A New Mechanism of Action in Human and Mouse Treg Cells: The Ke(y)to Suppression.

Little is known about how Treg cells mediate suppression, especially in tissues. In this issue of Immunity, Schmidleithner et al. report that Treg cells catabolize PGE2 into 15-keto-PGE2, a T cell-inhibitory metabolite, and that this mechanism controls inflammation in adipose tissue.

Eras of designer Tregs: Harnessing synthetic biology for immune suppression.

Since their discovery, CD4+ CD25hi FOXP3hi regulatory T cells (Tregs) have been firmly established as a critical cell type for regulating immune homeostasis through a plethora of mechanisms. Due to their immunoregulatory power, delivery of polyclonal Tregs has been explored as a therapy to dampen inflammation in the settings of transplantation and autoimmunity. Evidence shows that Treg therapy is safe and well-tolerated, but efficacy remains undefined and could be limited by poor persistence in vivo and lack of antigen specificity. With the advent of new genetic engineering tools, it is now possible to create bespoke "designer" Tregs that not only overcome possible limitations of polyclonal Tregs but also introduce new features. Here, we review the development of designer Tregs through the perspective of three 'eras': (1) the era of FOXP3 engineering, in which breakthroughs in the biological understanding of this transcription factor enabled the conversion of conventional T cells to Tregs; (2) the antigen-specificity era, in which transgenic T-cell receptors and chimeric antigen receptors were introduced to create more potent and directed Treg therapies; and (3) the current era, which is harnessing advanced genome-editing techniques to introduce and refine existing and new engineering approaches. The year 2022 marked the entry of "designer" Tregs into the clinic, with exciting potential for application and efficacy in a wide variety of immune-mediated diseases.

Tonic-signaling chimeric antigen receptors drive human regulatory T cell exhaustion.

Regulatory T cell (Treg) therapy is a promising approach to improve outcomes in transplantation and autoimmunity. In conventional T cell therapy, chronic stimulation can result in poor in vivo function, a phenomenon termed exhaustion. Whether or not Tregs are also susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. To "benchmark" exhaustion in human Tregs, we used a method known to induce exhaustion in conventional T cells: expression of a tonic-signaling chimeric antigen receptor (TS-CAR). We found that TS-CAR-expressing Tregs rapidly acquired a phenotype that resembled exhaustion and had major changes in their transcriptome, metabolism, and epigenome. Similar to conventional T cells, TS-CAR Tregs upregulated expression of inhibitory receptors and transcription factors such as PD-1, TIM3, TOX and BLIMP1, and displayed a global increase in chromatin accessibility-enriched AP-1 family transcription factor binding sites. However, they also displayed Treg-specific changes such as high expression of 4-1BB, LAP, and GARP. DNA methylation analysis and comparison to a CD8+ T cell-based multipotency index showed that Tregs naturally exist in a relatively differentiated state, with further TS-CAR-induced changes. Functionally, TS-CAR Tregs remained stable and suppressive in vitro but were nonfunctional in vivo, as tested in a model of xenogeneic graft-versus-host disease. These data are the first comprehensive investigation of exhaustion in Tregs and reveal key similarities and differences with exhausted conventional T cells. The finding that human Tregs are susceptible to chronic stimulation-driven dysfunction has important implications for the design of CAR Treg adoptive immunotherapy strategies.

Genetic engineering of regulatory T cells for treatment of autoimmune disorders including type 1 diabetes.

