In a recent study published in Cell, a group of researchers elucidated the shared immune tolerance mechanisms in cancer and pregnancy, focusing on the role of progestogen-induced B7 Homolog 4 (B7-H4) (an immune checkpoint protein) as an onco-fetal immune tolerance checkpoint (mechanisms allowing the immune coexistence of cancer and fetal cells).
Background
Immune checkpoint blockade (ICB) elicits durable responses across various cancers; however, most patients do not respond to current immunotherapy due to key immune-regulatory mechanisms fostering suppressive networks in the tumor microenvironment (TME). This leads to cancer immune evasion and resistance to ICB.
Pregnancy, an ideal model for exploring immune tolerance, shares critical immunosuppressive pathways with cancer, including Programmed Death-Ligand 1 (PD-L1), Human Leukocyte Antigen-G (HLA-G), Indoleamine 2,3-Dioxygenase (IDO), and Tregs. Further research is needed to fully understand the mechanisms and therapeutic potential of targeting B7-H4 in cancer and pregnancy-related immune tolerance.
About the study
In the animal studies, a spontaneous tumor model was induced using Medroxyprogesterone Acetate (MPA) and 7,12-Dimethylbenz[a]anthracene
(DMBA). Age-matched female Wild Type (WT) and B7-H4−/− mice were implanted with a pellet containing slow-releasing MPA subcutaneously. Mice were administered 1 mg of DMBA weekly by oral gavage for six doses.
Tumor growth was monitored, and evaluable tumor numbers were recorded. Tumor dimensions were measured with a caliper, and the tumor area was calculated. Tumor-free and overall survival were monitored. To establish transplantable tumors, primary cells were isolated from tumor nodules and inoculated into WT mice subcutaneously for in vivo passage. These cells were used to establish subcutaneous transplant tumors for therapy models using Mifepristone (RU486), BD-9136, or anti-PD-L1.
Mice were randomized into treatment groups and monitored for tumor-free and overall survival. B7-H4 expression on mouse endometrial epithelial cells (MEECs) was examined by administering RU486 to WT mice and harvesting the uteri for cell isolation.
In pregnancy models, female C57BL/6 (WT) and B7-H4−/− mice were mated with male Bagg Albino, C strain (BALB/c) (a common inbred strain of laboratory mouse) and C57BL/6 mice for allogeneic and syngeneic mating, respectively. Pregnant mice were euthanized at gestational day 13.5, and the number of all fetuses and resorbed fetuses was recorded. The fetus resorption rate was calculated. Cluster of Differentiation (CD)4 and CD8 T cells (Lymphocytes that identify and destroy infected or cancerous cells) were depleted using specific antibodies, and depletion efficiency was confirmed by flow cytometry. Embryo transfer was performed by treating WT and B7-H4−/− female mice with Pregnant Mare Serum Gonadotropin (PMSG) and Human Chorionic Gonadotropin (hCG), mating them with respective males and transferring two-cell embryos to recipient mice. Pregnancy outcomes were examined at gestational day 13.5 or 14.5.
Study results
To explore previously unappreciated onco-fetal immune tolerance mechanisms, 14 single-cell RNA-sequencing (scRNA-seq) datasets from various TME and placenta were cross-analyzed, focusing on the “non-self” cellular components of tumor and trophoblast cells. Given the importance of the major histocompatibility complex (MHC) and co-accessory molecules in the immune response, expression patterns of MHC, key B7 family members, and TNF family members were assessed.
The analysis confirmed previously reported key genes expressed in tumor and trophoblast cells. For instance, high Fibrinogen-like protein 1 (FGL1) expression was found in tumor cells, and high HLA-G expression in trophoblast cells in the placenta. Interestingly, B7-H4 V-Set Domain Containing T Cell Activation Inhibitor 1 (VTCN1) was ranked as the top gene highly expressed in tumor cells in the TME and trophoblast cells in the placenta. Thus, B7-H4 transcripts are expressed in the major cellular components of both TME and placenta.
The analysis was extended to the Tumor Immune Single-cell Hub 2 (TISCH2) database to compare the expression patterns of B7-H4 and PD-L1 (B7-H1, CD274). B7-H4 was primarily expressed by tumor cells, while different cell types, including myeloid cells, widely expressed PD-L1. Among many cancer types, the highest levels of B7-H4 transcripts were in cancers originating from female reproductive organs, such as breast and ovarian cancers.
This scRNA-seq data analysis aligned with bulk RNA-seq data from The Cancer Genome Atlas (TCGA), showing the highest levels of B7-H4 transcripts in breast, ovarian, and endometrial cancers. High levels of B7-H4 protein were detected in breast and endometrial cancers, with intermediate levels in cervical cancer. Hence, B7-H4 transcripts and proteins are highly expressed in pan-gynecological cancers.
At the maternal-fetal interface, B7-H4 was mainly expressed by trophoblast cells. Human trophoblast cells consist of anatomically and functionally different subsets, including extravillous trophoblast cells (EVTs), villous cytotrophoblast cells (VCTs), and syncytiotrophoblast (SCT). The expression of B7-H4 transcripts in trophoblast subsets from human first-trimester pregnancies, a critical stage for establishing maternal-fetal immune tolerance, was analyzed. HLA-G expression was mainly detected in EVTs, CD274 expression in EVTs and SCT, and VTCN1 in all three subsets of trophoblast cells. VTCN1 transcripts were also detected in in vitro differentiated EVTs.
Immunohistochemistry (IHC) staining of products of conception (POCs) from human first-trimester pregnancies showed that HLA-G+ EVTs expressed B7-H4 protein. Multiplex IHC staining revealed concurrent expression of B7-H4 and HLA-G in EVTs. The trophoblast cell line JEG-3 (a human placental choriocarcinoma cell line) also expressed both HLA-G and B7-H4. VCTs and SCT expressed varying levels of B7-H4 protein. Consistent with previous reports, amnion epithelial cells also expressed B7-H4, providing an additional B7-H4+ cell type at the human maternal-fetal interface.
Conclusions
To summarize, immune tolerance is crucial for both cancer progression and resistance to ICB, as well as for successful fetal development during pregnancy. Several immunosuppressive pathways, such as PD-L1, HLA-G, Indoleamine 2,3-Dioxygenase (IDO), and Tregs, are shared between the TME and the placenta.
This study identifies B7-H4 as a previously unrecognized onco-fetal immune tolerance checkpoint driven by progesterone signaling. B7-H4 supports local immune suppression in the TME and at the maternal-fetal interface, ensuring these sites remain immune-privileged during cancer progression and pregnancy. This molecular regulation contributes to a broader cellular immunosuppressive network, which is crucial for immune tolerance in both contexts.
