The spleen plays a central role in primary humoral alloimmunization to transfused mHEL red blood cells

Abstract

Background: Several differences exist between antigens on transfused red blood cells (RBCs) and other immunogens, including anatomical compartmentalization. Whereas antigens from microbial pathogens and solid organ transplants drain into local lymph nodes, circulating RBCs remain segregated in the peripheral circulation, where they are consumed by antigen-presenting cells (APCs) in the spleen and liver. Accordingly, it was hypothesized that the splenic APCs play a central role in primary alloimmunization to transfused RBCs.

Study design and methods: Recipient mice were splenectomized and transfused with transgenic RBCs expressing the membrane-bound hen egg lysozyme (mHEL) model RBC antigen. In some experiments, mHEL-specific CD4+ T cells were adoptively transferred into recipient mice to allow investigation of helper T-cell responses. Unmanipulated or sham-splenectomized mice served as controls. Recombinant murine cytomegalovirus expressing mHEL (mHEL-MCMV) was used as a control non-RBC immunogen. Humoral responses were measured by mHEL-specific enzyme-linked immunosorbent assay and flow cytometric–based RBC cross-match.

Results: Control animals synthesized detectable anti-HEL immunoglobulin (Ig)G after a single mHEL RBC transfusion. mHEL-specific CD4+ T cells underwent robust expansion, and adoptive transfer of CD4+ T cells resulted in a 1000-fold increase in anti-HEL IgG. In contrast, minimal anti-HEL IgG was detectable in splenectomized mice, mHEL-specific CD4+ T cells did not proliferate, and adoptive transfer did not increase anti-HEL IgG. However, anti-HEL IgG response after exposure to mHEL-MCMV was equivalent in control and splenectomized mice.

Discussion: Together, these findings illustrate the distinct properties of transfused RBCs as immunologic stimuli, with the spleen playing a critical role in primary RBC alloimmunization at the level of CD4+ T-cell activation.

Fig. 1

Splenectomy decreases immunization to an alloantigen on RBCs but not to the same antigen on a viral pathogen. Intact and splenectomized mice were transfused IV with 500 μL of a 20% solution of leukoreduced mHEL RBCs in PBS (A) or infected IP with 10⁶ pfu of mHEL-MCMV (B). Anti-HEL IgG response was measured by ELISA 2 weeks later. Representative experiments with four to five mice/group are shown, with sera at a 1:50 dilution. These experiments have been repeated three to four times with similar results (38 mice total in [A], 25 mice total in [B]).

Fig. 2

Increasing the precursor frequency of mHEL-specific CD4+ T cells in splenectomized mice does not increase alloimmunization after RBC transfusion. (A) Intact (—) and splenectomized (···) mice were adoptively transferred with 1.5 × 10⁶ mHEL-specific CD4+ T cells and transfused with leukoreduced mHEL RBCs. Anti-HEL IgG response was measured by ELISA 2 weeks later. A representative experiment with five mice/group is shown with sera titrated from 1:100 to 1:100,000; mean and SDs are depicted. This experiment has been performed four times (41 mice total) with similar results. (B) Flow cytometric cross-match of a representative mouse from each group, with sera at a dilution of 1 to 5, mixed with control C57BL/6 RBCs (gray line) and mHEL RBCs (black line).

Fig. 3

Antigen-specific CD4+ T cells fail to undergo division in splenectomized mice despite exposure to their antigen on transfused RBCs. Intact and splenectomized recipients were adoptively transferred with 1.5 × 10⁶ CFSE-labeled antigen-specific 3A9 × B6.PL-Thy1.1 CD4+ T cells, followed by transfusion with leukoreduced mHEL RBCs or no RBCs. Five days later division was assessed by gating on adoptively transferred CD4+ T cells (A = gate) and measuring CFSE dilution. The CFSE-positive gate was determined by gating on unlabeled adoptively transferred CD4+ T cells in control mice. Division was assessed by dilution of CFSE signal in the spleen, liver, or lymph nodes of an intact mouse (B, C, and E) or in the liver and lymph nodes of a splenectomized mouse (D and F). In all panels, gray shaded histograms represent control mice not transfused with mHEL RBCs. Staining from representative mice is shown; the y-axes represent cell number. This experiment was performed three times (15 mice total) with similar results.

Fig. 4

The requirement for a spleen is not due to lack of RBC antigen access to peripheral lymphatics. Intact (—) and splenectomized (- - -) mice were adoptively transferred with 1.5 × 10⁶ mHEL-specific CD4+ T cells, and injected IP with 500 μL of a 20% solution of leukoreduced mHEL RBCs in PBS. Anti-HEL IgG response was measured by ELISA 2 weeks later. A representative experiment with five mice/group is shown, with sera titrated from 1:50 to 1:50,000; mean and SDs are depicted. This experiment has been repeated twice, with similar results (20 mice total).