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Today it is generally accepted that B cells require cognate interactions with CD4+T cells to develop high-affinity antibodies against proteins. CD4+T cells recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4+T cells that can bind to the MHC class II–peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4+T-cell epitopes presented by HLA-DRB1*1501 to CD4+T cells during immune responses against FVIII. CD4+T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays.

Graft versus host disease (GVHD) is the primary cause of transplant related morbidity and mortality, limiting the widespread application of haemopoietic stem cell transplantation (HSCT). Evidence from murine models supports the role of CD4+CD25+regulatory T cells in the suppression of GVHD. Human evidence regarding the role of regulatory T cells in alloresponses is conflicting and may reflect the difficulty in defining and isolating the regulatory T cell population in humans. We have investigated the use of peripheral blood monocyte-derived dendritic cells (DCs) as stimulator cells in allogeneic mixed lymphocyte reactions (MLRs), as a means of assessing the in vitrosuppressive function of regulatory T cells in human alloresponses. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy adult volunteers. Magnetically isolated CD4+CD25+T cells were combined with 50×103autologous PBMCs or 20×103autologous CD4+T cells as responders, and 5×103allogeneic irradiated DCs. Proliferation was assessed by tritiated thymidine incorporation. The CD4+CD25+cells were anergic and demonstrated dose-dependent suppression of responder cell proliferation in the DC-driven allogeneic MLR. Greater than 50% suppression was seen with CD4+CD25+T cells co-cultured with responder PBMCs at ratios of 1:4 to 1:32. Furthermore, depletion of CD4+CD25+T cells from whole CD4+responder cells resulted in enhanced proliferation and an increase in the amplitude of the MLR. Flow cytometry indicated that the majority of the magnetically isolated CD4+CD25+T cells were FoxP3+on intracellular staining and demonstrated down-regulation of cell surface expression of the IL-7 receptor (CD127). The potent suppression demonstrated here by CD4+CD25+CD127−T cells at ratios of 1:32 responder cells, suggests that these cells have a potential role for suppressing alloresponses at physiological levels. Moreover, this assay provides the basis for future investigation into regulatory T cell function in patients post-HSCT.