Activating CTL precursors to reveal CTL function without skewing the repertoire byin 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‐2Dd tetramers and monoclonal antibodies (mAb) against Vβ, 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 isolatedcells) 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.

Protection against Lethal Vaccinia Virus Challenge in HLA-A2 Transgenic Mice by Immunization with a Single CD8+T-Cell Peptide Epitope of Vaccinia and Variola Viruses

“…Spontaneous release was determined from target cells incubated without effector cells, and maximum release was determined in the presence of 5% Triton X-100 (no. 3786; Research Products International, Mount Prospect, Ill.) (13,52).…”

Transcutaneous immunization induces mucosal CTLs and protective immunity by migration of primed skin dendritic cells

“…Maximum release was determined from supernatants of cells that were lysed by the addition of 5% Triton-X 100. Spontaneous release was determined from target cells incubated without added effector cells (34).…”

A Novel Functional CTL Avidity/Activity Compartmentalization to the Site of Mucosal Immunization Contributes to Protection of Macaques against Simian/Human Immunodeficiency Viral Depletion of Mucosal CD4+ T Cells

“…Immune cells were activated overnight at 0.5 ϫ 10 6 cells/well in a 12-well plate in the presence of 0.02 M synthetic CTL epitope peptides in complete T cell medium (56). Mamu-A*01 ϩ target cells were pulsed with peptide from 10 to 10 Ϫ4 M for 2 h during 51 Cr labeling and cultured with effectors at 37°C for 4 h. Specific release was calculated as described (7).…”