Malignant glioma cells use MHC class II transactivator (CIITA) promoters III and IV to direct IFN-gamma-inducible CIITA expression and can function as nonprofessional antigen presenting cells in endocytic processing and CD4(+) T-cell activation.

Malignant gliomas (MGs), lethal human central nervous system (CNS) neoplasms, contain tumor infiltrating lymphocytes (TIL). Although MHC class II molecules are frequently detected on MG cells, suggesting that they may be capable of antigen (Ag) presentation to CD4(+) T cells, deficiencies in CD4(+) T-cell activation are associated with these nonimmunogenic tumors. We evaluated regulation of the MHC class II transactivator (CIITA), the key intermediate that controls class II expression, in MG cells and tested whether MG cells could process native Ag. After interferon-gamma (IFN-gamma) stimulation, MG cells upregulated CIITA and class II molecules. IFN-gamma-inducible CIITA expression in MG cells, as well as primary human astrocytes, was directed by two CIITA promoters, pIV, the promoter for IFN-gamma-inducible CIITA expression in nonprofessional antigen-presenting cells (APC), and pIII, the promoter that directs constitutive CIITA expression in B cells. Both pIII and pIV directed CIITA transcription in vivo in MGs and ex vivo in IFN-gamma-activated primary MG cultures. We also demonstrate for the first time that MG cells can process native Ag for presentation to CD4(+) MHC class II-restricted Th1 cells, indicating that MG cells can serve as nonprofessional APC. CIITA may be a key target to modulate MHC class II expression, which could augment immunogenicity, Ag presentation, and CD4(+) T-cell activation in MG therapy.

Requirement for endocytic antigen processing and influence of invariant chain and H-2M deficiencies in CNS autoimmunity.

The role of processing in antigen (Ag) presentation and T cell activation in experimental allergic encephalomyelitis (EAE) was evaluated in wild-type mice, mice that selectively express either Ii p31 or p41, and mice completely deficient in Ii or H-2M. We demonstrate that processing of myelin oligodendrocyte glycoprotein (MOG) is required for presentation of the dominant encephalitogenic MOG epitope, p35-55. Ii p31- and p41-expressing mice developed EAE with similar incidence to wild-type mice, although p41 mice had a more severe course. Ag-presenting cells (APCs) from Ii- or H-2M-deficient mice could present p35-55, but not MOG, demonstrating that these APCs could not process native MOG. Ii- and H-2M-deficient mice were not susceptible to EAE by immunization with p35-55 or MOG or by adoptive transfer of encephalitogenic T cells. However, CD4+ T cells from p35-55-immunized H-2M-deficient mice proliferated, secreted IFN-gamma, and transferred EAE to wild-type, but not H-2M-deficient, mice. Thus, EAE resistance in H-2M-deficient mice is not due to an inability of APCs to present p35-55, or an intrinsic defect in the encephalitogenic T cell repertoire, but reflects a defect in APC function. Our results indicate that processing is required for initial Ag presentation and CNS T cell activation and suggest that autopathogenic peptides of CNS autoantigen may not be readily available for presentation without processing.

Differential expression of B7 co-stimulatory molecules by astrocytes correlates with T cell activation and cytokine production.

