Identification Of Cytotoxic T Lymphocytes Epitopes Research Articles

Revival of the identification of cytotoxic T-lymphocyte epitopes for immunological diagnosis, therapy and vaccine development

Immunogenic T-cell epitopes have a central role in the cellular immunity against pathogens and tumors. However, in the early stage of cellular immunity studies, it was complicated and time-consuming to identify and characterize T-cell epitopes. Currently, the epitope screening is experiencing renewed enthusiasm due to advances in novel techniques and theories. Moreover, the application of T-cell epitope-based diagnoses for tuberculosis and new data on epitope-based vaccine development have also revived the field. There is a growing knowledge on the emphasis of epitope-stimulated T-cell immune responses in the elimination of pathogens and tumors. In this review, we outline the significance of the identification and characterization of T-cell epitopes. We also summarize the methods and strategies for epitope definition and, more importantly, address the relevance of cytotoxic T-lymphocyte epitopes to clinical diagnoses, therapy and vaccine development.

Identification of MHC class II restricted T-cell-mediated reactivity against MHC class I binding Mycobacterium tuberculosis peptides

Major histocompatibility complex (MHC) class I restricted cytotoxic T lymphocytes (CTL) are known to play an important role in the control of Mycobacterium tuberculosis infection so identification of CTL epitopes from M. tuberculosis is of importance for the development of effective peptide-based vaccines. In the present work, bioinformatics technology was employed to predict binding motifs of 9mer peptides derived from M. tuberculosis for the 12 HLA-I supertypes. Subsequently, the predicted peptides were synthesized and assayed for binding to HLA-I molecules in a biochemically based system. The antigenicity of a total of 157 peptides with measured affinity for HLA-I molecules of K(D) ≤ 500 nM were evaluated using peripheral blood T cells from strongly purified protein derivative reactive healthy donors. Of the 157 peptides, eight peptides (5%) were found to induce T-cell responses. As judged from blocking with HLA class I and II subtype antibodies in the ELISPOT assay culture, none of the eight antigenic peptides induced HLA class I restricted CD8(+) T-cell responses. Instead all responses were blocked by pan-HLA class II and anti-HLA-DR antibodies. In addition, CD4(+) T-cell depletion before the 10 days of expansion, resulted in total loss of reactivity in the ELISPOT culture for most peptide specificities. FACS analyses with intracellular interferon-γ staining of T cells expanded in the presence of M. tuberculosis peptides confirmed that the responsive cells were indeed CD4(+). In conclusion, T-cell immunity against HLA-I binding 9mer M. tuberculosis-derived peptides might in many cases turn out to be mediated by CD4(+) T cells and restricted by HLA-II molecules. The use of 9mer peptides recognized by both CD8(+) and CD4(+) T cells might be of importance for the development of future M. tuberculosis peptide-based vaccines.

Identification of HLA-A*01- and HLA-A*02-restricted CD8+ T-cell epitopes shared among group B enteroviruses

Acute enteroviral infections ranging from meningitis, pancreatitis to myocarditis are common and normally well controlled by the host immune system comprising virus-specific CD8+ cytotoxic T lymphocytes (CTL). However, in some patients enteroviruses and especially coxsackieviruses of group B are capable of inducing severe chronic forms of diseases such as chronic myocarditis. Currently, it is not known whether divergences in the CTL-related immune response may contribute to the different outcome and course of enterovirus myocarditis. A pre-requisite for the study of CTL reactions in patients with acute and chronic myocarditis is the identification of CTL epitopes. In order to define dominant enterovirus CTL epitopes, we have screened, by using gamma interferon (IFN-gamma) ELISPOT, 62 HLA-A*01- and 59 HLA-A*02-positive healthy blood donors for pre-existing CTL reactions against 12 HLA-A*01 and 20 HLA-A*02 predicted CTL epitopes derived from coxsackieviruses of group B. Positive CTL reactions were verified by FACS analysis in a combined major histocompatibility complex-tetramer IFN-gamma staining. A total of 14.8% of all donors reacted against one of the three identified epitopes MLDGHLIAFDY, YGDDVIASY or GIIYIIYKL. The HLA-A*02-restricted epitope ILMNDQEVGV was recognized by 25% of all tested blood donors. For this peptide, we could demonstrate specific granzyme B secretion, a strong cytolytic potential and endogenous processing. All four epitopes were homologous in 36-92% of group B enteroviruses, providing a strong basis for monitoring the divergence of T-cell-based immune responses in enterovirus-induced acute and chronic diseases.

