DigitalCommons@Linfield

An In Vitro Reporter Cell System for Analysis of Functional Ly49 Receptor Binding of Cognate Ligands in Cis and Tran

Jereld R. Nicholson Library

Activation of natural killer (NK) cells requires the integration of stimulatory and inhibitory signals mediated in part by members of the allelic Ly49 receptor family. Inhibitory Ly49 receptors bind cognate MHC class I ligands in trans (ITIM activation) and in cis (no ITIM activation). We have developed an in vitro reporter cell system for analysis of the functional interaction between the activating Ly49H receptor (C57BL/6 strain, B6) and its murine cytomegalovirus (MCMV)-encoded ligand, m157. Since m157 also binds inhibitory Ly49 receptors, including Ly49I from 129 mice, we exploited our reporter cell system to determine: 1) whether Ly49H^B6 and/or Ly49I¹²⁹ bind the GPI-linked m157 ligand in cis, and 2) whether the induction of beta-galactosidase (b-gal) reporter activity of Ly49H-expressing HD12 cells could be inhibited or attenuated by the co-expression of Ly49I¹²⁹ (HD12-I129 cells) binding to the same m157 ligand. We found that Ly49H^B6, but not Ly49I¹²⁹, binds m157 in cis, as measured by flow cytometry and by activation of Ly49H reporter (HD12) cells. When Ly49I¹²⁹ is co-expressed in cis with Ly49H and m157, partial m157 staining is restored, possibly reflecting trogocytosis of m157 by Ly49I¹²⁹. When Ly49H is co-expressed with Ly49I¹²⁹ and m157, the mean fluorescence intensity of m157 is slightly reduced and correlates with a minor reduction in HD12 activation (in trans). We also show that co-expression of Ly49H^B6 and Ly49I¹²⁹ on HD12-I129 cells results in lower b-gal induction following co-incubation with m157-expressing stimulator cells compared with HD12 cells (expressing only Ly49H). These findings represent a novel demonstration of cis ligand binding for an activating Ly49 receptor, and also demonstrate the utility of this reporter cell system for analysis of other relevant inhibitory and activating Ly49 receptor interactions (e.g. Ly49G2 and H-2D^k).