Decreased antigen uptake by splenic and hepatic DCs following systemic IL-12 treatment.

<p>Leukocytes from IL-12 or vehicle control treated mice were incubated with 100 µg/ml DQ-OVA for 30 min at 37°C and analyzed by flow cytometric analysis gating on cDC (NKp46⁻CD11c⁺Class II⁺) and pDCs (NKp46⁻CD11c⁺Class II⁺mPDCA-1⁺) populations in the spleen (A) and liver (B). Left hand shows a representative histogram sample for an individual mouse displaying the antigen uptake and processing of DQ-OVA by cDC or pDCs (solid line). The shaded line is the respective isotype control. The MFI of a representative sample is shown in the upper right corner of each histogram. The right side graphically displays the mean ± SD of the DQ-OVA MFI from an independent experiment with 3–5 mice/group that has been repeated 3 times with similar results. *, <i>p</i><0.05; Mann-Whitney U test.</p

Hepatic T cells are differentially modulated compared to splenic T cells following IL-12 therapy in RENCA tumor bearing mice.

<p>(A) Schematic of the treatment cycle for orthotopic RENCA tumor bearing mice, where mice were injected with 1×10⁵ RENCA cells into the kidney capsule then treated with two cycles of IL-12 or VC injections. (B) Mice were euthanized on day 13 following treatment and the weight of the primary tumor was recorded. The activation of CD4 and CD8 T cells (CD3⁺DX5⁻) from the spleen and liver of VC (white) and IL-12 (black) treated RENCA tumor-bearing and VC (grey) and IL-12 (striped) treated non-tumor bearing mice is shown through expression of CD69 (C) and CD44high (D) Shown is the mean ± SEM from two independent experiments with 5 mice/group in each experiment. Purified splenic and hepatic T cells pooled from 2–5 mice/group from RENCA tumor-bearing mice were co-cultured with irradiated RENCA at a 10∶1 ratio for 96 hours followed by IFNγ detection E) and IL-10 (F) in the culture supernatants. The bar graphs represent the means ± SD assayed in triplicate. Shown is a representative experiment that has been repeated twice with similar results. NS not significant, **p<0.01; *** p<0.001; Mann Whitney U test.</p

Increased splenic and hepatic DC numbers following systemic IL-12 treatment.

<p>BALB/c mice were treated with 1 µg/mouse IL-12 (solid square) or vehicle control (VC; open triangle) for 4 consecutive days from day 0, as indicated with arrows, then leukocyte populations analyzed at various days post treatment. The total DC number (NKp46⁻CD11c⁺ Class II⁺), is shown for the spleen (A) and for the liver (B). The results shown are the mean ± SD from 3–5 mice/group at each time point and representative of 3 separate experiments. (C). Differences in DC subsets were examined in the spleen and liver at day 4 for CD11b⁺ (NKp46⁻CD11c⁺Class II⁺CD11b⁺), CD8α⁺ (NKp46⁻CD11c⁺Class II⁺CD8α⁺), and pDCs (NKp46⁻CD11c⁺Class II⁺B220⁺mPDCA-1⁺SiglecH⁺), respectively. Shown is the mean ± SD from 4–5 mice/group and representative of more than 3 independent experiments. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001; Mann Whitney U test.</p

Altered cytokine expression profile of splenic and hepatic DCs.

<p>Bulk lymphocytes from the spleen and liver were incubated with media alone (no treatment; no Tx), 25 µg/ml poly(I:C), 1 µg/ml LPS or 2.5 µg/ml CpG for 18 hours. Splenic DC (A) and hepatic DC (B) populations were gated and intracellular IL-12p40 expression examined by flow cytometric analysis. Shown is a representative sample from an individual mouse in an independent experiment repeated 3 times (3–5 mice/group). Purified splenic and hepatic DC pooled from 5–15 mice were cultured under the same conditions and culture supernatants harvested 48 hours later. Cytometric bead array was performed to detect in the supernatants the levels of IL-12p40 (D), IL-12p70 (D) and TNF (E) from splenic DC and hepatic DC. The bar graph displays the mean ± SEM expressed in pg/ml per 10⁶ cells derived from the pooled data obtained from 3 independent experiments. *, <i>p</i><0.05 (Mann Whitney U Test).</p

Enhanced T cell proliferation from hepatic DCs following systemic IL-12 treatment.

<p>(A) Differential HA TCR proliferation of naïve splenic and hepatic DC. Purified DC were peptide pulsed and incubated with 2×10⁴ HA-TCR T cells at a 1∶1 ratio for 72 hours and 1 µCi of [³H]thymidine added during the last 18 hours of culture. (B) Allogeneic mixed lymphocyte reaction. Purified splenic and hepatic DCs from BALB/c mice (H-2^d) were cultured at differing DC∶T cell ratios with 1×10⁵ purified T cells from C57BL/6 mice (H-2^b) for 72 hours with 1 µCi of [³H]thymidine added during the last 18 hours of culture. (C) HA TCR Tg model. Purified splenic and hepatic DCs were pulsed with varying doses of HA peptide for 2 hours then cultured with 2×10⁴ HA-TCR T cells at a 1∶1 ratio for another 72 hours. 1 µCi of [³H]thymidine was added to each well during the last 18 hours of culture. Shown is the mean ± SD done in quadruplicates from one of three independent experiments. *, <i>p</i><0.05; **, <i>p</i><0.01; *** <i>p</i><0.001; Student's T test.</p

Effects of 1D11 on 4T1 tumor growth and on the expansion of Tregs in vivo and in vitro.

