GENOMICS AND BIOMARKERS

R.J. Duquesnoy¹, M. Marrari¹, A. Mulder^2
1Pathology, University of Pittsburgh Medical Center, Pittsburgh/UNITED STATES OF AMERICA, ²Dept Of Immunohematology And Blood Transfusion, Leiden University Medical Centre, Leiden/NETHERLANDS

Body: Aim. Humoral sensitization affects transplant outcome and it is now apparent that HLA antibodies are specific for epitopes rather than antigens. Such epitopes can be structurally defined by HLAMatchmaker, an algorithm that considers patches of polymorphic amino acids within a three-Angstrom radius (eplets) as critical components of epitopes recognized by alloantibodies. This study addressed the question how mismatched HLA antigens induce specific antibodies in context with eplet differences with the antibody producer. Methods. We have studied so far 54 HLA class I specific human monoclonal antibodies (mAbs) tested in Luminex assays with single alleles using both Gen-Probe and One Lambda kits. These mAbs were derived from women sensitized during pregnancy. Their epitope specificity was determined from eplet differences between immunizing antigen and the HLA type of the antibody producer. Results. As expected, many mAbs (N=34) are specific for mismatched eplets predicted by HLAMatchmaker, many of which including 62GE, 144QL, 142TKH, 161D, 163RG and 166DG are on one or few alleles whereas others such as 41T, 65QIA, 79ERI, 82RL, 82RNR and 211W are shared by multiple alleles. In twenty cases, the specific epitope correspond with a mismatched eplet paired with a self-configuration present on the HLA antigen of the antibody producer. For instance, MUL2-C6 is specific for 144KR of the immunizing A11 and A1, A3, A24, A36 and A80 in the Luminex panel. Another mAb from the same producer (MUL-4C8) reacts with these antigens except A80 which has 151R rather than 151H shared between the reactive alleles. 151H is a self-residue on A1 and A25 of the producer of this antibody which is specific for 144KR+self151H. Identical reactivity patterns are seen with the 144KR-specific OK5-A3 and the 144KR+self151H specific BRO-11F6. Other examples of epitopes defined by pairs include 62QE+self56G (defined by OK2-H12), 71AAT+self90A (by MUL9-F4) 144KR+selff66NQ (by OK2-H12), 163LW+self73TE (by OK6-H10 and OK6-H12),163EW+self73TE (by WK1-D12), 142TKH+self149A,152V (by HA-5C2), 65QIA+self 76ERNG (by VTM3-A1) and 65RNA+self79ERI (by HDG4-1B1). It should be noted that in these pairs, the eplet and the self-configuration is between 7 and 15 Angstroms apart, a sufficient distance for contact by two separate complementarity-determining regions (CDR) of antibody. Conclusions. These findings demonstrate that immunizing antigens have mismatched eplets that can form antibody-reactive epitopes with self-configurations on the molecular surface. They seem to suggest that HLA antibodies are formed by autoreactive B-cells that have undergone receptor editing to accommodate the recognition of non-self eplets, the driving force of the humoral alloresponse. This concept enhances our understanding of structural epitope immunogenicity and the interpretation of antibody reactivity patterns with HLA panels.

Disclosure: All authors have declared no conflicts of interest.