T-cell receptor antagonists induce Vav phosphorylation by selective activation of Fyn kinase

T cell receptor (TCR) antagonists inhibit antigen-induced T cell activation and by themselves fail to induce phenotypic changes associated with T cell activation. However, we have recently shown that TCR antagonists are inducers of antigen-presenting cell (APC)–T cell conjugates. The signaling pathway associated with this cytoskeleton-dependent event appears to involve tyrosine phosphorylation and activation of Vav. In this study, we investigated the role played by the protein tyrosine kinases Fyn, Lck, and ZAP-70 in antagonist-induced signaling pathway. Antagonist stimulation increased tyrosine phosphorylation and kinase activity of Fyn severalfold, whereas little or no increase in Lck and ZAP-70 activity was observed. Second, TCR stimulation of Lck−, Fynhi Jurkat cells induced strong tyrosine phosphorylation of Vav. In contrast, minimal increase in tyrosine phosphorylation of Vav was observed in Lckhi, Fynlo Jurkat cells. Finally, study of T cells from a Fyn-deficient TCR transgenic mouse also showed that Fyn was required for tyrosine phosphorylation and activation of Vav induced by both antagonist and agonist peptides. The deficiency in Vav phosphorylation in Fyn-deficient T cells was associated with a defect in the formation of APC–T cell conjugates when T cells were stimulated with either agonist or antagonist peptide. We conclude from these results that Vav is a selective substrate for Fyn, especially under conditions of low-affinity TCR-mediated signaling, and that this signaling pathway involving Fyn, Vav, and Rac-1 is required for the cytoskeletal reorganization that leads to T cell–APC conjugates and the formation of the immunologic synapse.

Virus-specific CD8+ T cell numbers are maintained during γ-herpesvirus reactivation in CD4-deficient mice

The murine γ-herpesvirus 68 replicates in epithelial sites after intranasal challenge, then persists in various cell types, including B lymphocytes. Mice that lack CD4+ T cells (I-Ab−/−) control the acute infection, but suffer an ultimately lethal recrudescence of lytic viral replication in the respiratory tract. The consequences of CD4+ T cell deficiency for the generation and maintenance of murine γ-herpesvirus 68-specific CD8+ set now have been analyzed by direct staining with viral peptides bound to major histocompatibility complex class I tetramers and by a spectrum of functional assays. Both acutely and during viral reactivation, the CD8+ T cell responses in the I-Ab−/− group were no less substantial than in the I-Ab+/+ controls. Indeed, virus-specific CD8+ T cell numbers were increased in the lymphoid tissue of clinically compromised I-Ab−/− mice, although relatively few of the potential cytotoxic T lymphocyte effectors were recruited back to the site of pathology in the lung. Thus the viral reactivation that occurs in the absence of CD4+ T cells was not associated with any exhaustion of the virus-specific cytotoxic T lymphocyte response. It seems that CD8+ T cells alone are insufficient to maintain long-term control of this persistent γ-herpesvirus.

Dependence of lymphopenia-induced T cell proliferation on the abundance of peptide/ MHC epitopes and strength of their interaction with T cell receptors

Factors that affect naïve T cell proliferation in syngeneic lymphopenic hosts were investigated. 2C T cell receptor (TCR) transgenic T cells lacking both CD8 and CD4 survived but hardly proliferated. Proliferation of CD8+ 2C cells was proportional to the abundance of cognate peptide/MHC complexes and was severely inhibited by injection of anti-CD8 antibody. Weakly reactive self-peptides slightly enhanced CD8+ 2C cell proliferation whereas a potent agonist peptide promoted much more rapid proliferation, but inflammation-stimulating adjuvant had only a small effect on the rate of cell proliferation. The findings suggest that under uniform lymphopenic conditions, the widely different rates of proliferation of T cells expressing various TCR, or the same TCR in the presence or absence of CD8, reflect the strength of interaction between TCR and MHC associated with particular self-peptides.

Identification and modulation of a naturally processed T cell epitope from the diabetes-associated autoantigen human glutamic acid decarboxylase 65 (hGAD65)

T cell recognition of autoantigens is critical to progressive immune-mediated destruction of islet cells, which leads to autoimmune diabetes. We identified a naturally presented autoantigen from the human islet antigen glutamic acid decarboxylase, 65-kDa isoform (GAD65), by using a combination of chromatography and mass spectrometry of peptides bound by the type I diabetes (insulin-dependent diabetes mellitus, IDDM)-associated HLA-DR4 molecule. Peptides encompassing this epitope-stimulated GAD65-specific T cells from diabetic patients and a DR4-positive individual at high risk for developing IDDM. T cell responses were antagonized by altered peptide ligands containing single amino acid modifications. This direct identification and manipulation of GAD65 epitope recognition provides an approach toward dissection of the complex CD4+ T cell response in IDDM.

