Figure 7.

CpG(B)-STAT3dODN Treatment Results in B Cell-Lymphoma-Specific T Cell Immune Responses with Tumor Infiltration by CD8⁺ T Cells

Mice with s.c.-established A20 lymphoma were injected IT using 1 mg/kg CpG(B)-STAT3dODN, CpG-scrODN, or PBS 4 times every other day. Tumors were harvested and dispersed into single-cell suspensions for the phenotypic immune marker analysis using flow cytometry. (A) CpG(B)-STAT3dODN upregulates surface expression of MHC class-II, costimulatory CD40, CD80, and CD86 molecules. Data indicate means ± SEM (n = 5) from one of two independent experiments with similar outcomes. (B) CD8⁺ T cell infiltration in A20 lymphoma tumors visualized using immunohistochemical staining on FFPE tumor sections. Scale bars, 50 μm. (C) The infiltration by various T cell populations in A20 tumors were assessed using flow cytometry. Shown are percentages of total CD3⁺CD8⁺, CD3⁺CD4⁺ T cells, and CD3⁺CD4⁺Foxp3⁺ regulatory T cells (Tregs), as well as the ratio of CD3⁺CD8⁺ T cells to Tregs. (D) TLR9 stimulation combined with STAT3 inhibition generates A20 B cell-lymphoma-specific immune responses. Recall response to irradiated (100 Gy) A20 cells was assessed using single-cell suspensions prepared from tumor-draining lymph nodes from in vivo-treated mice as described above. Numbers of IFNγ-secreting cells were assessed using an ELISPOT assay. Upper panel: representative images; bottom panel: bar graphs representing results from one of two independent experiments. Data indicate means ± SEM (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p ≤ 0.0001; FFPE, formalin-fixed, paraffin embedded; MFI, mean fluorescence intensity; NS, not significant.