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ORFeome approach to the clonal, HLA allele-specific CD4 T-cell response to a complex pathogen in humans.
- Source :
-
Journal of immunological methods [J Immunol Methods] 2009 Aug 15; Vol. 347 (1-2), pp. 36-45. Date of Electronic Publication: 2009 Jun 09. - Publication Year :
- 2009
-
Abstract
- 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.
- Subjects :
- Animals
Antigen Presentation
Antigens, Viral immunology
CD4-Positive T-Lymphocytes virology
COS Cells
Chlorocebus aethiops
Cytokines metabolism
Genome, Viral
Humans
Hybridomas
Mice
Vaccinia virus immunology
Vaccinia virus pathogenicity
Antigens, Viral genetics
CD4-Positive T-Lymphocytes immunology
HLA-DQ Antigens immunology
HLA-DR Antigens immunology
Open Reading Frames
Proteomics methods
Smallpox Vaccine immunology
Vaccinia virus genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1872-7905
- Volume :
- 347
- Issue :
- 1-2
- Database :
- MEDLINE
- Journal :
- Journal of immunological methods
- Publication Type :
- Academic Journal
- Accession number :
- 19520082
- Full Text :
- https://doi.org/10.1016/j.jim.2009.05.011