Regulating the discriminatory response to antigen by T-cell receptor

The cell-mediated immune response constitutes a robust host defense mechanism to eliminate pathogens and oncogenic cells. T cells play a central role in such a defense mechanism and creating memories to prevent any potential infection. T cell recognizes foreign antigen by its surface receptors when presented through antigen-presenting cells (APCs) and calibrates its cellular response by a network of intracellular signaling events. Activation of T-cell receptor (TCR) leads to changes in gene expression and metabolic networks regulating cell development, proliferation, and migration. TCR does not possess any catalytic activity, and the signaling initiates with the colocalization of several enzymes and scaffold proteins. Deregulation of T cell signaling is often linked to autoimmune disorders like severe combined immunodeficiency (SCID), rheumatoid arthritis, and multiple sclerosis. The TCR remarkably distinguishes the minor difference between self and non-self antigen through a kinetic proofreading mechanism. The output of TCR signaling is determined by the half-life of the receptor antigen complex and the time taken to recruit and activate the downstream enzymes. A longer half-life of a non-self antigen receptor complex could initiate downstream signaling by activating associated enzymes. Whereas, the short-lived, self-peptide receptor complex disassembles before the downstream enzymes are activated. Activation of TCR rewires the cellular metabolic response to aerobic glycolysis from oxidative phosphorylation. How does the early event in the TCR signaling cross-talk with the cellular metabolism is an open question. In this review, we have discussed the recent developments in understanding the regulation of TCR signaling, and then we reviewed the emerging role of metabolism in regulating T cell function.     

Convergent synthesis of neoglycopeptides by coupling of 2-bromoethyl glycosides to cysteine and homocysteine residues in T cell stimulating peptides

The 2-bromoethyl -glycosides of the disaccharide galabiose [Gal(1-4)Gal] and the trisaccharides globotriose [Gal(1-4)Gal(1-4)Glc] and 3-sialyllactose [Neu5Ac(2-3)Gal(1-4)Glc] have been prepared by improved routes. The 2-bromoethyl glycosides were then used in cesium carbonate promoted alkylations of the sulfhydryl groups of cysteine and homocysteine residues in T cell stimulating peptides. This convergent and general approach was used to prepare 16 neoglycopeptides which were obtained in 52–95% yields after purification by HPLC. 1H NMR spectroscopy revealed that -elimination and epimerization of neoglycopeptide stereocentres did not occur during the synthesis.     

Presentation of antigenic peptides by products of the major histocompatibility complex

Molecules encoded by the major histocompatibility complex (MHC) are polymorphic integral membrane proteins adapted to the presentation of peptide fragments of foreign antigens to antigen-specific T-cells. The diversity of infectious agents to which an immune response must be mounted poses a unique problem for receptor–ligand interactions; how can proteins whose polymorphism is necessarily limited bind an array of peptides almost infinite in its complexity? Both MHC class I and class II determinants have achieved this goal by harnessing a limited number of peptide side chains to anchor the epitope in place while exploiting conserved features of peptide structure, independent of their primary sequence. While class I molecules interact predominantly with the N- and C-termini of peptides, class II determinants form an extensive hydrogen bonding network along the length of the peptide backbone. Such a strategy ensures high-affinity binding, while selectively exposing the unique features of each ligand for recognition by the T-cell receptor. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Suppression of the bacterial antigen-specific T cell response and the dendritic cell migration to the lymph nodes by osteopontin

Osteopontin (OPN) has been reported to enhance the interferon (IFN)-gamma-producing Th1-type T cell response through the induction of interleukin (IL)-12 and the suppression of IL-10. We therefore investigated whether OPN could enhance Th1 induction by vaccination against bacterial antigen in vivo. Unexpectedly, the co-inoculation of OPN suppressed the induction of IFN-gamma-producing CD4(+) T cells and T cell proliferative response after the subcutaneous heat-killed Listeria monocytogenes(HKLM) immunization. These results suggest that OPN down-regulates T cell priming. Since dendritic cells (DC) play a pivotal role in T cell priming, we next analyzed the effects of OPN on DC. The addition of OPN into the culture of either bone marrow-derived immature DC or an immature DC line JAWSII showed no effects on the expression of MHC class II, CD80, and CD86 molecules before and after HKLM stimulation. Consistently, in vitro OPN-treated DC showed a normal antigen-presenting function to an established Listeria-specific Th1-type T cells. However, when the DC were transferred into the footpad with HKLM and OPN, the migration of the transferred DC into the regional LN was suppressed in comparison to the DC transferred with HKLM alone. Furthermore, the addition of OPN into the culture of the DC line and HKLM severely suppressed the HKLM-induced expression of CCR7 chemokine receptor which is an important factor in the migration of DC into LN. All the results suggest the existence of an OPN-mediated negative feedback mechanism in the T cell immune response through the regulation of DC migration.     

