The use of purified intact soluble HLA proteins can be extremely useful in gaining insight into the complex nature of HLA-specific antibody recognition. The use of soluble inhibition in this study has highlighted the epitope specific nature of the anti- HLA response and has supported previous work that demonstrated how patterns of reactivity in the single antigen bead assay can be attributed to distinct patterns of amino acid substitutions [80, 147]. Soluble inhibition can also be used to support in silico
analysis of the mismatches between potential donor and recipient pairs [81]. Antibodies specific for potential epitope mismatches between donor and recipient can be assayed directly by soluble inhibition analysis simply by choosing one or more sHLA sharing the mismatched epitope, and depleting the reactivity of the patients’ HLA-specific antibody profile accordingly.
The analysis of HLA-A2 specific reactivity has indicated that the humoral response to HLA-A2 is highly complex and often individuals will produce antibodies to more than one epitope specific to the HLA-A2 protein. This is likely to have clinical implications, in particular in those cases where MFI reactivities are being monitored post-transplant. This data shows that the reported MFI values are often a sum of two or more antibodies competing for binding sites located very closely together on the same HLA molecule. It is entirely feasible that these competing antibodies will differ not only in their specificity, but also in affinity, and functional properties (complement, non- complement fixing). This competition may result in inhibition of binding and false low MFI values (the high-dose hook effect). In such cases it may be prudent, once epitope specificities have been identified, to monitor other bead specificities that carry one or other of the HLA-A2 epitopes individually. For example if we were to consider patient LT79, monitoring the changes in MFI of the HLA-A2 specific beads may not be the
best indicator of changes in antibody titre as there are at least three separate epitope specific antibodies competing for tightly clustered epitopes on the same molecule (Figure 4.7). By analysing the changes in the HLA-A69 bead specific for the 107W epitope and the HLA-B57 or B58 beads specific for the 62G epitope a much clearer picture of antibody level changes may emerge.
The nature of epitope specific binding and the fact that in a high proportion of cases there appears to be multiple antibodies binding to the same HLA molecules may have more fundamental implications. The ability of a given serum to activate complement via the classical pathway may not necessarily only require there to be sufficient levels of suitable HLA-specific IgG, but may also be reliant on the composition of the antibodies in the serum. A mixture of separate antibody populations specific for different epitopes on the same molecule may potentially be a very potent activator of complement via the classical pathway as this mechanism requires the co- localisation of IgG molecules such that C1q can be incorporated to form the first stage in complement activation. Given that in HLA incompatible transplantation, complement dependant cytotoxic crossmatch (CDC) positive transplants have much higher rates of antibody mediated rejection [91, 116] a more detailed understanding of the factors determining serum cytotoxicity would be of great clinical benefit.
Analysis of further samples allowed the identification of 12 specific epitopes from eight sera (table 4.2). This did not account for the total repertoire of HLA-reactive microbeads in each case, and many positive reactions were unexplained following sHLA inhibition. There are a number of possible reasons for this; firstly, the panel of sHLA specificities, although extensive, may not be broad enough to cover all epitopes at this stage. Secondly, antibody reactivity specific for any antigen may be a composite of many different epitope specific antibodies. The extensive sharing of epitopes
individual epitopes very difficult in complex sera such as those where there is likely to be high numbers of individual epitope specific antibodies in the same serum. Thirdly, there is a growing body of evidence which shows that many of the ‘positive’ reactions seen in the microbead assay are false and that the antibody profiles indicated are due in large part to the non-specific binding of serum immunoglobulin to denatured HLA antigen [126, 127]. These studies suggest that antibodies direct against denatured HLA or ‘crytpic’ epitopes are not likely to be significant to transplantation. Therefore the identification of these non-HLA antibodies is crucial, a prospective study using sHLA to determine ‘true positives’ in the single antigen microbead assay is indicated.
In summary, by using soluble HLA in the standard single antigen microbead assay it is often possible to identify the specific epitopes against which HLA-specific antibody is produced. Also, antibody to very common HLA antigens are often composed of a mixture of more than one epitope specific antibody and that this complexity may be crucial both to the accurate interpretation of MFI titre levels but also to the understanding of factors governing serum cytotoxicity. Finally, sHLA inhibition may help to address the problem of recognising antibodies directed against denatured HLA antigen.