Dynamics of antibody engagement of red blood cells in vivo and in vitro

Abstract

Exposure to allogenic red blood cells (RBCs), either through pregnancy or transfusion, can result in alloimmunization, which can lead to severe hemolytic transfusion reactions and pregnancy complications. Passively administered antibodies can be used to prevent alloimmunization, where steric hindrance of allogeneic epitopes has been postulated as one mechanism whereby antibody engagement may prevent RBC alloimmunization. However, the dynamics of antibody engagement on the RBC surface has remained difficult to study. To examine this, we leveraged the HOD (HEL, OVA and Duffy) model system and Fcγ receptor knockout recipients to define the dynamics of antibody engagement of the Duffy antigen in the absence of RBC clearance or antigen modulation. Using this approach, the on-rate of antibody engagement of HOD RBCs was very similar in vivo and in vitro, with high levels of antibody binding observed within minutes of HOD RBC exposure. In contrast, the off-rate of HOD RBC bound antibody was relatively slow, with appreciable dissociation not being observed for an hour. However, the dynamics of antibody interactions with HOD changed significantly when antibody decorated HOD RBCs were exposed to free antibody. Despite the presence of prebound antibody, free antibody rapidly associated with HOD RBCs, with the rate of free antibody association observed being faster in vivo than in vitro. Importantly, antibody association and dissociation occurred in the absence of any appreciable changes in RBC clearance, antigen modulation or complement deposition, suggesting that differences in antibody levels observed reflected actual differences in the dynamics of antibody binding. These results suggest that while antibodies appear to be relatively static on the cell surface once bound, antibody engagement can be quite dynamic, especially in the face of free antibody in solution. These results not only have implications in the mechanisms of antibody-mediated immunosuppression, but also the potential use of other antibody-based approaches designed to prevent hemolytic transfusion reactions or target antigens in vivo in general.

Keywords: AMIS; alloimmunization; antibody; antigen; red blood cell.

Figure 1

Alexa Fluor 660 or Alexa Fluor 647 labeled anti-Duffy antibodies exhibit distinct fluorescent patterns and similar binding characteristics toward HOD RBCs. (A) Schematic of HOD RBCs. (B) Schematic of binding evaluations of Alexa Fluor 660 (AF660-M29) or Alexa Fluor 647 (AF647-M29) anti-Duffy antibodies toward B6 or HOD RBCs. (C) Gating strategy used to identify RBC populations and binding profile following incubation with AF660-M29 or AF647-M29. (D) Binding profile of AF660-M29 shown as histograms toward B6 RBCs or HOD RBCs when using distinct spectral outputs to measure the relative fluorescence as indicated. (E) Binding profile of AF647-M29 shown as histograms toward B6 RBCs or HOD RBCs when using distinct spectral outputs to measure the relative fluorescence as indicated. (F) Examination of AF660-M29 or AF647-M29 binding to HOD RBCs over a range of concentrations as indicated plotted as maximal mean fluorescence intensity (MFI). Data are representative of 3 independent experiments.

Figure 2

Preincubation of HOD RBCs with anti-Duffy antibody reduces additional binding by free antibody in vitro. (A) Schematic of the experimental approach used to examine the impact of prebound Alexa Fluor 660 (AF660)-M29 antibody on free Alexa Fluor 647 (AF647-M29) anti-Duffy antibody association to HOD RBCs over the course of 30 minutes. (B) Flow cytometric analysis of AF660-M29 or AF647-M29 toward HOD RBCs at distinct ratios of AF647-M29 to AF660-M29. (C) Quantification of binding by each antibody as a function of maximal mean fluorescence intensity (MFI) over a range of AF660-M29 concentrations as indicated followed by incubating with a saturating concentration of AF647-M29. Data are representative of 3 independent experiments.

Figure 3

Free anti-Duffy antibodies rapidly associate with HOD RBCs even in the presence of prebound antibodies. (A) Flow cytometric gating strategy of differentially labeled HOD or B6 RBCs after in vivo or in vitro incubation. (B). Examination of precoated Alexa Fluor 660 (AF660-M29) binding on HOD RBCs over the time points indicated in vitro and in vivo. (C) Examination of Alexa Fluor 647 anti-Duffy antibody (AF647-M29) following exposure of HOD RBCs to free anti-Duffy antibody over the time points indicated in vitro and in vivo. (D) Examination of AF660-M29 or AF647-M29 anti-Duffy antibodies binding to HOD RBCs following exposure of HOD RBCs precoated with AF660-M29 and with free AF647-M29 over the time periods indicated in vitro or in vivo. Data are representative of 3 independent experiments *P < .05; **P < .01; ***P < .001; ****P < .0001; ns, not significant.

Figure 4

Anti-Duffy antibody engagement fails to induce detectable changes in HOD RBC survival or complement deposition on the HOD RBC surface. (A) Schematic of the experimental approach. (B) Flow cytometric gating strategy of HOD RBCs following transfusion of differentially labeled HOD or B6 RBCs for analysis of HOD RBC survival, total antibody binding, complement deposition or detection of antigen levels. (C) Survival of HOD RBCs in the presence of Alexa Fluor 660 anti-Duffy antibodies (AF660-M29), Alexa Fluor 647 anti-Duffy antibodies (AF647-M29), both or neither (PBS) as indicated. (D) Examination of bound antibody levels as histograms and mean fluorescent intensity (MFI) values at the time points indicated post-transfusion in the presence of AF660-M29, AF647-M29, both or neither as indicated. (E) Examination of C3 levels as histograms and MFI values at the time points indicated post-transfusion in the presence of AF660-M29, AF647-M29, both or neither as indicated. (F) Examination of C3b levels as histograms and MFI values at the time points indicated post-transfusion in the presence of AF660-M29, AF647-M29, both or neither as indicated. Data are representative of 3 independent experiments. ****P < .0001; ns, not significant.

Figure 5

Anti-Duffy antibody engagement fails to induce detectable changes in the levels of HEL, OVA, and Ter119. (A) Histograms of HEL antigen levels as measured by flow cytometry on HOD RBCs in the presence of Alexa Fluor 660 anti-Duffy antibodies (AF660-M29), Alexa Fluor 647 anti-Duffy antibodies (AF647-M29), both or neither (PBS) as indicated. (B) Quantification of HEL antigen levels as measured by flow cytometry on HOD RBCs in the presence of AF660-M29, AF647-M29, both or neither as indicated. (C) Quantification of OVA antigen levels as measured by flow cytometry on HOD RBCs in the presence of AF660-M29, AF647-M29, both or neither as indicated. (D) Quantification of Ter119 staining as measured by flow cytometry on HOD RBCs in the presence of AF660-M29, AF647-M29, both or neither as indicated. Data are representative of 3 independent experiments. ns, not significant.