Type 1 diabetes immunotherapy using polyclonal regulatory T cells.

Bluestone JA1, Buckner JH2, Fitch M3, Gitelman SE4, Gupta S2, Hellerstein MK3, Herold KC5, Lares A6, Lee MR6, Li K7, Liu W6, Long SA2, Masiello LM6, Nguyen V8, Putnam AL6, Rieck M6, Sayre PH9, Tang Q8.
Source: Med. Sci.
Publication Date: (2015)
Issue: 7: 315
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
T cell, human stim.
Species: human
Tissue Origin: blood
Culture Media:

T-cell expansion protocol: FACS-isolated cells were plated at ~2.5 × 105 Tregs per well inmultiplewells of a 24-well plate (Nunc) and activatedwithDynabeads CD3/
CD28 CTS anti-CD3/anti-CD28–coated microbeads  at a 1:1 bead/cell ratio. Cells were cultured either in X-VIVO 15 or in X-VIVO 15 customized by Lonza by substituting 100% of the glucose in the basemedium with D-glucose. supplemented with 10% human heat-inactivated pooled AB serum. At day 2, the culture volume
was doubled and IL-2 was added (Proleukin, 300 IU/ml; Prometheus). Cells were resuspended, fresh medium and IL-2 were added at days 5, 7, 9, and 12, and the cellswere transferred to cell culture plates and flasks (Nunc), and/or bags (Saint-Gobain) of increasing size to maintain a seeding density of ~2 × 105 to 3 × 105 cells/ml in plates or flasks and a concentration of 500,000/ml in bags.On day 9, cells were restimulated with fresh anti-CD3/anti-CD28–coated beads at a 1:1 ratio.


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 ß 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.