The IL-2-dependent maturation of CD4−CD8− double-negative T cells in transgenic 2C mice

Abstract

2C mice have a preponderance of 1B2+CD8+ T cells directed against L d. After in vitro sensitization, they become highly cytotoxic and will reject a tolerant L d+ heart graft. Anti-lymphocyte serum (ALS) eliminates the CD4+ and CD8+ T cells and a large CD4−CD8− double-negative (DN) transgenic cell population remains. To determine the activation characteristics of these 2C DN cells, peripheral T cells were analyzed by FACS for 1B2+, CD8+, and CD4+ before and 48 h after 0.5 mL ALS IP. In vitro, the response of these 2C DN cells were evaluated after 7 days of culture with BALB/c, IL-2, or BALB/c + IL-2. Cytotoxicity (CTL) was tested in a 4-h 51Cr-release assay. Following in vitro culture with BALB/c + IL-2, 2C DN cells were injected into B6 mice with a tolerant BALB/c heart (tolerization via anti-CD4 mAb and intrathymic BALB/c) to determine their immunogenicity. After the deletion of CD4+ and CD8+ T cells, DN cells increased to 96–99%. After 7 days of culture, the DN cells decreased again to 33–38%. CD8+ cells decreased to <0.1% after ALS treatment, but increased to 61–70% after in vitro culture. Untreated 2C cells responded to BALB/c or IL-2 equally well. However, after ALS treatment, DN cells responded to IL-2 and BALB/c + IL-2, but not BALB/c alone. In the periphery of transgenic 2C mice is a unique CD4−CD8− DN population of cells bearing the 1B2 transgenic marker. These noncytotoxic cells can be optimally stimulated to develop marked specific L d cytotoxicity in parallel with the expression of the CD8+ epitope. TABLE—ABSTRACT 75 Naı̈ve 2C ALS-2C (before culture) ALS-2C (after culture) %T cells by FACS DN 29–35% 96–99% 33–38% CD8+ 56–62% 0–0.1% 61–70% CD4+ 4–9% 0–1% 0% %CTL after culture with: Balb/c 51–54% — 0% IL-2 81–88% — 60–62% Balb/c + IL-2 86–91% — 88–90% ∗ ∗ DN T cells cultured for 7 days with Balb/c + IL-2 rejected the tolerant Balb/c heart in 5.3 ± 0.3 days (n = 4).