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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Immune homeostasis in the intestine is tightly controlled by FOXP3+ regulatory T cells (Tregs), defects of which are linked to the development of chronic conditions, such as inflammatory bowel disease (IBD). As a mechanism of immune evasion, several species of intestinal parasites boost Treg activity. The parasite Heligmosomoides polygyrus is known to secrete a molecule (Hp‐TGM) that mimics the ability of TGF‐β to induce FOXP3 expression in CD4+ T cells. The study aimed to investigate whether Hp‐TGM could induce human FOXP3+ Tregs as a potential therapeutic approach for inflammatory diseases. CD4+ T cells from healthy volunteers were expanded in the presence of Hp‐TGM or TGF‐β. Treg induction was measured by flow cytometric detection of FOXP3 and other Treg markers, such as CD25 and CTLA‐4. Epigenetic changes were detected using ChIP‐Seq and pyrosequencing of FOXP3. Treg phenotype stability was assessed following inflammatory cytokine challenge and Treg function was evaluated by cellular co‐culture suppression assays and cytometric bead arrays for secreted cytokines. Hp‐TGM efficiently induced FOXP3 expression (> 60%), in addition to CD25 and CTLA‐4, and caused epigenetic modification of the FOXP3 locus to a greater extent than TGF‐β. Hp‐TGM‐induced Tregs had superior suppressive function compared with TGF‐β‐induced Tregs, and retained their phenotype following exposure to inflammatory cytokines. Furthermore, Hp‐TGM induced a Treg‐like phenotype in in vivo differentiated Th1 and Th17 cells, indicating its potential to re‐program memory cells to enhance immune tolerance. These data indicate Hp‐TGM has potential to be used to generate stable human FOXP3+ Tregs to treat IBD and other inflammatory diseases. [ABSTRACT FROM AUTHOR]

Regulating the regulatory cells: Regulatory T cells (Tregs) are antigen-specific immunosuppressive cells that help prevent undesired immune responses. Chimeric antigen receptors (CARs) are engineered T cell receptors that allow direct recognition of a target antigen without requiring antigen presentation. CARs, most often, have been used with conventional T cells, directing them to attack cancer cells, for example. CAR Tregs have also been developed for treating autoimmune disorders or transplant rejection, but their biology is not as well understood. Dawson et al. addressed this knowledge gap by systematically studying and optimizing CAR signaling domains in Tregs and then applying them to a mouse model of graft-versus-host disease. Antigen-specific regulatory T cells (Tregs) engineered with chimeric antigen receptors (CARs) are a potent immunosuppressive cellular therapy in multiple disease models and could overcome shortcomings of polyclonal Treg therapy. CAR therapy was initially developed with conventional T cells, which have different signaling requirements than do Tregs. To date, most of the CAR Treg studies used second-generation CARs, encoding a CD28 or 4-1BB co-receptor signaling domain and CD3ζ, but it was not known if this CAR design was optimal for Tregs. Using a human leukocyte antigen–A2–specific CAR platform and human Tregs, we compared 10 CARs with different co-receptor signaling domains and systematically tested their function and CAR-stimulated gene expression profile. Tregs expressing a CAR encoding CD28wt were markedly superior to all other CARs tested in an in vivo model of graft-versus-host disease. In vitro assays revealed stable expression of Helios and an ability to suppress CD80 expression on dendritic cells as key in vitro predictors of in vivo function. This comprehensive study of CAR signaling domain variants in Tregs can be leveraged to optimize CAR design for use in antigen-specific Treg therapy. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is caused by immune-mediated destruction of insulin-producing β-cells. Insufficient control of autoreactive T cells by regulatory T cells (Tregs) is believed to contribute to disease pathogenesis, but changes in Treg function are difficult to quantify because of the lack of Treg-exclusive markers in humans and the complexity of functional experiments. We established a new way to track Tregs by using a gene signature that discriminates between Tregs and conventional T cells regardless of their activation states. The resulting 31-gene panel was validated with the NanoString nCounter platform and then measured in sorted CD4(+)CD25(hi)CD127(lo) Tregs from children with T1D and age-matched control subjects. By using biomarker discovery analysis, we found that expression of a combination of six genes, including TNFRSF1B (CD120b) and FOXP3, was significantly different between Tregs from subjects with new-onset T1D and control subjects, resulting in a sensitive (mean ± SD 0.86 ± 0.14) and specific (0.78 ± 0.18) biomarker algorithm. Thus, although the proportion of Tregs in peripheral blood is similar between children with T1D and control subjects, significant changes in gene expression can be detected early in disease process. These findings provide new insight into the mechanisms underlying the failure to control autoimmunity in T1D and might lead to a biomarker test to monitor Tregs throughout disease progression. [ABSTRACT FROM AUTHOR]

