NK cells colocalize with monocytes and DCs in the RA synovium and induce differentiation of CD14⁺ monocytes into DC_NK in vitro. (A) 15A11 staining pattern on cells isolated from SF of a patient with RA. The 3 gates (ie, R1, R2, and R3) set based on FSC and SSC profiles shown on the top contour plot, and the corresponding top 3 normalized histogram overlay plots show that 15A11 primarily detects cells within a lymphocyte gated (ie R1) population. The bottom contour plot shows CD56 and CD3 expression on cells gated on SF lymphocytes (ie R1), and the corresponding 3 normalized histogram overlay plots show that 15A11 binds to 51% of SF-NK cells (CD3⁻CD56⁺), whereas it shows minimal binding to SF-T cells (CD3⁺), or to CD3⁻CD56⁻ SF cells. In each histogram, the bold line indicates 15A11 staining, and the filled gray histogram indicates IgM-isotype control staining gated on respective population. An equal number of cells were used for staining with isotype and 15A11 versus CD3 and CD56. (B) The middle panel shows double staining of RA synovium using 15A11 mAb and anti-CD14 mAb. Red arrowheads indicate areas where NK cells (blue) and monocytes (red) are found in close apposition. The bar equals 50 μm. The right panel shows a higher magnification of the area marked with an asterisk in the central panel. The left panel shows staining of an adjacent RA section using isotype control antibodies. (C) Cell contact between NK cells and DCs in inflamed synovial tissue of a representative RA patient. Double staining using 15A11 mAb shows colocalization of 2 NK cells (red) with a DC-LAMP⁺ DC (brown). The tissue section has been counterstained with hematoxylin (blue) and the bar equals 50 μm. (D) CD14⁺ monocytes were cultured alone (- - -) or together with IL-15–stimulated SF NK cells (–). Cells were stained on day 3 for surface expression of CD14, CD86, CD80, DC-SIGN, CD40, and CD83 as indicated. DCs are gated based on their FSC:SSC profiles, and NK cells are excluded by gating on CD56⁺ cells. The filled gray histograms represent the isotype control staining. In each normalized histogram overlay, an equal number of cells were used for staining with isotype versus specific mAb. (E) NK-cell surface expression of GM-CSF and CD154 on PB CD56^bright NK cells (left 2 panels) and SF NK cells (right 2 panels). NK cells maintained in IL-15 were stained with a mAb against human GM-CSF or CD154 (bold lines, as indicated) or with an isotype control Ab (filled gray histogram).