Figure 3.

Non-specific PPI-tolDC-T-cell proliferation assay. (A) Schematic of non-specific T-cell stimulation experiments used for data in parts (B–D). For each group, 800K T cells were incubated with 200K DCs and analyzed after 72 h. Controls included T cells only (blank), T cells + untreated DCs + 0.5 μL/mL T-cell stimulation cocktail with PMA/ionomycin (positive), untreated DCs (naïve DCs), and DCs treated with 12.5 μg/mL R848 (R848 DC). All groups contained T cells, and all groups except blank were treated with 100 ng/mL αCD28/CD3. (B) Representative flow plot of unspecific T-cell proliferation for CD4⁺ T cells with gating for generation number. T-cell proliferation by generation number for each treatment was calculated for part (C). (C) Representative pie charts showing the breakdown of average T-cell generation for selected treatment groups. Top: CD4⁺ T cells Bottom: CD8⁺ T cells. Complete dataset can be found in Supplementary Figure S5. (D) Cells were further analyzed for CD4⁺ CD25⁺ FoxP3⁺ T cells (T_reg), and the percentage of T_reg per CD4⁺ cells was calculated. Gating strategy is found in Supplementary Figure S7. Significance was determined by a two-tailed student’s t-test with Tukey post-hoc test. ** indicates p < 0.01. All control samples were performed in duplicates, and experimental groups were performed in biological triplicates. Error bars are ±SD.