Cellular and Molecular regulators of RBC alloimmunization

The vast majority of RBC transfusion recipients do not generate antibodies against alloantigens expressed on the RBC surface. We therefore set out to understand why some RBC transfusions are more immunogenic than others. We hypothesized that underlying infections like cytomegalovirus (CMV) at the time of transfusion can lead to increased production of anti-RBC antibodies. Using mouse models, we found that CMV infections at the time of RBC transfusions led to enhanced production of anti-RBC antibodies. We then set out to understand the cytokine and cellular mechanism regulating enhanced antibody production. We found that type 1 IFN signaling was mostly not required for the generation of anti-RBC antibodies following CMV infection. In order to study other cytokines that might be regulating CMV-driven RBC alloimmunization, we first identified the cell types required for the generation of the anti-RBC antibody response. We found that CD4+ T cells were mostly required for generation of anti-RBC IgG. Furthermore, CMV infections led to increased expansion of anti-RBC CD4+ T cells and enhanced differentiation into helper cells. Given the importance of CD4+ T cells, we hypothesized that IL-21, a key T cell-derived cytokine might be regulating anti-RBC antibody production. Interestingly, we found that IL-21 signaling was only partially required for anti-RBC antibody generation following CMV infection, suggesting that other cytokines are regulating the antibody response. Given our mouse data, our collaborators in the Netherlands examined the association between CMV viremia and RBC alloimmunization in their patient dataset. They found that CMV positivity was associated with increased risk of RBC alloimmunization, which supported our mouse data. Our results show that CMV infections can lead to increased anti-RBC antibody responses.
We further studied anti-RBC antibody generation in a mouse model of storage-induced RBC alloimmunization. Though mouse models allow for the mechanistic exploration of class-switching, previous studies of RBC alloimmunization in mice have focused more on the total IgG response than the relative distribution, abundance, or mechanism of IgG subclass generation. Given this major gap, we compared the IgG subclass distribution generated in response to transfused RBCs relative to protein in alum vaccination and determined the role of STAT6 in their generation. WT mice were either immunized with Alum/HEL-OVA or transfused with HOD RBCs and levels of anti-HEL IgG subtypes were measured using end-point dilution ELISAs. To study the role of STAT6 in IgG class-switching, we first generated and validated novel STAT6 KO mice using CRISPR/cas9 gene editing. STAT6 KO mice were then transfused with HOD RBCs or immunized with Alum/HEL-OVA, and IgG subclasses were quantified by ELISA. When compared to antibody responses to Alum/HEL-OVA, transfusion of HOD RBCs induced lower levels of IgG1, IgG2b and IgG2c but similar levels of IgG3. Class switching to most IgG subtypes remained largely unaffected in STAT6 deficient mice in response to HOD RBC transfusion, with the one exception being IgG2b. In contrast, STAT6 deficient mice showed altered levels of all IgG subtypes following Alum vaccination. Our results show that anti-RBC class-switching occurs via alternate mechanisms when compared to the well-studied immunogen alum vaccination.