CD8 T-cell Epitopes Research Articles

Antigen-specific immunotherapy for autoimmune disease: fighting fire with fire?

The defining characteristic of an autoimmune disease is the existence of T- and B-cell autoreactivity directed against self proteins (autoantigens). The early events that precipitate autoreactivity in human autoimmune disease remain obscure. Once established, the autoreactive process itself may or may not be directly relevant to the pathological events involved in the disease. In patients with established rheumatoid arthritis, for example, on-going joint damage is most likely the result of a self-perpetuating inflammatory process that has few features of tissue-specific autoreactivity. In type 1 diabetes mellitus (DM), on the other hand, autoreactivity directed against the target cell in the pancreas reaches its peak of intensity around the time of clinical diagnosis and typically wanes only after the target cell has been destroyed. These are examples at opposite ends of a continuum but most theoretical models used to explain autoimmune disease invoke a period of autoreactivity against a specified protein autoantigen(s) as a critical phase in disease development. Work in animal models indicates that this phase may be susceptible to regulation by administration of the inciting autoantigen. In this review several key questions will be addressed in relation to antigen-specific immunotherapy (ASI) for autoimmune disease. First, does it work in animal models, and by what mechanism(s) of action? How should we best harness these effects for human disease? How will we know when it has worked? And finally, will it work in established disease or will prophylaxis be required? In terms of autoantigen administration, we will focus mainly upon systemic rather than mucosal routes, which have been discussed extensively in other recent reviews.1,2 A particular emphasis will be placed upon the issues to be faced in transposing ASI as a therapeutic option from animal into clinical studies.

Efficient identification of HLA-A*2402-restricted cytomegalovirus-specific CD8(+) T-cell epitopes by a computer algorithm and an enzyme-linked immunospot assay.

Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are useful tools for studying the CTL responses exclusively among those who own the major histocompatibility complex (MHC) class I molecules that present the peptides. For widening the application, an efficient strategy to determine such epitopes in the context of a given MHC is highly desirable. A rapid and efficient strategy is presented for the determination of CTL epitopes in the context of given MHC molecules of interest through multiple screenings consisting of a computer-assisted algorithm and MHC stabilization and enzyme-linked immunospot assays. A major cytomegalovirus (CMV)-specific CTL epitope, QYDPVAALF, in the amino acid sequence of its lower matrix 65 kd phosphoprotein (pp65) presented by HLA-A*2402 molecules was identified from 83 candidate peptides. The results indicate that the CMV-specific CTL response is highly focused to pp65 in the context of HLA-A*2402. Endogenous processing and presentation was confirmed using a peptide-specific CD8(+) T-cell clone as the effectors and autologous fibroblast cells infected with recombinant vaccinia virus expressing pp65 gene or CMV as antigen-presenting cells. Flow cytometric analysis of intracellular interferon-gamma production revealed 0.04% to 0.27% of CD8(+) T cells in peripheral blood of HLA-A24(+) and CMV-seropositive donors to be specific for the peptide. The tetrameric MHC-peptide complexes specifically bound to the reactive T-cell clone and 0.79% of CD8(+) T cells in peripheral blood from a seropositive donor. The peptide could be a useful reagent to study CTL responses to CMV among populations positive for HLA-A*2402.

Cytotoxic T cells to an epitope in the islet autoantigen IA-2 are not disease-specific.

Cytotoxic CD8 T lymphocytes (CTL) are effectors of pancreatic islet beta-cell destruction in type 1 diabetes but, with the exception of a single report, CTL to islet antigen peptides have not been identified. We used autologous blood monocyte-derived dendritic cells to elicit HLA-A2-restricted CTL to a peptide, MVWESGCTV (aa 797-805), that is contiguous with a dominant CD4 T-cell epitope in the islet antigen tyrosine phosphatase IA-2. IA-2 peptide-specific CTL activity measured as 51Cr release from autologous lymphoblasts was detected in 2/6 islet antibody-positive relatives at high risk for type 1 diabetes but also in 2/6 closely HLA-matched controls. All subjects had CTL activity to an HLA-A2-restricted Epstein-Barr virus peptide. CTL to the IA-2 self-peptide were therefore not disease-specific, consistent with other evidence that autoreactive T cells are present in healthy individuals.

Induction of polyomavirus-specific CD8(+) T lymphocytes by distinct dendritic cell subpopulations.