Whether astrocytes utilize B7:CD28 co-stimulation to activate T cells mediating CNS inflammatory disease is controversial. In this report, primary astrocytes and murine astrocyte lines, generated by immortalization at two different times, day 7 or 45 of culture, were examined for their capability to express B7 co-stimulatory molecules and to participate in B7:CD28 co-stimulation. Following exposure to IFN-gamma, primary astrocytes and astrocyte lines up-regulated MHC class II and B7-2 (CD86) molecules. However, B7-1 (CD80) expression was not inducible on primary astrocytes examined after IFN-gamma stimulation beginning on day 7 or on astrocyte lines immortalized on day 7. B7-1 expression was inducible on primary astrocytes examined later and could be up-regulated on astrocyte lines immortalized later. Unlike B7-1, temporal discordant expression of other co-stimulatory/adhesion molecules was not observed. Both B7-1(-)/B7-2(+) and B7-1(+)/B7-2(+) astrocyte lines were capable of stimulating proliferation of encephalitogenic Th1 cells, utilizing B7-2 for B7:CD28 co-stimulation. However, lines derived from immortalization later (B7-1(+)/B7-2(+)) were more effective in stimulating proliferation of naive myelin basic protein-specific CD4(+) T cells. Astrocyte lines that expressed both B7-1 and B7-2 also stimulated Thp cells to secrete proinflammatory Th1 cytokines, whereas lines that expressed B7-2 only stimulated Thp cells to produce a Th2 cytokine pattern. Thus, we demonstrate for the first time that individual astrocytes can differentially express B7-1 molecules, which may correlate with their ability to stimulate proinflammatory and regulatory patterns of cytokine production. These results suggest that astrocytes have potential for both promoting and down-regulating T cell responses, and that temporal differences in expression of B7 molecules should be considered when evaluating immune regulation by astrocytes.

Interferon-tau: prospects for clinical use in autoimmune disorders.

Interferon-tau (IFN-tau) is a type I IFN originally discovered for its role as a pregnancy recognition hormone in ruminant animals such as sheep and cows. IFN-tau possesses all of the biological properties ascribed to the other type I IFNs including antiviral, antiproliferative and immunomodulatory activities. However, IFN-tau differs in that it is relatively nontoxic to cells at high concentrations as compared to the toxicity normally associated with IFNs-alpha and -beta and the type II IFN, IFN-gamma. IFN-tau was examined for its ability to prevent the development of experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), in humans. IFN-tau prevents development of EAE as effectively as IFN-beta, a type I IFN currently being used for the treatment of MS. Unlike IFN-beta, however, IFN-tau treated mice did not develop leucopenia or experience bodyweight loss indicative of toxicity. Superantigens can induce relapses in EAE, similar to those that are observed in patients with relapsing-remitting MS; IFN-tau blocks superantigen reactivation of EAE. The inhibitory effect of IFN-tau on induction of EAE and reactivation by superantigen involves suppression of myelin basic protein and superantigen activation of T cells as well as suppressed induction of inflammatory cytokines such as tumour necrosis factor-alpha. In addition, IFN-tau has been shown to reduce immunologically mediated spontaneous fetal resorption. Thus, IFN-tau has considerable potential for treatment of autoimmune and immunologically mediated disorders, including MS.

Astrocytes express elements of the class II endocytic pathway and process central nervous system autoantigen for presentation to encephalitogenic T cells.

Astrocytes are nonprofessional APCs that may participate in Ag presentation and activation of pathogenic CD4+ T cells involved in central nervous system (CNS) inflammatory diseases. Using immortalized pure astrocytes as a complement to the study of primary astrocytes, we investigated whether these astrocytes express elements involved in the class II endocytic pathway and if they are capable of processing native myelin basic protein (MBP), a step that could be necessary for initiating or perpetuating T cell recognition of this self-Ag in vivo. Upon IFN-gamma-stimulation, primary and immortalized astrocytes up-regulate class II transactivator (CIITA), invariant chain (Ii) (p31 and p41), H-2Ma, and H-2Mb. Analysis of CIITA cDNA sequences demonstrated that CIITA transcription in astrocytes is directed by a promoter (type IV) that mediates IFN-gamma-inducible CIITA expression and encodes a CIITA protein that differs in its N-terminal sequence from CIITA reported in professional APC. Comparing live and fixed APC for Ag presentation, we show that Ag processing by APC is required for presentation of native MBP to autopathogenic T cells specific for the major MBP epitope, Acl-11. We have observed that primary astrocytes and some, but not all, astrocyte lines in the absence of contaminating microglia are capable of processing and presenting native MBP, suggesting that there may be heterogeneity. Our study provides definitive evidence that astrocytes are capable of processing CNS autoantigen, indicating that astrocytes have potential for processing and presentation of CNS autoantigen to proinflammatory T cells in CNS autoimmune disease.

Perspectives on FIV vaccine development.

