Autologous Human Cord Blood CD4+CD25+ T Cells Stimulate CD8+ T Cell Killing Activity, and Their Expression of NKG2D and Granzyme B.
Authors: Ruffin Pamela L. , Kim Young-June , Broxmeyer Hal E. 
Published: April 2020

Abstract
CD8+ T cells are crucial for host defense against viruses and tumor cells. Upon antigen recognition, CD8+ T cells proliferate and acquire effector functions. From mouse studies mainly, CD4+CD25+ T cells have been recognized to have suppressive immunomodulatory activity. Because of a concern that human CD4+CD25+ T cells contain both activated and regulatory T cells we chose to study this population from human cord blood (CB) which contains naïve cells, which are not activated. Human CB cells were isolated using magnetic cell separation of CD4+cells and subsequent purification by magnetic beads of these cells that co-expressed CD25. We assessed whether CD4+CD25+ T cells from cord blood influenced function of autologous effector CD8+ T cells. Upon co-culturing CB CD8+ T cells with autologous CD4+CD25+ cells, we found that CD4+CD25+ T cells positively promoted CD8+ T cell function. This included induction of Granzyme B (GrB), a protease crucial for killing target cells and a hallmark of CD8+ T cell effector function. GrB expression was detected by intracytoplasmic flow cytometry. We also assessed effects on NKG2D, a costimulatory receptor known to promote CD8+ T cell effector responses to viruses and tumor cells. It is believed that down-modulation of NKG2D expression is a major obstacle for CD8+ T cell-mediated tumor rejection often observed in cells from cancer patients. Ligation-induced down-regulation of NKG2D expression was observed after culturing CD8+ T cells on anti-NKG2D-coated plates; effects that were dose-dependent. We examined whether the CD4+CD25+ cells could enhance restoration of expression of NKG2D on CD8+ T cells by stimulating CD8+ T cells with anti-CD3 and IL-15 in the presence or absence of CD4+CD25+ T cells. Consistent with the previous noted positive effect of CD4+CD25+ T cells on GrB expression, they also enhanced NKG2D expression during anti-CD3 stimulation. We then determined if CD4+CD25+ T cells had effects on CD8+ T cell cytotoxicity against the C1R B lymphoblast cell line, that expresses major histocompatibility complex (MHC) class I related chain A (MICA), a ligand for NKG2D. Consistent with the positive effects of CD4+CD25+ T cells on expression of GrB and NKG2D, CD4+CD25+ T cells enhanced the cytotoxic activity of CD8+ T cells to MICA-expressing target cells. In summary, CD4+CD25+ T cells isolated from CB enhanced induction of GrB and NKG2D expression in autologous CD8+ T cells during anti-CD3 and IL-15 stimulation, which helps to explain the stimulatory effect of CD4+CD25+ T cells on autologous CD8+ T cell cytotoxicity. Our results with human CB CD4+CD25+ T cells may reflect actions of subpopulations of these cells. Further characterization of subsets of CD4+CD25+ T cells are necessary to better define the specific population with the stimulatory actions on autologous CD8+ T cells that we observed. More defined characterization of CD4+CD25+ T cells are warranted before such cells are used for clinical intervention in human disorders.