Fig. 2. A single-cell assay measures both T cell input accumulation and activation.

(A) TCR recognition of pMHC initiates an intracellular signaling cascade in T cells that culminates in nuclear translocation of the transcription factor NFAT and IL-2 production. PLC-γ, phospholipase C-γ; PIP₂, phosphatidylinositol 4,5-bisphosphate;IP3, inositol 1,4,5-triphosphate. (B) Experimental schematic of the single-cell pMHC-TCR binding input and the NFAT nuclear translocation response assay. (C) Images of NFAT-mCherry transduced T cell landing and spreading (green, dashed lines), binding pMHC (green circles), and NFAT nuclear translocation (purple bar) after interaction with a bilayer containing MCC-Atto647N-MHC. Cell landing was determined by a dark signature in RICM (yellow arrow), and activation time (t = 0) was defined as the first observation of NFAT translocation (purple dotted line). Images are representative of at least 30 independent experiments. Scale bar, 3 μm. (D) Analysis of NFAT activation determined by the ratio of mean nuclear intensity to mean cytosolic intensity. Epifluorescent images of an NFAT localization in the nucleus (purple line) or cytosol (cyan line) of an activating (left) and nonactivating (right) cell are representative of at least 30 independent experiments. (E) The distribution of single-cell NFAT-green fluorescent protein (GFP) abundance in T cells stimulated on an MCC-MHC bilayer as a function of activation state. Single-cell NFAT-FP intensity measurements at the time of initial T cell landing are pooled from 12 independent experiments.