Transfusion with Stored Antigen-Negative Blood Impairs T Cell Cross Priming to Red Cell Alloantigen in a Subsequent Transfusion
Authors: Weisberg Stuart P. , Shabro Aidin , Youssef Lyla A , Spitalnik Steven , Hod Eldad A. 
Published: October 2019

Abstract
BACKGROUND: Donor red blood cell (RBC) alloantigens are efficiently cross-presented by recipient antigen presenting cells (APCs), thereby priming recipient naive CD8 T cells; however, this process and its downstream immunological consequences are poorly understood. RBC antigen cross presentation has been linked to cytotoxic T cell responses driving bone marrow graft rejection, but in other systems it causes tolerance. The conventional dendritic cell (cDC) subset, cDC1, is required for cross-presentation of RBC antigen. Macrophages are insufficient for cross-presentation in the absence of cDC1s, but do promote RBC antigen cross-presentation by cDCs in vitro. After prolonged storage, a portion of transfused RBCs are rapidly cleared by the spleen. In murine models, transfused stored RBCs have multiple effects on splenic APCs, including architectural distortion, dendritic cell activation, and macrophage depletion via ferroptosis due to increased erythrophagocytosis. Thus, the antigen presentation machinery controlling alloimmunization to RBC antigens may be influenced by the storage duration of previously transfused RBCs.
AIMS: To better understand how recent transfusions may influence the immunogenicity of future transfusions, we used a mouse model to determine whether prior transfusion of stored, antigen-matched RBCs influences CD8 T cell cross priming to antigen mismatched RBCs in a subsequent transfusion.
METHODS: In an in vivo cross-priming assay, OVA-expressing RBCs from transgenic HOD mice (with RBC surface expression of hen egg lysozyme, ovalbumin (OVA), and Duffy antigens) were the transfused antigen source and naïve OT-1 CD8 T cells, which express an MHC class I restricted OVA-specific TCR transgene, were the responders. Magnetically-selected CD8+ OT-1 cells, labeled with CellTrace Far Red (CTFR), were adoptively transferred into MHC identical (H-2Kb) UBC-GFP C57BL6/J recipients. After 16 hours, these mice were transfused with fresh or stored (12 days in CPDA-1), leukoreduced UBC-GFP RBCs or PBS. Five hours after the initial transfusion, recipients were transfused again with fresh leukoreduced HOD RBCs. To determine the effect of stored RBC transfusion on T cell priming to a non-RBC associated antigen, groups of mice carrying adoptively transferred OT-1 T cells were transfused with fresh or stored RBCs and then challenged 5 hours later with soluble OVA protein (10 µg, IV). After 4 days, splenic OT-1 T cells were analyzed for proliferation (by CTFR dye dilution) and activation (by CD44 and CD122 expression).
RESULTS: Proliferation and activation of OT-1 T cells in response to challenge with soluble OVA protein were significantly increased in mice that had been previously transfused with stored RBCs compared to those transfused with fresh RBCs (Fig. 1A). In contrast, OT-1 T cell activation in response to HOD RBC transfusion was decreased in mice that had previously received a stored RBC transfusion, as compared to those receiving fresh RBCs or PBS (Fig. 1B).
CONCLUSION: The increased T cell priming after soluble OVA challenge in the mice previously transfused with stored vs. fresh RBCs may be related to the increased expression of co-stimulatory molecules on DCs after stored RBC transfusion. However, T cell priming to the RBC-associated antigen requires additional factors, which are disrupted by previous transfusion of stored, but not fresh, RBCs. Splenic red pulp macrophages potentiate OT-1 T cell cross priming by dendritic cells in vitro and become severely depleted after stored RBC transfusion in vivo. Thus, macrophage help may be needed for T cell cross priming to an RBC antigen in vivo, and stored RBC transfusions may disrupt macrophage function, thereby affecting the immunogenicity of subsequent RBC alloantigen exposure.
Figure 1. Effect of fresh and stored RBC transfusion on priming after antigenic challenge with soluble OVA or HOD RBCs.
Representative contour plots show proliferation-induced dye dilution and CD44 expression on OT-1 cells in the spleen 4 days after transfusion of fresh (left) or stored (right) RBCs followed 5 hours later with IV injection of soluble OVA protein (A) or HOD RBCs (B). Dot plots at the right of representative contour plots show compiled data from two independent experiments quantifying the percentage of activated and proliferated cells (CTFR diluted, CD44hi) in the OT-1 subgate (mean ± SEM, n = 4-9). *P < 0.05, **P < 0.01, unpaired t test.

Disclosures
No relevant conflicts of interest to declare.