Sensitive detection of GM1 lipid rafts and TCR partitioning in the T cell membrane.

The cholesterol-rich lipid rafts on T cell membrane play important role in the formation of T cell receptor (TCR) signalosome upon receptor ligation. Analytical studies on the kinetics of lipid rafts formation and recruitment of protein receptors to lipid rafts are still limited by the use of a large number of cells. Herein, we describe a strategy for detecting fine alterations in the amount and distribution of glycosphingolipid (GM1) lipid rafts, and in the formation of GM1-TCR complexes in detergent-insoluble and -soluble compartments of the T cell membrane from a relative low number of cells. Using this strategy, we found that the GM1 moiety was physically associated with TCR in both detergent-insoluble and -soluble fractions. Shortly after ligation of CD3/TCR complex with a soluble CD3- epsilon mAb, the TCR was found mainly in the detergent-soluble fraction of the T cell membrane.

Enzymatically mediated engineering of multivalent MHC class II-peptide chimeras.

We previously reported the genetic engineering of the first soluble, bivalent major histocompatibility complex (MHC) class II-peptide ligand for T-cell receptor (TCR). This ligand binds stably and specifically to cognate T-cells and exhibits immunomodulatory effects in vitro and in vivo. The increase in valence of MHC class II-peptide ligands was shown to parallel their avidity for cognate TCRs and potency in stimulating cognate T-cells. We describe a new enzymatic method to increase the valence of MHC-peptide ligands by cross-linking the N-glycan moieties of dimeric MHC II-peptide units through a flexible, bifunctional polyethylene glycol linker. Using this method, we generated covalently stabilized tetravalent and octavalent MHC II-peptide ligands which bound stably and specifically to cognate TCR and preserved their structural integrity in blood and lymphoid organs for 72 h. Depending on the TCR/CD4 occupancy and degree of TCR/CD4 co-clustering, the multivalent MHC II-peptide ligands polarized efficiently the antigen-specific CD4(+) T-cells toward type 2 cell differentiation or induced T-cell anergy and apoptosis. The enzymatically mediated engineering of multivalent MHC-peptide ligands for cognate TCRs may provide rational grounds for the development of new therapeutic agents endowed with strong modulatory effects on antigen-specific T-cells.

CpG motifs of DNA vaccines induce the expression of chemokines and MHC class II molecules on myocytes.

Determining how an immune response is initiated after in vivo transfection of myocytes with plasmids encoding foreign antigens is essential to understand the mechanisms of intramuscular (i. m.) genetic immunization. Since myocytes are facultative antigen-presenting cells lacking MHC class II and co-stimulatory molecules, it was assumed that their unique role upon DNA vaccination is to synthesize and secrete the protein encoded by the plasmid. Here we describe that i. m. injection of unmethylated CpG motifs induced the expression of chemokines (monocyte chemotactic protein-1) and MHC class II molecules on myocytes. Our results indicate that immunostimulatory DNA sequences (CpG motifs) of DNA vaccines augment synthesis of chemokine by myocytes with subsequent recruitment of inflammatory cells secreting IFN-gamma, a potent cytokine that up-regulates the expression of MHC class II molecules on myocytes. A myoblast cell line triple transfected with plasmids encoding MHC class II molecules and an immunodominant CD4 T cell epitope of influenza virus presented the endogenously synthesized peptide and activated specific T cells. These findings suggest that one mechanism for the immunogenicity of DNA vaccines consists in the presentation of peptides to CD4 T cells by in vivo plasmid-transfected myocytes.

Antigen-specific downregulation of T cells by doxorubicin delivered through a recombinant MHC II--peptide chimera.

As the number of drugs with potential therapeutic use for T-cell-mediated diseases increases, there is a need to find methods of delivering such drugs to T cells. The major histocompatibility complex (MHC)--peptide complexes are the only antigen-specific ligands for the T-cell receptor (TCR) expressed on T cells, and they may be an appropriate drug delivery system. We engineered a soluble bivalent MHC class II-peptide chimera on the immunoglobulin scaffold (I-E(d)alpha beta/Fc gamma 2a/HA110-120, DEF) that binds stably and specifically to CD4 T cells recognizing the HA110-120 peptide. Doxorubicin, a powerful antimitogenic anthracycline, was enzymatically assembled on the galactose residues of a DEF chimera. The DEF-gal-Dox construct preserved both the binding capacity to hemagglutinin (HA)-specific T cells, and the drug toxicity. Brief exposure of HA-specific T cells to DEF-gal-Dox construct in vitro was followed by drug internalization in the lysosomes, translocation to the nucleus, and apoptosis. Administration of DEF-gal-Dox to mice expressing the TCR-HA transgene reduced the frequency of TCR-HA T cells in the spleen and thymus by 27% and 42%, and inhibited HA proliferative capacity by 40% and 60%, respectively. It has not been demonstrated previously that pharmacologically active drugs able to modulate T-cell functions can be delivered to T cells in an antigen-specific manner by soluble, bivalent MHC II-peptide chimeras.

