Fas and Fas ligand expressed on cells of the immune system, not on the target tissue, control induction of experimental autoimmune uveitis.

The Fas-Fas ligand (FasL) interaction is important for maintaining lymphocyte homeostasis by signaling for activation-induced cell death. Mice homozygous for the lpr or gld mutations do not express functional Fas or FasL, respectively, and spontaneously develop progressive autoimmune symptoms. Recent studies implicated expression of FasL on immunologically privileged tissues in protection from immune-mediated damage. Conversely, tissue expression of Fas may facilitate damage. We evaluated the susceptibility of lpr and gld mice to induction of experimental autoimmune uveitis (EAU), a T cell-mediated autoimmune disease induced with retinal Ags, which targets the neural retina. gld as well as lpr mice immunized with a retinal Ag developed disease of lower incidence and severity than wild-type controls. Delayed hypersensitivity responses were not significantly different among immunized gld, lpr, or wild-type mice, although in vitro Ag-specific lymphocyte responses of the mutant mice were lower. To evaluate whether the diminished ability of gld and lpr mice to develop EAU was due to a defect at the level of the tissue or the immune system, radiation bone marrow chimeras constructed between wild-type and mutant mice were immunized to induce EAU. Mutant recipients of wild-type bone marrow, but not wild-type recipients of mutant bone marrow, developed normal disease scores. These results indicate that normal expression of Fas and of FasL on cells of the immune system is important for EAU expression. Unexpectedly, neither lack of Fas nor lack of FasL on the ocular tissues affected expression of EAU.

Interleukin 12 protects from a T helper type 1-mediated autoimmune disease, experimental autoimmune uveitis, through a mechanism involving interferon gamma, nitric oxide, and apoptosis.

Pathogenic effector T cells in experimental autoimmune uveitis (EAU) are T helper type 1-like, and interleukin (IL)-12 is required for their generation and function. Therefore, we expected that IL-12 administration would have disease-enhancing effects. Mice were immunized with a uveitogenic regimen of the retinal antigen interphotoreceptor retinoid-binding protein, treated with IL-12 (100 ng/d for 5 d), and EAU was assessed by histopathology. Unexpectedly, IL-12 treatment failed to enhance EAU in resistant strains and downregulated disease in susceptible strains. Only treatment during the first, but not during the second, week after immunization was consistently protective. High levels of interferon gamma (IFN-gamma) were present in the serum during IL-12 treatment, but subsequent antigen-specific IFN-gamma production in protected mice was diminished, as were IL-5 production, lymph node cell proliferation, and serum antibody levels. Treated mice had fewer cells and evidence of enhanced apoptosis in the draining lymph nodes. Unlike wild-type mice, IFN-gamma-deficient, inducible nitric oxide synthase (iNOS)-deficient, and Bcl-2(lck) transgenic mice were poorly protected by IL-12, whereas IL-10-deficient mice were protected. We conclude that administration of IL-12 aborts disease by curtailing development of uveitogenic effector T cells. The data are compatible with the interpretation that IL-12 induces systemic hyperinduction of IFN-gamma, causing activation of iNOS and production of NO, which mediates protection at least in part by triggering Bcl-2 regulated apoptotic deletion of the antigen-specific T cells as they are being primed.

Antitumor response elicited by a superantigen-transmembrane sequence fusion protein anchored onto tumor cells.

Superantigens stimulate T cells bearing certain TCR beta-chain variable regions when bound to MHC II molecules. We investigated whether the superantigen toxic shock syndrome toxin-1 (TSST1) could induce an antitumor immune response when anchored onto MHC II-negative tumor cells. Our approach was to facilitate association of TSST1 with cell membranes by fusing its coding region to the transmembrane region (TM) sequence of the proto-oncogene c-erb-B-2. TSST1-TM was expressed in bacteria with an N-terminal histidine tag and purified using nickel-agarose affinity chromatography. Purified TSST1-TM added to cultures of several different MHC II-negative tumor cells spontaneously associated with cell membranes, as detected by flow cytometry. Because superantigens can direct cell-mediated cytotoxicity against MHC II-positive cells, a TM fusion protein lacking the TSST1 MHC II binding domain (TSST(88-194)-TM) was also constructed. Tumor cells precoated with TSST1-TM or TSST(88-194)-TM stimulated proliferation of human peripheral blood lymphocytes in vitro whereas uncoated tumor cells did not. Mice preimmunized with TSST1-TM- or TSST(88-194)-TM-coated tumor cells mounted a systemic response that resulted in significant antitumor immunity as measured by regression of a parental tumor challenge. TSST1-TM and TSST(88-194)-TM fusion proteins represent a useful new strategy for attaching superantigens or potentially other proteins onto tumor cell surfaces without genetic manipulation.

Separation of function between the domains of toxic shock syndrome toxin-1.

Toxic shock syndrome toxin-1 (TSST1) is a superantigenic exotoxin produced by certain strains of Staphylococcus aureus Structurally, TSST1 is composed of two domains: residues determined by crystallography to directly interact with MHC II molecules reside within the N-terminal domain, while TSST1 residues critical for superantigenicity are within the C-terminal domain. In this study, we expressed the individual N- and C-terminal domains of TSST1 in Escherichia coli and studied their biologic activities. The TSST1 N-terminal domain (TSST(1-87)) did not induce proliferation of human PBLs or release of TNF-beta, but did induce TNF-alpha release. However, TSST1-elicited proliferation and release of both TNF isoforms were inhibited by a molar excess of TSST(1-87). The TSST1 C-terminal domain (TSST(88-194)) did not bind MHC II molecules, yet it elicited production of TNF-alpha and TNF-beta, and induced TCR Vbeta-specific proliferation similarly to intact TSST1. When covalently cross-linked to tumor cells, TSST(88-194) elicited a local in vivo antitumor response indistinguishable from TSST1. Although intact TSST1 causes lethal shock in vivo, the individual domains of this molecule may have therapeutic potential: the N-terminal domain to antagonize lymphocyte activation and TNF release during acute TSST1-precipitated toxic shock syndrome, and the C-terminal domain to stimulate antitumor responses without MHC II binding.

