Ligand Size Is Important for NK Signal Integration Figure 1. Elongation of MICA abrogates its ability to activate NK cells. (A) Normal-length and elongated forms of MICA-GFP were expressed at comparable levels in P815 cells. MICA was elongated by inserting 2, 3 or 4 Ig domains from CD2, CD22, or CD4. (B-E) Effect of MICA elongation on target cell lysis (B, D) and IFN-c production (C, E) by NKL (B, C) or IL-2 stimulated primary NK (D, E) cells. For lysis, NK cells were incubated with [35S]- Met labeled P815 cells expressing normal-length or elongated MICA for 5 h at the indicated E:T ratios. Lysis percentages are expressed as [35S]-Met release relative to total [35S]-Met. The data in B and D are representative results from three experiments. For IFN-c secretion, NK cells incubated for 16 h at the indicated E:T ratios (or E:T = 1:1 for panel E) with P815 cells expressing normal-length or elongated MICA. IFN-c secretion was quantified by ELISA. Data in C and E are representative results from four and two experiments respectively. Error bars are SD of triplicate measurements. In all panels B-E the same key applies for each bar, as indicated in panel B. (F) Effect of MICA elongation on NKG2D signaling. NKL cells were stimulated by P815 cells expressing normal-length and elongated MICA for 2 min. Cell lysates were analysed by Western-blot using an antibody specific for the phospho-tyrosine 113 of SLP-76. doi:10.1371/journal.pone.0015374.g001 termed 221), EBV-transformed B cells which lack endogenous class I MHC protein expression (Figure 3A) [44]. Expression of normal-length Cw6 on 221 cells strongly inhibited killing by YTS/KIR2DL1 cells (Figure 3B). In contrast, elongated forms of Cw6 were less effective inhibitors, with the extent of inhibition progressively decreased with increasing Cw6 length (Figure 3B). Similarly, whereas normal-length Cw6 strongly inhibited IFN-c secretion, elongated forms of Cw6 were far less inhibitory, even when expressed at high levels (Figure 3C). Similar results were obtained when intracellular IFN-c was measured (Figure S3). Elongated Cw6 can bind to KIR2DL1 One possible explanation for elongated Cw6 being less effective at inhibiting NK cell responses is that it could simply be unable to bind KIR2DL1. To test for this, we compared the binding of PLoS ONE | www.plosone.org 3 November 2010 | Volume 5 | Issue 11 | e15374