Nonhemolytic antigen loss from red blood cells requires cooperative binding of multiple antibodies recognizing different epitopes by James C. Zimring, Chantel M. Cadwell, Traci E. Chadwick, Steven L. Spitalnik, David A. Schirmer, Tao Wu, Charles A. Parkos, and Christopher D. Hillyer Blood Volume 110(6):2201-2208 September 15, 2007 ©2007 by American Society of Hematology

Non hemolytic antigen loss is not a general property of murine RBCs Non hemolytic antigen loss is not a general property of murine RBCs. (A) C57BL/6 mice were passively immunized with polyclonal anti-HEL antiserum or an anti-HEL IgG fraction from polyclonal antiserum. Non hemolytic antigen loss is not a general property of murine RBCs. (A) C57BL/6 mice were passively immunized with polyclonal anti-HEL antiserum or an anti-HEL IgG fraction from polyclonal antiserum. Control mice received no antibody. Mice were then transfused with a mixture of mHEL RBCs labeled with DiI and C57BL/6 RBCs labeled with DiO. Peripheral blood was obtained at the indicated time points and survival of the RBCs was determined by flow cytometry. (B) C57BL/6 mice were injected with the 10F7 anti-hGPA monoclonal antibody. Control mice received an IgG anti-HEL monoclonal antibody. Mice were then transfused with a mixture of hGPA RBCs labeled with DiI and C57BL/6 RBCs labeled with DiO and RBC survival was monitored. (C-H) Peripheral blood from the mice in panel A was stained with polyclonal anti-HEL antiserum followed by fluorescently labeled anti–mouse IgG and anti-HEL staining was measured by flow cytometry. Error bars in panels A and B represent standard deviation (SD). This experiment was reproduced 3 times with similar results. Representative flow plots and histograms are shown. In panels C, E, and G, the numbers in the upper left and lower right quadrants represent the percentage of circulating transfused B6 or mHEL RBCs, respectively. The numbers in the upper right quadrants represent the ratio of mHEL RBCs to B6 RBCs. James C. Zimring et al. Blood 2007;110:2201-2208 ©2007 by American Society of Hematology

Antigen loss occurs in multiple different murine strains. Antigen loss occurs in multiple different murine strains. C57BL/6, BALB/c or C3H mice were actively immunized with HEL/CFA. Mice were transfused with a mixture of mHEL RBCs labeled with DiI and C57BL/6 RBCs labeled with DiO. Four days after transfusion, peripheral blood was obtained and stained with polyclonal anti-HEL antiserum followed by fluorescently labeled anti–mouse IgG; anti-HEL staining was measured by flow cytometry. (A-C) Numbers in the upper left and lower right quadrants represent the percentage of circulating transfused B6 or mHEL RBCs, respectively. (D-F) B6 and mHEL histograms are shown individually and then overlaid. This experiment was reproduced 3 times with similar results. Representative flow plots and histograms are shown. James C. Zimring et al. Blood 2007;110:2201-2208 ©2007 by American Society of Hematology

Individual monoclonal anti-HEL antibodies do not induce antigen loss. Individual monoclonal anti-HEL antibodies do not induce antigen loss. C57BL/6 mice were passively immunized with the indicated anti-HEL monoclonal antibodies. Control mice received no antibody. Mice were transfused with a mixture of mHEL RBCs labeled with DiI and C56BL/6 RBC labeled with DiO. Three days after transfusion, peripheral blood was obtained, stained with polyclonal anti-HEL antiserum followed by fluorescently labeled anti-mouse IgG, and anti-HEL staining was measured by flow cytometry. This experiment was reproduced 3 times with similar results. Representative flow plots and histograms are shown. James C. Zimring et al. Blood 2007;110:2201-2208 ©2007 by American Society of Hematology

Antigen-loss requires the simultaneous binding of antibodies with different epitope specificities. Antigen-loss requires the simultaneous binding of antibodies with different epitope specificities. (A) C57BL/6 mice were injected with the indicated combinations of monoclonal antibodies. Mice were transfused with a mixture of mHEL RBCs labeled with DiI and C57BL/6 RBCs labeled with DiO. Six days after transfusion, peripheral blood was obtained and stained with polyclonal anti-HEL antiserum followed by fluorescently labeled anti–mouse IgG; anti-HEL staining was measured by flow cytometry. The combinations are arranged to maximize functional groupings of antibodies, and a complete table is presented that shows each condition twice but allows easier pattern analysis. Antigen loss is designated as “LOSS,” whereas no antigen loss is indicated by “ø.” The outcome of combining an antibody with itself, which is the same as injecting the antibody alone, was taken from the data with isolated antibodies in Figure 3. This experiment was reproduced in its entirety 2 times with identical results. Additional experiments tested smaller groups of antibody combinations with identical findings. (B) Each monoclonal antibody was directly conjugated to Alexa Fluor 647. mHEL RBCs were preincubated with the indicated unconjugated monoclonal antibodies followed by staining with the indicated conjugated antibodies. Blocking by unconjugated antibodies was measured by flow cytometry. Blocking in both directions is defined as epitope identity (I), failure to block as nonidentity (ø), and blocking in only one direction as partial identity (P). This experiment was reproduced 2 times with identical results. James C. Zimring et al. Blood 2007;110:2201-2208 ©2007 by American Society of Hematology

Model for molecular differences when 2 antibodies simultaneously bind to distinct epitopes on a single surface antigen. Model for molecular differences when 2 antibodies simultaneously bind to distinct epitopes on a single surface antigen. (A,B) A single IgG monoclonal antibody recognizing an epitope that occurs once on a surface molecule forms antigen dimers, but not multimolecular complexes. (C) The combination of 2 antibodies recognizing 2 distinct epitopes doubles the number of antibodies bound but also leads to multimolecular crosslinking. (D) The combination of intact IgG and F(ab′)2 fragments, each of which recognizes different epitopes, also induces multimolecular crosslinking but does not have an increased number of exposed Fc domains compared with a single IgG monoclonal antibody. James C. Zimring et al. Blood 2007;110:2201-2208 ©2007 by American Society of Hematology

The requirement for multiple antibodies binding different epitopes is not due to increased numbers of Fc domains on the RBC surface. The requirement for multiple antibodies binding different epitopes is not due to increased numbers of Fc domains on the RBC surface. A mixture of mHEL RBCs labeled with DiI and C57BL/6 RBCs labeled with DiO was incubated in vitro with the indicated monoclonal antibody and/or F(ab′)2 fragment combinations or PBS. After 30 minutes, the mixtures were transfused into C57BL/6 mice. Peripheral blood was obtained 3 days later, stained with polyclonal anti-HEL antisera followed by fluorescently labeled anti–mouse κ light chain, and anti-HEL staining was measured by flow cytometry. This experiment was reproduced 3 times with identical results. Representative histograms are shown. James C. Zimring et al. Blood 2007;110:2201-2208 ©2007 by American Society of Hematology