Amy L. Putnam

CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells

Regulatory T (T reg) cells are critical regulators of immune tolerance. Most T reg cells are defined based on expression of CD4, CD25, and the transcription factor, FoxP3. However, these markers have proven problematic for uniquely defining this specialized T cell subset in humans. We found that the IL-7 receptor (CD127) is down-regulated on a subset of CD4+ T cells in peripheral blood. We demonstrate that the majority of these cells are FoxP3+, including those that express low levels or no CD25. A combination of CD4, CD25, and CD127 resulted in a highly purified population of T reg cells accounting for significantly more cells that previously identified based on other cell surface markers. These cells were highly suppressive in functional suppressor assays. In fact, cells separated based solely on CD4 and CD127 expression were anergic and, although representing at least three times the number of cells (including both CD25+CD4+ and CD25−CD4+ T cell subsets), were as suppressive as the “classic” CD4+CD25hi T reg cell subset. Finally, we show that CD127 can be used to quantitate T reg cell subsets in individuals with type 1 diabetes supporting the use of CD127 as a biomarker for human T reg cells.

Human regulatory T cells: role in autoimmune disease and therapeutic opportunities

Summary The importance of regulatory T lymphocytes (Tregs) in the control of autoimmunity is now well established in a variety of experimental animal models. In addition, there are numerous studies suggesting that Treg deficits may be an underlying cause of human autoimmune diseases. The emergence of Tregs as an essential component of immune homeostasis provides a potential therapeutic opportunity for active immune regulation and long‐term tolerance induction. In this article, we summarize the core basic science and animal model studies of Tregs, review the status of multiple biologic and small molecule chemical compounds to promote Treg development in vivo, and discuss recent advances for the identification and expansion of polyclonal and antigen‐specific Tregs for adoptive immunotherapy. In summary, the review provides an in‐depth analysis and highlights the challenges and opportunities for immune intervention with Treg‐based therapeutics.

Expansion of Human Regulatory T-Cells From Patients With Type 1 Diabetes

OBJECTIVE—Regulatory T-cells (Tregs) have catalyzed the field of immune regulation. However, translating Treg-based therapies from animal models of autoimmunity to human clinical trials requires robust methods for the isolation and expansion of these cells—a need forming the basis for these studies. RESEARCH DESIGN AND METHODS—Tregs from recent-onset type 1 diabetic patients and healthy control subjects were isolated by fluorescence-activated cell sorting and compared for their capacity to expand in vitro in response to anti-CD3–anti-CD28–coated microbeads and IL-2. Expanded cells were examined for suppressive function, lineage markers and FOXP3, and cytokine production. RESULTS—Both CD4+CD127lo/− and CD4+CD127lo/−CD25+ T-cells could be expanded and used as Tregs. However, expansion of CD4+CD127lo/− cells required the addition of rapamycin to maintain lineage purity. In contrast, expansion of CD4+CD127lo/−CD25+ T-cells, especially the CD45RA+ subset, resulted in high yield, functional Tregs that maintained higher FOXP3 expression in the absence of rapamycin. Tregs from type 1 diabetic patients and control subjects expanded similarly and were equally capable of suppressing T-cell proliferation. Regulatory cytokines were produced by Tregs after culture; however, a portion of FOXP3+ cells were capable of producing interferon (IFN)-γ after reactivation. IFN-γ production was observed from both CD45RO+ and CD45RA+ Treg populations. CONCLUSIONS—The results support the feasibility of isolating Tregs for in vitro expansion. Based on expansion capacity, FOXP3 stability, and functional properties, the CD4+CD127lo/−CD25+ T-cells represent a viable cell population for cellular therapy in this autoimmune disease.

