Characterizing HLA-DQ Expression in Transplanted Kidneys with PhenoCycler Spatial Imaging

Aim: To characterize the longitudinal expression of HLA-DQ in serial kidney allograft biopsies before and after the onset of HLA-DQ de novo donor-specific antibodies (dnDSA) and antibody mediated rejection (AMR) utilizing Phenocycler digital imaging.

Methods: We performed a retrospective longitudinal review of kidney allograft biopsies from pediatric patients who developed HLA-DQ dnDSA and biopsy-proven AMR. This was coupled with clinical data and histologic reports. Formalin-fixed paraffin-embedded (FFPE) core needle biopsy tissue sections were stained to enable multiomic imaging of the whole tissue section at single-cell resolution. A customized 34-plex protein panel that labeled immune (including HLA-ABC, -DR, -DQA1) and structural markers was validated with tonsil and kidney controls. Stained slides were imaged using the Akoya PhenoCycler imaging system. QuPath image analysis was used to visualize HLA-DQ expression and assess spatial localization in relation to structural and infiltrating cell types.

Results: Paired allograft biopsies from a teenage patient with kidney failure due to renal dysplasia who developed HLA-DQ7, DQA1*03, DQA1*05dnDSA and AMR. Timepoint 1 (T1) obtained 28 months prior to DSA detection, lacked histologic AMR features. Timepoint 2 (T2) corresponded with AMR diagnosis per 2022 Banff Classification. HLA-DQ+ cells were detectable at low levels at T1, before DSA development. At T2, HLA-DQ expression was increased, primarily in CD31-/CD11c+ interstitial and peritubular capillary regions. Single-cell segmentation is being performed to distinguish and quantify true marker compression from adjacent cells with single-marker positivity.

Conclusion: These preliminary results suggest that the spatial and temporal expression of HLA-DQ is measurable with this technology. Further exploration of intragraft CD31-/CD11c+ cells may provide new insights into the mechanisms underlying AMR. Our future investigations will seek to expand the understanding of HLA-DQ expression in endothelial cells and other cell types for a more comprehensive risk analysis of immune-mediated injury. Emphasis will be placed on expanding the antibody panel to include additional surface markers to distinguish the CD31-/CD11c+ cell cluster, which may represent macrophages or dendritic cells from cell populations that share similar surface markers [1], but have distinct roles in allograft rejection.