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.


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+CD127(lo/-) and CD4+CD127(lo/-)CD25+ T-cells could be expanded and used as Tregs. However, expansion of CD4+CD127(lo/-) cells required the addition of rapamycin to maintain lineage purity. In contrast, expansion of CD4+CD127(lo/-)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)-gamma after reactivation. IFN-gamma 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+CD127(lo/-)CD25+ T-cells represent a viable cell population for cellular therapy in this autoimmune disease.
Putnam
Amy L
AL
Diabetes Center at the University of California, San Francisco (UCSF), San Francisco, California, USA.
Brusko
Todd M
TM
Lee
Michael R
MR
Liu
Weihong
W
Szot
Gregory L
GL
Ghosh
Taumoha
T
Atkinson
Mark A
MA
Bluestone
Jeffrey A
JA
D000328:Adult; D017668:Age of Onset; D015703:Antigens, CD / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D002455:Cell Division; D049109:Cell Proliferation; D016207:Cytokines / Q000378:metabolism; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000032:analysis; D006801:Humans; D007165:Immunosuppression Therapy; D015759:Ionomycin / Q000494:pharmacology; D008297:Male; D010641:Phenotype; D012016:Reference Values; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D055815:Young Adult
D015703:Antigens, CD; D016207:Cytokines; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D015759:Ionomycin; D013755:Tetradecanoylphorbol Acetate