Figure 3.

Natural killer (NK) cells restored the capacity of CTLA4-Igand interleukin (IL)-10 to inhibit CD4⁺ responder cells in the mixed lymphocyte reaction (MLR). (a) Miltenyi Microbead separation was used to purify CD4⁺ T cells and CD56⁺ NK cells from nylon wool T (NWT) cells. CD4⁺ T cells were isolated by negative selection and stained with anti-CD4-phycoerythrin (PE) (shaded) or negative control PE-conjugated monoclonal antibody (mAb) (unshaded). NK cells were positively selected with anti-CD56-fluorescein isothiocyanate (FITC)-conjugated mAb and captured by anti-FITC microbeads. The histogram shows the overlay of the positive fraction (shaded) against the negative fraction (unshaded). (b, c) NWT cells, CD4⁺ T cells or CD4⁺ T cells + 10% NK cells as the responder populations were added to either allogeneic immature dendritic cell (iDC) or mature dendritic cell (mDC) stimulators. A stimulator:responder (S:R) ratio of 1 : 100 was used. IL-10 (5 ng/ml) and CTLA4-Ig (20 ng/ml) were added alone or in combination to the MLR. Proliferation was measured by [³H] thymidine incorporation and results are expressed as the percentage inhibition of proliferation in comparison with untreated controls. P-values were determined by unpaired Student's t-test. Data shown are representative of three independent experiments.