Isolation and expansion of human natural T regulatory cells for cellular therapy.
Natural T regulatory cells (nTregs) play a key role in inducing and maintaining immunological tolerance. Cell-based therapy using purified nTregs is under consideration for several conditions, but procedures employed to date have resulted in cell populations that are contaminated with cytokine secreting effector cells. We have established a method for isolation and ex vivo expansion of human nTregs from healthy blood donors for cellular therapy aimed at preventing allograft rejection in organ transplants. The Robosep instrument was used for initial nTreg isolation and rapamycin was included in the expansion phase of cell cultures. The resulting cell population exhibited a stable CD4(+)CD25(++bright)Foxp3(+) phenotype, had potent functional ability to suppress CD4(+)CD25(negative) T cells without evidence of conversion to effector T cells including TH17 cells, and manifested little to no production of pro-inflammatory cytokines upon in vitro stimulation. Boolean gating analysis of cytokine-expressing cells by flow cytometry for 32 possible profile end points revealed that 96% of expanded nTregs did not express any cytokine. From a single buffy coat, approximately 80 million pure nTregs were harvested after expansion under cGMP conditions; these cell numbers are adequate for infusion of approximately one million cells kg⁻¹ for cell therapy in clinical trials.
Diabetes Research Institute, University of Miami, Leonard M. Miller School of Medicine, Miami, FL 33136, USA. rpahwa@med.miami.edu
Rajendra
R
Pahwa
Shashidhar
S
Jaggaiahgari
Savita
S
Pahwa
Luca
L
Inverardi
Andreas
A
Tzakis
Camillo
C
Ricordi
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods* / Q000592:standards; D002469:Cell Separation / Q000379:methods* / Q000592:standards; D016207:Cytokines / Q000096:biosynthesis; D005260:Female; D006084:Graft Rejection / Q000517:prevention & control; D006801:Humans; D016219:Immunotherapy, Adoptive; D018836:Inflammation Mediators / Q000378:metabolism; D017710:Lymphocyte Transfusion / Q000379:methods; D008297:Male; D016377:Organ Transplantation; D020123:Sirolimus / Q000494:pharmacology; D050378:T-Lymphocytes, Regulatory / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D014184:Transplantation, Homologous
D000900:Anti-Bacterial Agents; D016207:Cytokines; D018836:Inflammation Mediators; D020123:Sirolimus