Plasticity of Human Regulatory T Cells in Healthy Subjects and Patients with Type 1 Diabetes

Regulatory T cells (Tregs) constitute an attractive therapeutic target given their essential role in controlling autoimmunity. However, recent animal studies provide evidence for functional heterogeneity and lineage plasticity within the Treg compartment. To understand better the plasticity of human Tregs in the context of type 1 diabetes, we characterized an IFN-γ–competent subset of human CD4+CD127lo/−CD25+ Tregs. We measured the frequency of Tregs in the peripheral blood of patients with type 1 diabetes by epigenetic analysis of the Treg-specific demethylated region (TSDR) and the frequency of the IFN-γ+ subset by flow cytometry. Purified IFN-γ+ Tregs were assessed for suppressive function, degree of TSDR demethylation, and expression of Treg lineage markers FOXP3 and Helios. The frequency of Tregs in peripheral blood was comparable but the FOXP3+IFN-γ+ fraction was significantly increased in patients with type 1 diabetes compared to healthy controls. Purified IFN-γ+ Tregs expressed FOXP3 and possessed suppressive activity but lacked Helios expression and were predominately methylated at the TSDR, characteristics of an adaptive Treg. Naive Tregs were capable of upregulating expression of Th1-associated T-bet, CXCR3, and IFN-γ in response to IL-12. Notably, naive, thymic-derived natural Tregs also demonstrated the capacity for Th1 differentiation without concomitant loss of Helios expression or TSDR demethylation.

Type 1 diabetes immunotherapy using polyclonal regulatory T cells

Autologous regulatory T cells can be expanded and are well tolerated in patients with recent-onset type 1 diabetes. Regulating type 1 diabetes In patients with type 1 diabetes (T1D), immune cells attack the insulin-producing β cells of the pancreas. The resulting prolonged increase in blood sugar levels can lead to serious complications including heart disease and kidney failure. Regulatory T cells (Tregs) have been shown to be defective in autoimmune diseases. Now, Bluestone et al. report a phase 1 trial of adoptive Treg immunotherapy to repair or replace Tregs in type 1 diabetics. The ex vivo–expanded polyclonal Tregs were long-lived after transfer and retained a broad Treg phenotype long-term. Moreover, the therapy was safe, supporting efficacy testing in further trials. Type 1 diabetes (T1D) is an autoimmune disease that occurs in genetically susceptible individuals. Regulatory T cells (Tregs) have been shown to be defective in the autoimmune disease setting. Thus, efforts to repair or replace Tregs in T1D may reverse autoimmunity and protect the remaining insulin-producing β cells. On the basis of this premise, a robust technique has been developed to isolate and expand Tregs from patients with T1D. The expanded Tregs retained their T cell receptor diversity and demonstrated enhanced functional activity. We report on a phase 1 trial to assess safety of Treg adoptive immunotherapy in T1D. Fourteen adult subjects with T1D, in four dosing cohorts, received ex vivo–expanded autologous CD4+CD127lo/−CD25+ polyclonal Tregs (0.05 × 108 to 26 × 108 cells). A subset of the adoptively transferred Tregs was long-lived, with up to 25% of the peak level remaining in the circulation at 1 year after transfer. Immune studies showed transient increases in Tregs in recipients and retained a broad Treg FOXP3+CD4+CD25hiCD127lo phenotype long-term. There were no infusion reactions or cell therapy–related high-grade adverse events. C-peptide levels persisted out to 2+ years after transfer in several individuals. These results support the development of a phase 2 trial to test efficacy of the Treg therapy.

The effect of costimulatory and interleukin 2 receptor blockade on regulatory T cells in renal transplantation.

Regulatory T cells (Treg) are critical regulators of immune tolerance. Both IL‐2 and CD28‐CD80/CD86 signaling are critical for CD4+CD25+FOXP3+ Treg survival in mice. Yet, both belatacept (a second‐generation CTLA‐4Ig) and basiliximab (an anti‐CD25 monoclonal antibody) are among the arsenal of current immunotherapies being used in kidney transplant patients. In this study, we explored the direct effect of basiliximab and belatacept on the Tregs in peripheral blood both in the short term and long term and in kidney biopsies of patients with acute rejection. We report that the combined belatacept/basiliximab therapy has no long‐term effect on circulating Tregs when compared to a calcineurin inhibitor (CNI)‐treated group. Moreover, belatacept‐treated patients had a significantly greater number of FOXP3+ T cells in graft biopsies during acute rejection as compared to CNI‐treated patients. Finally, it appears that the basiliximab caused a transient loss of both FOXP3+ and FOXP3− CD25+ T cells in the circulation in both treatment groups raising important questions about the use of this therapy in tolerance promoting therapeutic protocols.

