Ligand Size Is Important for NK Signal Integration Figure 6. The size of Cw6 and MICA influences their localization within the synapse and the integration of their receptor signals. (A) A panel of P815 transfectants were produced expressing matching levels of normal-length or elongated MICA and Cw6. (B) NKL cells expressing the inhibitory receptor KIR2DL1 were mixed with P815 cells (E:T 20:1) and cytotoxic activity determined as in Figure 1B. To augment cytotoxicity triggered by elongated MICA, IL-2 (100 U/ml) and IL-12 (10 ng/ml) were added exogenously. Data shown is representative of three independent experiments (mean 6 SD of triplicates). (C, D) Organization of normal-length and elongated Cw6 and MICA in immune synapses. 221 cells expressing normal- length or elongated Cw6-mCherry (red) and MICA-GFP (green) were incubated with KIR2DL1-expressing NKL cells for 30 min before imaging by live- cell confocal microscopy. Scale bar represents 10 mm. From acquired image stacks, en-face views of the contact regions were reconstructed and the Pearson correlation coefficients (r) between GFP and mCherry fluorescence were determined. Data points represent individual synapses and the mean and SD are shown (p = 4.161027 for MICA plus Cw6 vs. MICA plus Cw6-CD4; 1.361027 for MICA plus Cw6 vs. MICA-CD4 plus Cw6; 0.05 for MICA plus HLA vs. MICA-CD4 plus Cw6-CD4 (2-tailed t-test). doi:10.1371/journal.pone.0015374.g006 Our demonstration of the functional importance of matching dimensions of NKG2D/MICA and KIR2DL1/Cw6 raises the question as to whether this is required for signal integration between other activating and inhibitory NK receptors. In support of this the available structural information suggests that inhibitory receptor/ligand complexes all span similar dimensions (10– 15 nm), despite significant variation in inhibitory receptor structure and length. For example although the LIR-1 (ILT-2) receptor has 4 Ig domains compared with the 2 Ig domains of most KIR molecules, it binds the membrane-proximal rather than the membrane-distal portion of MHC class I molecules [53], so that LIR-1/MHC class I and KIR2D/MHC class I complexes span similar distances. Furthermore, whereas KIR3D receptors have one more Ig domain than KIR2D receptors the two membrane-proximal Ig domains of both receptors are involved in binding [54], and so KIR3D-MHC class I and KIR2D-MHC class I complexes span a similar size. The similarity in the dimensions of activating and inhibitory NK receptor-ligand complexes, despite variation in primary structure and mode of binding, strongly suggests that this is of functional importance. In conclusion, we have shown that elongation of the NKG2D ligand MICA abrogates NKG2D signaling, consistent with a role for the kinetic-segregation mechanism in signal transduction through NK receptors. We have also shown that the dimensions of complexes KIR2DL1-HLA-Cw6 and NKG2D-MICA need to be matched for optimal signal integration and colocalization in the NK cell IS. Our results suggest that size-dependent organisation of molecules within immune synapses is an important general principle for immune cell recognition. Materials and Methods Cell lines The HLA-A/B/C negative human B-cell line 721.221 [55] [subsequently abbreviated as 221], the murine mastocytoma line P815 (ATCC, catalogue number TIB-64), and the EBV-immortal- ized human NK cell line YTS [42] were cultured in DMEM supplemented with 10% heat-inactivated FCS, 100 mg/ml strepto- mycin, 100 U/ml penicillin (all Invitrogen, Paisley, UK). The human NK cell leukemia line NKL [56], was cultured in RPMI- 1640 supplemented with 10% heat-inactivated FCS, 100 mg/ml streptomycin, 100 U/ml penicillin, L-glutamine, b-mercaptoetha- nol, non-essential amino acids (all Invitrogen) and 100 U/ml IL-2 (Roche, Welwyn, UK). Primary human NK cells were isolated from healthy donor blood by negative magnetic selection (Miltenyi, Bergisch Gladbach, Germany) and cultured as described previously PLoS ONE | www.plosone.org 8 November 2010 | Volume 5 | Issue 11 | e15374