An Intact Spleen Is Required for Alloimmunization to Transfused Red Blood Cells Due to Intrasplenic Activation of CD4+ T Cells.

Background: Factors influencing rates of alloimmunization to antigens on transfused red blood cells (RBC alloimmunization) are poorly defined. In particular, the role of the spleen with respect to alloantibody formation is unclear, with conflicting clinical reports in the literature. Moreover, the complexities of multiply mismatched antigens and antigen priming due to transfusions prior to splenectomy make human studies difficult to interpret. To better define the role of the spleen in RBC alloimmunization, we utilized a murine model of transfusion medicine (with the model RBC antigen mHEL (membrane bound hen egg lysozyme)).

Methods: Cohorts of splenectomized and non-splenectomized mice (C57BL/6 × B10.BR) were transfused with the human dose equivalent of 1 unit of leukoreduced mHEL RBCs. RBC alloimmunization was assessed by anti-HEL IgG specific ELISA. The role of antigen-specific CD4+ T cells was studied by the adoptive transfer of 1.5 × 106 HEL-specific CFSE-labeled CD4+ T cells from 3A9 TCR transgenic donors. Adoptively transferred cells were visualized using a congenic marker (Thy1.1); enumeration and division of these cells were monitored by flow cytometry in liver, spleen (if applicable), and lymph node preparations.

Results: Splenectomy dramatically decreased RBC alloimmunization; 14 of 14 splenectomized mice (from 3 experiments) had undetectable to very low levels of anti-HEL IgG following transfusion with mHEL RBCs (average 6.3 fold less than non-splenectomized mice, 95% C.I. 4.6). Moreover, ten of ten splenectomized mice failed to make detectable levels of anti-HEL IgG even following the adoptive transfer of HEL-specific CD4+ T cells. In comparison, elevating the precursor frequency of HEL-specific CD4+ T cells increased RBC alloimmunization by 10,000 fold in non-splenectomized mice. Proliferation and division of CD4+ T cells were detectable in both spleen and liver preparations of non-splenectomized mice by day 3 following transfusion; in contrast, no expansion nor division of CD4+ T cells was seen in liver nor lymphatic preparations of splenectomized animals.

Conclusions: The low level of RBC alloimmunization seen in non-splenectomized mice in this system is limited by existing CD4+ helper T cell responses, as increasing the naive helper T cell precursor frequency dramatically increased RBC alloimmunization. Furthermore, the spleen itself is critical to CD4+ helper T cell function during alloimmunization given that, in splenectomized mice, adoptively transferred HEL-specific CD4+ T cells fail to expand, divide, or stimulate production of detectable alloantibody. Ongoing studies are investigating the phenotype of HEL-specific CD4+ T cells (as effector cells, anergic cells, or regulatory cells) in splenectomized and non-splenectomized mice. These studies have implications for preventing alloimmunization in transfusion-dependent patient populations.