Abstract
Background: RBC transfusion can induce alloimmune responses that lead to hemolysis. However, only a subset of transfused patients become alloimmunized, and there is heightened interest in identifying factors that promote or inhibit alloimmunization. The frequency of alloimmunization has recently been shown to be increased in patients with inflammatory disease. In mouse models, inflammation induced by the viral RNA mimetic, polyinosinic:polycytidylic acid (poly(I:C)), profoundly enhances alloimmune antibody responses. Poly(I:C) induces production of inflammatory cytokines, including Type 1 interferon (IFNαβ) and monocyte chemoattractant protein 1 (MCP-1), which recruits CCR2 expressing monocyte-derived dendritic cells (MoDCs) from bone marrow. We hypothesize that IFNαβ promotes inflammation-induced RBC alloimmunization by regulating MoDC-mediated consumption of transfused allogeneic RBCs.
Methods: To investigate the role of inflammation in alloimmunization to a human RBC antigen, we utilized a novel mouse model that expresses high levels of the human KEL glycoprotein specifically on RBCs (KELhi). Poly(I:C) was administered to wildtype (WT), CCR2-/-, and IFNAR1-/- mice, which lack the IFNαβ receptor, at varying time points relative to transfusion of leukoreduced KELhi RBCs. The alloimmune response was assessed by measuring KEL-specific IgG via flow cytometric crossmatch. For dendritic cell activation and RBC consumption experiments, RBCs labeled with fluorescent DiO were transfused following poly(I:C) injection. DiO-containing phagocytes, including Mo-DCs (CD11c+ CD11bhi Ly6C+ F4/80+ MHCIIlo), and activation markers were detected by flow cytometry 6hrs after transfusion.
Results: WT Mice treated with poly(I:C) 3 hrs prior to RBC transfusion generated anti-KEL glycoprotein alloantibodies. However, alloantibodies were not detectable when poly(I:C) was withheld or administered at days -7, -1, +1, +4, or +7 in relation to RBC transfusion, indicating that inflammation may promote antigen consumption. Poly(I:C) treatment 3 hrs prior to transfusion of DiO-labeled KELhi RBCs led to elevated activation marker expression and marked erythrophagocytosis by MoDCs in the spleen and peripheral blood. MoDC recruitment and RBC consumption were respectively mediated by CCR2 and IFNAR1-dependent mechanisms. Additionally, compared to WT controls, alloimmunization in the presence of poly(I:C) was significantly reduced in CCR2-/- mice and completely abrogated in IFNAR1-/- mice (Figure 1).
Conclusions: Although inflammatory stimuli, including poly(I:C), have been shown to enhance alloimmunization, mechanisms underlying these results have been poorly understood. Here, we demonstrate an important role for MoDC erythrophagocytosis and a critical role for IFNαβ signalingin inflammation associated alloimmunization to a T-dependent human antigen expressed on murine RBCs. Although it is unclear whether these findings will apply to other RBC antigens in mice and humans, MCP-1 and IFNαβ are produced in inflammatory diseases associated with alloimmunization. Thus, identifying risk factors for inflammatory cytokine production in transfusion recipients may allow for personalized transfusion protocols for at risk patients.
Figure 1 Monocyte-derived dendritic cells and IFNαβ signaling promote alloimmunization to KELhi RBCs. a) WT mice were treated with or without 100µg poly(I:C) 3 hours prior to transfusion with 75µL leukoreduced DiO+ KELhi RBCs. Plots show CD11bhi MoDCs in peripheral blood (Ter119-TCRb-CD19-CD11c+Ly6C+F480+MHClo). Numbers on plots indicated percent of gated cells. b) Cumulative data of a. c) Anti-KEL specific IgG in serum of WT mice following transfusion of KELhi RBCs in the presence or absence of poly(I:C). d) Anti-KEL IgG in serum of indicated mice pretreated with poly(I:C) and transfused with KELhi RBCs. Anti-KEL IgG antibodies in serum were measured by flow cytometric cross-match, completed by incubating serum from transfused mice with KELhi RBCs, and subsequently staining for RBC bound IgG. The adjusted MFI was calculated by subtracting the reactivity of serum with syngeneic WT RBCs from the reactivity of serum with KELhi RBCs. Data show the peak antibody response. Open circles indicate data from individual mice. Figure 1. Monocyte-derived dendritic cells and IFNαβ signaling promote alloimmunization to KELhi RBCs. a) WT mice were treated with or without 100µg poly(I:C) 3 hours prior to transfusion with 75µL leukoreduced DiO+ KELhi RBCs. Plots show CD11bhi MoDCs in peripheral blood (Ter119-TCRb-CD19-CD11c+Ly6C+F480+MHClo). Numbers on plots indicated percent of gated cells. b) Cumulative data of a. c) Anti-KEL specific IgG in serum of WT mice following transfusion of KELhi RBCs in the presence or absence of poly(I:C). d) Anti-KEL IgG in serum of indicated mice pretreated with poly(I:C) and transfused with KELhi RBCs. Anti-KEL IgG antibodies in serum were measured by flow cytometric cross-match, completed by incubating serum from transfused mice with KELhi RBCs, and subsequently staining for RBC bound IgG. The adjusted MFI was calculated by subtracting the reactivity of serum with syngeneic WT RBCs from the reactivity of serum with KELhi RBCs. Data show the peak antibody response. Open circles indicate data from individual mice.
Disclosures
No relevant conflicts of interest to declare.
Generation of iPSC-Derived Human Megakaryocytes for Flow Cytometric Detection of Platelet-Specific Alloantibodies
