Bioengineering strategies to model and modulate immune cells

Abstract

Optimal activation of immune cells is necessary to elicit a protective response against pathogens and foreign objects. In dysregulated immune systems, cells are aberrantly activated or exhausted. Understanding biochemical and mechanical aspects that modulate immune responses helps us design biomaterials-based in vitro research and drug screening models for immune pathologies. Furthermore, materials science principles can be applied to synthesize immunomodulatory biomaterials to treat immune pathologies. In this study, a 3D bioprinted platform was developed to study the influence of physical microenvironment on T cell activation, roles of calcium in T cell activation in 2D and 3D and validate the principle in vivo, and the in vitro and in vivo immunomodulatory effects of 2D antimonene nanosheets were investigated. First, we developed a 3D bioprinted platform with tuneable mechanical properties to culture and activate T cells in different stiffnesses and porosities. T cells were bioprinted in gelatin methacryloyl (GelMA) hydrogels using a digital light processing (DLP)-based 3D bioprinter. The hydrogels recapitulated physiologically and pathologically relevant stiffnesses of a lymph-node mimetic tissue construct as evidenced by hydrogel characterization. The biomechanical properties of the 3D bioprinted hydrogel influence T cell activation. Some cellular responses of the 2D and 3D cultures in a soft matrix (19.83 ± 2.36 kPa) were comparable; however, they differed in a stiff matrix (52.95 ± 1.36 kPa). Furthermore, primary mouse T cells activated with PMA and ionomycin were 1.35-fold more viable in the soft matrix than in the stiff matrix. T cells bioprinted in a soft matrix and a stiff matrix released 7.4-fold and 5.9-fold higher amounts of IL-2 than 2D cultured cells, respectively. Furthermore, Th1/Tc1 cytokines were induced upon T cell activation as a function of changes in the mechanical properties, whereas Th2 cytokine production was not induced as a function of changes in the mechanical properties of the microenvironment. Second, we studied the effects of high intracellular calcium levels during in vitro T cell activation and validated the principle in vivo in a DSS-induced colitis model in mice. We investigated the effects of high intracellular calcium amounts using in vitro T cell receptor (TCR)-independent and TCR-dependent activation models. High intracellular calcium amounts increased the production of reactive oxygen species (ROS), and thereby decreased the activation-associated proliferation of T cells. This high calcium-induced inhibition of T cell proliferation could be rescued by treatment with an antioxidant, N-acetyl cysteine (NAC). We also found that high intracellular calcium inhibited T cell activation in the 3D bioprinted T cell culture platform we developed. We also tested the universality of the principle by studying the effects of tert-Butylhydroquinone (tBHQ), a SERCA inhibitor and Nrf2 activator. While tBHQ alone did not increase intracellular calcium amounts, they did so upon a combination treatment with PMA. Also, tBHQ inhibited T cell activation-associated proliferation in a dose-dependent manner. To validate this principle in vivo, we intraperitoneally injected tBHQ in mice with DSS-induced colitis. tBHQ ameliorated DSS-induced colitis in mice as evidenced by rescue of colon length shortening and lower disease activity index. Third, we synthesized, characterized, and tested the immunomodulatory effects of a two-dimensional nanomaterial, antimonene. Antimonene nanosheets were biocompatible and inhibited nitric oxide and IL6 production in thioglycollate-elicited peritoneal exudate cells. Furthermore, antimonene ameliorated DSS-induced colitis in mice, as evidenced by rescue of colon length shortening, less tissue damage, and decreased serum pro-inflammatory cytokine levels. In summary, the studies present T cell responses in an in vitro model of melanoma tumor microenvironment and suppression of innate immune cell responses to ameliorate DSS-induced colitis using 2D antimonene nanosheets.