CD4+ T cell recognition of MHC class II-restricted epitopes from NY-ESO-1 presented by a prevalent HLA DP4 allele: Association with NY-ESO-1 antibody production

NY-ESO-1 is a tumor-specific shared antigen with distinctive immunogenicity. Both CD8+ T cells and class-switched Ab responses have been detected from patients with cancer. In this study, a CD4+ T cell line was generated from peripheral blood mononuclear cells of a melanoma patient and was shown to recognize NY-ESO-1 peptides presented by HLA-DP4, a dominant MHC class II allele expressed in 43–70% of Caucasians. The ESO p157–170 peptide containing the core region of DP4-restricted T cell epitope was present in a number of tumor cell lines tested and found to be recognized by both CD4+ T cells as well as HLA-A2-restricted CD8+ T cells. Thus, the ESO p157–170 epitope represents a potential candidate for cancer vaccines aimed at generating both CD4+ and CD8+ T cell responses. More importantly, 16 of 17 melanoma patients who developed Ab against NY-ESO-1 were found to be HLA-DP4-positive. CD4+ T cells specific for the NY-ESO-1 epitopes were generated from 5 of 6 melanoma patients with NY-ESO-1 Ab. In contrast, no specific DP4-restricted T cells were generated from two patients without detectable NY-ESO-1 Ab. These results suggested that NY-ESO-1-specific DP4-restricted CD4+ T cells were closely associated with NY-ESO-1 Ab observed in melanoma patients and might play an important role in providing help for activating B cells for NY-ESO-1-specific Ab production.

Localization of CD4+ T cell epitope hotspots to exposed strands of HIV envelope glycoprotein suggests structural influences on antigen processing

The spectrum of immunogenic epitopes presented by the H2-IAb MHC class II molecule to CD4+ T cells has been defined for two different (clade B and clade D) HIV envelope (gp140) glycoproteins. Hybridoma T cell lines were generated from mice immunized by a sequential prime and boost regime with DNA, recombinant vaccinia viruses, and protein. The epitopes recognized by reactive T cell hybridomas then were characterized with overlapping peptides synthesized to span the entire gp140 sequence. Evidence of clonality also was assessed with antibodies to T cell receptor Vα and Vβ chains. A total of 80 unique clonotypes were characterized from six individual mice. Immunogenic peptides were identified within only four regions of the HIV envelope. These epitope hotspots comprised relatively short sequences (≈20–80 aa in length) that were generally bordered by regions of heavy glycosylation. Analysis in the context of the gp120 crystal structure showed a pattern of uniform distribution to exposed, nonhelical strands of the protein. A likely explanation is that the physical location of the peptide within the native protein leads to differential antigen processing and consequent epitope selection.

Protein quantification from complex protein mixtures using a proteomics methodology with single-cell resolution

We have developed an extremely sensitive technique, termed immuno-detection amplified by T7 RNA polymerase (IDAT) that is capable of monitoring proteins, lipids, and metabolites and their modifications at the single-cell level. A double-stranded oligonucleotide containing the T7 promoter is conjugated to an antibody (Ab), and then T7 RNA polymerase is used to amplify RNA from the double-stranded oligonucleotides coupled to the Ab in the Ab-antigen complex. By using this technique, we are able to detect the p185her2/neu receptor from the crude lysate of T6–17 cells at 10−13 dilution, which is 109-fold more sensitive than the conventional ELISA method. Single-chain Fv fragments or complementarity determining region peptides of the Ab also can be substituted for the Ab in IDAT. In a modified protocol, the oligonucleotide has been coupled to an Ab against a common epitope to create a universal detector species. With the linear amplification ability of T7 RNA polymerase, IDAT represents a significant improvement over immuno-PCR in terms of sensitivity and has the potential to provide a robotic platform for proteomics.

