Programmable DNA-augmented hydrogels for controlled activation of human lymphocytes

Abstract

Contractile forces within the planar interface between T cell and antigen-presenting surface mechanically stimulate T cell receptors (TCR) in the mature immune synapses. However, the origin of mechanical stimulation during the initial, i.e., presynaptic, microvilli-based TCR activation in the course of immune surveillance remains unknown and new tools to help address this problem are needed. In this work, we develop nucleic acid nanoassembly (NAN)-based technology for functionalization of hydrogels using isothermal toehold-mediated reassociation of RNA/DNA heteroduplexes. Resulting platform allows for regulation with NAN linkers of 3D force momentum along the TCR mechanical axis, whereas hydrogels contribute to modulation of 2D shear modulus. By utilizing different lengths of NAN linkers conjugated to polyacrylamide gels of different shear moduli, we demonstrate an efficient capture of human T lymphocytes and tunable activation of TCR, as confirmed by T-cell spreading and pY foci.

Keywords: Mechanobiology; T cell; TCR mechanosensing.

Figure 1.. Artificial immune synapse with an introduced DNA-based mechanics-controlling module.

(A) T-cell’s cognate TCR-pMHC axis: (1) Human primary T cells engage with their cognate antigen peptides, exposed on antigen-presenting cells (APC) via TCR-decorated microvilli^,. The antigen-engaged microvilli secure early cognate T-cell-APC interactions and facilitate the formation of the mature immune synapse. (2) TCR-pMHC interactions within the IS form a 13 nm-long molecular axis between APC and T-cell surfaces. (3) T-cell cytoskeletal centripetal flow and actomyosin contractility within the IS exert shear stresses onto the TCR that results in the mechanical 3D force momentum (torque) and bending of the TCR-pMHC axis. 3D force momentum (torque), along with the shear and possibly other forces, facilitate the mechanosensory activation of the TCR downstream signaling via the Nck-Lck-Zap70 pathway. Note that the interaction energy between T-cell and antigen is modulated by the „catch-bond’ mechanism within the TCR-pMHC axis in addition to TCR downstream mechanosensing signaling. (B) CD3ε TCR complex subunit is a relay for the mechanical forces from the extracellular TCR-pMHC axis to the intracellular TCR signaling apparatus. Bypassing the „catch-bond’-modulated variability within the TCR-pMHC axis via the CD3ε subunit targeted by the OKT3 mAb allows for standardized studying of the TCR’s downstream torque mechanosensing. Note the introduction of the additional controlling module as the bundled dsDNA between the elastic polyacrylamide gel surface and the TCR complex.

Figure 2.. Presynaptic antigen surveillance and engagement of T cells via microvilli TCR foci.

(A) TCR foci-decorated membranous linkers, i.e., microvilli, facilitate antigen surveillance of T cells with a larger pMHC scanning radius, but require non-contractile pre-synaptic force generation and pMHC mechanosensing mechanisms. Laser confocal microscopy. Freshly isolated, human naïve (resting) CD4+ T-cells (green, myosin IIa) are presented to the OKT3 dots micropattern (red), printed on the plasma treated glass (1–3). Contractile (+DMSO) and non-contractile (+Blebbistatin, 50 μM) spherical human T cells (4–5) quickly engage multiple OKT3 dots (1 μm each with linear distances between dot centers equal 5 μm). T cell engagement happens without prerequisite T-cell spreading and retraction (short term, 10 minutes), and without external mechanical stimuli (no vibration, no hydrodynamic flow). Quick engagement of the multiple distant OKT3 dots occurs via microvilli, accompanied with their immediate (10 minutes) structural thickening. (B) For T-cell microvilli tips, growing TCR microclusters accumulate innate deformational stresses and 3D bending forces, that provide the mechanical stimulation for TCR complex (1). Mature immune synapse features both TCR foci-driven torques and actomyosin contractility- and lamellipodial polymerization-depolymerization turnover dynamics-driven 2D tangential shear stresses (2). However, due to the experimental constraints, OKT3 dots do not support the formation of a planar tensile IS.

Figure 3.. 1D spreading of human CD4+ T-cells depends on lengths of OKT3-PAG linkers.

(A) Schematic views of OKT3 linkage to the PAG substrate. (B) Comparison of 1D T-cell spreading lengths during the course of one hour along the OKT3 attached via long linker, short linker, or direct adsorption onto a plasma-treated activated glass surface (C) Fluorescent micrographs (iSIM) of the hCD4+ T-cells (blue/green) spreading along OKT3 lanes (red).

Figure 4.. Multi-step strategy for PAG surface micropatterning with NANs.

