ABSTRACT
BACKGROUND: Early response to treatment has been shown to be a predictor of later clinical outcomes in eating disorders (EDs). Specifically, early weight gain trajectories in anorexia nervosa (AN) have been shown to predict higher rates of later remission in inpatient treatment. However, no study has, as of yet, examined this phenomenon within outpatient treatment of first episode cases of AN or in emerging adults. METHODS: One hundred seven patients with AN, all between the ages of 16 and 25 and with an illness duration of < 3 years, received treatment via the first episode rapid early intervention in eating disorders (FREED) service pathway. Weight was recorded routinely across early treatment sessions and recovery outcomes (BMI > 18.5 kg/m2 and eating psychopathology) were assessed up to 1 year later. Early weight gain across the first 12 treatment sessions was investigated using latent growth mixture modelling to determine distinct classes of change. Follow-up clinical outcomes and remission rates were compared between classes, and individual and clinical characteristics at baseline (treatment start) were tested as potential predictors. RESULTS: Four classes of early treatment trajectory were identified. Three of these classes (n = 95), though differing in their early change trajectories, showed substantial improvement in clinical outcomes at final follow-up. One smaller class (n = 12), characterised by a 'higher' start BMI (> 17) and no early weight gain, showed negligible improvement 1 year later. Of the three treatment responding groups, levels of purging, depression, and patient reported carer expressed emotion (in the form of high expectations and low tolerance of the patient) determined class membership, although these findings were not significant after correcting for multiple testing. A higher BMI at treatment start was not sufficient to predict optimal clinical outcomes. CONCLUSION: First episode cases of AN treated via FREED fit into four distinct early response trajectory classes. These may represent subtypes of first episode AN patients. Three of these four trajectories included patients with substantial improvements 1 year later. For those in the non-response trajectory class, treatment adjustments or augmentations could be considered earlier, i.e., at treatment session 12.
A key feature of anorexia nervosa (AN) is an unhealthily low body weight. Previous studies show that more weight gained early in inpatient treatment leads to better outcomes. This study tried to see if this was also true for outpatients receiving treatment for the first time. All participants were emerging adults between the ages of 16 and 25 who had been ill for less than 3 years. Weight was recorded across the first 12 weekly treatment sessions. Statistics showed that the patients fit roughly into four different groups in early treatment, each with different starting weights and rates of weight gain in the first 12 treatment sessions. The group a patient belonged to could sometimes be predicted by vomiting behaviours, level of depression, and patients' perception of parental tolerance and expectations at the start of treatment. Out of the four groups, three did relatively well 1 year later, but one small group of patients did not. This small group had a higher starting weight than many of the other groups but did not gain any weight across the first 12 sessions. These patients could benefit from a change or increase in the amount or intensity of treatment after the first 12 treatment sessions.
ABSTRACT
OBJECTIVE: A cognitive interpersonal maintenance model of anorexia nervosa (AN) was first proposed in 2006 and updated in 2013 (Schmidt and Treasure, J Br J Clin Psychol, 45, 343-366, 2006; Treasure and Schmidt, J Eat Disorders, in press.). The aim of this study was to test the interpersonal component of this model in people with AN requiring intensive hospital treatment (inpatient/day patient). METHOD: On admission to hospital women with AN or eating disorder not otherwise specified (AN subtype; n = 152; P) and their primary carers (n = 152; C) completed questionnaires on eating symptoms (P), depression and anxiety (P, C), accommodation and enabling (C), and psychological control (C). Structural equation modeling was used to examine relationships among these components. RESULTS: Carers' expressed emotion and level of psychological control were significantly related to carers' distress, which in turn, was related to patients' distress. This pathway significantly predicted eating symptoms in patients. DISCUSSION: The cognitive interpersonal maintenance model of eating disorders (EDs) was confirmed in part and suggests that interventions targeting interpersonal maintaining factors such as carer distress might impact on patient outcomes.
