![]() 4 The amino acid sequence of this RhD protein was deduced from complementary DNA (cDNA) analysis, 5, 6 and transfection experiments confirmed that it does indeed encode the RhD antigen. With the development of modern immunochemical techniques, it was demonstrated that anti-D antiserum or mAbs immunoprecipitate a nonglycosylated protein of approximately 30 kd that is exposed on the surface of RhD-positive, but not RhD-negative, RBCs. The major focus for research on the alloimmune response to RhD has been characterization of the red blood cell (RBC) membrane antigen that is recognized by anti-D alloantibodies. 1, 3 There is therefore an urgent need to more fully understand the immune response to RhD in order to design better strategies to prevent HDN. Although monoclonal antibodies (mAbs) specific for RhD have been developed, it remains unclear whether these preparations will be safe, efficacious, or affordable replacements for polyclonal anti-D immunoglobulin. Furthermore, despite the use of anti-D immunoglobulin, alloimmunization still occurs in 1% to 1.5% of women at risk and cannot be reversed once an immune response has been initiated. 2 In particular, fears about the transmission of viral disease and the spongiform encephalopathies have recently resulted in difficulties in recruiting volunteers for immunization programs, or cessation of such programs, with potential for shortages of anti-D immunoglobulin. Although infant mortality due to HDN has been dramatically reduced by passive administration of human anti-D immunoglobulin to women at risk of alloimmunization, this prophylactic program is threatened by growing ethical concerns over the deliberate immunization of RhD-negative volunteers to produce human blood products. 1 Not only is this antigen routinely considered in transfusion medicine, but RhD incompatibility between mother and fetus is the classic cause of hemolytic disease of the newborn (HDN). The most immunogenic and clinically important blood group borne by the Rhesus complex is the Rhesus D (RhD) antigen. 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. 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. 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. 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. ![]() 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. ![]() The PBMCs were stimulated with a panel of up to 68 overlapping synthetic 15-mer peptides spanning the complete sequence of 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. Therefore, the aim of this study was to map alloreactive T-cell epitopes on the RhD 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.
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