Anke Fuchs

Background: FOXP3 ⁺ regulatory T cells (Tregs) play a pivotal role in maintaining immune homeostasis and self-tolerance. Despite advances in Treg-based immunosuppressive therapies, precise identification of human Tregs facilitating their isolation with high purity remains challenging because canonical markers such as FOXP3 and CD25 are also induced in activated CD4 ⁺ effector T cells (Teffs). This study aims to leverage adult peripheral blood, umbilical cord blood, and pediatric thymic tissue to precisely characterize human Tregs and to gain deeper insights into heterogeneity across sources and developmental stages. Methods: We conducted extensive flow cytometric analysis of 31 extra- and intracellular markers expressed by human Tregs, followed by an in-depth comparison of Tregs and Teffs, as well as between Tregs derived from all three sources. Results: Our results showed that, while most markers were shared with Teffs, the transcription factor Helios, the co-inhibitory receptors CTLA-4 and TIGIT, and the glycoprotein receptor GPA33 were expressed by a higher proportion of Tregs than Teffs across sources. Contrary, a consistently higher proportion of Teffs than Tregs expressed the co-stimulatory receptors CD26 and CD226. Thymocytes displayed marked heterogeneity, containing Tregs at distinct developmental stages and recirculating peripheral Tregs. The proportion of CD25 ⁺ FOXP3 ^lo/– CD4 single positive (SP) precursor cells expressing Treg specific markers Helios, TIGIT and CTLA-4 was significantly lower than of CD25 ⁺ FOXP3 ⁺ double-positive (CD4 ⁺ CD8 ⁺ ) Tregs and mature CD25 ⁺ FOXP3 ⁺ CD4SP Tregs. These findings challenge the previously held notion that CD25 ⁺ FOXP3 ^lo/– precursors uniformly mature into CD25 ⁺ FOXP3 ^+/hi Thy-Tregs. As this subset differs from mature Thy-Tregs primarily by FOXP3 expression - a marker unsuitable for live-cell sorting - conventional isolation methods fail to exclude this immature subset. Importantly, our study identified the surface markers CD45RA/CD45RO, GPA33, TIGIT, and PD-1 to exclusively distinguish mature Thy-Tregs from these precursors. Moreover, our study provides a detailed characterization of highly activated recirculating peripheral Tregs within the thymocytes. Among the three sources examined, cord blood-derived Tregs exhibited the greatest phenotypic uniformity compared with adult blood- and thymus-derived Tregs. Conclusion: Overall, this study provides highly detailed insights into the heterogeneity of Tregs across distinct developmental stages and therapeutic sources, while also contributing towards improved isolation strategies for therapeutic approaches.

Background: Adoptive transfer of regulatory T cells (Tregs) has demonstrated safety, feasibility and early signs of efficacy in promoting immunological tolerance in inflammatory conditions such as graft-versus-host disease (GvHD). Chimeric antigen receptor (CAR)-engineered Tregs offer localized activation and suppression compared to polyclonal Tregs, but their clinical translation is limited by high manufacturing costs, lengthy developing times and fixed single-antigen specificity. To address these limitations, we employed the universal adapter Reverse CAR (RevCAR) system, which harbors a peptide epitope lacking intrinsic antigen specificity but provides flexibility in targeting through the use of an antigen-specific RevCAR Target Module (RevTM). As a proof-of-concept, we used a RevTM targeting carcinoembryonic antigen (CEA), which is highly expressed in the gastrointestinal (GI) tract, as a potential strategy to achieve localized immunosuppression in GI acute GvHD. Methods: To support clinical translation, we established an automated, GMP-compatible, clinical-scale manufacturing process. Tregs were magnetically enriched from leukapheresis using the CliniMACS ^® Plus, followed by high-purity sorting on the MACSQuant ^® Tyto ^® . The sorted cells were virally transduced and the RevCAR Tregs were expanded on the CliniMACS Prodigy ^® to obtain clinically relevant cell numbers. The harvested products were evaluated for phenotype, stability, antigen specificity and suppressive function. Results: Across five manufacturing runs, Tregs (CD4 ⁺ CD25 ^high CD127 ^low FOXP3 ⁺ ) with a median initial purity of 94% were expanded to achieve a median therapeutic yield of 602 × 10 ⁶ cells. The final product maintained a high purity (median: 91.9%) and exhibited high RevCAR expression (median: 60% RevCAR ⁺ ). Mass cytometry analysis revealed that expanded RevCAR Tregs predominantly exhibited a central memory phenotype with high expression of functional and homing markers. Under experimental pro-inflammatory conditions, the cells maintained stable FOXP3 and Helios expression with minimal pro-inflammatory cytokine production. Importantly, RevCAR Tregs showed antigen-specific activation upon target engagement via the CEA-specific RevTM and robust, dose-dependent suppression. Conclusion: The study establishes a scalable, GMP-compatible process for manufacturing pure, stable and functional universal RevCAR Tregs for clinical applications. Furthermore, the RevCAR system offers a promising approach toward an allogenic, off-the-shelf Treg therapy capable of treating diverse immune-mediated diseases with spatial precision.

Regulatory T cells (Tregs) play a pivotal role in modulating excessive immune responses and maintaining immune homeostasis in humans. Notably, therapeutic strategies employing autologous and allogeneic Tregs have shown promising signs of efficacy in the treatment and prevention of graft versus-host disease (GvHD), transplant rejection and autoimmune diseases. Treg cells are typically obtained from peripheral blood or umbilical cord blood, but the largely antigen-experienced memory state of peripheral blood Tregs and the limited number of Tregs that can be isolated from cord blood remain obstacles. However, recent studies have identified the thymus as a novel and promising source of Tregs, overcoming the abovementioned limitations. Currently, human thymus-isolated regulatory T cells (thyTregs) are being investigated in phase 1/2 clinical trials to assess their safety and efficacy in both autologous and allogeneic settings. This review provides a comprehensive overview of the different manufacturing processes for isolation and expansion of thymus-derived regulatory T cells, their clinical relevance and current ongoing clinical trials investigating the therapeutic potential of this novel class of Tregs.

The initial idea of a distinct group of T-cells responsible for suppressing immune responses was first postulated half a century ago. However, it is only in the last three decades that we have identified what we now term regulatory T-cells (Tregs), and subsequently elucidated and crystallized our understanding of them. Human Tregs have emerged as essential to immune tolerance and the prevention of autoimmune diseases and are typically contemporaneously characterized by their CD3⁺CD4⁺CD25^high CD127^lowFOXP3⁺ phenotype. It is important to note that FOXP3⁺ Tregs exhibit substantial diversity in their origin, phenotypic characteristics, and function. Identifying reliable markers is crucial to the accurate identification, quantification, and assessment of Tregs in health and disease, as well as the enrichment and expansion of viable cells for adoptive cell therapy. In our comprehensive review, we address the contributions of various markers identified in the last two decades since the master transcriptional factor FOXP3 was identified in establishing and enriching purity, lineage stability, tissue homing and suppressive proficiency in CD4⁺ Tregs. Additionally, our review delves into recent breakthroughs in innovative Treg-based therapies, underscoring the significance of distinct markers in their therapeutic utilization. Understanding Treg subsets holds the key to effectively harnessing human Tregs for immunotherapeutic approaches.

Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications.