Quantifying Dynamic T-Cell Function with Incucyte and iQue Analysis
T-cells are critical in adaptive immunity, with unique surface receptors that allow T cells to sense and respond to diverse types of pathogenic organisms as well as for defense against unwanted target cells, such as emergent tumor cells. T-cell activation and proliferation is fundamental to regulating the effect and extent of the immune response, requiring dynamic models in order to understand T-cell biology in fighting infection or disease.
The Incucyte® Live-Cell Analysis System and iQue Advanced Flow Cytometry Platform can be used to evaluate and quantitate key functions of T cells, including activation and clustering, antibody internalization assays, chemotaxis, cytokine production, as well as the evaluation of complex co-culture models, such immune cell killing and transendothelial migration assays.
Visualization of PBMC activation using phase-contrast imaging and live-cell ICC (anti-CD71 Fabfluor-488). PBMCs (30K/well) were treated with anti-CD3 and IL-2, or vehicle control, and labeled using anti-CD71-Fabfluor-488. a) Label-free phase-contrast imaging detects morphological differences between non-activated and activated PBMCs; phase-contrast imaging coupled with live-cell ICC shows upregulated CD71+ in activated cells. b) Activation induces increased cell size, increased cell eccentricity and upregulation of CD71+ in larger vs smaller subsets of cells.
T cell activation assessment with the iQue Advanced Flow Cytometry Platform and ForeCyt software. Dose and temporal responses in the percentage of activated T Cells and the levels of secreted IFNγ among different treatments. Unique patterns of T cell activation markers observed for each compound tested.
Visualize immune cell/tumor cell interplay using Incucyte immune cell killing assays. (1) Physical contact between a small cytotoxic T cell and a larger labeled tumor cell (red). T lymphocyte division. (2) Tumor cells under attack from a cytotoxic T lymphocyte: The "kiss of death". (3) Tumor cell cytoplasmic granulation immediately followed by caspase 3/7 labeling (green), nuclear condensation and cell death. (4) Tumor cell mitosis: One cell becomes two.
Measure tumor cell death and proliferation (optional) in real time using Incucyte immune cell killing assays. User-friendly Incucyte® software enables direct image-based detection of apoptotic tumor cells (green objects, outlined in yellow) and real-time counting of live tumor cells (Incucyte® NucLight red labeled nuclei, outlined in blue). Kinetic readouts reveal the time dependence of treatment effects.
Temporal Target Cell and Cytokine Analysis. SKOV-3 NucLight Green cells were weeded with PMBCS and activated with CD3/CD28 Dynabeads. (a) Target cell count timecourse analyzed with Incucyte. Temporal cytokine data for (B) IFNγ and (C) TNFα using only 10µl/ day of supernatant samples were quantified using Qbead component of TCA kit (Qbeads or Assay Builder: IFNγ, option 1 and TNFα option 2)/ (D) Concentration response curves for AUC from the time-courses of target cell count (Incucyte) and cytokines (iQue3).
CD3xCD19 BiTE antibody induces cytotoxicity and target cell clustering. Ramos NucLight Green cells were seeded with PBMCs and activated with BiTE antibody (anti-hCD3xCD19) or control antibody (anti-hCD3xβGAL). Representative images at 72 h in the presence of (A) control or (B) BiTE antibody. Incucyte® time-course data (images every 3 h) tracked the cell quantity (C) and clustering (E) of target cells. The fold change in green integrated intensity metric, which accounts for both brightness and size (GCU x μm2) was used to quantify target cells. Concentration-response curves at 72 h for target cell killing (D) and clustering (F) in the presence of the BiTE antibody compared with the control antibody.
Impact of activated PBMCs on tumor spheroid proliferation. A549 tumor cells stably expressing nuclear restricted RFP were seeded in a round bottom ULA 96-well plate and allowed to form spheroids for 3 d. Once formed, spheroids were co-cultured with freshly isolated PBMCs in the presence or absence of an Anti-CD3 and IL-2 antibody cocktail. Incucyte® HD phase and fluorescence images compare the effect of PBMCs on spheroid proliferation in the absence (non-activated, top panel) and presence (activated, bottom panel) of Anti-CD3 and IL-2 antibodies. Note marked loss of fluorescence intensity in spheroids in the presence of activated PBMCs. Time course plot shows spheroid cytotoxicity quantified as a loss of fluorescence intensity over time. Data were collected over 7 d at 6 h intervals. Each data point represents mean ± SEM, n=3 wells.
Visualization of leukocyte extravasation. CD3/CD28 Dynabead activated primary T cells were seeded on a HUVEC monolayer cultured on fibronectin and images were acquired every minute using the Incucyte® System. Yellow and orange arrows indicated leukocytes moving between HUVEC cells and eventually down the pores (blue circles) of the ClearView insert.
Primary T cells extravasation toward CXCL12. CD3/CD28 Dynabead activated primary T-cells were seeded on a HUVEC monolayer cultured on fibronectin. The insert containing T cell:HUVEC monolayer co-cultures was then exposed to CXCL12 gradients at the indicated concentrations. Images were acquired every 30 minutes using the Incucyte® ZOOM and phase analysis was performed. Analysis of pharmacological response was performed at t=6 hr. Each data point represents mean ± SEM, n=4.
Zumwalde et al, 2013 J Immunol. October 1; 191(7): 3681–3693. doi:10.4049/jimmunol.1201954.
Immune cell killing
McCormack et al, 2013 Cancer Immunol Immunother. Apr; 62(4): 773–785. doi:10.1007/s00262-012-1384-4