Integrative multi-omics identifies regulatory and exhausted T cell types and novel immunotherapy targets in CLL lymph nodes

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) using 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 their differentiation state trajectories. In comparison to blood and bone marrow where T cell phenotypes were similar, T cells in CLL lymph nodes were most distinct. We identified a disease-specific accumulation of regulatory T cell subsets and CD8 + T cells harboring different stages of exhaustion, including precursor exhausted T cells (T PEX ) and terminally exhausted (T EX ) exclusively in the CLL lymph node tissue. Integration of T cell receptor sequencing data revealed a clonal expansion of T PEX , suggesting their reactivity for CLL cells. Interactome analyses identified novel potential immunotherapy targets for CLL, including the TIM3 ligand Galectin-9. Targeting Galectin-9 slowed down disease development and reduced the number of TIM3 expressing T cells in a CLL mouse model. Galectin-9 expression correlated with shorter survival of patients with CLL, renal cell carcinoma or glioma. It therefore likely contributes to cancer immune escape and represents a novel target for immunotherapy.