Activation and Effector Phenotype of CD8+ T Cells Crossprimed by a Minor Histocompatibility Antigen Expressed on Red Blood Cells
Authors: Desmarets Maxime , Zimring James C. 
Published: April 2020

Abstract
Background: Transfusion immunology research has traditionally focused on humoral immunity to RBC antigens, with study of CD4+ T cell and B cell responses. However, minor histocompatibility antigens (mHAs) on transfused RBCs in theory can also be cross-presented by recipient APCs to CD8+ T cells. This has clinical relevance in the context of transfusion patients who undergo transplantation, as several lines of evidence suggest that immunization to mHAs can lead to rejection of bone marrow and solid organ transplants. To test the hypothesis that mHAs on RBCs are capable of inducing immune responses through cross priming of CD8+ T cells, we created a novel transgenic mouse (HOD mouse), which expresses Ovalbumin (OVA) on RBCs but neither on leukocytes nor platelets.
Methods: 1×107 OT-I splenocytes (TCR transgenic for the OVA derived peptide SIINFEKL presented by H-2Kb) were adoptively transferred into B6.Thy1.1 mice. 100μl of packed HOD RBCs from whole or leukoreduced blood were then transfused intravenously. Control mice were transfused with FVB whole blood (HOD mice are on a FVB background). Three to 4 days post transfusion the OT-I T cells were visualized in the spleen by staining with anti-CD8 antibody and the Kb/SIINFEKL tetramer. Proliferation was evaluated by CFSE dilution. The activation phenotype was studied by staining with antibodies against CD44 and CD62L. Expression of effector molecules was studied by staining with antibodies against CD107a, Granzyme B, IFNγ, and IL-2.
Results: Compared to background analysis of OT-I T cells in control mice that received FVB blood, OT-I T cells in HOD recipients had a maximal expansion 24 fold higher in mice transfused with HOD leukoreduced blood (p=.0001), and 26 fold in mice transfused with HOD whole blood (p=.0672). Compared to naïve OT-I T cells, cross-primed OT-I T cells were CD44high, CD62Llow, CD107ahigh, suggesting activation and degranulation. Expression of IFNγ was also increased. However, cross-primed OT-I T cells were Granzyme Blow and did not have increased IL-2 expression. Lack of increased Granzyme B and IL-2 was not an artifact of the assay not working, as strong expression of both Granzyme B and IL-2 were detected in OT-I T cells stimulated with OVA in vitro.
Discussion: Overall these results demonstrate that RBC antigens can be cross-primed into the MHC class I pathway by recipient APCs. However, activation of CD8+ T cells specific for the RBC antigen demonstrates differentiation into an incomplete effector phenotype. The effect is unlikely due to contaminating donor leukocytes in the transfused unit since OVA is not expressed by WBCs. Moreover, we did not see any differences in proliferation or activation phenotype between the mice transfused with whole blood and leukoreduced blood. Activation was not due to direct presentation by residual cells, as the HOD donor mice are on an FVB (H-2q) background and cannot directly present mHAs to the H-2b restricted OT-I T cells. The reason for an incomplete effector profile is unclear, but may be because transfused RBCs are a sterile introduction of foreign antigen. These results also suggest better leukoreduction is not a viable solution to the problem of transfusion-induced immunization to mHAs, as the antigen is coming from the RBCs themselves. Matching for relevant mHAs or use of immunomodulatory treatments may be required.