A molecular approach to the identification of cytotoxic T-lymphocyte epitopes within equine herpesvirus 1

Equine herpesvirus 1 (EHV-1) causes respiratory and neurological disease and abortion in horses. Animals with high frequencies of cytotoxic T lymphocytes (CTL) show reduced severity of respiratory disease and frequency of abortion, probably by CTL-mediated control of cell-associated viraemia. This study aimed to identify CTL epitopes restricted by selected major histocompatibility complex (MHC) class I alleles expressed in the equine leukocyte antigen (ELA) A3 haplotype. Effector CTL were induced from EHV-1-primed ponies and thoroughbreds with characterized MHC class I haplotypes and screened against P815 target cells transfected with selected EHV-1 genes and MHC class I genes. Targets that expressed EHV-1 gene 64 and the MHC B2 gene were lysed by effector CTL in a genetically restricted manner. There was no T-cell recognition of targets expressing either the MHC B2 gene and EHV-1 genes 2, 12, 14, 16, 35, 63 or 69, or the MHC C1 gene and EHV-1 genes 12, 14, 16 or 64. A vaccinia virus vector encoding gene 64 (NYVAC-64) was also investigated. Using lymphocytes from ELA-A3 horses, the recombinant NYVAC-64 virus induced effector CTL that lysed EHV-1-infected target cells; the recombinant virus also supplied a functional peptide that was expressed by target cells and recognized in an MHC-restricted fashion by CTL induced with EHV-1. This construct may therefore be used to determine the antigenicity of EHV-1 gene 64 for other MHC haplotypes. These techniques are broadly applicable to the identification of additional CTL target proteins and their presenting MHC alleles, not only for EHV-1, but for other equine viruses.

The use of Epstein-Barr virus-transformed B lymphocyte cell lines in a peptide-reconstitution assay: identification of CEA-related HLA-A*0301-restricted potential cytotoxic T-lymphocyte epitopes.

In the development of cytotoxic T lymphocyte (CTL)-mediated immunotherapy, the identification of CTL epitopes is of crucial importance. Binding of a peptide to major histocompatibility complex (MHC) class I molecules is one of the prerequisites for its function as a CTL epitope. We describe the technique, validation, and application of a simple cellular assay, intended for the screening of peptides for binding, that can be applied to any human leukocyte antigen (HLA) allele. Reconstitution of peptides in MHC class I molecules after elution by acid treatment was previously shown to be possible in specially engineered cell lines expressing only one type of MHC class I, and was applied for the HLA-A*0201 allele. We now report the optimal conditions for application of this type of binding assay to the HLA-A*0301 allele. The adaptations that were necessary to make the technique operational for HLA-A*0301 are shown in detail. These consisted of lowering the pH during acid treatment to 2.9 and lengthening the duration of elution to 90 s. Furthermore, immediate aspiration of eluted peptides appeared to be essential for this allele. We found also that the use of Epstein-Barr virus (EBV)-transformed B cell lines (B-LCL) yields results similar to those of the use of cell lines expressing only one specific MHC class I allele. Homozygosity for the desired HLA allele improves the sensitivity of the assay, but heterozygous cells can also be employed. Finally, we applied this technique to a search for HLA-A*0301 binding peptides derived from carcinoembryonic antigen (CEA). Of a set of 34 CEA-specific peptides that fit with a specified HLA-A*0301-binding motif, we identified a set of six peptides with high binding affinity to this allele. These peptides can be regarded as potential CTL epitopes.