Alicia N. McMurchy

Inducible reprogramming of human T cells into Treg cells by a conditionally active form of FOXP3

FOXP3 is required for the development of Treg and its expression is often used as a surrogate marker of functional suppression. However, it is now known that activated human T effector cells can also express FOXP3 without acquiring regulatory activity. To more closely examine the requirements for FOXP3 to reprogram human T cells into Treg, we developed a conditionally active form of FOXP3 and show here that full acquisition of Treg phenotype and function is strictly dependent on the amount of active FOXP3 a T cell expresses. In addition, the phenotypic and functional alterations induced by FOXP3 are only fully manifested following prolonged induction of protein activity. Induction of FOXP3 activity does not upregulate EBI3 or p35 mRNA, providing evidence that secretion of IL‐35 does not substantially contribute to the suppressive mechanism of human Treg. These data represent the first formal evidence that FOXP3 acts as a quantitative regulator rather than a simple molecular switch for Treg.

Moving to tolerance: clinical application of T regulatory cells.

Decreasing the incidence of chronic rejection and reducing the need for life-long immunosuppression remain important goals in clinical transplantation. In this article, we will review how regulatory T cells (Treg) came to be recognized as an attractive way to prevent or treat allograft rejection, the ways in which Treg can be manipulated or expanded in vivo, and the potential of in vitro expanded/generated Treg for cellular therapy. We will describe the first regulatory T cell therapies that have been or are in the process of being conducted in the clinic as well as the safety concerns of such therapies and how outcomes may be measured.

TH17 Cells in Autoimmunity and Immunodeficiency: Protective or Pathogenic?

In 2005 a newly discovered T helper cell subset that secreted interleukin (IL)-17 became the center of attention in immunology. Initial studies painted Th17 cells as the culprit for destruction in many different autoimmune and auto-inflammatory diseases. Subsequently, the discovery of patients with primary immunodeficiencies in the IL-17 pathway taught us that Th17 cells have a critical role in defense against certain fungal and bacterial infections. Moreover, the paradoxical exacerbation of Crohn’s disease in the clinical trials of a Secukinumab (AIN457), a fully human neutralizing antibody to IL-17A, has cast into doubt a universal pro-inflammatory and harmful role for Th17 cells. Evidence now suggests that depending on the environment Th17 cells can alter their differentiation program, ultimately giving rise to either protective or pro-inflammatory cells. In this review we will summarize the evidence from patients with immunodeficiencies, autoimmune, or auto-inflammatory diseases that teaches us how the pro-inflammatory versus protective function of Th17 cells varies within the context of different human diseases.

Suppression assays with human T regulatory cells: a technical guide.

The suppression of inappropriate immune responses by Treg cells is one of the major ways that the body maintains immune tolerance and homeostasis. Since defects in the suppressive capacity of Treg cells underlie many different immune‐mediated diseases, there is great interest in developing ways to track the number and function of Treg cells as biomarkers of tolerance and in devising ways to enhance their function therapeutically. However, the methods of studying human Treg cells are fraught with technical challenges that can often lead to misinterpretation. The most common way to determine the suppressive capacity of human Treg cells is to measure their ability to suppress the proliferation of responding CD4+ T cells. Here, we discuss the technical considerations that must be taken into account when performing suppression of T‐cell proliferation assays with human Treg cells. We also consider how T cells may falsely appear suppressive because of dying cells in the system, improper resting of T‐cell lines prior to the assay, or insufficient proliferation of the responding T cells. We propose that, in the future, classification of a population of cells as “regulatory” should rely on more than a simple test for blockade of CD4+ T‐cell proliferation.

A novel function for FOXP3 in humans: intrinsic regulation of conventional T cells

The role of forkhead box P3 (FOXP3) is well-established in T-regulatory cells, but the function of transient FOXP3 expression in activated human conventional T (Tconv) cells is unknown. In the present study, we used 2 approaches to determine the role of FOXP3 in human Tconv cells. First, we obtained Tconv clones from a female subject 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 RNAagainst FOXP3. We found that FOXP3-deficient Tconv cells proliferate more and produce more cytokines than wild-type Tconv cells and have differential expression of 274 genes. We also investigated the role of FOXP3 in Th1 and Th17 cells and found that the expression of activation-induced FOXP3 was higher and more sustained in Th17 cells compared with Th1 cells. Knocking down FOXP3 expression in Th17 cells significantly increased the production of IFN-γ and decreased the 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.

Point mutants of forkhead box P3 that cause immune dysregulation, polyendocrinopathy, enteropathy, X-linked have diverse abilities to reprogram T cells into regulatory T cells

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 T(H)17 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.

