CO41 | Monocyte activation by CAR-T cells: role of extracellular vesicles and secretome in immune modulation

Abstract

Chimeric Antigen Receptor-T (CAR-T) cell therapy has transformed treatment of CD19⁺ B cell malignancies, often causing immune-related toxicities with unclear mechanisms. The monocyte-macrophage lineage appears key, but CAR-T effects on monocytes are not fully known. We investigated for the first time how CAR-T cells interact with monocytes and trigger immune activation.
CAR-T cells (axicabtagene ciloleucel, axi-cel, and tisagenlecleucel, tisa-cel) were isolated from infusion bag washes of eight non-Hodgkin Lymphoma patients with low-grade CRS/ICANS (1-2). To investigate the role of CAR-T–released particles in monocyte activation, cells were co-cultured for 1-3h with CD19⁺ SU-DHL-8 lymphoma cells. Extracellular vesicles (EVs) and the complete secretome (SEC) were collected and used to treat THP-1 monocytes for 1, 3, and 24h. Monocyte-to-macrophage transition (MMt) was analyzed morphologically and molecularly. EV uptake was assessed by fluorescence microscopy, and cytokine expression quantified by digital PCR.
Neither EVs nor SEC significantly affected THP-1 proliferation in either axi-cel or tisa-cel products. When treated for 1-3h with both EVs and SEC, monocytes adopted an irregular shape, with pseudopods formation. This aspect was more evident in cells treated with axi-cel-derived EVs and SEC (Figure 1A) and wasn’t appreciable after 24h. To assess if these changes were actually related to the monocyte’s differentiation, the PPM1A transcript was quantified 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 1-3h post-treatment. Surprisingly, cells treated with tisa-cel-derived EVs and SEC showed an opposite trend, with an increased expression after 1h of treatment with EVs and SEC, not observed after 24h. Collectively, these findings suggest that CAR-T-derived factors promote monocyte differentiation toward a macrophage-like phenotype rather than proliferation. Fluorescence microscopy confirmed EVs uptake by monocytes, indicating their key role in cells activation (Figure 1B). Cytokine analysis revealed significant over-expression of IL-8 after 1-3h with axi-cel-derived EVs and SEC, but not after 24h. In tisa-cel context just the treatment with EVs and SEC obtained after 3h of co-culture induce cytokines overexpression after 1-3h, but not after 24h (Figure 1C). TNF-α, ICAM1, and IL-1β transcripts show the same trend. No effects were observed using EVs or SEC from naive SU-DHL-8 or non-activated CAR-T cells.
For the first time, we show that CAR-T cells release factors capable of inducing MMt and transient pro-inflammatory activation. These findings highlight a key role of CAR-T-derived EVs/SEC in modulating innate immune responses. Understanding their mechanisms may help identify potential targets to modulate immune activation and improve clinical outcomes in CAR-T-treated patients.