(P-CB-23) Transfused RBCs are Weak Inducers of IgG Class-Switching Due to Inefficient Induction of CD4+ T Cell Help

Background/Case Studies: Red blood cell (RBC) alloimmunization to non-ABO antigens is a significant clinical complication for chronically transfused patients. In response to transfused RBCs that express foreign antigens, a subset of patients will make IgG antibodies that target the foreign antigen, posing a significant clinical barrier to future transfusion. For those patients who instead produce a predominately IgM response to the non-ABO antigen (e.g. most patients who make anti-M antibodies), the consequences of alloimmunization are much less severe. Despite the clinical importance of transfusion induced class-switching from IgM to IgG, the cellular and molecular regulators of this key process are largely unexplored.

Study

Design/Methods: To better understand the IgG class-switching induced by transfused RBCs, we directly compared the class-switching induced by transfusion with that induced by the well-studied vaccination approach of protein in aluminum hydroxide (Alum) adjuvant. Wild type mice were 1) either transfused with RBCs expressing the HOD antigen (containing Hen Egg Lysozyme, Ovalbumin, Duffy proteins) or 2) were vaccinated with HEL-OVA antigen (Hen Egg Lysozyme and Ovalbumin proteins) emulsified with widely used adjuvant Alum. Both loss-of-function and gain-of-function CD4+ T cell help experiments were performed using CD4 depleting antibodies, CD40L blocking antibodies, CD40 stimulatory antibodies, or antigen-specific CD4+ T cell adoptive transfers.

Results/Findings: By directly comparing the antibody levels generated in response to HOD transfusion with those generated in response to standard Alum vaccination, we demonstrate that HOD transfusion is a weak inducer of IgG class-switching. Furthermore, loss-of-function experiments using CD40L blockade and CD4+ T cell depletion demonstrated that T cell help is required for class-switching after transfusion, with no effect on IgM production. Most importantly, providing supra-physiological level of T cell help by using either a CD40 stimulating antibody or adoptively transferring antigen-specific CD4+ T cells resulted in significant increases in class-switching in a dose-response manner after transfusion, with no effect on class-switching after vaccination.

Conclusions: These results support a model in which IgM is the default antibody induced by transfused RBCs and its production is independent of T cell help. In contrast, IgM production is enhanced by T cell help in the vaccine setting. Most importantly, transfused RBCs are poor inducers of class-switched IgG due in large part to suboptimal induction of CD4+ T cell help. This stands in stark contrast to standard protein in Alum vaccination, where T cell help is saturated at baseline and cannot be enhanced further.

Importance of research: Despite the significant clinical difference between IgM and IgG antibodies to non-ABO RBC antigens, our understanding of how class switching from IgM to IgG is induced by transfused RBCs is remarkably limited. Herein we demonstrate that transfused RBCs are poor inducers of class-switched IgG when compared to vaccination. We go on to demonstrate that this is due to the fact that transfused RBCs fail to induce optimal CD4+ T cell help.