Shared Molecular Mechanism in Cancer and Pregnancy: Suppressing Immune System

Researchers clarified the immune tolerance mechanisms common to both pregnancy and cancer in a recent study published in Cell. They specifically examined the function 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 fetal cells and cancer).

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Background

Immune checkpoint blockage (ICB) produces long-lasting effects on a variety of cancer types. Nevertheless, because of important immune-regulatory systems that support suppressive networks in the tumor microenvironment (TME), the majority of patients do not react to current immunotherapy. Cancer immune evasion and ICB resistance result from this.

A perfect model to study immunological tolerance, pregnancy shares several immunosuppressive mechanisms with cancer, such as Indoleamine 2,3-Dioxygenase (IDO), Human Leukocyte Antigen-G (HLA-G), and Programmed Death-Ligand 1 (PD-L1). To completely comprehend the processes and therapeutic possibilities of targeting B7-H4 in immunological tolerance associated to pregnancy and cancer, more research is required.

About the Study

In the animal research, 7,12-Dimethylbenz[a]anthracene and medroxyprogesterone acetate (MPA) were used to create a spontaneous tumor model.

(DMBA). Age-matched female B7-H4−/− and Wild Type (WT) mice were subcutaneously implanted with a pellet containing slow-releasing MPA. For six doses, mice received one milligram of DMBA once a week via oral gavage.

Evaluable tumor counts were noted, and tumor growth was tracked. Using a caliper, the tumor’s measurements were determined, and the tumor’s area was computed. Both overall survival and tumor-free status were tracked. Primary cells were extracted from tumor nodules and subcutaneously injected into WT mice for in vivo passage in order to create transplantable tumors. These cells were utilized to create subcutaneous transplant tumors in order to create therapeutic models for anti-PD-L1, BD-9136, or mifepristone (RU486).

The tumor-free and overall survival of the mice were observed after they were randomly assigned to treatment groups. RU486 was given to WT mice in order to study B7-H4 expression on mouse endometrial epithelial cells (MEECs), and the uteri were then harvested for cell isolation.

Female C57BL/6 (WT) and B7-H4−/− mice were used in pregnancy models, and for allogeneic and syngeneic mating, respectively, male Bagg Albino, C strain (BALB/c), a common inbred strain of laboratory mouse, and C57BL/6 mice were mated. At gestational day 13.5, pregnant mice were put to death, and the number of fetuses overall and those that had been resorbed was noted. The rate of fetal resorption was computed. Using certain antibodies, CD4 and CD8 T cells—lymphocytes that recognize and eliminate malignant or infectious cells—were reduced, and the effectiveness of the depletion process was verified using flow cytometry. Pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were administered to WT and B7-H4−/− female mice, who were then mated with their respective males and given two-cell embryos to be transferred to recipient mice. Results of pregnancies were assessed at either gestational day 13.5 or 14.5.

Study findings
Fourteen single-cell RNA-sequencing (scRNA-seq) datasets from different TME and placenta were cross-analyzed, with an emphasis on the “non-self” cellular components of tumor and trophoblast cells, in order to investigate hitherto unidentified onco-fetal immune tolerance mechanisms. The major histocompatibility complex (MHC) and co-accessory molecules play a significant role in the immune response; therefore, the expression patterns of MHC, important members of the B7 family, and TNF family members were evaluated.

The analysis validated important genes that were previously reported to be expressed in trophoblast and tumor cells. For example, there was high expression of HLA-G in placental trophoblast cells and high expression of Fibrinogen-like protein 1 (FGL1) in tumor cells. It’s interesting to note that VTCN1, or B7-H4 V-Set Domain Containing T Cell Activation Inhibitor 1, was found to be the most highly expressed gene in both placental trophoblast cells and tumor cells in the TME. Thus, the main cellular components of the placenta and TME both express B7-H4 transcripts.

The analysis was expanded to analyze the expression patterns of B7-H4 and PD-L1 (B7-H1, CD274) using the Tumor Immune Single-cell Hub 2 (TISCH2) database. Tumor cells were the main cells that expressed B7-H4, but myeloid cells and other cell types expressed PD-L1 extensively. Breast and ovarian cancers were among the several cancer kinds that had the greatest levels of B7-H4 transcripts. These malignancies originated from female reproductive organs.

The largest quantities of B7-H4 transcripts were found in breast, ovarian, and endometrial malignancies, according to this analysis of scRNA-seq data that was linked with bulk RNA-seq data from The Cancer Genome Atlas (TCGA). Breast and endometrial malignancies were shown to have high quantities of B7-H4 protein, but cervical cancer had moderate levels. Therefore, B7-H4 proteins and RNAs are strongly expressed in all types of gynecological malignancies.

The majority of B7-H4 expression at the maternal-fetal contact was seen in trophoblast cells. There are several morphologically and functionally distinct subsets of human trophoblast cells, such as syncytiotrophoblast (SCT), villous cytotrophoblast cells (VCTs), and extravillous trophoblast cells (EVTs). In human first-trimester pregnancies, trophoblast subsets were examined for the expression of B7-H4 transcripts, a crucial stage for building maternal-fetal immunological tolerance. EVTs were the primary cells to express HLA-G, SCT and EVTs were the primary cells to express CD274, and all three trophoblast cell subsets expressed VTCN1. Additionally, transcripts for VTCN1 were found in vitro differentiated EVTs.

Products of conception (POCs) from human first-trimester pregnancies stained with immunohistochemistry (IHC) revealed that B7-H4 protein was expressed by HLA-G+ EVTs. B7-H4 and HLA-G were found to be expressed concurrently in EVTs by multiplex IHC staining. HLA-G and B7-H4 were also expressed by the human placental choriocarcinoma cell line JEG-3, which is a trophoblast cell line. B7-H4 protein was expressed at different amounts by VCTs and SCTs. Amnion epithelial cells expressed B7-H4 as well, which is consistent with other results and adds another B7-H4+ cell type at the human maternal-fetal interface.

In conclusion
In summary, immunological tolerance plays a critical role in the development of a healthy fetus during pregnancy, the advancement of cancer, and resistance to ICB. The placenta and the TME share a number of immunosuppressive mechanisms, including PD-L1, HLA-G, Indoleamine 2,3-Dioxygenase (IDO), and Tregs.

This work identified B7-H4 as a progesterone-driven onco-fetal immune tolerance checkpoint that was previously unknown. In order to maintain the immune-privileged status of the TME and the maternal-fetal interface throughout the course of cancer and pregnancy, B7-H4 promotes local immune suppression in these areas. Immune tolerance in both situations depends on a larger cellular immunosuppressive network, which is facilitated by this molecular control.

For more information: Progestogen-driven B7-H4 contributes to onco-fetal immune tolerance, Cell, https://doi.org/10.1016/j.cell.2024.06.012