title: Activating CTL precursors to reveal CTL function without skewing the repertoire by in vitro expansion.
abstract: Detection of the functional CD8(+) CTL response usually requires in vitro restimulation. The differences between the CD8(+) CTL repertoire in freshly isolated precursor cells and CD8(+) CTL after short-term in vitro expansion have been generally assumed to be minimal, but have never been defined experimentally. Using staining with P18-I10/H-2D(d) tetramers and monoclonal antibodies (mAb) against Vbeta, we show the surprising result that there was significant skewing of the CD8(+) CTL repertoire after just 7 days of stimulation. In contrast, we found that overnight incubation of precursor cells with peptide allows the functional assessment of CD8(+) CTL (which cannot be detected ex vivo from freshly isolated cells) without changing the absolute number of antigen-specific CTL as measured by tetramer staining or the repertoire of TCR analyzed with mAb. This study affords a better understanding of the differences between the ex vivo and in vitro stimulated CTL repertoire, and provides an approach to reveal a more faithful representation of the functional in vivo CTL response without skewing of the repertoire of T cells detected.
authors.affiliation: Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1578, USA. Belyakov@box-b.nih.gov
authors.forename: I M
authors.initials: IM
authors.lastname: Belyakov
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: R
authors.initials: R
authors.lastname: Koka
authors.forename: J D
authors.initials: JD
authors.lastname: Ahlers
authors.forename: J T
authors.initials: JT
authors.lastname: Snyder
authors.forename: R
authors.initials: R
authors.lastname: Tse
authors.forename: J
authors.initials: J
authors.lastname: Cox
authors.forename: J S
authors.initials: JS
authors.lastname: Gibbs
authors.forename: D H
authors.initials: DH
authors.lastname: Margulies
authors.forename: J A
authors.initials: JA
authors.lastname: Berzofsky
mesh_terms: D000818:Animals; D005260:Female; D019302:HIV Envelope Protein gp160 / Q000276:immunology; D015497:HIV-1 / Q000276:immunology; D006412:Hematopoietic Stem Cells / Q000276:immunology*; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000032:analysis; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D019302:HIV Envelope Protein gp160; D016693:Receptors, Antigen, T-Cell, alpha-beta