Feline immunodeficiency virus (FIV), discovered a decade ago, is the causative agent of feline immunodeficiency syndrome (FAIDS), a chronically degenerative, fatal disease in domestic cats. Our understanding of the immunopathogenesis of FIV has improved but the development of an effective therapy and prophylaxis has been slow, reflecting the remarkable adaptability of the virus to modern medical intervention. FIV vaccine development has had its successes and failures similar to those encountered in human immunodeficiency virus (HIV) vaccine research. This review summarizes the status of FIV vaccine research, including trials of conventional, recombinant subunit and recombinant vector-based vaccines, and potential mechanisms of vaccine protection. The lessons learned from the FIV model should provide new insights for the approaches toward the development of HIV vaccines.

Oral feeding of interferon tau can prevent the acute and chronic relapsing forms of experimental allergic encephalomyelitis.

IFN tau is a member of the type I IFN family but unlike IFN alpha and IFN beta, IFN tau lacks toxicity at high concentrations. Recently, ovine IFN tau was shown to prevent acute induction and superantigen reactivation of experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). In this report, we examined the ability of IFN tau when administered by oral feeding to block development of EAE. Oral feeding of INF tau prevented paralysis in the acute form of EAE in NZW mice and chronic-relapsing EAE in SJL/J mice. In addition, oral feeding of IFN tau at 10(5) U/dose was as effective as intraperitoneal (i.p.) injection in preventing chronic-relapsing EAE, and both forms of IFN tau administration resulted in IL10 production. Histological examination revealed no inflammatory lymphocytic infiltration to the CNS in IFN tau treated animals as compared to controls. Prolonged treatment of IFN tau was shown to be necessary for chronic-relapsing EAE since removal of IFN tau treatment by either oral feeding or i.p. injection resulted in onset of disease. Lastly, sera from SJL/J mice which received prolonged IFN tau treatment by oral feeding exhibited little to no development of anti-IFN tau antibodies. Thus, oral feeding of ovine IFN tau may be a successful form of IFN tau administration for treatment of autoimmune diseases such as MS and may circumvent potentially debilitative antibody responses.

CD4 T suppressor cells mediate interferon tau protection against experimental allergic encephalomyelitis.

Interferon tau is a type I IFN that was originally identified as a pregnancy recognition hormone produced by trophoblast cells. It is as potent an antiviral agent as IFN alpha and IFN beta, but lacks the toxicity associated with high concentrations of these IFNs in tissue culture and in animal studies. We recently showed that IFN tau, like IFN beta, can prevent the development of experimental allergic encephalomyelitis (EAE). We report here that IFN tau prevents EAE in mice by induction of suppressor cells and suppressor factors. Suppressor cells can be induced by IFN tau in tissue culture, and in vivo by either intraperitoneal injection or by oral administration to mice. Incubation of suppressor cells with myelin basic protein (MBP)-sensitized T cells blocked or delayed the MBP-induced proliferation. Further intraperitoneal injection of suppressor cells into mice blocked induction of EAE by MBP. Suppressor cells possessed the CD4 T cell phenotype, and produced soluble suppressor factors that inhibited MBP activation of T cells from EAE mice. The suppressor factors were found to be IL-10 and TGF beta, which acted synergistically to inhibit the MBP activation of T cells from EAE mice. These findings are important for understanding the mechanism(s) by which type I IFNs protect against autoimmune disease.

Superantigens as virulence factors in autoimmunity and immunodeficiency diseases.

Virulence factors are microbial products that are known to be harmful to the host and may assist in the pathogenesis of the micro-organism. Superantigens, including those produced by bacteria and viruses, clearly act as virulence factors. The clinical effects of superantigens can be not only acute but also chronic and complex. Recent evidence suggests that superantigens may play a central role in the pathogenesis of autoimmune and immunodeficiency disorders. It is our contention that superantigens, as environmental factors, can change a controllable disease into one that becomes relentless for susceptible individuals. To illustrate the detrimental effects of superantigens on disease outcome, modulation of experimental allergic encephalomyelitis by superantigen, as well as the potential role of superantigens in human immunodeficiency virus pathogenesis will be discussed. The information presented may provide valuable insight into the role of superantigens in autoimmunity and human immunodeficiency virus infection.

Superantigens: structure and relevance to human disease.

Superantigens are a class of immunostimulatory molecules produced by bacteria and viruses. Their potent immune effects are due to their unique ability to bind to the major histocompatibility complex (MHC) outside the antigen-binding cleft and to stimulate T cells in a T-cell receptor (TCR) Vbeta-specific manner. Structural studies have revealed the binding sites involved in the MHC/superantigen/TCR complex. The bacterial superantigens are responsible for a number of syndromes, including food poisoning and toxic shock syndrome, but their effects may be not only acute but also chronic and complex. Recent evidence suggests that superantigens may be relevant to the pathogenesis of autoimmune and immunodeficiency disorders. To illustrate the detrimental effects of superantigens on disease outcome, evidence demonstrating the modulation of experimental allergic encephalomyelitis, an animal model for multiple sclerosis, by superantigen, as well as the potential role of superantigens in HIV pathogenesis of AIDS, will be presented. The information presented may provide valuable insight into the role of superantigens in autoimmunity and HIV infection.

The IFN pregnancy recognition hormone IFN-tau blocks both development and superantigen reactivation of experimental allergic encephalomyelitis without associated toxicity.

Multiple sclerosis (MS) is an inflammatory demyelinating autoimmune disease if the central nervous system (CNS). Recently, the type I IFN, IFN-beta-1b was demonstrated to be a useful immunotherapy for MS. During treatment with IFN-beta-1b, toxicity at higher doses has been observed. IFN-tau, discovered for its role in the reproductive cycle, possesses all of the functions normally ascribed to the type I IFNs but lacks the toxicity normally associate with IFN treatment in vitro. We have examined the effects of IFN-tau treatment on experimental allergic encephalomyelitis (EAE), an animal model useful for the study of MS. EAE is a model of Ag-induced autoimmunity that can be modulated by bacterial superantigen to resemble the relapsing-remitting pattern of autoimmune disease observed in MS. IFN-tau was able to prevent development of EAE as effectively as IFN-beta but without associated toxicity such as lymphocyte suppression and weight loss. In addition, IFN-tau was able to prevent superantigen reactivation of EAE akin to the reduction in disease exacerbations observed in IFN-beta-1b treated MS patients. Mechanisms by which IFN-tau may prevent EAE include reduced proliferation in response to the autoantigen myelin basic protein and reduced TNF-alpha production. Thus, IFN-tau may prove to be a promising new IFN therapy for MS in light of its ability to prevent EAE and the lack of toxicity exhibited by this novel IFN.

Accelerated induction of experimental allergic encephalomyelitis in PL/J mice by a non-V beta 8-specific superantigen.

Superantigens such as the staphylococcal enterotoxins can play an important role in exacerbation of autoimmune disorders such as experimental allergic encephalomyelitis (EAE) in mice. In fact, superantigens can reactivate EAE in PL/J mice that have been sensitized to rat myelin basic protein (MBP). The T-cell subset predominantly responsible for disease in PL/J mice bears the V beta 8+ T-cell antigen receptor (TCR). The question arises as to whether T cells bearing other V beta specificities are involved in induction or reactivation of EAE with superantigen. Thus, we have investigated the ability of a non-V beta 8-specific superantigen, staphylococcal enterotoxin A (SEA) (V beta specificities 1, 3, 10, 11, and 17), to induce EAE in PL/J mice that have been previously protected from disease by anergy and deletion of V beta 8+ T cells. PL/J mice were first pretreated with the V beta 8-specific superantigen staphylococcal enterotoxin B (SEB) and then immunized with MBP. These mice exhibited V beta 8-specific anergy and depletion and did not develop EAE, even when further treated with SEB. However, administration of SEA to these same mice induced an initial episode of EAE which was characterized by severe hindleg paralysis and accelerated onset of disease. In contrast to SEB pretreatment, PL/J mice pretreated with SEA did develop EAE when immunized with MBP, and after resolution of clinical signs of disease these mice were susceptible to relapse of EAE induced by SEB but not by SEA. Thus, superantigens can activate encephalitogenic MBP-specific non-V beta 8+ T cells to cause EAE in PL/J mice. These data suggest that superantigens can play a central role in autoimmune disorders and that they introduce a profound complexity to autoimmune diseases such as EAE, akin to the complexity seen in multiple sclerosis.

