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In vaccine development, a major objective is to induce strong, specific T cell responses. This might be obtained by targeting antigen to cell surface molecules that efficiently channel the antigen into endocytic compartments for loading of MHC molecules. Antibodies have been used to deliver antigen; however, it is important to define optimal targets on antigen-presenting cells (APC) for efficient delivery. For this purpose, we have established a T cell readout that can be used to screen large numbers of different mAb for their ability to load MHC class II molecules. The novel human CD4+ T cell clone is specific for mouse Ig C kappa (40-48) and restricted by HLA-DR4 (DRA1,B1*0401). DR4 apparently presents both mouse and human C kappa 40-48, but there is no cross-reaction at the T cell level. B cells from DR4 transgenic mice spontaneously process and present the mouse C kappa peptide. The mouse C kappa -specific T cell readout was used to demonstrate that mouse mAb specific for human dendritic cell (DC)-specific ICAM-grabbing non-integrin (DC-SIGN), a novel DC-specific molecule, were 10- to 1000-fold more potent at inducing kappa-specific human CD4+ T cell proliferation compared to control mAb. Consistent with this finding, DC-SIGN-specific mAb were rapidly internalized upon binding and found in intracellular vesicles. These results strongly argue that DC-SIGN-specific mAb are channeled into the MHC class II presentation pathway. Thus, DC-SIGN could be an efficient target for antibody-mediated delivery of T cell epitopes in vaccine development.

To investigate the genetic background of anti-F(ab′)2autoantibodies and the mechanism behind their production we have analyzed 10 human monoclonal antibodies directed against IgG F(ab′)2and IgG Fab. They were all derived from peripheral blood by the EBV/hybridoma technique. Eight were from three healthy individuals and two from two patients with primary Sjögren's syndrome (pSS). They react with epitopes on distinct regions of IgG, including epitopes present on or near the hinge of IgG, epitopes on the Fdγ, and an antigenic determinant(s) present on λ light chains. These determinants are either exposed on the intact IgG molecule or revealed following pepsin or papain digestion. The VHgermline gene repertoire used is diverse and with considerable overlap with that used by rheumatoid factors (RF). The two IgG antibodies from normals are extensively mutated (13 and 24 mutations/VH), but with a replacement to silent mutation ratio in the CDRH1 + 2 of only 3.7. The IgM antibodies from normals are also heavily mutated (mean 10 mutations/VH). This suggests that anti-F(ab′)2from normals are generated by an antigen-driven somatic hypermutation mechanism. In contrast, the two IgM antibodies from pSS are virtually unmutated in both VHand VL. Together with published data of pSS RF and anti-Ro 52-kDa sequences (1–3), this suggests that there is an expanded population of naı̈ve B cells with autoantibody specificities in the peripheral blood of pSS patients.

To clone and characterize human anti-Ro/SSA autoantibodies from a patient with primary Sjögren's syndrome (pSS). Monoclonal antibodies (mAb) were raised from the peripheral blood of a patient with pSS using Epstein-Barr virus transformation and a hybridoma technique. Specificity was determined using cell extracts, recombinant Ro 52-kd, Ro 60-kd, and La proteins as well as Ro 52-kd peptides in enzyme-linked immunosorbent assay (ELISA) and Western blot. The immunofluorescence pattern was analyzed using cultured human and mouse cell lines. Complementary DNA was amplified by polymerase chain reaction, and Ig variable (V)–region genes were directly sequenced. Two human anti–Ro 52-kd mAb of IgM isotype, denoted SG1 and SG3, were cloned from the peripheral blood of a patient with pSS. The 2 mAb reacted with the Ro 52-kd antigen in cell extracts of human cell lines and mouse cell lines, and with purified human recombinant Ro 52-kd protein in ELISA and Western blot. SG1 reacted specifically with 1 peptide, amino acids 136–156, of the Ro 52-kd protein, and SG3 was mapped to react with a recombinant fragment representing amino acids 136–292. Immunofluorescence studies revealed cytoplasmic staining with both mAb. Both were encoded by VH3-family genes. SG1 was highly homologous to the DP-77 germ-line gene, with 2 replacement mutations and 1 silent. It utilized the DPL-11 germ-line gene from the Vλ2-family gene, with 1 silent mutation. SG3 was 100% homologous to the DP-47 germ-line gene, combined with a Vκ1-family gene that was 100% homologous to the A30 germ-line gene. Two human mAb were demonstrated to be specific for the Ro 52-kd protein and to be directed against 2 different epitopes, 1 linear and 1 conformation-dependent, within a region previously described to be immunodominant. Somatic hypermutation appeared to be of minor importance in generating these 2 specificities.