Alloimmunization to transfused HOD red blood cells is not increased in mice with sickle cell disease

Abstract

Background: Increased rates of red blood cell (RBC) alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease.

Study design and methods: Transgenic sickle mice, which express human α and β(S) globin, were transfused with fresh or 14-day-stored RBCs containing the HOD (hen egg lysozyme, ovalbumin, and human Duffy(b) ) antigen; some recipients were inflamed with poly(I : C) before transfusion. Anti-HOD alloantibody responses were subsequently measured by enzyme-linked immunosorbent assay and flow crossmatch; a cohort of recipients had posttransfusion serum cytokines measured by bead array.

Results: Both Berkeley and Townes homozygous (SS) and heterozygous (AS) mice had similar rates and magnitude of anti-HOD RBC alloimmunization after fresh HOD RBC transfusion compared with control animals; under no tested condition did homozygous SS recipients make higher levels of alloantibodies than control animals. Unexpectedly, homozygous SS recipients had blunted cytokine responses and lower levels of anti-HOD alloantibodies after transfusion of 14-day stored RBCs, compared with control animals.

Conclusions: In sum, homozygous β(S) expression and the ensuing disease state are not alone sufficient to enhance RBC alloimmunization to transfused HOD RBCs in two distinct humanized murine models of sickle cell disease under the conditions examined. These data suggest that other factors may contribute to the high rates of RBC alloimmunization observed in humans with sickle cell disease.

Figure 1. Berkeley SS mice do not have increased alloimmunization to transfused HOD RBCs

Berkeley SS or 50% AS recipients, as well as C57BL/6 recipients, were transfused with 1 “unit” (100 µL) of packed HOD RBCs. Anti-HEL IgG was measured by ELISA 2 weeks following transfusion. A compilation of data from 6 independent experiments (n=76 recipients total) is shown; p>0.05 by ANOVA for responses in SS, 50% AS, and C57BL/6 recipients.

Figure 2. Berkeley SS mice clear transfused HOD RBCs more rapidly than control recipients

Berkeley SS or C57BL/6 recipients were transfused with 1 unit of HOD RBCs, and recovery of the transfused RBCs was monitored by staining of recipient RBCs with MIMA 29 (anti-Fy3) at 10 minutes, 90 minutes, and 24 hours after transfusion (representative curves are shown).

Figure 3. The response to transfused HOD RBCs in the presence of poly (I:C) is not greater in Berkeley SS than control recipients

Berkeley SS or C57BL/6 recipients were pretreated with 25 micrograms of poly (I:C) i.p. and transfused 4 hours later with 1 unit of HOD RBCs; alloimmunization was measured by anti-HEL ELISA 2 weeks later. A representative experiment (n=16 recipients total) with mean +1SD is depicted; p>0.05 for groups of poly (I:C) transfused recipients by Mann-Whitney U test.

Figure 4. Cytokine and alloimmunization responses to 14-day stored HOD RBCs are not greater in Berkeley SS than control recipients

Berkeley SS, 50% AS, or C57BL/6 recipients were transfused with fresh or 14-day stored leukoreduced HOD RBCs. Serum KC, IL-6, and MCP-1 were measured 90 minutes after transfusion (A,B,C) and alloimmunization was measured 2 weeks later by ELISA (D). 2 representative experiments (with mean +1SD) are shown); this experiment has been repeated 2 times (n=38 recipients total) for cytokine analysis and 3 times (n=55 recipients total) for alloimmunization analysis, with similar results.

Figure 5. Robust responses to Balb/c splenocytes are observed in Berkeley Homozygous SS, 50% AS, and C57BL/6 mice

1 million Balb/c splenocytes were adoptively transferred to Berkeley SS (black bars), 50% AS (white bars), or C57BL/6 (grey bars) recipients, and anti-Balb antibodies were measured 1 and 2 weeks post-immunization by flow cytometric crossmatch using Balb/c or control C57BL/6 splenocyte targets. A representative experiment (n=15 recipients total) with mean +1SD is shown; p>0.05 by ANOVA with Bonferroni post-test at 1 and 2 week points; this experiment has been repeated twice with similar results.

Figure 6. The rate and magnitude of response to transfused HOD RBCs are similar in Townes “knock-in” homozygous SS, heterozygous AS, and control AA recipients

Townes SS, AS, or AA recipients were transfused with 1 unit of packed HOD RBCs. Anti-HEL IgG was measured by ELISA 2 weeks following transfusion (A). A compilation of data from 3 independent experiments (n=37 recipients total) is shown; p>0.05 by ANOVA with Bonferroni post-test for responses in SS, AS, and AA recipients. Representative flow cytometric histograms (B) with sera from representative transfused homozygous SS recipient (left panel), transfused heterozygous AS recipient (middle panel), and untransfused control (right panel), crossmatched with target HOD RBCs (black line) and control FVB RBCs (grey line).