LAUSR

Aim Transplant recipients developing donor specific HLA antibodies are at risk for antibody-mediated rejection . DSA contribute to the process of AMR by binding to the HLA molecules expressed on endothelial cells and triggering signal networks. HLA molecules lack signal motifs in their short cytoplasmic tail. We previously reported that HLA class I recruits integrin beta 4 to induce EC proliferation. However, this signal complex does not regulate HLA-I Ab-stimulated P-selectin expression and monocyte adhesion. We hypothesized that failure of B*44:03 to stimulate monocyte adhesion was due to its inability to recruit TLR4. Methods For HLA I-induced protein complex formation, HAEC were stimulated with F 2 fragment of anti-HLA I mAb W6/32 or mouse IgG as a control and cell lysates were immunoprecipitated with a mouse anti-HLA-B12 mAb recognizing both B*44:02 and B*44:03 alleles. Complex formation between class I and TLR4 and protein phosphorylation were detected by Western Blot. Gene silencing was determined with siRNA transfection. P-selectin cell surface expression was detected by cell-based ELISA. Monocyte adhesion was assessed by CFSE labeling, measured by fluorescence microscopy and analyzed with CellProfiler. Results Treatment of HAEC with anti-B12 mAb stimulated an increase in phosphorylation of FAK, mTOR, S6K, S6RP, ERK and Akt in B*44:02, but not in B*44:03 HAEC. Co-ligation of HLA-A and -B in B*44:03 inhibited HLA-A Ab-triggered activation of signal networks. Co-immunoprecipitation experiments showed complex formation between HLA I and TLR4 in B*44:02 EC, but not in B*44:03. Furthermore, transfection of EC with TLR4 siRNA inhibited HLA-B12 mAb-induced phosphorylation of Src, FAK, PI3K/Akt, mTOR, S6K, S6RP, and ERK, P-selectin expression, and monocyte adhesion. Conclusions Our results demonstrate that HAEC carrying B*44:03 fail to transduce signals because they are unable to recruit TLR4 to form a signal complex. These data provide insights into the mechanisms of AMR and may explain different outcomes in transplant recipients producing Ab to different HLA alleles. Disrupting complex formation between TLR4 and HLA I may provide a new therapeutic strategy to prevent AMR.