymic Aire. Together, these results suggest that thymic expression of insulin plays a key role in trimming and removing high-affinity insulin-specific T cells from the repertoire to help promote tolerance.

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

Regulatory T (Treg) cells are essential for maintaining peripheral tolerance, preventing autoimmunity, and limiting chronic inflammatory diseases. This small CD4+ T cell population can develop in the thymus and in the peripheral tissues of the immune system through the expression of an epigenetically stabilized transcription factor, FOXP3. Treg cells mediate their tolerogenic effects using multiple modes of action, including the production of inhibitory cytokines, cytokine starvation of T effector (e.g., IL-2), Teff suppression by metabolic disruption, and modulation of antigen-presenting cell maturation or function. These activities together result in the broad control of various immune cell subsets, leading to the suppression of cell activation/expansion and effector functions. Moreover, these cells can facilitate tissue repair to complement their suppressive effects. In recent years, there has been an effort to harness Treg cells as a new therapeutic approach to treat autoimmune and other immunological diseases and, importantly, to re-establish tolerance. Recent synthetic biological advances have enabled the cells to be genetically engineered to achieve tolerance and antigen-specific immune suppression by increasing their specific activity, stability, and efficacy. These cells are now being tested in clinical trials. In this review, we highlight both the advances and the challenges in this arena, focusing on the efforts to develop this new pillar of medicine to treat and cure a variety of diseases.

Context:Hypophysitis is a known immune-related adverse event (irAE) of immune checkpoint inhibitors (CPIs), commonly associated with CTLA-4 inhibitors and less often with PD-1/PD-L1 inhibitors. Objective:We aimed to determine clinical, imaging, and HLA characteristics of CPI-induced hypophysitis (CPI-hypophysitis). Methods:We examined the clinical and biochemical characteristics, magnetic resonance imaging (MRI) of the pituitary, and association with HLA type in patients with CPI-hypophysitis. Results:Forty-nine patients were identified. Mean age was 61.3 years, 61.2% were men, 81.6% were Caucasian, 38.8% had melanoma, and 44.5% received PD-1/PD-L1 inhibitor monotherapy while the remainder received CTLA-4 inhibitor monotherapy or CTLA-4/PD-1 inhibitor combination therapy. A comparison of CTLA-4 inhibitor exposure vs PD-1/PD-L1 inhibitor monotherapy revealed faster time to CPI-hypophysitis (median 84 vs 185 days, P < .01) and abnormal pituitary appearance on MRI (odds ratio 7.00, P = .03). We observed effect modification by sex in the association between CPI type and time to CPI-hypophysitis. In particular, anti-CTLA-4 exposed men had a shorter time to onset than women. MRI changes of the pituitary were most common at the time of hypophysitis diagnosis (55.6% enlarged, 37.0% normal, 7.4% empty or partially empty) but persisted in follow-up (23.8% enlarged, 57.1% normal, 19.1% empty or partially empty). HLA typing was done on 55 subjects; HLA type DQ0602 was over-represented in CPI-hypophysitis relative to the Caucasian American population (39.4% vs 21.5%, P = 0.01) and CPI population. Conclusion:The association of CPI-hypophysitis with HLA DQ0602 suggests a genetic risk for its development. The clinical phenotype of hypophysitis appears heterogenous, with differences in timing of onset, changes in thyroid function tests, MRI changes, and possibly sex related to CPI type. These factors may play an important role in our mechanistic understanding of CPI-hypophysitis.

Post-acute sequelae of COVID-19 (PASC) represent an emerging global crisis. However, quantifiable risk factors for PASC and their biological associations are poorly resolved. We executed a deep multi-omic, longitudinal investigation of 309 COVID-19 patients from initial diagnosis to convalescence (2-3 months later), integrated with clinical data and patient-reported symptoms. We resolved four PASC-anticipating risk factors at the time of initial COVID-19 diagnosis: type 2 diabetes, SARS-CoV-2 RNAemia, Epstein-Barr virus viremia, and specific auto-antibodies. In patients with gastrointestinal PASC, SARS-CoV-2-specific and CMV-specific CD8+ T cells exhibited unique dynamics during recovery from COVID-19. Analysis of symptom-associated immunological signatures revealed coordinated immunity polarization into four endotypes, exhibiting divergent acute severity and PASC. We find that immunological associations between PASC factors diminish over time, leading to distinct convalescent immune states. Detectability of most PASC factors at COVID-19 diagnosis emphasizes the importance of early disease measurements for understanding emergent chronic conditions and suggests PASC treatment strategies.