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. Author manuscript; available in PMC: 2015 Mar 1.
Published in final edited form as: Cancer Immunol Res. 2013 Dec 31;2:207–216. doi: 10.1158/2326-6066.CIR-13-0121

Figure 1. GVAX immunization combined with disruption of CD8 Treg activity in B6-DK mice significantly potentiates antitumor immunity.

Figure 1

A) B6-WT mice were challenged with 2×105 B16 cells s.c. and immunized with irradiated 1×106 GVAX (grey box) or not (black box) on days 5, 8 and 11. Qa-1 expression was determined on gated subsets of splenocytes and TILs 20–24h after the last immunization. Data are representative of 2 independent experiments and presented as ratio of mean fluorescence intensity (MFI) of specific Qa-1 staining and MFI of background (bg) staining with streptavidin only. B) CD8 Treg efficiently infiltrate into tumors. Mice were challenged with 5×105 B16 cells. The lymphoid fraction of collagenase/dispase digested tumors was collected by gradient centrifugation on days 10, 20, and 30 after challenge. The proportions of CD8 Treg (CD122+Ly49+) were determined by FACS as percent (%) of CD8+CD3+ cells. A representative FACS plot from day 20 after challenge is shown on the left. Tumor growth is shown on the right. C) B6-WT (■) or B6-DK (□) mice were challenged with 2×105 B16 and immunized on the same day with irradiated 1×105 GVAX followed by immunization with irradiated 5×105 B16 and 5×105 GVAX s.c. on day 7 and 14, n=15 mice per group. Data are presented as cumulative survival (left panel) or tumor growth (right panel) of B16 challenged mice from two independent experiments. D) B6-WT (■) or B6-DK (grey □) mice were treated as in B). Percent (%; left panel) or absolute number (right panel) of CD4 TFH cells (CD200+ICOS+PD-1hi gated from CD4+CD3+) from either spleens or tumors (Tm) of treated animals was determined 3 days after the last immunization. ICOS expression by tumor-infiltrating CD3+CD4+ lymphocytes was determined by FACS 3 days after the last immunization. Data are representative of at least 3 independent experiments. E) Cellular analysis of tumor-bearing B6-WT (■) or B6-DK (grey □) mice immunized with GVAX. The proportion and absolute number of CD8, Ly49+ CD8, NK (NK1.1+CD3−) or CD4 Treg (CD25+FoxP3+ gated from CD3+CD4+) lymphocytes from the spleens, tumor draining lymph nodes (dLN) or tumors (Tm) was determined by FACS 3 days after the last immunization. Data are representative of at least 2 independent experiments.