Isolation and Characterization of Human CD4+CD25+ T Regulatory Cells for Early Phase Clinical Trials in Hematopoietic Cell Transplantation.
Authors: Hou Jing-Zhou , Baker Jeanette , Sheehan Kelvin , Negrin Robert S. 
Published: April 2020

Abstract
Naturally occurring CD4+CD25+ T regulatory cells (Tregs) are a subpopulation of CD4+ T cells vital to homeostasis and maintenance of tolerance. Tregs develop in the thymus and are characterized by expression of the FoxP3 transcription factor. Tregs suppress the proliferation and expansion of conventional CD25-CD4+ and CD8+ T cells through direct cell-cell contact. Defect in Tregs results in multi-organ autoimmune disorders. Adoptive transfer of Tregs cured diabetes in NOD mice and inflammatory bowel disease in murine models. Co-transplantation of Tregs with conventional CD4+ and CD8+ T cells has been shown to control acute graft versus host diseases (GVHD) without abrogating graft versus tumor (GVT) effects in murine bone marrow transplant models.Translating this novel strategy to suppress acute GVHD without loss of GVT holds great promise not only in clinical hematopoietic cell transplantation (HCT), but also in adoptive cellular therapy beyond HCT. Large scale isolation of sufficient human Tregs for therapeutic application remains challenging. Tregs comprise 2–10% of CD4+ T cells. In contrast to rodent Tregs, human CD4+CD25+ cells are more heterogeneous because CD25 is also expressed at lower level by activated non-suppressive conventional T cells and B cells. In addition, immunophenotype of human Tregs is less well studied. Here, we explore the isolation and characterization of human CD4+CD25+ Tregs from peripheral blood mononuclear cells of healthy blood donors and from CD34+ cell depleted fraction of allogenic donors. CD25+ cells were isolated by three different approaches: 1) CD4+ enrichment followed by CD25+ selection, 2) CD19 depletion followed by CD25+ selection, 3) CD25+ selection without prior enrichment or depletion, using magnetic beads. The purities of isolated CD4+CD25+ cells were 95%, 93%, and 93% respectively by FACS. The percentage of CD25 high expressing cells was also similar among the three approaches. More than 90% purity of CD4+CD25+ cells was consistently obtained by CD25+ selection alone using CliniMACS® CD25 microbeads via double column separation utilizing the AutoMACS®. The yield of CD25+ cells varied based upon the ratio of cells/buffer/microbeads. The isolated CD25+ cells were then sorted into CD4+CD25bright (top 25–30%) and CD4+CD25dim (bottom 25–30%) by FACS, fixed and stained for intracellular expression of FoxP3. More than 95% of CD4+CD25bright cells expressed FoxP3. In contrast, only 40% of CD4+CD25dim cells expressed FoxP3. No FoxP3 expression was detected in CD4+CD25− cells. Murine Tregs express a variety of co-stimulatory molecules, however, expression of these molecules is less well characterized on human CD4+CD25+ cells. Using multi-color FACS, we observed that human FoxP3+CD4+CD25bright cells express high levels of CD27 (98%), CD28 (100%), and CD30 (50%) and low levels (&lt;10%) of CTLA-4, GITR, 4-1BB, PD-1 and ICOS on the cell surface. However, CTLA-4, GITR, PD-1 and ICOS were expressed intracellularly in more than 50% of FoxP3+CD4+CD25bright cells. OX40, TRAIL, ICOS ligand and FasL were not detected on the cell surface or in the cytoplasm of FoxP3+CD4+CD25bright cells. FoxP3+CD4+CD25bright cells also express CD62L and CCR5. In summary, highly purified CD4+CD25+ Treg cells can be isolated by magnetic beads and cell sorting. Importantly, the majority of the cells express FoxP3. Utilizing this technique, the clinical application of Tregs appears feasible.