Neutrophils Contribute to RBC Alloantibody Production
Authors: Richards Amanda L , Zimring James C. , Hudson Krystalyn E. 
Published: October 2019

Abstract
Background: Red blood cell (RBC) alloimmunization occurs in up to 10% of transfusion recipients (excluding RhD). Factors influencing alloimmunity are poorly defined; however, transfusion into an inflamed recipient significantly enhances alloimmunization rates in mouse models, and is associated with increased alloimmunization in some human studies. Our lab has recently reported that poly (I:C)-elicited inflammation leads to a significant increase in numbers of neutrophils in the spleen. Additionally, transfusion of syngeneic RBCs into an inflamed recipient leads to enhanced erythrophagocytosis by plasmacytoid dendritic cells (pDCs) and, unexpectedly, neutrophils. Although neutrophils typically eliminate and/or neutralize microorganisms, they can also secrete proinflammatory cytokines, present peptides to T cells through expression of MHC and costimulatory molecules, and elicit antibodies from B cells. Thus, given neutrophil participation in erythrophagocytosis, we hypothesized that neutrophils contribute to RBC-specific alloantibody production in an inflammatory setting.
Methods: Recipient C57Bl/6 (B6) mice were treated with 0.5mg anti-Ly6G to deplete neutrophils (clone 1A8) - control mice received PBS. For long-term experiments, anti-Ly6G antibody was given every 48 hours to sustain neutrophil reduction. Post initial neutrophil reduction, recipient mice were given 200ug poly (I:C) to elicit inflammation followed by transfusion of 100uL of packed allogeneic HOD RBCs [HOD RBCs express a transgenic alloantigen (HOD), which is the only difference between syngeneic B6 and HOD RBCs]. For RBC consumption experiments, HOD RBCs were labeled with DiO, and erythrophagocytosis was analyzed (18-24 hours post transfusion) assessing DiO consumption by flow cytometry in different leukocyte subsets (visualized by staining with lineage specific antibodies). The presence of anti-HOD alloantibodies was evaluated in serum (14 days post-transfusion) via indirect immunoflourescence with both B6 and HOD RBC targets.
Results: Anti-HOD alloantibodies (IgG2c and IgG3 subtypes) were significantly increased in serum of mice transfused with HOD RBCs in the context of poly (I:C)-elicited inflammation, compared to PBS controls. Neutrophil-depleted recipients that received a HOD RBC transfusion in the presence of poly (I:C) made significantly less anti-HOD alloantibodies (p<0.05), compared to their neutrophil-replete counterparts. In the absence of inflammation, red pulp macrophages (RPM) clear the bulk of RBCs. However, in an inflamed recipient, allogeneic HOD RBC consumption is significantly enhanced in pDCs and neutrophils (rates remain unchanged in RPM). Additionally, neutrophils upregulate MHC-II and costimulatory markers, compared to transfusion in the absence of inflammation. There was a significant increase in RPM numbers upon neutrophil depletion, but RBC consumption rates were unaltered; rather, neutrophil-depleted inflamed transfusion recipients had a significant decrease in overall RBC phagocytes (p<0.05), suggesting a lack of compensatory RBC clearance mechanism. However, neutrophil depletion also resulted in downregulation of MHC-II on RPMs and a slight, but statistically insignificant, increase in RBC consumption by pDCs.
Conclusions: These data demonstrate that in an inflamed recipient, neutrophils consume transfused RBCs and express MHC-II and costimulatory molecules. Moreover, depletion of neutrophils significantly decreases alloantibody responses. Together, these findings suggest that consumption of transfused RBCs by neutrophils may play a previously unappreciated role in RBC alloimmunization. Neutrophils may play a role through direct antigen presentation; however, because alterations in other phagocytic populations were also observed with neutrophil depletion, alternate mechanisms cannot be ruled out. Together, these data indicate that while RPMs are the major consumer of transfused RBCs, they may not be the major antigen presenting cell. These findings strongly implicate neutrophils as having a functional contribution to RBC alloimmunization, either through serving directly as antigen presenting cells and/or by affecting other more traditional APC populations.

Disclosures
Zimring: BloodworksNW: Patents & Royalties: Patent Application filed on technology in this abstract - no royalties; Immucor Inc.: Research Funding.