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Blood, Vol. 112, Issue 12, 4420-4424, December 1, 2008
Dendritic cell and natural killer cell cross-talk: a pivotal role of CX3CL1 in NK cytoskeleton organization and activation
Blood Pallandre et al.
112: 4420
Supplemental materials for: Pallandre et al
Files in this Data Supplement:
- Figure S1. CX3CL1 prevents KIR activation and SHP-1 recruitment in human peripheral NK cell interactions with autologous mDC (JPG, 74.3 KB)
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To confirm the role of CX3CL1 on KIR signaling following interactions with their cognate MHC Class I molecules on autologous mDC, human peripheral NK cell subsets were sorted according to KIR2DL1 expression using fluorescence activated cell sorting (with anti-KIR2DL1 clone 143211, R&Dsystems). (A) KIR2DL1+ and KIR2DL1− resting peripheral human NK and HLA-Cw4 mDC were cultured in the presence of CX3CL1-blocking mAb (at a 1:10 DC-NK ratio) in 96- well plates for 24 hours. CCL3 mAb was used as a control. NK-derived IFN-γ production was assessed in culture supernatants. The data represent means of triplicates ± SE. Three independent experiments were performed with similar results. (B) The influence of CX3CL1 in cognate KIR and MHC Class I interactions during DC and NK cell cross-talk was investigated on human peripheral NK cells. KIR2DL1+ and KIR2DL1− resting human NK were incubated with autologous HLA-Cw4 mDC (ratio 2:1) during 0, 3 and 10 minutes at 37°C and immunoprecipitated with protein G beads coated with anti-KIR2DL1 mAb (T-20 clone, Santa-cruz). The immunoprecipitate was run on an SDS-PAGE, transferred to a membrane and then immunoblotted with anti-SHP-1 mAb (upper panel), anti-phosphotyrosine mAb (middle panel) and anti-KIR2DL1 mAb (clone 2F9, Abcam; lower panel). A representative experiment out of three is shown.
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