OR37 Antibody ligation of Hla B*44:03 fails to stimulate endothelial cell proliferation due to a failure to recruit integrin beta4

Abstract

Aim Antibody-mediated rejection (AMR) is an important clinical problem after solid organ transplantation. Recipients producing posttransplant HLA antibodies (Ab) are at a higher risk for acute and chronic AMR. Notably, not all patients producing post-transplant Ab develop AMR. We postulated that distinct HLA class I alleles may differ in their capacity to transduce signals. Our previous data showed that HLA-B*44:03 was less efficient than other B locus molecules, including B*44:02, to stimulate signal transduction networks in endothelial cells (EC) despite having a similar binding capacity. We tested the hypothesis that failure of B*44:03 to stimulate signal transduction and EC proliferation was due to its inability to recruit integrin β 4. Method Primary human aortic EC (HAEC) were stimulated with human HLA-B12 allele specific mAb, and phosphorylation of signaling proteins was detected by Western Blot. For protein:protein complex formation, HAEC were stimulated with F(ab’)2 fragment of anti-HLA class I (HLA I) mAb W6/32 or mouse IgG as a control. Cell lysates were immunoprecipitated with a mouse anti-HLA-B12 mAb recognizing both B*44:02 and B*44:03 alleles and complex formation between class I and integrin β 4 was detected by Western Blot. Gene silencing was determined with siRNA transfection. EC proliferation was measured by BrdU incorporation and analyzed by flow cytometry. Results Treatment of HAEC with anti-B12 mAb stimulated a increase in phosphorylation of Src, mTOR, ERK and Akt in B*44:02, but not in B*44:03 HAEC. Co-immunoprecipitation experiments showed complex formation between HLA I and integrin β 4 in B*44:02 HAEC, but not in HAEC carrying B*44:03. Furthermore, transfection of EC with integrin β 4 siRNA inhibited HLA-B12 mAb-induced protein phosphorylation and cell proliferation. Conclusions Our results demonstrate that HAEC carrying B*44:03 fail to transduce proliferative signals because they are unable to recruit integrin β 4 into a signaling complex. Disrupting complex formation between integrin β 4 and HLA I may provide a new therapeutic target to prevent AMR.