Background In the context of Chimeric Antigen Receptor T-cell (CAR-T) therapy, understanding the dynamic interactions between CAR-T cells and target cells is essential for optimizing treatment efficacy. Analysis of CAR-T cell interaction with dimly expressed fluorescent protein in target cells presents a major compensation challenge especially amidst high numbers of non-target cells. Further, very short and transient interaction of the CAR-T cell with the target cell, makes it more challenging to observe and characterize various CAR-T cell candidates for cell-cell interactions.

Methods Ramos cells were transfected with mCherry fluorescent protein tagged target and was sorted using a BigFoot^TMSpectral Cell Sorter assess target protein expression fidelity. They were then co-cultured with Cyan Fluorescent Protein (CFP) labelled non-target U2OS cells. The ability of CellTrace^TM Far Red dye labelled CAR-T cells to target and interact with low frequency mCherry positive Ramos Cells in the presence of CFP labelled U2OS was studied at different timepoints and with different CAR-T:Ramos cell ratios. CellEvent^TM Caspase 3/7 AF488 and SYTOX AADvanced^TM ReadyFlow^TM reagents were used to identify target cells in early apoptosis (AF488) and late apoptosis/dead (AAD), respectively. BrightComp eBeads^TM compensation beads in CFP and mCherry were used to compensate the fluorescent proteins and were analysed using an Attune^TM CytPix^TM imaging cytometer. Automated image analysis parameters were set to capture Ramos and CAR-T cell doublets and to eliminate random doublets from true interactions.

Results In the absence of a distinct cell-based compensation control, the BrightComp eBeads were able to clearly distinguish mCherry and YFP labelled cells from the other fluorescent parameters. Increased Caspase-3/7 AF488 signal in only the mCherry positive cells indicated a time-dependent cell-mediated cytotoxicity. The high-resolution images in conjunction with flow cytometry data and enabled the simultaneous characterization of various cellular parameters, including cell morphology, fluorescent protein expression and apoptosis progression. Although apoptosis increased overtime, the frequency of true CAR-T and Target cell interaction did not change with time, however, these interactions were greatly altered by altering the CAR-T:target ratios.

Conclusions This comprehensive methodology, employed in our study aims to facilitate a gain of deeper understanding of CAR-T cell and target cell interactions, particularly in terms of apoptosis induction. The findings from this study can contribute to the optimization and development of CAR-T cell therapy approaches for improved treatment outcomes.

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