Dendritic cells are pivotal antigen-presenting cells for generating adaptive T-cell responses. Here, we show that dendritic cells belonging to either the myeloid-related or lymphoid-related subset are permissive for infection by mouse polyomavirus and, when loaded with a peptide corresponding to the immunodominant anti-polyomavirus CD8(+) T-cell epitope or infected by polyomavirus, are each capable of driving expansion of primary polyomavirus-specific CD8(+) T-cell responses in vivo.

New insights into evaluating effective T-cell responses to HIV.

Towards addressing questions related to HIV pathogenesis and vaccine design we are fortunate to have the availability of the SIV-infected rhesus macaque model. The strengths of this model which include a rapid rate of progression to AIDS and knowledge of the dose route and strain of the infecting virus complement studies in HIV-infected patients in which the reagents host genetics and access to samples are more extensive and better defined. Unfortunately there is currently still too little known about the antiviral immune responses in either system to directly and accurately compare their similarities and differences and to draw any definitive conclusions. Therefore the data and views presented herein will simply reflect what has recently been discovered in both humans and non-human primate studies. (excerpt)

T-cell vaccination alters the course of murine herpesvirus 68 infection and the establishment of viral latency in mice.

Diseases caused by gammaherpesviruses such as Epstein-Barr virus are a major health concern, and there is significant interest in developing vaccines against this class of viral infections. However, the requirements for effective control of gammaherpesvirus infection are only poorly understood. The recent development of the murine herpesvirus MHV-68 model provides an experimental tool to dissect the immune response to gammaherpesvirus infections. In this study, we investigated the impact of priming T cells specific for class I- and class II-restricted epitopes on the acute phase of the infection and the subsequent establishment of latency and infectious mononucleosis. The data show that vaccination with either major histocompatibility complex class I- or class II-restricted T-cell epitopes derived from lytic cycle proteins significantly reduced lung viral titers during the acute infection. Moreover, the peak level of latently infected spleen cells was significantly reduced following vaccination with immunodominant CD8(+) T-cell epitopes. However, this vaccination approach did not prevent the long-term establishment of latency or the development of the infectious mononucleosis-like syndrome in infected mice. Thus, the virus is able to establish latency efficiently despite strong immunological control of the lytic infection.

Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library.

Type 1 diabetes is an autoimmune disease in which the insulin-producing pancreatic beta cells are destroyed at an early age by an immune process that involves both CD4 and CD8 T lymphocytes. The identification of autoantigens in diabetes is very important for the design of antigen-specific immunotherapy. By screening a pancreatic islet cDNA library, we have identified the autoantigen recognized by highly pathogenic CD8 T cells in the non-obese diabetic mouse, one of the best animal models for human diabetes. This is the first identification, to our knowledge, of a CD8 T-cell epitope in an autoimmune disease. The peptide recognized by the cells is in the same region of the insulin B chain as the epitope recognized by previously isolated pathogenic CD4 T cells. This has very important implications for the potential use of insulin in preventative therapy.

Infection with cytotoxic T-lymphocyte escape mutants results in increased mortality and growth retardation in mice infected with a neurotropic coronavirus.

C57BL/6 mice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating encephalomyelitis several weeks after inoculation. Previously, we showed that mutations in the immunodominant CD8 T-cell epitope (S-510-518) could be detected in nearly all samples of RNA and virus isolated from these mice. These mutations abrogated recognition by T cells harvested from the central nervous systems of infected mice in direct ex vivo cytotoxicity assays. These results suggested that cytotoxic T-lymphocyte (CTL) escape mutants contributed to virus amplification and the development of clinical disease in mice infected with wild-type virus. In the present study, the importance of these mutations was further evaluated by infecting naive mice with MHV-JHM variants isolated from infected mice and in which epitope S-510-518 was mutated. Compared to mice infected with wild-type virus, variant virus-infected animals showed higher mortality and morbidity manifested by decreased weight gain and neurological signs. Although a delay in the kinetics of virus clearance has been demonstrated in previous studies of CTL escape mutants, this is the first illustration of significant changes in clinical disease resulting from infection with viruses able to evade the CD8 T-cell immune response.

Different Endosomal Proteolysis Requirements for Antigen Processing of Two T-cell Epitopes of the M5 Protein from ViableStreptococcus pyogenes

We studied endosomal proteolysis of the surface fibrillar M5 protein from viable Streptococcus pyogenes as an essential step involved in major histocompatibility complex class II-restricted antigen processing of two immunodominant CD4(+) T-cell epitopes (17-31/Ed and 308-319/Ad). Intracellular proteolysis of viable streptococci for presentation of 17-31, bound by serine proteinase cleavage sites, was mediated by serine proteinases, whereas processing of soluble recombinant M5 protein required in addition cysteine proteinases. Furthermore, processing of 17-31 was resistant to ammonium chloride and thus was not dependent on endosome acidification. Cysteine and serine proteinase cleavage sites were located adjacent to 308-319, and its processing was dependent on serine, cysteine, and aspartic proteinases, as well as on endosomal acidification. The data suggest that antigen processing of two major T-cell epitopes on streptococcal M5 protein occurred in different endosomal compartments by different classes of intracellular proteinases.