Type I interferon inhibition of superantigen stimulation: implications for treatment of superantigen-associated disease.

The interferons (IFNs) are a family of secretory glycoproteins possessing potent antiviral, antiproliferative, antimicrobial, and immunomodulatory activities. It has been shown that the IFNs and superantigens have an important effect on the course of certain autoimmune disorders, and thus we have examined the effect of the type I and type II IFNs on superantigen-induced stimulation. The type I IFNs, alpha, beta, and tau, inhibited induction of T cell proliferation by several staphylococcal enterotoxin superantigens; the type II IFN, gamma, was without effect. The type I IFNs inhibited T cell proliferation to the same extent, approximately 50% at 10(3) units of IFN/ml, and in a dose-dependent manner. Consistent with inhibition of proliferation, the type I IFNs also inhibited IL-2 production as well as levels of IL-2 receptor expression. Inhibition was not increased by using the IFNs in combination, suggesting that they inhibited proliferation by the same mechanism. IFNs alpha and beta, but not IFN-tau, were toxic to cells at high concentrations (> or = 10(4) units/ml). Thus, the mechanism by which type I IFNs inhibit cell proliferation differs from that associated with their toxic effects. A partial reduction of V beta-specific superantigen-induced T cell expansion by type I IFNs was also demonstrated using flow cytometry. We recently showed that superantigens play an important role in the reactivation of experimental allergic encephalomyelitis. The potent antiproliferative activities of the type I IFNs strongly suggest the further study of their use as therapies for superantigen-associated diseases, such as multiple sclerosis and other autoimmune disorders, as well as toxic shock syndrome.

The C-terminus of IFN gamma is sufficient for intracellular function.

We have previously shown that murine interferon gamma (IFN gamma) and its C-terminal peptide, muIFN gamma (95-133), bind to a region on the cytoplasmic domain of the IFN gamma receptor contained in the synthetic peptide, MIR(253-287). This region of the murine receptor bears considerable homology (approximately 80%) to its human counterpart. Here we report that not only do human IFN gamma and the human IFN gamma C-terminal peptide, huIFN gamma(95-134), bind to the cytoplasmic domain of the human IFN gamma receptor, but also that this interaction is species non-specific. MuIFN gamma(95-133) binds to human IFN gamma receptor cytoplasmic peptide HIR(252-291), and huIFN gamma(95-133) binds to MIR(253-287). Furthermore, treatment of murine macrophage cell lines with C-terminal peptides of either murine or human IFN gamma results in 10-fold upregulation of MHC class II molecule expression and increased resistance to infection with vesicular stomatitis virus (VSV) (10(6)-10(9)-fold reduction in yield). These data suggest a direct role for the C-terminus of IFN gamma in the initiation of intracellular signalling processes and may be indicative of a more general mechanism of action for extracellular signalling molecules.

Can superantigens trigger sudden infant death?

A majority of sudden infant death syndrome (SIDS) victims have respiratory or gastrointestinal infections prior to death. This has led to an investigation of the role of pathogenic bacteria and the potentially lethal toxins they produce as triggers for sudden infant death. A small group of bacteria have been consistently identified in SIDS victims as compared to controls, and remarkably, three of these produce superantigenic toxins. Superantigens exert a powerful effect on the immune system, stimulating T-cells, which subsequently induces the formation of large amounts of cytokines. Generation of an overwhelming inflammatory response may lead to death by shock, or other, as yet unrecognized effects of the toxin on the respiratory or cardiac systems. A SIDS/superantigen model is proposed which may explain many of the pathological characteristics of SIDS and establish quantifiable markers for SIDS.

Multiple binding sites on the superantigen, staphylococcal enterotoxin B, imparts versatility in binding to MHC class II molecules.

