Temporal assessment of phenotype and receptor profile of in vitro expanded healthy natural killer cells using irradiated feeder cells

Abstract

Abstract Allogeneic natural killer (NK) cell therapy is an emerging approach in treating hematological malignancies. NK cells recognize tumor cells in an MHC-independent manner via activating receptors, which include Natural Killer group 2, member D (NKG2D), DNAX accessory molecule-1 (DNAM-1), 2B4, and Natural Cytotoxicity Receptors (NCRs) (NKp30, NKp44, NKp46). They also target tumor cells via antibody-dependent cell cytotoxicity using the Fc receptor (CD16). Methods to expand purified cytotoxic NK cells for immunotherapy continue to evolve. Therefore, it is critical to evaluate the dynamics of phenotypes of NK cells and expression of activating receptors during NK cell expansion. This study expanded healthy donor NK cells using irradiated feeder cells, followed by evaluation of their phenotype and activating receptor(s) expression in short- and long-term cultures. Healthy adult donors signed the informed consent. The study was conducted according to the Declaration of Helsinki, and it was approved by the medical ethics committee of Aga Khan University (No. 2024-8539-27830). Peripheral blood mononuclear cells were isolated followed by isolation of live CD56+CD3- NK cells by fluorescence-activated cell sorting. Expansion of NK cells was performed in the presence of irradiated Daudi cells (Epstein-Barr virus lymphoblastoid cell line) (Berg et al Cytotherapy 2009) for 14-17 days (short-term) (N=6) and 24-30 days (long-term) (N=3) with 500 IU/ml recombinant human (rhu) IL-2 and NK MACS media containing human AB plasma. NK cells frozen were retrieved and incubated overnight in rhu IL-2. Live CD56+CD3- cells were analyzed for the expression of CD16, NKG2D, DNAM-1, NKp30, NKp44, NKp46, and 2B4 by flow cytometry. A similar analysis of resting NK cells (referred to as R-NK) (N=3) from healthy donors at day 0 was performed. Data was analyzed by FlowJo software using fluorescent minus one (FMO) control for gating. Data is reported as the mean percentage of cells. Relative mean fluorescence intensity (RMFI) was calculated using unstained control. We found 7-to 191-fold and 23-to 45-fold expansion of CD56+CD3-NK cells in short-and long-term cultures respectively. Live CD56+CD3-NK cells were found to be 98.02% in all cultures. R-NK cells showed 78.5% CD16+NK cells, while it declined to 56.5% and 24.2% in short-and long-term cultures respectively. CD16 RMFI was found to be high in R-NK cells (39,318) compared to short-(15,810) and long-term (7,254) cultures. NKG2D+NK cells decreased in long-term cultures (66.8%) compared to R-NK cells (94.97%) and short-term cultures (91.73%). NKG2D RMFI increased in short-term cultures (8,744) compared to R-NK cells (3,992) and long-term cultures (4,469). DNAM+NK cells and DNAM RMFI increased in both short-(96.47%) (RFMI 29,127) and long-term (95.73%) (RFMI 27,973) expansions compared to R-NK cells (43.63%) (RFMI 8,541).The number of 2B4+NK cells was 99% in all groups; however, 2B4 RMFI increased in both short- (21,035) and long-term cultures (19,925) compared to R-NK cells (12,603). NKp30+NK cells increased upon expansion compared to R-NK cells (R-NK:67.30%; short-term culture: 88.37%; long-term culture:77.9%). NKp30 RMFI increased in short-term cultures (7,354) compared to R-NK cells (2,400) and long-term cultures (3,828). NKp44+NK cells were found to be increased in short-term (50.77%) cultures compared to R-NK cells (0.53%) and long-term cultures (12.77%). NKp44 RMFI was found to be increased in both short-(22,096) and long-term (25,795) cultures compared to R-NK cells (4,881). The number of NKp46+NK cells and NKp46 RMFI were noted to be increased in both short-(80.03%) (RMFI 14,847) and long-term (82.53%) (RMFI 13,318) cultures when compared to R-NK cells (46.07%) (RFMI 10,278). In summary, the number of CD16+NK cells and CD16 RFMI decreased upon expansion. DNAM+, NKp30+, and NKp46+NK cells increased in both short- and long-term cultures. The number of NKG2D+ and NKp44+NK cells increased in short-term cultures but decreased in long-term cultures. RMFI of DNAM, NKp46, NKp44, and 2B4 on NK cells increased in both short- and long-term NK expansion, while the RMFI of NKG2D and NKp30 increased in short-term cultures but decreased in long-term cultures. These data demonstrate that the expression of activating receptors on NK cells changes during the process of expansion. These changes could alter the effectiveness of allogeneic NK cells therapy used alone or in combination with engaging molecules.