Transfusion in the absence of inflammation induces antigen-specific tolerance to murine RBCs

Abstract

Most human transfusion recipients fail to make detectable alloantibodies to foreign RBC antigens ("nonresponders"). Herein, we use a murine model to test the hypothesis that nonresponders may be immunologically tolerant. FVB mice transfused with RBCs expressing transgenic human glycophorin A (hGPA) antigen in the absence of inflammation produced undetectable levels of anti-hGPA immunoglobulins, unlike those transfused in the presence of polyinosinic:polycytidylic acid-induced inflammation. Mice in the nonresponder group failed to produce anti-hGPA after subsequent transfusions in the presence of polyinosinic:polycytidylic acid, whereas anti-hGPA levels increased in the responder group. This tolerance was antigen specific, because nonresponders to hGPA produced alloantibodies to RBCs that expressed a different transgenic antigen. This tolerance was not an idiosyncrasy of the hGPA antigen nor of the recipient strain, because B10.BR mice transfused with membrane-bound hen egg lysozyme antigen-transgenic RBCs also demonstrated induced nonresponsiveness. These data demonstrate that RBCs transfused in the absence of inflammation can induce tolerance.

Figure 1

hGPA or mHEL RBCs are nonimmunogenic (or weakly immunogenic) when transfused into murine recipients in their baseline states. (A) Anti-hGPA response by flow cross-matching in FVB recipients transfused once with 1 “unit” of hGPA RBCs (100 μL of packed RBCs) in the presence or absence of poly(I:C); compilation of 3 representative experiments, 30 recipients total. Adjusted mean fluorescence intensity (MFI) was 0 for the hGPA group and 262 for the poly(I:C) hGPA group (P < .0001). (B) Anti-hGPA response by flow cross-matching in FVB recipients (10 total) after each of 3 hGPA transfusions given 3 weeks apart in the presence or absence of poly(I:C). Mean adjusted MFI for hGPA group after 1, 2, and 3 transfusions: 0. Mean adjusted MFI for poly(I:C) hGPA group after 1, 2, and 3 transfusions: 477.2, 1141, and 1887, respectively. SD depicted by error bars. (C) Anti-HEL response by ELISA in B10.BR recipients transfused once with 1 “unit” of leukoreduced mHEL RBCs in the presence or absence of poly(I:C). Representative experiment with 13 mice total; mean optical density 0.03 for the mHEL group and 0.77 for the poly(I:C) mHEL group (P < .005). This anti-HEL response in poly(I:C) mHEL–immunized recipients was not boosted by additional poly(I:C) mHEL transfusions. OD 415 indicates optical density at 415 nm.

Figure 2

Antigen-specific “nonresponsiveness” persists after subsequent RBC transfusion in the presence of inflammation. (A) Six weeks after hGPA or wild-type FVB RBC transfusion, recipients were challenged with hGPA RBCs in the presence of poly(I:C), with anti-hGPA levels assessed by flow cross-matching after the final transfusion. Compilation of 3 experiments, 29 recipients total. Adjusted mean fluorescence intensity (MFI) was 119 after poly(I:C) hGPA transfusion in the group initially not transfused, 103 in the group initially transfused with hGPA RBCs, and 1 in the group initially transfused with FVB RBCs. P < .0001 between the group initially not transfused and the hGPA group; P = .6 between the hGPA and FVB transfused groups. No anti-hGPA was produced in any group 2 weeks after the initial transfusion. (B) FVB recipients were either not transfused or transfused with hGPA RBCs, then transfused again 6 weeks later with HOD or HOD × hGPA F1 RBCs in the presence of poly(I:C), with anti-HOD assessed by flow cross-matching after the final transfusion. Compilation of 4 experiments, 40 recipients total. Mean adjusted MFI was 56 after poly(I:C) HOD or HOD × hGPA F1 in the group initially not transfused compared with 44 in the group initially transfused with hGPA RBCs (P = .65). No anti-hGPA or anti-HOD was detected in either group after the initial hGPA transfusion, and no anti-hGPA was detected after the final transfusion. (C) B10.BR recipients were transfused with mHEL or control C57BL/6 RBCs and then boosted 6 weeks later with mHEL RBCs in the presence of poly(I:C), with anti-HEL assessed by ELISA 2 weeks after the first and last transfusions. Compilation of 2 experiments with 18 recipients. Mean optical density (OD) was 0.45 before and 0.33 after poly(I:C) mHEL transfusion in the group initially transfused with mHEL RBCs; OD was 0.1 before and 1.1 after poly(I:C) mHEL transfusion in the group initially transfused with C57BL/6 RBCs (P = .004 between groups after the last transfusion). (D) B10.BR recipients were transfused with mHEL or control C57BL/6 RBCs and then injected subcutaneously 6 weeks later with HEL protein emulsified in complete Freund's adjuvant (CFA), with anti-HEL assessed 2 weeks after this immunization by flow cross-matching. Compilation of 2 experiments, 21 recipients total. Adjusted MFI was 3 after HEL/CFA treatment in the group initially transfused with mHEL RBCs compared with 38 in the group initially transfused with C57BL/6 RBCs (P = .0006).