Cytotoxic T-cell-resistant variants arise at early times after infection in C57BL/6 but not in SCID mice infected with a neurotropic coronavirus.

Under certain conditions, C57BL/6 mice persistently infected with mouse hepatitis virus strain JHM (MHV-JHM) develop clinical disease and histological evidence of demyelination several weeks after inoculation with virus. In a previous report, we showed that mutations in the RNA encoding an immunodominant CD8 T-cell epitope within the surface glycoprotein (epitope S-510-518) were present in all persistently infected animals and that these mutations abrogated recognition by virus-specific cytotoxic T cells (CTLs) in direct ex vivo cytotoxicity assays. To obtain further evidence that these mutations were necessary for the development of clinical disease, the temporal course of their appearance was determined. Mutations in the epitope were identified by 10 to 12 days after inoculation, and in some mice, virus containing mutated epitope was the dominant species detected by 15 days. In addition, most mice that remain asymptomatic at 80 days after inoculation, a time after which clinical disease almost never develops, were infected with only wild-type virus. Finally, analysis of virus isolated from mice with severe combined immunodeficiency (SCID) revealed the presence only of wild-type epitope S-510-518. These results, by showing that mutations are not selected in SCID mice and occur at early times after inoculation in C57BL/6 mice, support the view that they result from immune pressure and contribute to virus persistence and demyelination in mice infected persistently with MHV-JHM.

Analysis of the human T-cell response to picornaviruses: identification of T-cell epitopes close to B-cell epitopes in poliovirus.

Little is known about the nature and specificity of T-cell-mediated responses to picornaviruses in humans. In this study, the nature of the T-cell response to seven picornaviruses, including polioviruses, coxsackieviruses B3 and B4, human rhinovirus 14, and encephalomyocarditis virus, was determined. Twenty-nine individuals responded to poliovirus type 3, coxsackievirus B3, and encephalomyocarditis virus by proliferation of T cells, and from such cultures, 130 virus-specific T-cell lines were established. T-cell lines generated in response to encephalomyocarditis virus were exclusively strain specific. However, the majority of T-cell lines established in response to viruses, other than encephalomyocarditis virus, were cross-reactive to each other. Their cross-reactivity was confirmed in 2 of the 30 picornavirus-specific clonally derived T-cell lines from two subjects, but the majority of these lines were serotype specific. T-cell epitopes adjacent to each of the B-cell antigenic sites in VP1 of poliovirus type 3 were identified. The response to the region adjacent to B-cell antigenic site 1 (residues 97 to 114) was dominant between individuals. The localization of this major CD4 T-cell epitope may permit the construction of chimeric viruses utilizing the natural picornavirus T-cell response to augment production of antibody specific for inserted sequences.

Synthetic asset swaps

<h3>Abstract</h3> Genotype 1 of the hepatitis C virus (HCV) is the most prevalent of the variants of this virus. Its two main subtypes, HCV-1a and HCV-1b, are associated to differences in epidemic features and risk groups, despite sharing similar features in most biological properties. We have analyzed the impact of positive selection on the evolution of these variants using complete genome coding regions, and compared the levels of genetic variability and the distribution of positively selected sites. We have also compared the distributions of positively selected and conserved sites considering different factors such as RNA secondary structure, the presence of different epitopes (antibody, CD4 and CD8), and secondary protein structure. Less than 10% of the genome was found to be under positive selection, and purifying selection was the main evolutionary force in both subtypes. We found differences in the number of positively selected sites between subtypes in several genes (<i>Core</i>, HVR2 in <i>E2, P7</i>, helicase in <i>NS3</i> and <i>NS4a).</i> Heterozygosity values in positively selected sites and the rate of non-synonymous substitutions were significantly higher in subtype HCV-1b. Logistic regression analyses revealed that similar selective forces act at the genome level in both subtypes: RNA secondary structure and CD4 T-cell epitopes are associated with conservation, while CD8 T-cell epitopes are associated with positive selection in both subtypes. These results indicate that similar selective constraints are acting along HCV-1a and HCV-1b genomes, despite some differences in the distribution of positively selected sites at independent genes.