Real-time analysis of cell surface HLA class I interactions

Characterisation of the kinetics of assembly and dissociation of the HLA class I heterotrimeric complex provides valuable insights into the relative contributions of each element to complex stability. However, to date there has been no real-time binding analysis on whole cells. Hen we have developed an optical biosensor model to investigate the binding of class I HLA complexes on whole cells to human beta(2)-microglobulin (beta(2)m) and the effects of different HLA-specific peptides on this binding. We immobilised beta(2)m on an IAsys(TM) biosensor surface and established conditions to analyse the binding of this to HLA-A2 expressing cells (T2 cells). Using 721.221 cells as an HLA negative control we showed that HLA-A2 binding was optimal using a) a carboxymethylated dextran surface and b) no growth factors or supplements in the culture medium at the binding event. Using these conditions we verified specificity of binding by inhibition of the reaction with free beta(2)m and determined the dissociation rate constant for T2 cell binding to beta(2)m (0.03 s(-1)). In addition, we demonstrated the ability of different HLA specific peptides to modulate cellular HLA-A2 binding to beta(2)m This is the first time that interactions of cell surface HLA class I molecules has been investigated using realtime analysis. Furthermore, our peptide analysis has shown that this model can be used to characterise peptide specific HLA-binding responses on the whole cell surface in real-time. (C) 1998 Elsevier Science S.A. All rights reserved.