gms | German Medical Science

Objective: Natural Killer (NK) cells have proven to be a promising candidate for the immunotherapy of tumours. As NK cells have a different cytokine-production profile compared to T cells, they do not bear the risk of inducing cytokine linked adverse side effects. Glioblastomas express elevated levels of HLA-E which is recognized by the inhibiting NKG2A receptor, blocking the natural cytotoxicity of NK cells. HLA-E is also recognized by the activating NKG2C receptor, albeit with a lower affinity. NKG2C+ NK cells therefore represent a potential candidate for the immunotherapy of GBM. In HCMV seropositive donors, the number of NKG2C+ NK cells is elevated. Nevertheless, the numbers are still too low for a therapeutic setup. Hence, an efficient and selective expansion method is needed. To this end, we want to validate feeder cell lines with expression of an artificial β-2-microglobulin-HLA-E trimer for the selective expansion of the NKG2C+ subset from peripheral blood of HCMV seropositive donors. In this project, we aim to (I) establish a selective ex vivo expansion protocol for NKG2C+ NK cells and (II) to prove their cytotoxic activity.

Methods: Primary NK cells from buffy coats of healthy donors were expanded for 14 days ex vivo using the newly developed feeder cell lines. NK cells were characterized concerning their expression of various activating and inhibitory surface receptors in addition to maturation and exhaustion markers. The characterization was performed by flow cytometry analysis. The cytotoxicity of the expanded NKG2C+ NK cells was analysed using chrome release assays and cytokine ELISA.

Results: The selective expansion of NKG2C+ NK cells using the modified feeder cell lines resulted in a NK cell product containing up to 95% single NKG2C+ NK cells. It was noted that such cells developed from CD56bright NK cells and developed through NKG2A+/NKG2C- early and NKG2A+/NKG2C+ intermediate states. A median expansion factor for single NKG2C+ NK cells of 104 respectively 115 could be achieved. Expanded NK cells were found to be mature and active with an increased expression of activating and inhibitory receptors, while maintaining their cytotoxicity.

Conclusion: Our results demonstrated an efficient ex vivo expansion of primary human NK cells, with a selective expansion of NKG2C+ NK cells. Expanded NK cells maintained their cytotoxicity. For further studies the ex vivo expansion must be adapted to reach clinically relevant numbers for immunotherapy of glioblastoma.