Immu-43. in-depth single-cell analysis of the immune landscape in chordomas reveals t-cell exhaustion and informs rational immunotherapeutic intervention

Abstract Chordomas are rare tumors that arise from remnants of the notochord. The standard of care for chordoma remains only surgery and radiotherapy. Though checkpoint inhibition therapy shows promise, recent immunotherapy trials failed to reach endpoints. This emphasizes the need to comprehensively understand the tumor and peripheral immune landscape of chordomas to establish a rationale for immunotherapeutic intervention. Here, we aimed to investigate the composition and functionality of the tumor and peripheral immunological landscape of chordomas using single-cell RNA-sequencing, TCR profiling, and multiplex immunofluorescence. We generated a comprehensive single-cell atlas of chordomas incorporating paired single-cell transcriptomic samples from the tumor and blood of five patients and single-cell TCR sequencing data to analyze the T-cell clonality. Furthermore, we analyzed and quantified the immune cell composition (CD3+, CD4+, CD8+, CD20+, and CD163+ cells) of 33 chordomas (14 clival and 19 spinal) through multiplex immunofluorescence. Our findings revealed that the CD8+ T-cell antitumoral immunity originates in the periphery, characterized by clonal expansion of cytotoxic and memory T cells in the blood. However, this immunity undergoes progressive exhaustion following tumor infiltration. Furthermore, we observed that CTLA-expressing central memory and Tregs constitute most of the CD4 T-cell tumoral landscape. Within the B cell compartment, regulatory B cells and plasma cells were the most abundant. Hierarchical clustering analysis of 33 chordomas identified 3 distinct clusters based on the relative abundance of T cells, B cells, and macrophages. Notably, a higher abundance of CD3+ T cells was found in newly diagnosed chordomas as opposed to recurrent cases. Our study provides crucial insights into the complex immune landscape of chordoma. The results of this work may be of foundational importance to understanding chordoma immunology and tailoring immunotherapeutic strategies for these tumors.