Fig 2: The effects of different dosage and timing protocols. Mice (n = 5) were injected with TC-1/A9 cells and immunized by a gene gun. Mice received combinations of ODN1826, a-GalCer and a-Tim-3 3 times on the days of immunization (A), 5 times with two additional doses on days 13 and 17 (B) and 3 times with a one-week delay following DNA immunization (i.e., on days 10, 13 and 17) (C). Bars: ±SEM; ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical significance refers to the comparison with the group immunized with the PADRE.E7GGG gene. The experiment was repeated with similar results.

Fig 3: LSD1 depletion preserves intratumoral TCF1+PD-1int progenitor exhausted CD8+ T cells.a, b Frequencies (a) and PD-1 MFI (b) of CD8+PD-1+ TILs on day 18 after MC38 tumor implantation (Lsd1f/f group, n = 4; Cd4-Cre+Lsd1f/f group, n = 5). c–e Representative flow plots (c), percentages (d) and cell numbers (e) of TCF1+PD-1int, TCF1+PD-1hi and TCF1-PD-1hi cells in CD8+ TILs analyzed by flow cytometry on day 12 and 18 after MC38 tumor implantation (Lsd1f/f d12 group, n = 5; Cd4-Cre+Lsd1f/f d12 group, n = 5; Lsd1f/f d18 group, n = 4; Cd4-Cre+Lsd1f/f d18 group, n = 4). f Percentages of TCF1+PD-1int, TCF1+PD-1hi and TCF1-PD-1hi cells in CD8+Tetramer+ TILs on day 18 after MC38-OVA tumor implantation (Lsd1f/f group, n = 14; Cd4-Cre+Lsd1f/f group, n = 10). g, h, Slamf6 (g) and Tim3 (h) expression by different subpopulations of CD8+ TILs analyzed by flow cytometry (Lsd1f/f group, n = 8; Cd4-Cre+Lsd1f/f group, n = 6). i, j, Representative flow plots of Ly6C expression (i) and percentages of Ly6C+ cells (j) in different subpopulations of CD8+ TILs (n = 4 per group). k, l GzmB (k) and Ki-67 (l) expression by different subpopulations of CD8+ TILs analyzed by flow cytometry on day 18 (n = 4 per group). m, n Cell apoptosis analysis by Annexin V staining (m) and active Casp3/7 staining (n) of CD8+ TILs isolated from MC38 tumors on day 14 (n = 5 per group). Data represent two independent experiments (a–e, g–n) or are pooled from three independent experiments (f) and are presented as mean ± SEM (a–n). Statistical significance was determined by two-sided unpaired t test (a–n). Source data are provided as a Source data file.

Fig 4: Expression of T-cell activation/exhaustion markers on the CD8+ TIL. a Expression of Nrp-1, PD-1, LAG-3, CTLA-4 and Tim-3 on CD8+ T cells from B16F10 TIL isolated at day 15. Right: percentages of Nrp-1 among CD8+ T cells expressing or not PD-1 (n = 26), Tim-3 (n = 19), LAG-3 (n = 19) or CTLA-4 (n = 16). b Expression of Nrp-1 on CD8+ T cells correlated with that of PD-1 (n = 38). c Expression of Nrp-1 on CD8+ TIL is restricted to a PD-1hi T-cell population. Left, Expression of Nrp-1 and PD-1 on CD8+ TIL; identification of three T-cell subsets: Nrp-1+PD-1hi, Nrp-1-PD-1+and Nrp-1-PD-1-. Right: expression of exhaustion/activation markers, functional proteins and transcription factors in Nrp-1+PD-1hi, Nrp-1-PD-1+ and Nrp-1-PD-1- CD8+ T-cell subsets. Heat maps including percentages of cells positive for expression of PD-1, Nrp-1, Tim-3, CTLA-4, LAG-3, granzyme B (GrzB) and Ki-67, and gMFI for NFATc1, IRF-4, Helios, Blimp-1 and T-bet (n = 5). d gMFI of PD-1 on Nrp-1+PD-1hi, Nrp-1-PD-1+ and Nrp-1-PD-1- TIL subsets (n = 16). Results are representative of three to five independent experiments. Means ± SEM two-tailed Student’s unpaired t test a or one-way ANOVA test with Bonferroni correction d. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file c

Fig 5: Targeting LSD1 enhances long-lasting response to anti-PD-1 treatment.a Experimental design of administering GSK2879552 and PD-1 blocking antibodies. b Tumor growth curves of wildtype mice inoculated with MC38 tumor cells and treated with GSK2879552 or vehicle control in combination with anti-PD-1 or isotype control. c Individual tumor growth curves over time in response to anti-PD1 treatment alone or in combination with GSK2879552. d, e Percentages of TCF1+PD-1int and TCF1-PD-1hi cells (d) or GzmB+ cells (e) in CD8+ TILs (Vehicle + Isotype, n = 6; GSK + Isotype, n = 6; Vehicle + anti-PD-1, n = 6; GSK + anti-PD-1, n = 7). f, g, Percentages (f, n = 9, 10, 6, 9, respectively) and cell numbers (g, n = 8, 9, 6, 5, respectively) of tumor-infiltrating CD3+CD4+ T cells and CD3+CD8+ T cells in gated CD45+ leukocytes. h, i Frequencies (h, n = 9, 10, 6, 9, respectively) and cell numbers (i, n = 8, 9, 6, 5, respectively) of CD8+TCF1-TIM-3+ TILs. Data represent two independent experiments (b, d–i) or are pooled from two independent experiments (c), and are presented as mean ± SEM (b, d–i). Sample sizes are as indicated. Statistical significance was determined by two-way ANOVA (b) or two-sided unpaired t test (d–i). Source data are provided as a Source data file.