Abstract
Uncovering bispecific antibody mechanisms via 3D superresolution imaging of T cell-tumor cell membrane contacts Introduction: Bispecific antibodies (BsAbs) enable T cells to attach to cancer cells, forming specialized membrane contacts, known as the synapse, that allow T cells to exert cytotoxic effects. Despite advances in the biological design of BsAbs, the biophysical characteristics of the synapse formed between T cells and cancer cells remain poorly understood. Characterizing membrane contacts takes into consideration the unique cell shape defined by its plasma membrane, where BsAbs targets are located. Furthermore, when tumor cells occupy specific anatomical locations, the mechanisms by which T cells navigate and anchor to these cells, and how BsAbs enhance this process, remain largely unexplored. Lack of such insights confounds our understanding of why some tumor cells fail to respond to BsAbs, whereas gaining this knowledge can inform the BsAb design in addition to biological considerations. A key challenge in visualizing membrane contacts is the lack of high spatial resolution and an efficient membrane labeling method that is quick, durable, suitable for primary cells, and compatible with multiplexed imaging. To address these limitations, we employed a pan-membrane-protein labeling approach to covalently tag membrane proteins on cancer cells and murine T cells with distinct small-molecule dyes. We further characterized their membrane contacts using conventional fluorescence and volumetric superresolution imaging in the context of BsAbs.
Methods: Naive T cells isolated from BALB/c mouse spleens were pan-membrane-protein labeled using an NHS-ester-based technique. For the landing of T cells on the glass surfaces, the surfaces were prepared by incubating the surface with either poly-L-lysine (PLL), 50 ng/mL CD20xCD3 murine BsAb, or 50 ng/mL anti-mouse CD3 antibody. Labeled cells were added to the well for 30 minutes, fixed, and imaged using TIRF microscopy. For the coculture of T cells and cancer cells, murine A20 lymphoma cells and BALB/c primary T cells were pan-membrane-protein labeled with two distinct colors and cocultured for 13 hours with and without 50 ng/mL CD20xCD3 bispecific antibody. Cells were then fixed and imaged using epifluorescence and volumetric multispot structured illumination microscopy.
Results: We first used activating glass surfaces to study how BsAbs affect T cell interaction with surfaces coated with different ligands. The monovalent anti-CD3 arm of the CD20xCD3 BsAb caused similar cell spreading upon landing on the surface compared to bivalent CD3 antibodies at the same concentration. Cell spreading signals engagement with the T cell receptor and subsequent T cell activation, unlike cells landing on a PLL surface, which did not show this behavior. Next, we co-cultured lymphoma cells and primary T cells. In the absence of BsAbs, we found that T cells engaged with A20 cells through juxtaposed membrane contact. Superresolution imaging further revealed that T cells also interacted with the membrane protrusions of A20 cells. In the presence of BsAbs, more pronounced deformation of the T cell membrane was observed. Moreover, an accumulation of membrane proteins from both T and A20 cells was observed at the synapse. Superresolution imaging revealed the T cell membrane closely conforming to the A20 cell membrane, as well as protruding out to make direct contact with the A20 cell. In addition, superresolution images revealed the presence of A20 cell membrane proteins on the membrane of some T cells away from the contact site, suggesting potential trogocytic activity between the T and A20 cells. However, its implications regarding BsAbs require further investigation.
Conclusions: Our membrane labeling and imaging technique revealed a propensity of T cells to conform to A20 cancer cells, forming specialized synapses in the presence of BsAbs. The highly sensitive imaging capability also revealed the involvement of membrane processes of A20 cells in capturing T cells and the transfer of membrane proteins from A20 to T cells. Our technological advancements open new avenues for deepening our understanding of T cell behavior, cellular heterogeneity, and complex membrane topology in the context of BsAb-guided T cell cytotoxicity and potential resistance mechanisms employed by cancer cells.
