Differences in Regulatory T Cell Function in Alloimmunized Versus Non-Alloimmunized Mice Following Multiple Red Cell Transfusions.

Red blood cell (RBC) alloimmunization remains a major complication for transfusion-dependent patients such as those with sickle cell disease. Alloimmunization rates in transfusion recipients lacking the antigen can be as high as 70% for the RhD antigen or much lower for minor antigens. Several factors including the host’s immune status have been shown to influence the recipient’s ability to react to RBC alloantigens. However, it is not clear whether inherent differences in immune response can be identified between those who have or have not developed RBC alloantibodies following multiple transfusions. Increasing evidence indicates that CD4⁺ regulatory T cells (Tregs) characterized by co-expression of CD25 and the forkhead/winged helix transcription factor (Foxp3) are key regulators of a wide spectrum of immune responses. We have recently found that manipulation of Treg numbers affects the rate and the frequency of RBC alloimmunization in mouse models. We hypothesized that Treg number or function may be reduced in multi-transfused recipients who develop alloantibodies compared to those who do not. To test this, we established a transfusion regimen in which B6 mice were first injected with RBCs from mice transgenic for human glycophorin A (huGPA) antigen plus CpG dinucleotides as an inflammatory signal and thereafter on a weekly basis for three weeks with GPA RBCs alone. Using this protocol, about 50% of mice (n=23, three separate experiments) developed anti-huGPA alloantibodies (IgM and IgG) which were pathogenic based on red cell survival studies. No significant differences in the frequency of Foxp3⁺CD25⁺ splenic Treg population between mice that produced alloantibodies (n=12) versus those that did not (n=11) (9.6% ± 0.7% vs. 9.5% ± 0.7%, p=0.9) as measured by intracellular staining of CD4⁺ T cells was observed. In addition, sorted splenic CD4⁺CD25⁺ Tregs isolated from mice that had made allo-anti-huGPA and those that did not were equally anergic following stimulation (p≥0.2), a characteristic of normal Tregs whereas effector CD4⁺CD25⁻ cells from both groups of mice were highly proliferative. In contrast, when co-cultured at different suppressor/effector ratios (1:4, 1:8 and 1:16), Tregs from transfused immunized mice had an overall twofold less ability in suppressing autologous CD4⁺CD25⁻ than Tregs from transfused non-immunized mice (p<0.05, n=3 separate experiments). To distinguish whether the reduced Treg suppressive activity of mice that had made alloantibodies was due to impaired suppressor function of the Tregs or a result of increased resistance to suppression by the CD4⁺CD25⁻ T cells, we compared the suppressive effects of Tregs of these two groups of mice on proliferation of autologous and allogeneic CD4⁺CD25⁻ cells (n=3). We found that CD4⁺CD25⁺ Tregs from transfused non-immunized mice inhibited the proliferation of alloimmunized and non-alloimmunized CD4⁺CD25⁻ effector cells to the same degree (suppression=27% at suppressor/effector ratio 1:8). In contrast, CD4⁺CD25⁺ Tregs from transfused alloimmunized mice were less effective suppressors of CD4⁺CD25⁻ cells from either immunized or non-immunized mice (suppression=55% at suppressor/effector ratio 1:8). Altogether our data suggest differences in functional capacities of Tregs in multi-transfused mice which have developed alloantibodies versus those who have not. This may underlie the differences in immune response in transfused recipients which consequently dictate the outcome of the induction of red cell alloantibodies.