<p>(A-D) Mice were treated with 0.1 mg 1D11 or mouse IgG1 (i.p., 3×week), starting at day 1 of tumor inoculation. (A) Kinetics of tumor growth. (B) Weight of tumors isolated at 14 days after inoculation. (C-E) Effect of 1D11 on Tregs. CD4 cells and Tregs was analyzed with FACS at 14 days after tumor inoculation. (C, D) Typical FACS analysis of CD4 cells and Tregs. Number represents the percentage of CD4⁺ cells in total tumor infiltrating CD45⁺ leukocytes (C) or Foxp3⁺ cells in intratumoral CD45⁺CD4⁺ cells (D). (E) Summary of proportion of Foxp3⁺ cells in CD4 cells present in the tumor, spleen, mesenteric LNs and axillary/inguinal LNs (N = 3∼7). (F-G) TGFβ inhibits, while 1D11 promotes, proliferation of Tregs in vitro. (F) CD4⁺Foxp3/gfp⁺ Tregs were stimulated in the presence of TNF with increasing concentrations of rhTGFβ1. (G) Tregs were stimulated in the presence of TNF or medium alone with increasing concentration of 1D11. After incubation for 72 hours, the proliferation of Tregs was determined by [³H] thymidine incorporation. By compared with respective control (without rhTGFβ1or 1D11), *p<0.05, ** p<0.01. N = 3. The data are representatives of three separate experiments with same results.</p

Combination treatment of 1D11 and CY reduces the number of splenic MDSCs and promotes their re-differentiation.

<p>Four weeks after 4T1 tumor inoculation, cell suspensions were prepared from spleen and MDSCs were analyzed by FACS, gating on live CD45⁺ cells. (A-B) Proportion and number of Gr1⁺CD11b⁺ cells in the spleens. Typical FACS plots are shown in (A, gating on total live splenic CD45⁺ cells) and summary of data pooled from three experiments are shown in (B, percent of PBS control group, Means±SEM, N = 10∼13). (C) Absolute number of Gr1⁺CD11b⁺ cells in the spleen (N = 11, pooled from two separate experiments). (D-E) Expression of I-A/I-E and CD80 on Gr1⁺CD11b⁺ splenic cells. (D) Typical FACS plots (gating on Gr1⁺CD11b⁺ cells) and (D) summary of data pooled from three separate experiments (Means±SEM, N = 9). Comparison of indicated groups, *p<0.05, ** p<0.01, ***p<0.001.</p

Effect of combination of 1D11and reduced dose of CY on primary tumor growth and pulmonary metastasis.

<p>Three days after tumor inoculation, the mice were i.p. treated with single dose of CY (2 mg) or 1D11 (0.1 mg, 3×week), or combination of CY and 1D11 or mouse IgG1. Mice were sacrificed ∼4 wks after tumor inoculation. (A) Kinetics of tumor growth. The data are representatives of three separate experiments with similar results (N = 10, data shown as means±SEM). (B) Weight of tumors after 4 wks of inoculation (N = 17, pooled from two separate experiments). (C) The number of grossly visible metastatic nodules in the lung (N = 14, pooled from two separate experiments). Comparison of indicated groups, * p<0.05, ** p<0.01, *** p<0.001.</p

Combination treatment of 1D11 and CY promotes tumor infiltration of IFNγ-producing T cells.

<p>Four weeks after 4T1 tumor inoculation, cell suspension was prepared from tumor tissues. (A-B) Proportion of TCRβ⁺ T cells in total tumor infiltrating CD45⁺ leukocytes. Typical flow plots are shown in (A), and summary of data from three separate experiments is shown in (B, Means±SEM, N = 14∼20). (C-F) IFNγ expression by CD8 and CD4 TILs. Intracellular expression of IFNγ was analyzed by FACS, gating on live CD45⁺TCRβ⁺CD8⁺ cells (C-D) or CD45⁺TCRβ⁺CD4⁺ cells (E-F). Data shown are typical FACS plots (C, E) and summary of data (D, F) from three separate experiments (Means±SEM, N = 9). Comparison of indicated groups, * p<0.05, ** p<0.01. <b>(</b>G, H<b>)</b> Normal WT Balb/c mice and IFNγ KO mice were inoculated with 4T1 cells and treated with 1D11+CY in the same manner. (G) Incidence of 4T1 tumor in mice treated with PBS. (H) Incidence of 4T1 tumor in mice treated with 1D11 and CY. Data are shown as percent tumor free mice (%, KO mice N = 3, WT mice N = 5), which are representatives of two separate experiments with similar results.</p

Combination treatment of 1D11 and CY increases survival of mice with 4T1 lung metastasis in an orthotopic implantation/resection format.

<p>4T1 cells were inoculated into the mammary fat pad and primary tumors were surgically removed at day 12 after inoculation. 1D11 or Mu IgG1 (13C4) control antibody (5 mg/Kg, i.p.) were administered three times per week for the first two weeks and followed by once a week, starting from 7 days after tumor inoculation. A single subtherapeutic dose of CY (50 mg/Kg, i.p.) or vehicle was given on day 14, two days following surgical resection of the primary tumor. The therapeutics was treated alone or combined as indicated. (A) Survival curves for the different treatment groups. Survival curves are significantly different between the groups (p<0.0007; Log-Rank (Mantel-Cox) Test). (C) Representative images from lungs at gross and (D) in histological cross-section (H&E stained).</p