T Cell Landscape Definition by Multi-Omics Identifies Galectin-9 as Novel Immunotherapy Target in Chronic Lymphocytic Leukemia

Failure of immunotherapy after applying checkpoint inhibitors or CAR-T cells is linked to T cell exhaustion. Here, we explored the T cell landscape in chronic lymphocytic leukemia (CLL) by single-cell omics analyses of blood, bone marrow and lymph node samples of patients and spleen samples of a CLL mouse model. By single-cell RNA-sequencing, mass cytometry (CyTOF), and multiplex image analysis of tissue microarrays, we defined the spectrum of phenotypes and transcriptional programs of T cells and and their differentiation state trajectories. We identified disease-specific accumulation of distinct regulatory T cell subsets and T cells harboring an exhausted phenotype exclusively in the CLL lymph node tissue. Integration of TCR data revealed a clonal expansion of CD8⁺ precursor exhausted T cells, suggesting their reactivity for CLL cells. Interactome analyses identified the TIM3 ligand Galectin-9 as novel immunoregulatory molecule in CLL. Blocking of Galectin-9 in CLL-bearing mice slowed down disease development and reduced the number of TIM3 expressing T cells. Galectin-9 expression correlated with shorter survival of CLL patients. Thus, Galectin-9 contributes to immune escape in CLL and represents a novel target for immunotherapy.

Note:

Funding Information: This study was supported by a research grant of the German Cancer Aid (Deutsche Krebshilfe) to MS, by grants from FNRS-Télévie to IFB (7.4529.19, 7.6603.21), MW (7.6504.18), GP (7.4501.18, 7.6518.20), SG (7.4502.19, 7.6604.21), and from the Luxembourg National Research Fund (FNR), Fondation Cancer and Plooschter Projet to EM and JP (C20/BM/14582635, and C20/BM/14592342). The authors acknowledge support by the state of Baden-Württemberg through bwHPC and the German Research Foundation (DFG) through grant INST 35/1597-1 FUGG. MC, KB and DS are supported by the German Federal Ministry of Education and Research (BMBF 01ZZ2004).

Declaration of Interests: None to declare.

Ethics Approval Statement: Tissue microarrays and lymph node sections were provided by the Tissue Bank of the National Center for Tumor Diseases (NCT) Heidelberg, Germany in accordance with the regulations of the tissue bank and the approval of the ethics committee of Heidelberg University.

Samples from patients and heathy controls were obtained after informed consent and according to the guidelines of the Ethics Committees of the involved University hospitals and the Declaration of Helsinki.

All experiments involving laboratory animals were conducted in pathogen-free animal facilities at the German Cancer Research Center in Heidelberg or the Luxembourg Institute of Health in Luxembourg with the approval of the Regierungspräsidium Karlruhe (G-77/19 and G-112/21) and the Luxembourg Ministry for Agriculture (#LUPA 2019/21), respectively. Mice were treated in accordance with the European guidelines.

Keywords: CLL; single-cell RNA-seq; mass cytometry; T cell exhaustion; tumor microenvironment; Galectin-9; TIM3

Suggested Citation: Suggested Citation

Llao Cid, Laura and Wong, John K.L. and Fernandez Botana, Iria and Paul, Yashna and Wierz, Marina and Flörchinger, Alessia and Gonder, Susanne and Pagano, Giulia and Chazotte, Margot and Bestak, Kresimir and Schifflers, Christoph and Iskar, Murat and Roider, Tobias and Mallm, Jan-Philipp and Cosma, Antonio and Campton, DE and Gerhard-Hartmann, Elena and Rosenwald, Andreas and Colomer, Dolors and Campo, Elias and Schapiro, Denis and Dietrich, Sascha and Lichter, Peter and Moussay, Etienne and Paggetti, Jérôme and Zapatka, Marc and Seiffert, Martina, T Cell Landscape Definition by Multi-Omics Identifies Galectin-9 as Novel Immunotherapy Target in Chronic Lymphocytic Leukemia. Available at SSRN: https://ssrn.com/abstract=4314156

This version of the paper has not been formally peer reviewed.