The activation of pigeon cytochrome c-specific T cell hybridomas by antigenic peptides is influenced by non-native sequences at the amino terminus of the determinant

We recently demonstrated that the sequence 95-104 contains all the residues necessary for direct recognition of the I-Ek restricted pigeon cytochrome c determinant but that residues located in sequences to the amino-terminal side of residue 95 improve the ability of peptides containing the sequence 95-104 to stimulate Ag-specific T cell clones. In this study we use synthetic peptides with amino-terminal leader sequences containing residues that differ with respect to their conformational stabilizing effects, charge, and hydrophilicity to examine the mechanism by which they modulate T cell recognition. Our findings indicate that the role of these residues in T cell stimulation is not related to their ability to stabilize alpha-helical secondary structure, nor do they appear to be processed differently. The leader sequences do not differentially influence the ability of the peptides to be presented by APC displaying Ia molecules of related haplotype, i.e., E alpha kE beta k, E alpha kE beta b, and E alpha kE beta s, to T cells which recognize the pigeon cytochrome c determinant on such presenting cells. Because antigenic potency correlates with the inclusion of hydrophobic residues and positively charged residues in the leader sequences, we discuss our findings with reference to the possibility that they non-specifically enhance the interaction of the antigenic peptides with the APC membrane.     

一种新型T细胞免疫原的原核表达及对口蹄疫疫苗的免疫增强作用

主要探讨了T细胞免疫原TI对口蹄疫疫苗的免疫增强作用。设计并原核表达产生了一种包含口蹄疫病毒VP1,VP4,3A和3D蛋白上多个T细胞表位与两个通用T细胞表位的T细胞免疫原,命名为TI;同时表达了O和Asia 1两个型口蹄疫病毒 VP1 蛋白的串联编码基因,表达产物命名为OA-VP1。将上述T细胞免疫原分别与OA-VP1和口蹄疫灭活疫苗按不同剂量组合免疫小鼠,于免疫后不同时间测定各组小鼠的体液与细胞免疫应答情况。采用微量中和试验检测小鼠O型和Asia1型中和抗体,采用流式细胞检测技术和测定γ-干扰素的水平来分析不同免疫组小鼠细胞免疫的水平。结果显示,与灭活疫苗或OA-VP1单独免疫组相比,添加TI抗原后灭活疫苗 (P＜0.01) 和OA-VP1免疫组(P＜0.05)小鼠均能产生高水平的特异性中和抗体;且CD4+ T细胞数量显著增多,IFN-γ产生水平显著升高 (P＜0.01)。说明TI抗原具有很好的诱导特异性体液与细胞免疫应答的作用,是一种很好的免疫增效剂,可作为口蹄疫蛋白亚单位疫苗和灭活疫苗中的一种有效成分,以提高疫苗的免疫效果。     

T-cell responses to multiple antigens presented by RNA-transfected APCs: a possible immunomonitoring tool

The increasingly deeper understanding of how the immune system recognizes and destroys tumors promises to enable the development of new approaches for gene therapy and immunotherapy. However, a treatment that induces safe and potentially beneficial antitumor responses is expected to require stepwise refinements. As part of this challenge, assays are needed to measure specific antitumor immune responses in patients. This becomes problematic because most tumors express unknown tumor antigens and it is often difficult to obtain sufficient amounts of viable tumor material for in vitro assays. Recently it was demonstrated that RNA derived from tumor cells stimulated T cells in an antigen-specific manner. These studies have formed the basis for the development of dendritic cell vaccines that express tumor antigens following translation of tumor RNA. Therefore, it occurred to us that antigen-presenting cells transfected with total tumor RNA might also be valuable in monitoring the antitumor responses induced in patients who participate in clinical trials. To test this hypothesis, we developed a model in which Epstein-Barr virus (EBV)–transformed lymphoblastoid cell lines were used as a source of RNA. Since this RNA encodes for known EBV antigens, it was possible to determine whether the expected responses were observed. Our results show for the first time that T cells primed to APC transfected with RNA isolated from EBV-infected lymphocytes exhibited a fine specificity that enabled them to recognize individual EBV antigens.This work was supported by a Developmental Research Grant from the Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine.     

