B CELLS AND ANTIBODY RESPONSE

K. Iwasaki¹, Y. Miwa¹, M. Haneda¹, H. Ogawa², K. Uchida³, A. Takeda³, A. Nakao¹, T. Kobayashi^1
1Applied Immunology, Nagoya University School of Medicine, Nagoya/JAPAN, ², Obihiro University of Agriculture and Veterinary Medicine, Obihiro/JAPAN, ³, Nagoya Daini Red Cross Hospital, Nagoya/JAPAN

Body:
[Introduction]
It has been observed that a graft organ continues to survive It has been observed that a graft organ continues to survive and function normally even in the presence of anti-graft antibodies. However,the mechanisms behind acquirement of this condition, termed ‘accommodation’, remain unknown. In ABO-incompatible (ABO-i) transplantation, we frequently observedC4d deposition on organ without any injury, while C4d binding is related to graft rejection in HLA-incompatible (HLA-i) transplantation. Thus, we speculated there might exist the mechanisms tonavigate accommodation, particularly in ABO-i Tx. Raf/MEK/ERK signaling pathway is widely conserved among eukaryote. Numerous experiments have suggested that ERK activation strongly correlated withendothelial activation, followed by graft organ rejection. In current experiment, we compared signaling events in endothelial cells between anti-ABO and anti-HLA antibody binding and tried toelucidate the mechanisms of accommodation.
[Method]
The human endothelial-like immortalized cell line EA.hy926, derived from the fusion of human umbilical vein endothelial cells and the lung carcinoma cell line A549, was used. Human blood type A or Bantigen-expressing EA.hy926 cells were established by introduction of A or B transferase gene. Western blot analysis was carried out using antibody against ERK, phospho-ERK, MEK, phospho-MEK, c-Rafand phospho-c-Raf. CD55 and CD59 proteins were monitored by FCM using anti-CD55-FITC and anti-CD59-FITC antibodies. Blood human serum was obtained from O type healthy volunteer. anti-HLA W6/32 andanti-A/B antibodies were used in current experiments.. The cells viability was determined by MTT assay.
[Results]
First, we successfully established A/B antigen-presenting cells. Blood type O serum stimulation showed IgG, IgM, C4c, and C3d binding. Western blotting experiment showed that anti-A/B associationreduced ERK activation as half and increased complement regulatory protein, CD55 (about 2 fold) and CD59 (about 2.5 fold). In contrast, anti-HLA association on those cells increased ERKphosphorylation about 3 fold and showed approximately 30% reduction of complement regulatory proteins. ERK inhibitor itself induced those genes about 2 fold each. Finally, pre-incubation with anti-AIgM for 24 to 48 hrs enhanced the protection against complement attack, while anti-HLA did not.
[Conclusion]
The signaling events after antibody association on endothelial cells depend on the type of antibody and its concentration. ERK pathway has been proven to be associated with the expression ofcomplement regulatory proteins. Thus, if it would be possible to control ERK activation timely in transplantation, to obtain accommodation might be possible strategy.

Disclosure: All authors have declared no conflicts of interest.