Rapamycin/IL-2 combination therapy in patients with type 1 diabetes augments Tregs yet transiently impairs β-cell function

Abstract

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 × 10(6) 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.

Trial registration: ClinicalTrials.gov NCT00525889.

Figures

FIG. 1.

Transient decrease in β-cell function with rapamycin/IL-2 combination therapy. Trial design is shown in A for which T1D patients were treated with 4.5 × 106 IU IL-2 s.c. three times per week for 1 month and rapamycin 2–4 mg/day orally for 3 months with target blood rapamycin trough levels of 5–10 ng/mL (see Supplementary Fig. 1). β-Cell function was monitored using an MMTT, and PBMCs were collected for mechanistic studies on the indicated days. B: Peak C-peptide values from the MMTT were assessed for all subjects over time. Statistical significance was determined using a paired Student t test comparing screening and day 84 and days 84–168.

FIG. 2.

Rapamycin/IL-2 combination therapy transiently increases nTregs in the peripheral blood. Thawed PBMCs were stained for viability, CD4, CD25, CD127, and FOXP3. A and B: The frequency of CD4+ cells that are CD25+CD127lo and the number of cells that are CD3+CD4+FOXP3+ within PBMCs are shown for each patient. C: nTregs quantified using real-time PCR analysis of the Treg-specific demethylated region of the FOXP3 gene relative to the methylated region are expressed as percentage of CD3 cells (www.epiontis.com). D: PBMCs were stimulated with PMA/ionomysin in the presence of brefeldin A/myosin prior to staining for FOXP3 and IFN-γ. Means ± SD are shown in D. Statistical significance was determined by a one-way ANOVA and is shown in each graph. Significant differences from time 0 to each time point were determined using a paired Student t test and are indicated with an asterisk. *P ≤ 0.05 and **P ≤ 0.01.

FIG. 3.

Rapamycin/IL-2 combination therapy results in a persistent enhancement of IL-2 responsiveness in CD4+CD25+ T cells. Thawed PBMCs were stimulated with media alone or 100 IU/mL IL-2 prior to fixation, permeabilization, and staining for CD4, CD25, and pSTAT5. Representative dot plots of CD4+ cells are shown in A. B: The frequency of pSTAT5+ cells of CD4+CD25+ cells (A, top) is shown for control (n = 5) and T1D (n = 5) subjects matched to subjects in the trial (noted by ITN R + 2 Trial) by HLA, age, sex, and genotype (PTPN221858 or PTPN2rs1893217). Day 0 and 28 analyses are shown for patients in the trial. Statistical significance was determined between control and T1D subjects (matched + trial) using a two-sample Student t test and between day 0 and 28 in the trial using a paired Student t test. C: The frequency and fold increase (FI) of pSTAT5 in CD4+CD25+ cells are shown over time for patients in the trial. Statistical significance was determined by a one-way ANOVA and is shown in both graphs. Significance from time 0 to each time point was determined using a paired Student t test and is indicated with an asterisk. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

FIG. 4.

Rapamycin/IL-2 combination therapy modulates pAkt, but not phosphorylated (p)Erk or pS6, in CD4+ T cells. Thawed PBMCs were stimulated with media alone or cross-linking of anti-CD3/anti-CD28–bound antibodies with Fab x-linker for 20, 30, or 10 min for Erk, S6, and Akt analysis, respectively. Cells were then fixed, permeabilized, and stained for CD4, CD8, pAkt, pErk, and pS6. Representative histograms are shown for pErk (A), pS6 (B), and pAkt (C) with media (gray line) and anti-CD3/anti-CD28 (black line) stimulation. Fold increase (FI) was determined by the equation (MFI with CD3/CD28 stim) ÷ (MFI with media) for each sample and is compared between day 0 and 84 for pErk (A), pS6 (B), and pAkt (C). Data from all subjects receiving full dosing of rapamycin are included except for pAkt for subjects 1 and 2, for which pAkt data were not acquired. Statistical significance was determined by a paired Student t test.

FIG. 5.

Rapamycin/IL-2 combination therapy did not alter CD4 helper subset differentiation or CD8 memory composition. Thawed PBMCs were stained for viability, CD4, CD8, CD45RO, CXCR3, and CRTH2. A: The frequency of memory CD45RO+ (solid square) and naive CD45RO− (open circles) in the CD4+ (left) and CD8+ (right) T-cell populations is shown. B: The frequency of CXCR3 and CRTH2 of CD4+CD45RO+ cells is shown as approximate markers of Th1 and Th2 cells, respectively. C: PBMCs were stimulated for 6 h with PMA/ionomysin in the presence of brefeldin A/myosin prior to staining for CD4, CD45RO, IL-17, and IFN-γ. Frequencies of IFN-γ+ (open squares, left axis) and IL-17+ (solid circles, right axis) cells of CD4+CD45RO+-gated cells are shown. Means ± SD are shown for all subjects assayed. Statistical significance was determined by a one-way ANOVA and is shown in each plot.

FIG. 6.

Rapamycin/IL-2 combination therapy increased eosinophils and sIL-2RA while decreasing serum TGF-β. A: The frequency of eosinophils in whole blood was determined in a standard hematologic assay and is shown for each subject over time. B: Serum analytes were measured using a multiplex Searchlight chemiluminiscent assay. Averages of duplicates for all subjects are shown for day 0 and 84, and subjects 1, 2, 3, 5, and 7 are shown for time points between days 14 and 70. Of the 22 cytokines and cytokine receptors tested, data are shown where statistical differences were observed. Measures are shown for each subject over time, and statistical significance was determined by a one-way ANOVA, noted in the graph. Significance from time 0 to each time point was determined using a paired Student t test and is indicated with an asterisk. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

FIG. 7.

Rapamycin/IL-2 combination therapy transiently augments NK cell frequency. Thawed PBMCs were stained for viability, CD56, CD3, and CD16. A: The number of CD56+CD3− NK cells of PBMCs are shown for individual subjects. B: The average ± SD frequencies for CD56hiCD16+ (open squares), CD56hiCD16− (closed triangles), and CD56loCD16+ (closed circles) subsets of CD56+ NK cells within the live lymphocyte gate are shown. Statistical significance was determined by a one-way ANOVA, noted in the graph. Significance from time 0 to each time point was determined using a paired Student t test and is indicated with an asterisk. *P ≤ 0.05, **P ≤ 0.01.