Quantitative extrapolation from single-tags (QuEST) immunofluorescence microscopy to derive TCR signalosome stoichiometries in human primary T cells

Upon T cell receptor (TCR) engagement, a T cell forms an immunological synapse (IS) with an antigen-presenting cell (APC), which can be mimicked by purified ligands on supported lipid bilayers (SLBs)1,2. Microvilli actively scan the surface; upon initial engagement, F-actin-dependent TCR microclusters form, and the central supramolecular activation cluster (cSMAC) sustains TCR signaling in CD8 T cells3,4. Although signaling activities within the IS have been observed qualitatively through total internal reflection immunofluorescence microscopy5-7, the stoichiometric relationships among the components of the TCR signalosome remain unknown. In this study, we employed a two-step approach to quantify the components of the TCR signalosome. First, Jurkat cell lines expressing GFP-tagged proteins on a knockout background were used to calibrate fluorescence intensity (IF) signals against molecular copy numbers, based on measurements of single-tag signals and multiple corrections. In the second step, this calibration was applied to determine the stoichiometries of key TCR signalosome components, including TCR, CD8, CD28, CD45, PD-1, Lck, ZAP-70, LAT, and PLC{gamma}1, across scanning, early activation, and sustained activation states in human primary T cells. We refer to the method as quantitative extrapolation from single-tags (QuEST) immunofluorescence microscopy. Applying the QuEST, we were surprised to find that the ZAP-70:TCR ratio in microclusters and the cSMAC was 1:1, far from the potential 10:1 ratio. Nanoscale structures of the TCR signalosome were further captured using direct stochastic optical reconstruction microscopy (dSTORM), confirming that ZAP-70 was strongly co-localized with the TCR. Moreover, we applied QuEST to confirm the presence of T cell intrinsic CD28 recruitment, independent of CD80 or CD86 on SLBs, during TCR activation. This T cell intrinsic CD28 recruitment could be disrupted through engagement of PD-1 with PD-L1 on SLBs. This shows that PD-1 engagement can disrupt T cell intrinsic CD28 costimulation. QuEST provides a broadly applicable pipeline for quantitative analysis of TCR signalosomes in human primary cells, enabling a quantitative basis for the rational manipulation and engineering of the TCR signalosome in immunotherapies.