Fig 2: Long-term immune-memory effects.(A) Schematic illustration of bacteria-based PTT therapy to inhibit cancer relapse. (B) Tumor growth curves of rechallenged tumors inoculated 40 days after elimination of their first tumors (eight mice per group). Data are presented as the mean ± SEM. (C) Morbidity-free survival of different groups of mice with rechallenged CT26 tumors after various treatments indicated to eliminate their primary tumors (10 mice per group). Bacteria-based PTT, even in the absence of anti–CTLA-4 blockade, could trigger strong enough immune responses to reject rechallenged tumor. (D) Quantification of proportions of CD8+ effector memory T cells (TEM) gated on CD3+ cells in the spleen analyzed at day 40 right before rechallenging mice with secondary tumors. (E) Cytokine levels, including TNF-α and IFN-γ, in sera from mice isolated at day 47. Data are presented as the mean ± SD. Statistical analysis was performed using the Student’s two-tailed t test (***P < 0.001, **P < 0.01, and *P < 0.05). (F) Schematic illustration of bacteria-based photothermal immunotherapy to inhibit the growth of primary tumors, abscopal metastatic tumors, and rechallenged tumors.

Fig 3: Atlas of NK cells in Cyp26a1‐MO‐treated mice. (A) Representative macroscopic views of uteri from Control‐MO and Cyp26a1‐MO treated mice. Arrows represent abnormal implantation sites. (B) CYP26A1 expression in uterus from Cyp26a1‐MO knockdown mouse model analysed by western blotting. (C) Workflow for single‐cell transcriptome profiling of NK cells from Control‐MO or Cyp26a1‐MO‐treated mouse uterus. CD45+CD3−CD122+ cells were enriched by FACS and then used for scRNA‐seq. (D) t‐SNE plots of NK cells, showing four main clusters for Cyp26a1‐MO treatment and control‐MO mice. Colours indicate different cell clusters. (E) Violin plots displaying marker genes (top row) for four main NK cell subsets. The first two columns represent the names of the NK cell clusters and the corresponding numbers of cells

Fig 4: DHM administration promoted the activation of hepatic NK cells in mice. a–c The frequency of intra-hepatic NK cells (cells were gated by NK1.1+ CD3−) and NKG2D expression (cells were gated by NK1.1+ CD3− NKG2D+) which in intra-hepatic NK cells were analyzed by FCM. d–e Frequencies of intra-hepatic NK cells and IFN-γ expression (cells were gated by NK1.1+ CD3− IFN-γ +) in intra-hepatic NK cells were analyzed by FCM. Experiments were independently repeated at least three times. *p < 0.05, **p < 0.01, compared to the control group; #p < 0.05, ##p < 0.01, compared to the CCl4 group. FCM, flow cytometry; IFN-γ, interferon gamma; NK cells, natural killer cells; NKG2D, NK cell receptor natural killer group 2 member D

Fig 5: Obesity-accelerated ageing is associated with enhanced osteoclastogenic potential and BM adipocyte accumulation. The levels of BM CD3−B220−Ter119−Ly6G−Ly6Chigh CD11blow OCPs were determined as the frequency of single cells (FoS) expressed as % live BM cells at the indicated timepoints in (A) male and (B) female mice fed Chow or HCD, the latter cohort treated with either PBS or ES-62. The data are presented as the mean ± SEM values for the indicated group, where for male mice, at d56: n=6; d340: Chow, n=6; PBS, n=7; ES-62, n=8; d500, Chow, n=5; PBS, n=5; ES-62, n=5 individual mice and for female mice, at d56: n=6; d340: Chow, n=6; PBS, n=8; ES-62, n=8; d500, Chow, n= 3; PBS, n=5; ES-62, n=6 individual mice. Significant differences are indicated by *=p<0.05, male Chow v male PBS or ES-62 (A) and female Chow v female PBS (B) and **=p<0.01, female Chow v female ES-62 (B) groups. (C–F) OC differentiation at day 5 was assessed by TRAP staining and the size of multinucleated OCs (>3 nuclei) determined by Image J analysis. Representative images (x4 magnification) of OCs of the indicated groups of male (C) and female (D) mice are shown and the mean group OC size ± SEM determined from the mean values of triplicate cultures of individual male mice at each time point where for male mice (E), at d56: n=6; d340: Chow, n=6; PBS, n=3; ES-62, n=4; d500, Chow, n=5; PBS, n=5; ES-62, n=5 individual mice and for female mice (F), at d56: n=6; d340: Chow, n=6; PBS, n=3; ES-62, n=4; d500, Chow, n=4, PBS, n=5; ES-62, n=5 individual mice. Significant differences are indicated by *=p<0.05, Chow v PBS or ES-62 for male (E) and female (F) mice. (G–I) BM adipocytes were visualised (x10 magnification, scale bars 400 µm) and quantitated by Image J analysis where the mean group number/mm2 ± SEM is determined from the mean values of triplicate fields of view (FoV) of the individual mice at each time point. For male mice (G), d500 data scale expanded in insert), d56: n=6; d340: Chow, n=6; PBS, n=6; ES-62, n=6; d500, Chow, n=3; PBS, n=3; ES-62, n=3 individual mice and for female mice (H), d56: n=6; d340: Chow, n=5, PBS, n=5; ES-62, n=6; d500, Chow, n=3; PBS, n=3; ES-62, n=3 individual mice. Significant differences are indicated by blue**=p<0.01 for male PBS v male ES-62 (G) and red*=p<0.05, female PBS v female ES-62 (H) groups. Representative images are shown for each group (I).