Fig 2: Combined PD-L1 + PD1 Blockade Promotes Tumor Eradication and T Cell Persistence in the Absence of Antigen(A) Radiance of tumor growth in mice treated with either aPD-L1 or aPD1, combination aPD-L1 + aPD1 (dual). Gray box indicates treatment period. Each line is an independent mouse. Arrow indicates unexpected death.(B) Number of tetramer+ T cells per milliliter in blood and mean tumor radiance. Symbol indicates that some mice were euthanized because of tumor growth. Data are mean ± SEM for tetramer number. Data are mean for tumor radiance.(C) Kaplan-Meier survival curve of mice in (A) and (B). n = 4 or 5 mice per group.(D) Proportion of tetramer+ T cells (gated on live CD45+CD8+ T cells) in blood at the weeks (W) post-tumor. LTS, long-term survivors.(E) Proportion of intratumoral tetramer+ T cells that express KLRG1 and/or Lag3 at day 14. Single, aPD-L1 only.(F) Quantification of groups in (E). Data are mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.0005 (one-way ANOVA with a Tukey post hoc test).(G) Proportion of CD8+ T cells that produce TNFa following CB101–109 peptide re-stimulation ex vivo.(H) Proportion of CD8+ T that produce the indicated cytokines from tumors in mice treated with single (aPD-L1) or dual (aPD-L1 + aPD-1) blockade at day 14. Data are mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.0005 (one-way ANOVA with a Tukey post hoc test).(I) Number of CD8+ T cells per gram that produce TNFa from mice treated with single or dual therapy at day 14. Each dot is an independent mouse. Data are mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.0005 (one-way ANOVA with a Tukey post hoc test). See also Figure S5.(J) Tumor radiance following delayed single (aPD-L1) or delayed dual (aPD-L1 + aPD-1) blockade beginning day 14 post-tumor implantation. Significant difference in tumor size at day 21 was assessed using an unpaired two-tailed Student’s t test. Data are mean ± SEM.(K) Kaplan-Meier survival curve of mice in treated as in (J). Control group is the same animals shown in (C). Survival prolongation for delay dual (MST 42 days) versus untreated (MST 22 days) was determined by a log rank test. See also Figure S5.

Fig 3: Rapid Intratumoral and Progressive Systemic Dysfunction of Neoantigen-Specific T Cells(A) Frequency of PD-1, Lag3, Tim3, and TIGIT expression by tetramer+ T cells in the spleen (Spl) and tumor (Tum) at day 22 post-tumor implantation. Each dot is an independent mouse, and data are mean ± SEM. *p < 0.05, **p < 0.05, and ***p < 0.005 (one-way ANOVA, with a post hoc test to correct for multiple comparisons).(B) Representative plots of markers quantified in (A).(C) Mean frequency of CB101–109:H-2Db-specific CD8+ T cells that express none (0), one (PD-1), or co-express 2, 3, or 4 inhibitory receptors at day 22.(D) Proportion of total CD8+ T cells producing IFN? following ex vivo re-stimulation with CB101–109 peptide at day 22.(E) Number of tetramer+ T cells producing IFN? at day 22. Data are mean ± SEM, and each dot is an independent mouse. *p < 0.05 (one-way ANOVA, with a post hoc test to correct for multiple comparisons).(F) Progressive phenotypic and functional changes in splenic tetramer+ CD8+ T cells in mice bearing KPC2a tumors ± aPD-L1. Data are mean ± SEM. n = 4 or 5 mice per group.(G) Progressive phenotypic and functional changes in intratumoral tetramer+ CD8+ T cells. Data are mean ± SEM. *p < 0.05 (unpaired two-tailed Student’s t test). n = 4 or 5 mice per group.(H) Analysis of the indicated antigens on circulating tetramer+ T cells following aPD-L1 treatment. Data are mean ± SEM. n = 4 or 5 mice per group. See also Figure S4.

Fig 4: Lycorine treatment inhibits T cell infiltration and function in cardiac allografts. On day 7 posttransplant, allograft-infiltrated leukocytes were isolated from PBS-treated mice (Vehicle) or lycorine-treated mice (5 mg kg-1, b.i.d. Lycorine) were isolated and analyzed by flow cytometry. A) Counts of allograft-infiltrated cells and CD45+ cells in each group (n = 6). B) Numbers of allograft-infiltrated CD4+ T cells, CD8+ T cells, B cells and NK cells (n = 6). C) The expression of membrane CD69, CD25, OX40, and GITR on the surface of allograft-infiltrated CD4+ T cells and CD8+ T cells was measured (n = 5). Values for mean fluorescence intensity (MFI) are shown. D) Bar graphs depict MFI values for PD1, CTLA4, LAG3, and TIM3 on T-cell subsets (n = 5). E) Allograft-infiltrated leukocytes were stimulated with a Cell Stimulation Cocktail (plus protein transport inhibitors) and intracellular staining was performed. Percentages of IFN-? + and TNF-a + T-cell subsets are shown (n = 5). F) Levels of CD107a expression on allograft-infiltrating CD4+ T cells and CD8+ T cells from each group (n = 5). The graphs show the means ± SEM of three independent experiments. *P < 0.05, and **P < 0.01, Student's t-test.