The inhibition of the CD161-CLEC2D interaction as immune checkpoint blockade in glioblastoma multiforme

• Applicant: Dr. Sascha Marx
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Immunology ; Clinical Neurology; Neurosurgery and Neuroradiology
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 419826340

Glioblastoma multiforme (GBM) is the most common brain tumor in adults. Despite standard treatment the mean overall survival is frequently only a little over 1 year. Immunotherapy is not as well developed in GBM compared to other malignancies outside the central nervous system (CNS), because there is lack of knowledge about immunological processes in GBM. The goal of the present project is to close gaps in the understanding of T-cell function in GBM. Preliminary single cell RNA-sequencing data could identify clonally expanded T cell populations based on their TCR chain sequences. In this analysis it was seen that the CD161 (KLRB1) is overexpressed by such T-cells. The CD161 ligand, CLEC2D, is expressed at the cell surface of human GBM cell lines. CD161 is expressed by CD4+ und CD8+ T-cells with an effector gene expression and inactivation of the KLRB1 gene in primary human T cells greatly enhances their effector function in a humanized mouse model of GBM. It is therefore hypothesized that the CD161 – CLEC2D pathway inhibits the anti-tumor function of both CD8 and CD4 effector T cell populations in GBM.The current project aims to study the CD161-CLEC2D interaction in human GBM in more detail to accelerate a translation into the clinic. Initially a genetic approach will be used to study this inhibitory receptor – ligand pair by inactivating the KLRB1 gene in primary T-cells or the CLEC2D gene in GBM cells. Blocking monoclonal antibodies specific for human CD161 and CLEC2D will also be generated to examine the therapeutic potential of antibody-mediated inhibition of this pathway. In vivo experiments will be conducted mainly in humanized mouse models, in which human T-cells (with GBM-specific CAR or TCR) will be injected in immunodeficient mice after stereotactic implantation of GBM-cells into the CNS. Furthermore, the therapeutic potential will be tested in fully immunocompetent mice harboring GBM. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing approaches will be used to define the functional changes in T cell populations when the CD161 – CLEC2D pathway is inhibited.Furthermore analysis of tumor-infiltrating T cells in GBM patients enrolled in a clinical trial (personalized neoantigen peptide vaccination plus PD-1 blockade) will be done. Tumors both at initial surgery and relapse will be investigated in order to determine how expression of these inhibitory receptors and their ligands changes following therapy. CD161 expression in T-cells (as well as other inhibitory receptors) and CLEC2D on tumor cells and myeloid cells (as well as other inhibitory ligands) will be addressed, given that T-cell exhaustion is accompanied with expression of these receptors and ligands. It is hypothesized that CD161 is an important inhibitory receptor for intra-tumoral T-cells in GBM and that inhibition of the CD161-CLEC2D pathway is able to enhance T-cell effector function in GBM.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Dr. Kai Wucherpfennig