The role of antimicrobial peptides in animal defenses

It is becoming clear that the cationic antimicrobial peptides are an important component of the innate defenses of all species of life. Such peptides can be constitutively expressed or induced by bacteria or their products. The best peptides have good activities vs. a broad range of bacterial strains, including antibiotic-resistant isolates. They kill very rapidly, do not easily select resistant mutants, are synergistic with conventional antibiotics, other peptides, and lysozyme, and are able to kill bacteria in animal models. It is known that bacterial infections, especially when treated with antibiotics, can lead to the release of bacterial products such as lipopolysaccharide (LPS) and lipoteichoic acid, resulting in potentially lethal sepsis. In contrast to antibiotics, the peptides actually prevent cytokine induction by bacterial products in tissue culture and human blood, and they block the onset of sepsis in mouse models of endotoxemia. Consistent with this, transcriptional gene array experiments using a macrophage cell line demonstrated that a model peptide, CEMA, blocks the expression of many genes whose transcription was induced by LPS. The peptides do this in part by blocking LPS interaction with the serum protein LBP. In addition, CEMA itself has a direct effect on macrophage gene expression. Because cationic antimicrobial peptides are induced by LPS and are able to dampen the septic response of animal cells to LPS, we propose that, in addition to their role in direct and lysozyme-assisted killing of microbes, they have a role in feedback regulation of cytokine responses. We are currently developing variant peptides as therapeutics against antibiotic-resistant infections.

Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells.

Complementing reporter genes provide biological indicators of coincident expression of proteins in cells. We have adapted intracistronic complementation of the Escherichia coli lacZ gene for use in mammalian cells. Enzymatic activity detectable by quantitative biochemical assay, flow cytometry, or microscopy is produced upon convergent expression of two distinct mutant lacZ peptides within single cells, or upon fusion of cells expressing such mutants. A novel fluorescent substrate for beta-galactosidase (Fluor-X-Gal) increases detection and permits simultaneous microscopic visualization of other fluorescent markers. The enzymatic complementation described here should facilitate studies of cell fusion, cell lineage, and signal transduction, by producing activity only when two proteins are expressed at the same time and place in intact cells.

The natural killer cell receptor Ly-49A recognizes a peptide-induced conformational determinant on its major histocompatibility complex class I ligand.

Natural killer (NK) cells are inhibited from killing cellular targets by major histocompatibility complex (MHC) class I molecules. In the mouse, this can be mediated by the Ly-49A NK cell receptor that specifically binds the H-2Dd MHC class I molecule, then inhibits NK cell activity. Previous experiments have indicated that Ly-49A recognizes the alpha 1/alpha 2 domains of MHC class I and that no specific MHC-bound peptide appeared to be involved. We demonstrate here that alanine-substituted peptides, having only the minimal anchor motifs, stabilized H-2Dd expression and provided resistance to H-2Dd-transfected, transporter associated with processing (TAP)-deficient cells from lysis by Ly-49A+ NK cells. Peptide-induced resistance was blocked only by an mAb that binds a conformational determinant on H-2Dd. Moreover, stabilization of "empty" H-2Dd heavy chains by exogenous beta 2-microglobulin did not confer resistance. In contrast to data for MHC class I-restricted T cells that are specific for peptides displayed MHC molecules, these data indicate that NK cells are specific for a peptide-induced conformational determinant, independent of specific peptide. This fundamental distinction between NK cells and T cells further implies that NK cells are sensitive only to global changes in MHC class I conformation or expression, rather than to specific pathogen-encoded peptides. This is consistent with the "missing self" hypothesis, which postulates that NK cells survey tissues for normal expression of MHC class I.

Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex.

Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide-MHC complexes have been less well characterized. We have used a complete set of singly substituted analogs of a mouse MHC class I, Kk-restricted peptide, influenza hemagglutinin (Ha)255-262, to address the binding specificity of this MHC molecule. Using the same peptide-MHC complexes we determined the fine specificity of two Ha255-262-specific, Kk-restricted T cells, and of a unique antibody, pSAN, specific for the same peptide-MHC complex. Independently, a model of the Ha255-262-Kk complex was generated through homology modeling and molecular mechanics refinement. The functional data and the model corroborated each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody.

Expression and analysis of presenilin 1 in a human neuronal system: localization in cell bodies and dendrites.

Mutations in the recently identified presenilin 1 gene on chromosome 14 cause early onset familial Alzheimer disease (FAD). Herein we describe the expression and analysis of the protein coded by presenilin 1 (PS1) in NT2N neurons, a human neuronal model system. PS1 was expressed using recombinant Semliki Forest virions and detected by introduced antigenic tags or antisera to PS1-derived peptides. Immunoprecipitation revealed two major PS1 bands of approximately 43 and 50 kDa, neither of which were N-glycosylated or O-glycosylated. Immunoreactive PS1 was detected in cell bodies and dendrites of NT2N neurons but not in axons or on the cell surface. PS1 was also detected in BHK cells, where it was also intracellular and colocalized with calnexin, a marker for the rough endoplasmic reticulum. A mutant form of PS1 linked to FAD did not differ from the wild-type protein at the light microscopic level. The model system described here will enable studies of the function of PS1 in human neurons and the role of mutant PS1 in FAD.

Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L.

Proliferation of dispersed plant cells in culture is strictly dependent on cell density, and cells in a low-density culture can only grow in the presence of conditioned medium (CM). No known plant hormones have been able to substitute for CM. To quantify the mitogenic activity of CM, we examined conditions for the assay system using mechanically dispersed mesophyll cells of Asparagus officinalis L. and established a highly sensitive bioassay method. By use of this method, the mitogenic activity of CM prepared from asparagus cells was characterized: it was heat-stable, susceptible to pronase digestion, and resistant to glycosidase treatment. On the basis of these results, the mitogenic activity in CM was purified 10(7)-fold by column chromatography, and two factors named phytosulfokine-alpha and -beta (PSK-alpha and PSK-beta) were obtained. By amino acid sequence analysis and mass spectrometry, the structures of these two factors were determined to be sulfated pentapeptide (H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH) and sulfated tetrapeptide (H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-OH). PSK-alpha and PSK-beta were prepared by chemical synthesis and enzymatic sulfation. The synthetic peptides exhibited the same activity as the natural factors, confirming the structure for PSK-alpha and PSK-beta mentioned above. This is the first elucidation of the structure of a conditioned medium factor required for the growth of low-density plant cell cultures.

Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance induction.

Vaccination with synthetic peptides representing cytotoxic T lymphocyte (CTL) epitopes can lead to a protective CTL-mediated immunity against tumors or viruses. We now report that vaccination with a CTL epitope derived from the human adenovirus type 5 E1A-region (Ad5E1A234-243), which can serve as a target for tumor-eradicating CTL, enhances rather than inhibits the growth of Ad5E1A-expressing tumors. This adverse effect of peptide vaccination was rapidly evoked, required low doses of peptide (10 micrograms), and was achieved by a mode of peptide delivery that induces protective T-cell-mediated immunity in other models. Ad5E1A-specific CTL activity could no longer be isolated from mice after injection of Ad5E1A-peptide, indicating that tolerization of Ad5E1A-specific CTL activity causes the enhanced tumor outgrowth. In contrast to peptide vaccination, immunization with adenovirus, expressing Ad5E1A, induced Ad5E1A-specific immunity and prevented the outgrowth of Ad5E1A-expressing tumors. These results show that immunization with synthetic peptides can lead to the elimination of anti-tumor CTL responses. These findings are important for the design of safe peptide-based vaccines against tumors, allogeneic organ transplants, and T-cell-mediated autoimmune diseases.

Amyloid beta-protein reduces acetylcholine synthesis in a cell line derived from cholinergic neurons of the basal forebrain.

The characteristic features of a brain with Alzheimer disease (AD) include the presence of neuritic plaques composed of amyloid beta-protein (Abeta) and reductions in the levels of cholinergic markers. Neurotoxic responses to Abeta have been reported in vivo and in vitro, suggesting that the cholinergic deficit in AD brain may be secondary to the degeneration of cholinergic neurons caused by Abeta. However, it remains to be determined if Abeta contributes to the cholinergic deficit in AD brain by nontoxic effects. We examined the effects of synthetic Abeta peptides on the cholinergic properties of a mouse cell line, SN56, derived from basal forebrain cholinergic neurons. Abeta 1-42 and Abeta 1-28 reduced the acetylcholine (AcCho) content of the cells in a concentration-dependent fashion, whereas Abeta 1-16 was inactive. Maximal reductions of 43% and 33% were observed after a 48-h treatment with 100 nM of Abeta 1-42 and 50 pM of Abeta 1-28, respectively. Neither Abeta 1-28 nor Abeta 1-42 at a concentration of 100 nM and a treatment period of 2 weeks was toxic to the cells. Treatment of the cells with Abeta 25-28 (48 h; 100 nM) significantly decreased AcCho levels, suggesting that the sequence GSNK (aa 25-28) is responsible for the AcCho-reducing effect of Abeta. The reductions in AcCho levels caused by Abeta 1-42 and Abeta 1-28 were accompanied by proportional decreases in choline acetyltransferase activity. In contrast, acetylcholinesterase activity was unaltered, indicating that Abeta specifically reduces the synthesis of AcCho in SN56 cells. The reductions in AcCho content caused by Abeta 1-42 could be prevented by a cotreatment with all-trans-retinoic acid (10 nM), a compound previously shown to increase choline acetyltransferase mRNA expression in SN56 cells. These results demonstrate a nontoxic, suppressive effect of Abeta on AcCho synthesis, an action that may contribute to the cholinergic deficit in AD brain.