BCL6 regulates the endothelial pro-immunogenic phenotype relevant to organ transplant rejection

Abstract

AbstractBackgroundIFNγ induces an endothelial cell pro-immunogenic phenotype through the JAK/STAT1 pathway, which can influence alloreactive leukocytes in transplant rejection. Numerous endogenous suppressors of JAK and STAT activation have been described, but regulation of STAT1 at the level of transcription is not well-understood. In immune cells, the DNA binding protein BCL6 controls transcription of lineage and inflammatory genes, including STAT-dependent responses. The goal of this study was to determine if BCL6 also modulates the IFNγ-induced immunogenic phenotype in endothelium.MethodsBCL6 binding to target genes and similarity to STAT1 motifs was analyzedin silicousing public datasets.In vitro, primary human aortic endothelial cells were tested for expression of IFNγ-inducible costimulatory molecules, HLA and cytokines, under BCL6 overexpression, depletion and pharmacological targeting. Gene expression was measured by RNA-Seq and protein expression was confirmed by flow cytometry, Luminex and ELISA. Paired biopsies from stable and rejecting human cardiac allografts were compared for expression of BCL6, PD ligands, CXCL chemokines and HLA-DR in the vasculature.ResultsBCL6 expression is increased within human cardiac transplants during rejection, which is positively correlated with expression of interferon response gene HLA-DR in donor blood vessels. Further, BCL6 is IFNγ-inducible via JAK1/2 in endothelium. Next, the consensus DNA binding motif of BCL6 is highly similar to that of STAT1, and numerous interferon response genes harbor BCL6 DNA binding motifs. Depletion of BCL6 in endothelium results in augmentation, while overexpression causes suppression, of MHC class II, chemokine, and PD ligand expression. Unexpectedly, pharmacological targeting of the corepressor domain BTB domain of BCL6 also repressed many interferon response genes, particularly HLA class II, CXCR3 chemokines and PD-L2. On the other hand, BCL6 targeting did not impact inducible expression of any HLA class I genes, PD-L1 or CD40; and the effect is correlated with the presence of BCL6 binding motifs in or near affected genes.ConclusionOur results show for the first time that the transcriptional repressor BCL6 selectively controls the endothelial response to IFNγ. A better understanding of the endogenous mechanisms that regulate donor endothelial activation has the potential to discover new avenues to dampen transplant rejection, with broader relevance to other vascular inflammatory diseases.