Subject(s)
Anorexia Nervosa/psychology , Caregivers/psychology , Depression/psychology , Interpersonal Relations , Models, Psychological , Adolescent , Adult , Age Factors , Anorexia Nervosa/diagnosis , Caregivers/statistics & numerical data , Confounding Factors, Epidemiologic , Depression/epidemiology , Feeding and Eating Disorders/diagnosis , Feeding and Eating Disorders/psychology , Female , Humans , Male , Middle Aged , Psychometrics , Social Control, Informal , Socioeconomic Factors , Surveys and Questionnaires , United Kingdom/epidemiology , Young AdultABSTRACT
The anti-Hu syndrome is the most common paraneoplastic neurologic syndrome but the exact mechanism of immune mediated neuronal injury remains unknown. Anti-Hu antibodies do not appear to play a pivotal role in the pathogenesis of the disease. To assess cell-mediated immunity, we selected 51 peptides from the Hu-D sequence and tested their ability to bind to six common HLA class I molecules. Stable complexes with purified HLA molecules were obtained with 19/51 (37%) selected peptides. Subsequently, the ability of the 19 HLA-binding peptides to stimulate T cells from 10 patients and 10 control subjects was evaluated by detecting IFN-gamma secretion. An anti-peptide T-cell response was observed in 7/10 Hu-positive patients but also in 3/10 control subjects. Overall, a significant T-cell activation occurred in response to 74% (14 out of 19) of the selected peptides in the Hu-positive patients vs. 16% (3 out of 19) in the control group (p < 0.001). In addition, T cells of patients tested within 3 months of the onset of anti-Hu syndrome responded to 82% (14 out of 17) of assessed Hu-D peptides vs. 37% (7 out of 19) in patients tested 1 year or more after developing the syndrome (p < 0.01). Thus, the present study suggests a role of cellular immunity during the course of anti-Hu syndrome.
Subject(s)
Carcinoma, Small Cell/immunology , Immunity, Cellular/immunology , Nerve Tissue Proteins/immunology , Paraneoplastic Syndromes, Nervous System/immunology , RNA-Binding Proteins/immunology , T-Lymphocytes/immunology , Carcinoma, Small Cell/complications , Cells, Cultured , ELAV Proteins , HLA-A Antigens/immunology , HLA-A Antigens/metabolism , Humans , Interferon-gamma/metabolism , Nerve Tissue Proteins/metabolism , Paraneoplastic Syndromes, Nervous System/complications , Peptide Fragments/immunology , RNA-Binding Proteins/metabolism , Statistics, Nonparametric , Syndrome , T-Lymphocytes/metabolismABSTRACT
Cytotoxic T-lymphocyte (CTL) responses directed to different human immunodeficiency virus (HIV) epitopes vary in their protective efficacy. In particular, HIV-infected cells are much more sensitive to lysis by anti-Gag/p17(77-85)/HLA-A2 than to that by anti-polymerase/RT(476-484)/HLA-A2 CTL, because of a higher density of p17(77-85) complexes. This report describes multiple processing steps favoring the generation of p17(77-85) complexes: (i) the exact COOH-terminal cleavage of epitopes by cellular proteases occurred faster and more frequently for p17(77-85) than for RT(476-484), and (ii) the binding efficiency of the transporter associated with antigen processing was greater for p17(77-85) precursors than for the RT(476-484) epitope. Surprisingly, these peptides, which differed markedly in their antigenicity, displayed qualitatively and quantitatively similar immunogenicity, suggesting differences in the mechanisms governing these phenomena. Here, we discuss the mechanisms responsible for such differences.
Subject(s)
Antigen Presentation/immunology , Cysteine Endopeptidases , Epitopes, T-Lymphocyte/immunology , Gene Products, gag/immunology , HIV Antigens/immunology , HIV Infections/immunology , HIV Reverse Transcriptase/immunology , HIV-1/immunology , HLA-A2 Antigen/immunology , Immunodominant Epitopes/immunology , Multienzyme Complexes , T-Lymphocytes, Cytotoxic/immunology , Viral Proteins , Amino Acid Sequence , Biological Transport , Endopeptidases/metabolism , Epitopes, T-Lymphocyte/metabolism , Gene Products, gag/metabolism , HIV Antigens/metabolism , HIV Reverse Transcriptase/metabolism , Humans , Immunodominant Epitopes/metabolism , Major Histocompatibility Complex , Molecular Sequence Data , Proteasome Endopeptidase Complex , Protein Precursors/metabolism , Proteins/metabolism , gag Gene Products, Human Immunodeficiency VirusABSTRACT