(A) Fluorescently labeled streptavidin-acrylamide conjugate micropattern is imprinted into polymerized bulk PAG from intermediate glass surfaces. (1) Cured gel is then incubated with biotin-5’-DNA/RNA heteroduplex. (2) Passivation of remaining streptavidin reactivity: open streptavidin biotin-binding sites are blocked with three-fold excess free biotin, then (3) rinsed from non-reacted biotin. (4–5) Isothermic reassociation of PAG-biotin-dsDNA-biotin linker by addition of cognate biotin-5’-DNA/RNA heteroduplex, followed by rinsing of the dsRNA byproducts. Resulting dsDNA linker exposes a free and available biotinylated distal end, without self-locking onto the PAG-streptavidin surface. (6–7) dsDNA-biotin linker functionalized with fluorescently labeled streptavidin-OKT3. (8) Biotinylated dsDNA and streptavidin-OKT3 conjugates self-assemble into the tent-like molecular nanopillars - a rigid multipod nanostructure that facilitates T-cell adhesion via CD3ε-OKT3 interaction. (B) (Top left) - Micrographic views of fluorescent streptavidin lanes, imprinted into the PAG surface. (Bottom left) - Streptavidin-conjugated, fluorescent OKT3 (Alexa Fluor® 555), adsorbed onto streptavidin-biotin-dsDNA-biotin PAG lanes (G’=2.3 kPa). (Right) - Human CD4+ T cell spread along the resulting OKT3 lane (G’=50 kPa).

Figure 5.. 1D spreading of human CD4+ T cells recorded as a function of the TCR-OKT3 molecular torque resistance.

(A) Comparison of OKT3-PAG linker-facilitated clearance for 21 nm-long CD45 extracellular domain length (ECD-CD45). All linker components are shown to scale: streptavidin - 5 nm, OKT3 antibody molecule - 15 nm, 39 bp dsDNA - 13.3 nm, 66 bp dsDNA - 22.5 nm, 93 bp dsDNA - 31.6 nm, biotin and acryloyl links - below 1 nm. The OKT3-acryloyl linker system accrues to the length of ~15 nm, which when linked to the 3 nm-long extracellular CD3ε domain, results in ~18 nm clearance (below ECD-CD45 length). All other OKT3 linker configurations exceed the ECD-CD45 length, allowing for the CD45 clearance, and thus independence from TCR-CD45 kinetic segregation (KS). (B) Three PAG surfaces with G’ of 2.3, 8.6, and 50 kPa are tested and each G’ value features either long linker (Fig. 2A, low torque resistance), or short linker (Fig. 2A, intermediate torque resistance), or multiple lengths dsDNA linkers (high torque-resistance). Graphs show T-cell spreading lengths measured along the 1 μm-wide OKT3 lanes and corresponding brightness of OKT3 lanes proportional to the relative OKT3 surface concentrations. High torque-resistance 39 bp dsDNA nanopillar within the OKT3-PAG system increases TCR activation at G’=8.6 kPa when compared to the lower torque resistance linkers for the G’. Overall early TCR activation-induced 1D T-cell spreading is reduced with increased dsDNA linker lengths and overall molecular flexibility. (C) 3D micrographic views of the human CD4+ T cells spreading along OKT3-decorated PAG lanes with various G’ and different molecular linker systems. (D) T-cell 1D spreading along PAG lanes with 66, 81, and 93 bp dsDNA linkers decorated with OKT3.

Figure 6.. Formation of pY foci in human CD4+ T cells in response to linker force momentum resistance and antigen density.

(A) T cell pY foci response is measured by the number of pY foci, for short linker (green) and 39 bp dsDNA linker (red, (4)). Direct immobilization of OKT3 antibodies increases (~40x) surface density of OKT3 compared to dsDNA linkers (1). Increased OKT3 density correlates with increased pY foci response compared to dsDNA lanes of similar G’= 8.6, or 50 kPa (2). The lane, G’ of 50 kPa, with normalized OKT3 density (diluted, short linker) provides negligible pY foci response (2) and no spreading (3) compared to dsDNA lanes. In addition, OKT3 lanes with 39 bp dsDNA linkers demonstrate increased pY response on gels with increased shear force modulus (2). (B) Micrographic views. T-cell pY foci development along PAG lanes with G’ of 8.6, and 50 kPa for OKT3 antibody molecule immobilized with short linkers (green), and 39 bp dsDNA linkers (red). The 39 bp dsDNA linker (G’ 50 kPa) provides more pY foci compared to short linker (G’ 50 kPa) with normalized OKT3 density (views 3 and 5).