Insights into the pathogenesis of type 1 diabetes: a hint for novel immunospecific therapies.

Type 1 diabetes is an organ-specific autoimmune disease whose incidence is increasing worldwide. At present, there is no effective therapy to prevent or cure this disease. The genetic background (MHC and non-MHC genes) and environmental factors (pathogens, drugs, and diet) are critical for the initiation of the autoimmune response against the pancreatic beta-cells. Recognition of the pancreatic autoantigens by T cells in a predetermined environment of antigen-presenting cells, costimulation, and cytokines is crucial for the selective activation of diabetogenic or protective/regulatory T cells. Once the autoimmune process is triggered, epitope spreading and sustaining the autoimmune responses by continuous antigen stimulation leads to expansion of effector cells, which launch the attack on the beta-cells. Despite of some controversy, most of the studies in humans and animal models suggest that CD4 (Th1) T cells are directly involved in the autoimmune attack by secretion of pro-inflammatory cytokines and recruitment of cytotoxic CD8 T cells. Secretion of anti-inflammatory cytokines by Th2 cells is protective against the disease. Therapy with peptides derived from major target antigens, such as glutamic acid decarboxylase 65 or proinsulin, can prevent the disease in animal models by rising protective Th2 cells. Herein, we review the recent progress in the immunopathogenesis of Type 1 diabetes and insights into the development of new diagnostic tools and antigen-specific immunomodulators, such as MHC-peptide chimeras.

Modulation of CD4 T cell function by soluble MHC II-peptide chimeras.

Peptides antigens of 8 to 24 amino acid residues in length that are derived from processing of foreign proteins by antigen presenting cells (APC), and then presented to T cells in the context of major histocompatibility complex molecules (MHC) expressed by APC, are the only physiological ligands for T cell receptor (TCR). Co-ligation of TCR and CD4 co-receptor on T cells by MHC II-peptide complexes (signal 1) leads to various T cell functions depending on the nature of TCR and CD4 co-ligation, and whether costimulatory receptors (signal 2) such as CD28, CTLA-4, CD40L are involved in this interaction. Recently, the advance of genetic engineering led to the generation of a new class of antigen-specific ligands for TCR, i.e., soluble MHC class I-, and MHC class II-peptide chimeras. In principle, these chimeric molecules consist of an antigenic peptide which is covalently linked to the amino terminus of alpha-chain in the case of MHC I, or beta-chains in the case of MHC II molecules. Conceptually, such TCR/CD4 ligands shall provide the signal 1 to T cells. Since soluble MHC-peptide chimeras showed remarkable regulatory effects on peptide-specific T cells in vitro and in vivo, they may represent a new generation of immunospecific T cell modulators with potential therapeutic applicability in autoimmune and infectious diseases. This review is focused on the immunomodulatory effects of soluble, MHC class II-peptide chimeras, and discuss these effects in the context of the most accepted theories on T cell regulation.

T-cell tolerance and autoimmune diabetes.

Herein we describe the major signaling events that occur in T-cells upon T-cell receptor (TCR) engagement, and the mechanisms responsible for the induction of T-cell anergy that may ultimately lead to the development of immunospecific therapies in T-cell mediated autoimmune diseases. A new type of antigen presenting molecule (dimeric MHC class-II/peptide, DEF) endowed with antigen-specific immunomodulatory effects such as induction of Th2 polarization and T-cell anergy is also described as a potential antidiabetogenic agent. According to our preliminary results, the MHC II/peptide-based approach may provide rational grounds for further development of antigen-specific immunotherapeutic agents such as human-like MHC lI/peptide chimeras endowed with efficient down-regulatory effects in CD4 T-cell-mediated autoimmune diseases such as Type 1 diabetes, multiple sclerosis, primary biliary cirrhosis, and rheumatoid arthritis.

Kinetics of anti-fibrillin-1 autoantibodies in MCTD and CREST syndrome.

