ORFeome approach to the clonal, HLA allele-specific CD4 T-cell response to a complex pathogen in humans

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• 作者：Lichen Jing ; Stella Mayo McCaughey ; D. Huw Davies ; Tiana M. Chong ; Phillip L. Felgner ; Stephen C. De Rosa ; Christopher B. Wilson ; David M. Koelle
• 关键词：T-cell ; CD4 ; Antigen ; Epitope ; Specificity ; Combinatorial
• 刊名：Journal of Immunological Methods
• 出版年：2009
• 出版时间：15 August 2009
• 年：2009
• 卷：347
• 期：1-2
• 页码：36-45
• 全文大小：1101 K

文摘

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.