The human G1m1 allotype associates with CD4+ T-cell responsiveness to a highly conserved IgG1 constant region peptide and confers an asparaginyl endopeptidase cleavage site.

The human G1m1 allotype comprises two amino acids, D12 and L14, in the CH3 domain of IGHG1. Although the G1m1 allotype is prevalent in human populations, ~40% of Caucasiods are homozygous for the nG1m1 allotype corresponding to E12 and M14. Peptides derived from the G1m1 region were tested for their ability to induce CD4+ T-cell proliferative responses in vitro. A peptide immediately downstream from the G1m1 sequence was recognized by CD4+ T cells in a large percentage of donors (peptide CH315-29). CD4+ T-cell proliferative responses to CH315-29 were found at an increased frequency in nG1m1 homozygous donors. Homozygous nG1m1 donors possessing the HLA-DRB1*07 allele displayed the highest magnitudes of proliferation. CD4+ T cells from donors homozygous for nG1m1 proliferated to G1m1-carrying Fc-fragment proteins, whereas CD4+ T cells from G1m1 homozygous donors did not. The G1m1 sequence creates an enzymatic cleavage site for asparaginyl endopeptidase in vitro. Proteolytic activity at D12 may allow the presentation of the CH315-29 peptide, which in turn may result in the establishment of tolerance to this peptide in G1m1-positive donors. Homozygous nG1m1 patients may be more likely to develop CD4+ T-cell-mediated immune responses to therapeutic antibodies carrying the G1m1 allotype.

CD4+ T-cell epitope determination using unexposed human donor peripheral blood mononuclear cells.

The engineering of protein therapeutics to improve their stability, their efficacy, or to create "humanized" versions introduces changes to the amino acid sequence that are potential T-cell epitopes. Until now, there has been no available assay to detect primary T-cell responses to novel epitopes in humans. Currently available in vitro protocols for epitope determination rely on peripheral blood lymphocytes from environmentally exposed or disease-bearing donors. This severely limits the opportunity to confirm T-cell epitopes in novel proteins, because exposed donors are not available to novel or engineered proteins. Other methods for determining T-cell epitopes are either computer-modeled predictions based on potential binding to HLA molecules or the identification of peptides presented by HLA molecules removed from the surface of tumor cells or protein-pulsed antigen-presenting cells. Because HLA binding is necessary, but not sufficient, for T-cell responses, these methods must be validated by in vitro presentation assays. The authors describe a dendritic cell-based assay that identifies CD4+ T-cell epitopes in novel proteins using unexposed donors. Predicted T-cell epitopes in the protein of interest were confirmed using cells from two verified exposed donors. The major CD4+ T-cell epitope of the novel protein examined in this study associated with the expression of HLA DRb1*15. This assay reflects de novo priming in vitro, and it accurately identifies primary T-cell epitopes. This assay is a powerful tool for determining relevant immunostimulatory T-cell epitopes for all types of immunoregulatory applications.

Antigen-specific human antibodies from mice comprising four distinct genetic modifications.

Human sequence monoclonal antibodies, which in theory combine high specificity with low immunogenicity, represent a class of potential therapeutic agents. But nearly 20 years after Köhler and Milstein first developed methods for obtaining mouse antibodies, no comparable technology exists for reliably obtaining high-affinity human antibodies directed against selected targets. Thus, rodent antibodies, and in vitro modified derivatives of rodent antibodies, are still being used and tested in the clinic. The rodent system has certain clear advantages; mice are easy to immunize, are not tolerant to most human antigens, and their B cells form stable hybridoma cell lines. To exploit these advantages, we have developed transgenic mice that express human IgM, IgG and Ig kappa in the absence of mouse IgM or Ig kappa. We report here that these mice contain human sequence transgenes that undergo V(D)J joining, heavy-chain class switching, and somatic mutation to generate a repertoire of human sequence immunoglobulins. They are also homozygous for targeted mutations that disrupt V(D)J rearrangement at the endogenous heavy- and kappa light-chain loci. We have immunized the mice with human proteins and isolated hybridomas secreting human IgG kappa antigen-specific antibodies.

