*Purpose: Allorecognition of donor HLA and induction of humoral alloimmunity is a major cause of graft loss after solid-organ transplantation. Current approaches to study molecular HLA mismatch at the patient level are often compromised due to multitude of variables that influence humoral responses in clinical transplantation (e.g. sensitisation events, immunosuppression). Herein we examined the capacity of an improved Electrostatic Mismatch Score (EMS3D), along with current sequence-based immunogenicity algorithms, to predict HLA-DQ DSA development in a unique clinical transplant cohort.

*Methods: Recipients who received a kidney from a donor with two HLA-DQ mismatches, developed a consistent DSA response against one of the donor HLA-DQ alleles (immunogenic) but not the other (less immunogenic), follow up >3yrs, were studied; N=18. DSA were detected using Luminex single-antigen-beads and serial serum dilutions (reported as titer). HLA immunogenicity was assessed using currently available B-cell and T-cell algorithms – HLAMatchmaker (eplets), Cambridge HLA Immunogenicity algorithm (amino acid mismatches; AAMM), Electrostatic Mismatch Score 3D (EMS3D), and predicted indirectly recognised epitopes (PIRCHE).

*Results: Within individual patients, DSA responses were more likely against the donor HLA-DQ mismatch with the higher immunogenicity score (p<0.01). This was true in 15/18 (83%) of cases using eplet and PIRCHE scoring and in 16/18 (89%) of cases using AAMM. The eplet score was highly correlated to the AAMM score (rho=0.98). Analysis of donor HLA-DQ immunogenicity using the structural score EMS3D correctly identified DSA positive mismatches in all patients (18/18, independent of sequence polymorphism), although in four cases the score was equivalent between DSA positive and negative HLA mismatches. Notably, in this cohort, donor HLA alleles with EMS3D scores below 0.25 were not immunogenic (possible EMS3D score range 0-0.5). All scores correctly identified HLA-DQ mismatches that induced moderate/high titer DSA (≥1:32).

*Conclusions: We describe a unique cohort for studying donor HLA immunogenicity where, within each patient, all variables that might influence alloimmunity, other than donor HLA, are controlled. This approach allows robust investigation of donor specific humoral alloimmunity in the clinical setting. Results in this cohort suggest that the structural EMS3D approach may be advantageous compared to sequence-based immunogenicity algorithms. Investigation of a larger cohort is required to fully explore the translational potential of HLA immunogenicity algorithms.

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