An unfolded protein response is the initial cellular response to the expression of mutant matrilin-3 in a mouse model of multiple epiphyseal dysplasia

Multiple epiphyseal dysplasia (MED) can result from mutations in matrilin-3, a structural protein of the cartilage extracellular matrix. We have previously shown that in a mouse model of MED the tibia growth plates were normal at birth but developed a progressive dysplasia characterised by the intracellular retention of mutant matrilin-3 and abnormal chondrocyte morphology. By 3 weeks of age, mutant mice displayed a significant decrease in chondrocyte proliferation and dysregulated apoptosis. The aim of this current study was to identify the initial post-natal stages of the disease. We confirmed that the disease phenotype is seen in rib and xiphoid cartilage and, like tibia growth plate cartilage is characterised by the intracellular retention of mutant matrilin-3. Gene expression profiling showed a significant activation of classical unfolded protein response (UPR) genes in mutant chondrocytes at 5 days of age, which was still maintained by 21 days of age. Interestingly, we also noted the upregulation of arginine-rich, mutated in early stage of tumours (ARMET) and cysteine-rich with EGF-like domain protein 2 (CRELD2) are two genes that have only recently been implicated in the UPR. This endoplasmic reticulum (ER) stress and UPR did not lead to increased chondrocyte apoptosis in mutant cartilage by 5 days of age. In an attempt to alleviate ER stress, mutant mice were fed with a chemical chaperone, 4-sodium phenylbutyrate (SPB). SPB at the dosage used had no effect on chaperone expression at 5 days of age but modestly decreased levels of chaperone proteins at 3 weeks. However, this did not lead to increased secretion of mutant matrilin-3 and in the long term did not improve the disease phenotype. We performed similar studies with a mouse model of Schmid metaphyseal chondrodysplasia, but again this treatment did not improve the phenotype.     

Human herpesvirus-6-specific interleukin 10-producing CD4+ T cells suppress the CD4+ T-cell response in infected individuals

Human herpesvirus‐6 (HHV‐6) infection normally persists for the lifetime of the host and may reactivate with immunosuppression. The mechanism behind HHV‐6 latent infection is still not fully understood. In this study, we observed that decreased proliferation of CD4⁺ T cells and PBMCs but not CD8⁺ T cells from HHV‐6‐infected individuals was stimulated with HHV‐6‐infected cell lysates. Moreover, HHV‐6‐stimulated CD4⁺ T cells from HHV‐6‐infected individuals have suppressive activity on naïve CD4⁺ T and CD8⁺ T cells from HHV‐6‐uninfected individuals. However, no increased proportion of CD4⁺ CD25⁺ Treg cells from HHV‐6‐infected individuals contributed to the suppressive activity of the HHV‐6‐stimulated CD4⁺ T cells from HHV‐6‐infected individuals. Transwell experiments, ELISA and anti‐IL‐10 antibody blocking experiment demonstrated that IL‐10 may be the suppressive cytokine required for suppressive activity of CD4⁺ T cells from HHV‐6‐infected individuals. Results of intracellular interleukin (IL)‐10 and IL‐4 further implicated the HHV‐6‐speciflc IL‐10‐producing CD4⁺ T cells in the suppressive activity of CD4⁺ T cells from HHV‐6‐infected individuals. Results of intracellular interferon (IFN)‐γ demonstrated a decreased frequency of HHV‐6‐speciflc IFN‐γ‐producing CD4⁺ T, but not CD8⁺ T cells in HHV‐6‐infected individuals, indicating that it was the CD4⁺ Th1 responses in HHV‐6‐infected individuals that were selectively impaired. Our findings indicated that HHV‐6‐specific IL‐10‐producing CD4⁺ T cells from HHV‐6‐infected individuals possess T regulatory type 1 cell activity: immunosuppression, high levels of IL‐10 production, with a few cells expressing IFN‐γ, but none expressing IL‐4. These cells may play an important role in latent HHV‐6 infection.     

