Fig 2: Anti-tumor activity of Nb-CAR.BiTE-?dT cells in the orthotopic PBMC-CDX-NSG mouse models. A) Protocol for evaluating the anti-tumor activity of mRNA-driven Nb-CAR.BiTE-?dT cells in the PBMC-CDX-NSG mice bearing orthotopic HLA-Ghigh/PD-L1high TNBC or PD-L1 overexpressing lung tumors. Seven days after inoculation with 1 × 106 tumor cells, the mice received tail vein injection with PBMCs (5 × 106 cells per mouse). Three days later, the parental, Nb-CAR or Nb-CAR.BiTE-?dT cells (1 × 107 cells/mouse) were injected through tail vein once a week for 4 weeks. B-G) Superior anti-tumor activity of Nb-CAR.BiTE-?dT cells in the PBMC-CDX-NSG mouse model. The tumor growth of the orthotopically inoculated B,C) MDA-MB-231 (n = 10) and E,F) PD-L1-overexpressing A549 cells (n = 5) were monitored weekly through IVIS. D,G) Survival rates were recorded. These results suggested that Nb-CAR.BiTE-?dT is effective against HLA-G/PD-L1-double positive solid tumors as well as PD-L1-overexpressing solid tumors in vivo. Results are representative of three independent experiments. Data represent the mean ± SD; *p < 0.05; **p < 0.01; and ***p < 0.001 based on the Kaplan–Meier method and log-rank test.

Fig 3: Characterization of the Nb-CAR.BiTE construct. A) Schematic representation of mRNA and lentiviral HLA-G Nb-CAR, with or without combined secretable PD-L1/CD3e-targeted Nb-BiTE constructs. The bi-epitopic Nb-CAR comprises two tandem extracellular HLA-G Nbs fused with a CD8a hinge/TM, followed by a modified 4-1BB cytosolic domain (4-1BB/Tyk) and a modified CD3? intracellular domain (CD3?/ITAM) linked to a self-cleavage peptide P2A to separate the secretable bivalent Nb-BiTE, which includes two PD-L1 Nbs linked to a CD3? Nb. B) The blocking activity of HLA-G Nbs. The blockade activity of HLA-G Nb clones #1 and #2 or HLA-G mAb 87G to LILRB1/HLA-G (upper panels) and KIR2DL4/HLA-G (bottom panel) were determined by competitive ELISA. C) HLA-G Nb moiety capable to bind to all HLA-G isoforms. The binding activity of HLA-G Nb clones and it derived bi-epitopic Nb moiety to recombinant HLA-G-ß2M (corresponding to HLA-G G1 and G5), HLA-G a1a3 domains (G2 and G6), HLA-G a1a2 domains (G4) and HLA-G a1 domain (G5) were examined by ELISA-based binding assay. D) PD-L1 blockade Nbs. The activity of PD-L1 Nb clones #1 and #2 was determined by PD-L1/PD-1 blockade bioassay. E) Biofunction and affinity of CD3e Nb. PBMCs (1 × 106 cells) were treated with/ without 100 ng CD3e Nb or OKT3. The enriched ?dT cells from PBMCs were supplemented with 1000 IL mL-1 IL-2 and then treated with or without 100 ng CD3e Nb or OKT3. After 6 d, the images were taken (upper panel), Scale bar = 20 µm, and the cell numbers were counted and normalized to the CD3+ cell populations (bottom panel). Evaluation of the novel CAR signaling cassette. F) Bi-epitopic HLA-G Nb-CARs were engineered using 4-1BB-CD3? (BB-3z) or modified signaling cassettes (IFNAR/BB-3z or BBTyk-3z). G) BB-3z-, IFNAR/BB-3z-, or BBTyk-3z-based HLA-G Nb-CAR lentiviral particles (MOI = 3) were transduced into ?dT cells, and after three days, the expression of Nb-CAR was determined by flow cytometry. H,I) Parental and BB-3z-, IFNAR/BB-3z-, or BBTyk-3z-Nb-CAR-?dT cells were cocultured with MDA-MB-231 cells at an E:T ratio of 3:1. H) After 2 h, the phosphorylation status of STAT2 and Syk/ZAP70 in the CAR-?dT cells was determined; and I) after 24 h, the induced cytotoxicity was determined using a LIVE/DEAD cell-mediated cytotoxicity assay (upper left panel); CD107a expression in CAR-?dT cells was detected by flow cytometry using specific antibodies (bottom left panel); and their supernatants were harvested to detect the contents of granzyme B, IFN-?, TNF-a, and IL-17A by ELISA. These data demonstrated that the elements for engineering the Nb-CAR.BiTE construct were functional and may be superior than that of the conventional CAR design. Results are representative of at least three independent experiments. Data represent the mean ± SD, n = 3; *p < 0.05; **p < 0.01; and ***p < 0.001 based on paired Student's t-test.

Fig 4: REP patients retain almost complete secretory epithelial cells. (A) Subpopulation analysis of epithelial cells UMAP diagram. Colours represent different subpopulations. HPlEpc, human placental epithelial cells, Violin plots of marker genes of each cell subpopulation. (B) Functional annotation of each epithelial cell subpopulation. (C) Cell proportions of epithelial cell subpopulations in REP, AEP, and IP groups. Colours represent identified subtypes. (D) Immunofluorescence for HLA‐G (EVT marker) and OVGP1 (secretory epithelial cells marker) in the maternal‐foetal interface of AEP and REP samples (representative images from AEP = 8, REP = 8). Green represents OVGP1, red represents HLA‐G, and blue represents DAPI (nucleus marker). Scale bars, 100 μm. AEP, abortive tubal ectopic pregnancy; IP, intrauterine pregnancy; REP, ruptured tubal ectopic pregnancy.

Fig 5: Secretory epithelial cells for macrophage accumulation and EVTs invasion by CSF1‐CSF1R regulatory axe. (A) Dot plot of predicted ligand‐receptor interactions. The circle size represents the p‐value, and the colour is the gene level indicating the interaction. (B) Immunofluorescence for OVGP1 (secretory epithelial cells marker) and CSF1 in the maternal‐foetal interface of REP. Green represents OVGP1, red represents CSF1, and blue represents DAPI (nucleus marker). Scale bars, 100 μm. (C) Immunofluorescence for HLA‐G (EVTs marker) and CSF1R in the maternal‐foetal interface of REP. Green represents CSF1R, red represents HLA‐G, and blue represents DAPI (nucleus marker). Scale bars, 100 μm. (D) Representative images showed the migration of HTR8/SVneo cells in the differential treatments (0 h, 18 h). The migration of HTR8/SVneo cells was compared between 0 and 18 h. Scale bars, 50 μm. Data are represented as mean ± SD. (E) Representative images of invaded HTR8/SVneo cells treated with different concentrations of CSF1 or GW2580. Scale bars = 100 μm. The number of invaded cells was compared in different groups. Data are represented as mean ± SD. (F) The EVT growth area of the explants was recorded by microscopy after 24 and 48 h, respectively. Scale bars, 200 μm. The EVT growth area in the CSF1 group was significantly larger than that in the GW2580 group after 24 and 48 h. Data are represented as mean ± SEM. (G) Real‐time qPCR analysis of MMP2 expression in HTR8/SVneo cells treated with CSF1 compared to control. Data are represented as mean ± SD. AEP, abortive tubal ectopic pregnancy; IP, intrauterine pregnancy; REP, ruptured tubal ectopic pregnancy. . *p < 0.05, **p < 0.01. ***p < 0.001. ****p < 0.0001.