Type 1 diabetes immunotherapy using polyclonal regulatory T cells

Abstract

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(+)CD127(lo/-)CD25(+) polyclonal Tregs (0.05 × 10(8) to 26 × 10(8) 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(+)CD25(hi)CD127(lo) 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.

Fig. 1. Study Design

(A) Dose escalation plan. Subjects were enrolled in 4 cohorts with target doses ranging from 0.05 to 26 × 10⁸, where the dose was escalated by 8-fold for each subsequent cohort. The first subject in each cohort received a single infusion of polyclonal Tregs and was observed for a minimum of 3 weeks for dose limiting toxicities (DLT) after which time clinical data was extracted from the database and study team safety review was conducted. If no grade 3 or higher adverse event was observed, treatment of subsequent subjects in that cohort could proceed. Otherwise, treatment would be suspended for DSMB review. Following treatment of the last subject in each cohort, subjects were observed for a minimum of 13 weeks. The study team reviewed cumulative data to assess for any grade 3 or higher related adverse event, any related serious adverse event, undetectable C-peptide in a MMTT at week 13 in 2 subjects, or any other significant safety concerns based on other considerations. The study team’s review decision was reported to the DSMB for approval prior to proceeding to the next dosing cohort. (B) Subject schedule of events. Blood (target of 400 ml) for Treg manufacturing was drawn at week −2, and Treg infusion was given on day 0. Subjects were seen for follow-up assessments on day 4, then weekly for the first 4 weeks, then every 13 weeks for the first year, then every 6 months for 2 years, and then contacted by phone every 6 months for years 3–5 to assess for adverse events.

Fig. 2. Increased expression of CD45RO, CCR7, and CD38 on Tregs after in vitro expansion

CD4⁺CD25⁺CD127^loTregs, as shown in Supplemental Figure S6, were stained for (A) CD45RO, (B) CCR7, and (C) CD38 before and after expansion. Representative FACS plots are shown for each of these stains on the left and graphs plotting data for all subjects (n=4) on the right. Data points for each subject are represented in a unique and consistent symbol (blue upright triangle, red circle, orange inverted triangle, and magenta square). Significance was determined by paired t test and indicated with an asterisk. **P = 0.0014, ***P = 0.0001, and ****P < 0.0001

Fig. 3. Treg identity and function

(A) Percent of expanded cells expressing FOXP3 protein as determined by flow cytometry and percent of DNA demethylated at the FOXP3 Treg Specific Demethylation Region (TSDR) was determined by Epiontis as described in Methods section. (B) Percent of STAT5 phosphorylation in response to IL-2 stimulation in CD4⁺CD25⁺ Tregs pre- and post-expansion in healthy controls (n=5) and subjects with T1D (n=10). Staining and gating was done as previously described (25). Significance was determined by Mann Whitney test. (C) In an in vitro culture, CFSE labeled Teff cells were cultured for 4 days in the presence of anti-CD3/CD28 antibody coated beads in the presence or absence of expanded/natural Tregs of same donor. T-cell proliferation in these cultures was analyzed by flow cytometry for CFSE dilution as previously described (41). Each condition was set-up in duplicate wells and compared to cultures with Teff alone. Data are represented as mean + SEM for n=4 healthy individuals. Statistical difference between non-expanded and expanded Tregs was determined by t test, where the difference seen at 1:2 is P=0.0050, 1:4 is P=0.0025, and 1:8 is P=0.0354 (*P < 0.05 and **P < 0.01). (D) Suppression assays using CFSE-labeled CD4⁺CD127⁺CD25⁻ cells sorted from standard PBMC as Teff cells cultured alone or activated with anti-CD3/anti-CD28 coated beads, and/or co-incubated with Treg cells (from patients enrolled in this trial) show a consistent level of suppression. Comparison of mean of % proliferating CFSE+ cells with increasing ratio of Treg:Teff is shown for non- expanded Treg as compared to expanded Treg (n=3). Statistical difference between non-expanded and expanded Tregs from T1D patients was determined by t test, where the difference seen at 1:8 is P=0.0195, 1:16 is P=0.0112, and 1:32 is P=0.0333 (*P < 0.05). (E) Treg functional markers CD25, CD39, CD45RA, CD45RO, CTLA-4, and LAP were analyzed on expanded Tregs (solid squares) and non-expanded Tregs (open circles) from same healthy control donor by flow cytometry (n>4 individuals as depicted). Representative plots from the FACS analysis can be seen in Supplemental Figure S7. Significance was determined by t test.

