The Critical Role of the NKG2D Receptor in CD8+CTL and CD8+CD56+ NKT Cell Cytotoxicity.

Abstract

We previously demonstrated the ability to grow and ex vivo expand mobilized peripheral blood mononuclear cells (PBMNC) from myeloma patients into aggressive cytotoxic effector cells (Blood 102; 422b, 2003). These experiments were designed to test the function and mechanism of tumor cell killing of these cells. Peripheral blood stem cells (PBSC) were collected from myeloma pts after mobilizing with cyclophosphamide and rhG-CSF and cultured in Aim-V serum-free medium at 37 and 5% CO2. After 2 hrs, the non-adherent cells were removed and placed in culture with Aim-V, IL-2 (50 IU/ml) and OKT-3 (50 ng/ml) for 7 days. Cytotoxicity of the expanded cells was tested on Day 0 and Day 7 using a chromium release assay. To identify the cytotoxic potential of cell subsets, cell populations were depleted using the Auto MACS Magnetic Cell Sorter (Miltenyi Biotec Auburn, CA) and cytotoxicity assays were repeated. Since the CD8+ cell(s) were the most cytotoxic, the CD8+ cells were isolated and their mechanisms of tumor cell killing were evaluated by testing killing through MHC Class I, T cell receptor or the NKG2D receptor. The ex vivo expanded population was extremely cytotoxic and killed RPMI 8226 myeloma cells at 60% lysis (+/− 1.6%) (E:T of 100:1) when compared to 3.9 % on day 0 (+/− 0.8%). CD8+ or CD8+CD56+ cell subsets contributed to > 83.3 % (+/− 1.5%) of the killing. Blocking the TCR pathway (Redirected Cytotoxicity Assay) had no effect on killing and blocking the MHC Class I molecules decreased cytotoxicity by only 6%. When the NKG2D receptor was blocked, cytotoxicity by the CD8+ cells decreased by 48% (+/− 2%), demonstrating the critical role of the NKG2D in these CD8+ cell populations. The expanded cytotoxic effector cells aggressively lyse myeloma tumor cells in a MHC and non-MHC restricted fashion. These ex vivo expanded CD8+ cells likely acquire the NKG2D receptor and kill tumor cells in a non-MHC restricted manner. Since MHC expression is often low or absent on myeloma cells and the NKG2D ligands are fairly specific to tumor cells, the infusion of these ex vivo expanded cells following transplant may improve clinical outcomes.