Clinical Grade Manufacturing of Human Alloantigen-Reactive Regulatory T Cells for Use in Transplantation

Regulatory T cell (Treg) therapy has the potential to induce transplantation tolerance so that immunosuppression and associated morbidity can be minimized. Alloantigen‐reactive Tregs (arTregs) are more effective at preventing graft rejection than polyclonally expanded Tregs (PolyTregs) in murine models. We have developed a manufacturing process to expand human arTregs in short‐term cultures using good manufacturing practice‐compliant reagents. This process uses CD40L‐activated allogeneic B cells to selectively expand arTregs followed by polyclonal restimulation to increase yield. Tregs expanded 100‐ to 1600‐fold were highly alloantigen reactive and expressed the phenotype of stable Tregs. The alloantigen‐expanded Tregs had a diverse TCR repertoire. They were more potent than PolyTregs in vitro and more effective at controlling allograft injuries in vivo in a humanized mouse model.

Human Antigen-Specific Regulatory T Cells Generated by T Cell Receptor Gene Transfer

Background Therapies directed at augmenting regulatory T cell (Treg) activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects, including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments, with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However, current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover, FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific, whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition. Methodology/Principal Findings To overcome these limitations, we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR) gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs, and maintained the capacity to suppress conventional T cell responses directed against tyrosinase, as well as bystander T cell responses. Using this methodology in a model tumor system, murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff) activity as determined by tumor cell growth and luciferase reporter-based imaging. Conclusions/Significance These results support the feasibility of class I-restricted TCR transfer as a promising strategy to redirect the functional properties of Tregs and provide for a more efficacious adoptive cell therapy.

Rapid assessment of in vitro expanded human regulatory T cell function.

Human regulatory T cells (Treg) are able to actively suppress autoreactive immune responses. Phenotypically, they are broadly characterized as CD4+, CD25+, CD127(lo/⁻) and FoxP3+. CD45RA can be used to further differentiate the population into naïve (CD45RA(+)) and induced (CD45RA⁻) Treg. The functional potential of Treg is routinely determined by assessing their ability to suppress T cell function in 3-5day proliferation assays. Since Treg are being explored for therapeutic use, a short-term functional assay could serve as a valuable tool for evaluating the potency of Treg. Therefore, an assay designed to measure Treg suppression of activation marker expression by responder T cells in 7 to 20h has been examined in this report. Using flow cytometry, expression of CD69 and CD154 on T cells, in response to stimulation with CD3/CD28 beads, was used as a measure of activation in the assay. Treg from healthy volunteers were sorted as CD4+CD25+CD127(lo/⁻)CD45RA+ cells with a BD FACSAria™ II. The highly purified Treg were then expanded in vitro and their function was assessed in short term activation marker suppression assays using autologous PBMC as responder cells. The data suggest that this short term suppression assay could be a reliable surrogate for assessing Treg functional potential.

The role of X-linked FOXP3 in the autoimmune susceptibility of Turner Syndrome patients

Turner Syndrome patients have an absent second sex chromosome and a predisposition to autoimmune disease. We hypothesized that the autoimmune susceptibility in Turner Syndrome may be due to an alteration in the expression of the X-linked FOXP3 gene. FOXP3 is important in the development of regulatory T cells, and complete loss of FOXP3 expression has been shown to result in severe autoimmunity. To test this hypothesis, we characterized the regulatory T cells and performed immunophenotyping on the peripheral blood leukocytes of a cohort of Turner Syndrome patients. These patients retained regulatory T cell frequency and function despite an increased prevalence of autoimmunity. Immunophenotyping revealed a decrease in the ratio of CD4 to CD8 lymphocytes. These findings suggest that the autoimmune predisposition in Turner Syndrome is not due to alterations in regulatory T cells but may be associated with a change in the proportion of T cell subsets.