Chimeric Antigen Receptor-T (CAR-T) cell therapy have changed the treatment paradigms in several hematological malignancies, especially in CD19+ B cell lymphomas. CAR-T are known to be accompanied by varying types and grades of immune-mediated toxicities, with the underlying mechanism is still not yet elucidate. In this context, the monocyte-macrophage lineage appears to play a significant role, even if the role of CAR-T on monocytes activation has not yet been described. For the first time we investigated the monocytes' engagement by CAR-T cells and the mechanisms triggering this immunological activation.

CAR-T cells (axicabtagene ciloleucel, axi-cel, and tisagenlecleucel, tisa-cel) were isolated from infusion bags wash of 8 patients: 4 with Diffuse Large B-Cell Lymphoma and 4 with Follicular Lymphoma, characterized by low-grade toxicities (CRS grade 1–2, ICANS grade 1). We firstly co-cultured CAR-T cells for 1h and 3h with an in vitro model of CD19+ lymphoma, SU-DHL-8. With the aim to evaluate the potential role of particles released by activated CAR-T cells in monocytes' activation, Extracellular Vesicles (EVs) released during co-culture and complete secretome (SEC) were isolated. Secondly, both of them were administered to a monocyte in vitro model (THP-1 cells) for 1, 3, and 24h to evaluate their effects. Untreated THP-1 cells served as control. The proliferation of THP-1 cells was assessed via Ki67 transcript digital PCR (dPCR) quantification. Monocyte-to-macrophage transition was evaluated by morphological and molecular analysis. Lymphoma and CAR-T derived EVs uptake was evaluated using fluorescence microscopy by differentially labeling their membrane.

Compared to untreated cells, neither EVs nor SEC significantly altered THP-1 proliferation rate in both axi-cel and tisa-cel products. Conversely, the activation of monocytes was strongly appreciable via morphological analysis. Untreated cells maintained a round shape with an intact membrane, preserving these peculiarities during cultures. However, when treated 1 or 3h with both EVs and SEC, cells adopted an elongated and irregular shape, with pseudopods formation. These cells appeared rich in cytoplasmic granules and exhibited an altered membrane structure. This phenomenon was more evident in cells treated with axi-cel-derived EVs and SEC. To assess if these changes were actually related to the monocyte's differentiation, the quantification of PPM1A transcript, a key phosphatase that coordinates this process, was performed via dPCR. EVs and SEC obtained after 1h of co-culture of axi-cel products induced PPM1A overexpression after 24h of treatment, while EVs and SEC obtained after 3h of co-culture induced the overexpression also after 1 and 3h post-treatment. Surprisingly, cells treated with activated tisa-cel-derived EVs and SEC showed an opposite trend, with a statistically significant increased expression of PPM1A after 1h of treatment with EVs and SEC obtained after 1h of co-culture, not observed after 24h. Taken together, these results suggest that the cells are transitioning into macrophages rather than proliferating. To note that the type of lymphoma resulted not influencing the data.

To confirm that these insights were EVs-driven, their uptake by monocytes was evaluated by fluorescence microscopy. The results confirmed that EVs are the key of the monocytes' activation process because of their post-treatment localization into monocytes' cytoplasm. Both CAR-T and lymphomas' EVs seems to be uptaken. To exclude that the observed effects were due to particles released by lymphoma cells, THP-1 were treated with EVs and SEC isolated from naive SU-DHL-8. No morphological and molecular differences were observed. The same negative results were obtained when monocytes were treated with EVs and SEC of non-activated CAR-T cells.For the first time, we demonstrated that CAR-T cells, upon activation via CD19 antigen recognition, release factors able to induce monocyte-to-macrophage transition and supporting the immune system engagement. These pivotal observations open to further characterization of CAR-T cells-derived EVs/SEC to identify potential targets for the modulation of the immune system's engagement and support the previously suggested (PMID: 39576958) macrophage-based interventions to improve clinical outcomes in those patients.

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2025
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