Activating CTL precursors to reveal CTL function without skewing the repertoire by in vitro expansion.
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.
Belyakov
I M
IM
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
Wang
J
J
Koka
R
R
Ahlers
J D
JD
Snyder
J T
JT
Tse
R
R
Cox
J
J
Gibbs
J S
JS
Margulies
D H
DH
Berzofsky
J A
JA
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*
D019302:HIV Envelope Protein gp160; D016693:Receptors, Antigen, T-Cell, alpha-beta