To determine MHC class II molecule binding regions of staphylococcal enterotoxin B (SEB), we employed a structurally based approach in which eight overlapping peptides of the entire SEB molecule were synthesized to encompass discrete secondary structures based on the SEB crystalline structure. SEB peptides encompassing amino acid residues 1-33, 31-64 and 179-212 successfully competed with [125I]SEB for binding to DR1 transfected L cells. In contrast, SEB peptides encompassing amino acid residues 1-33, 124-154, 150-183 and 179-212 successfully competed with [125I]SEB for binding to Raji cells (HLA-DR3, DRw10, DQw1 and DQw2). In addition, the SEB peptide (124-154) inhibited the mitogenic function of SEB. Thus, we have identified multiple regions, including the C-terminus, of SEB that are involved in binding to MHC class II and have shown that these interactions are complex and dependent on the haplotype of the MHC class II molecule.

Staphylococcal enterotoxins can reactivate experimental allergic encephalomyelitis.

Staphylococcal enterotoxins (SEs) are one member of a unique group of molecules known as superantigens. They are potent T-cell activators and stimulate a large number of T cells bearing specific T-cell-receptor beta-chain variable regions. It has been proposed that superantigens may trigger autoimmune disorders by stimulation of autoreactive T cells with restricted beta-chain variable-chain usage. We investigated the effects of SEs B and A (SEB and SEA) on the reactivation of experimental allergic encephalomyelitis, an animal model for multiple sclerosis. We report that SEB can reinduce encephalitis multiple times in PL/J mice that had previously recovered from an acute episode. SEB was also able to induce encephalitis in mice previously immunized with myelin basic protein but did not show clinical signs of disease. In addition, it was observed that T cells from PL/J mice that had been previously activated by myelin basic protein in complete Freund's adjuvant or in complete Freund's adjuvant alone were resistant to the induction of anergy by SEB. To determine whether reactivation of experimental allergic encephalomyelitis was specific for SEB, another superantigen, SEA, was employed. It was found that SEA was also able to reinduce experimental allergic encephalomyelitis in mice previously recovered from an acute episode and those that had been previously immunized with myelin basic protein but did not show clinical signs of disease. These results indicate that SEs are capable of reactivating autoreactive T cells and inducing autoimmune disease.

Treatment of PL/J mice with the superantigen, staphylococcal enterotoxin B, prevents development of experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE), an antigen induced autoimmune disease, is mediated by V beta 8+ CD4+ T cells in PL/J mice after injection with the autoantigen, myelin basic protein (MBP). Recently the superantigen, staphylococcal enterotoxin B (SEB), has been shown to peripherally anergize and delete T cells in a V beta specific manner. By treatment of PL/J mice with SEB, we have been able to protect PL/J mice from the development of EAE. Two-color FACS analysis of the spleens of SEB treated mice showed depletion of V beta 8+ CD4+ T cells. Consistent with this observation, spleen cells of SEB treated mice that did not show signs of EAE could not be stimulated in vitro with SEB but did respond to SEA. Thus, V beta specific superantigens may prove to be a preventative therapy for autoimmune diseases mediated by V beta specific T lymphocytes.

Identification of binding domains on the superantigen, toxic shock syndrome-1, for class II MHC molecules.

Toxic shock syndrome toxin-1 (TSST-1) is a member of the staphylococcal enterotoxin superantigen family. In order to determine the regions on the TSST-1 molecule involved in binding to class II MHC, seven overlapping peptides of the entire TSST-1 molecule were synthesized and tested for their ability to compete with 125I-TSST-1 for binding to class II MHC on murine A20 cells and HLA on Raji cells. Peptides corresponding to N-terminal amino acid residues 39 through 68 and C-terminal residues 155 through 194 competed with 125I-TSST-1 for binding to class II MHC. Also, binding studies with class II MHC beta-chain peptides indicate that regions encompassed by I-A beta b(30-60) and I-A beta b(60-90) are binding regions for TSST-1. Thus, we have identified binding domains on the TSST-1 molecule for class II MHC molecule receptors on antigen presenting cells.