Fig. 4

Reversal of immunosuppression within the MB49^luc bladder TME following NHS-muIL12 treatment. MB49^luc bladder tumors (n = 3) from control Ig– and NHS-muIL12–treated mice were isolated 5 days post–final NHS-muIL12 treatment (day 20, see Fig. 1, panel a). CD45⁺ cells were purified from tumor homogenates and cocultured with CD4⁺ and CD8⁺ T cells (effectors) purified from the spleen of C57BL/6 mice in the presence of soluble αCD3 and αCD28 for 48 h as described in Methods. Following in vitro stimulation, cells were stained for T cell (CD3⁺, CD4⁺, CD8⁺), activation (CD44) and proliferative (Ki-67) markers. a Representative FACS plots showing the CD44 and Ki67-expressing CD4⁺ (top row) and CD8⁺ (bottom row) T cells after co-incubation with CD45⁺ tumor-derived cells (ratio 1:2) from MB49^luc bladder from control Ig– and NHS-muIL12–treated mice. FACS plots indicated as “T cells only” were from cultures containing indicated splenic CD4⁺ or CD8⁺ T cells from naïve B6 mice (i.e., no addition of bladder tumor-derived CD45⁺ cells). Numbers within each FACS plot are the percentage of CD4⁺ and CD8⁺ T cells co-expressing CD44 and Ki67. b, c Frequency of CD4⁺ (b) and CD8⁺ (c) T cell proliferation reported as a percentage of Ki67-expressing cells using CD45⁺ cells from tumors of control Ig– (solid squares) and NHS-muIL12–treated mice (open squares) at ratios CD45⁺ tumor-derived cells to T cells of 1:2 to 1:128. Dashed line in b and c represents the average proliferation as measured by Ki67-expressing T cells in the absence of tumor-derived CD45⁺ cells. Student’s t-test; *P < 0.05 (vs. control Ig–treated mice). Data are from a single experiment