the spleen plays a central role in primary humoral alloimmunization to transfused mhel red blood cells 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 antigenpresenting cells apcs in the spleen and liver accordingly it was hypothesized that the splenic apcs play a central role in primary alloimmunization to transfused rbcsstudy design and methods recipient mice were splenectomized and transfused with transgenic rbcs expressing the membranebound hen egg lysozyme mhel model rbc antigen in some experiments mhelspecific cd4 t cells were adoptively transferred into recipient mice to allow investigation of helper tcell responses unmanipulated or shamsplenectomized mice served as controls recombinant murine cytomegalovirus expressing mhel mhelmcmv was used as a control nonrbc immunogen humoral responses were measured by mhelspecific enzymelinked immunosorbent assay and flow cytometricbased rbc crossmatchresults control animals synthesized detectable antihel immunoglobulin igg after a single mhel rbc transfusion mhelspecific cd4 t cells underwent robust expansion and adoptive transfer of cd4 t cells resulted in a 1000fold increase in antihel igg in contrast minimal antihel igg was detectable in splenectomized mice mhelspecific cd4 t cells did not proliferate and adoptive transfer did not increase antihel igg however antihel igg response after exposure to mhelmcmv was equivalent in control and splenectomized micediscussion 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 tcell activation