OA1-AM24-TU-14 - CD47 Expression on Transfused RBCs Impacts Antibody-Mediated Clearance and Antigen Loss in a Mouse Model

CD47KO mice were crossbred with HOD mice (which express the transgenic triple fusion protein hen egg lysozyme (HEL), ovalbumin, and human Duffy b antigen), to generate HOD CD47 wild type (WT), HOD CD47 heterozygous (Het), and HOD CD47 knockout (KO) mice.  B6 or FcγR KO recipients passively immunized with anti-HEL IgG monoclonal antibodies (clones 2F4 and 4B7) or saline were transfused with DiI-labeled B6 RBCs and either DiO-labeled HOD CD47WT, HOD CD47Het, or HOD CD47KO RBCs.  Transfused HOD RBCs were analyzed using a flow cytometer to determine clearance from the circulation and to measure surface IgG, complement component C3, and HOD antigen levels.  Ten recipients were included in each group and were analyzed using a one-way ANOVA with Tukey's multiple comparisons test.  IACUC approval was obtained prior to experimentation. 

Results/Findings:

HOD CD47WT RBCs have approximately twice the CD47 of HOD CD47Het RBCs, while HOD CD47KO RBCs are completely deficient in CD47.  HOD antigen expression is similar despite differences in CD47 level.  In the absence of passive antibody administration, HOD CD47WT and HOD CD47Het RBCs had similar clearance rates, while the HOD CD47KO RBCs rapidly cleared from the circulation in B6 recipients. While HOD CD47WT RBCs had similar clearance in the absence or presence of a combination of 2F4 and 4B7 anti-HEL antibodies, there was accelerated clearance of HOD CD47Het and HOD CD47KO RBCs in the presence of 2F4 and 4B7 anti-HEL antibodies (P < 0.01).  The accelerated clearance was FcyR-dependent.  HEL antigen levels decreased significantly in all recipients in the presence of combined 2F4 and 4B7 anti-HEL antibodies. However, similar HEL antigen levels were observed on HOD CD47WT and HOD CD47HET RBCs in the presence or absence of 2F4 alone or 4B7 alone, while HOD CD47KO RBCs had significantly less HEL antigen in the presence of either 2F4 or 4B7 antibody alone (P < 0.01).

Conclusions: CD47 on RBCs can impact RBC antigen modulation and clearance in an incompatible transfusion mouse model. This suggests that the clinical impact of CD47 loss from RBCs during storage is a critical factor that should be considered for effective transfusion practice.