Figure 7. Depletion/inhibition of immune cell subtypes abrogates triple combination therapy.

A. C57Bl/6 mice implanted with (2 × 10⁴) 005 GSCs on day 0 and treated with G47Δ-mIL12 or PBS injected IT on day 8 and anti-CTLA-4 and anti-PD-1 antibodies or isotype control IgG (5 mg/kg hamster IgG and 10 mg/kg rat IgG) injected IP on days 8, 11 and 14 (n=6/group; upward arrows). Depletion antibodies against CD4 or CD8 (10 mg/kg) or clodronate liposomes (Clod; first injection 50 mg/kg followed by 25 mg/kg) were injected IP on days 4, 7, 10, 13, 20, and 27 (downward arrows), or BLZ945 (BLZ; 200 mg/kg) was gavaged for two cycles from days 6–10 and days 12–16 in triple therapy mice. Median survival of mice was determined: Mock (PBS/IgG/liposome/20% captisol), 35.5 days or triple therapy +αCD4 antibody, 32.5 days; +BLZ, 41.5 days; +αCD8 antibody, 45 days; +Clod, 43 days; +PBS (IgG/liposome/20% captisol). +PBS was compared to +BLZ (p=0.004), +Clod (p=0.02), +αCD4 (p=0.0007), +αCD8 (p=0.02), or Mock (p=0.0006) by Log-rank analysis. Similarly, Mock was compared to +PBS (p=0.0006), +BLZ (p=0.06), +Clod (p=0.01), +αCD4 (p=0.4), or +αCD8 (p=0.0006). B. C57Bl/6 mice implanted with 005 GSCs (2 × 10⁴) on day 0 and treated with G47Δ-mIL12 (5 × 10⁵ pfu) or PBS injected IT on day 18 and anti-CTLA-4 and anti-PD-1 antibodies or isotype control IgGs (5 mg/kg hamster IgG and 10 mg/kg rat IgG) injected IP on days 18, 21 and 24 (n=2/group). Depletion antibodies (αCD4, αCD8; 10 mg/kg) or Clod (first injection 50 mg/kg followed by 25 mg/kg) were injected IP on days 14, 17, 20, and 23, or BLZ (200 mg/kg) gavaged from days 16–20 and 22–25. Twenty-four hr after the last immune checkpoint injection or 8 hr after the last BLZ gavage, animals were sacrificed on day 25 and brains collected. Brain tumor sections (2 sections/mouse, at least 200 μm apart from each other) were stained for CD4, CD8, CD68, and F4/80. Positive cells were counted (5 fields/section for CD4⁺ and CD8⁺, 8 fields/section for CD68⁺, and 6 fields/section for F4/80⁺) and presented as mean ± SEM. Data were assessed by Student’s t test between indicated groups *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Only significant differences between Mock or triple therapy and other treatments indicated. See also Figure S7.