While red blood cells (RBCs) transfusion can provide life saving therapy, patients who require chronic transfusion therapy may develop RBC alloantibodies that limit the availability of compatible RBCs for future transfusion and increase the risk of hemolytic transfusion reactions. However, not all patients generate alloantibodies following RBC exposure. Among factors that potentially influence RBC alloantibody formation, several studies suggest that a recipient’s MHC class II repertoire may predict an individual’s likelihood of responding to a particular RBC alloantigen. However, whether MHC class II antigens are required for efficient alloantibody formation following RBC transfusion remains unknown. As a result, we examined the potential role of MHC class II in the development of RBC alloantibodies following transfusion in a murine model of KEL alloimmunization.
RBCs transgenically expressing the human KEL antigen specifically under a β-globin promoter (KEL RBCs) were transfused into C57BL/6, C57BL/6 MHC class II knock out (KO) or KEL transgenic control recipients. Following transfusion, blood was harvested on days 3, 5, 7, 14, 21 and 28 following transfusion and serum was analyzed for IgM or IgG anti-KEL antibodies by indirect immunofluorescence using flow cytometry with KEL and control C57BL/6 RBCs as targets. To deplete CD4 T cells, mice were injected with anti-CD4 (clone GK1.5) 4 and 2 days prior to transfusion. As a control, additional C57BL/6 recipients were similarly injected with an isotype control. C57BL/6 recipients were also injected in parallel with GK1.5 or isotype control followed by splenocyte examination for CD4 T cell depletion using anti-CD3 and an anti-CD4 clone that recognizes a different CD4 epitope than GK1.5 (clone RM4-5). All experiments were completed at least three times with 3–5 recipients per group per experiment.
Transfusion of KEL RBCs resulted in significant IgM anti-KEL antibody formation that peaked approximately 5 days following transfusion in both C57BL/6 and C57BL/6 MHC class II KO recipients. Similarly, IgG anti-KEL antibodies could also be detected in C57BL/6 or C57BL/6 MHC class II KO as early as 7 days following transfusion and continued to rise to similar peak levels within 14 to 21 days following KEL RBC transfusion. Injection of GK1.5, but not isotype control antibody, depleted CD4 T cells to less than 1% of their original level. Transfusion of KEL RBCs into C57BL/6, CD4 depleted C57BL/6 or isotype control treated C57BL/6 resulted in similar levels of IgM anti-KEL antibody that peaked approximately 5 days following transfusion. Likewise, transfusion of KEL RBCs induced similar levels of IgG anti-KEL antibodies within 7 days following transfusion that also peaked between 14 and 21 days in C57BL/6, CD4 depleted C57BL/6 or isotype control treated C57BL/6 recipients. (All the above differences achieved a p value of <0.05)
Despite the potential role of CD4 T cells in facilitating RBC alloantibody formation, these results suggest that significant IgG RBC alloantibody can occur independent of MHC class II or CD 4 T cells. Although it remains possible that CD4 T cells become activated following RBC alloantigen exposure, the lack of CD4 T cell requirement in this model suggests that some patients may be capable of mounting a clinically significant immune response following RBC transfusion in the absence of CD4 T cell help. As a result, MHC antigen presentation of unique RBC alloantigens may not be necessary for RBC alloimmunization to occur.
Zimring:Immucor Inc.: Research Funding; Terumo: Research Funding; Haemonetics: Consultancy; Cerus: Honoraria.
Asterisk with author names denotes non-ASH members.