Figure 2. In vitro generated CD8⁺NKG2D^high cells are highly activated, cytotoxic effector T cells.

CD8⁺ T cells were pre-activated with anti-CD3 (plate-bound, 1 μg/ml, 24 h) and stimulated with IL-6 (1 ng/ml, 8 d), IL-15 (20 ng/ml, 8 d) or IL-6 and IL-15 as indicated. A. IL-15 stimulated CD8⁺ T cells show upregulated surface expression of NKG2D (CD8⁺NKG2D^high) as assessed by flow cytometry (n = 3). B. NKG2D expression on CD8⁺ T cells following IL-15 stimulation. C. Proportions of CD28⁺ and CD28^null cells within the CD8⁺NKG2D^high cell population under basal conditions and after IL-15 stimulation. Left panel: exemplary staining and gating strategy, right panel: quantitative evaluation (n = 9) D. DAP10 gene expression under long-term IL-15 stimulation, d0 = ex vivo expression (n = 10). E. CD8⁺NKG2D^high cells are generated by IL-15 and to much lesser extents by IL-6 stimulation, with no synergistic effects for the combination of IL-15 and IL-6. Only IL-15 generated CD8⁺NKG2D^high cells express high levels of markers indicative for cytotoxic propensity (F., n = 6) and activation (G., n = 3), produce high amounts of the pro-inflammatory cytokines IFNγ and TNFα (H.: flow cytometry, n = 6), low but detectable levels of IL-6 (I: ELISA, n = 6) and are skewed to an effector memory phenotype (J; Tem, T effector memory, CD44⁺CD62L⁻; Tcm, T central memory, CD44⁺CD62L⁺; naïve, naïve T cells CD44⁻CD62L⁺) (n = 3). * p < 0.05, ns = not significant.