Identification of alloreactive T-cell epitopes on the Rhesus D protein

Although considerable effort has been devoted to characterizing alloantibodies specific for the Rhesus D (RhD) blood group antigen, virtually nothing is known about the helper response that drives their production. Therefore, the aim of this study was to map alloreactive T-cell epitopes on the RhD protein. Peripheral blood mononuclear cells (PBMCs) were obtained from 22 RhD-negative volunteers in whom anti-D alloantibodies had developed after deliberate immunization or RhD-incompatible pregnancy. The PBMCs were stimulated with a panel of up to 68 overlapping synthetic 15-mer peptides spanning the complete sequence of the RhD protein. One or more peptides elicited proliferative responses by PBMCs from all 22 of the alloimmune volunteers but from only 2 of 8 alloantibody-negative control donors. Proliferation of PBMCs from the alloimmune donors was mediated by major histocompatibility complex class II-restricted T cells expressing the CD45RO marker of previous activation or memory. The number of peptides that induced proliferative responses was unrelated to either the frequency of, or time since, exposure to RhD-positive red blood cells, but it correlated strongly (R(s) = 0.75; P <.003) with the level of anti-D antibodies in deliberately immunized donors. The patterns of stimulatory peptides varied among alloimmune volunteers, but particular sequences were commonly recognized, with 4 peptides each eliciting a response in more than 50% of these donors. Identification of such peptides containing dominant alloreactive helper epitopes is the first step in the development of improved or new approaches to preventing hemolytic disease of the newborn that are based on modulating the T-cell response to the RhD protein.