ORFeome approach to the clonal, HLA allele-specific CD4 T-cell response to a complex pathogen in humans.
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.
Jing
Lichen
L
Department of Medicine, University of Washington, 1616 Eastlake Avenue East, Suite 251, Seattle, WA 98102, USA. lchjing@u.washington.edu
McCaughey
Stella Mayo
SM
Davies
D Huw
DH
Chong
Tiana M
TM
Felgner
Phillip L
PL
De Rosa
Stephen C
SC
Wilson
Christopher B
CB
Koelle
David M
DM
D000818:Animals; D017951:Antigen Presentation; D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000821:virology; D019556:COS Cells; D002522:Chlorocebus aethiops; D016207:Cytokines / Q000378:metabolism; D016679:Genome, Viral; D006683:HLA-DQ Antigens / Q000276:immunology*; D006684:HLA-DR Antigens / Q000276:immunology*; D006801:Humans; D006825:Hybridomas; D051379:Mice; D016366:Open Reading Frames; D040901:Proteomics / Q000379:methods*; D012900:Smallpox Vaccine / Q000276:immunology*; D014616:Vaccinia virus / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity
D000956:Antigens, Viral; D016207:Cytokines; C078786:DryVax vaccine; D006683:HLA-DQ Antigens; D006684:HLA-DR Antigens; D012900:Smallpox Vaccine