Fig 2: The crosstalk between fetus and mothers leads to high levels of RANKL/RANK expression at the maternal–fetal interface. (a) RANKL expression in villi and decidua of normal pregnancy (n=12) by immunohistochemistry. Original magnification: × 200. (b) RANKL secretion by primary trophoblasts (1 × 105 cells per well) and DSCs (1 × 105 cells per well) (n=6) from normal pregnant women by ELISA after culture for 24–96 h (left). RANKL production by trophoblasts alone, DSCs alone and the co-culture of trophoblasts and DSCs (n=6) for 48 h (right). (One-way ANOVA). (c) We isolated primary PBMCs from peripheral blood (n=6) and DLC from deciduas of normal pregnant women (n=6), and then analyzed RANK expression on pMo and dMφ from normal pregnant women by labeling with anti-CD14, RANK and CD45 antibodies. (Student’s t-test). (d) Further analysis of the phenotype of RANK+ and RANK− pMo and dMφ from normal pregnant women (n=24) by FCM. (One-way ANOVA). (e and f) Trophoblasts, DSCs and/or dMφ (n=6) were co-cultured at a 1 : 1 : 1 ratio for 48 h, and then RANKL expression on CK7+ trophoblasts and CK7− DSCs, and RANK on CD14+ dMφ were evaluated by FCM, respectively. (One-way ANOVA). Tro: human trophoblasts. pMo: human peripheral blood monocytes; dMφ: human dMφ. Data are expressed as the mean±S.E.M. *P<0.05, **P<0.01 and ***P<0.001. NS: no statistical difference

Fig 3: Impaired FAO and LD accumulation in elderly-derived monocytes(A–I) Blood monocytes were isolated from young and old mice. (A) Western blot analysis of p-AMPK and p-mTOR expression in each group. Representative blots of three independent experiments are shown. (B) ATP concentrations were tested using an ATP colorimetric/fluorometric assay and normalized by the protein concentration (n = 4 per group). (C) The OCR was tested in XF-96 assay medium and normalized by protein concentration. Continuous OCR values (pmoles/min/μg protein) are shown. Each repetition involved three mice per group, and three replicates were performed. (D) Western blot analysis of ACADVL, HADHA, and ACADM expression in each group. Representative blots of three independent experiments are shown. (E) FAO enzyme abundance in monocytes was detected by flow cytometry. Representative mean fluorescent intensity (MFIs) and the mean MFIs are shown (n = 4 per group). (F) Morphological analysis of young and old murine monocytes by electron microscopy. (G) LDs in monocytes were stained with BODIPY 493/503 dye and then imaged under a confocal microscope before 3D rendering using Imaris software (version 9.0). (H) The mean LD volume of 50 analyzed cells. (I) LD density in monocytes from young (red line) and old (blue line) mice were analyzed by flow cytometry.(J) Human blood monocytes were isolated by using two-step procedures, and LDs were identified in CD14+ cells by BODIPY 493/503 immunofluorescence staining.(K) BODIPY 493/503 MFI values in CD14+/CD16+ blood monocytes isolated from young and elderly human subjects.(L) Correlation analysis between age and monocytes, BODIPY MFI values. (n = 30).The resulting Spearman's correlation coefficient (r) and corresponding p value are reported. All data represent mean ± SD. ∗∗p < 0.01, ∗∗∗p < 0.001; t test (B, E, and H).

Fig 4: Monkey CCR4+ Treg depletion for animals M11016 and 11216 in the peripheral blood using the CCR4 immunotoxin. (A) Representative flow cytometry analysis of the CCR4+ cell depletion in the peripheral blood using the antibodies against human CD4 and CCR4. (B) CCR4+ cell depletion in the peripheral blood was monitored by flow cytometry using the antibodies against human CD4 and CCR4 (CD4+ CCR4+). (C) Representative flow cytometry analysis of the CCR4+Foxp3+ Treg depletion in the peripheral blood using the antibodies against human CCR4 and Foxp3 (CCR4+Foxp3+ among the gated CD4+ cells). (D) CCR4+ Treg depletion in the peripheral blood was monitored by flow cytometry using the antibodies against human CCR4 and Foxp3 (CCR4+Foxp3+ among the gated CD4+ cells). (E) The other CD4+ cells in the peripheral blood were monitored by flow cytometry using antibodies against human CD4 and CCR4 (CD4+ CCR4−). (F) The CD8+ T cells in the peripheral blood were monitored by flow cytometry using the antibodies against human CD3, CD4, and CD8 (CD3+ CD4− CD8+). (G) The B cells in the peripheral blood were monitored by flow cytometry using antibodies against human CD20, CD3, CD16, and HLA‐DR (CD20+ CD3− CD16− HLA‐DR +). (H) The NK cells in the peripheral blood were monitored using antibodies against human CD3, CD16, and NKp80 (CD3− CD16+ NKp80+). (I) Monocytes in the peripheral blood were monitored by flow cytometry using antibodies against human CD14, CD16, and CD11b (CD14+ CD11b+ or CD14+ CD16+, PBMC gating). (J) The effector Tregs in the peripheral blood were monitored by flow cytometry using antibodies against CD45RA and Foxp3 (CD45RA ‐Foxp3+ among the gated CD4+ cells).

Fig 5: M6903 efficiently saturated TIM-3 in vitro in human whole blood and demonstrated expected pharmacokinetics in marmoset monkeys. a) TO increased with increased concentrations of M6903. Serial dilutions of M6903 were incubated with fresh human whole blood for 1 hour. The unoccupied TIM-3 on CD14+ cells was measured by flow cytometry with anti-TIM-3 (2E2)-APC, which competes with M6903 for TIM-3 binding. The graph shows 4 representative donors (KP46318, KP46315, KP46231 and KP46233) out of the 10 total donors. b) M6903 PK profile in marmoset monkeys in vivo following a single iv administration. The graph shows serum concentrations (ng/mL) of M6903 at 0, 1, 6, 24, 48, 72, 96, 168, 336, 504, and 672-hour time points after dosing with 0.20, 1.0, 5.0, or 20.0 mg/kg doses.