Be0003 1

The dose of PIP-CpG was 18.2 nmol (250 μg) and was administered IV via tail vein in 100 μL PBS. For T cell depletion, anti-CD4 (100 μg; BioXCell: BE0003–1; clone GK1.5, rat IgG2b) and anti-CD8a (100 μg; BioXCell: BE0061; clone 2.43, rat IgG2b) antibodies were injected intraperitoneally 2 days before therapy, on the day of the first treatment, and at 6, 13, and 20 days post-treatment. T cell depletion in blood was validated by flow cytometry. Treatments (vehicle or PIP-CpG) and tumor growth measurements were initiated when tumor(s) became palpable (typically around 50 days old; usually 2–3 tumors per mouse). For all experiments, mice were methodically assigned to treatment groups to ensure mice in different groups were well-matched on the first day of treatment (i.e. similar number of tumors, total tumor burden, age). The size of each tumor, expressed as volume (length × width × height), was monitored continuously with digital calipers (Mitutoyo) until the euthanasia end point was reached (criteria: when any tumor reached 1.5 cm in the largest diameter). Mice were naired regularly to facilitate tumor measurements. To minimize potential confounders, mice from different treatment groups were housed together.

In order to study whether effects on primary tumor delay were immune related, in a second experiment, mice were treated with anti-CD4 and anti-CD8 antibodies (BE0003–1 and BP0004–1, BioXcell, NH, USA). Depletion of CD4+ and CD8+ T-cell subsets was done by i.p. injections at 100 μg/mouse on the 4th, 5th and 6th day after tumor inoculation and subsequently on every 6th day until sacrifice as was described by Demaria et al. [18 ].

Mouse-specific antibodies were obtained from Bio X Cell including anti-mouse PD-1 (RMP1–14, Catalog #BE0146) and anti-mouse 4-1BB (LOB12.3, Catalog # BE0169). For PD-1 and 4-1BB antibody treatments, 200 μg of each antibody was diluted with PBS for a total injection volume of 200 μL. Mice received intraperitoneal (IP) injections of antibody every 3 days, starting on day 9 after tumor implantation, for a total of 3–4 treatments. Control treatments consisted of 200 μL PBS. CD8 and CD4 depletion was performed as described previously (26 (link)). In brief, CD8 or CD4 T cells were depleted via IP injection of 200ug anti-CD8 (53–6.7, BioXCell Catalog #BE0004) or 200ug anti-CD4 (GK1.5, BioXCell Catalog #BE0003–1) at day 5, 6, 7, and 14 following tumor implantation for early CD8 depletion or at day 12, 13, and 14 for late-start CD8 depletion.