Fig. 2: Immunoprofiling of tumor-infiltrating immune cells after treatment with engineered SAM.

a Experimental schedule for SAM treatment of CT26 subcutaneous tumor (s.c)-bearing mice. Mice received the indicated amounts of SAM by intravenous (i.v) injection when their tumors had reached approximately 120 mm³ (day 0). Bacteria-treated mice were then given Doxy (orally, 1.7 mg/kg) daily from day 3. ( + ) means Doxy was administered. Tumors, spleens, tumor-draining lymph nodes (TdLNs), and blood were isolated on day 9 for flow cytometry (FACS) analysis. Ex vivo isolated tumor-infiltrating lymphocytes were restimulated with PMA/ionomycin in the presence of brefeldin A. b–g Frequency of isolated intratumoral M1-like macrophages (F4/80⁺CD86⁺) (b), M2-like macrophages (F4/80⁺CD206⁺) (c), activated DCs (CD11c⁺CD86⁺MHCII^Hi) (d), Tregs (CD3⁺CD4⁺CD25⁺FOXP3⁺) (e), activated CD4⁺ T cells (CD4⁺FOXP3⁻IFN-γ⁺) (f), activated CD8⁺ T cells (GzmB⁺CD8⁺) (g) (n = 7 mice examined from two independent experimental replicates; ****P < 0.0001; ns not significant; unpaired two-tailed t-test). h, i Frequencies of L-2Ld MuLV gp70 peptide tetramer-bound (Tet⁺) CD8⁺ T cells in peripheral blood (left), TdLNs (middle), and tumors (right) (h); and exhausted/activated CD8⁺ T cells (PD-1⁺CD8⁺) (i) n = 7 mice examined from two independent experimental replicates; ****P < 0.0001; ns, not significant; unpaired two-tailed t-test). j, k Frequencies of central memory CD4⁺ T cells (CD4⁺CD44^HiCD62^Hi) (j) and central memory CD8⁺ T cells (CD8⁺CD44^HiCD62^Hi) (k) in TdLNs (n = 7 mice examined from two independent experimental replicates; ****P < 0.0001; ns not significant; unpaired two-tailed t-test). l Experimental scheme for SAM-FC treatment of CT26 tumor-bearing BALB/c mice with T cell depletion. The mice were injected intravenously with 2 × 10⁷ cfu SAM-FC(+). T cells were depleted by intraperitoneal administration of anti-CD4 (αCD4) or anti-CD8 (αCD8) antibodies (200 µg/mouse, i.p injection, twice per week for 5 times). IgG isotype was used as a control. m Average tumor growth curves (left) and representative images (right) of CT26 tumor-bearing BALB/c mice with or without T cell depletion after SAM-FC(+) treatment as described in (l) (n = 5 mice/group, from one experiment; ****P < 0.0001; ns, not significant; two-way ANOVA with Tukey’s multiple comparisons test).