Selective Expansion of Human Regulatory (CD4+CD25+CD127LowFOXP3+) Cells to High Purity by Inhibiting Expansion of CD4+CD25+CD127HighFOXP3-Conventional T Cells with Rapamycin.
Authors: Hou Jing-Zhou , Zambricki Elizabeth , Zeiser Robert , Negrin Robert S. 
Published: October 2019

Abstract
CD4+CD25+FOXP3+ natural occurring T regulatory (Treg) cells possess great therapeutic potential as adoptive cellular therapy for controlling autoimmune diseases and acute GVHD. However, the clinical application of human Treg is limited by the difficulty of obtaining sufficient numbers of CD4+CD25+FOXP3+ cells. This study explored a strategy to expand human Treg in high purity and high numbers. By comparing expression of cell surface molecules on sorted CD4+CD25High versus CD4+CD25- cells using mAb microarray, we identified CD127 was highly expressed in CD4+CD25- and CD4+CD25Low cells in human PBMC. This was confirmed by two recent reports. Incorporating this new discovery, CD4+CD25+ cells were isolated by CD25+ selection from PBMC of healthy blood donors. The selected CD25+ cells were sorted into CD4+CD25HighCD127Low and CD4+CD25LowCD127High cells. FOXP3 were positive in 94% of CD4+CD25HighCD127Low cells, and 5% in CD4+CD25LowCD127High cells. The sorted cells were expanded with and without rapamycin at concentrations between 1–100ng/ml in X-vivo medium containing IL-2 and anti-CD3/CD28 beads. The anti-CD3/CD28 beads were removed at day 7 and cells were cultured for a total of 21 days. In the absence of rapamycin, CD4+CD25+CD127Low cells expanded 28 folds, yet only 60% were FOXP3 positive at day 7. At day 14 and 21, only 30% and 20% of the expanded cells were positive for FOXP3, although cells expanded 156 and 1560 folds respectively. In the presence of rapamycin between 1ng/ml and 20ng/ml, sorted CD4+CD25+CD127Low cells remain FOXP3+ in 96%, 60% and 56% at days 7, 14 and 21. However, the number of cells increased only 19, 36, and 21 folds at days 7, 14 and 21 respectively. To overcome the insufficient expansion of CD4+CD25+CD127Low cells in the presence of rapamycin, anti-CD3 mAb (OKT3) or anti-CD3/CD28 beads was added to the media at day 7 after the initial beads were removed and cultured until day 21. At day 14, 66% of the cells were FOXP3 positive with rapamycin and anti-CD3 and 80% for cells with rapamycin and ani-CD3/CD28 beads. The numbers of cells were expanded: 13 folds with rapamycin and anti-CD3, and 238 folds with rapamycin and ani-CD3/CD28 beads. At day 21, the numbers of cells were expanded: 34 folds with rapamycin and anti-CD3, and 2856 folds with rapamycin and ani-CD3/CD28 beads. The suppressive function of expanded cells on allogeneic proliferation of conventional CD4 T cells in the mixed lymphocytes reaction (MLR) was positively correlated with the percentage of FOXP3 positive cells added to the culture. In contrast, the sorted CD4+CD25LowCD127High cells expanded in the same condition as controls had much lower percentages of FOXP3+ cells with or without rapamycin and in the presence or absence of coupled anti-CD3/CD28 beads although the numbers of cells were expanded much higher. In summary, sorted human CD4+CD25HighCD127Low cells were 94% FOXP3+. Without rapamycin, the FOXP3 negative cells expanded much faster and dominated the expanded cell population at day 14 and 21. In the presence of rapamycin, CD4+CD25HighCD127LowFOXP3+ cells were preferentially expanded with IL-2 and anti-CD3/CD28 beads.
Continued stimulation from anti-CD3/CD28 beads enhanced the expansion of CD4+CD25HighCD127LowFOXP3+ cells. The expanded cells are functionally suppressive on allogeneic proliferation of conventional CD4+ T cells.