Figure 4.

EnnA antitumor activity against E0771 tumors is mediated by CD8⁺ T cells

(A) Immunofluorescence analysis showing that EnnA induces recruitment of CD8β⁺ T cells to the E0771 tumor site and increases tumor infiltration compared to control. The scale bar represents 300 μm for 4x and 20 μm for 40x.

(B) Average of 180 cells from three different fields of control and EnnA-treated tumors (n = 3). Data are represented in the percentage of mean ± S.E.M.

(C) Immunofluorescence analysis of 4T1 tumors treated with EnnA (10 mg/kg/48 h) or 17-AAG (25 mg/kg/48 h) stained with anti-CD45 and anti-CD8α. The scale bar represents 50 μm.

(D) Average of 250 cells from three different fields of control and EnnA-treated tumors (n = 3). Data are represented in the percentage of mean ± S.E.M.

(E) Immunofluorescence analysis of E0771 tumors from control and EnnA-treated animals showing that CD8β+ T cells also express granzyme b. The scale bar represents 300 μm for 4x and 50 μm for 20x.

(F) Average of 135 cells from three different fields of control and EnnA-treated tumors (n = 3).

(G) Flow cytometric analysis of cells from tumors treated with EnnA or DMSO. Data are represented in the percentage of mean ± S.E.M.

(H) Depletion of CD8⁺ T cells from mice using a monoclonal antibody (InVivoMAb anti-mouse CD8α, clone 53–6.7) abrogates antitumor activity of EnnA. Data are represented in the percentage of mean ± S.E.M. n represents the number of mice per group.

(I) EnnA treatment increases the number of CD8⁺ T cells in spleens, which is abrogated by an anti-CD8 antibody. Data are represented as mean ± S.E.M. ∗p < 0.05; ∗∗p < 0.01 by one-way ANOVA or unpaired tailed Student’s t test and are a representation of at least two independent studies.