Figure 4. Cytolytic activity against autologous melanoma of NK cell subsets expressing no inhibitory receptors for self-HLA class I.

The cytolytic activity of NK cell subsets expressing no inhibitory receptors for self-HLA class I (“HLA non-inhibited”) was compared with the counterpart expressing at least one receptor for self-HLA. A: Cytolytic activity was assessed by CD107a degranulation assay for total “HLA-inhibited” and total “HLA non-inhibited” NK cell subsets. A representative experiment is shown. Briefly, triple fluorescence staining with a mixture of PE-conjugated anti-KIR2DL1 mAb and a mixture of other anti-KIR (KIR2DL2/3 and KIR3DL1) and anti-NKG2A mAbs allowed to gate on the KIR⁻/NKG2A⁻ subset plus NK cell subset expressing receptors for HLA class I alleles not expressed by the related patient. On the other hand, the staining permitted also to gate on all “HLA-inhibited” subsets. Thus, as shown in panel A, CD107a expression of the whole “HLA non-inhibited” subset was analyzed and compared with that of whole “HLA-inhibited” subset, in the presence of absence of anti-HLA class I blocking mAb. In agreement with the absence of inhibitory receptors for self HLA, “HLA non-inhibited” NK cell subset did not increase their cytotyitc activity in the presence of anti-HLA class I masking mAb. A summary of all experiments performed in each patients is shown in figure 3B(ii,iii,iv) . B: The effectiveness of “HLA non-inhibited” NK cell subsets in killing autologous melanoma was also assessed by a standard ⁵¹chromium release assay. The whole “HLA non-inhibited” NK cell populations derived by three representative patients were sorted out by flow cytometry as double negative cells and compared with the unstained total bulk NK populations. The comparison was performed with the unstained total NK cells, rather than with HLA-inhibited NK cell subets, to avoid possible interference caused by antibody binding to functional receptors. Results shown are from three patients and data correspond to mean values±SD of experiments performed in triplicate.