In many types of cancer, p53 frequently accumulates in tumor cells and anti-p53 antibodies can be detected. However, only four CD8(+) T-cell epitopes from p53 have been identified in humans so far. To further analyze the development of a T-cell response against p53, peptides having binding motifs specific for HLA-A1, -A2, -A3, -A24, -B7, -B35, -B44, and -B51 molecules have been defined. The HLA-binding capacity of those peptides was tested, and the stability of formed complexes was defined. Thirteen peptides that bound to HLA-A24 and -B44 molecules are presented. The positive peptides were then used to detect the anti-p53 response of CD8(+) T lymphocytes from patients with bladder cancer. Six peptides, presented by HLA-A2, -B51, or -A24, were able to stimulate T cells from two patients (among 16) with tumor cells that strongly accumulated p53. On the contrary, p53 peptides systematically failed to stimulate T cells from healthy donors or patients with low or undetectable levels of p53 in their tumor cells. These results have led to the identification of four new potential T CD8(+) epitopes from p53: 194-203 associating with HLA-B51 and 204-212, 211-218, and 235-243 associating with HLA-A24.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Peptides/immunology , Tumor Suppressor Protein p53/immunology , Urinary Bladder Neoplasms/immunology , Cells, Cultured , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/metabolism , HLA-A Antigens/immunology , HLA-A Antigens/metabolism , HLA-A2 Antigen/immunology , HLA-A2 Antigen/metabolism , HLA-A24 Antigen , HLA-B Antigens/immunology , HLA-B Antigens/metabolism , HLA-B51 Antigen , Histocompatibility Antigens Class I/immunology , Humans , Lymphocyte Activation , Peptides/metabolism , Protein Binding , Tumor Suppressor Protein p53/chemistry , Urinary Bladder Neoplasms/therapyABSTRACT
First and foremost among the many factors that influence epitope presentation are the degradation of Ag, which results in peptide liberation, and the presence of HLA class I molecules able to present the peptides to T lymphocytes. To define the regions of HIV-1 Nef that can provide multiple T cell epitopes, we analyzed the Nef sequence and determined that there are 73 peptides containing 81 HLA-binding motifs. We tested the binding of these peptides to six common HLA molecules (HLA-A2, -A3, -A24, -B7, -B8, and -B35), and we showed that most of them were efficient binders (54% of motifs), especially peptides associating with HLA-A3, -B7/35, and -B8 molecules. Nef peptides most frequently recognized by T cells of HIV-1-infected individuals were 90-97, 135-143, 71-81, 77-85, 90-100, 73-82, and 128-137. The frequency of T cell recognition was not directly related to the strength of peptide-HLA binding. The generation of Nef epitopes is crucial; therefore, we investigated the digestion by the 20S proteasome of a large peptide, Nef(66-100). This fragment was efficiently cleaved, and NH(2)-terminally extended precursors of epitope 71-81 were recognized by T cells of an HIV-1-infected individual. These results suggest that a high frequency of T cell recognition may depend on proteasome cleavage.
Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Cysteine Endopeptidases/metabolism , Epitopes, T-Lymphocyte/metabolism , Gene Products, nef/immunology , Gene Products, nef/metabolism , HIV-1/immunology , HLA Antigens/metabolism , Multienzyme Complexes/metabolism , Peptide Fragments/immunology , Amino Acid Motifs/immunology , Amino Acid Sequence , Antigen Presentation , CD8-Positive T-Lymphocytes/enzymology , CD8-Positive T-Lymphocytes/immunology , Cell Line, Transformed , HIV Seropositivity/enzymology , HIV Seropositivity/immunology , HIV-1/enzymology , Histocompatibility Antigens Class I/metabolism , Humans , Hydrolysis , Immunodominant Epitopes/metabolism , Molecular Sequence Data , Peptide Fragments/metabolism , Proteasome Endopeptidase Complex , Protein Binding/immunology , nef Gene Products, Human Immunodeficiency VirusSubject(s)
Cytotoxicity, Immunologic/physiology , HLA Antigens/immunology , Histocompatibility Antigens Class I/immunology , Killer Cells, Natural/immunology , Recombinant Fusion Proteins/pharmacology , Cells, Cultured , Clone Cells , Cloning, Molecular , Cytotoxicity, Immunologic/drug effects , Escherichia coli , HLA Antigens/pharmacology , HLA-G Antigens , Histocompatibility Antigens Class I/pharmacology , Humans , K562 Cells , Killer Cells, Natural/drug effects , Polymerase Chain Reaction , Recombinant Fusion Proteins/isolation & purificationABSTRACT
Backbone modifications have been introduced into the melanoma derived peptide MART-1(27-35) to increase its binding to class I major histocompatibility complex HLA-A2 molecule, and ultimately to enhance its immunogenicity. Each analogue was obtained by replacing one peptide bond at a time in the natural epitope by the aminomethylene (CH2-NH) surrogate. All analogues displayed an increased resistance to proteolysis. Interestingly, the comparative results showed that five analogues bound more efficiently to HLA-A2 than the parent peptide. On the other hand, two pseudopeptide/HLA-A2 complexes were recognized by one melanoma-specific T cell clone. Close examination of the impact of such modifications at the molecular level provides useful supports for the rational design of stable compounds with applications in anti-tumour specific immunotherapy and in vaccine development.
