PL4-SN-08 - Toll-like Receptor 9 on Donor RBCs Enhances Transfusion Induced Alloimmunity in Mice

Recipient inflammation at the time of transfusion has been shown to increase RBC alloimmunization in both humans and mice.  Because inflammation is known to regulate immune responses through direct activation of cells of the adaptive immune system, mechanistic studies on inflammation and RBC alloimmunization have focused almost exclusively on cellular components of adaptive immunity (e.g. dendritic cells, T cells, and B cells).  TLR9 is a receptor that recognizes CpG motifs found in bacteria and activates the innate immune system.   Recently, it has been reported that TLR9 is also expressed on RBCs.  Moreover, RBC expressed TLR9 has been shown to bind to circulating CpG, which the RBCs help clear to mitigate inflammation.  Given the central role of inflammation in RBC alloimmunization, we hypothesized that TLR9 on RBCs regulates alloimmunization to transfused RBCs.  

Study

Design/Methods: RBCs expressing the well-studied HOD model alloantigen were transfused into HOD negative recipients and alloantibodies to the HOD antigen were measured by flow-cytometry based crossmatch. HOD donor mice were crossed with TLR9 -/- mice to generate HOD RBCs missing TLR9 (HOD.TLR-/-).  Recipient mice were either wild-type (B6), mice with a global deletion of TLR9 (TLR9-/-), or an RBC specific TLR9 deletion generated by crossing RBC specific CRE transgenic mice with mice that have a floxed conditional TLR9 knockout locus (TLR9^fl/flCre+).   Prior to transfusion, mice were treated with either CpG (ODN 1826) or PBS (vehicle control). Anti-HOD IgG was measured day 21 post-transfusion.

Results/Findings: CpG pre-treatment caused a greater than 4-fold increase in anti-HOD IgG compared to control mice at day 21 (p=0.0005).  In contrast, no significant enhancement by CpG was observed when HOD.TLR9-/- RBCs were transfused.  Global deletion of TLR9 in recipients eliminated the enhancement of alloimmunization to HOD RBCs by CpG.  No change in CpG mediated enhancement was observed when recipients had an RBC specific deletion of TLR9.

Conclusions: TLR9 expressed on RBCs plays a mechanistic role in enhancement of alloimmunization to transfused RBCs by CpG, which is a model for recipient inflammation in response to microbial infection.  Because no effect was observed when TLR9 was selectively eliminated from recipient RBCs, we conclude that the role of TLR9 is not a general RBC role but is particular to the transfused RBCs expressing alloantigen.  CpG is known to act on TLR9 expressed by B cells to enhance activation and differentiation if the B cell receptor (BCR) is simultaneously engaged.  As such, we posit that TLR9 on RBCs binds to and delivers CpG to B cells while alloantigen on the same RBC selectively activates the BCR of B cells that encode an immunoglobulin specific for the alloantigen.