Expression and purification of recombinant toxicshock-syndrome toxin I.

Toxic-shock-syndrome toxin I (TSSTI), an exotoxin produced by certain strains of Staphylococcus aureus, has been closely associated with the pathogenesis of toxic shock syndrome. Outside the context of its staphylococcal host, TSSTI may offer therapeutic uses. We report here a strategy for high-level expression and simplified purification of TSSTI. We have subcloned the coding region for TSSTI into a vector containing an inducible T7 promoter sequence and expressed the protein in an Escherichia coli host strain. The recombinant TSSTI protein contained ten sequential histidine residues (Histag) at its N-terminus, which enabled its efficient purification using nickel-agarose-affinity resin. Histag-TSSTI (H-TSSTI) was further purified to homogeneity using a size-exclusion column. By this system, 80 mg of highly purified H-TSSTI can be consistently obtained per litre of culture in under 3 days. H-TSSTI retained biological activity and was unaffected by the presence of the Histag, as measured in lymphocyte proliferation assays.

Expression and radiolabeling of recombinant proteins containing a phosphorylation motif.

Radiolabeled proteins are useful in basic and clinical research. Current methods available for radiolabeling proteins involve chemical derivatization, resulting in multiple additions of radionuclides at random sites. A method designed to specifically localize the radionuclide to a unique site will offer advantages of control and predictability in radiolabeling. We have studied the usefulness of a prokaryotic expression vector by incorporating the coding sequence of a consensus phosphorylation motif (Kemptide) for the cAMP-dependent protein kinase A immediately upstream to the multiple cloning site. This vector was used to express five different recombinant proteins with a phosphorylation site at the amino terminus. In addition, the phosphorylation motif was introduced into two other proteins and expressed in yeast. The genetically engineered proteins were purified to homogeneity by affinity chromatography and radiolabeled with [gamma32P]ATP in vitro. All seven proteins used in this study could be expressed with the phosphorylation sequence at their amino terminus and specifically labeled without loss of biological activity. This strategy allows the option of labeling proteins to high or low specific radioactivity and holds potential for in vitro binding and in vivo localization studies.

Myasthenia gravis: effect on antibody binding of conservative substitutions of amino acid residues forming the main immunogenic region of the nicotinic acetylcholine receptor.

In Myasthenia Gravis most anti-acetylcholine receptor (AChR) antibodies are against a highly conserved area of the AChR alpha-subunit called the Main Immunogenic Region (MIR). Amino acid residues critical for MIR formation have been located within the sequence alpha 67-76. In the present study, binding of anti-AChR monoclonal antibodies (mAbs) to synthetic peptide analogues of the sequence alpha 67-76 of human and Torpedo AChRs containing conservative single-residue substitutions identified the amino acid residues most important to the antigenicity of the MIR sequence, and offered clues to its tridimensional structure. Conservative substitutions of residues Asn68 and Asp71 greatly diminished mAb binding, identifying them as critical contact residues for anti-MIR mAbs. Substitutions at Asp70 and Tyr72 moderately affected binding. Cross-reactive mAbs originally raised against Electrophorus AChR bound single residue-substituted synthetic peptides in a manner consistent with the possibility that Electrophorus AChR may have a glutamic acid residue at position alpha 70 or alpha 71. Substitutions at residues Asp/Ala70 and Val/Ile70 between human and Torpedo alpha-subunits may be size-compensating, suggesting these amino acids in the native AChR may be in closer proximity than proposed in previous models of the MIR.

Amino acid residues within the sequence region alpha 55-74 of Torpedo nicotinic acetylcholine receptor interacting with antibodies to the main immunogenic region and with snake alpha-neurotoxins.

The sequence region 55-74 of the alpha-subunit of the acetylcholine receptor (AChR) from Torpedo californica electroplax comprises the amino-terminal end of a sequence segment--residues alpha 67-76--forming the main immunogenic region (MIR), which is most frequently recognized by anti-AChR autoantibodies in myasthenia gravis. The synthetic sequence alpha 55-74 of Torpedo AChR binds alpha-bungarotoxin (alpha BTX), suggesting that amino acid residues within this sequence region may contribute to formation of an alpha BTX binding site. Using single-residue substituted synthetic analogues of the sequence alpha 55-74 of Torpedo AChR, in which each residue was sequentially substituted by either glycine or alanine, we sought identification of the amino acids involved in interaction with alpha-neurotoxins and with three different anti-MIR monoclonal antibodies (mAbs 6, 22, and 198). Substitution of Arg55, Arg57, Trp60, Arg64, Leu65, Arg66, Trp67, or Asn68 strongly inhibited alpha-toxin binding, whereas substitutions of Ile61, Val63, Pro69, Ala70, Asp71, or Tyr72 had marginal effects. Substitutions within the region alpha 68-72 significantly diminished binding of anti-MIR mAbs, although residue preferences differed among mAbs. Further, substituting Trp60 substantially reduced binding of mAb 198, and moderately affected binding of mAb 6, and substitution of Asp62 slightly but consistently affected binding of mAbs 6 and 22.