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 γ (IFN-γ) were present in the serum during IL-12 treatment, but subsequent antigen-specific IFN-γ 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-γ–deficient, inducible nitric oxide synthase (iNOS)-deficient, and Bcl-2lck 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-γ, 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.

Respiratory syncytial virus (RSV) causes bronchiolitis, the main cause of infantile hospitalization. Immunity against reinfection is poor, and there is great interest in boosting vaccine responses using live vectors expressing host cytokines. We therefore constructed a recombinant RSV expressing murine interleukin 18 (RSV/IL-18), a cytokine capable of inducing strong antiviral immune responses. In vitro RSV/IL-18 replicated at wild-type levels and produced soluble IL-18. In naïve BALB/c mice, RSV/IL-18 infection significantly increased both IL-18 mRNA and protein and attenuated the peak viral load 3-fold. Despite a reduced viral load, RSV/IL-18 infection caused a biphasic weight loss at days 2 and 6 postinfection that was not seen in wild-type infection. Day 2 disease was associated with enhanced pulmonary natural killer (NK) cell numbers and activity and was prevented by NK cell depletion during infection; day 6 disease was correlated with CD8 T-cell recruitment and was enhanced by NK cell depletion. IL-18 expression during priming also enhanced RSVspecific antibody responses and T-cell responses on secondary RSV infection. Therefore, while IL-18 boosted antiviral immunity and reduced the viral load, its coexpression worsened disease. This is the first recombinant RSV with this property, and these are the first studies to demonstrate that NK cells can induce pathology during pulmonary viral infections.Human respiratory syncytial virus (RSV) is the major cause of infantile viral bronchiolitis worldwide (27). RSV infection results in lower respiratory tract illness (LRTI) in 25 to 40% of children, with 0.5 to 2% requiring hospitalization. Immunity against RSV is short-lived and incomplete, and reinfection with the same strain can occur regularly throughout life. In elderly persons, RSV causes morbidity and mortality that match those resulting from influenza A virus infection in those vaccinated against seasonal influenza; there is currently no RSV vaccine. The relative roles of the virus and the immune response in causing disease are much debated (9).The proinflammatory cytokine interleukin 18 (IL-18) is produced by a wide range of cells, including macrophages, neutrophils, and airway epithelial cells, and is a potent promoter of immune responses. It induces gamma interferon (IFN-␥) production from T cells without the requirement for T-cell receptor (TCR) engagement, an effect that is greatly enhanced by the presence of IL-12. Together, these cytokines enhance T helper cell type 1 (Th1) responses (15,25,32). IL-18 also directly promotes NK cell activation and proliferation and has been shown to drive antiviral immunity in a number of situations (18,24,26). In the presence of IL-12, IL-18 is also capable of preventing IgE production (34), but in the absence of IL-12 (or with an abundance of IL-2 or IL-4), it promotes the differentiation of Th2 cells and induces nonspecific IgE production (33, 35). Increased RSV titers are seen in IL-18 knockout mice (2), and polymorphisms in the IL-18 promoter are associated with ...

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.

No abstract

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.

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.

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-β, but did induce TNF-α 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-α and TNF-β, and induced TCR Vβ-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.

Superantigens stimulate T cells bearing certain TCR β-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 (TSST88–194-TM) was also constructed. Tumor cells precoated with TSST1-TM or TSST88–194-TM stimulated proliferation of human peripheral blood lymphocytes in vitro whereas uncoated tumor cells did not. Mice preimmunized with TSST1-TM- or TSST88–194-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 TSST88–194-TM fusion proteins represent a useful new strategy for attaching superantigens or potentially other proteins onto tumor cell surfaces without genetic manipulation.