Fig 2: Effect of intratumoral α-AED treatment in the immune cell infiltration of secondary lymphoid organs in the 4T1 tumor model. Determination of innate and adaptive immune subpopulations by flow cytometry in lymph nodes. (a) T lymphocytes CD3+. (b) CD8+ T cytotoxic lymphocytes. (c) CD4+ T helper lymphocytes. (d) CD4+/Foxp3+ T lymphocytes. (e) F4/80+ macrophages. (f) NKp46+ cells. (g) CD19+ B lymphocytes. (h) Plasmatic cells (CD138+). (i) Plasmablasts (CD138+/CD19+). Graphs represent the mean ± SD of the data from three independent experiments. Gates from 10,000 cells were collected. Statistical significance was calculated using a Kruskal–Wallis test (*, p ≤ 0.05).

Fig 3: Innate and adaptive immune cells in the tumor microenvironment. Determination of innate immune subpopulations by flow cytometry. (A) F4/80+ macrophages. (B) NKp46+ cells. Flow cytometry analysis of adaptive immune cells within the tumor. (C) T CD3+. (D) CD19+ B lymphocytes. (E) CD4+ T helper lymphocytes. (F) CD8+ T cytotoxic lymphocytes. (G) CD4+/Foxp3+ T lymphocytes. (H) CD8+/Foxp3+ T lymphocytes. Graphs represent the mean ± SD of the data from two independent experiments (4T1, n = 10; 4T1+T. canis, n = 10). Representative contour plots of the cytometric analysis of the corresponding population (below). Gate from 10,000 cells was collected. Statistical significance was calculated using a t-test (*p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001).

Fig 4: Effect of α-AED local injection on the humoral-related cell infiltration in the 4T1 tumor model. Humoral-related cell subpopulations by flow cytometry of the tumor. (A) Plasmatic cells (CD138+). (B) Plasmablasts (CD138+/CD19+). (C) B cells (CD19+). (D) Representative dot plots of the cytometric analysis of the corresponding population tumor cells. Graphs represent the mean ± SD of the data from three independent experiments. Gates from 10,000 cells were collected. Statistical significance was calculated using a Kruskal–Wallis test (*, p ≤ 0.05; ***, p ≤ 0.001).

Fig 5: Humoral response to T. canis infection in BALB/c mice. (A) Splenic weight. (B) Representative images of a spleen from a mouse with tumors (4T1 group) and of a spleen from a mouse with tumors and infection (4T1+T. canis). Millimetric grid as a background. Black bar = 0.5 cm. (C) Anti-T. canis IgG serum levels measured by indirect ELISA. (D) Frequencies of CD19+ B lymphocytes in the spleen. (E). Frequencies of CD19+ B lymphocytes in PLNs. Graphs represent the mean ± SD of the data from two experiments (4T1, n = 10; 4T1+T. canis, n = 10). Statistical significance was calculated using a t-test (***p ≤ 0.001; ****p < 0.0001).