Suppression of pathogenic immune responses is a major goal in the prevention and treatment of type 1 diabetes. Adoptive cell therapy using regulatory T cells (Tregs), a naturally suppressive immune subset that is often dysfunctional in type 1 diabetes, is a promising approach to achieving localised and specific immune suppression in the pancreas or site of islet transplant. However, clinical trials testing administration of polyclonal Tregs in recent-onset type 1 diabetes have observed limited efficacy despite an excellent safety profile. Several barriers to efficacy have been identified, including lack of antigen specificity, low cell persistence post-administration and difficulty in generating sufficient cell numbers. Fortunately, the emergence of advanced gene editing techniques has opened the door to new strategies to engineer Tregs with improved specificity and function. These strategies include the engineering of FOXP3 expression to produce a larger source of suppressive cells for infusion, expressing T cell receptors or chimeric antigen receptors to generate antigen-specific Tregs and improving Treg survival by targeting cytokine pathways. Although these approaches are being applied in a variety of autoimmune and transplant contexts, type 1 diabetes presents unique opportunities and challenges for the genetic engineering of Tregs for adoptive cell therapy. Here we discuss the role of Tregs in type 1 diabetes pathogenesis and the application of Treg engineering in the context of type 1 diabetes.

Acidity promotes the differentiation of immunosuppressive regulatory T cells.

The metabolic milieu is emerging as a major contributing factor in the maintenance of the immunosuppressive microenvironment within tumors. In particular, the presence of lactic acid produced by highly glycolytic cancer cells is known to suppress antitumor immune cell subsets while promoting immunosuppressive cell populations, such as regulatory T cells (Tregs). Unlike conventional T cells, Tregs have a unique, potent ability to take up lactic acid to fuel both mitochondrial metabolism and gluconeogenesis, thus supporting suppressive function and proliferation. In this issue of the European Journal of Immunology [Eur. J. Immunol. 2023.53:2250258], Rao et al. uncover a novel mechanism by which lactic acid can support Treg accumulation within tumors in mice. This study shows that lactic acid, through a pH-dependent mechanism rather than lactate itself, promotes TGFß-induced differentiation of Tregs from conventional CD4+ T cells. These findings build on the already multifaceted role of lactic acid in maintaining an immunosuppressive tumor microenvironment.

Targeting regulatory T cell metabolism in disease: Novel therapeutic opportunities.

Regulatory T cells (Tregs) are essential for immune homeostasis and suppression of pathological autoimmunity but can also play a detrimental role in cancer progression via inhibition of anti-tumor immunity. Thus, there is broad applicability for therapeutic Treg targeting, either to enhance function, for example, through adoptive cell therapy (ACT), or to inhibit function with small molecules or antibody-mediated blockade. For both of these strategies, the metabolic state of Tregs is an important consideration since cellular metabolism is intricately linked to function. Mounting evidence has shown that targeting metabolic pathways can selectively promote or inhibit Treg function. This review aims to synthesize the current understanding of Treg metabolism and discuss emerging metabolic targeting strategies in the contexts of transplantation, autoimmunity, and cancer. We discuss approaches to gene editing and cell culture to manipulate Treg metabolism during ex vivo expansion for ACT, as well as in vivo nutritional and pharmacological interventions to modulate Treg metabolism in disease states. Overall, the intricate connection between metabolism and phenotype presents a powerful opportunity to therapeutically tune Treg function.

Preclinical assessment of antigen-specific chimeric antigen receptor regulatory T cells for use in solid organ transplantation.

A primary goal in transplantation medicine is the induction of a tolerogenic environment for prevention of transplant rejection without the need for long-term pharmacological immunosuppression. Generation of alloantigen-specific regulatory T cells (Tregs) by transduction with chimeric antigen receptors (CARs) is a promising strategy to achieve this goal. This publication reports the preclinical characterization of Tregs (TR101) transduced with a human leukocyte antigen (HLA)-A*02 CAR lentiviral vector (TX200) designated to induce immunosuppression of allograft-specific effector T cells in HLA-A*02-negative recipients of HLA-A*02-positive transplants. In vitro results demonstrated specificity, immunosuppressive function, and safety of TX200-TR101. In NOD scid gamma (NSG) mice, TX200-TR101 prevented graft-versus-host disease (GvHD) in a xenogeneic GvHD model and TX200-TR101 Tregs localized to human HLA-A*02-positive skin transplants in a transplant model. TX200-TR101 persisted over the entire duration of a 3-month study in humanized HLA-A*02 NSG mice and remained stable, without switching to a proinflammatory phenotype. Concomitant tacrolimus did not impair TX200-TR101 Treg survival or their ability to inhibit peripheral blood mononuclear cell (PBMC) engraftment. These data demonstrate that TX200-TR101 is specific, stable, efficacious, and safe in preclinical models, and provide the basis for a first-in-human study.