Subject(s)
Epitopes/chemistry , Neoplasm Proteins/chemistry , Neoplasm Proteins/chemical synthesis , Amino Acid Sequence , Amino Acids/chemistry , Cell Line , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Epitopes/immunology , Fluorenes/chemistry , HLA-A2 Antigen/metabolism , Humans , Melanoma/immunology , Melanoma/metabolism , Molecular Sequence Data , Neoplasm Proteins/immunology , Peptides/chemistry , Protein Binding , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
CD8(+) T lymphocytes, which are major immune effectors, require primary stimulation by dendritic cells (DC) presenting MHC class I molecule-bound epitopes. Sensitization to exogenous protein epitopes that are not synthesized in DC, such as cross-priming, is obtained through pathways leading to their association with MHC class I. To follow class I-restricted pathways in human DC, we have tracked a lipopeptide derived from the conserved HLA-A*0201-restricted HIV-1 reverse transcriptase 476-484 epitope, by N-terminal addition of an Nepsilon-palmytoyl-lysine. Indeed, lipopeptides elicit cytotoxic responses from CD8(+) T lymphocytes, whereas peptides without a lipid moiety do not. The lipopeptide and its parent peptide were labeled unequivocally by rhodamine to study their entry into immature monocyte-derived human DC by confocal microscopy. The lipid moiety induced endocytosis of the lipopeptide, assessed by rapid entry into vesicles, colocalization with Dextran-FITC and dependence on energy. Internalization occurred even when actin filaments were depolymerized by Cytochalasin B. This internalization induced functional stimulation of specific CD8(+) T lymphocytes in IFN-gamma ELISPOT assays. The peptide alone was not visualized inside the DC and was presented through direct surface association to HLA-A*0201. Therefore, lipopeptides are a unique opportunity to define precisely the pathways that lead exogenous proteins to associate with MHC class I molecules in DC. The results will also be useful to design lipopeptide vaccines.
Subject(s)
Antigen Presentation , CD8-Positive T-Lymphocytes/immunology , Cell Communication/immunology , Dendritic Cells/immunology , HIV Antigens/immunology , HIV Infections/immunology , HIV-1/immunology , CD8-Positive T-Lymphocytes/cytology , Cell Line , Dendritic Cells/cytology , Endocytosis/immunology , Histocompatibility Antigens Class I/immunology , Humans , Lymphocyte ActivationABSTRACT
The design of heteroclytic antigens with high MHC binding capacity is of particular interest to overcome the weak immunogenicity of peptide epitopes derived from tissue antigens expressed by tumors. In the present study, double-substituted peptide analogues of the tumor-associated antigen MART-1(27-35) incorporating a substitution at a primary anchor residue and a beta-amino acid residue at different positions in the sequence were synthesized and evaluated for binding to the human histocompatibility class I molecule HLA-A2 and for recognition by tumor-infiltrating lymphocytes. Interestingly, by combining a Leu for Ala substitution at P2 (which alone is deleterious for antigenic activity) with a beta-amino acid substitution at a putative TCR contact residue, recognition by tumor-infiltrating lymphocytes was partially restored. The analogue [Leu(28),beta-HIle(30)]MART-1(27-35) displays both a higher affinity to HLA-A2 and a more prolonged complex stability compared to [Leu(28)]MART-1(27-35). Overall, these results suggest that double-substitution strategies and beta-amino acid replacements at putative TCR contact residues might prove useful for the design of epitope mimics with high MHC binding capacity.
