Source: National Cancer Institute; creator Rhoda Baer, photographer

Scientists at Dana-Farber Cancer Institute, Massachusetts General Hospital, and the Broad Institute of MIT and Harvard, reported the discovery of a potential new target for immunotherapy of malignant brain tumors. The team used single-cell RNA sequencing (RNA-seq) to study gene expression and the clonal landscape of tumor-infiltrating T cells across more than 30 patients with diffuse gliomas and glioblastoma. Their work indicated that the immune cell surface receptor, CD161, suppresses the cancer-fighting activity of immune T cells, and is activated by its ligand, CLEC2D, on tumor cells and immune-suppressing cells in the brain. Their studies showed that genetically deleting the KLRB1 that codes for CD161, or using antibodies to block CD161, increased T cell-mediated killing of glioma cells in vitro and anti-tumor function in vivo.

The studies also indicated that the same pathway may be pertinent to other forms of cancer. “… we showed that this pathway is also relevant in a number of other major human cancer types,” including melanoma, lung, colon, and liver cancer, said Kai Wucherpfennig, MD, PhD, director of the Center for Cancer Immunotherapy Research at Dana-Farber. Wucherpfennig is corresponding author of the report along with Mario Suva, MD, PhD, of Massachusetts General Hospital; Aviv Regev, PhD, of the Broad Institute, and David Reardon, MD, clinical director of the Center for Neuro-Oncology at Dana-Farber.

The team described its work in Cell, in a paper titled, “Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis.” They concluded: “Our comprehensive atlas of T cell expression programs across the major classes of diffuse gliomas thus identifies the CD161-CLEC2D pathway as a potential target for immunotherapy of diffuse gliomas and other human cancers.”

Immunotherapy drugs against cancer are designed to disable immune checkpoints, effectively the brakes exploited by cancer cells to suppress the body’s defensive response by T cells against tumors. Disabling these checkpoints unleashes the immune system to attack cancer cells, but such checkpoint inhibitors aren’t effective against all cancers. One of the most frequently targeted checkpoints is PD-1, but recent trials of drugs that target PD-1 in glioblastomas (GBM) have failed to benefit patients. “Although blockade of the inhibitory PD-1 and/or CTLA-4 receptors represent major therapeutic advances in several cancer types, a recent Phase III clinical trial of anti-PD-1 therapy failed to demonstrate benefit in recurrent GBM patients,” the authors wrote.

Gliomas are brain cancers that include glioblastoma, the most aggressive and incurable type of brain tumor. Prior to the newly reported study, little was known about the expression of genes and the molecular circuits of immune T cells that infiltrate glioma tumors, but fail to halt their growth. “T cells are critical effectors of cancer immunotherapies, but little is known about their gene expression programs in diffuse gliomas,” the investigators stated.

Kai Wucherpfennig, MD, PhD, Dana-Farber Cancer Institute [Dana-Farber Cancer Institute]
To open a window on these T cell circuits, the investigators applied single-cell RNA-seq technology to glioma-infiltrating T cells from fresh tumor samples from 31 patients. They used the resulting analyses to create an “atlas” of pathways that regulate T cell function. They then used this T cell atlas to identify potentially new pathways regulating T cell function in diffuse gliomas. They focused on subsets of cytotoxic glioma-infiltrating T cells that co-expressed natural killer (NK) cell genes, which may play a role in anti-tumor immunity. In analyzing the RNA-seq data, the researchers identified the CD161 protein, encoded by the KLRB1 gene, as a potential inhibitory receptor. “These analyses highlighted the NK gene KLRB1 (encoding CD161) as a potential inhibitory receptor,” they noted. “CLEC2D, the ligand for CD161, is a surface molecule expressed by myeloid cells and malignant cells, suggesting a ligand-receptor pathway for immunotherapy.”

To determine if blocking the CD161 pathway could restore the T cells’ ability to attack the glioma cells, the researchers disabled it either by knocking out the KLRB1 that codes for CD161, or by the use of antibodies to block the CD161-CLEC2D pathway. In an animal model of gliomas, this strategy strongly enhanced the killing of tumor cells by T cells, and improved survival. The researchers were also encouraged by the finding that blocking the inhibitory pathway appeared to reduce T-cell exhaustion—a loss of cell-killing function in T cells that has represented a major hurdle in immunotherapy.

The scientists also found that fewer T cells from gliomas contained PD-1 than CD161. As a result, they said, “CD161 may represent an attractive target, as it is a cell surface molecule expressed by both CD8 and CD4 T cell subsets [the two types of T cells involved in response against tumor cells] and a larger fraction of T cells express CD161 than the PD-1 protein.” They used CRISPR/Cas9 gene-editing technology to inactivate the KLRB1 gene in T cells and showed that CD161 inhibits the tumor cell-killing function of T cells.

“Our comprehensive atlas of T cell expression programs across the major classes of diffuse gliomas thus identifies the CD161-CLEC2D pathway as a potential target for immunotherapy of diffuse gliomas and other human cancers,” the authors commented. “The identification of largely similar subsets of T cells across different classes of diffuse gliomas suggests that common strategies could be leveraged for T cell-mediated immunity.”

They carried out tests in two different animal models that were created by implanting “gliomaspheres”—3-dimensional clusters of tumor cells from human patients—into rodents, which developed aggressive tumors that invaded the brain. The scientists subsequently injected T cells with the KLRB1 gene edited out into the cerebrospinal fluid of some of the animals, and T cells that hadn’t had the KLRB1 gene deleted. Transfer of the gene-edited T cells slowed the growth of the tumors and “conferred a significant survival benefit,” in both of the animal models of gliomas, the scientists said.

Their studies also found that KLRB1 and its associated transcriptional program are expressed by T cell populations in other human cancers. “The significance of these findings may extend beyond diffuse gliomas, as illustrated by expression of KLRB1 in tumor-infiltrating T cells from four other common human cancers,” they concluded. “Our study highlights the significance of ‘‘NK cell’’ receptors that are expressed by tumor-infiltrating T cells that possess diverse TCR repertoires … Inhibitory and activating NK cell receptors expressed by tumor-infiltrating T cells may offer opportunities for

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