Clonotypic analysis of T cells cultured in extracellular matrices of different viscoelasticies

The efficacy of adoptive T cell therapy is highly dependent on the generation of T cell populations that are able to both provide immediate effector function and long-term protective immunity. Inspired by the emerging consensus that T cell phenotype and function are inherently linked to their tissue localization, we present an approach to generate functionally distinct T cell populations via the physical properties of their surrounding matrix. A collagen type I based extracellular matrix (ECM) was engineered to allow for independent tuning of matrix stiffness and viscoelasticity, two features that characterize the mechanical properties of tissues. To assess whether phenotypic differences imparted by matrix viscoelasticity are due to the selection and persistence of specific T cell clones, we performed single cell RNA-sequencing with TCR sequencing Overall design: T cells cultured in fast-relaxing and slow-relaxing matrices, plate-cultured T cells and the parent T cell population were separately stained with TotalSeq C 0252 anti-human Hashtag 2 (Biolegend # 394663), TotalSeq C 0253 anti-human Hashtag 3 (Biolegend # 394665), TotalSeq C 0254 anti-human Hashtag 4 (Biolegend # 394667) and TotalSeq C 0251 anti-human Hashtag 1 (Biolegend # 394661), in addition to TotalSeq C 0063 anti-human CD45RA (Biolegend # 304163). After staining, the T cells were pooled together for sequencing.