Subject(s)
Epitopes/chemistry , HLA-A2 Antigen/metabolism , Lymphocytes, Tumor-Infiltrating/metabolism , Melanoma/chemistry , Neoplasm Proteins/chemistry , Peptide Fragments/chemical synthesis , Chromatography, High Pressure Liquid , Electrophoresis, Capillary , Humans , Isoantigens/chemistry , Isoantigens/metabolism , Magnetic Resonance Spectroscopy , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tumor Cells, CulturedABSTRACT
Human papilloma virus type 16 (HPV-16) is the HPV most frequently associated with cervical carcinoma in humans. For the prevention or treatment of cervical carcinoma, the E6 and E7 oncoproteins appear to be good targets for vaccine-induced cytotoxic T lymphocytes (CTL). Lipopeptide vaccination is an efficient way of stimulating cellular responses. However, to synthesize effective lipopeptides, it is necessary to define which epitopes are immunogenic. In this study we first determined that peptide 80 - 88 of the E6 protein was recognized by CTL from a healthy donor in association with the HLA-B18 molecule. We then defined the HLA-B18 anchoring peptide motif by testing the binding of various short peptides with the HLA-B18 molecule and showed that it was related to the HLA-A1-specific peptide motif. Furthermore, in analyzing the potential E7 epitopes susceptible to associating with HLA-B18, we demonstrated that peptide E7 44 - 52 gave the strongest binding. It could also be recognized by CTL from peripheral blood mononuclear cells (PBMC) of the same healthy donor. Finally, with PBMC from a patient with a cervical intraepithelial neoplasia grade 3, we found CTL which recognized the E6 80 - 88 epitope. We have hence identified two peptides encoded by the E6 and E7 proteins which are presented by the HLA-B18 molecule and could be included in a vaccine against HPV-16.
Subject(s)
HLA-B Antigens/metabolism , Oncogene Proteins, Viral/immunology , Repressor Proteins , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Binding Sites , HLA-B18 Antigen , Humans , Molecular Sequence Data , Papillomavirus E7 Proteins , Peptide Fragments/immunologyABSTRACT
There is considerable evidence that peptides corresponding to MHC class I-restricted epitopes can be used as immunogens or immunomodulators. Pseudopeptides containing isosteric replacements of the amide bond provide more stable analogues, which may even have enhanced biologic activity. But there have been very few studies on the use of pseudopeptides to initiate or modulate the cellular immune response. This study describes the immunogenicity of a partially modified retro-inverso pseudopeptide of an influenza virus epitope and shows that this pseudopeptide modulates the cytokine profile expressed by CD8+ CTL generated from primed precursors. Moreover, the pseudopeptide is much more efficient at low concentration than the wild-type epitope to stimulate IFN-gamma secretion by CD8+ T effector cells. These results are analyzed with reference to changes in the conformation of the MHC molecule/peptide complex deduced from molecular modeling. The findings support the idea that partially modified retro-inverso analogues can be used as altered peptide ligands to enhance the stimulation of natural epitope-specific CTL and to modify their functional properties. Hence, pseudopeptide ligands might be promising tools for use in immunotherapy.
Subject(s)
Cytokines/biosynthesis , Epitopes, T-Lymphocyte/immunology , Influenza A virus/immunology , Peptide Fragments/pharmacology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Viral Matrix Proteins/pharmacology , Cell Line , Cytokines/genetics , Cytotoxicity Tests, Immunologic , Enzyme-Linked Immunosorbent Assay , Epitopes, T-Lymphocyte/pharmacology , HLA-A2 Antigen/chemistry , HLA-A2 Antigen/metabolism , Humans , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Lymphocyte Activation/drug effects , Models, Molecular , Peptide Fragments/chemical synthesis , Peptide Fragments/metabolism , RNA, Messenger/analysis , T-Lymphocytes, Cytotoxic/chemistry , Viral Matrix Proteins/chemical synthesis , Viral Matrix Proteins/metabolismABSTRACT
Recent studies have shown that transfusions of HLA-compatible donor lymphocytes may induce complete remission in marrow-grafted patients with relapses of acute myeloblastic leukemia (AML). We investigated the in vitro generation of antileukemia T-cell clones obtained from the peripheral blood mononuclear cells of a partially HLA-compatible donor (HLA-A2 and B7 molecules in common with the leukemic blasts) after stimulation with a pool of naturally processed peptides extracted from leukemic blast cells collected at diagnosis from a patient with hyperleucocytosis AML. We recovered a significant quantity of peptides that bound to the HLA-A2 or HLA-B7 molecules that were able to induce cytolytic T-lymphocyte (CTL) lines and clones specific for the eluted AML peptides and restricted to the HLA-A2 or B7 molecules. Such CTL line did not recognize the patient's nonleukemic cells, and one clone was able to interact with the leukemic blasts from which the naturally processed peptides had been eluted. Such T-cell clones might provide a rationale for the development of adoptive immunotherapy and could be used to improve the efficiency of HLA-compatible T-lymphocyte transfusions and the graft-versus-leukemia response in patients with AML.
