Background: Although much is known about the structure and immunogenicity of red blood cell (RBC) antigens, little is known about their processing and presentation by antigen presenting cells (APC). Red blood cells are a unique immunogen, in that they are given intravenously, without inflammation, and typically don’t enter peripheral tissues and lymphatics. Unlike pathogens, which cause an immune response in the majority of patients, only a small minority of chronically transfused patients develop alloantibodies to RBC antigens. In a murine model of RBC transfusion, we have previously reported that recipient inflammation, induced by Poly (I:C) (a double-stranded RNA that mimics viral inflammation), significantly enhances alloimmunization to RBC antigens. In this report, we explore the role of antigen presenting cells in the immune response to antigens on transfused RBCs, in an uninflammed state as well as in the presence of Poly (I:C).
Methods: 3, 3-dihexadecyloxacarbocyanine perchlorate (DiO) was used as a fluorescent RBC label. Labeled RBCs were transfused into C57BL/6 recipient mice, in the absence or presence of inflammation with poly (I:C). 24 hours post-transfusion, APCs were analyzed in the spleen, liver, and lymph nodes. Macrophages (F4/80+) and dendritic cells (DC) (CD11c+) were gated on by flow cytometry, as were T cells (CD3+), B cells (CD19+) and RBCs (Terr 119+). RBC consumption was assessed by measuring DiO fluorescence in these cell populations.
Results: In the absence of inflammation, the majority of RBCs are consumed by macrophages in the spleen, with 3 fold less consumption by liver macrophages and no consumption by lymph node macrophages. Both splenic and liver DCs consume 3 fold fewer RBCs than splenic macrophages. Recipient inflammation with Poly (I:C) alters this pattern, with a significant increase in consumption by both splenic and liver DCs and a decrease in consumption by splenic macrophages. As a negative control, no RBC consumption was seen after gating on non-phagocytic T cells or B cells. Likewise, measures of RBC consumption were not an artifact of RBC sticking to the APC surface, as staining for TER119 was negative.
Discussion: Red blood cells are a unique immunogen, in that they circulate for many days, don’t enter lymphatics, and often don’t cause a detectable alloantibody response. These studies demonstrate that recipient inflammation with Poly (I:C), which we have previously reported enhances alloimmunization to transfused RBCs, significantly increases DC consumption of transfused RBCs. As DCs are typically considered to be more potent APCs than macrophages, and as we have previously shown that Poly (I:C) signficantly induces co-stimulatory molecule expression on DCs, these findings provide one potential mechanism by which inflammation enhances RBC alloimmunization. Ongoing studies are directly assessing the relative potency of these different APCs in their ability to activate CD4+ T cells specific for RBC antigens.
Splenic Dendritic Cells Survey Red Blood Cells for Missing Self-CD47 to Trigger Adaptive Immune Responses