Background: BK virus-associated nephropathy (BKVN) is a significant complication in renal transplantation that leads to allograft dysfunction or failure. BKV is thought to reactivate as a result of over-immunosuppression and current treatment is reduction of immunosuppression. The role of adaptive and innate immune responses to BKV have only been partially explored and requires further elucidation. Previous studies have characterized variable degrees of heterogeneous immune infiltrates, including macrophages, plasma cells, eosinophils, NK cells, and lymphocytes in BKVN, but these studies were limited to detection of only 1 cell type per stain/section.We have used a novel multiplexed immunofluorescence technique to define spatial relationships and colocalization among immune cell phenotypes within the kidney microenvironment. We hypothesized that there will be a distinct signature of immune cell infiltrates in BKVN in transplant recipients.

Methods: Co-Detection by inDEXing (CODEX) employs DNA barcoded antibodies to enable high parameter profiling of tissues using a single stain, multiple image approach. We performed CODEX on a set of SV40 positive and negative tissue using an immune panel including SV40 large T antigen, immune cell subsets (T cells, B cells, NK cells, macrophages, plasma cells, neutrophils) and structural markers (blood vessels, renal tubules).

Results: CODEX is able to identify structural features of the kidney, such as renal epithelial cells, tubules, blood vessels, and glomeruli. SV40 Large T antigen expressing epithelial cells are detectable in BKVN positive samples. Dense lymphocytic infiltrate surrounding a renal tubule consisting of CD4 and CD8 T cells, some of which co-expression CD45RA. CD45RA expression may suggest migration of naïve T cells into the kidney in response to BK virus infection. Interestingly, few NK cells or dendritic cells were present.

Conclusions: Our preliminary studies show promising data that CODEX can identify important spatial relationships and phenotypic characterization of immune cells in BKVN. Further studies using CODEX are needed to characterize the immune signature of the various stages in BKVN. These findings can identify potential immune cells of interest and cellular markers that can translate to the design of future diagnostic and therapeutic approaches in BKVN.

CITATION INFORMATION: Fajardo C., Grimm P., Kambham N., Nolan G., Samusik N., Maltzman J. Multiplexed Immunofluorescence to Investigate the Immune Response to BK Virus in Pediatric Kidney Transplant Recipients Am J Transplant. 2017;17 (suppl 3).

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