Subject(s)
Antigen Presentation , Antigens, Neoplasm/immunology , Leukemia, Myeloid, Acute/immunology , T-Lymphocytes, Cytotoxic/immunology , HLA-A2 Antigen/immunology , HLA-B7 Antigen/immunology , Humans , Leukemia, Myeloid, Acute/pathology , Lymphocyte Activation , Peptides/immunology , Tumor Cells, CulturedABSTRACT
Tumor cells can have different morphologic or metabolic phenotypes and display genetic instability. Thus they could also vary in their ability to present epitopes to the immune system. We have analyzed the presentation of H-2 Kb- and Db-restricted cytotoxic T lymphocyte (CTL) epitopes of a tumor-associated antigen by three cell lines derived from hepatocarcinomas developed in vivo by mice transgenic for SV40 T targeted to the liver. SV40 T is the obvious tumor-specific antigen and epitopes derived from this antigen were therefore studied. The study included four already known epitopes that can be presented by SV40-transformed kidney cells and two new CTL epitopes that were identified in the present work. CTL lines specific for each epitope were obtained from C57BL/6 mice and were used to map the presentation of SV40 T peptides by the hepatocarcinoma cells. These tumor cells were derived from the same tissue, induced by the same agent and all naturally presented peptide p232-240 from p53. Despite these common features, they all had different patterns of spontaneous presentation of SV40 T CTL epitopes. The mechanisms underlying this disparity are discussed, together with the possible consequences for establishing immunotherapeutic strategies.
Subject(s)
Antigens, Polyomavirus Transforming/immunology , Epitopes, T-Lymphocyte/immunology , Liver Neoplasms, Experimental/immunology , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Animals , Antigens, Polyomavirus Transforming/metabolism , Epitope Mapping , H-2 Antigens/immunology , H-2 Antigens/metabolism , Liver Neoplasms, Experimental/metabolism , Male , Mice , Mice, Inbred C57BL , Peptides/immunology , Peptides/metabolism , Tumor Cells, CulturedABSTRACT
This study investigates the generation of primary melanoma cell-specific cytotoxic T lymphocytes (CTLs) in vitro. Induction of peptide-specific CTLs from unfractionated naive peripheral blood mononuclear cells from HLA-A2 healthy donors was assessed using 2 recently described 9-mer epitopes from the melanoma tumor antigen Melan-A/MART-1. The need for help from CD4+ T lymphocytes for the long-lasting induction of CTLs and the capacity of the peptide-induced CTL lines to recognize many melanoma cells were evaluated. CTL lines were obtained reproducibly when CD4+ T-lymphocyte help was provided during the primary stimulation either in an autologous way, in the case of tetanus toxoid antigen (TT) responder donors, or with allogeneic TT-activated T-helper cells, separated by an insert well, in the case of tetanus toxoid non-responder donors. We also investigated helper T-cell-derived factors that are produced by TT-activated lymphocytes. Our results strongly suggest that a complex network of cytokines like interleukin-2 (IL-2), interferon-gamma, IL-6 and IL-1 exerts stimulatory effects for the initiation process of CTLs. In contrast, cytokine-like IL-4 might inhibit generation of cytolytic activity if provided by TT-activated T cells at early stages of induction. Our approach can be used to generate CTLs of a desired specificity for clinical use in adoptive immunotherapy protocols.
