Background.PVG.RT1urats develop a strong CD4 T cell-dependent alloantibody response to class I major histocompatibility complex (MHC) Aaantigen, during which CD4 T helper cells recognize and respond to Aa-derived peptides presented by recipient class II MHC (indirect allorecognition). On the basis of evidence that CD4 T cells that encounter antigen presented by resting B cells become tolerant, we have targeted synthetic Aa-derived allopeptides for in vivo presentation to class I MHC-disparate CD4 T cells by resting recipient B cells.Methods.PVG.RT1urats were treated with two peptides, P1 and P2, corresponding to the α-helical regions of Aa(residues 57-80 and 143-163), which were conjugated via an N-terminal cysteine residue to monovalent Fab fragments of OX60 monoclonal antibody, which labels membrane IgD-positive B cells.Results.RT1urats primed with free (nonconjugated) P1 or P2 emulsified in complete Freund's adjuvant produced strong peptide-specific antibody responses and a heightened anti-Aaantibody response to an Aa-disparate PVG.R8 heart graft, confirming that each peptide encompasses one or more major T cell determinant for B cell help. Pretreatment of PVG.RT1urats with a mixture of OX60-Fab-P1/P2 conjugates markedly reduced their ability to mount an Aaantibody response when challenged with either Aa-disparate blood transfusion or an Aa-disparate heart graft, although PVG.R8 heart graft survival was not prolonged.Conclusions.In this report, we show that synthetic Aa-derived allopeptides are able, when targeted for in vivo presentation to CD4 T cells by resting B cells, to impair the ability of RT1urats to mount an antibody response to Aaantigen. All subclasses of IgG anti-Aaalloantibody were profoundly reduced, suggesting that the responsible mechanism is more likely to be CD4 T helper cell unresponsiveness rather than Th1/Th2 T cell polarization.