Fig. 4. Metabolic assessments. (Left column)

C-peptide area under the curve (AUC). C-peptide AUC is reported for fasting 4-hour mixed meal tolerance test without carbohydrate restriction for 3 days preceding test. The target glucose level at the start of the test was between 70 and 200 mg/dL. Regular insulin or short acting insulin analogues was allowed up to 6 and 2 hours before the test, respectively, to achieve the desired glucose level. The baseline blood samples (−10 minutes and 0 minutes) were drawn, and then subjects drank Boost High Protein Nutritional Energy Drink® (Nestle Nutrition) at 6 kcal/kg (1 kcal/mL) to a maximum of 360 mL Blood was drawn at 15, 30, 60, 90, 120, 150, 180, 210, and 240 minutes following Boost dose. C-peptide AUC was calculated using the trapezoid rule. (Middle Column) Hemoglobin A1c (HbA1c). (Right Column) Insulin Use. Subjects self-reported insulin use for the 3 days immediately preceding the scheduled visit. The average total insulin (long acting + short acting) use per day normalized to weight is reported.

Fig. 5. Changes in T lymphocyte subsets after Treg infusion

PBMC samples collected from subjects on days 0, 7, 28, and 91 were frozen from all patients then thawed simultaneously before being stained with CD4, CD45RO, CD25, CD127, CCR7, and CD38 for FACS analysis. Representative FACS plots for all analyses are shown in Supplemental Figure S6. All subjects shown are from cohort 4 (n=4), as detailed in Table 1. (A) An increase in %CD38⁺ cells was seen within CD4⁺CD45RO⁺ T cells subset post Treg infusion. (B) Within the CD4⁺CD127^lo/−CD25⁺ Tregs the percentage of CCR7⁺ Tregs increased after infusion of expanded Tregs. (C) MFI of the patients’ CD4⁺CD127^lo/−CD25⁺Treg population increased upon addition of expanded Tregs. For all plots comparisons were made to day 0 and significance was determined by paired t test.

Fig. 6. Survival of infused polyclonal Tregs

During ex vivo expansion, the ²H label from deuterated glucose (²H2-glucose) contained in the cell culture medium is incorporated into the deoxyribose moiety in replicating DNA through the de novo purine nucleotide synthesis pathway. Three subjects (002–015, 007–102, and 002–017) were treated with a single dose of ²H-labeled Tregs at a target dose of 3.2 ×10⁸cells, and four subjects (002–018, 002–019, 007–103, and 002–022) were treated with a target dose of 26 ×10⁸ cells that were approximately 60% enriched for the ²H-label. Peripheral blood was collected on days 1, 4, 7, 14, 28, 91, 182, and 364 days post infusion, and Tregs were sorted from the peripheral blood. Following isolation and hydrolysis of genomic DNA, the ²H isotopic enrichment of the purine deoxyribonucleosides in Tregs sorted from whole blood was assessed by gas chromatography/mass spectrometry. Background enrichment of unlabeled Tregs was ≤0.1% for each of the seven subjects.

Fig. 7. Changes in Tregs phenotypes after transfer

Frozen samples of expanded Tregs or PBMC collected at time points on days 7–91 post infusion were FACS sorted for CD4⁺CD25⁺CD127^lo Tregs. These Tregs were further subsetted and sorted based on expression of CCR7, CD38, CD45RA, and CD45RO. Collected Tregs subsets were then analyzed by mass- spectrometry for ²H-label, which was incorporated into the infused Tregs during the expansion. (A) CCR7+CD45RO+CD38+ Tregs (black bars) were analyzed for %enrichment, which represented an increasingly smaller proportion of total Treg ²H enrichment over time. (B) Tregs were subsetted into CCR7⁻ (black bars), CCR7⁺CD38⁺ (small grid bars), and CCR7⁺CD38⁻ (large grid bars) populations for ²H enrichment analysis pre and post infusion. (C) Tregs were subsetted into CCR7⁻ (black bars), CCR7⁺CD45RA⁻CD45RO⁺ (white bars), CCR7⁺CD45RA^intCD45RO^int (speckled bars), and CCR7⁺CD45RA⁺CD45RO⁻ (striped bars) populations for ²H enrichment analysis pre and post infusion. All plots shown are representative of data collected from subjects in cohort 4. “% of Total Tregs with Label” is defined is Supplemental Figure S8.