Intracellular CD3 and surface CD8β distinguish activated human T cells from natural killer cells after αCD3/αCD28...

Many studies of human T cell function involve activating primary T cells in vitro to assess functional outcomes. A common model of human T cell activation is polyclonal stimulation with αCD3/αCD28 antibodies. However, subsequent identification of these stimulated T cells in a mixed population is complicated by surface CD3 loss. In particular, both cytotoxic T cells and natural killer (NK) cells can appear CD3^-CD8α⁺, leading to misidentification of NK cells as cytotoxic T cells. To address this limitation, we investigated incorporating intracellular CD3 and surface CD8β staining following αCD3/αCD28 stimulation to improve discrimination between cytotoxic T cells and NK cells. We found that intracellular CD3 staining completely recovers lost CD3 expression. When intracellular staining is not amenable to downstream applications in which live cells are required, we found that CD8β more accurately identifies cytotoxic T cells while excluding NK cells than does the more common CD8α staining. We validated CD8β as a more faithful marker of cytotoxic T cells by comparing cytokine profiles of CD8α or CD8β gated PBMC to those of purified T cells, showing that CD8β most closely mimics the cytokine profile of cytotoxic T cells within a purified T cell population. We conclude that i) intracellular CD3 staining is ideal when cell fixation and permeabilization is possible and ii) surface CD8β staining is best when intracellular CD3 staining is not possible, e.g., when recovery of live cells from fluorescence-activated cell sorting is necessary.