CD28-B7 interactions allow the induction of CD8+ cytotoxic T lymphocytes in the absence of exogenous help.

The activation requirements for the generation of CD8+ cytotoxic T cells (CTL) are poorly understood. Here we demonstrate that in the absence of exogenous help, a CD28-B7 interaction is necessary and sufficient for generation of class I major histocompatibility complex-specific CTL. Costimulation is required only during the inductive phase of the response, and not during the effector phase. Transfection of the CD28 counter receptor, B7, into nonstimulatory P815 cells confers the ability to elicit P815-specific CTL, and this response can be inhibited by anti-CD28 Fab or by the chimeric B7-binding protein CTLA4Ig. Anti-CD28 monoclonal antibody (mAb) can provide a costimulatory signal to CD8+ T cells when the costimulatory capacity of splenic stimulators is destroyed by chemical fixation. CD28-mediated signaling provokes the release of interleukin 2 (IL-2) from the CD8+ CTL precursors, as anti-CD28 mAb could be substituted for by the addition of IL-2, and an anti-IL-2 mAb can block the generation of anti-CD28-induced CTL. CD4+ cells are not involved in the costimulatory response in the systems examined. We conclude that CD8+ T cell activation requires two signals: an antigen-specific signal mediated by the T cell receptor, and an additional antigen nonspecific signal provided via a CD28-B7 interaction.

Two distinct immunoglobulin heavy chain isotypes in a primitive, cartilaginous fish, Raja erinacea.

Immunoglobulin heavy chain genes in Raja erinacea (little skate) are organized in clusters consisting of VH, DH, JH segments and CH exons (1). An immunoglobulin heavy chain mu-like isotype that exhibits 61-91% nucleotide sequence identity in coding segments to the Heterodontus francisci (horned shark) mu-type immunoglobulin is described. The overall length of the mu-type clusters is approximately 16 kb; transmembrane exons (TM1 and TM2) are located 3 to CH exon 4 (CH4). In three of four TM-containing genomic clones, a significant deletion is present in TM1. A second isotype of Raja immunoglobulin heavy chain genes has been detected by screening a spleen cDNA library with homologous Raja VH- and CH1-specific probes complementing the respective regions of the mu-like isotype. Weak hybridization with VH-specific probes and no discernable hybridization with C mu-specific probes were considered presumptive evidence for a second immunoglobulin isotype that nominally is designated as X-type. The Vx region of the X-type cDNA is approximately 60% identical at the nucleotide (nt) level to other Raja VH segments and thus represents a second VH family. Putative Dx and Jx sequences also have been identified. The constant region of the X-type immunoglobulin heavy chain gene consists of two characteristic immunoglobulin domains and a cysteine-rich carboxy terminal segment that are only partially homologous with the mu-like isotype. Genomic Southern blotting indicates that the V and C segments of both immunoglobulin heavy chain isotypes are encoded by complex multigene families. Vx- and different Cx-specific probes hybridize to different length transcripts in northern blot analyses of Raja spleen RNA suggesting that the regulation of expression of the X-type genes may involve differential RNA processing.

Teratogen metabolism: thalidomide activation is mediated by cytochrome P-450.

A metabolite of thalidomide generated by hepatic microsomes inhibited the attachment of tumor cells to concanavalin A-coated polyethylene. Evidence that metabolite formation is mediated by microsomal cytochrome P-450 is presented. Microsomes incubated with thalidomide underwent a type I spectral shift. Metabolite formation was reduced or eliminated by carbon monoxide, SKF-525A, metyrapone, and N-octylamine. Superoxide dismutase treatment had no effect. Metabolite formation required microsomes and NADPH and was dependent on the length of 37 degrees C incubation. The metabolite could be isolated by successive hexane and chloroform extractions. It is likely the inhibitory thalidomide metabolite was generated by a minor cytochrome P-450 species. Whether this thalidomide metabolite is involved in the drug's teratogenic activity remains to be shown.