Figure 1.

Activated NK cells cause damage of autologous BEC and release of autoantigen-containing microparticles. A. Cytotoxicity of activated NK cells against autologous BEC and EC. SpMC isolated from 2 PBC patients and 5 non-PBC patients were cultured in vitro with a mixture of TLR3-L and TLR4-L for 3 days, washed and then used for isolation of NK cells, which was assayed for cytotoxicity against autologous BEC or EC target cells at serial effector/target (E/T) ratios in a standard ⁵¹Cr release assay. The assay was performed in triplicate and results expressed as mean +/− S.D. Representative data from one PBC patient are shown. B. Activated NK cells were added to the co-cultures, in triplicate, to bring the NK/BEC ratio to the indicated levels for a standard ⁵¹Cr release assay, in the presence or absence of NKG2D Ab. C. Western blot analysis for detecting PDC-E2 using a mAb against PDC-E2. The whole lysate of NK-lysed BEC, or microparticles (MP) isolated by filtration and ultracentrifugation from cell lysates recovered after induction of cytolysis with E/T ratio of 50 or 10, were analyzed. D. Isolated microparticles (MP) were re-suspended in PBS and sterilized by filtration, then used as stimulating antigen in a standard ³H-TdR proliferation assay for the PDC-E2 specific CD4⁺ T cell clone TCC independent 1 (31). For the purpose of control, the T cell clone was incubated with the PDC-E2 163-176 peptide (P) or without the peptide (−). HLA DR53 matched irradiated monocyte derived dendritic cells were included in the assay as APC. E. Production of IFN-γ by the PDC-E2 specific CD4⁺ T cell clone stimulated with microparticles from BEC or EC as described in C. Results are presented as mean +/− S.D. *, significant differences (p<0.01) between Ag (−) versus PDC-E2 peptide or MP from BEC; **, significant differences (p<0.05) between anti NKG2D Ab treatment is present or absent.