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Antigen modulation as a potential mechanism of anti-KEL immunoprophylaxis in mice

Jingchun Liu, Manjula Santhanakrishnan, Prabitha Natarajan, David R. Gibb, Stephanie C. Eisenbarth, Christopher A. Tormey, Alexa J. Siddon, Sean R. Stowell, Donald R. Branch and Jeanne E. Hendrickson

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Immunoprophylaxis with anti-KEL sera prevents alloimmunization in wild-type, FcγR KO, and C3 KO mice, but not in FcγR KO × C3 KO (double-KO) mice. (A) General experimental design. Recipients were passively immunized with anti-KEL sera in some experiments, followed by transfusion of murine RBCs expressing the human KEL glycoprotein. Alloimmune responses were measured in serum after transfusion. (B) Total anti-KEL IgG measured in the serum of recipients on day 28 posttransfusion. (C) Total anti-KEL IgG measured in the serum of recipients treated with anti-KEL immunoprophylaxis prior to RBC exposure; P < .05 by 1-way ANOVA between double-KO and all other recipients. These data are a compilation of 2 independent experiments with 2 or 3 mice per group per experiment.

  • Figure 2.

    Immunoprophylaxis with anti-KEL sera leads to less RBC clearance in FcγR KO × C3 KO (double-KO) recipients compared with wild-type, FcγR KO, or C3 KO recipients. Posttransfusion clearance curves of KEL RBCs in (A) wild-type, (B) FcγR KO, (C) C3 KO, and (D) FcγR KO × C3 KO (double-KO) recipients, in the absence (solid line) or presence (dashed line) of immunoprophylaxis, to 24 hours. These data are representative of 2 or 3 independent experiments with 3 to 5 mice per group per experiment; error bars indicate SD between individual mice. (E) Comparison of percentage of KEL RBCs remaining between 10 minutes and 24 hours after transfusion (left) or 10 minutes and 5 days after transfusion (right) in the presence of anti-KEL immunoprophylaxis; *P < .05 by 2-way ANOVA between double-KO and all other recipients.

  • Figure 3.

    In vitro phagocytosis of antibody-coated KEL RBCs is observed in wild-type, FcγR KO, and C3 KO, but not FcγR KO × C3 KO (double-KO), splenocytes. Opsonized murine KEL RBCs were incubated in vitro for 15 minutes with splenocytes from (A) wild-type (C57BL/6) mice, (B) FcγR KO mice, (C) C3 KO mice, or (D) double-KO mice lacking FcγR and C3. Scanning was initially completed at low power, and then 30 high-power fields were evaluated in duplicate wells. The microscope used was an Olympus BX40, with an Olympus PLAN 100× objective and a numerical aperture of 1.25 under oil emersion. Images were captured using a SPOT Insight CCD camera (model 14.2) using SPOT basic software (version 4.7).

  • Figure 4.

    Modulation of the KEL antigen on transfused RBCs in the presence of immunoprophylaxis requires FcγRs or C3. KEL RBCs labeled with the lipophilic dye DiO were recovered from recipients transfused in the absence or presence of anti-KEL immunoprophylaxis 10 minutes, 1 hour, and 24 hours after transfusion (A) and incubated with anti-KEL sera followed by fluorescently conjugated anti–mouse IgG (schematic shown in B). (C) Detection of the KEL antigen on recovered lipophilically labeled KEL RBCs in wild-type, FcγR KO, C3R KO, and FcγR × C3 KO (double-KO) recipients, transfused in the presence or absence of anti-KEL immunoprophylaxis (P < .05 at 1 hour and 24 hours after transfusion by 2-way ANOVA between double-KO and other recipients treated with immunoprophylaxis). These data are representative of 2 or 3 independent experiments with 3 mice per group per experiment; error bars indicate SD between mice.

  • Figure 5.

    Near-complete removal of the KEL glycoprotein antigen on RBCs occurs in wild-type, but not FcγR KO × C3 KO (double-KO), recipients treated with anti-KEL immunoprophylaxis. (A) KEL RBCs labeled with the lipophilic dye DiO were recovered from recipients transfused in the absence or presence of anti-KEL immunoprophylaxis 10 minutes, 1 hour, and 24 hours after transfusion and incubated directly with fluorescently conjugated anti–mouse IgG (P < .05 at 1 hour and 24 hours after transfusion by 2-way ANOVA between double-KO and other recipients treated with immunoprophylaxis). (B) Side-by-side comparison of the KEL antigen detection signal versus the signal of RBCs incubated directly with fluorescently conjugated antibody 24 hours after transfusion, in FcγR × C3 (double-KO) recipients treated with anti-KEL immunoprophylaxis. (C) Western blot data of membrane preps from RBCs recovered from wild-type (B6) or FcγR × C3 (double-KO) mice 24 hours after transfusion, in the absence or presence of treatment with anti-KEL immunoprophylaxis. The data in A and B are representative of 2 to 3 independent experiments with 3 mice per group per experiment; error bars indicate SD. The data in C are representative of 2 independent experiments with 2 mice per group per experiment.

  • Figure 6.

    Total C3 detected on transfused KEL RBCs in wild-type and FcγR KO mice. Posttransfusion measurements of total C3 on lipophilically labeled KEL RBCs in different strains of recipients, transfused in the absence or presence of anti-KEL immunoprophylaxis. These data, shown at 10 minutes, 1 hour, and 24 hours after transfusion, are representative of 2 independent experiments with 3 mice per group per experiment; error bars indicate SD.