Sica, Ilona
(2024)
T cell activation and function investigated using fully-defined 3D culture.
PhD thesis, University of Nottingham.
Abstract
3D culture is gaining recognition as the advanced in vitro culture system to study cells. It bridges planar culture (2D culture) and in vivo studies, which could overcome oversimplified environment settings and genetic/physical differences to humans by animal models. For example, planar culture plates lack the architecture of tissues with extracellular matrix (ECM), whereas mouse tumours are poor representations of human ones. T cells are part of the immune system, and various studies have been conducted to understand their role in healthy or diseased tissues and the efficacy of therapies targeting these cells. To get more constructive in vitro results, by avoiding oversimplified culture (or in some cases animal models), 3D culture could be used by selecting appropriate biomaterial to study cells. Various biomaterials were tested for 3D T cell culture, enabling observation of cell-cell interaction, migration, effector function, and the effect of the microenvironment (i.e. oxygen gradient, stiffness) on cells. However, some of them have limitations such as modified environment (e.g. collagen hydrogel), tumour origins (e.g. Matrigel), poor stiffness level control or even immunogenic properties which could activate an unwanted immune response. In this case, self-assembling peptide gel offers solutions to those common biomaterial problems and makes it a great candidate for T cell 3D culture.
Self-assembling peptide gel is composed of FEFEFKFK octapeptide (F, phenylalanine, K, lysine, E, glutamic acid); under correct temperature, pH and salt concentration makes the hydrogel. The whole gelation process relies on electrostatic interactions between amino acids, avoiding complex and toxic crosslinking methods for gel formation. Primary human T cells were encapsulated in the peptide gel to analyse the biomaterial’s biocompatibility and suitability as the 3D culture system for cells.
T cells were encapsulated in the peptide gel; their viability and effector functions over 7 days were observed. T cells were encapsulated with antigen presenting cells (APCs) to simulate their activation process as found in the peripheral tissues. APCs were either artificial (antibody CD3 and CD28 coated polystyrene beads) or actual cells such as dendritic cells (DC) isolated from the healthy human donors. The purpose of these experiments was to test their ability to respond to stimuli and deliver an appropriate response in the hydrogel. The material itself was modified to test the microenvironment effect on T cell response from the soft to the harder material, representing lymph node to tumour-like properties, respectively. In addition, the hydrogel was functionalized with extracellular matrix (ECM) proteins, fibronectin and collagen type I, by adsorption process to indicate whether proteins influence T cell behaviour (i.e. co-stimulation effect) as described in the literature. All of these distinctive experiments were set up to test the peptide gel's ability to support these complex immune cells, and whether future immune studies could benefit from this novel in vitro platform. Cells were monitored by proliferation assays (3H Thymidine, Cell-Trace CFSE), IFNγ and IL-10 cytokine secretion (ELISA) and phenotype marker expression (flow cytometry).
T cells showed better growth in the softer gels (average 283 Pa) than the rigid ones, which restrained expanding proliferating clusters of cells. They responded to various stimuli provided by artificial and actual pro-inflammatory APC the peptide gel. Furthermore, functionalized gels with matrix proteins demonstrated a co-stimulatory effect on cells, which reflected inflammatory tissue scenarios experienced by cells. The peptide gel was also used to test the immunomodulatory effect of p38 inhibitor (BIRB196 drug) treated CD1c cells, which under 2D culture settings enhance T cells' immune response. T cells under 3D culture demonstrated similar results to the published 2D culture studies, indicating peptide gel compatibility as the novel in vitro culture. However, in the presence of fibronectin, the p38-treated CD1c effect was diminished on T cells, and this could represent a possible scenario of glioblastoma tumour.
In conclusion, peptide gel demonstrated its ability to support basic T cell function. Adding additional parameters to the gel that reflect tissue microenvironments - demonstrated different cell behaviour, which is most of the time misrepresented by 2D cultures. The material itself is inert and showed no immunogenic properties that could mislead immune-related studies. This conducted research demonstrates the need for 3D culture-like studies to build a better understanding of immune cells, and self-assembling peptide gel is a good start to advance these in vitro studies.
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