Marginal Zone B Cells Facilitate RBC Alloimmunization Independent of Direct CD4⁺ T Cell Activation

Background: Blood transfusions decrease complications in patients with bone marrow failure syndromes, hemoglobinopathies, or neoplasia. However, patients who receive this often life-saving treatment can develop alloantibodies against non-ABO red blood cell (RBC) antigens. RBC alloimmunization can have severe clinical consequences, including increased morbidity and mortality secondary to difficulty obtaining compatible RBCs for future transfusion and an increased risk for hemolytic transfusion reactions.Aside from Rh(D) immune globulin, there are no prophylactic treatments available to prevent RBC alloimmunization. Improved understanding of the immune mechanism underlying RBC alloimmunization is central in developing additional prophylactic treatments. Recent studies suggest that marginal zone (MZ) B cells are critical for immune responses to RBC alloantigens. As a result, we sought to determine mechanisms whereby MZ B cells mediate RBC alloimmunization.

Methods: RBCs expressing the HOD antigen [hen egg lysozyme, ovalbumin (OVA), and human Duffy] were labeled with DiI and transfused into C57BL/6 recipients, followed by confocal examination for HOD RBC localization by staining sections for CD1d, MadCAM and B220. HOD RBCs were also transfused into C57BL/6 recipients treated with anti-CD4 T cell (clone: GK 1.5), anti-MZ B cell (anti-CD11a and anti-CD49d) or isotype control followed by examination of anti-HOD IgM and IgG antibodies on days 5 and 14 post-transfusion by flow cross-match. To determine the impact of B cells in general and MZ B cells in particular on CD4⁺ T cell activation following HOD RBC transfusion, CFSE labeled splenocytes from OTII transgenic mice with CD4⁺ T cells specific for HOD RBCs were adoptively transferred into B-cell deficient μMT mice, MZ B cell depleted C57BL/6 recipients, C57BL/6 controls, and mice genetically deficient in MZ B cells followed by transfusion of HOD RBCs. On days 1 and 3 post-transfusion, OTII proliferation, activation and cytokine profile were assessed using flow cytometric analysis. To examine the role of MZ B cells in antigenic trafficking, C57BL/6 recipients were administered FTY720, which displaces MZ B cells from the marginal sinus, or PBS, followed by HOD RBC transfusion and analysis for anti-HOD antibody formation.

Results: HOD RBCs initially localized within the marginal sinus following transfusion, where they displayed significant co-localization with MZ B cells. While depletion of MZ B cells inhibited anti-HOD IgM and IgG antibody formation, CD4⁺ T cell depletion only prevented anti-HOD IgG antibody development. Despite the requirement of MZ B cells in anti-HOD antibody formation, CFSE-labeled OTII CD4⁺ T cells showed proliferation as well as evidence of key markers of T cell activation following transfusion of HOD RBCs into MZ B cell depleted, genetically deleted or B cell deficient μMT recipients, strongly suggesting that the requirement of MZ B cells for IgG antibody formation does not reflect MZ B cell-mediated CD4⁺ T cell activation. Given the localization of HOD RBCs with MZ B cells and the ability of CD4⁺ T cells to proliferate in the absence of MZ B cells, we next determined whether the requirement of MZ B cells for anti-HOD IgG antibody formation may actually reflect MZ B cell-mediated trafficking of HOD RBCs to the B cell follicle. Consistent with this possibility, MZ B cell displacement from the marginal sinus to the splenic follicle, which prevents MZ B cell-mediated trafficking of antigen, prevented formation of anti-HOD IgM and IgG following HOD RBC transfusion.

Conclusion: The ability of CD4⁺ T cells to undergo proliferation and activation following HOD RBC transfusion in the absence of MZ B cells suggests that the requirement of MZ B cells for antibody formation does not reflect MZ B cell-mediated CD4⁺ T cell activation. In contrast, the localization of HOD RBCs with MZ B cells, coupled with the ability of MZ B cell displacement to prevent HOD RBC-induced alloimmunization, strongly suggests that MZ B cells regulate RBC alloimmunization by initially trapping and the trafficking RBC alloantigens to the B cell follicle. In doing so, MZ B cells appear to be a key initiator and overall regulator of RBC alloimmunization and therefore a potential target for prophylactic measures designed to inhibit this process.