S. Alice Long

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.

Defects in IL-2R Signaling Contribute to Diminished Maintenance of FOXP3 Expression in CD4+CD25+ Regulatory T-Cells of Type 1 Diabetic Subjects

OBJECTIVE In humans, multiple genes in the interleukin (IL)-2/IL-2 receptor (IL-2R) pathway are associated with type 1 diabetes. However, no link between IL-2 responsiveness and CD4+CD25+FOXP3+ regulatory T-cells (Tregs) has been demonstrated in type 1 diabetic subjects despite the role of these IL-2–dependent cells in controlling autoimmunity. Here, we address whether altered IL-2 responsiveness impacts persistence of FOXP3 expression in Tregs of type 1 diabetic subjects. RESEARCH DESIGN AND METHODS Persistence of Tregs was assessed by culturing sorted CD4+CD25hi natural Tregs with IL-2 and measuring FOXP3 expression over time by flow cytometry for control and type 1 diabetic populations. The effects of IL-2 on FOXP3 induction were assessed 48 h after activation of CD4+CD25− T-cells with anti-CD3 antibody. Cytokine receptor expression and signaling upon exposure to IL-2, IL-7, and IL-15 were determined by flow cytometry and Western blot analysis. RESULTS Maintenance of FOXP3 expression in CD4+CD25+ Tregs of type 1 diabetic subjects was diminished in the presence of IL-2, but not IL-7. Impaired responsiveness was not linked to altered expression of the IL-2R complex. Instead, IL-2R signaling was reduced in Tregs and total CD4+ T-cells of type 1 diabetic subjects. In some individuals, decreased signal transducer and activator of transcription 5 phosphorylation correlated with significantly higher expression of protein tyrosine phosphatase N2, a negative regulator of IL-2R signaling. CONCLUSIONS Aberrant IL-2R signaling in CD4+ T-cells of type 1 diabetic subjects contributes to decreased persistence of FOXP3 expression that may impact establishment of tolerance. These findings suggest novel targets for treatment of type 1 diabetes within the IL-2R pathway and suggest that an altered IL-2R signaling signature may be a biomarker for type 1 diabetes.

Rapamycin/IL-2 Combination Therapy in Patients with Type 1 Diabetes Augments Tregs yet Transiently Impairs β-Cell Function

Rapamycin/interleukin-2 (IL-2) combination treatment of NOD mice effectively treats autoimmune diabetes. We performed a phase 1 clinical trial to test the safety and immunologic effects of rapamycin/IL-2 combination therapy in type 1 diabetic (T1D) patients. Nine T1D subjects were treated with 2–4 mg/day rapamycin orally for 3 months and 4.5 × 106 IU IL-2 s.c. three times per week for 1 month. β-Cell function was monitored by measuring C-peptide. Immunologic changes were monitored using flow cytometry and serum analyses. Regulatory T cells (Tregs) increased within the first month of therapy, yet clinical and metabolic data demonstrated a transient worsening in all subjects. The increase in Tregs was transient, paralleling IL-2 treatment, whereas the response of Tregs to IL-2, as measured by STAT5 phosphorylation, increased and persisted after treatment. No differences were observed in effector T-cell subset frequencies, but an increase in natural killer cells and eosinophils occurred with IL-2 therapy. Rapamycin/IL-2 therapy, as given in this phase 1 study, resulted in transient β-cell dysfunction despite an increase in Tregs. Such results highlight the difficulties in translating therapies to the clinic and emphasize the importance of broadly interrogating the immune system to evaluate the effects of therapy.

CD44 Costimulation Promotes FoxP3+ Regulatory T Cell Persistence and Function via Production of IL-2, IL-10, and TGF-β

Work by our group and others has demonstrated a role for the extracellular matrix receptor CD44 and its ligand hyaluronan in CD4+CD25+ regulatory T cell (Treg) function. Herein, we explore the mechanistic basis for this observation. Using mouse FoxP3/GFP+ Treg, we find that CD44 costimulation promotes expression of FoxP3, in part through production of IL-2. This promotion of IL-2 production was resistant to cyclosporin A treatment, suggesting that CD44 costimulation may promote IL-2 production through bypassing FoxP3-mediated suppression of NFAT. CD44 costimulation increased production of IL-10 in a partially IL-2-dependent manner and also promoted cell surface TGF-β expression. Consistent with these findings, Treg from CD44 knockout mice demonstrated impaired regulatory function ex vivo and depressed production of IL-10 and cell surface TGF-β. These data reveal a novel role for CD44 cross-linking in the production of regulatory cytokines. Similar salutary effects on FoxP3 expression were observed upon costimulation with hyaluronan, the primary natural ligand for CD44. This effect is dependent upon CD44 cross-linking; while both high-molecular-weight hyaluronan (HA) and plate-bound anti-CD44 Ab promoted FoxP3 expression, neither low-molecular weight HA nor soluble anti-CD44 Ab did so. The implication is that intact high-molecular weight HA can cross-link CD44 only in those settings where it predominates over fragmentary LMW-HA, namely, in uninflamed tissue. We propose that intact but not fragmented extracellular is capable of cross-linking CD44 and thereby maintains immunologic tolerance in uninjured or healing tissue.