Evidence for induction of DNA double strand breaks in the bystander response to targeted soft X-rays in CHO cells

This study investigated the role of DNA double strand breaks and DNA base damage in radiation-induced bystander responses in Chinese hamster ovary (CHO) cell lines. Two CHO repair-deficient clones, xrs5 (DNA double strand break repair-deficient) and EM9 (DNA base excision repair-deficient) were used in addition to the wild type (CHO). The Gray Cancer Institute ultrasoft X-ray microprobe is a powerful tool for investigating the bystander response, because it permits the irradiation of only a single nucleus of a cell, as reported previously. In order to investigate the bystander effect in each repair-deficient cell line, we irradiated a single cell within a population and scored the formation of micronuclei. When a single nucleus in the population was targeted with 1 Gy, elevated numbers of micronuclei were induced in the neighbouring unirradiated cells in the EM9 and xrs5 cell lines, whereas induction was not observed in CHO. The induction of micronuclei in xrs5 was significantly higher than that in EM9. Under these conditions, the surviving fraction in the neighbouring cells was significantly lower in xrs5 than in the other cell lines, showing a higher cell killing effect in xrs5. To confirm that bystander factors secreted from irradiated cells caused these effects, we carried out medium transfer experiments using conventional X-irradiation. Medium conditioned for 24 h with irradiated cells was transferred to unirradiated cells and elevated induction of micronuclei was observed in xrs5. These results suggest that DNA double strand breaks rather than base damage are caused by factors secreted in the medium from irradiated cells.     

Biological response modifiers (BRM) as antigens

In order to investigate tumoricidal effector cells in therapy by biological response modifiers (BRM) such asPropionibacterium acnes, bacillus Calmette-Guérin (BCG),Streptococcus pyogenes and a protein-bound polysaccharide (PSK), we established T cell lines specific for each BRM from BALB/c mice immunized with the corresponding BRM. These T cell lines proliferated and produced interleukin-2-(IL-2) and/or IL-4, but only in the presence of the relevant BRM and BALB/c spleen cells as the antigen and antigen-presenting cells respectively. Cross-functional experiments indicated that each BRM acts as a nominal antigen, but not as a non-specific immunostimulator. In addition, the T cell lines killed Ia-positive syngeneic B lymphoma cells, but only in the presence of the relevant BRM. These experiments excluded the possibility of cytotoxic effects by each BRM. The T cell lines and clones also killed Ia-negative bystander target cells, but only in the presence of both a relevant antigen and antigen-presenting cells. The T cell clones specific forS. pyogenes orP. acnes tested were Thy1⁺, L3T4⁺ and Lyt2^–. These results indicate that some BRM exert tumoricidal activity by inducing T cells that recognize them as an antigen and kill tumor cells in an antigen-specific manner. The T cells killed tumor targets in either a tumor-necrosis-factor(TNF)-dependent or a TNF-independent manner. The mediator of the latter pathway remains to be elucidated.     

Dissecting the role of peptides in the immune response: theory, practice and the application to vaccine design.

Analytical biochemistry and synthetic peptide based chemistry have helped to reveal the pivotal role that peptides play in determining the specificity, magnitude and quality of both humoral (antibody) and cellular (cytotoxic and helper T cell) immune responses. In addition, peptide based technologies are now at the forefront of vaccine design and medical diagnostics. The chemical technologies used to assemble peptides into immunogenic structures have made great strides over the past decade and assembly of highly pure peptides which can be incorporated into high molecular weight species, multimeric and even branched structures together with non-peptidic material is now routine. These structures have a wide range of applications in designer vaccines and diagnostic reagents. Thus the tools of the peptide chemist are exquisitely placed to answer questions about immune recognition and along the way to provide us with new and improved vaccines and diagnostics.     

