(C-119) Ceramide is transferred from artificial antigen-presenting cells to T-cells via trogocytosis

Immune therapy with adoptive T-cell transfer is gaining ground as a potent cancer-directed treatment and requires highly functional antigen-specific T cells. A biomaterial-based system was previously developed in our lab, termed APC-mimetic scaffolds (APCms). The material consists of mesoporous silica rods coated with lipid bilayers, that are functionalized to engage and activate T-cells. The APCms system mediates rapid and controlled T-cell expansion and can be utilized for ex-vivo manipulation of T-cells for adoptive cell therapy. 

Trogocytosis is a process in which one cell acquires a membrane fraction of another cell in a contact-dependent manner. This phenomenon enables recipient cells to gain novel functions by acquiring donor-cell molecules. In the immune system, trogocytosis was shown to play important roles, such as the transfer of pMHC molecules from antigen-presenting cells (APC)  to B and T-cells, providing them with antigen-presentation abilities. 

Previous data gathered in our lab suggests that trogocytosis can occur between APCms and T-cells, enabling the transfer of membrane-bound molecules between the biomaterial and the T-cell. It is unclear, however, whether lipids can be transferred between APCms and T-cells by trogocytosis. 

Ceramide is a signaling lipid involved in receptor clustering and has been shown to enhance T-cell functions. The aim of this study was to investigate whether transfer of ceramide from APCms to T-cells can occur via trogocytosis. If trogocytosis of lipids were observed, it might be utilized as a novel method to transfer molecular payload to T-cells, in order to improve their phenotype and fitness for adoptive cell therapy.

The methods employed in the study were aimed to track the transfer of fluorescently-labeled ceramide from APCms to T-cells using confocal microscopy.  

APCms were fabricated as previously described and functionalized with activating anti-CD3 and anti-CD28 antibodies. The experimental group consisted of APCms fabricated with NBD-labeled ceramide in their lipid bilayer. Control groups consisted of APCms formulations lacking ceramide labeling or lacking ceramide altogether. Murine CD8 T cells were negatively selected from splenocytes using magnetic beads. After isolation, T-cells were incubated with the different formulations of APCms in a 48-well plate, in the presence of myogenic cytokines (e.g., interleukine-7 and interleukine-15).  Following  24-hour incubation, T-cells were partially separated from the APCms and stained with Alexa Fluor 647 anti-CD3 antibody. Cells were then fixed and stained with DAPI for nuclear stain. Cell preparations were imaged on Zeiss LSM 710 confocal microscope in x100 magnification. Images were analyzed by IMARIS software. The cellular distribution of ceramide was assessed relative to controls.

Analysis of confocal images demonstrated the presence of NBD-labeled ceramide in T-cells. Two distinct patterns of ceramide distribution in the T-cell were observed: In isolated cells and cells interacting with APCms, ceramide was distributed evenly in the cell membrane, where it was co-localized with CD3. Some ceramide was also identified in the cytoplasm. In contrast, in T-cells that were in contact with one another, ceramide was condensed to a small area of the membrane, in close proximity to the cellular-adhesion area. In these cases, it was also co-localized with CD3.
Intriguingly, CD3 signal was also detected on the APCms, likely indicating membrane transfer from T-cells to APCms. 

Our results demonstrate that ceramide molecules can be transferred from artificial antigen-presenting cells to T-cells by trogocytosis. Moreover, the presence of CD3 receptors on the APCms suggests that similarly to trogocytosis between two native cells, trogocytosis between T-cells and the biomaterial is also bi-directional. Adoptive T-cell therapy requires highly specific T-cells that are functionally fit to perform robust cancer-cell killing. To this end, efforts are being made to achieve optimal T-cell phenotype during ex-vivo T-cell manipulations. One such approach could be the enrichment of T-cells with bioactive lipids. Our study focused on ceramide since it is involved in receptor clustering, and can promote T-cell activation and tumor control. Our results demonstrate that the enrichment of T-cells with ceramide could be achieved using trogocytosis, which enables the trafficking of ceramide between the biomaterial and T-cells. These results might open a window of opportunity for harnessing trogocytosis in ex-vivo cell manipulation with biomaterials to improve the fitness of T-cells and optimize tumor control.