Subject(s)
Antigens, Neoplasm/immunology , Cytokines/biosynthesis , Leukocytes, Mononuclear/immunology , Melanoma/immunology , Neoplasm Proteins/immunology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Helper-Inducer/immunology , Cells, Cultured , Cytotoxicity, Immunologic , HLA-A2 Antigen/biosynthesis , Humans , Interferon-gamma/biosynthesis , Leukocytes, Mononuclear/cytology , Lymphocyte Activation , MART-1 Antigen , Molecular Sequence Data , Neoplasm Proteins/chemistry , Oligopeptides/chemistry , Oligopeptides/pharmacology , Polymerase Chain Reaction , Receptor-CD3 Complex, Antigen, T-Cell/analysis , Receptors, Antigen, T-Cell, alpha-beta/analysis , T-Lymphocytes, Cytotoxic/cytology , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
Chronic myeloid leukemia (CML) is characterized cytogenetically by a t(9;22) translocation which generates a hybrid bcr-abl gene, encoding a p210(bcr-abl) fusion protein. The induction in vitro of leukemia-specific T cells reactive with p210(bcr-abl) is a strategy developed for an immunological therapeutic approach in CML. Peptides from the junction region of this chimeric protein have been considered as potential targets for a cytotoxic response against leukemic cells. However, only a few peptides encompassing the two p210(bcr-abl) breakpoints have been shown to bind to the most common HLA class I molecules, which limits the number of patients who could benefit from this approach. We assume that the presence of chimeric BCR-ABL protein in leukemic cells may affect processing and delivery of peptides, possibly giving rise to new epitopes at the cell surface. We selected 162 peptides from the whole sequence of this protein, including 14 peptides of the b2a2 and b3a2 junctions, which had an anchor motif for a common HLA class I molecule. We tested their ability to bind to eight HLA class I molecules (HLA-A1, -A2, -A3, -A11, -B7, -B8, -B27, -B44). We identified 48 peptides from outside the junction region, with intermediate or strong binding capacities to these HLA class I molecules contrasting with only six junction peptides with a moderate binding capacity to HLA-A3/A11, -B8, or -B44 molecules. Moreover, cytotoxic T lymphocyte lines specific for various peptides outside the junction were generated from peripheral blood mononuclear cells of HLA-A2 or -B7 healthy donors and from one CML patient. These results contribute to evaluation of immunity to the BCR-ABL chimeric protein. Further studies are required to investigate whether such epitopes are correctly processed and presented by leukemic cells.
Subject(s)
Fusion Proteins, bcr-abl/immunology , HLA Antigens/metabolism , Histocompatibility Antigens Class I/metabolism , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Binding Sites/genetics , Cell Line , Cell Line, Transformed , Fusion Proteins, bcr-abl/genetics , HLA-B7 Antigen/metabolism , Herpesvirus 4, Human , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/immunology , Peptide Fragments/genetics , Peptide Fragments/immunology , Protein Binding , Tumor Cells, CulturedABSTRACT
RAS oncogenic proteins are frequently found mutated in human cancers, where they are known to be implicated in the tumoral process. Mutations occur preferentially at positions 12, 13 or 61. Identification of potential T cell epitopes is the first step to determine it RAS mutated proteins can generate tumor specific antigens which could be further used as targets for cancer immunotherapy protocols. We have investigated the capacity of synthetic wild-type and mutant RAS derived peptides encompassing positions 12 and 13 to bind to three frequent HLA-A alleles: HLA-A*0201, HLA-A*0301 and HLA-A*1101. Binding was evaluated by two methods using TAP-defective cell lines: a cytometric assay based on HLA molecules stabilization at the cell surface, and an assembly assay detecting interactions between solubilized HLA molecules and peptides. Positive HLA binding was observed for two sets of synthetic peptides, one specific for HLA-A*0201 allele (RAS 5-14), and the other one specific for HLA-A*0301 and HLA-A*1101 alleles (RAS 8-16). Interestingly, the different substitutions at positions 12 and 13 were not equivalent for HLA binding. These observations will be useful for the in vitro generation of restricted CD8+ T lymphocytes specific for mutated RAS proteins and recognizing tumoral cells expressing such RAS mutations.
Subject(s)
Alleles , HLA-A Antigens/genetics , Peptide Fragments/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Cell Line , Humans , Mutation , Structure-Activity Relationship , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
The human T-cell leukemia virus type 1 (HTLV-1) is a human retrovirus associated with two diseases for which no successful treatment is yet available; the development of a vaccine is therefore an important issue. Since HTLV-1 is a persistent virus, an efficient vaccine will probably require a cytotoxic T-lymphocyte (CTL) response in addition to the production of antibodies. To identify potential CTL epitopes, we have selected, within all of the HTLV-1 proteins, nonapeptides containing anchor residues required for association with HLA-A2 molecules (residues at positions 2 and 9), which is the most frequently occurring A allele in all human populations. A set of 111 peptides was synthetized and tested in vitro in two assembly assays using processing-defective T2 cells. Anchor motifs selected were those containing two major anchor residues (L2/M2/12-V9/L9/I9) (one letter amino-acid code) and those including tolerated anchor residues (V2/A2/T2 and/or A9/M9/T9). The analysis of the binding capacity of the peptides confirms the high efficiency of the L2-V9 anchor motif and shows that a systematic research of potential binding peptides should exclude peptides containing known detrimental residues rather than select only peptides with known favored residues. We show that 39 peptides representative of all the HTLV-1 proteins are able to bind to HLA-A2 molecules. Strong binder peptides which are very likely good CTL epitopes were identified in three HTLV-1 proteins, Tax, envelope, and polymerase. Three of the strong binder peptides correspond to previously described HLA-A2-restricted CTL epitopes in the Tax protein, and two others are localized in a domain of the viral envelope recognized by natural neutralizing antibodies. This latter result has important implications for the development of an anti-HTLV-1 vaccine.
