Evaluating Plasma Extracellular Vesicles As Biomarkers of Inflammation and Outcome in Diffuse Large B-Cell Lymphoma Patients Undergoing CAR-T Therapy
Authors: Muntion Sandra , Navarro-Bailon Almudena , Gomez-Redondo Martin , Preciado Silvia , López-Corral Lucía , Lopez Parra Miriam , Villaron Eva M , Martin Lopez Ana Africa , Pérez López Estefanía , Sanchez-Guijo Fermin 
Published: December 2024

Introduction: Extracellular vesicles (EVs) are nanoparticles released by eukaryotic cells and capable of transferring microRNAs, mRNAs and proteins to neighboring cells modifying their functionality. Their potential as biomarkers in certain diseases is being evaluated, although their role in the context of CAR-T therapy has not yet been extensively studied.
Aim: To isolate and multiparametrically characterize circulating EVs from patients with diffuse large B-cell lymphoma (DLBCL) treated with CAR-T cells and to identify their role as biomarkers of response and inflammatory complications (e.g. cytokine-release syndrome-CSR-). In addition, to evaluate in vitro the effect of plasma EVs from patients who developed CRS on macrophages, as key mediators of post-CAR-T inflammatory events.
Methods: EVs were isolated from peripheral blood plasma (PB) of 21 patients with DLBCL undergoing CAR-T therapy at three time points (pre-infusion, day +7 and day +28). Quantification and characterization of EVs was performed using the novel ExoView® technology (NanoView Biosciences) and transmission electron microscopy (TEM). EVs' microRNA content was studied by sequencing using QIAseq® miRNA Library from QIAGEN For Illumina® NGS. Circulating cytokines at the three time points were studied by Luminex®. For the study of the inflammatory response, monocytes (CD14+) were isolated from buffy coats and differentiated to macrophages with GM-CSF for 5 days. After that, these cells were activated with EVs from plasma of the patients obtained at the time of CRS. After 48h, the immunophenotypic profile (M1/M2) of the macrophages was studied by flow cytometry (FC) and their cytokines by Luminex.
Results: By ExoView®, we confirmed that EVs were smaller than 1mm and expressed specific tetraspanines (CD63, CD81 and CD9) in all cases. The presence or absence of the CAR receptor on EVs was found to be related to treatment response. The day +7 was the time-point with the highest concentration of circulating EVs in plasma. The content of EVs in microRNAs (Responders n=11 vs Progression n=10) showed over-expression of 19, 10 and 14 microRNAs at the respective time points (0, +7, +28). Expression of hsa-miR-27a was significantly downregulated in plasma samples from patients with DLBCL who achieve complete response compared to those who progressed. In studies of the inflammatory response after co-culture of macrophages with EVs -CRS, we observed that macrophages had a predominantly M2 phenotype, which was more heterogeneous in progressing patients. In addition, EVs from progressing patients who developed grade II CRS elicited a significantly higher proportion of proinflammatory macrophages compared to those who developed grade I CRS.
Conclusion: Circulating EVs increase in patients with DLBCL after treatment with CAR T-cell therapy and there are differences in their content of microRNAs in responding patients compared to those who progress. EVs may be important mediators in macrophage responses after CRS.
Funding: Partially supported by competitive grants GLD21/00162 from Gilead, GRS2393/A/21 from Gerencia Regional de Salud de Castilla y León and IFEQ22/00125 from Instituto de Salud Carlos III (ISCIII) from European Recovery, Transformation and Resilience Plan