Current Projects

2MM1DSA/dnDSA

Historically, histocompatibility between donor and recipient was determined by comparing the HLA antigens possessed by the two individuals and counting the number of mismatched antigens, as viewed by the recipient. This meant that the highest number of mismatches a donor/recipient pair could have at each locus was 2 (1 per allele); with UNOS focusing on matching at the HLA-A, -B, -DR loci, and the NMDP adding one or more of the other loci – HLA-C, DQ, DP. More recently, molecular mismatch analysis has been proposed as a more granular method of quantifying the degree of incompatibility among antigen-mismatched pairs, aiming to predict the likelihood of donor-specific antibodies (DSA) development, antibody-mediated rejection (ABMR) and cell-mediated rejection (TCMR). Approaches for molecular mismatch analyses include eplet mismatch load, amino acid mismatch load, and electrostatic mismatch score. While these approaches have been successful in correlating mismatch load and de novo DSA development at the population level, many exceptions exist (i.e. DSA development despite low mismatch load, or no DSA development in recipients with high mismatch loads, even many years post-transplant), indicating that these methods may not necessarily incorporate all the required information to provide information  at the level of an individual donor/recipient pair. We postulate that differences in the immunogenicity of mismatches may play a role in these observations, though the factors that contribute toward HLA immunogenicity are still unclear. In order to study this, we have developed a new model for interrogating mismatches that lead to de novo DSA development and those that do not, in a cohort we have named the “2 mismatch 1 DSA” (2MM1DSA) cohort. Specifically, 2MM1DSA patients are those who have been transplanted across 2 HLA-DQ mismatches and developed de novo DSA against only one of the mismatches, but not against the other. These patients provide a unique environment to study the immunogenic properties of HLA-DQ mismatches while controlling for patient-specific variables such as adequacy of immunosuppression or other factors like other diseases that can affect the immune system, etc. Importantly, it also allows us to view each mismatch through the lens of the recipient’s immune system, which is critical when considering a more personalized approach to histocompatibility.

HLA-DQ Antibody Adsorption

Despite the prevalence of antibodies directed against donor HLA antigens, HLA epitopes are not yet well defined. Several approaches have been published in recent years, mostly utilizing tools like the HLA Matchmaker or other software that interrogate the amino acid sequences (2D approach). Our approach takes advantage of one of the known of the single antigen bead assay, namely – inhibition (colloquially known as prozone). Inhibition is a common phenomenon observed in serum of highly-sensitized patients in which MFI values of antibodies in undiluted serum do not correlate with antibody strength (revealed either with the use of EDTA or dilution studies) due to inhibitory factors present in the serum..

Dilution studies allow us to visualize the full extent of inhibition across all positive HLA specificities. A closer look at such studies reveals numerous instances of distinct “patterns” of inhibition, the cause of which may help us better understand HLA epitopes and antibody kinetics. We speculate that each pattern is likely associated with a single antibody, recognizing a specific epitope that is shared among the group of specificities within that unique patters. We have a unique bank of serum samples with unique inhibition patterns of HLA-DQ directed antibodies that are used to perform adsorption/elution experiments. As targets we use both DQ-homozygous lymphocytes and “single single-” antigen beads with the goal to elucidate immunological drivers of inhibition and better characterize HLA epitopes.