Fig. 6

Adoptive transfer of bi-specific scFv antibody (BsAb)-secreting CAR-T cells modulate intratumoral cytokines and the proportions of infiltrating immune cells, MDSCs, and M2 TAMs. (A) Representative flow cytometry images and bar summary plots (right) showing the percentage of infiltrating CD8 + CD3 + T cells in tumor-bearing mice. Analysis of cell suspensions extracted from tumors by flow cytometry detecting infiltrating T cells originally gated as CD45 + cells. (B-C) Representative flow cytometry images and bar summary plots (right) showing the percentage of infiltrating MDSCs (B) and M2 TAMs (C) in tumor-bearing mice. Tumor cell suspensions were examined by flow cytometry to detect infiltration of MDSCs marked by CD11b + GR1 + and M2 TAMs marked by F4/80 + CD206+, initially gated as CD45 + cells. (D-E) Representative flow cytometry images and bar summary plots (right) showing the generation of perforin and granzyme B in tumor-infiltrating CD8-positive T cells. The percentages of CD8-positive T cells generating granzyme B (D) and perforin (E) in the TME were analyzed by flow cytometry. (F-I) Intratumoral cytokine evaluation of IL-12, TNF-α, IFN-γ, and IL-15 by ELISA. Data are expressed as means ± SD; * P < 0.05; **P < 0.01, by unpaired Student’s t test. scFv, single-chain fragment variable; CAR-T, chimeric antigen receptor-modified-T; MDSCs, myeloid-derived suppressor cells; TAMs, tumor associated macrophages; CD, cluster of differentiation; TME, tumor microenvironment; IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; ELISA, enzyme-linked immunosorbent assay