Wild-type FOXP3 is selectively active in CD4+CD25hi regulatory T cells of healthy female carriers of different FOXP3 mutations

Forkhead box P3 (FOXP3) is constitutively expressed by CD4(+)CD25(hi) regulatory T cells (nTregs). Mutations of FOXP3 cause a severe autoimmune syndrome known as immune dysregulation polyendocrinopathy enteropathy X-linked, in which nTregs are absent or dysfunctional. Whether FOXP3 is essential for both differentiation and function of human nTreg cells remains to be demonstrated. Because FOXP3 is an X-linked gene subject to X-chromosome inactivation (XCI), we studied 9 healthy female carriers of FOXP3 mutations to investigate the role of wild-type (WT) versus mutated FOXP3 in different cell subsets. Analysis of active WT versus mutated (mut)-FOXP3 allele distribution revealed a random pattern of XCI in peripheral blood lymphocytes and in naive and memory CD4(+)T cells, whereas nTregs expressed only the active WT-FOXP3. These data demonstrate that expression of WT-FOXP3 is indispensable for the presence of a normal nTreg compartment and suggest that FOXP3 is not necessary for effector T-cell differentiation in humans.

Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells.

Regulatory T cell (Treg)–based therapy is a promising approach to treat many immune‐mediated disorders such as autoimmune diseases, organ transplant rejection, and graft‐versus‐host disease (GVHD). Challenges to successful clinical implementation of adoptive Treg therapy include difficulties isolating homogeneous cell populations and developing expansion protocols that result in adequate numbers of cells that remain stable, even under inflammatory conditions. We investigated the potential of discarded human thymuses, routinely removed during pediatric cardiac surgery, to be used as a novel source of therapeutic Tregs. Here, we show that large numbers of FOXP3+ Tregs can be isolated and expanded from a single thymus. Expanded thymic Tregs had stable FOXP3 expression and long telomeres, and suppressed proliferation and cytokine production of activated allogeneic T cells in vitro. Moreover, expanded thymic Tregs delayed development of xenogeneic GVHD in vivo more effectively than expanded Tregs isolated based on CD25 expression from peripheral blood. Importantly, in contrast to expanded blood Tregs, expanded thymic Tregs remained stable under inflammatory conditions. Our results demonstrate that discarded pediatric thymuses are an excellent source of therapeutic Tregs, having the potential to overcome limitations currently hindering the use of Tregs derived from peripheral or cord blood.

Molecular Regulation ofCellular Immunity by FOXP3

The immune system is responsible for not only eliminating threats to the body, but also for protecting the body from its own immune responses that would cause harm if left unchecked. Forkhead box protein 3 (FOXP3) is a forkhead family member with an important role in the development and function of a type of CD4+ T cell called T regulatory cells that is fundamental for maintaining immune tolerance to self. This chapter reviews the structure of FOXP3 and how its role in the immune system was discovered. Studies of patients with mutations in FOXP3 who suffer from a syndrome known as IPEX (immune dysregulation, polyendocrinopathy, enteropathy, x-linked) are also discussed. Investigation into how expression of FOXP3 is regulated and how it interacts with other proteins have recently provided considerable insight into mechanisms by which the lack of this protein could cause disease. We also discuss how FOXP3 is involved in the reciprocal development ofT regulatory cells and proinflammatory T-cells that produce IL-17. A better understanding of how FOXP3 is regulated and the molecular basis for its function will ultimately contribute to the development ofT regulatory cell-based cellular therapies that could be used to restore dysregulated immune responses.

Effect of ex vivo Expanded Recipient Regulatory T Cells on Hematopoietic Chimerism and Kidney Allograft Tolerance Across MHC Barriers in Cynomolgus Macaques

BackgroundInfusion of recipient regulatory T (Treg) cells promotes durable mixed hematopoietic chimerism and allograft tolerance in mice receiving allogeneic bone marrow transplant (BMT) with minimal conditioning. We applied this strategy in a Cynomolgus macaque model. MethodsCD4+ CD25high Treg cells that were polyclonally expanded in culture were highly suppressive in vitro and maintained high expression of FoxP3. Eight monkeys underwent nonmyeloablative conditioning and major histocompatibility complex mismatched BMT with or without Treg cell infusion. Renal transplantation (from the same BMT donor) was performed 4 months post-BMT without immunosuppression to assess for robust donor-specific tolerance. ResultsTransient mixed chimerism, without significant T cell chimerism, was achieved in the animals that received BMT without Treg cells (N = 3). In contrast, 2 of 5 recipients of Treg cell+ BMT that were evaluable displayed chimerism in all lineages, including T cells, for up to 335 days post-BMT. Importantly, in the animal that survived long-term, greater than 90% of donor T cells were CD45RA+ CD31+, suggesting they were new thymic emigrants. In this animal, the delayed (to 4 months) donor kidney graft was accepted more than 294 days without immunosuppression, whereas non–Treg cell BMT recipients rejected delayed donor kidneys within 3 to 4 weeks. Early CMV reactivation and treatment was associated with early failure of chimerism, regardless of Treg cell administration. ConclusionsOur studies provide proof-of-principle that, in the absence of early CMV reactivation (and BM-toxic antiviral therapy), cotransplantation of host Treg cell can promote prolonged and high levels of multilineage allogeneic chimerism and robust tolerance to the donor.