The major histocompatibility complex (MHC) class II region is assumed to influence autoimmune diseases such as rheumatoid arthritis. In the mouse, the H-2q haplotype is associated with susceptibility to collagen-induced arthritis, while the H-2p haplotype is not. The class II A molecules of these haplotypes differ by only four amino acids in the first domain of the beta chain. To test if this difference accounts for the MHC influence on susceptibility to collagen-induced arthritis, H-2p mice were made transgenic with an Abp gene altered to resemble the Abq gene. The transgenic A beta chain hybridized with the A alpha p chain and was shown to be physiologically expressed by testing antigen-presentation capacity to Aq-restricted T cell hybridomas and with FACS analyses. These transgenic mice developed an autoimmune response to type II collagen and also collagen-induced arthritis. The data unequivocally suggest the Ab gene as a major genetic susceptibility locus for autoimmune collagen-induced arthritis.

The Aq major histocompatibility complex (MHC) class II molecule is associated with susceptibility to murine collagen-induced arthritis (CIA), whereas the closely related H-2Ap molecule is not. To understand the molecular basis for this difference, we have analyzed the ability of H-2Aq and H-2Ap molecules (referred to as Aq and Ap) to bind and present collagen type II (CII)-derived glycosylated and non-glycosylated peptides. T cell clones specific for the immunodominant CII 256-270 peptide and restricted to both Aq and Ap molecules were identified. When these clones were incubated with CII protein and either Aq- or Ap-expressing antigen-presenting cells (APC), only Aq-expressing APC were able to induce stimulation. With the use of A(beta) transgenic mice this could be shown to be solely dependent on the MHC class II molecule itself and to be independent of other MHC- or non-MHC genes. Peptide binding studies were performed using affinity-purified MHC class II molecules. The CII 256-270 peptide bound with lower affinity to the Ap molecule than to the Aq molecule. Using a set of alanine-substituted CII 256-270 peptides, MHC class II and T cell receptor (TCR) contacts were identified. Mainly the side chains of isoleucine 260 and phenylalanine 263 were used for binding both the Aq and Ap molecule, i.e. the peptide was orientated similarly in the binding clefts. The major TCR contact amino acids were lysine 264, which can be posttranslationally modified, and glutamic acid 266, which is the only amino acid in the heterologous peptide which differs from the mouse sequence. Glycosylation at positions 264 and 270 of the CII 256-270 peptide did not change the anchor positions used for binding to the Aq or Ap molecules. The autologous form of the peptide (with aspartic acid at position 266) bound with lower affinity to the Aq molecule as compared with the heterologous peptide. The variable affinity displayed by the immunodominant CII 256-270 peptide for different MHC class II molecules, the identification of MHC and TCR contacts and the significance of glycosylation of these have important implications for the understanding of the molecular basis for inherited MHC class II-associated susceptibility to CIA and in turn, for development of novel treatment strategies in this disease.

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The expression of genes specifically in B cells is of great interest in both experimental immunology as well as in future clinical gene therapy. We have constructed a novel enhanced B cell-specific promoter (Igk-E) consisting of an immunoglobulin kappa (Igk) minimal promoter combined with an intronic enhancer sequence and a 3 0 enhancer sequence from Ig genes. The Igk-E promoter was cloned into a lentiviral vector and used to control expression of enhanced green fluorescent protein (eGFP). Transduction of murine B-cell lymphoma cell lines and activated primary splenic B cells, with IgK-E-eGFP lentivirus, resulted in expression of eGFP, as analysed by flow cytometry, whereas expression in non-B cells was absent. The specificity of the promoter was further examined by transducing Lin À bone marrow with Igk-E-eGFP lentivirus and reconstituting lethally irradiated mice. After 16 weeks flow cytometry of lymphoid tissues revealed eGFP expression by CD19 + cells, but not by CD3 + , CD11b + , CD11c + or Gr-1 + cells. CD19 + cells were comprised of both marginal zone B cells and recirculating follicular B cells. Activated human peripheral mononuclear cells were also transduced with Igk-E-eGFP lentivirus under conditions of selective B-cell activation. The Igk-E promoter was able to drive expression of eGFP only in CD19 + cells, while eGFP was expressed by both spleen focusforming virus and cytomegalovirus constitutive promoters in CD19 + and CD3 + lymphocytes. These data demonstrate that in these conditions the Igk-E promoter is cell specific and controls efficient expression of a reporter protein in mouse and human B cells in the context of a lentiviral vector.

The pig major histocompatibility complex DRB genes were studied by polymerase chain reaction (PCR) amplification of exon 2 from eight domestic pigs and two European wild boars. Sequence comparisons together with a phylogenetic analysis showed the existence of at least three DRB genes of which only one appears to be expressed. The two putative DRB pseudogenes contained deletions in exon 2, making it possible to confirm the presence of three non-allelic DRB genes by analyzing the length polymorphism of the amplified PCR products. The expressed gene shows allelic polymorphism at the same positions as in the human DRB1 gene. In addition, this pig gene shows extensive allelic polymorphism at positions 84-88, whereas, e.g., human DRB genes do not. Surprisingly, the two putative DRB pseudogenes also display a considerable amount of allelic polymorphism, albeit of a different character as compared with the expressed DRB gene. Short stretches of sequences are shared between individual alleles at different loci. These sequence similarities cannot be due to natural selection, since two of the three DRB genes involved are polymorphic pseudogenes constituting allelic series that have diverged after the inactivation event. Instead, the results indicate that the sequences have been exchanged between the DRB genes by intergenic recombination.