Aggregating the human leukocyte antigen (HLA) Class I antigens on the endothelial membrane has been known to elicit an activation, an underlying mechanism of chronic rejection in organ transplant recipients. The current study aims at examining the endothelial responses using HLA typed microvascular cultures from human adipose tissues upon exposure to the serum that contain corresponding antibodies collected from mismatched transplant recipients.
We have successfully cultured 30 microvascular cultures and typed their HLAs. They are functionally competent to respond to inflammatory TNF-α stimulation and the aggregating monoclonal antibody against HLA Class I. The post-transplantation serum was collected either from the recipients with pathologically diagnosed chronic rejection or from the recipients without rejection. We determined their activation either by double-staining the endothelial cells in crude cultures with flow cytometry or by quantifying cytokine releases in purified endothelial cells using ELISA.
Under our current protocol, adipose tissue cultures are functionally intact in regard to its responses to TNF-alpha and anti-HLA Class I antibody. We observed that the post-transplantation serum with rejection contained the pathogenic antibodies and led to proinflammatory activation, as demonstrated by not only increased CD54+/CD31+ and CD106+/CD31+ cell counts but also inflammatory cytokine releases including MCP-1, IL-8 and RANTES.
This methodological study provides the feasibility of examining the pathogenicity of the alloantibodies in mis-transplant serum. Potentially, the endothelial activation elicited as a result of exposure can be used as an alternative readout for chronic rejection.
We prototype an ex vivo model that enables us to examine whether allogenic antibodies from the recipient can functionally activate microvascular endothelial cells from the donor adipose tissues. This system can be further developed as crossmatch using cellular responses as readouts for chronic rejection for post-transplant surveillance.

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