Helios is a marker, not a driver, of human Treg stability.

Treg therapy holds promise as a potentially curative approach to establish immune tolerance in transplantation and autoimmune disease. An outstanding question is whether therapeutic Tregs have the potential to transdifferentiate into effector T-cells and, thus, exacerbate rather than suppress immune responses. In mice, the transcription factor Helios is thought to promote Treg lineage stability in a range of inflammatory contexts. In humans, the role of Helios in Tregs is less clear, in part, due to the inability to enrich and study subsets of Helios-positive versus Helios-negative Tregs. Using an in vitro expansion system, we found that loss of high Helios expression and emergence of an intermediate Helios (Heliosmid )-expressing population correlated with Treg destabilization. We used CRISPR/Cas9 to genetically ablate Helios expression in human naive or memory Tregs and found that Helios-KO and unedited Tregs were equivalent in their suppressive function and stability in inflammation. Thus, high Helios expression is a marker, but not a driver, of human Treg stability in vitro. These data highlight the importance of monitoring Helios expression in therapeutic Treg manufacturing and provide new insight into the biological function of this transcription factor in human T-cells.

PTEN is required for human Treg suppression of costimulation in vitro.

Regulatory T-cell (Treg) therapy is under clinical investigation for the treatment of transplant rejection, autoimmune disease, and graft-versus-host disease. With the advent of genome editing, attention has turned to reinforcing Treg function for therapeutic benefit. A hallmark of Tregs is dampened activation of PI3K-AKT signaling, of which PTEN is a major negative regulator. Loss-of-function studies of PTEN, however, have not conclusively shown a requirement for PTEN in upholding Treg function and stability. Using CRISPR-based genome editing in human Tregs, we show that PTEN ablation does not cause a global defect in Treg function and stability; rather, it selectively blocks their ability to suppress antigen-presenting cells. PTEN-KO Tregs exhibit elevated glycolytic activity, upregulate FOXP3, maintain a Treg phenotype, and have no discernible defects in lineage stability. Functionally, PTEN is dispensable for human Treg-mediated inhibition of T-cell activity in vitro and in vivo but is required for suppression of costimulatory molecule expression by antigen-presenting cells. These data are the first to define a role for a signaling pathway in controlling a subset of human Treg activity. Moreover, they point to the functional necessity of PTEN-regulated PI3K-AKT activity for optimal human Treg function.

Guidelines for standardizing T-cell cytometry assays to link biomarkers, mechanisms, and disease outcomes in type 1 diabetes.

Cytometric immunophenotyping is a powerful tool to discover and implement T-cell biomarkers of type 1 diabetes (T1D) progression and response to clinical therapy. Although many discovery-based T-cell biomarkers have been described, to date, no such markers have been widely adopted in standard practice. The heterogeneous nature of T1D and lack of standardized assays and experimental design across studies is a major barrier to the broader adoption of T-cell immunophenotyping assays. There is an unmet need to harmonize the design of immunophenotyping assays, including those that measure antigen-agnostic cell populations, such that data collected from different clinical trial sites and T1D cohorts are comparable, yet account for cohort-specific features and different drug mechanisms of action. In these Guidelines, we aim to provide expert advice on how to unify aspects of study design and practice. We provide recommendations for defining cohorts, method implementation, as well as tools for data analysis and reporting by highlighting and building on selected successes. Harmonization of cytometry-based T-cell assays will allow researchers to better integrate findings across trials, ultimately enabling the identification and validation of biomarkers of disease progression and treatment response in T1D.