ORFeome approach to the clonal, HLA allele-specific CD4 T-cell response to a complex pathogen in humans
| Autor: | Stella Mayo McCaughey, David M. Koelle, Stephen C. De Rosa, Phillip L. Felgner, Christopher B. Wilson, Lichen Jing, Tiana M. Chong, D. Huw Davies |
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| Rok vydání: | 2009 |
| Předmět: |
CD4-Positive T-Lymphocytes
Proteomics Immunology Vaccinia virus Human leukocyte antigen Genome Viral Biology Epitope Article chemistry.chemical_compound Mice Open Reading Frames Antigen HLA-DQ Antigens Chlorocebus aethiops HLA-DR Immunology and Allergy Animals Humans ORFeome Antigens Viral Genetics Antigen Presentation Hybridomas HLA-DQ Antigen HLA-DR Antigens Virology Open reading frame chemistry COS Cells Cytokines Vaccinia Smallpox Vaccine |
| Zdroj: | Journal of immunological methods. 347(1-2) |
| ISSN: | 1872-7905 |
| Popis: | The CD4 T-cell response to vaccinia promotes antibody and long-term CD8 responses. HLA class II molecules present microbial epitopes to CD4 T-cells. In humans, at least 3 loci encode cell-surface peptide-binding HLA class II heterodimers. Using intracellular cytokine cytometry (ICC) assays, we determined that HLA DR had the strongest contribution to vaccinia antigen presentation. Among panels of vaccinia-restricted T-cell clones, most were DR-restricted but rare DQ-restricted clones were also recovered. Vaccinia has over 200 open reading frames (ORFs), providing a significant bottleneck to assigning fine specificity. To overcome this, we expressed each predicted vaccinia ORF using in vitro transcription and translation. Array-based pool proteins were used to rapidly assign fine specificity to each DQ-restricted clone and to a sample of HLA DR-restricted clones. Reactivity was confirmed using synthetic peptides for selected CD4 T-cell clones. This method should be broadly applicable to the study of large-genome, sequenced pathogens, and could also be used to investigate T-cell responses to cDNAs expressed in neoplastic and autoimmune disorders in which CD4 responses might be adaptive or harmful. |
| Databáze: | OpenAIRE |
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