Regulatory T Cells (Tregs) Can Be Isolated from G-CSF Mobilized PBSC after Monocyte Depletion and Inhibit Anti-Stem Cell T Cell Alloreactivity

Small numbers of human CD4+CD25+FoxP3+ Tregs can be isolated from normal peripheral blood, thus their potential clinical application is limited. In this study we tested whether Granulocyte Colony-Stimulating Factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) from healthy donors can represent a useful source of CD4+CD25+ cells with regulatory activity. We utilized antibodies conjugated to microbeads (Miltenyi Biotec Inc, Auburn, CA) to immunomagnetically separate the cells on a MidiMACS device (Miltenyi) and checked the purity after isolation by flow cytometry. Starting from on average 4.0±1.8 × 10⁸ unseparated PBSC (n=3) we positively selected 2.4±0.8 × 10⁶ CD34+ cells (>90% purity). We then utilized the CD34− cell fraction to isolate Tregs. Due to the large content of CD4^dim monocytes in the initial cell product, we initially depleted the PBSC of CD14+ cells and then utilized a two-step process that includes a CD4+ cell negative selection (using a cocktail of biotin-conjugated antibodies against CD8, CD14, CD19, CD16, CD36, CD56, CD123, TCR g/δ and Glycophorin-A) followed by a positive selection of CD25+ cells (Treg isolation kit, Miltenyi). This process allowed us to obtain 1.2±1 × 10⁶ CD4+CD25+ cells with a purity of >70%. Intracellular expression of FoxP3 was also detected in purified CD4+CD25+ cells by flow cytometry. Primary mixed leukocyte cultures (MLC) were performed with irradiated CD34+ cells isolated from PBSC and HLA mismatched blood CD3+ responders for 6 days and T cell response was measured by a 3H-thymidine uptake assay. Tregs isolated from PBSC were added to the MLC at 1:2 Treg:responder ratio to test their regulatory function. Control experiments were performed using CD4+CD25− cells. Addition of Tregs isolated from PBSC resulted in 76±17% inhibition of anti-CD34 T cell alloreactivity (cpm: 19000±530 vs 4590±1880) (n=3), while control CD4+CD25 neg cells did not show suppressive activity. These findings show that after isolation of CD34+ cells, adequate numbers of Tregs can be obtained from the CD34− cell fraction of PBSC by using a three-step process. In addition, since Tregs isolated from PBSC suppressed in-vitro T cell alloreactivity against CD34+ cells, these findings will prompt the design of pre-clinical studies to test the combination of PBSC-derived CD34+ cells and Tregs in HLA mismatched transplantation.