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: Expression of PD-L1 in NSCLC and TNBC cells interferes with their sensitivity to HLA-G targeted CAR-?dT cells. A,B) HLA-G and PD-L1 were frequently expressed in the same lung (n = 24) and TNBC (n = 30) tumor lesions. HLA-G and PD-L1 expressions were determined by 2-plexed IHC staining using specific antibodies (left). Scale bar = 20 µm of. HLA-G and PD-L1 expression levels in tumor lesions and normal adjacent tissues were shown as a tissue cytometry dot-plot (middle panel) and the quantified data are displayed (right panel). C,D) Expression levels of HLA-G and PD-L1 in lung cancer and breast cancer cell lines. C) A549 and H1975; D) MCF-7, T47D and MDA-MB-231 cells were harvested to evaluate the levels of HLA-G and PD-L1 by immunoblotting (left panels) and flow cytometry (right panels). E) PD-L1 blockade re-enforced cytotoxicity induced by Nb-CAR-?dT cells in HLA-Ghigh/PD-L1high tumor cells. MDA-MB-231 and H1975 cells were pretreated with or without 10 µg mL-1 atezolizumab for 15 min and then cocultured with parental or Nb-CAR-?dT cells (E:T = 3:1) for 48 h. F) PD-L1 antagonized HLA-G targeted CAR-?dT-induced cytolysis. Mock or PD-L1-overexpressed A549 cells were treated with or without 10 µg mL-1 atezolizumab for 15 min and then co-incubated with parental or Nb-CAR-?dT cells (E:T = 3:1) for 48 h. Specific lysis was performed by the LIVE/DEAD cell-mediated cytotoxicity assay using flow cytometry analysis. These results suggested that elevated PD-L1 levels on tumor cells may be a risk of immune escape from Nb-CAR-?dT therapy in solid tumors. Results are representative of at least three independent experiments. Data represent the mean ± SD, n = 3–4, *p < 0.05; **p < 0.01; and ***p < 0.001 based on paired Student's or Student's t-tests.

Fig 4: 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.

Fig 5: The differences in proportion and transcriptome of EVT between AEP and REP. (A) Workflow for single‐cell transcriptome profiling of fallopian tube and placenta. Numbers in parentheses indicate the number of individuals analysed. AEP (n = 2), REP (n = 3), and IP (n = 2). (B) Placental and fallopian tubal cell clusters from 10× Genomics scRNA‐seq analysis visualized by UMAP. Colours indicate cluster. TB, trophoblast cell. Immune, immune cells. Epithelial, Epithelial cells. Fibroblasts/Endo, Fibroblastic cells, stromal cells (IP), or Endothelial cells. (C) UMAP visualization of trophoblast cell analysis. Each point represents one cell that is coloured by its cell type as shown in the legend. CTB, cytotrophoblast, EVT, extravillous trophoblast; p, proliferative; STB, syncytiotrophoblast. (D) The expression level of each cluster‐defining markers and percentage of positive cells in each cluster. (E) Comparison of each trophoblastic type of number and proportion in three groups (IP, AEP and REP). (F) Expression of genes related to invasion or epithelial‐mesenchymal transition in trophoblast cells in three groups (IP, AEP and REP). AEP, abortive tubal ectopic pregnancy. IP, intrauterine pregnancy; REP, ruptured tubal ectopic pregnancy. (G) Immunochemical staining for HLA‐G (EVTs marker) and MMP2 (invasive marker) (representative images from AEP = 3, REP = 3). Scale bars, 100 μm.