Integration of intermittent calcium signals in T cells revealed by temporally patterned optogenetics

Abstract

T cells become activated following one or multiple contacts with antigen-presenting cells. Calcium influx is a key signaling event elicited during these cellular interactions; however, it is unclear whether T cells recall and integrate calcium signals elicited during temporally separated contacts. To study the integration of calcium signals, we designed a programmable, multiplex illumination strategy for temporally patterned optogenetics (TEMPO). We found that a single round of calcium elevation was insufficient to promote nuclear factor of activated T cells (NFAT) activity and cytokine production in a T cell line. However, robust responses were detected after a second identical stimulation even when signals were separated by several hours. Our results suggest the existence of a biochemical memory of calcium signals in T cells that favors signal integration during temporally separated contacts and promote cytokine production. As illustrated here, TEMPO is a versatile approach for dissecting temporal integration in defined signaling pathways.

Keywords: Biochemistry; Biological sciences; Immunological methods; Immunology.

Graphical abstract

Figure 1

TEMPO: a dedicated approach for programmable, multiplexed photoactivation sequences TEMPO (temporally patterned optogenetics) is an approach designed to characterize the impact of a multiple photoactivation sequences on cells expressing specific optogenetic actuators. The experimental design includes the plating of cells expressing an optogenetic actuator of interest in 96-well plates and the programming of up to 18 photoactivation sequences (controlling pulse durations and intervals) with a simple-to-use graphical interface. Blue-LED illumination is performed directly in the cell incubator. After the photoactivation and additional culture period if needed, cells and supernatants can be analyzed using a variety of assays including colorimetric assays, flow cytometry, or ELISA.

Figure 2

Defining the cellular response to photoactivation using the eOS1 calcium actuator (A) Generation of ZART T cells. The B3Z T cell hybridoma (expressing β−Galactosidase under the control of the NFAT elements of the IL-2 promoter) was retrovirally transduced to express the FRET-based calcium reporter Twitch2B and the eOS1 (enhanced OptoSTIM-1) calcium actuator. (B) Schematic representation of the consequence of photoactivation in ZART T cells, including calcium elevation, β-galactosidase activity, and cytokine production. (C and D) The calcium response triggered by a single round of photoactivation in ZART T cells was evaluated using live imaging using two-photon excitation. (C) Representative time-lapse images showing the kinetics of intracellular calcium elevation in ZART T cells following a 5s LED illumination using the TEMPO device. (D) The mean calcium signal was quantified over time, 5 min before and up to 60 min after photoactivation. Data are representative of 2 independent experiments. (E) Monitoring the calcium response after repeated photoactivation. ZART T cells were subjected to a single photoactivation (5s pulse) or two rounds of photoactivation (5s pulses) 1 h apart using TEMPO. Cell aliquots were collected at various time points after each photoactivation and analyzed by time-resolved flow cytometry to monitor calcium levels using the Twitch2B fluorescent reporter. Each cell aliquot was acquired for 15 min and immediately replaced by a new aliquot to generate an almost continuous signal curve by concatenation. Aliquot switching can results in minor changes in calcium index. Note that the responses after the first or the second photoactivation are similar in intensity and duration. Data are representative of 3 independent experiments. (F and G) Evaluating the minimal duration of calcium signal to elicit NFAT transcriptional activity in ZART T cells. (F) ZART T cells or control B3Z T cells were subjected to TEMPO using from 0 to 6 rounds of photoactivation every 20 min. (G) After 15 h, β-galactosidase activity was quantified in the cell lysate. Some ZART T cells were treatd with thapsigargin during the whole assay to estimate the maximal response and used to normalize the data (expressed as % of maximal response). Data are representative of 4 independent experiments.

Figure 3

Cells can integrate calcium signals separated by several hours ZART T cells were subjected to TEMPO to evaluate their capacity to integrate intermittent calcium signals. Cells were exposed (or not) to one or two pulses (5s) of photoactivation delivered with the indicated time interval. After 15 h, β-galactosidase activity was quantified in the cell supernatant. (A) Experimental set-up illustrating the different sequences of photoactivation programmed in the TEMPO device. (B) Bulk NFAT transcriptional responses were evaluated by monitoring β-galactosidase activity in cell lysates for the indicated photoactivation sequences. (C) Single-cell assessment of NFAT transcriptional activity using the flow cytometric FDG assay to reveal β-galactosidase activity. Results are representative of 4 independent experiments.

Figure 4

Integration of intermittent calcium signals promotes the production of multiple cytokines ZART T cells were subjected to TEMPO to evaluate cytokine production following intermittent calcium signals. Cells were exposed (or not) to one or two pulses (5s) of photoactivation delivered with the indicated time interval. After 15 h, cell supernatants were collected and subjected to a multiplex cytokine assay. (A) Experimental set-up illustrating the different sequences of photoactivation programmed in the TEMPO device. (B) Cytokine/chemokine (IL-2, IL-4, IL-5, IL-13, IL-18, GM-CSF, TNF-α, CCL5, and CCL3) concentrations measured with the indicated patterns of photoactivation. Results are representative of 2 independent experiments. ∗∗, p < 0.01; ∗∗∗, p < 0.001; ns, not significant (one-way ANOVA).