Final Report Summary - SYNINTER (Smart interrogation of the immune synapse by nano-patterned and soft 3D substrates)
The immunological synapse, formed at the interface between a T-lymphocyte and an antigen presenting cell (APC), has been the target of intense multidisciplinary research in the last decade. Its possible key role in a host of diseases of the immune system has motivated medical research whereas its assembly, arising partly from simple underlying thermodynamic and elastic considerations has fascinated physicists. Studies point to a crucial role for adhesion mediated by protein clusters for the stability and activity of the synapse. One way to address the question of how spatial organization of ligands and receptors influence the synapse is by interrogating the synapse with artificial substrates, exhibiting motifs down to the cluster length-scale, which closely mimic an APC. The aim of SYNINTER was to design smart substrates and suitable detection techniques to understand better the dynamics and spatial organization found in the immunological synapse, with special focus on activation clusters, and taking into account possible mechanosensitivity of the process.
In the course of SYNINTER, we developed such substrates, using tools of nano-technology including unconventional novel bottom-up techniques based on self-organization, to control the placement of the adhesion molecules and the antigens. We created APC-mimetic innovative synthetic substrates to study the impact of ligand clustering on T cell activation and spreading. The substrates exhibit ligands in the form of an array of sub micrometric dots surrounded by a fluid supported lipid bilayer (SLB) which may itself be functionalized with another bio-molecule. Alternatively, the dots are surrounded by a passive polymer layer. In both cases, we showed that for T cell adhesion mediated by T cell receptor (TCR) alone, global cell scale parameters like cell area depend only on the average density of the ligands, whereas local parameters like TCR /ZAP-70 organization, the actin distribution and membrane topography are severely altered on patterned as compared to homogeneously distributed ligands. We show that in the patterned but not in the homogeneous case, the TCR, ZAP-70 and actin are present in the form of dots. Remarkably, in presence of additional adhesion ligands (against the T cell integrin LFA1) on the SLB, while the TCR is still clustered by clustering of its ligands, global parameters are now also impacted. The actin organization changes to a peripheral ring, resembling the classical actin distribution seen on homogeneous substrates, the patterned membrane topography disappears and the membrane is flat, and the cell area increases significantly. We conclude that clustering of TCR is important but only under additional stimulation. .
In addition, we showed that T cells adhering via the TCR-complex respond to environmental stiffness in an unusual biphasic fashion. As the stiffness increases, adhesion and spreading first increase, then decrease, attaining their maximal values on an optimally stiff surface, with stiffness comparable to certain antigen presenting cells. Remarkably, in presence of additional ligands for the integrin LFA-1, spreading increases monotonously with stiffness up to a saturation value. We propose a mesoscopic semi-analytical model linking spreading to molecular characteristics of bonds that identifies force sensitivity of the off-rate and the effective bond stiffness as the crucial parameters that determine monotonic or biphasic mechanosensitive behavior.
In the course of SYNINTER, we developed such substrates, using tools of nano-technology including unconventional novel bottom-up techniques based on self-organization, to control the placement of the adhesion molecules and the antigens. We created APC-mimetic innovative synthetic substrates to study the impact of ligand clustering on T cell activation and spreading. The substrates exhibit ligands in the form of an array of sub micrometric dots surrounded by a fluid supported lipid bilayer (SLB) which may itself be functionalized with another bio-molecule. Alternatively, the dots are surrounded by a passive polymer layer. In both cases, we showed that for T cell adhesion mediated by T cell receptor (TCR) alone, global cell scale parameters like cell area depend only on the average density of the ligands, whereas local parameters like TCR /ZAP-70 organization, the actin distribution and membrane topography are severely altered on patterned as compared to homogeneously distributed ligands. We show that in the patterned but not in the homogeneous case, the TCR, ZAP-70 and actin are present in the form of dots. Remarkably, in presence of additional adhesion ligands (against the T cell integrin LFA1) on the SLB, while the TCR is still clustered by clustering of its ligands, global parameters are now also impacted. The actin organization changes to a peripheral ring, resembling the classical actin distribution seen on homogeneous substrates, the patterned membrane topography disappears and the membrane is flat, and the cell area increases significantly. We conclude that clustering of TCR is important but only under additional stimulation. .
In addition, we showed that T cells adhering via the TCR-complex respond to environmental stiffness in an unusual biphasic fashion. As the stiffness increases, adhesion and spreading first increase, then decrease, attaining their maximal values on an optimally stiff surface, with stiffness comparable to certain antigen presenting cells. Remarkably, in presence of additional ligands for the integrin LFA-1, spreading increases monotonously with stiffness up to a saturation value. We propose a mesoscopic semi-analytical model linking spreading to molecular characteristics of bonds that identifies force sensitivity of the off-rate and the effective bond stiffness as the crucial parameters that determine monotonic or biphasic mechanosensitive behavior.