Characterization of Antigen-Specific CD4⁺ T Cell Responses to the Transfusion of Red Blood Cells

To study the antigen-specific CD4⁺ T cell responses to transfused red blood cells (RBCs) in vivo, we have utilized the adoptive transfer of naïve RBC-specific CD4⁺ T cells at low precursor frequencies. Our approach combines the readily available OT-II T cell receptor transgenic mouse, which recognizes ovalbumin, with the Hen egg lysozyme, Ovalbumin, and the human Duffy (HOD) RBC alloimmunization model transgenically expressing a chimeric antigen comprised of hen egg lysozyme, ovalbumin, and human Duffy protein selectively on RBCs. OT-II mice have been crossed to Pep Boy mice to allow for the use of the CD45.1 congenic marker to readily identify antigen-specific cells in a C57/Black6 (C57/B6) CD45.2 background. As both IL-21 and IL-4 are known to support B cell activation, class switching, and differentiation into antibody-secreting plasmablasts, we have further crossed OT-II/CD45.1 mice with mice expressing knock-ins of two separate fluorescent reporters that read out both IL-21 and IL-4 RNA expression. This approach allows for the use of commonly employed multiparameter flow cytometric analysis to characterize the activation, proliferation, and phenotype of RBC antigen-specific T cells, as well as their subsequent cytokine production in response to transfused allogeneic RBCs. Using these approaches, we present examples of antigen-specific T cell responses induced by HOD transfusion in wild-type (WT) C57/B6 recipients.