Data_Sheet_1_HMGN1 and R848 Synergistically Activate Dendritic Cells Using Multiple Signaling Pathways.pdf

High mobility group nucleosome-binding protein 1 (HMGN1 or N1) is a Th1-polarizing alarmin, but alone is insufficient to induce antitumor immunity. We previously showed that combination of N1 and R848, a synthetic TLR7/8 agonist, synergistically activates dendritic cells (DCs) and induces therapeutic antitumor immunity, however, it remained unclear how N1 and R848 synergistically activate DCs. Here, we show that co-stimulation with N1 and R848 of human monocyte-derived DCs (MoDCs) markedly upregulated DC's surface expression of CD80, CD83, CD86, and HLA-DR, as well as synergistic production of pro-inflammatory cytokines including IL-12p70, IL-1β, and TNF-α. This combination also synergistically activated NF-κB and multiple MAPKs that are involved in DC maturation. Moreover, N1 and R848 synergistically increased nuclear translocation of interferon (IFN) regulatory transcription factors (e.g., IRF3 and IRF7) and promoted the expression of type 1 IFNs such as IFN-α2, IFN-α4, and IFN-β1. Similar signaling pathways were also induced in mouse bone marrow-derived DCs (BMDCs). RNA-seq analysis in human MoDCs revealed that N1 plus R848 synergistically upregulated the expression of genes predominantly involved in DC maturation pathway, particularly genes critical for the polarization of Th1 immune responses (e.g., IL12A, IL12B, and IFNB1, etc.). Overall, our findings show that (1) N1 synergizes with R848 in activating human and mouse DCs and (2) the synergistic effect based on various intracellular signaling events culminated in the activation of multiple transcriptional factors. These findings have important implications for future clinical trials since N1 and R848 synergistically promoted optimal Th1 lineage immune responses resulting in tumor rejection in mice.</p

1D11 in combination with CY potently inhibits the development of mouse 4T1 tumor and induces anti-tumor immunity.

<p>Three days after tumor inoculation, the mice were i.p. treated with single dose of CY (4 mg) or 1D11 (0.1 mg, 3×week), or combination of CY and 1D11 or mouse IgG1. (A) Percent tumor-free mice (%). (B) Survival of tumor inoculated mice. (C) Tumor size in groups treated with PBS, or CY alone or 1D11 alone. (D) Tumor size in groups treated with CY+1D11 or CY+Mu IgG1. Two weeks after last 1D11 treatment (60 days after initial tumor inoculation), the tumor-free mice (designated as pre-treated) were re-inoculated with 4T1 cells into the right thoracic mammary fat pad, and CT26 cancer cells were inoculated (s.c.) into the left flank. For comparison, age- and gender-matched normal Balb/c mice (designated as untreated) were inoculated with 4T1 and CT26 tumor cells in the same manner. (E) Incidence of 4T1 and CT26 tumor development on day 18 after tumor inoculation. (F) Growth of 4T1 tumor and (G) growth of CT26 tumor. Data shown in C, D, F and G are means±SEM (N = 5∼10). Comparison of two groups: * p<0.05; **p<0.01. The data are representatives of three 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

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

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 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

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

Increased MCP-1 expression in 4T1 cells has no effect on spontaneous lung metastasis in MCP-1^−/− mice, but increases lung metastasis after intravenous injection.

<p>A. 1×10⁵ 4T1-L10 cells were injected into a mammary pad of WT or MCP-1^−/− mice. The size of each primary tumor was measured and the area was calculated. <i>n = 4</i> for WT, <i>n = 3</i> for MCP-1^−/− mice. B. 1×10⁵ 4T1-L10 cells were injected into a mammary pad of WT or MCP-1^−/− mice. All mice were euthanized 4 weeks after the injection and the number of metastatic tumor nodules on the surface of each lung was counted by eye. C. Total RNA was extracted from each tumor and the expression of MCP-1 mRNA was examined by Northern analysis. Ten µg of total RNA was used. D. Sera were collected 2 weeks after the injection of 4T1-L10 cells and MCP-1 concentrations were measured by ELISA. E. 4T1-L5 or 4T1-L10 cells (5×10⁴ cells in 0.2 ml PBS) were intravenously injected into WT (left panel) or MCP-1^−/− mice (right panel). Two weeks later, mice were euthanized and the number of metastatic tumor nodules on the lung was counted. The results are the summary of two independent experiments. <i>n = 8</i> for WT, and <i>n = 8</i> (4T1-L5) or <i>n = 9</i> (4Y1-L10) for MCP-1^−/− mice.</p

MCP-1-deficiency in tumor stroma results in early necrosis, reduced macrophage infiltration and reduced angiogenesis.

<p>A. 1×10⁵ 4T1 cells were injected into a mammary pad of WT or MCP-1^−/− mice. Primary tumors were excised two weeks after 4T1 cell injection and fixed in formalin. Tissue sections were prepared from the center of each tumor, stained by H&E, and the area of necrosis was measured. % of necrotic area was calculated by using a formula, area of necrosis/area of tumor×100. <i>n = 5</i> for each group. B. H&E section of tumor tissue away from necrosis. C. Immunohistochemical examination of tumor sections. Green, cytokeratin; Red, F4/80 or CD31.</p

The absence of MCP-1 in tumor stroma reduces the lung metastasis of 4T1 cells and prolongs survival.

<p>A. 1×10⁵ 4T1 cells were injected into a mammary pad of WT or MCP-1^−/− mice. The size of each primary tumor was measured and the area was calculated. <i>n = 9</i> for WT, <i>n = 8</i> for MCP-1^−/− mice. B. Primary tumors were excised from WT and MCP-1^−/− mice 31 days after tumor cell injection and weighed. <i>n = 9</i> for WT, <i>n = 8</i> for MCP-1^−/− mice. C. Mice were euthanized on day 31, and lungs were harvested and fixed in Bouin's solution. The number of metastatic tumor nodules on the surface of lungs of each mouse was counted by eye. <i>n = 9</i> for WT, <i>n = 8</i> for MCP-1^−/− mice. D. Ten thousand 4T1 cells were injected in a mammary pad of each mouse and the survival of each mouse was examined. <i>n = 8</i> for WT, <i>n = 8</i> for MCP-1^−/− mice.</p