Comparison of Surface Phenotype, Conjugation, and Regulatory Proteins within Natural Killer (NK) Populations Derived from Umbilical Cord Blood (UCB) and Adult Blood (AB).
Authors: Masri O. R. , Huang Y. , Fanning L. , Finney M. , Kozik M. , Banks J. , Lam M. , Duam-Woods K. , Laughlin M. J. 
Published: October 2019

Abstract
NK cells mediate innate immune system responses in early defense through cytolysis and the production of regulatory cytokines and chemokines. We sought to investigate whether there are differentiating characteristics of NK cells derived from neonatal peripheral blood and adult peripheral blood. These analyses included surface phenotype, confocal imaging of lipid rafting, measurement of target conjugation, and Western blot analyses of regulatory proteins. Immunophenotyping of UCB grafts (n=40) revealed 14.1% (SEM 0.9%) of UCB mononuclear cells (MNC) expressed NK phenotypes (CD56+ CD3−). Lipid raft migration within NK cells was achieved through stimulation with K562 targets via brief centrifugation. One million NK and target cells were placed in a 1:1 and 1:2 respective ratio. Lipid rafts were stained with anti-cholera toxin B and cholera-toxin B Alexa Flour-594, which bind to cellular surfaces via the pentasaccharide chain of ganglioside GM1. In addition, K562 plasma membranes were stained with concanavalin A (FITC), which selectively binds to alpha-mannopyranosyl and alpha-glucopyranosyl residues. In order to excite appropriate wavelengths and acquire pictures, Argon (488 nm) and Helium-Neon (543 nm) lasers were utilized in conjunction with a Zeiss 510 laser scanning confocal microscope. Lipid raft recruitment and formation of immunological synapse of stimulated AB and UCB NK cells were observed in the presence of K562 targets. These confocal images were verified by quantification of NK/target conjugates through flow cytometry to assess the percent fold change of conjugation between AB NK cells co-cultured with self targets in comparison to third party HLA (KIR) disparate targets. The percentage of conjugation between AB NK cells in third party HLA disparate targets compared with HLA matched MNC targets demonstrated an average increase in fold change of 7.2 (1.6) (n=7). Next, NFATc2 (NFAT1) protein expression in UCB and AB was measured by Western blot (anti-NFAT1, BD Transduction Laboratories, San Diego, CA). This transcription factor is of particular interest due its known regulation of cytokines and chemokines including IFN-gamma and TNF-alpha. NK cells were isolated from UCB or AB by magnetic separation (AutoMacs, Miltenyi Biotech, Auburn, CA). Surface phenotype and purity were verified by flow cytometry including CD3, CD4, CD8, CD19, and CD56. Average purity was 81.5% (3.7%) CD56+CD3− NK (n=4) from UCB MNC and 76.9% (2.9%) (n=4) from AB MNC. Western blotting demonstrated the intensity of NFATc2 (NFAT1) protein expression in AB NK relative to UCB NK was 1495, showing higher expression in AB NK.
Ongoing experiments include further analysis of UCB NK cells by confocal imaging and measurement of rates of conjugation by multi-color flow cytometry as well as measurement of regulatory proteins including Zap-70, LcK, Syk, PKC-theta, and LAT comparing UCB and AB NK. In conclusion, neonatal peripheral blood CD56+CD3− NK differ from their adult counterpart in expression of transcription regulatory proteins including NFATc2 (NFAT1), as well as rates of conjugation and lipid rafting. These observed differences in innate immune responses of neonatal NK cells compared with that of adults has important implications in immune tolerance.