NKG2D is a potent costimulatory receptor which is involved in activating various CD8+ T cell responses. Cells undergoing various stresses including tumor formation and virus infection express MHC class I related chain (MIC), a ligand for NKG2D. Thus, MIC acts as a signature molecule of the target cells for CD8+ T cells to recognize in killing. In contrast, MIC also plays a role in impairing effector CD8+ T cell responses because it rapidly internalizes NKG2D from the cell surface. It is unclear how these opposing effects of NKG2D ligation on CD8+ T cell fate are regulated. In this study, we demonstrated that 4-1BB, another costimulatory receptor, might be an important regulator for induction and maintenance of NKG2D expression after its internalization, thereby enhancing effector CD8+ T cell responses. Whereas NKG2D is constitutively expressed on naïve human CD8+ T cells, 4-1BB is induced only by activation. Costimulation of NKG2D with anti-NKG2D and suboptimal anti-CD3 eliminated NKG2D but efficiently induced 4-1BB on naïve CD8+ T cells isolated from human umbilical cord blood, even in the presence of TGF-ß1 which inhibited 4-1BB expression in the absence of NKG2D costimulation. In vitro chromium release assays indicated that the CD8+ T cells costimulated with NKG2D remained low in their cytotoxic activity against C1R B lymphoma cell line expressing MIC which was used as target cells. We found these results despite the fact that the CD8+ T cells were fully activated and expressed 4-1BB and granzyme B at high levels. This is correlated with a lack of NKG2D on the cell surface. Subsequent 4-1BB costimulation restored NKG2D to the cell surface, even in the presence of TGF-ß1. The resulting CD8+ T cells expressing both NKG2D and 4-1BB showed extremely potent cytotoxic activity. Importantly, the NKG2D co-expressed with 4-1BB was unique for being highly refractory to the downregulation by TGF-ß1 and internalization from the cell surface. IL-4 markedly promoted downregulation of NKG2D and 4-1BB expression induced by TGF-ß1, generating a CD8+ T cell type lacking both NKG2D and 4-1BB on the cell surface. These cells were inert and unable to gain cytotoxic as well as proliferative activity, suggesting that CD8+ T cells lacking both NKG2D and 4-1BB may represent terminally incapacitated cells. In summary, NKG2D may be involved in inducing 4-1BB expression and in turn 4-1BB may play a critical role for stabilizing surface NKG2D expression to overcome tumor immune evasion especially in tumor microenvironments enriched with soluble MIC and TGF-ß1. Due to such coupled supports, co-expression of NKG2D and 4-1BB may be a key biomarker for defining effective tumor infiltrating CD8+ T cells that can be used in a clinical setting of immune therapeutic strategies.

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