*Purpose: Molecular mismatch load (MML) analysis has been a proposed approach to assess donor/recipient compatibility in support of organ allocation algorithms. While conceptually appealing, MML is a programmed approach that only provides the number of mismatched amino acids or eplets between donor and recipient. Indeed, higher MML correlates statistically with greater risk of developing donor-specific antibodies (DSA) and rejection at the population level. Yet, we previously demonstrated that patients can develop DSA despite low MML and postulate that compatibility is dependent on the immunogenic nature of the mismatch rather than the total number of mismatches. In order to better understand HLA epitopes, we utilized CRISPR-Cas9 editing of HLA-DQ molecules and studied the impact of the mutation on antibody binding.

*Methods: DQA1*05:05/DQB1*03:01 (DQ7) homozygous B-Lymphoblastoid cells (SWEIG007) were obtained from the European Collection of Authenticated Cell Cultures. CRISPR-Cas9 gene editing was used to introduce a mutation in a conserved region of HLA-DQB1 resulting in a deletion-insertion variant c.280_286delinsA (p.Asn62_Gln64delinsLys) (SWEIG-Mut). SWEIG007 and SWEIG-Mut cells were used as targets for adsorption elution experiment using either HLA-DQ monoclonal antibodies (mAb: REA303, 1a3, and HLADQ1) or patients’ sera containing DQ7 antibodies. Antibody profiles pre- and post- adsorption were analyzed on single antigen bead assays.

*Results: As expected, SWEIG007 cells adsorbed all three monoclonal antibodies tested. Conversely, the SWEIG-Mut cells adsorbed 2 mAbs but not clone HLADQ1, suggesting that the mutation in positions 62-64 affected the HLADQ1 epitope but not those recognized by the other mAbs. Similarly, using sera from patients with de-novo DSA, SWEIG007 cells were able to adsorb DQ7 antibodies from all sera samples. SWEIG-Mut cells showed varying ability to adsorb antibodies from the same sera. Thus, patients’ sera that lost the ability to be adsorbed by SWEIG-Mut recognize an epitope containing position 62-64 while the others recognize a different epitope of the DQ7 molecule, not affected by the mutagenesis. Importantly, MML analysis suggested many more eplets than required to explain the observed results.

*Conclusions: MML provides enumeration of mismatched eplets/amino acids between donor and recipient but does not provide information on the identity of the immunogenic epitope recognized by the antibody. Our results demonstrate that not all mismatches have the same impact on antibody recognition, confirming our assertion that some mismatches are more immunogenic than others. Adsorption elution experiments using cells expressing genetically modified HLA antigens may assist in better understanding the immunogenicity of HLA epitopes, and thus provide the required knowledge to support compatibility determination for the purpose of organ allocation.

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