Treg CTCL cells inhibit normal control antigen-stimulated cytokine production. Normal control lymphocytes (2 × 105/well) were stimulated to proliferate with tetanus toxoid. CTCL cells were induced to assume a Treg phenotype, as previously described, and were added at graded doses to the normal control lymphocytes. Supernatants were harvested at day 5 and were tested for cytokine concentration by ELISA. Results are presented as the mean ± SD of 3 replicate wells. Coexpression of membrane Vβ8/cytoplasmic CTLA-4 was determined in responding CTCL cells by flow cytometry. (A) Nonpulsed DCs cocultivated with Vβ8+ CTCL cells overnight. (B) Responding CTCL cells cocultured with DCs pulsed with CD3-treated apoptotic CTCL. (C) DCs pulsed with CD4-treated viable CTCL cells and cultured with responding CTCL cells. (D) CTCL cells that were induced to assume a Treg phenotype by cocultivation with tumor-loaded DCs (B) or noninduced CTCL cells cultured with viable CTCL cells (C) were added to antigen-stimulated normal control cells (1:1, 1:10, 1:100 CTCL cells/normal cells), and the production of IL-2 was measured by ELISA. Significant suppression was obtained when Treg CTCL cells were added compared with the addition of the same number of noninduced CTCL cells (P ≤ .001). (E) Adding a 1:1 ratio of Treg CTCL cells to antigen-stimulated normal control cells significantly reduced the production of IFN-γ (P ≤ .001) compared with the addition of the same number of noninduced CTCL cells. For panels D and E, ▪ indicates no antigen; ▨, tetanus.
