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

ABSTRACT: 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

ORGANISM(S): Homo sapiens

PROVIDER: GSE230629 | GEO | 2023/05/02

REPOSITORIES: GEO

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Project description:The fibroblast-populated 3D collagen matrix has been used to study the effect of mechanical stress on cell fate; this process is relevant to the fields of wound healing and tissue engineering. Gene array data was generated from mechanically stressed vs. stress-released matrices. The parameters of the collagen matrix model were: collagen type = bovine type I; collagen concentration = 1.5 mg/mL; initial matrix volume = 0.2 mL; initial matrix diameter = 11 mm (cultured in 24-well plates); cell type = human foreskin fibroblast, passage <10; initial matrix cell concentration = 1,000,000 cell/mL (200,000 cell/matrix); culture medium = 5% FBS in DMEM with 1 M-BM-5g/mL ascorbate. Matrices (n = 6 per experimental group) were incubated for 24 hr in the attached state; the released groups then underwent matrix detachment from the culture plate (defined as t = 0), while the attached groups were left undisturbed. RNA was isolated from attached and released matrices 6 and 24 hr after t = 0. Gene expression in the attached vs. released condition at 6 or 24 hr then was analyzed by hybridizing the anti-sense RNA derived from attached and released matrices at a given time point onto a single chip. Refer to the attached Figure 1 for the experimental design. The index experiment was defined as the comparison of gene expression in attached vs. released collagen matrices in a single strain of human foreskin fibroblasts at 6 and 24 hr after stress-release (i.e., after t = 0). Each experiment utilized two mechanical conditions (attached and released) at two time points (6 and 24 hr). So with each condition utilizing 6 matrices, each index experiment required a total of 24 matrices. In each index experiment, the chip hybridizations were: (i) 6 hr attached vs. 6 hr released, and (ii) 24 hr attached vs. 24 hr released (i.e., two gene chips per index experiment). Each hybridization was done using a 10K spotted gene chip manufactured in the UNMC Microarray Core Facility. The index experiment was performed on three fibroblast strains, meaning that expressional data was derived from three foreskin donors (nonpooled samples). Dye-swap was not performed; dye assigned to attached vs. released remained constant among all chips. Since the index experiment was performed three times, the total number of gene chips used for this entire dataset was six.

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

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