The role of forkhead box P3 (FOXP3) is well-established in regulatory T cells, but the function of transient FOXP3 expression in activated human conventional T (Tconv) cells is unknown. We used two approaches to determine the role of FOXP3 in human Tconv cells. First, we obtained Tconv clones from a female who is hemizygous for a null mutation in FOXP3, allowing the comparison of autologous T cell clones that do or do not express FOXP3. Second, we knocked down activation-induced FOXP3 in Tconv cells from healthy donors with small interfering RNA against FOXP3. We found that FOXP3-deficient Tconv cells proliferate more, produce more cytokines than wild type Tconv cells, and have differential expression of 264 genes. We also investigated the role of FOXP3 in Th1 and Th17 cells and found that in comparison to Th1 cells, expression of activation-induced FOXP3 was higher and sustained in Th17 cells. Knocking down FOXP3 expression in Th17 cells significantly increased production of IFN-γ and decreased expression of CCR4, but had no effect on IL-17 expression. These data reveal a novel function of FOXP3 in Tconv cells and suggest that expression of this protein is important in the function of multiple CD4+ T cell lineages. [ABSTRACT FROM AUTHOR]

Background: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is a primary immunodeficiency with autoimmunity caused by mutations in forkhead box P3 (FOXP3), which encodes a transcription factor involved in regulatory T (Treg) cell function. The mechanistic basis for how different mutations in FOXP3 cause distinct manifestations of IPEX remains unclear. Objective: To determine whether 3 different point mutants of FOXP3 that cause severe or mild IPEX differ in their ability to reprogram conventional T cells into Treg cells. Methods: Human CD4+ T cells were transduced with wild-type or point mutant forms of FOXP3, and changes in cell surface marker expression, cytokine production, proliferation and suppressive capacity were assessed. Ex vivo TH17 cells were also transduced with different forms of FOXP3 to monitor changes in IL-17 production. Results: The forkhead mutant F373A failed to upregulate CD25 and CCR4, did not suppress cytokine production, and induced suppressive activity less effectively than wild-type FOXP3. In contrast, although the forkhead mutant R347H was also defective in upregulation of CD25, it suppressed the production of cytokines, conferred suppressive capacity on CD4+ T cells, and suppressed IL-17 production. F324L, a mutant outside the forkhead domain associated with mild IPEX, was equivalent to wild-type FOXP3 in all aspects tested. Conclusion: Mutations in FOXP3 that cause IPEX do not uniformly abrogate the ability of FOXP3 to regulate transcription and drive the development of Treg cells. These data support the notion that factors in addition to functional changes in Treg cells, such as alterations in conventional T cells, are involved in the pathogenesis of IPEX. [ABSTRACT FROM AUTHOR]

Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2-specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases. [ABSTRACT FROM AUTHOR]

T regulatory cells (Tregs) control immune homeostasis by preventing inappropriate responses to self and nonharmful foreign antigens. Tregs use multiple mechanisms to control immune responses, all of which require these cells to be near their targets of suppression; however, it is not known how Treg-to-target proximity is controlled. Here, we found that Tregs attract CD4+ and CD8+ T cells by producing chemokines. Specifically, Tregs produced both CCL3 and CCL4 in response to stimulation, and production of these chemokines was critical for migration of target T cells, as Tregs from Ccl3-/- mice, which are also deficient for CCL4 production, did not promote migration. Moreover, CCR5 expression by target T cells was required for migration of these cells to supernatants conditioned by Tregs. Tregs deficient for expression of CCL3 and CCL4 were impaired in their ability to suppress experimental autoimmune encephalomyelitis or islet allograft rejection in murine models. Moreover, Tregs from subjects with established type 1 diabetes were impaired in their ability to produce CCL3 and CCL4. Together, these results demonstrate a previously unappreciated facet of Treg function and suggest that chemokine secretion by Tregs is a fundamental aspect of their therapeutic effect in autoimmunity and transplantation. [ABSTRACT FROM AUTHOR]