Figure 5.

NKG2D, DNAX accessory molecule 1 (DNAM-1), NKp44 and NKp46 are involved in Nishi natural killer (NK) cell degranulation towards rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). (a) RA-FLS were seeded at 1 × 10⁴ cells/well in 48-well plates, grown to confluency (approximately 4·8 × 10⁴ cells/well) for 72 hr, and Nishi were added at the indicated ratios overnight. Adherent cells were fixed with 4% paraformaldehyde and stained with eosin. An ImmunoSpot Image Analyzer was used to take images and quantify % of well area covered. Figure and graph are duplicates of one donor, and representative of n = 2 donors. (b) RA-FLS were seeded at 2 × 10⁴ cells/well in 96-well plates, the following day 6 × 10³ cells/well Nishi were added to the approximately 4·8 × 10⁴ RA-FLS/well giving an effector : target ratio of 1 : 4. RA-FLS and Nishi were co-cultured overnight. Wells were analysed as described. Representative of n = 3. (c, d) RA-FLS were seeded at 3 × 10⁴ cells/well in 96-well plates, the following day 9 × 10⁴ Nishi/well were added. Nishi and RA-FLS were co-cultured as described and blocked with monoclonal antibodies (mAbs) or isotype-matched control immunoglobulin as indicated. Nishi were identified as viable, single CD3⁻ CD16⁻ CD56⁺ cells. Data points are a combination of several experiments and a total of n = 8 donors. (e) RA synovial fluid mononuclear cells (SFMC) were enriched for NK cells by antibody-coated bead separation and cultured in interleukin-15 (IL-15) for up to 3 weeks. RA-FLS were seeded at 1·5 × 10⁴ cells/well in 96-well plates, the following day 4·5 × 10⁴ autologous enriched CD3⁻ CD56⁺ RA SF NK cells were added per well. NK cells and FLS were co-cultured as described. Results from two donors are shown. NK cells were identified as viable, single CD3⁻ CD56⁺ cells.