Mice rendered deficient in lymphotoxin (LT) by gene targeting in embryonic stem cells have no morphologically detectable lymph nodes or Peyer's patches, although development of the thymus appears normal. Within the white pulp of the spleen, there is failure of normal segregation of B and T cells. Spleen and peripheral blood contain CD4+CD8- and CD4-CD8+ T cells in a normal ratio, and both T cells subsets have an apparently normal lytic function. Lymphocytes positive for immunoglobulin M are present in increased numbers in both the spleen and peripheral blood. These data suggest an essential role for LT in the normal development of peripheral lymphoid organs.

Complement receptor 1 (CR1, CD35) and complement receptor 2 (CR2, CD21) have been implicated as regulators of B-cell activation. We explored the role of these receptors in the development of humoral immunity by generating CR1-and CR2-deficient mice using gene-targeting techniques. These mice have normal basal levels of IgM and of IgG isotypes. B-and T-cell (14)(15)(16)(17)(18). Experiments using the T-cell-dependent antigens from sheep red blood cells (SRBC) and keyhole limpet hemocyanin have suggested that the primary immune response deficit in mice treated with anti-mouse CR1 and CR2 mAb can be overcome by using high doses of the immunogen (14).Here we have further explored the role of these receptors in the development of humoral immunity by generating CR1-and CR2-deficient mice using gene targeting. Our data show that CR1 and CR2 play important roles in the B-cell response and are necessary for appropriate B-cell activation and Ab production at both low and high doses of antigen. In addition, we demonstrate that germinal center formation is retained in the absence of these proteins. These mice should provide an excellent model to study in detail the specific roles of CR1 and CR2 in the immune response.

SummaryWe have investigated whether sequence 67 to 74 shared by B chains of rheumatoid arthritis (RA)-associated HLA-DR molecules imparts a specific pattern of peptide binding. The peptide binding specificity of the RA-associated molecules, DRBI*0401, DRBI*0404, and the closely related, RA nonassociated DRBI*0402 was, therefore, determined using designer peptide libraries. The effect of single key residues was tested with site-directed mutants of DRBI*0401. The results have demonstrated striking differences between RA-linked and unlinked DR allotypes in selecting the portion of peptides that interacts with the 67-74 area. Most differences were associated with a single amino acid exchange at position 71 of the DR ~ chain, and affected the charge of residues potentially contacting position 71. The observed binding patterns permitted an accurate prediction of natural protein derived peptide sequences that bind selectively to RA-associated DR molecules. Thus, the 67-74 region, in particular position 71, induces changes of binding specificity that correlate with the genetic linkage of RA susceptibility. These findings should facilitate the identification of autoantigenic peptides involved in the pathogenesis of RA.

Human T-cell-mediated autoimmune diseases are genetically linked to particular alleles of MHC class II genes. Susceptibility to pemphigus vulgaris (PV), an autoimmune disease of the skin, is linked to a rare subtype of HLA-DR4 (DRB1*0402, 1 of 22 known DR4 subtypes). The PV-linked DR4 subtype differs from a rheumatoid arthritisassociated DR4 subtype (DRB1*0404) only at three residues (DR,3 67, 70, and 71). The disease is caused by autoantibodies against desmoglein 3 (DG), and T cells are thought to trigger the autoantibody production against this keratinocyte adhesion molecule. Based on the DRB1*0402 binding motif, seven candidate peptides of the DG autoantigen were identified. T cells from four PV patients with active disease responded to one of these DG peptides (residues 190-204); two patients also responded to DG-(206-220). T-cell clones specific for DG-(190-204) secreted high levels of interleukins 4 and 10, indicating that they may be important in triggering the production of DG-specific autoantibodies. peptide was presented by the disease-linked DRB1*0402 molecule but not by other DR4 subtypes. Site-directed mutagenesis of DRB1*0402 demonstrated that selective presentation of , which carries a positive charge at the P4 position, was due to the negatively charged residues of the P4 pocket (DR,8 70 and 71). DR8 71 has a negative charge in DRB1*0402 but a positive charge in other DR4 subtypes, including the DR4 subtypes linked to rheumatoid arthritis. The charge of the P4 pocket in the DR4 peptide binding site therefore appears to be a critical determinant of MHC-linked susceptibility to PV and rheumatoid arthritis.