Low-dose interleukin-2 fosters a dose-dependent regulatory T cell tuned milieu in T1D patients

Most autoimmune diseases (AID) are linked to an imbalance between autoreactive effector T cells (Teffs) and regulatory T cells (Tregs). While blocking Teffs with immunosuppression has long been the only therapeutic option, activating/expanding Tregs may achieve the same objective without the toxicity of immunosuppression. We showed that low-dose interleukin-2 (ld-IL-2) safely expands/activates Tregs in patients with AID, such HCV-induced vasculitis and Type 1 Diabetes (T1D). Here we analyzed the kinetics and dose-relationship of IL-2 effects on immune responses in T1D patients. Ld-IL-2 therapy induced a dose-dependent increase in CD4(+)Foxp3(+) and CD8(+)Foxp3(+) Treg numbers and proportions, the duration of which was markedly dose-dependent. Tregs expressed enhanced levels of activation markers, including CD25, GITR, CTLA-4 and basal pSTAT5, and retained a 20-fold higher sensitivity to IL-2 than Teff and NK cells. Plasma levels of regulatory cytokines were increased in a dose-dependent manner, while cytokines linked to Teff and Th17 inflammatory cells were mostly unchanged. Global transcriptome analyses showed a dose-dependent decrease in immune response signatures. At the highest dose, Teff responses against beta-cell antigens were suppressed in all 4 patients tested. These results inform of broader changes induced by ld-IL-2 beyond direct effects on Tregs, and relevant for further development of ld-IL-2 for therapy and prevention of T1D, and other autoimmune and inflammatory diseases.

ECM components guide IL-10 producing regulatory T-cell (TR1) induction from effector memory T-cell precursors

We describe a role for ECM as a biosensor for inflammatory microenvironments that plays a critical role in peripheral immune tolerance. We show that hyaluronan (HA) promotes induction of Foxp3- IL-10–producing regulatory T cells (TR1) from conventional T-cell precursors in both murine and human systems. This is, to our knowledge, the first description of an ECM component inducing regulatory T cells. Intact HA, characteristic of healing tissues, promotes induction of TR1 capable of abrogating disease in an IL-10–dependent mouse colitis model whereas fragmentary HA, typical of inflamed tissues, does not, indicating a decisive role for tissue integrity in this system. The TR1 precursor cells in this system are CD4+CD62L−FoxP3−, suggesting that effector memory cells assume a regulatory phenotype when they encounter their cognate antigen in the context of intact HA. Matrix integrity cues might thereby play a central role in maintaining peripheral tolerance. This TR1 induction is mediated by CD44 cross-linking and signaling through p38 and ERK1/2. This induction is suppressed, also in a CD44-dependent manner, by osteopontin, a component of chronically inflamed ECM, indicating that CD44 signaling serves as a nexus for fate decisions regarding TR1 induction. Finally, we demonstrate that TR1 induction signals can be recapitulated using synthetic matrices. These results reveal important roles for the matrix microenvironment in immune regulation and suggest unique strategies for immunomodulation.

Combination of rapamycin and IL-2 increases de novo induction of human CD4(+)CD25(+)FOXP3(+) T cells.

The immune system protects itself from autoreactivity by maintaining a balance between effector and Treg responses. Peripheral induction of Treg is one mechanism by which this balance may be maintained. Thus, it is important to understand factors that influence de novo generation of CD4(+)CD25(+)FOXP3(+) Treg. Here, we focus on the effects of cytokines and the cell cycle inhibitor rapamycin. The cytokines IL-2 and IL-7, but not IL-4, increased initial activation induced FOXP3 expression, increased proliferation and sustained expression of FOXP3(+) cells throughout the culture. Addition of rapamycin to cultures containing IL-2 further increased the frequency and absolute number of functional CD4(+)CD25(+)FOXP3(+) Treg. This increase was not due to selective proliferation of FOXP3 cells, but was instead, the result of an increase in the frequency of FOXP3(+) cells induced in G0 through delayed activation while the addition of IL-2 promoted survival and proliferation of the FOXP3(+) population. Thus, combination of rapamycin and IL-2 may provide improved treatment options in transplantation and autoimmunity by promoting induction, survival, and expansion of functional iTreg from CD4(+)CD25(-) cells.