Subject(s)
Gene Products, tax/immunology , HLA-A2 Antigen/biosynthesis , HTLV-I Infections/immunology , Human T-lymphotropic virus 1/immunology , T-Lymphocytes, Cytotoxic/immunology , Viral Envelope Proteins/immunology , Amino Acid Sequence , Epitope Mapping , HLA-A2 Antigen/immunology , Human T-lymphotropic virus 1/metabolism , Humans , Receptors, Antigen, T-Cell/immunologyABSTRACT
Syntheses of a series of partially modified retro-inverso analogues of the antigenic peptide M58-66 derived from the influenza virus matrix protein are reported. The retro-inverso modification phi(NH-CO) was obtained by replacement of two successive amino acid residues with a 2-substituted malonate derivative and gem-diaminoalkyl residue. The resulting compounds 1-8 were tested for their binding to the human histocompatibility class I molecule HLA-A2 in an assembly assay using lysates of peptide transporter-deficient cells T2. Specific peptide-dependent HLA-A2 assembly was revealed by an enzyme-linked immunosorbent assay. Significant HLA-A2 assembly was detected in the presence of analogues [gGly58-(S)mLeu59]-M58-66 (1a), [gGly61-(R,S)mPhe62]M58-66 (4), [gVal63-(R,S)mPhe64]M58-66 (6), and [gPhe64-(R,S)mAla65]M58-66 (7). The introduction of the retro-inverso modification between P2-P3, P3-P4, P5-P6, and P8-P9 (compounds 2, 3, 5, and 8, respectively) however led to a dramatic reduction in peptide binding to HLA-A2. Interestingly, compound 1a which contains modification between P1-P2 was found to be the most potent analogue, being able to retain the original HLA-A2 binding profile of the parent peptide M58-66. Taken together, these results and recent binding data obtained in the context of murine MHC class I molecule H-2Kd suggest that the incorporation of peptide bond surrogates in MHC class I-restricted epitopes is a useful approach to design molecules having both increased stability and high MHC-binding capacity. Depending on their agonist or antagonist effects at the T-cell receptor, such non-natural MHC ligands are likely to find many applications in the development of peptide-based vaccines or as potential therapeutic agents in the treatment of allergies and autoimmune diseases.
Subject(s)
HLA-A2 Antigen/metabolism , Orthomyxoviridae/metabolism , Peptides/metabolism , Viral Matrix Proteins/metabolism , Amino Acid Sequence , Humans , Ligands , Molecular Sequence Data , Peptides/chemistry , Protein Binding , Spectrometry, Mass, Fast Atom Bombardment , Tumor Cells, Cultured , Viral Matrix Proteins/chemistryABSTRACT
The peptide-binding motif of HLA-A29, the predisposing allele for birdshot retinopathy, was determined after acid-elution of endogenous peptides from purified HLA-A29 molecules. Individual and pooled HPLC fractions were sequenced by Edman degradation. Major anchor residues could be defined as glutamate at the second position of the peptide and as tyrosine at the carboxyl terminus. In vitro binding of polyglycine synthetic peptides to purified HLA-A29 molecules also revealed the need for an auxiliary anchor residue at the third position, preferably phenylalanine. By using this motif, we synthesized six peptides from the retinal soluble antigen, a candidate autoantigen in autoimmune uveoretinitis. Their in vitro binding was tested on HLA-A29 and also on HLA-B44 and HLA-B61, two alleles sharing close peptide-binding motifs. Two peptides derived from the carboxyl-terminal sequence of the human retinal soluble antigen bound efficiently to HLA-A29. This study could contribute to the prediction of T-cell epitopes from retinal autoantigens implicated in birdshot retinopathy.