Fig 2: 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).

Fig 3: Anticancer activity of HMME/R837@Lip-augmented SDT plus anti-PD-L1 therapy in murine colorectal cancer models. a Schematic illustration of the experiment design to assess the antitumour immune responses against mimic distant tumours and the immunological memory response triggered by HMME/R837@Lip-augmented SDT and anti-PD-L1 combination therapy; b, c primary (b) and mimic distant (c) tumour growth curves of different groups of tumour-bearing mice after various treatments as indicated in the figure. Error bars are based on SD (n = 6); d time-dependent body-weight surveillance of mice (n = 6) after different treatments; e morbidity-free survival of mice after different treatments (n = 6); f tumour-growth curves of the rechallenged tumours in the corresponding groups were stopped when the first mouse died. Error bars are based on SE (n = 8); g morbidity-free survival of mice after the indicated treatment (n = 8), statistical significance was calculated via the log-rank (Mantel–Cox) test; h TNF-a and IFN-? levels in serum isolated from mice of the treatment group and native group 7 days after the second tumour was introduced. Error bars are based on SE (n = 5 for the native group, n = 4 for the combined treatment group); i representative flow-cytometry plots of splenic lymphocytes of CT26 tumour-bearing mice treated with the combined immunotherapy at day 29 right before mice rechallenged with the secondary tumours (gating on CD3+ cells); j, k absolute quantification of naive T cells, TCM and TEM in the spleen. TCM central memory T cells, TEM effector memory T cells. Error bars are based on SD (n = 4). Statistical significances were calculated via Student’s t test. *p < 0.05, **p < 0.01 and ***p < 0.001

Fig 4: FoxP3-Specific Deletion of Blimp1 Leads to Dysregulated GC Responses(A) Histology of splenic GCs, and quantification of GL7+ GC areas from 4- to 5-month-old FoxP3Cre (WT) and Prdm1fl/flFoxP3Cre (KO) mice (n = 3–4/group).(B) Flow cytometry of splenic TFH (CD4+CD3+ICOShiCXCR5+FoxP3-) and GC B (B220+GL7+Fas+) from 4- to 5-month-old mice.(C) (Left) Serum ANA levels from 4- to 5-month-old mice (n = 4/group). (Right) Serum IgE levels from 1.5- and >4-month-old WT and KO mice (n = 7–11/group).(D–G) Flow cytometry and frequency of splenic TFH and GC B from 6-week-old mice before (D and E) or after (F and G) immunization with NP-KLH in complete Freund’s adjuvant (CFA) (n = 4/group).(H) Serum anti-NP23 Ig, anti-NP4 Ig, IgG1, and IgE levels from 6-week-old mice after immunization with NP-KLH in CFA (n = 4/group).p.i., post immunization; -, before immunization. For (B)–(H), the data represent one of four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (unpaired two-tailed Student’s t test). Error bars indicate means ± SEMs.See also Figure S1.

Fig 5: Mechanism of Blimp1-Dependent Control of TFR Differentiation: Contribution of the IL-23R-STAT3 Axis and CXCR5-CCR7 Expression(A) WT and KO mice (6 weeks old) were immunized with NP-OVA in CFA. Seven days later, TFR cells (CD4+CD3+PD-1+CXCR5+YFP+) were sorted for microarray analysis. Differential gene expression in TFR from WT and KO mice (>1.5-fold) is shown.(B) Pathway analysis revealed control of genes associated with TH cell differentiation by Blimp1.(C) Genes related to cytokine-cytokine receptor interaction by DAVID.(D) IL-23R expression by donor TFR cells in Tcra-/- hosts after tamoxifen-induced deletion of Blimp1, as in Figure 4.(E) IL-23R expression in the CD25-expressing TFR subsets from WT and KO mice (6–8 weeks old) 10 days post-immunization.(F) FoxP3-GFP reporter mice were immunized with NP-OVA in CFA. Seven days later, Treg (CD4+CD3+GFP+CD25+) and CD4+ naive T cells (CD4+CD3+GFP-CD44-) were sorted, chromatin prepared, and ChIP-PCR analyses performed for Blimp1, acetylated H3 (AcH3), and H3K27me3 at Blimp1-binding sites at the 3rd intron of Il23r, the 1st intron of Cxcr5, the 5' distal element of CTLA-4, the 3rd intron of Ccr7, the FoxP3 CNS2, the 1st intron of Il2ra, and a non-specific region (C, control) of Il2ra. Data are shown as the percentage of input. Naive cells were used as controls for the anti-Blimp1 assay. Rabbit IgG isotype (R-iso) and mouse IgG isotype (M-iso) served as controls for the anti-AcH3 assay and anti-H3K27 assay, respectively.(G) Prdm1fl/fliCre- (WT) and Prdm1fl/fliCre+ (Del) mice (8 weeks old) were treated with tamoxifen at day 0, followed by immunization with NP-OVA in CFA at day 1 and injection of tamoxifen daily for 4 days. Expression of pSTAT3 and pSTAT5 in TFR cells at day 7 post-immunization. At right, pSTAT3:pSTAT5 ratios in TFR cells are shown (n = 4/group).In (D), (E), and (G), the data represent one of two experiments. In (F), the data represent one of three independent experiments. **p < 0.01 (unpaired two-tailed Student’s t test). Error bars indicate means ± SEMs.See also Figure S4.