Plates were washed, incubated with biotin-labeled secondary antibodies for 1 h at 4C, washed again, and incubated for an additional 1 h at room temperature with Eu3+-labeled streptavidin (Wallac) in assay buffer (Wallac). complexes when antigen-presenting cells (APCs) were pulsed with recombinant MBP. In addition, MK16 inhibited interleukin 2 secretion by two transfectants that expressed human MBPspecific T cell receptors. Analysis of the structural requirement for MK16 binding demonstrated that the two major HLA-DR2 anchor residues of MBP 8599 and the COOH-terminal Vamp5 part of the peptide, in particular residues Val-96, Pro-98, and Arg-99, were important for binding. Based on these results, the antibody was used to determine if the HLA-DR2MBP peptide complex is presented in MS lesions. The antibody stained APCs in MS lesions, in particular microglia/macrophages but also in some cases hypertrophic astrocytes. Staining of APCs was only observed in MS cases with the HLA-DR2 haplotype but not in cases that carried other haplotypes. These results demonstrate that HLA-DR2 molecules in MS lesions present a myelin-derived self-peptide and suggest that microglia/macrophages rather Lapatinib (free base) than astrocytes are the predominant APCs in these lesions. Keywords:multiple sclerosis, antigen presentation, myelin basic protein, autoimmunity, microglia == Introduction == Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) with unknown etiology. Inherited susceptibility to MS is associated with the HLA class II genes DRB1*1501, DRB5*0101, and DQB1*0602, which are all contained in the DR2 haplotype1. Because these three Lapatinib (free base) genes are in strong linkage disequilibrium, it has not been possible to determine which of them are the principal MS risk genes. Any of these three genes or combinations of them could mediate the MHC class IIassociated susceptibility to MS. The exact role for the disease-associated MHC class II molecules in the pathogenesis of MS is not clear. One prevailing hypothesis is that they present self-antigens to autoaggressive T cells that enter the CNS and induce an inflammatory response that leads to clinical manifest disease23. The target autoantigens are unknown in MS, but, largely based on their encephalitogenicity in animal models for this disease, myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein are the major candidates (for review see references 3 and4). Investigations of the immune response against CNS-derived proteins in MS patients have largely focused on MBP as a candidate antigen. The MBP 84103 peptide has been defined as the strongest binder of all MBP peptides to the disease-associated HLA-DR2 molecules and is recognized by T cell clones from patients and normal individuals with the HLA-DR2 haplotype567. Several lines of evidence indicate, however, that T cells recognizing this epitope are only in vivo activated in MS patients68910. The minimal epitope for HLA-DR2restricted T cell recognition has been mapped to residues 8599 of MBP7. T cell lines against other regions of MBP have also been generated, although less frequently561112131415. The identity of the cells in the CNS that present myelin-derived autoantigens to CD4+T cells is not clear. Phenotypic and functional analyses have implicated both microglia/macrophages and astrocytes (for review see references161718). To directly examine MHC class IIrestricted presentation of a myelin antigen in MS lesions, we have generated an mAb (MK16) that is specific for the HLA-DR2 (DRB1*1501)-bound MBP 8599 peptide. Here we describe the generation and a Lapatinib (free base) detailed characterization of this antibody and demonstrate how it can be used to detect HLA-DR2MBP 8599 peptide complexes intra- and extracellularly when APCs have been incubated with native MBP. This antibody was also used to examine antigen presentation in MS lesions and normal brain tissue. The antibody specifically labeled microglia/macrophages and in some cases hypertrophic astrocytes from patients with the HLA-DR2 haplotype, whereas these cells were not stained in lesions from HLA-DR2negative patients. == Materials and Methods == == Cell Lines. == Drosophila melanogasterSchneider 2 (S2) cell transfectants expressing either DRA1*0101/DRB1*1501 (DRB1*1501) or DRA1*0101/DRB1*0401 (DRB1*0401) molecules were grown at 25C in Schneider’s medium (Sigma Chemical Co.) supplemented with 10% heat-inactivated FCS, 2 mMl-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin. The following transfectants and T cell hybridomas were all grown at 37C in DMEM (Sigma Chemical Co.) supplemented with 10% heat-inactivated FCS, 2 mMl-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 50 M -ME, and 1% nonessential amino acids: L cell transfectants expressing either DRA1*0101/DRB5*0101 (DRB5*0101) or DRB1*1501 molecules, the mouse BW 58 TCR-/ T cell hybridoma19, and four T cell transfectants expressing.