The Role of the Immunological Synapse in Differential Effects of APC Subsets in Alloimmunization to Fresh, Non-stored RBCs

Abstract

Background: Each year, over 5 million red blood cell (RBC) transfusions are administered to patients in the USA. Despite the therapeutic benefits of RBC transfusions, there are associated risks. RBC-specific alloantibodies may form in response to antigenic differences between RBC donors and recipients; these alloantibodies can be a problem as they may mediate hemolysis or pose barriers to future transfusion support. While there is currently no reliable way to predict which RBC recipients will make an alloantibody response, risk factors such as inflammation have been shown to correlate with increased rates of RBC alloimmunization. The underlying mechanisms behind how inflammation mediates alloantibody production are incompletely defined.Methods: To assess erythrophagocytosis, mice were treated with PBS or inflammatory stimuli followed by a transfusion of allogeneic RBCs labeled with a lipophilic dye. At multiple time points, RBC consumption and expression of activation makers by leukocytes was evaluated. To determine which antigen presenting cell (APC) subset(s) were capable of promoting allogeneic T cell activation, sorted leukocyte populations (which had participated in erythrophagocytosis) were co-cultured in vitro with allogeneic CD4+ T cells; T cell proliferation and ability to form immunological synapses with APCs were determined.Results: Upon transfusion of fresh allogeneic RBCs, multiple APCs consumed transfused RBCs. However, only CD8+ and CD11b+ dendritic cells formed productive immunological synapses with allogeneic T cells and stimulated proliferation. Importantly, allogeneic T cell activation and RBC alloantibody production occurred in response to RBC transfusion alone, and transfusion in the context of inflammation enhanced RBC consumption, the number of immune synapses, allogeneic T cell proliferation, and the rate and magnitude of alloantibody production.Conclusions: These data demonstrate that regardless of the ability to participate in RBC consumption, only a subset of APCs are capable of forming an immune synapse with T cells thereby initiating an alloantibody response. Additionally, these data provide mechanistic insight into RBC alloantibody generation.

Structure and function of the immune system in the spleen

“…Both splenic DCs and macrophages phagocytose allogeneic murine RBCs (90, 91) and because splenic macrophages clear endogenous aged RBCs, it had been assumed that macrophages also regulate the adaptive immune response (92). However, more recent studies have concluded that DCs are the primary APC for initiating the allogeneic response to RBCs (47, 93).…”

Autoantigen presentation by splenic dendritic cells is required for RBC‐specific autoimmunity

“…Multiple studies have shown that macrophages (from the spleen and liver) consume the majority of RBCs in circulation 5–7 . However, despite significant erythrophagocytosis, RBC‐containing macrophages do not stimulate an immune response; indeed, even in response to allogeneic RBCs, macrophages fail to form productive synapses with T cells and do not elicit activation or proliferation 15 . As such, it is not surprising that macrophages do not play a significant role in priming OTII CD4 + T cells to HOD RBC‐derived autoantigens.…”

“…Splenocytes were harvested and processed 18 to 24 hours after transfusion, stained with antibodies to delineate APCs, and CD8 + and CD8 − DCs were sorted. Sorted DiO + DC subsets were cocultured at a 10:1 ratio with enriched OTII CD4 + T cells, as described previously 15 . OTII CD4 + T cells cultured in media alone served as a negative control, whereas stimulation with phorbol 12‐myristate 13‐acetate (PMA; 10 ng/mL) plus ionomycin (1 μg/mL) was a positive control for proliferation.…”

“…To address this complexity, we undertook a genetic approach to delete MHC‐II from particular APCs; while the MHC locus of mice contains two functional MHC‐II molecules, I‐A and I‐E, C57BL/6 (B6) mice (and all murine strains with the b haplotype) express only I‐A, as I‐E is nonfunctional 13,14 . The HOD antigen contains the ovalbumin 323‐339 epitope, which is presented in I‐A b of B6 mice and is recognized by OTII CD4 + T cells 11,15 . Taking advantage of this, we generated multiple murine lines whereby I‐A b (ie, the only functional MHC‐II molecule in B6 mice) expression was modulated on different APCs by breeding HOD mice (which are on a B6 background) with I‐A b floxed (referred to as I‐Ab fl/fl herein) and Cre‐expressing transgenic mice (HOD + I‐Ab fl/fl xCre + ).…”

“…13,14 The HOD antigen contains the ovalbumin 323-339 epitope, which is presented in I-A b of B6 mice and is recognized by OTII CD4 + T cells. 11,15 Taking advantage of this, we generated multiple murine lines whereby I-A b (ie, the only functional MHC-II molecule in B6 mice) expression was modulated on different APCs by breeding HOD mice (which are on a B6 background) with I-A b floxed (referred to as I-Ab fl/fl herein) and Creexpressing transgenic mice (HOD + I-Ab fl/fl xCre + ). With this approach, we targeted APCs previously shown to participate in RBC consumption and utilized Cretransgenic mice with promoter-driven Cre recombinase, including: (a) Lyz2 cre+ which is expressed by macrophages, monocytes and neutrophils, 16 (b) CD11c cre+ which is expressed by multiple dendritic cell (DC) subsets, 17 (c) CD19 cre+ expressed by B cells, 18 (d) Alb cre+ expressed in liver hepatocytes, 19 and (e) CMV cre+ for ubiquitous expression (negative control).…”