Fig. 7: ISV with FOLactis formed long-term immunological memory in a syngeneic tumor model and synergized with anti-PD1 antibody.

Representative flow cytometry images of PD1⁺CD8⁺ T cells seven days after treatments in CT26 treated tumors (a) and their flow cytometric analysis (b) (n = 4). c Immunohistochemistry analysis of PD1 expression in CT26 treated tumors seven days after treatments (n = 3). Scale bars, Left, 200 µm; Right, 100 µm. d Representative flow cytometry images of effector memory T cells (T_EM, CD3⁺CD8⁺CD44⁺CD62L⁻) and central memory T cells (T_CM, CD3⁺CD8⁺CD44⁺CD62L⁺) seven days after treatments in the spleen (n = 4) and their flow cytometric analysis (e, f). g Splenocytes of mice in the NS or FOLactis group were incubated with CFSE labeled CT26 cells at effector-to-target ratio (E: T) of 5:1, 10:1, and 20:1. PI was added six hours after incubation and the percentage of dead cells was analyzed by flow cytometry (n = 3). h Schematic diagram of administration route for FOLactis combined with anti-PD1 in tumor suppression experiment. Created with BioRender.com. Average tumor growth curves (n = 6) (i) and survival data (n = 10) (j) of tumor-bearing mice. The dose of PD1 was 100 μg for each mouse in every administration. Tr: treated; NT: nontreated. k Schematic illustration of evaluating the immune memory effect. Mice cured of CT26 tumor were rechallenged subcutaneously (s.c.) 60 days later with 5 × 10⁵ tumor cells. Created with BioRender.com. l Tumor growth curves of each mouse following the rechallenge (n = 5). m Survival data of two groups of mice (n = 5). n Pictures of representative mice were taken 15 days after rechallenge. The tumors of each mouse were circled. For the experiments in b, e, f, and g, the error bars represented mean ± s.e.m. statistical significance was calculated by two-tailed unpaired Student’s t-tests. ns represented p > 0.05. For the experiments in i, data were the mean ± s.e.m. p-values were determined by two-way ANOVA with Tukey’s multiple comparisons test. ns represented p > 0.05. Differences in survival were determined by using the Kaplan–Meier method, and the p value was determined via the log-rank (Mantel–Cox) test. Source data are provided as a Source Data file.