Quantifying NK cell growth and survival changes in response to cytokines and regulatory checkpoint blockade helps identify optimal culture and expansion conditions

Abstract

NK cells are innate lymphocytes critical for immune surveillance, particularly in eradication of metastatic cancer cells and acute antiviral responses. In contrast to T cells, NK cell‐mediated immunity is rapid, with spontaneous cytotoxicity and cytokine/chemokine production upon pathogen detection. The renaissance in cancer immunology has cast NK cell biology back into the spotlight with an urgent need for deeper understanding of the regulatory networks that govern NK cell antitumor activity. To this end, we have adapted and refined a series of quantitative cellular calculus methods, previously applied to T and B lymphocytes, to dissect the biologic outcomes of NK cells following stimulation with cytokines (IL‐15, IL‐12, IL‐18) or deletion of genes that regulate NK cell proliferation (Cish), survival (Bcl2l11), and activation‐induced‐cell‐death (AICD; Fas). Our methodology is well suited to delineate effects on division rate, intrinsic apoptosis, and AICD, permitting variables such as population half‐life, rate of cell division, and their combined influence on population numbers in response to stimuli to be accurately measured and modelled. Changes in these variables that result from gene deletion, concentration of stimuli, time, and cell density give insight into the dynamics of NK cell responses and serve as a platform to dissect the mechanism of action of putative checkpoints in NK cell activation and novel NK cell immunotherapy agents.

Deconvoluting global cytokine signaling networks in natural killer cells

“…During extended stimulation, IL-15 was the main driver of proliferation, whereas IL-12 inhibited proliferation in this in vitro setting devoid of antigen (Extended Data Fig. 6c), in contrast to MCMV infection 25 , consistent with previous studies 40,41 . Together, these studies highlight a division of labor across cytokine conditions that support NK cell activity; however, additional conditions present in vivo are required to fully optimize their effector potential.…”

The Antitumor Effect of Heparin is not Mediated by Direct NK Cell Activation

“…Murine splenic NK cells were exposed to heparin and assayed for cell proliferation kinetics as previously described [ 12 , 31 ]. Interestingly, while heparin does not appear to affect NK cell division rates ( Figure 2 A), using total cohort number analysis to estimate survival of NK cells [ 31 ], we observed that heparin was able to enhance the survival of NK cells in vitro ( Figure 2 B).…”

“…Murine splenic NK cells were exposed to heparin and assayed for cell proliferation kinetics as previously described [ 12 , 31 ]. Interestingly, while heparin does not appear to affect NK cell division rates ( Figure 2 A), using total cohort number analysis to estimate survival of NK cells [ 31 ], we observed that heparin was able to enhance the survival of NK cells in vitro ( Figure 2 B). Addition of heparin in cell cultures for enhanced manufacturing of NK cell products has previously been considered [ 36 ] as it is an effective replacement for stroma [ 37 ], and this might be because of the survival stimulus that we showed here.…”

“…Time endpoints (0, 24, 48, 72, 96, and 120 h) were assessed on a BD FACS Verse cytometer (BD Biosciences, San Jose, CA, USA). Flow cytometry analysis was performed using FlowJo X (BD Bioscience) software, and division numbers were determined using the precursor cohort-based method [ 12 , 31 ].…”

IKAROS and AIOLOS activate AP-1 transcriptional complexes and are essential for natural killer cell development

“…Invitrogen) as described 79 . Briefly, NK cells were resuspended at no more than 2 × 10 AAVS1, IKZF1, IKZF2, Synthego) was formed and mixed together with NK cells and Cas9 enhancer, followed by electroporation (4D-Nucleofector X Unit, Lonza) 80 .…”