Figure 3.
Figure 3. Proliferative response to DCs loaded with apoptotic CTCL cells. (A) CTCL cells (2 × 106/well) cocultivated overnight alone without DCs did not proliferate substantially (0). Adding purified CTCL cells (2 × 106/well) to autologous DCs (1 × 105/well) overnight induced a small proliferative response (auto DC), whereas adding allogeneic DCs (allo DC, 1 × 105/well) did not stimulate proliferation. When autologous DCs were pulsed overnight with CD3 treated apoptotic CTCL cells (1 × 106/well) and then cocultivated with freshly purified CTCL cells (2 × 106/well), a significant (P ≤ .001) proliferative response was obtained. The proliferative response was determined by measuring the counts per minute (CPM) incorporated after an 18-hour pulse of 3[H]-thymidine. Results presented are the mean ± SD of 5 replicate cultures. (B) CTCL cells mobilize calcium stores after stimulation with tumor-loaded DCs. Purified CTCL cells (107) were loaded with indo-1 am and flowed through a cytometer equipped with a UV laser. DCs (3 × 105) loaded overnight with apoptotic CTCL cells (1 × 106) were injected into the flow stream, and induction of a calcium flux was measured over time. Controls included DCs loaded with viable CTCL cells or γ-irradiated normal control CD4 T cells.

Proliferative response to DCs loaded with apoptotic CTCL cells. (A) CTCL cells (2 × 106/well) cocultivated overnight alone without DCs did not proliferate substantially (0). Adding purified CTCL cells (2 × 106/well) to autologous DCs (1 × 105/well) overnight induced a small proliferative response (auto DC), whereas adding allogeneic DCs (allo DC, 1 × 105/well) did not stimulate proliferation. When autologous DCs were pulsed overnight with CD3 treated apoptotic CTCL cells (1 × 106/well) and then cocultivated with freshly purified CTCL cells (2 × 106/well), a significant (P ≤ .001) proliferative response was obtained. The proliferative response was determined by measuring the counts per minute (CPM) incorporated after an 18-hour pulse of 3[H]-thymidine. Results presented are the mean ± SD of 5 replicate cultures. (B) CTCL cells mobilize calcium stores after stimulation with tumor-loaded DCs. Purified CTCL cells (107) were loaded with indo-1 am and flowed through a cytometer equipped with a UV laser. DCs (3 × 105) loaded overnight with apoptotic CTCL cells (1 × 106) were injected into the flow stream, and induction of a calcium flux was measured over time. Controls included DCs loaded with viable CTCL cells or γ-irradiated normal control CD4 T cells.

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