Abstract. Goodpasture's disease is a severe nephritis characterized by autoantibodies to the ␣3 chain of type IV collagen, ␣3(IV)NC1, in the glomerular basement membrane. The disease is very strongly associated with HLA-DR15, the affinities of ␣3(IV)NC1 peptides for DR15 are known, and elution experiments have identified major naturally processed sequences. Here, the fine specificity and cytokine profile of ␣3(IV)NC1-reactive T cells from patients with Goodpasture's disease is defined. Peripheral blood mononuclear cells from patients at diagnosis proliferated in response to significantly more peptides ( 2 ϭ 8.6, P ϭ 0.004) from a panel spanning the sequence of ␣3(IV)NC1 than did those from control DR15-positive donors and were highly focused (P ϭ 0.0002, binomial distribution) on two peptides, ␣3 71-90 and ␣3 131-150 .

RhD is a major blood group and the most important target antigen in hemolytic disease of the newborn (HDN). The aims of this study were to establish a humanized mouse model of responses to the RhD protein and to test whether these could be prevented by the induction of immune tolerance. HLA-DR15 is a major restricting element for human T-helper (Th) cells specific for RhD protein, and expression of this HLA-DR transgene was found to confer on mice the ability to respond to immunization with purified RhD protein. Synthetic peptides containing dominant IntroductionThe RhD antigen is a highly immunogenic and clinically important human blood group. Approximately 10% of pregnancies are at risk for hemolytic disease of the newborn (HDN) caused by RhD incompatibility, 1 and current prophylaxis is dependent on passive RhD immune globulin. Fuller understanding of the immune response to RhD will enable the design of alternative strategies to prevent HDN.Most immunoglobulin G (IgG) antibody responses are dependent on T-cell help, and the production of antibodies specific for red blood cells (RBCs), 2 including anti-D, 3 is no exception. T-helper (Th) cells recognize short antigen-derived peptides displayed by specialized antigen-presenting cells (APCs), and it is now clear that the context in which such recognition takes place determines whether a specific immune response is activated or tolerized. 4,5 Methods to manipulate Th recognition of the RhD protein to favor tolerance are of particular interest because this would permit antigen-specific intervention to prevent HDN, without the risks associated with the use of blood products. Mucosal administration of helper epitopes is an approach that has been used to induce systemic tolerance to antigens of pathogenic relevance in models of transplantation, 6 autoimmunity, 4,5 and asthma. 7 In many cases, the induction of such tolerance has been attributed to active mechanisms of immune regulation, particularly the stimulation of regulatory T cells. 6,8,9 Dominant epitopes from the RhD protein that induce the proliferation of Th cells from alloimmunized donors in vitro have previously been mapped by us. 3 In particular, peptides RhD 52-66 , RhD [97][98][99][100][101][102][103][104][105][106][107][108][109][110][111] were each able to stimulate Th cells in vitro from more than 50% of alloimmunized donors, with responses to at least 1 sequence in every donor. The question arises as to whether administering these peptides by a tolerogenic route could prevent responses to the entire RhD protein in vivo. To address this possibility, we first developed a humanized model of responsiveness to the RhD protein. The HLA-DRB1*1501 allele is significantly overrepresented (47.6%; 2 ϭ 4.269; P ϭ .039) in RhD-negative donors who have produced anti-D antibodies in response to exposure to RhD-positive RBCs, 10 and HLA-DR is the major restricting locus for Th cells specific for RhD protein epitopes. 3 Mice transgenic for HLA-DR15 were selected for this study, predicting that the RhD protein would...

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Regulatory T (Tr) cells have the potential to treat immune-mediated disease, but cloning such cells for study from patients with autoimmune disease has proven difficult. Here, we describe autoantigen-specific, interleukin-10 (IL-10)–secreting Tr cell clones recovered ex vivo from a patient with autoimmune hemolytic anemia (AIHA) and characterize their phenotype, origin, and regulatory function. These IL-10+ Tr cells recognized a peptide, 72H-86L, derived from the Rh red blood cell autoantigen and shared phenotypic characteristics with both natural and inducible Tr cells. The clones also expressed different Tr markers depending on activation state: high levels of CD25 and LAG-3 when expanding nonspecifically, but FoxP3 after activation by the autoantigen they recognize. Despite a discrete Tr phenotype, these cells stably expressed the T helper 1 (Th1) signature transcription factor T-bet, suggesting they derive from Th1 T cells. Finally, the contribution of CTLA-4 in activating these IL-10+ Tr cells was confirmed by analyzing responses to transgenic B7.1-like molecules that preferentially bind either CD28 or CTLA-4. Overall, these Tr cells have a functional phenotype different from those described in previous studies of human Tr populations, which have not taken account of antigen specificity, and understanding their properties will enable them to be exploited therapeutically in AIHA.