Ligand Size Is Important for NK Signal Integration Figure 4. Elongated HLA-Cw6 binds KIR2DL1. (A) Binding of KIR2DL1 beads. 221 cells expressing the indicated form of Cw6 were incubated with fluorescent beads coated with KIR2DL1-CD4d34 or CD4d34. Bead binding was analysed by flow cytometry and expressed as percentage of cells forming conjugates to bead. A representative of two experiments is shown, the error bars represent the SD of triplicates. (B) P815 cells expressing normal-length or elongated Cw6 were allowed to form conjugates with equal numbers of KIR2DL1-expressing NKL cells for 30 min. The number of conjugates was determined as in Figure 2B. (1-tailed t-test: p = 0.025 for P815-Cw6 vs. P815, p = 0.037 for P815-Cw6-CD4 vs. P815 (C) Clustering of Cw6 and KIR2DL1 in YTS-221 immune synapses. 221 cells expressing the indicated Cw6-mCherry were allowed to form contacts with KIR2DL1-GFP - expressing YTS cells for 30 min and imaged live by confocal microscopy. Clustering was quantified as fold-enrichment of GFP brightness within the contact area versus outside the contact area. Each point represents an individual cell/cell contact with the mean and SD shown as lines, p = 0.11 (2- tailed t-test). The scale bar represents 10 mm. doi:10.1371/journal.pone.0015374.g004 Discussion The ectodomains of leukocyte cell surface molecules vary greatly in size [21,46]. Here, we set out to test for the first time whether the size of ligands for activating and inhibitory NK cell receptors influences NK cell responses. We first tested the functional consequence of elongating the extracellular domain of MICA by insertion of 2–4 domain spacers. While normal-length MICA was sufficient to trigger activation of NKL or primary NK cells, elongated forms of MICA were less effective, and the functional response was inversely correlated with MICA length. We showed that elongation did not abrogate NKG2D binding to MICA but did abrogate early signaling, as measured by SLP-76 phosphorylation. Taken together these findings suggest that elongation of MICA abrogates NKG2D triggering. The kinetic- segregation model predicts that elongation of a receptor/ligand complex would abrogate signaling because the increased inter- membrane distance leads to less effective segregation of the engaged receptor from tyrosine phosphatases with large ectodo- mains. This is consistent with very recent data showing that elongation of mouse ligands for NKG2D also abrogated their efficacy in triggering NK cell activation [47]. Thus, the results presented here, showing that elongation of MICA abrogates NKG2D triggering, is consistent with NKG2D signaling involving the kinetic-segregation mechanism. There are many activating receptors other than NKG2D expressed on NK cells, with 16 identified in humans [2]. While they vary in structure they have in common the fact that they are small and, where identified, their cell-surface ligands are also small [2]. Also, with the probable exception of DNAM-1 (CD226), they all signal by tyrosine phosphorylation of their cytoplasmic domains or the cytoplasmic domains of associated signaling subunits such as DAP10, DAP12 or CD3f, and this phosphorylation is thought to be mediated Src-like tyrosine kinases [2]. Thus, taken together with our finding that activation through NKG2D is inhibited by elongation its ligand, kinetic-segregation might be important for signaling by many if not all activating NK receptors. We next investigated whether recognition by an inhibitory NK cell receptor was also dependent on ligand size. To do this we expressed a single-chain trimer form of HLA-Cw6 (Cw6) incorporating a peptide predicted to be compatible with KIR2DL1 binding. We used the single chain trimer to rule out any effect of elongation on peptide loading, as this could indirectly affect KIR recognition [48,49]. Expression of normal-length Cw6 on 721.221 cells inhibited killing by KIR2DL1-expressing YTS cells. Elongation of Cw6, while not affecting binding to KIR2DL1, reduced its ability to inhibit NK cell-mediated lysis, with inhibition broadly inversely correlated with Cw6 length. It has also recently been reported that elongation of a mouse MHC class I molecule on target cells abrogates its inhibitory effect on mouse NK cell killing [47]. The inhibitory effect of KIR2DL1 requires phos- phorylation of cytoplasmic tyrosine residues. One possible mechanism by which elongation of Cw6 abrogates KIR2DL1 function is that it inhibits signaling. Given the small size of the PLoS ONE | www.plosone.org 6 November 2010 | Volume 5 | Issue 11 | e15374