IVIg Versus A Anti-HLA-E MAb TFL007 That Mimics HIA-I Reactivities Of IVIg On HLA Antibodies Secretion

Intravenous immunoglobulin (IVIg) is one of the most useful therapeutic drugs used to desensitize those patients with high panel reactive antibody (PRA) before kidney transplantation and to treat humoral antibody mediated rejection after kidney transplantation. Although it is very powerful in immunomodulation, the mechanism in reduce anti-human leukocyte antigen (HLA) antibody is far to clear. In addition, there are some other disadvantages for IVTg, such as the expensive price, shortage in clinic because of plasma limit, verified clinical effect based on different lot. It becomes an urgent issue to investigate the mechanism of IVIg and produce surrogate uniformed item that is cheap and could be used in clinic.Here we give a reasonable hypothesis to explain the effect of IVIg shown in clinic from a brand new point associated with the relationship between IVIg and HLA-E antibody. Then we verified HLA-E monoclonal antibody TFL007 could serve a better effect on inhibiting HLA antibodies secretion with two different cell culture systems in vitro. The first part of this thesis is to verify the relationship between IVIg and HLA-E antibody. We found all IVIg we used in experiment could bind to a broad of beads coated with HLA class Ia (A/B/Cw) antigens and HLA class Ib (E/F/G) antigens. And the tilter reacted with HLA class Ib was very high. This phenomenon showed that there were HLA antibodies in IVIg. We did further study by adsorbing the HLA-E antibody from IVIg and found the profile IVIg showed to bind HLA class I antigens disappeared along with HLA-E antibody adsorption. This means there are HLA-E antibodies in IVIg, and it is the reason that IVIg bind to all HLA class I antigens. By such findings, we speculate HLA-E antibody may serve the similar effect to reduce the HLA antibody secretion since it mimics the HLA binding profile of IVIg.In the second part of this thesis, we generate the method to stimulate purified B cells from peripheral blood then secret HLA antibody. The blood we took was from a healthy volunteer who got HLA immunized during her first pregnancy. The specific allo-HLA antibodies keep presented in her peripheral blood for over 20 years without exposure to allo-HLA antigen, which suggested there are long-life memory B cells in her blood. We compared two regimens to stimulate B cells, and take one with interleukin 4 (IL-4) to do further following studies according the difference in significant plasma cell differentiation and verified trend of HLA antibodies secretion.In the third part of this thesis, we analyzed the distinct effect of IVIg and HLA-E monoclonal antibody TFL007 on HLA secretion. Firstly, we used the system we built with purified human peripheral B cells. We found IVIg could only inhibit the primary HLA DRB 1*0101 alloantibody significantly, but not for secondary HLA DRB alloantibodies. More importantly, it could enhance the secretion of other secondary HLA DRB alloantibodies except HLA DRB1*0102 alloantibody. In contrast, and HLA-E monoclonal antibody TFL007 could inhibit all primary and secondary allo-HLA DRB antibodies secretion. And the inhibition effect is stronger than IVIg even for the primary HLA DRB1*0101 alloantibody. By cooperation with Japanese Hokkaido red-cross blood center, we verified our finding by using immortalized B cells from another multi-pregnant woman. In this separate study, IVIg did not show significant inhibition on HLA class I antibodies. However, TFL007 showed a remarkable effect to inhibit HLA antibodies secretion. Of note, the inhibition is dose-dependent, which showed the strongest inhibition in the highest concentration group and the inhibition became weak along with the reduced TFL007 concentration.In this thesis, we studied the relationship between IVIg and HLA-E antibody. Then we verified HLA-E monoclonal antibody TFL007 could serve a better effect on inhibiting HLA antibodies secretion with two different cell culture systems in vitro. As a uniform and easy to made preparation, HLA-E monoclonal antibody TFL007 has the possibility to replace IVIg in clinic to decrease the high PRA condition before kidney transplant and to treat humoral antibody mediated rejection after kidney transplantation.