Figure 4
Figure 4. Impacts of initial step 1 DC conditioning treatments upon subsequent culture of T cells from tumor-bearing mice. Tumor-pulsed DC preparations and co-cultures were performed as in Figure 3B. (A) T-cell fold-expansion 12 days after initial exposure to tumor-pulsed DCs or to anti-CD3; labels indicate prior DC conditioning treatments. As distinguished in the legend, the displayed T-cell expansions were performed either with no added cytokine, added rIL-2 only (24 IU/mL beginning day 2 of T-cell culture), or IL-2 + IL-7 + IL-15 (50 ng IL-7, 5 ng IL-15). Data are representative of 3 experiments. (B) Coculture of T cells from tumor-bearing mice with Flt3L + IL-6– conditioned DCs results in superior expansion of tumor-specific T cells. Step 1 DC conditioning was performed with Flt3L + IL-6, rest of DC preparation and T-cell cocultures as in Figure 3B. After a 12-day coculture, T cells were harvested and replated either alone or with irradiated MCA-203 or MCA-105 as stimulator cells. Monensin (Golgistop) was added after 5 hours, and cells analyzed after an additional 12 hours for intracellular IFNγ production. Dot plots show T cells recultured after expansion in IL-2 + IL-7 + IL-15, but coculture with tumor pulsed, Flt3L + IL-6–conditioned DCs yielded virtually identical tumor-specificity even when no cytokines were added during the 12-day coculture (not shown). (Bi) Dot plots display CD4 versus IFNγ staining; the percentages shown in each dot plot is that of total CD4 cells producing IFNγ. (Bii) Dot plots display CD8 versus IFNγ staining; the percentages in each dotplot is that of total CD8 cells producing IFNγ. This is representative of 6 experiments. (C) T cells driven with Flt3L + IL-6–conditioned DCs are highly effective as adoptive therapy. Five-day established MCA-203 subcutaneous tumors were treated intravenously with T cells from MCA-203 bearers after 12-day culture driven by Flt3L + IL-6–conditioned, tumor-pulsed DCs or by anti-CD3. Conventional nonmyelablative total body irradiation (500 cGy) was given as an adjunct prior to T cells. Cure rates were 0/5 (A, No treatment); 2/5 (B, 5 million anti-CD3 driven T cells); 5/5 (C, 5 million Flt3L + IL-6 DC-driven T cells); 5/5 (D, same as C but 2 million T cells). Treatment outcome A versus C/D, P < .008; A versus B, P < .141; B versus C/D, P = .04. T-cell cultures driven by Flt3L + IL-6–conditioned DCs were also highly effective against more advanced tumors (Figure S6). Data are representative of 4 experiments. Error bars represent SD.

Impacts of initial step 1 DC conditioning treatments upon subsequent culture of T cells from tumor-bearing mice. Tumor-pulsed DC preparations and co-cultures were performed as in Figure 3B. (A) T-cell fold-expansion 12 days after initial exposure to tumor-pulsed DCs or to anti-CD3; labels indicate prior DC conditioning treatments. As distinguished in the legend, the displayed T-cell expansions were performed either with no added cytokine, added rIL-2 only (24 IU/mL beginning day 2 of T-cell culture), or IL-2 + IL-7 + IL-15 (50 ng IL-7, 5 ng IL-15). Data are representative of 3 experiments. (B) Coculture of T cells from tumor-bearing mice with Flt3L + IL-6– conditioned DCs results in superior expansion of tumor-specific T cells. Step 1 DC conditioning was performed with Flt3L + IL-6, rest of DC preparation and T-cell cocultures as in Figure 3B. After a 12-day coculture, T cells were harvested and replated either alone or with irradiated MCA-203 or MCA-105 as stimulator cells. Monensin (Golgistop) was added after 5 hours, and cells analyzed after an additional 12 hours for intracellular IFNγ production. Dot plots show T cells recultured after expansion in IL-2 + IL-7 + IL-15, but coculture with tumor pulsed, Flt3L + IL-6–conditioned DCs yielded virtually identical tumor-specificity even when no cytokines were added during the 12-day coculture (not shown). (Bi) Dot plots display CD4 versus IFNγ staining; the percentages shown in each dot plot is that of total CD4 cells producing IFNγ. (Bii) Dot plots display CD8 versus IFNγ staining; the percentages in each dotplot is that of total CD8 cells producing IFNγ. This is representative of 6 experiments. (C) T cells driven with Flt3L + IL-6–conditioned DCs are highly effective as adoptive therapy. Five-day established MCA-203 subcutaneous tumors were treated intravenously with T cells from MCA-203 bearers after 12-day culture driven by Flt3L + IL-6–conditioned, tumor-pulsed DCs or by anti-CD3. Conventional nonmyelablative total body irradiation (500 cGy) was given as an adjunct prior to T cells. Cure rates were 0/5 (A, No treatment); 2/5 (B, 5 million anti-CD3 driven T cells); 5/5 (C, 5 million Flt3L + IL-6 DC-driven T cells); 5/5 (D, same as C but 2 million T cells). Treatment outcome A versus C/D, P < .008; A versus B, P < .141; B versus C/D, P = .04. T-cell cultures driven by Flt3L + IL-6–conditioned DCs were also highly effective against more advanced tumors (Figure S6). Data are representative of 4 experiments. Error bars represent SD.

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