Label-free single-cell live imaging reveals fast metabolic switch in T lymphocytes

T-cell activation induces a metabolic switch generating energy for proliferation, survival, and functions. We used noninvasive label-free two-photon fluorescence lifetime microscopy (2P-FLIM) to map the spatial and temporal dynamics of the metabolic NAD(P)H co-enzyme during T lymphocyte activation. This provides a readout of the OXPHOS and glycolysis rates at a single-cell level. Analyzes were performed in the CD4+ leukemic T cell line Jurkat, and in human CD4+ primary T cells. Cells were activated on glass surfaces coated with activating antibodies mimicking immune synapse formation. Comparing the fraction of bound NAD(P)H between resting and activated T cells, we show that T-cell activation induces a rapid switch toward glycolysis. This occurs after 10 min and remains stable for one hour. Three-dimensional analyzes revealed that the intracellular distribution of fraction of bound NAD(P)H increases at the immune synapse in activated cells. Finally, we show that fraction of bound NAD(P)H tends to negatively correlate with spreading of activated T cells, suggesting a link between actin remodeling and metabolic changes. This study highlights that 2P-FLIM measurement of fraction of bound NAD(P)H is well suited to follow a fast metabolic switch in three dimensions, in single T lymphocytes with subcellular resolution.

Corresponding maps of fraction of bound NAD(P)H of the T cells from the same region of interest.We note that dead cells have a typical fraction of bound NAD(P)H that is higher than viable cells.

Figure S1
: Assessing phototoxicity in primary T cells during longitudinal FLIM imaging of NAD(P)H using a viability dye.-Figure.S2: Workflow of 3D FLIM for single cell analysis -Figure S3: Example of single cell longitudinal analysis of the fraction bound NAD(P)H.-Figure S4: Longitudinal analysis of the fraction of bound NAD(P)H in non-activated (Control) and activated Jurkat T cells.-Figure S5: Metabolic shift during Jurkat T cell activation revealed by NAD(P)H FLIM.-Figure S6: Metabolic shift during primary T cell activation revealed by NAD(P)H FLIM.-Figure S7: Metabolic trajectory in Jurkat T cells following drug treatment.-Figure S8: Metabolic trajectory in primary T cells following drug treatment.-Figure S9: 3-dimensional (3D) visualization of mitochondria and nucleus in Jurkat T cells.-Figure S10: Simultaneous analysis of primary T cell spreading and metabolic shift during activation, presented donor by donor.

Figure S1 :
Figure S1: Assessing phototoxicity in primary T cells during longitudinal FLIM imaging of NAD(P)H using a viability dye. A. Representative images of dead cells stained with a viability dye in a ROI with primary T cells activated and imaged at 10min, 30min, and 1h during the 3D FLIM imaging protocol.After 1 hour the T cells that were longitudinally imaged were still viable.The dye was used to identify and exclude dead cells from the statistical analysis.B. Corresponding maps of fraction of bound NAD(P)H of the T cells from the same region of interest.We note that dead cells have a typical fraction of bound NAD(P)H that is higher than viable cells.

Figure S2 :
Figure S2: Workflow of 3D FLIM for single cell analysis.A. NAD(P)H FLIM and LifeAct-mCherry Z-stack images of Jurkat T cells.B. The Z projection is used to create single cell masks.C. 3D of the fraction of bound NAD(P)H.D. The single cell mask is applied to the 3D FLIM raw data of fraction of bound NAD(P)H to extract the FLIM data of single cell for every Z plane.

Figure S4 :Fraction
Figure S4: Longitudinal analysis of the fraction of bound NAD(P)H in non-activated (Control) and activated Jurkat T cells.A. Quantification of fraction of bound NAD(P)H of Jurkat T cells over time (10 min, 30 min and 1 hour) in control and activated condition.Each dot represents one cell.Mean with standard deviation range are shown.T test:**P ≤ 0.01, ***P<0.001.N= 2 experiments.

Figure S5 :
Figure S5: Metabolic shift during Jurkat T cell activation revealed by NAD(P)H FLIM. A. Representative images of phase lifetime (tauP) and B. modulation lifetime (tauM) of NAD(P)H of Jurkat T cells in control and activated condition at three time points (10 min, 30 min and 1 hour).Quantification of tauP (C) and tauM (D) of NAD(P)H over time of control and activated Jurkat T cells (10 min, 30 min and 1 hour).Each dot is one cell, data are presented by mean with standard deviation range.T-test: **P ≤ 0.01, ***P<0.001.