Antibody-Mediated Immunosuppression Can Result from RBC Antigen Loss Independent of Fcγ Receptors in Mice

Background: Prophylaxis against red blood cell (RBC) alloimmunization can be achieved by Rh immune globulin (RhIg), a polyclonal anti-RhD antibody preparation used to prevent de novo anti-RhD alloantibody formation, a process commonly termed antibody-mediated immunosuppression (AMIS). As the availability of RhIg donors can be limited and the only available immunoprophylaxis is against RhD, there is a critical need to develop monoclonal antibody alternatives to RhD and immunoprophylaxis strategies in general to other RBC alloantigens. As different monoclonal antibodies to the RhD antigen can have differential immunological outcomes,understanding the mechanism regarding how monoclonal antibodies induce AMIS can facilitate the development and accurate assessment of monoclonal antibodies against RhD and other RBC alloantigens for appropriate therapeutic activity. Recent studies suggest that AMIS may occur through alterations in the target antigen, otherwise known as antigen modulation. However, several studies suggest that AMIS may occur independent of antigen modulation. In particular, AMIS to RBCs that transgenically express the HOD (hen egg lysozyme (HEL), ovalbumin and Duffy) antigen have been shown to occur independent of the activating Fcγ receptors thought to be required for modulation of the HEL antigen. Therefore, we sought to determine whether anti-HEL monoclonal antibodies can induce antigen modulation and whether AMIS to HOD RBCs occurs via monoclonal antibody-induced alterations to the target antigen.

Methods: Using RBCs transgenically express the HOD antigen, we transferred HOD RBCs into C57BL/6 wild-type (WT) or FcγR-chain knockout recipients (FcγR KO) in the presence or absence of anti-HEL monoclonal antibodies individually or in combination. Serum was collected at days 3, 5, 7, 14, 21 and 28 post-transfusion. Antibody development in WT or FcγR KO recipients was examined by flow crossmatch, where serum was incubated with HOD RBCs. Following incubation, RBCs were washed and incubated with fluorescently-tagged secondary antibody and analyzed using flow cytometry. To determine the impact of antibody engagement, RBCs were sampled at 10 minutes and 2 hours, along with days 1, 2, 3 and 5 post-transfusion. The level of detectable HEL antigen, complement deposition, HOD RBC survival and bound antibody to the RBC surface was measured by flow cytometry.

Results: A combination of anti-HEL antibodies suppressed anti-HOD IgM formation more effectively than monoclonal anti-HEL antibodies alone, which correlated with the rate of decrease in bound antibody and detectable HEL on HOD RBCs over time. Unexpectedly, combination or individual anti-HEL monoclonal antibodies not only induced AMIS, but also likewise induced antigen modulation in FcγR KO recipients. Loss of detectable antigen did not appear to reflect anti-HEL induced complement fixation and subsequent complement-mediated masking of the HEL antigen, as no complement could be detected on HOD RBCs when evaluated in parallel. Furthermore, regardless of anti-HEL antibody exposure, in combination or individually, anti-HEL antibodies failed to induce significant RBC clearance in WT or FcγR KO recipients. Pre-exposure of HOD RBCs to anti-HEL antibodies reduced antigen levels and likewise suppressed anti-HOD antibody formation following HOD RBC exposure.

Conclusion: In summary, these results suggest that antibody-mediated antigen modulation may reflect a mechanism of AMIS that can occur independent of activating FcγRs and may provide a surrogate to identify antibodies capable of inducing AMIS against different RBC alloantigens.