Influence of irradiated PBMCs on both ex vivo cell proliferation of natural killer cells and change in cellular property

The harnessing of natural killer cells (NK cells) as adoptive anti-cancer therapy relies on sufficient numbers of functional NK cells. The use of autologous peripheral blood mononuclear cells (PBMCs) from respective allogeneic donors to boost NK cell proliferation ex vivo, has shown to be a suitable strategy to obtain high NK cell numbers for clinical applications. However, the mechanisms underlying the effect of PBMCs on NK cell proliferation have not been completely characterized. Thus, in this study, we performed a thorough analysis of the influence of autologous PBMCs on NK cell proliferation. We used irradiated autologous CD56-negative PBMCs stimulated with OKT3 that induced significantly increased expansion of purified CD56+CD3- NK cells, already after 5 days of culture (3.2 mean fold, n= 9) compared to standard cultures with only IL-2 supplement (1.3 mean fold n=90 proliferation, as observed when NK cells were cultured separately from the PBMCs through a permeable membrane. Depletion of B cells had no influence on the enhancement of NK cell proliferation, observing no changes in terms of fold expansion or cytokine profile, exemplified by high levels of TNF-α, IFN-γ, IL-6 and GM-CSF. In contrast the absence of T cells or monocytes reduced dramatically the proliferation of NK cells and abrogated cytokine production. Moreover, we analyzed differences in gene expression between proliferating and non-proliferating NK cells within this co-culture set up, to sought to determine changes in signalling pathways or phenotype related to a high proliferative status. The transcriptome analysis indicated that actively proliferating NK cells developed a differential activation response towards the stimuli provided during the co-culture compared to non-proliferating NK cells. In particular, differences in phenotype, expression of mitogen-activated protein kinases and zinc finger transcription factors could be demonstrated. In summary, our findings give rise to improve ex vivo expansion protocols of NK cells for future clinical applications. A total of 15 samples were included in the study. Briefly, three different samples of natural killer (NK) cells were obtained from 5 different donors. These three samples from each donor consisted on freshly isolated NK cells (resting, day 0), 5-days highly proliferating NK cells (proliferating) and 5-days non-proliferating NK cells (non-proliferating). 5-days cultured NK cells were obtained from the same sample of NK cells co-culture with respecitve autologous irradiated feeder cells. On day 0 NK cells were labeled with a cell trace dye, and on day 5, after exclusion of dead cells and gating on CD3negative/CD56+ cells, proliferating and non-proliferating NK cells (CD3negative/CD56+ cells) were sorted according to the cell trace dye brightness. Therefore proliferating NK cells are defined as dim due to a higher dilution of the dye after several number of cell divisions completed, and non-proliferating NK cells are defined as bright due to a lower dilution of the dye and higher brightness thereof.