Red Blood Cell Alloimmunization Is Affected by Depletion of Donor White Cell Subsets.

Red blood cell (RBC) alloimmunization leads to therapeutic limitations due to difficulties in obtaining compatible RBC components. Better understanding of immune mechanisms responsible for alloantibody formation are necessary in order to develop ways of preventing or treating alloimmunization in high risk patients. A murine model of RBC alloimmunization was developed using B6CBA-F1-Tg Fy^b mice that express the human Fy^b blood group as donors, and recipient mice of the B6CBA-F1 strain. RBC were obtained from donor mice that were anesthetized and exsanguinated by cardiac puncture. Donor whole blood was collected into sterile syringes containing CPDA-1 (Sigma Aldrich, St. Louis, MO) and was then centrifuged to produce packed RBCs. Recipient mice were transfused 0.3 mL via the tail vein once a week for two weeks with donor RBC that had 10⁸ spleen cells added which were then depleted of either CD4, CD8, CD19 or natural killer (NK) cell subsets (n=6 in each group). Depletions were performed using immunodensity negative selection with murine anti-CD4, CD8, CD19 and DX5 antibodies (StemCell Technologies, Vancouver, BC). Two control groups were transfused with either washed buffy coat depleted RBC (leukodepletion to fewer than 5 × 10⁶ remaining white blood cells (WBC)) or RBC with spleen cells added that was processed through immunodensity negative selection without the addition of antibody (no leukodepletion with 1 × 10⁸ WBC). Serum was collected from recipient mice once a week for four weeks post-transfusion. Circulating IgG anti-Fy^b titers were measured by flow cytometry using donor RBC and staining with fluorescein isothiocyanate conjugated rat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA); target cells were gated on by size and side scatter parameters using a FACScan flow cytometer (Becton Dickenson, Franklin Lakes, NJ). Serum samples from week 4 post transfusion demonstrated a mean anti-Fy^b titer of 64 in the CD4 depleted group. Mean titers of 256, 512 and 512 were observed in the CD8, CD19 and NK depletion groups, respectively. The leukodepletion control group had a mean anti-Fy^b titer of 16, and the non-leukodepletion control group showed a mean titer of 64. These results indicate that donor contaminating WBC affect RBC alloimmunization. CD4 cell removal led to an anti-Fy^b titer that was the same as the non-leukodepletion control group. CD8, CD19 and NK cell removal increases anti-Fy^b titer and may indicate a variety of inhibitory roles of these cell types.