Using a highly sensitive Radioimmunoassay (RIA), the kinetics of synthesis of anti-fibrillin (Fbn-1) autoantibodies were studied in 17 patients with mixed connective tissue disease (MCTD) and two with CREST syndrome calcinosis, Raynaud's oesophageal dismotility, sclerodectyly and teleangiectasis who were found to be positive for this autoimmune response. IgG autoantibodies specific for recombinant Fbn-1 (rFbn-1) (aa 369-425) were found in all patients excepting one with MCTD, multiple sclerosis, and dermatomyositis. IgM were found in fewer cases. Several kinetics patterns of anti-Fbn-1 autoantibodies were observed: a) long lasting persistence of IgG and IgM autoantibodies up to 14 years; b) fluctuation of antibodies during various periods up to 16 years; c) disappearance of antibody response after several years, and d) patients producing IgG but not IgM autoantibodies. No differences in the synthesis of autoantibodies were observed between MCTD patients with a stable disease, and those developing during the course features of systemic sclerosis (SSc), Sjogren's syndrome, or rheumatoid-like arthritis. In one patient displaying a lupus-like syndrome for 3 years, the appearance of anti-Fbn-1 autoantibodies coincided with the occurrence of MCTD and scleroderma. While the detection of anti-Fbn-1 autoantibodies may be clinically useful in differential diagnosis or eventual prognosis of patients with connective tissue diseases, their role in the pathogenesis of scleroderma syndromes requires further investigation.

Escape from self-tolerance leads to neonatal insulin-dependent diabetes mellitus.

Double transgenic (dTg) mice expressing the hemagglutinin (HA) of influenza virus under the insulin promoter and the TCR specific for the immunodominant CD4 T cell epitope of HA (HA110-120) develop insulin-dependent diabetes mellitus (IDDM). In order to gain information on the breaking down of neonatal self-tolerance we studied the occurrence of IDDM after birth. Our results showed that newborn mice develop fulminant IDDM characterized by occurrence of insulitis as early as 3 days after birth, followed by hyperglycemia by 7 days, and significant hypoinsulinemia by 28 days. The neonatal breakdown of self-tolerance of T cells positively selected in the thymus is supported by the facts that: (i) peripheral HA110-120 specific T cells from neonates are fully functional and proliferated upon stimulation with the nominal peptide, and (ii) peptide-specific T cells were accumulated in the pancreas of dTg mice as early as 3 days after birth. Our results demonstrate that diabetes occurring in young dTg mice is due to early activation of self-reactive T cells immediately after birth. Accumulation of specific T cells in the target organ leads to destruction of pancreatic beta-cells and IDDM. These mice may provide a useful model to evaluate new strategies for the prevention of diabetes.

Antigen-specific signaling by a soluble, dimeric peptide/major histocompatibility complex class II/Fc chimera leading to T helper cell type 2 differentiation.

Interaction between a T cell receptor (TCR) and various ligands, i.e. , anti-TCR antibodies, superantigens, peptides, or altered peptide ligands in the context of major histocompatibility complex (MHC) molecules can trigger different T helper cell (Th) effector functions. Herein, we studied the T cell response induced by a soluble, dimeric peptide/MHC class II chimera, namely hemagglutinin (HA)110-120/I-E(d)alphabeta/Fcgamma2a (DEF). We have previously demonstrated that the soluble DEF molecule binds stably and specifically to HA110-120-specific TCRs expressed by a T cell hybridoma. Administration of DEF in vivo induced differentiation of resting and activated peptide-specific T cells toward a Th2 response, as indicated by the increase of interleukin (IL)-4, IL-10, and specific immunoglobulin (Ig)G1 antibodies and decrease of IL-2, specific IgG2a antibodies, and cytotoxic T lymphocyte activity. In contrast to HA110-120 peptide presented by the DEF molecule to T cells, the nominal synthetic peptide induced a predominant Th1 response, and the PR8 virus-derived HA110-120 peptides induced a mixed Th1/Th2 response. Independent of antigen processing, soluble DEF was almost 2 logs more potent in stimulating cognate T cells than the nominal peptide. Polarization of cognate T cells toward the Th2 response occurred upon interaction of soluble DEF with TCR and CD4 molecules followed by early activation of p56(lck) and ZAP-70 tyrosine kinases, and negative signaling of the signal transducer and activator of transcription (STAT)4 pathway of Th1 differentiation. DEF-like molecules may provide a new tool to study the mechanisms of signaling toward Th2 differentiation and may also provide a potential immunotherapeutic approach to modulate autoreactive T cells toward protective Th2 immune responses.

