Primed CD4 T Cells to an Intracellular Alloantigen Facilitate Alloimmunization Following Subsequent Transfusion
Authors: Patel Seema R , Bennett Ashley L , Zerra Patricia E , Chonat Satheesh , Maier Cheryl , Stowell Sean R 
Published: April 2021

Abstract
Background: While red blood cell (RBC) alloantibodies can increase the probability of transfusion-related complications, not all patients become alloimmunized following transfusion. Several factors have been hypothesized to govern susceptibility to alloimmunization, including general differences in immune function and the potential impact of recipient inflammation at the time of transfusion. However, as individuals that do generate alloantibodies appear to experience an increased rate of additional alloantibody formation following subsequent transfusion, distinct immunological responses induced following exposure to certain antigens expressed by transfused RBCs may directly facilitate the development of alloantibodies following subsequent transfusion. Thus, while CD4 T cell help classically occurs through direct recognition of a peptide that resides within a target B cell antigen, these clinical observations suggest that CD4 T cells that respond to one RBC antigen may directly facilitate immunity to a completely distinct RBC alloantigen following subsequent transfusion. As RBCs express a variety of possible polymorphic antigens, both inside RBCs and at the RBC surface, exposure to alloantigens within RBCs may possess the capability to induce a CD4 T cell response that would be undetectable by clinical serological analysis, but which may facilitate subsequent alloimmunization. To test this, we determined whether cellular adaptive immunity to an intracellular alloantigen might enhance alloimmunization following subsequent RBC transfusion.
Methods: B6 recipients were immunized three times a week apart against an intracellular antigen, green fluorescent protein (GFP). Two weeks following the last immunization, recipients were transfused with RBCs expressing the model antigen HOD (a fusion protein consisting of hen egg lysozyme fused to ovalbumin and human Duffy b) and GFP (HOD x GFP RBCs) or RBCs expressing the human Glycophorin A (hGPA) and GFP antigens (hGPA x GFP RBCs). Serum was collected at days 7, 14, 21 and 28 post-transfusion and the levels of anti-HOD or anti-GPA antibodies were determined by incubation of serum with HOD RBCs, GPA RBCs or B6 RBCs, followed by detection with fluorescently labeled anti-IgG antibodies. The adjusted mean fluorescent intensity (MFI) for anti-HOD or anti-GPA antibodies was calculated by subtracting the MFI observed following incubation with B6 RBCs from the MFI observed following incubation with HOD or GPA RBCs, respectively.
Results: Recipients that underwent GFP immunization experienced a statistically significant enhancement of anti-HOD IgG compared to non-GFP immunized recipients following subsequent transfusion of HOD x GFP RBCs (p &lt; 0.05), strongly suggesting that prior exposure to a single intracellular antigenic determinant can enhance antibody formation following subsequent exposure to RBCs expressing both the intracellular antigen and a clinically relevant surface antigen. To determine whether immunological priming toward an intracellular antigen can impact RBC alloimmunization toward other cell surface RBC alloantigens, we likewise transfused recipients that had been previously immunized against GFP with RBCs that express hGPA and GFP. Similar to the impact of prior GFP immunization on HOD alloimmunization, prior GFP immunization rendered recipients responsive to the hGPA antigen following transfusion of hGPA x GFP RBCs. Importantly, non-GFP immunized recipients were non-responsive to the hGPA antigen regardless of being expressed on the same RBCs as GFP (p &lt; 0.05), suggesting that prior immunization toward an intracellular antigen may not only enhance subsequent alloimmunization in previously alloimmunized individuals, but also render non-responding recipients responsive to RBC-induced alloimmunization.
Conclusion: These results demonstrate that immunity to an intracellular alloantigen can directly influence the immunological outcome following exposure to a subsequent extracellular RBC alloantigen. Moreover, these findings suggest a mechanism whereby alloantibody responders may exhibit an increased rate of additional alloantibody formation and highlight a previously under appreciated mechanism by which cellular adaptive immunity can impact the ability of an individual to respond to unrelated immunogens.

Disclosures
Chonat: Agios Pharmaceuticals: Honoraria.