SMALL EXTRACELLULAR VESICLES IN THE LEUKEMIA MICROENVIRONMENT SUSTAIN CLL PROGRESSION BY HAMPERING T CELL-MEDIATED ANTI-TUMOR IMMUNITY

GARGIULO, ERNESTO; ELODIE VIRY; MORANDE PABLO; LARGEOT, ANNE; SUSANNE GONDER; FENG XIAN; NIKOLAOS IONNOU; MOHANED BENZARTI; FELIX KLEINE BORGMANN; MICHEL MITTELBRONN; GUNNAR DITTMAR ; PETR V. NAZAROV; JOHANNES MEISER; BASILE STAMATOPOULOS; ALAN G. RAMSAY; ETIENNE MOUSSAY; JEROME PAGGETTI

Viena

Congreso; EHA congress 2022; 2022

EHA

Background: Small extracellular vesicles (sEV) are nano-sized particles released by every cell and found in all biofluids.Given their composition and abundance, sEV are commonly involved in cell-to-cell communication through the transfer ofgenetic material and proteins. Furthermore, sEV possess direct functions carried out by sEV-ligands capable to affect thebiological functions of targeted cells. In cancer, tumor-derived sEV are involved in the re-education of microenvironment(ME) cells promoting tumor proliferation, immune escape and metastasis. We previously demonstrated that leukemiaderivedsEV are involved in the re-education of surrounding cells and increased immune escape. Indeed, chroniclymphocytic leukemia (CLL)-derived sEV induce stromal cell conversion into cancer-associated fibroblasts (Paggetti et al., Blood, 2015), and modulate PD-L1 expression in monocytes (Haderk et al. Science Immunology, 2017).Aims: The goal of the present work was to characterize leukemia ME-derived sEV (LME-sEV) and to evaluate their rolein the disease development and progression in vivo.Methods: To obtain a biological representation of sEV in CLL microenvironment, we isolated LME-sEV directly fromspleens of leukemic mice, obtaining a complex mix of sEV released by both CLL and ME cells alike. Small EVcharacterization was performed using a wide range of techniques, including qPCR, mass spectrometry and single sEVflow cytometry (FC). The effect on target cells was evaluated both ex vivo and in vivo using high-throughput techniques,FC, qPCR and cytotoxic assay. Small EV impact on CLL development in vivo was evaluated by generating a novelpreclinical mouse model in which sEV release is genetically impaired due to Rab27a/b knock-out. Finally, we analyzedthe expression of sEV-related genes in a cohort of 144 CLL patients using qPCR followed by regression analysis.Results: LME-sEV showed a distinct proteome (A) and RNA contents compared to healthy counterparts (HCME-sEV),including miRNA enriched in the plasma of CLL patients. Furthermore, FC-based immune checkpoint (ICP) screeningshowed the presence of multiple ICP ligands anchored on CLL-derived sEV (CD20+ subset of LME-sEV) (B), while highexpression of the corresponding ICP receptors was found on T cells from matching LME. We also found that LME-sEVare internalized by different T cell subsets, thus we performed in vivo and ex vivo functional studies to assess sEV impacton T cells. High-throughput analysis of cells isolated from spleens of control mice treated with LME-sEV revealedconsiderable physiological changes mainly in CD8+ T cells. Indeed, CD8+ T cells showed alterations in theirtranscriptome, proteome and metabolome leading to cell exhaustion, decreased functions and survival. In line with this,absence of sEV dramatically delayed CLL progression in vivo. This effect was due to CLL inability to escape immunesurveillance in absence of sEV and this was rescued by LME-sEV treatment (C). Finally, we identified a consistent sEVgene signatures in CLL patients correlating with treatment-free survival, overall survival, and with unfavorable clinicalparameters routinely used in CLL diagnosis and prognosis (D).