Characterization of mutated protein encoded by partially duplicated fibrillin-1 gene in tight skin (TSK) mice.

Fibrillin-1 (Fbn-1) is a ubiquitous protein present in the extracellular matrix of various organs and it is a major component of microfibrils embedded in the core of elastic fibers. In humans, mutations or deletions of the Fbn-1 gene are associated with several genetic diseases. In addition, several microsatellite alleles near Fbn-1 gene were found associated with diffuse scleroderma. In TSK/+ mice, which develop a scleroderma-like syndrome, the Fbn-1 gene exhibits an inframe duplication of exons 17-40. In this study, we report that the synthesis and secretion of wild-type Fbn-1 in TSK/+ is higher than that of the mutated Fbn-1 protein excluding the possibility that TSK genetic defect is due to a loss of the wild allele. We also demonstrate for the first time that TGF-beta, which plays a crucial role in skin fibrosis, binds to both wild-type and mutated Fbn-1. The amount of bound TGF-beta was higher in mutated than wild-type Fbn-1 and appears related to the number of TGF-beta binding motifs.

Doxorubicin-induced cell death in highly invasive human gliomas.

Glioblastoma is the most invasive form of primary brain tumors, and is often refractory to chemotherapy. Herein, we provide evidence that two highly invasive human glioma cell lines U-87 MG and U-373 MG, entered apoptosis after 48 hours following 24 h growth arrest induced by Doxorubicin (10 micrograms/2 x 10(5) cells/ml). Apoptosis depended solely on the level of intracellular drug accumulation, and it was not related to a functional p53 tumor suppressor factor. The multidrug resistance gene 1 (mdr-1) encoded P-glycoprotein (P-gp) was weakly expressed in these cells upon exposure to Doxorubicin, and exerted no influence on the extent of cellular drug efflux. Drug efflux occurred only in U-373 MG glioma cells subsequent to physical damage of the membrane upon exposure to Doxorubicin. Pretreatment of tumor cells with 10 micrograms/ml Doxorubicin precluded tumor formation on the chorioallantoic membrane (CAM) of embryonated hen eggs. Single-dose application of 0.4 microgram Doxorubicin on CAM/U-87 MG and CAM/U-373 MG tumor transplants inhibited tumor invasion in CAM tissue by 40 to 50%. These data suggest that highly invasive glioblastomas can be driven to apoptosis following growth arrest induced by Doxorubicin, providing that intracellular drug accumulation suffices cytotoxic levels.

Antineoplastic efficacy of doxorubicin enzymatically assembled on galactose residues of a monoclonal antibody specific for the carcinoembryonic antigen.

We have developed a novel procedure to couple enzymatically the antineoplastic agent doxorubicin (Dox) on the galactose residues of a monoclonal antibody specific for the tumor-associated carcinoembryonic antigen. The synthesis of the immunoconjugate consists of covalent attachment of the NH2 terminus of Dox to oxidized galactose residues of desialylated monoclonal antibody, followed by concurrent stabilization of Schiff bases by mild reduction with pyridine borane. The immunoconjugate preserved both antibody specificity and drug cytotoxicity. At equimolar concentrations, the immunoconjugate was 8 times more cytotoxic against two carcinoembryonic antigen-expressing carcinoma cell lines, LoVo and SW-480, than Dox alone. The intracellular drug accumulation was 8-8.5 times higher than that obtained with free Dox, and >50% of the drug delivered by the conjugate was retained for 24 h in the tumor cells. Only 4 days after treatment with a single dose of immunoconjugate carrying 2.5 ng of Dox, LoVo and SW-480 tumor transplants on the chorioallantoic membrane of embryonated hen eggs showed reduced tumor-induced angiogenesis and tumor progression by half, with no detectable damage to surrounding tissues. In contrast, the same amount of free drug induced insignificant changes in tumor progression and tumor-induced angiogenesis. Enzymatically mediated, glycosidic coupling of antineoplastic agents to antibodies specific for tumor-associated antigens may represent a novel platform for the development of more efficient anticancer agents with reduced side effects.

Towards development of T-cell vaccines.

Recent studies on the recognition of antigens by CD4+ and CD8+ T cells have revealed new ways of preparing efficient T-cell vaccines. Here, Constantin Bona and colleagues discuss several approaches for the development of T-cell vaccines, with applications ranging from the induction of protective immunity against intracellular parasites to the development of therapeutic agents against autoimmune disorders, allergic diseases and cancer.

