Fig 2: Increased CD4 T cell infiltration adjacent to NOD tumoursTumours from B6.TAg+ and NOD.TAg+ mice at 12 weeks of age were assessed by histology. Representative images of n = 3. A. Immunofluorescence for CD4, Foxp3 and DAPI, in the tumour (left) and directly adjacent tissue (right). B. Immunofluorescence for CD8 and DAPI, in the tumour (left) and directly adjacent tissue (right). Scale bar = 100μm.

Fig 3: Immunohistochemistry of exemplary 4T1 tumors. Representative tumor sections after 7-channel staining of DAPI (blue), Ly-6C (green), F4/80 (yellow), S100A9 (orange), CD8 (red), CD11b (purple) and CD4 (white) with correlating merged images. Sections of implanted 4T1 wt tumors in wt (a) and S100A9 ko (b) mice as well as 4T1 ko tumors in wt (c) and S100A9 ko (d) mice.

Fig 4: C-agarose+CpG Treatments Change the Ratio of Immune Cell Subsets and Cytokine Levels in the TDLN from a Murine 4T1 Breast Cancer Model(A) Lymph node cells were stained with PE-CD4 (CD4+ T cells), APC-CD8 (CD8+ T cells), APC-CD11c (DCs), or PE-CD49b (NK cells) and examined by flow cytometry. (B) The ratio of CD4+ T cells, CD8+ T cells, DCs, and NK cells. (C) The homogenates of lymph nodes were harvested to determine the levels of IL-1α, IL-1β, TNF-α, CXCL9, CXCL10, IL-2, IFN-γ, and granzyme B by ELISA. n = 10 mice per group. Data are representative results from five independent experiments and are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. m-ODN, mismatched oligonucleotide; TDLN, tumor-draining lymph node.

Fig 5: Analysis of the immune cell composition in livers of Ctrl and Stab1 KO without metastases or with melanoma metastases of B16F10 luc2 or Wt31. Livers of Ctrl or Stab1 KO without metastases (A) or with metastases of Wt31 (B) or B16F10 luc2 (C) were analyzed for T cells (CD3+CD4, CD3+CD8), macrophages (CD11b+F4/80), dendritic cells (CD11c+MHCII), or regulatory T cells (CD4+FOXP3) by immunofluorescence. By whole slide imaging, the numbers of respective immune cells were objectively quantified. The numbers of cells were set in relation to a liver area of 1 × 106 µm2. Metastases were excluded from the analysis. (A) Untreated livers of Ctrl or Stab1 KO were analyzed. CD3+CD4: p = 0.8857; CD3+CD8: p = 0.8857; CD11b+F4/80: p = 0.6857; CD11b+MHCII: p = 0.4206; CD4+FOXP3: p = 0.7302; (B) Livers of Ctrl or Stab1 KO with Wt31 melanoma metastases were analyzed. CD3+CD4: p = 0.2000; CD3+CD8: P = 0.2286; CD11b+ F4/80: p = 0.2000; CD11c + MHCII: p ≥ 0.9999; CD4+FOXP3: p = 0.7000; (C) Livers of Ctrl or Stab1 KO T cells with B16F10 luc2 melanoma metastases were analyzed. CD3+CD4: p = 0.5556; CD3+CD8: p > 0.9999; CD11b+F4/80: p > 0.9999; CD11c+MHCII: p = 0.9048; CD4+FOXP3: p = 0.4127. Data information: ns = not significant.