Activation of guanylate cyclase by natriuretic peptides in mouse pituitary AtT20 cells is influenced by phosphorylation of ANP

The discovery of free and membrane-bound ectokinases raises the question whether phosphorylation is another mechanism to modulate the action of distinct neuropeptides. Atrial-natriuretic-peptide (ANP) which is widespread found in the central nervous system (CNS) and involved in the modulation of stress reactions and emotional states like anxiety contains a recognition-motif for cAMP-dependent protein kinase A. We investigated the effect of phosphorylation of ANP and C-type natriuretic peptide (CNP), a related peptide without phosphorylation site, on their ability to activate their receptors in mouse pituitary AtT20 cells by measuring the formation of cyclic guanosinmonophosphate (cGMP). Phosphorylation with protein kinase A inactivated ANP. Coincubation experiments adding adenosintriphosphate (ATP), ATP-analogues or inhibitors of protein kinases to the medium pointed to the presence of an intrinsic protein kinase A like ectokinase-activity on AtT20 cells. The activity of CNP was unaffected in these experiments. Phosphorylation by ectokinases may be a physiological mechanism to regulate the biological activity of ANP in different tissues, such as pituitary and CNS.     

Kinetic and conformational properties of a novel T-cell antigen receptor transmembrane peptide in model membranes

Core peptide (CP; GLRILLLKV) is a 9-amino acid peptide derived from the transmembrane sequence of the T-cell antigen receptor (TCR) alpha-subunit. CP inhibits T-cell activation both in vitro and in vivo by disruption of the TCR at the membrane level. To elucidate CP interactions with lipids, surface plasmon resonance (SPR) and circular dichroism (CD) were used to examine CP binding and secondary structure in the presence of either the anionic dimyristoyl-L-alpha-phosphatidyl-DL-glycerol (DMPG), or the zwitterionic dimyristoyl-L-alpha-phoshatidyl choline (DMPC).Using lipid monolayers and bilayers, SPR experiments demonstrated that irreversible peptide-lipid binding required the hydrophobic interior provided by a membrane bilayer. The importance of electrostatic interactions between CP and phospholipids was highlighted on lipid monolayers as CP bound reversibly to anionic DMPG monolayers, with no detectable binding observed on neutral DMPC monolayers.CD revealed a dose-dependent conformational change of CP from a dominantly random coil structure to that of beta-structure as the concentration of lipid increased relative to CP. This occurred only in the presence of the anionic DMPG at a lipid : peptide molar ratio of 1.6:1 as no conformational change was observed when the zwitterionic DMPC was tested up to a lipid : peptide ratio of 8.4 : 1.     

Generation of specific antitumor reactivity by the stimulation of spleen cells from gastric cancer patients with MAGE-3 synthetic peptide

The induction of cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) using MAGE peptide has been investigated in order to use MAGE antigens immunotherapeutically. We therefore developed a simplified method for inducing peptide-specific CTL that kill tumor cells expressing MAGE from the PBMC of either healthy donors or even cancer patients. Since the spleen is a major lymphoid organ, we used a simple method to examine the capacity of spleen cells to generate MAGE-specific CTL by in vitro stimulation with MAGE peptide in gastric cancer patients. The CTL responses could thus be induced from unseparated spleen cells in HLA-A2 patients with gastric carcinoma expressing MAGE-3 by stimulating these cells with autologous spleen cells pulsed with HLA-A2-restricted MAGE-3 peptide as antigen-presenting cells and by using keyhole limpet hemocyanin and interleukin-7 for the primary culture. The induced CTL were thus able to lyse HLA-A2-positive carcinoma cells transfected with MAGE-3 and expressing MAGE-3, as well as the target cells pulsed with the peptide, in an HLA-class-I or -A2-restricted manner. Since MAGE-specific CTL could be induced from the spleen cells of gastric cancer patients, the spleen appears to play an important role in either clinical tumor vaccination or the treatment of cancer patients by adoptive immunotherapeutic approaches using the MAGE peptide. Received: 3 December 1998 / Accepted: 30 March 1999     

Synthesis of linear and comb-like peptide constructs containing up to four copies of a T cell epitope and their capacity to stimulate T cells.

Polypeptide constructs containing up to four copies of the T cell epitope 306-318 of influenza virus haemagglutinin have been synthesized on solid phase. Between the copies, a non-natural PEG-based spacer amino acid has been introduced. The oligomeric epitopes were analysed by RP-HPLC and ES-MS. The arrangement of the epitopes within the peptide constructs was either linear or comb-like. The proliferative response in a T helper cell assay induced by these oligomerized epitopes has been tested, showing that the linearly arranged epitopes are more effective than the comb-like oligomers.