P014 | CAR-T CELLS FROM NON-HODGKIN LYMPHOMAS PATIENTS SECRETE FACTORS ABLE TO ACTIVATE THE MONOCYTIC-MACROPHAGIC TRANSITION: AN IN VITRO MODEL

The biological mechanisms of toxicity associated with Chimeric Antigen Receptor-T (CAR-T) cell therapy and the cells involved in it are not yet fully elucidated. Among these, the monocyte-macrophage axis appears to play a key role. We investigate the role of extracellular vesicles (EVs) and secretome released by CAR-T cells in promoting the monocyte-to-macrophage transition using the monocytic cell line THP-1. CAR-T cells (axi-cel), isolated from infusion bags of patients with DLBCL or follicular lymphoma that had grade 1-2 CRS, were co-cultured for 1 hour with a CD19+ lymphoma cell line, SU-DHL-8, to activate CAR-T cells and stimulate EVs release. After being isolated from the culture medium, the EVs were administered to THP-1 cells for 1 and 3 hours to evaluate their effect on monocytes. Part of the complete medium was also administered to assess the contribution of the total secretome. The activation of monocytes into macrophages was evaluated by morphological and molecular analysis. Treatment with EVs and secretome induced morphological changes in THP-1 cells. Untreated cells maintained a round shape with an intact membrane, preserving their peculiarities during culture. However, when treated, they began to adopt a more elongated and irregular shape. These cells appeared rich in cytoplasmic granules and exhibited an altered membrane structure (Fig. 1A). Molecular analyses based on the quantification of the PPM1A transcript, a macrophage-specific marker, confirmed the activation of monocytes to macrophages. This effect is increased when stimulated CAR-T cells-derived EVs are investigated (Fig. 1B), while it’s not appreciable in non-stimulated ones (Fig. 1C). dPCR evaluation of the Ki67 transcript, a proliferation marker, showed a partial reduction in transcript levels across all conditions, appreciable in cells treated with EVs and the secretome after 1 hour and with secretome after 3 hours. These differences were not statistically significant. The same trend was confirmed by CCK8 assay. This result suggests that the cells are transitioning into macrophages rather than proliferating. Even though the selected patients had a low-grade CRS, their CAR-T cells, upon activation via CD19 antigen recognition, released factors capable of promoting the transition without altering the metabolic activity of the cells. For the first time, these results suggest that CAR-T cells partially engage other immune cells by secreting specific factors.