Activation and degranulation of CAR-T cells using engineered antigen-presenting cell surfaces

• Published in: PloS one 2020-09, Vol.15 (9), p.e0238819-e0238819
• Main Authors: Dirar, Qassim, Russell, Teal, Liu, Lumei, Ahn, Sarah, Dotti, Gianpietro, Aravamudhan, Shyam, Conforti, Laura, Yun, Yeoheung
• Format: Article
• Language: English
• Subjects: Adoptive Transfer - methods; Antibodies; Antigen presenting cells; Antigen-Presenting Cells - immunology; Antigens; Antigens, CD19 - metabolism; Antigens, Surface - metabolism; B-Lymphocytes - immunology; Bioengineering; Biology and Life Sciences; Calcium (intracellular); Calcium ions; Cancer; CD19 antigen; Cell activation; Cell Line, Tumor; Cell surface; Chimeric antigen receptors; Clinical trials; Cytology; Degranulation; Engineering and Technology; Health aspects; Humans; Immunology; Immunotherapy, Adoptive - methods; Laboratories; LAMP-1 protein; Lymphocytes; Lymphocytes T; Lymphoma, B-Cell - immunology; Lymphoma, B-Cell - therapy; Medicine and Health Sciences; Membrane proteins; Morphology; Phosphorylation; Physiological aspects; Plasma; Proteins; Receptors, Antigen, T-Cell - metabolism; Receptors, Chimeric Antigen - metabolism; Research and Analysis Methods; Synapses; Synaptogenesis; T cells; T-Lymphocytes - immunology; Tumor cells; Tumors
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0238819

Adoptive cell transfer of Chimeric Antigen Receptor (CAR)-T cells showed promising results in patients with B cell malignancies. However, the detailed mechanism of CAR-T cell interaction with the target tumor cells is still not well understood. This work provides a systematic method for analyzing the activation and degranulation of second-generation CAR-T cells utilizing antigen-presenting cell surfaces. Antigen-presenting cell surfaces composed of circular micropatterns of CAR-specific anti-idiotype antibodies have been developed to mimic the interaction of CAR-T cells with target tumor cells using micro-contact printing. The levels of activation and degranulation of fixed non-transduced T cells (NT), CD19.CAR-T cells, and GD2.CAR-T cells on the antigen-presenting cell surfaces were quantified and compared by measuring the intensity of the CD3ζ chain phosphorylation and the Lysosome-Associated Membrane Protein 1 (LAMP-1), respectively. The size and morphology of the cells were also measured. The intracellular Ca2+ flux of NT and CAR-T cells upon engagement with the antigen-presenting cell surface was reported. Results suggest that NT and CD19.CAR-T cells have comparable activation levels, while NT have higher degranulation levels than CD19.CAR-T cells and GD2.CAR-T cells. The findings show that antigen-presenting cell surfaces allow a quantitative analysis of the molecules involved in synapse formation in different CAR-T cells in a systematic, reproducible manner.