Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

Medical University of Vienna

Obesity and hepatic steatosis are frequently associated with the development of a non-alcoholic steatohepatitis (NASH). The mechanisms driving progression of a non-inflamed steatosis to NASH are largely unknown. Here, we investigated whether ingestion of peroxidized lipids, as being present in Western style diet, triggers the development of hepatic inflammation.

Food-derived peroxidized fatty acids may trigger hepatic inflammation: a novel hypothesis to explain steatohepatitis.

Blockade of the T cell coinhibitory molecules CTLA-4 and PD-1 has clinical utility to strengthen T cell responses. In addition to these immune checkpoints an ever-growing number of molecules has been implicated in generating coinhibitory signals in T cells. However, investigating coinhibitory molecules in primary human cells is complicated by the restricted expression and promiscuity of both coinhibitory receptors and their ligands. Here we have evaluated the potential of fluorescence-based transcriptional reporters based on the human Jurkat T cell line in conjunction with engineered T cell stimulator cell lines for investigating coinhibitory pathways. CTLA-4, PD-1, TIGIT, BTLA and 2B4 expressing reporter cells were generated and activated with T cell stimulator cells expressing cognate ligands of these molecules. All accessory molecules tested were functional in our reporter system. Engagement of CTLA-4, PD-1, BTLA and TIGIT by their ligands significantly inhibited T cell activation, whereas binding of 2B4 by CD48 resulted in enhanced responses. Mutational analysis revealed intracellular motifs that are responsible for BTLA mediated T cell inhibition and demonstrates potent reporter inhibition by CTLA-4 independent of cytoplasmic signaling motifs. Moreover, considerably higher IC50 values were measured for the CTLA-4 blocker Ipilimumab compared to the PD-1 antibody Nivolumab. Our findings show that coinhibitory pathways can be evaluated in Jurkat-based transcriptional reporters and yield novel insights on their function. Results obtained from this robust reductionist system can complement more time consuming and complex studies of such pathways in primary T cells.

A cellular platform for the evaluation of immune checkpoint molecules.

T cell antigen recognition requires T cell antigen receptors (TCRs) engaging MHC-embedded antigenic peptides (pMHCs) within the contact region of a T cell with its conjugated antigen-presenting cell. Despite micromolar TCR:pMHC affinities, T cells respond to even a single antigenic pMHC, and higher-order TCRs have been postulated to maintain high antigen sensitivity and trigger signaling. We interrogated the stoichiometry of TCRs and their associated CD3 subunits on the surface of living T cells through single-molecule brightness and single-molecule coincidence analysis, photon-antibunching-based fluorescence correlation spectroscopy and Förster resonance energy transfer measurements. We found exclusively monomeric TCR-CD3 complexes driving the recognition of antigenic pMHCs, which underscores the exceptional capacity of single TCR-CD3 complexes to elicit robust intracellular signaling.

Monomeric TCRs drive T cell antigen recognition.

The main function of T cells is to identify harmful antigens as quickly and precisely as possible. Super-resolution microscopy data have indicated that global clustering of T cell antigen receptors (TCRs) occurs before T cell activation. Such pre-activation clustering has been interpreted as representing a potential regulatory mechanism that fine tunes the T cell response. We found here that apparent TCR nanoclustering could be attributed to overcounting artifacts inherent to single-molecule-localization microscopy. Using complementary super-resolution approaches and statistical image analysis, we found no indication of global nanoclustering of TCRs on antigen-experienced CD4 T cells under non-activating conditions. We also used extensive simulations of super-resolution images to provide quantitative limits for the degree of randomness of the TCR distribution. Together our results suggest that the distribution of TCRs on the plasma membrane is optimized for fast recognition of antigen in the first phase of T cell activation.

TCRs are randomly distributed on the plasma membrane of resting antigen-experienced T cells.

TIM-3 has been considered as a target in cancer immunotherapy. In T cells, inhibitory as well as activating functions have been ascribed to this molecule. Its role may therefore depend on the state of T cells and on the presence of interaction partners capable to perform functional pairing. Carcinoembryonic antigen-related cell adhesion molecule (CEACAM1) has been proposed to bind TIM-3 and to regulate its function. Using a T cell reporter platform we confirmed CEACAM1-mediated inhibition, but CEACAM1 did not functionally engage TIM-3. TIM-3 and CEACAM1 coexpression was limited to a small subset of activated T cells. Moreover, results obtained in extensive binding studies were not in support of an interaction between TIM-3 and CEACAM1. Cytoplasmic sequences derived from TIM-3 induced inhibitory signaling in our human T cell reporter system. Our results indicate that TIM-3 functions are independent of CEACAM1 and that this receptor has the capability to promote inhibitory signaling pathways in human T cells.

TIM-3 and CEACAM1 do not interact in cis and in trans.

Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and temporal behavior are still poorly defined. We here report a FRET-based sensor equipped either with a TCR-reactive single chain antibody fragment or peptide-loaded MHC, the physiological TCR-ligand. The sensor was tethered to planar glass-supported lipid bilayers (SLBs) and informed most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5 pN and force loading rates on the TCR of 1.5 pN per second. When facing fluid-phase SLBs instead, T-cells still exerted tensile forces yet of threefold reduced magnitude and only prior to but not upon activation.

Florian Kellner

Institute for Hygiene and Applied Immunology,
Center for Pathophysiology,
Infectiology and Immunology,
Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology

Medical University of Vienna

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Florian Kellner

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute for Hygiene and Applied Immunology,Center for Pathophysiology,Infectiology and Immunology,Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology

Medical University of Vienna

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Institute for Hygiene and Applied Immunology,
Center for Pathophysiology,
Infectiology and Immunology,
Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology