Characterizing NK cell Response to Cytokine Combinations
Shilan Dong MSa , Jason Cahoona, Rachit Ohri PhDa,*
aEnable Life Sciences LLC, 100 Barber Avenue, Worcester MA, USA 01606
*Correspondence: R Ohri, email@example.com
Introduction: Cancer immunotherapies provide new opportunities for novel combinations 1, with
Natural Killer (NK) cells providing an ideal platform given their centrality in both innate and adaptive immune system functions 2. Combination cytokine treatments of NK cells was evaluated for interleukin (IL) 2 and IL12, given the synergy between the two for NK cell production of IFNγ 3 - a cytokine which plays a central role in coordinating immune responses to tumors and Granzyme B - a protease for mediating apoptosis in target cells.
Materials and Methods: NK92 (ATCC) cells were first cultured in RPMI 1640 media with 20% FBS, 1% Penicillin/Streptomycin and 100 U/ml IL2. Following IL2 starvation for 12 h, cells were plated at a density of 1.5×10 5 cells / well in 24-well plates, then exposed over 24 h to two cytokines (0-1000 U/ml for IL2 and 0-100 U/ml for IL12). For 1000 U/ml IL2 and 10 U/ml IL12, a time-course study was pursued over 0, 1, 2, 4, 6, 8, 24, 32, 48 and 56 h. IFNγ levels in cell supernatants were detected using ELISA (Enzyme-Linked ImmunoSorbent Assay). Cells were first lyzed and the cell lysate were detected for Granzyme B using ELISA.
Results : The NK92 cell activating combination of 1000 U/ml IL2 and 10 U/ml IL12 yielded an optimal concentration combination for maximal IFNγ production over 24 h, with ~90% of the highest IFNγ levels with less IL12 required. The time-course data suggests plateauing of IFNγ levels between 24 h and 56 h, and also suggests a rapid increase in IFNγ levels between 8 h and 24 h. In comparison to the IFNγ expression, intracellular Granzyme-B expression levels is mainly dependent on the IL2 concentration alone and reached their plateau at ~32 h rather than 24 h under the same conditions (1000 U/ml IL2 and 10 U/ml IL12).
Discussion: Our work establishes a standardized cell-culture based assay for NK cell activation,
which can be leveraged for evaluating cytokine combination therapies and also for optimizing
individualized cytokine dosages for NK cell based personalized immunotherapy regimen.