Fig 2: 5Aza potentiates macrophage-dependent T-cell activation. A, Percentage of IFNγ+ cells in visceral fat. Six-week-old male mice were intraperitoneally injected with MC38 cells (1.0×106 cells in 100 µL PBS) and immediately treated with DMSO or 5Aza. Then, the SVF cells of the epididymal fats were isolated on day 5. After 6-8 h activation by cell activation cocktail, IFNγ+ T cells (DAPI-CD45+CD3+ IFNγ+), IFNγ+ CD8+ T cells (DAPI-CD45+CD3+CD8+CD4-IFNγ+), and IFNγ+CD4+T cells (DAPI-CD45+CD3+CD8-CD4+ IFNγ+) were dynamically calculated by flow cytometry. This experiment was repeated three times. Each tested sample was pooled from 3 individual ones. Representative result was shown. B-D, Calculation of IFNγ+ T cells (B), IFNγ+CD4+ T cells (C) and IFNγ+CD8+ T cells (D) in SVF cells as described above in A (n = 3). E-H, Frequencies of IFNγ+ T cells (F), IFNγ+CD4+ T cells (G) and IFNγ+CD8+ T cells (H) in peripheral blood of the mice as described above in A. Representative result was shown. I-L, Frequencies of IFNγ+ T cells (J), IFNγ+CD4+ T cells (K) and IFNγ+CD8+ T cells (L) in spleen of the mice as described above in A. Representative result was shown. M, Schematic diagram of lymphocytes stimulated by macrophages in vitro. After 72 h of thioglycolate induction, mice were sacrificed and peritoneal macrophages (PMs) were harvested and treated with DMSO or 5Aza for 48 h. Then, SVFs of the epididymal fats were added to co-culture and the expression of IFNγ+ T cells were analyzed. N-P, Calculation of IFNγ+ T cells (N), IFNγ+CD8+ T cells (O) and IFNγ+CD4+ T cells (P) as described above in M (n = 3). Q-S, Calculation of IFNγ+ T cells. The SVFs of the epididymal fats from macrophageko were treated with DMSO or 5Aza (4 µM), and then the levels of IFNγ+ cells (Q), IFNγ+CD8+ T cells (R) and IFNγ+CD4+ T cells (S) were analyzed by flow cytometry (n = 3). Representative result was shown. All the data were shown as the mean ± s.e.m. All data were analyzed with Student's t test. (n.s., not significant; *P < 0.05, **P < 0.01, and ***P < 0.005).

Fig 3: oHSV2-aPD1 Therapy Reduces Inhibitory Immune Cells in the SpleenC57BL/6J-Pdcd1 mice were subcutaneously inoculated with 2 × 105 B16R and treated as described in the Materials and Methods. Tumor-bearing mice were euthanized 5 days after the last treatment. (A–G) Spleens were collected and analyzed to determine levels of Tregs (defined as CD4+CD25+FoxP3+) (A), MDSCs (defined as CD45+CD11b+Gr-1+) (B), CD45+CD11b+F4/80+ (C), and CD8+ T cell exhaustion (D–G). Data are presented as individual replicates. *p < 0.05, **p < 0.01. ns, not significant.

Fig 4: Agpat4 silencing in CRC suppresses T cell-dependent tumor progression. a Eight-week-old male NCG mice were intraperitoneally inoculated with sh-Agpat4- or sh-NC-transfected MC-38 cells (4 × 106 cells in 100 μl of PBS per mouse). Twelve days later, the mice were sacrificed, and the tumor nodules were collected and weighed. (n = 6). b Agpat4-silenced MC-38 cells increased the percentage of CD8+ T cells in epididymal fats. Six-week-old mice were intraperitoneally engrafted with sh-Agpat4- or sh-NC-transfected MC-38 cells (4 × 106 cells in 100 μl of PBS per mouse) on day 0 and were sacrificed for the analysis of total T cells, CD4+ T cells and CD8+ T cells in the stromal vascular fraction (SVF) of epididymal fats on days 0, 3, and 5. T cells were marked by the phenotype CD45+CD3+CD11b−. CD4+ T cells were defined by the phenotype CD45+CD3+CD11b−CD8−CD4+. CD8+ T cells were defined by the phenotype CD45+CD3+CD11b−CD4−CD8+. Representative results are shown. c Percentage of IFN-γ+ cells in the CD8+ T cells described above in b. (n = 5). d Six-week-old male C57BL/6 mice were intraperitoneally inoculated with MC-38 cells (4 × 106 cells in 100 μl of PBS per mouse) on day 0 and then treated with anti-CD8 antibody or IgG as a control 3 times (days 2, 4, and 6) at a dose of 100 μg/day. Mice were sacrificed for tumor observation on day 14. (n = 5). The data in a–d represent the means ± s.e.ms. (***P < 0.005; Student’s t test)

Fig 5: Treg depletion improved antitumor response with anti-PD-1 + 90Y-NM600. Tramp-C1 tumors were collected from animals (n=3 per group) treated with IgG alone, TRT+anti-PD-1+non Treg depleting anti-CTLA-4 (non-depleter), or TRT+anti-PD-1+Treg-depleting anti-CTLA-4 (depleter). Anti-PD-1 was given on Day 0 and anti-CTLA-4 was given on days 0, 3, and 6 following TRT. Each antibody treatment used 200 µg antibody delivered i.p. (A) Study schema. Tumors were digested and evaluated by flow cytometry 7 days after TRT for (B) Tregs (CD4 +CD25+FoxP3+) cells as a percent of CD45 +cells, and (C) Effector memory cells (CD44 +CD27-CD62L-) as a percent of CD8 +T cells. TRAMP-C1 tumor-bearing mice (n=7 per group) were treated with high-dose TRT, anti-PD-1, and anti-CTLA-4 as before and followed for (D) tumor growth and (E) survival to a tumor volume of 2000 mm3. *P<0.05, **p<0.01, ***p<0.001. Error bars represent mean±SE. Results shown are from one experiment and are representative of two independent experiments. TRT, targeted radionuclide therapy.