Functional islet-specific Treg can be generated from CD4 + CD25 - T cells of healthy and type 1 diabetic subjects

CD4+CD25+FOXP3+ Treg cells require TCR engagement for suppressive function, thus ensuring that suppression occurs only in the presence of specific antigens; however, to date no studies have addressed the function of self‐antigen‐specific Treg in humans. These studies were designed to determine whether peripheral generation and function of islet antigen‐specific adaptive Treg are defective in human subjects with type 1 diabetes (T1D). Islet antigen‐specific adaptive Treg were induced in vitro by activation of CD4+FOXP3− T cells with glutamic acid decarboxylase and islet‐specific glucose‐6‐phosphate catalytic subunit‐related protein peptides in the context of T1D‐associated HLA‐DRβ alleles. Antigen‐specific Treg were characterized using flow cytometry for FOXP3 and class II tetramer and assessed for the ability to inhibit proliferation. These adaptive Treg were then compared with influenza‐specific Treg from the same study population. The function of tetramer+ cells that expressed FOXP3 was similar for both influenza and islet antigens generated from control and T1D subjects. In fact, the potency of suppression correlated with FOXP3 expression, not antigen specificity. Thus, these data suggest that development of functional adaptive Treg can occur in response to islet antigens and activation of islet‐specific Treg may potentially be used as a targeted immunotherapy in T1D.

Multiple Autoimmune-Associated Variants Confer Decreased IL-2R Signaling in CD4+CD25hi T Cells of Type 1 Diabetic and Multiple Sclerosis Patients

IL-2 receptor (IL-2R) signaling is essential for optimal stability and function of CD4+CD25hiFOXP3+ regulatory T cells (Treg); a cell type that plays an integral role in maintaining tolerance. Thus, we hypothesized that decreased response to IL-2 may be a common phenotype of subjects who have autoimmune diseases associated with variants in the IL2RA locus, including T1D and MS, particularly in cells expressing the high affinity IL-2R alpha chain (IL-2RA or CD25). To examine this question we used phosphorylation of STAT5 (pSTAT5) as a downstream measure of IL-2R signaling, and found a decreased response to IL-2 in CD4+CD25hi T cells of T1D and MS, but not SLE patients. Since the IL2RArs2104286 haplotype is associated with T1D and MS, we measured pSTAT5 in controls carrying the rs2104286 risk haplotype to test whether this variant contributed to reduced IL-2 responsiveness. Consistent with this, we found decreased pSTAT5 in subjects carrying the rs2104286 risk haplotype. Reduced IL-2R signaling did not result from lower CD25 expression on CD25hi cells; instead we detected increased CD25 expression on naive Treg from controls carrying the rs2104286 risk haplotype, and subjects with T1D and MS. However the rs2104286 risk haplotype correlated with increased soluble IL-2RA levels, suggesting that shedding of the IL-2R may account in part for the reduced IL-2R signaling associated with the rs2104286 risk haplotype. In addition to risk variants in IL2RA, we found that the T1D-associated risk variant of PTPN2rs1893217 independently contributed to diminished IL-2R signaling. However, even when holding genotype constant at IL2RA and PTPN2, we still observed a significant signaling defect in T1D and MS patients. Together, these data suggest that multiple mechanisms converge in disease leading to decreased response to IL-2, a phenotype that may eventually lead to loss of tolerance and autoimmunity.

IL-2 therapy in type 1 diabetes: "Trials" and tribulations.

IL-2 facilitates immunity or tolerance depending on its availability. In model systems, it is well established that low dose IL-2 promotes selective expansion of regulatory T cells (Treg), an IL-2 responsive cell type known to control autoimmunity. Moreover, many autoimmune diseases are marked by defects in Treg and/or IL-2/IL-2 receptor signaling. Thus, patients with immune-mediated diseases were treated with IL-2 with the goal of increasing Treg and controlling autoimmunity. In graft versus host disease, HCV-induced vasculitis and type 1 diabetes (T1D), Treg numbers increased with IL-2 therapy. Yet there was no relationship between Treg number and clinical outcome. In fact, in T1D subjects treated with rapamycin and IL-2 therapy there was no measureable clinical benefit. In this review, we compare results from IL-2 treatment of patients with immune-mediated diseases, discuss possible mechanisms of IL-2 treatment and suggest future directions for use of IL-2 therapy in T1D.