Clearance of Incompatible RBC Is Compromised during Persistent Viral Infection

Background: While immune-mediated destruction of red blood cells (RBCs) can occur following incompatible transfusion, the development of a hemolytic transfusion reaction is not the invariable outcome of an incompatible transfusion.  Although a variety of factors may impact the likelihood of RBC clearance following incompatible RBC transfusion, chronic infection-induced immune complex formation may inhibit Fc gamma receptor-mediated removal of antibody coated RBCs. To examine this possibility, we tested the effect of persistent viral infection on the clearance of RBCs following incompatible transfusion.

Methods: For chronic viral infection, C57BL/6J recipients were injected intravenously (iv) with 2 x 10⁶ pfu of LCMV clone-13 strain.  Total IgG, anti-LCMV specific IgG or circulating immune complex formation was assessed by ELISA against anti-mouse Ig, LCMV lysate, or human C1q respectively.  At 45 days post infection, LCMV or non-infected recipients were immunized with anti-Fy3 or injected with PBS. RBCs expressing the HEL, OVA and Duffy chimeric antigen (HOD), were labeled with chloromethylbenzamido 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI ), while WT RBC were labeled with fluorescently distinct 3,3’-dihexadecyloxacarbocyanine perchlorate (DiO) to facilitate detection of antigen positive RBCs post-transfusion. HOD RBCs were then transfused into anti-Fy3 immunized or non-immunized LCMV infected or non-infected recipients, followed by analysis for RBC clearance at 10 min, 1h, 2h, 4h, 24h and 48h following transfusion.  In addition, RBCs were stained for potential bound antibody, complement and possible alterations in the HOD antigen followed by flow cytometric analysis. 

Results:  Recipients chronically infected with LCMV clone-13 displayed high levels of total and LCMV specific antibody in the serum. Furthermore, large immune complexes could be observed following the development of chronic infection, which persisted 45 days following initial LCMV inoculation. While HOD RBCs transfused into control non-infected, yet anti-Fy3-immunized C57BL/6J recipients displayed rapid clearance following incompatible transfusion, clearance of HOD RBCs failed to occur at similar levels in LCMV clone-13 infected recipients 45 days following LCMV exposure despite immunization with anti-Fy3. Regardless of the LCMV infection state, binding of anti-Fy3 to HOD RBCs was unaffected.  However, HOD antigen decreased following the initial transfusion into non-infected, anti-Fy3-immunized recipients, while significant alterations in the HOD antigen failed to occur in anti-Fy3 immunized recipients also infected with LCMV or any recipients lacking anti-Fy3.

Conclusion: Differences in recipient clearance capacity following incompatible RBC exposure in non-infected mice and LCMV clone-13 infected mice suggests that rapid phagocytic removal of RBCs can be blocked by the production of immune complexes secondary to chronic LCMV infection. While antibody binding to HOD cells was not decreased in chronically infected mice, antigen modulation was almost completely abrogated, suggesting that engagement of unimpeded clearance pathways may be important for antigen modulation. Taken together, these data demonstrate that chronic viral infection can lead to significantly altered outcomes of RBC clearance and antigen modulation following incompatible transfusion.