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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE92512 | GEO | 2017/09/04

SECONDARY ACCESSION(S): PRJNA357845

REPOSITORIES: GEO

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Project description:Natural Killer cells (NK), a major constituent of innate immune system, have the ability to kill the transformed and infected cells without prior sensitization; can be put to immunotherapeutic use against various malignancies. NK cells discriminate between normal cells and transformed cells via a balance of inhibitory and activating signals induced by interactions between NK cell receptors and target cell ligands. Present study investigates whether expansion of NK cells could augment their anti-myeloma (MM) activity. For NK cell expansion, peripheral blood mononuclear cells from healthy donors and myeloma patients were co-cultured with irradiated K562 cells transfected with 4-1BBL and membrane-bound IL15 (K562-mb15-41BBL). A genome-wide profiling approach was performed to identify gene expression signature in expanded NK (ENK) cells and non-expanded NK cells isolated from healthy donors and myeloma patients. A specific set of genes involved in proliferation, migration, adhesion, cytotoxicity, and activation were up regulated post expansion, also confirmed by flow cytometry. Exp-NK cells killed both allogeneic and autologous primary MM cells more avidly than non-exp-NK cells in vitro. Multiple receptors, particularly NKG2D, natural cytotoxicity receptors, and DNAM-1 contributed to target lysis, via a perforin mediated mechanism. In summary, vigorous expansion and high anti-MM activity both in vitro and in vivo provide the rationale for testing exp-NK cells in a clinical trial for high risk MM. Differential gene expression profile in expanded natural killer (ENK) cells and non-expanded natural killer (NK) cells from healthy donors and myeloma patients Eight healthy donor and eight myeloma patients were used in the study. Non-expanded natural killer (NK) cells were isolated from PBMCs of healthy donors and myeloma patients. Expanded natural killer (ENK) cells were generated from same set of samples as mentioned in expansion protocol. All ENK and NK cells were used for gene expression profiling.

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

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