Antigen presentation by dendritic cells after immunization with DNA encoding a major histocompatibility complex class II-restricted viral epitope.

Intramuscular and intracutaneous immunization with naked DNA can vaccinate animals to the encoded proteins, but the underlying mechanisms of antigen presentation are unclear. We used DNA that encodes an A/PR/8/34 influenza peptide for CD4 T cells and that elicits protective antiviral immunity. DNA-transfected, cultured muscle cells released the influenza polypeptide, which then could be presented on the major histocompatibility complex class II molecules of dendritic cells. When DNA was injected into muscles or skin, and antigen-presenting cells were isolated from either the draining lymph nodes or the skin, dendritic, but not B, cells presented antigen to T cells and carried plasmid DNA. We suggest that the uptake of DNA and/or the protein expressed by dendritic cells triggers immune responses to DNA vaccines.

Presentation of a viral peptide assembled on the carbohydrate moieties of immunoglobulin does not require processing.

We have previously demonstrated that an immunodominant CD4 T cell epitope, HA110-120 of the hemagglutinin (HA) of the A/PR/8/34 influenza virus, enzymatically assembled on the carbohydrate moieties of self immunoglobulins (Ig) primed the precursors of peptide-specific T cells and induced efficient proliferation in vivo of naive lymphocytes from transgenic mice expressing the peptide-specific T cell receptor. Here, we show that an immuno-galacto-peptide construct, IgG-gal-HA, does not require intracellular or extracellular processing to present the peptide to the specific T cells. The presentation occurs following the binding of the IgG-gal-HA construct to Fc gamma receptor on the surface of antigen-presenting cells (APC), with concurrent interaction of the peptides to their neighboring major histocompatibility complex class II molecules. This mechanism of peptide presentation may harness the immune response in vivo by the engagement of APC with a low capacity of antigen processing, such as neonatal B cells. In addition, the enzymatic method of assembling various aminated compounds on the sugar moieties of Ig may offer novel perspectives on immuno-targeting of antagonist peptides, cytostatic drugs, and biologically active ligands of therapeutic use.

Immunogenicity of a contiguous T-B synthetic epitope of the A/PR/8/34 influenza virus.

A contiguously linked T-B synthetic viral epitope (110HA120-150HA159,T-B) was investigated for its potency in inducing humoral and cellular immune responses in vivo. The T-cell epitope 110HA120 corresponds to the site 1 hemagglutinin (HA) of the A/PR/8/34 (PR8) influenza virus and is recognized by CD4 T cells in association with I-Ed class II major histocompatibility complex molecules. The 150HA159 represents a major B-cell epitope of the HA protein. T-B dipeptide emulsified in Freund's complete adjuvant was able to induce strong antiviral antibody titers and a high frequency of specific T-cell precursors after a single inoculation in BALB/c mice. In contrast, immunization under identical conditions with equimolar mixtures of T and B peptides did not elicit antibody titers or a cellular immune response. As indicated by the isotypes of antiviral antibodies, the T-B dipeptide preferentially induced a Th1-like immune response. Challenge with T-B dipeptide, but not with T or B peptide alone, stimulated peptide-specific T memory cells in mice previously primed with PR8 virus or with T-B dipeptide. As a consequence, 71 and 57% of these mice, respectively, survived infection with two 100% lethal doses of PR8 virus. Our results suggest that, inasmuch as contiguity between T- and B-cell epitopes provides enough signaling capacity to trigger the mechanisms of T-B-cell cooperation in vivo, a T-B contiguous epitope may well represent a minimal built-in subunit vaccine. Aside from their potential bioavailability, the T-B contiguous epitopes may also represent attractive tools for investigating the molecular mechanisms of T-B-cell cooperation responsible for antiviral protection.

Protective immunity elicited by vaccination with DNA encoding for a B cell and a T cell epitope of the A/PR/8/34 influenza virus.

Numerous reports have demonstrated that immunization with plasmids bearing influenza virus hemagglutinin (HA) or nucleoprotein (NP) genes elicits humoral and cellular protective responses. Herein we describe the generation of a plasmid (pVH-TB) encoding for a VH region of a self-Ig in which both the major B cell epitope HA150-159 and the immunodominant CD4 T cell epitope HA110-120 of HA of the A/PR/8/34 influenza virus were genetically inserted in the CDR2 and CDR3 loops, respectively. Our results demonstrate unequivocally that i.m. injection of pVH-TB plasmid in BALB/c mice elicited specific cellular and humoral immune responses able to protect against infection with lethal doses of A/PR/8/34 influenza virus.