Endothelial cell activation and microvascular inflammation are hallmarks of antibody-mediated rejection and expression of activation associated transcripts by intragraft endothelial cells is observed whether or not complement activation is indicated. HLA expression is strongly upregulated by the allograft microvasculature during rejection and is a target for Donor Specific Antibody (DSA) binding. DSA specific for HLA antigens are prevalent, are deleterious to the allograft  and both complement-dependent and -independent pathways of DSA-mediated damage have been reported.
We have developed an in vitro model of human microvascular endothelial cells  to reveal that inflammation-induced HLA-DR expression results in the activation and differentiation of allogeneic CD4⁺-T into both IL-6-dependent pro-inflammatory Th17 and anti-inflammatory Treg (Taflin C. 2011). Endothelial binding of HLA-II-specific antibodies further increased IL-6 secretion and amplification of pro-inflammatory Th17 and Th1 (Lion J. 2016, Cross AR. 2019). In the current study we have evaluated whether a humanized monoclonal anti-IL-6 antibody (clazakizumab) can alter the outcome of HLA-specific antibody binding to endothelial cells.
Human microvascular endothelial cells cells (HMEC-1 and primary renal glomerular cells) were cultured in the presence or absence of non-HLA matched PBMC  as described (Taflin C 2011). Soluble factors were assesed by ELISA assay and CD4⁺-T sub-populations identified by intracellular cytokine labelling. C4d and C5b-9 deposition on endothelial cells were measured in the presence or absence of complement-containing human AB serum. Antibodies specific for HLA antigens were either monoclonal or purified DSA from transplant patient serum or plasma-exchange products.
When endothelial cells were co-cultured with PBMC from non-HLA matched donors, addition of clazakizumab  strongly reduced IL-6 at 24hrs and the decrease was maintained even after 72hrs. MCP-1 levels were also reduced. Activation of endothelial cells by HLA class I and II antibodies prior to co-culture further increased IL-6 and this was also decreased by clazakizumab. The cell-surface phenotype of endothelial cells  in co-cultures was unaltered by clazakizumab whereas differentiation of Th17 and Th1 were both decreased (particularly IFN_Ү⁺Th1). HLA-specific antibody binding activated complement, and clazakizumab did not have a direct effect on C4d or C5b-9 deposition on endothelial cells Neither steady-state expression of the complement regulatory proteins CD46, CD55 and CD59 nor their expression under conditions of complement activation were altered by clazakizumab.
These data indicate that anti-IL-6 antibody may limit endothelial damage mediated by DSA binding to HLA class I or II.  The results reveal the ability of clazakizumab to alter endothelial immunogenicity leading to reduced differentiation of pro-inflammatory CD4⁺-T. Moreover the decreased level of MCP-1 may limit recuitment of myeloid cells within the allograft.