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. 2013 Feb 21;38(2):237-49.
doi: 10.1016/j.immuni.2012.09.012. Epub 2013 Jan 11.

The Transcription Factor NFAT Exhibits Signal Memory During Serial T Cell Interactions With Antigen-Presenting Cells

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The Transcription Factor NFAT Exhibits Signal Memory During Serial T Cell Interactions With Antigen-Presenting Cells

Francesco Marangoni et al. Immunity. .
Free PMC article

Abstract

Interactions with antigen-presenting cells (APCs) interrupt T cell migration through tissues and trigger signaling pathways that converge on the activation of transcriptional regulators, including nuclear factor of activated T cells (NFAT), which control T cell function and differentiation. Both stable and unstable modes of cognate T cell-APC interactions have been observed in vivo, but the functional significance of unstable, serial contacts has remained unclear. Here we used multiphoton intravital microscopy in lymph nodes and tumors to show that while NFAT nuclear import was fast (t(1/2 max)∼1 min), nuclear export was slow (t(1/2)∼20 min) in T cells. During delayed export, nuclear NFAT constituted a short-term imprint of transient TCR signals and remained transcriptionally active for the T cell tolerance gene Egr2, but not for the effector gene Ifng, which required continuous TCR triggering for expression. This provides a potential mechanistic basis for the observation that a predominance of unstable APC interactions correlates with the induction of T cell tolerance.

Figures

Fig. 1
Fig. 1
NFAT-GFP nuclear translocation is a sensitive readout of TCR triggering. (A) Domain structure of full-length murine NFAT1 and NFAT1(1-460)-GFP (“NFAT-GFP”). TAD: N-terminal transactivation domain. The 6-aa linker (DPPVAT) is shown in orange. (B) Frequency of HA-CTL expressing both NFAT-GFP and H2B-mRFP after transduction and selection. Similar results were obtained for HA-TCM. (C) HA-CTL expressing NFAT-GFP (green) and H2B-mRFP (red) were co-cultured with B cells pulsed with 10 μM HA peptide or not (Ctrl). Scale bar=10 μm. (D) Percentage of HA-CTL (red symbols) and HA-TCM (black symbols) with visually scored nuclear NFAT upon exposure to B cells pulsed with a range of peptide doses. Each data point represents ≥100 cells. Lines are sigmoid curve-fits. Numbers are EC50 values. One experiment representative of two is shown.
Fig. 2
Fig. 2
Kinetics of NFAT nucleocytoplasmic shuttling in T cells in vivo. (A) Experimental setup: HA-TCM expressing NFAT-GFP (green) and H2B-mRFP (red) were transferred i.v. into mice with 7-day-old CT26 tumors implanted in the dorsal foot in order to maximize TCM recruitment to the draining popliteal LN. Two days later, target B cells pulsed with HA-peptide (blue) or not (white) were injected i.v., and MP-IVM was started immediately. (B) Intravital micrograph depicting a typical LN preparation. Collagen visualized through second harmonic generation (blue) outlines the LN capsule on the left. Scale bar=50 μm. See also movie S1. (C and D) Enlarged image sequences from the same (C) and a similar recording (D) as in B, showing NFAT localization in HA-TCM upon contact (arrow) with HA peptide-pulsed B cells (C) and upon interruption (arrow) of contact (D). Arrowheads in D identify disengaged TCM. See also movie S2. Time in min:sec. Scale bar=10 μm. (E) Representative traces of HA-TCM depicting the color-coded NFAT SI and the distance (between cell centroids) to the nearest HA peptide-pulsed (Ag+) or control B cell. Dashed lines indicate 8 μm of distance, at which physical contact was typically observed. Grey symbols represent ambiguous measurements when the recorded TCM made contact with both control and HA-peptide-pulsed B cells. Traces selected from 581 recorded in 7 movies from 2 independent experiments. (F) NFAT kinetics in vivo. For nuclear import, t=0 represents the time of initial contact with an Ag+ B cell (11 tracks). For nuclear export, t=0 represents the time of detachment from an Ag+ B cell (18 tracks at t=0). Mean±SEM of NFAT signaling indices are shown. Red lines are sigmoid curve-fits; t1/2 max: Mean time to half-maximal response.
Fig. 3
Fig. 3
NFAT nuclear export after calcineurin blockade in vitro. (A) Experimental design. (B) Image sequence of an HA-CTL expressing NFAT-GFP (green) and H2B-mRFP (red) conjugated to HA-expressing CT26 tumor cells (expressing H2B-Cerulean, blue) after addition of CsA. See also movie S3. Time in min:sec. Scale bar=10 μM. (C) Average NFAT signaling indices over time, in presence (red symbols) or absence (black symbols) of CsA. Error bars represent SD. Lines show sigmoid curve-fits. One experiment representative of two is shown.
Fig. 4
Fig. 4
NFAT is transcriptionally active during nuclear export and promotes Egr2, but not Ifng expression. (A) Experimental design to measure NFAT-dependent transcription after PMA and ionomycin stimulation and calcineurin inactivation. BFA: brefeldin A; ActD: actinomycin D; CHX: cycloheximide. (B) Intracellular IFN-γ in CTL treated with CHX, CsA, or with ActD starting at different time-points during the measurement phase. Dashed line indicates background expression after addition of ActD at time 0. Mean ± SD of triplicates is shown. (C) “NFAT memory time” is derived by relating expression above background of IFN-γ after CsA (red symbol) to expression during various time intervals of stimulation in absence of CsA, as shown in B. (D) Experimental design to measure NFAT-dependent transcription of IFN-γ and Egr2 after blockade of TCR triggering. ConA: concanavalin A; αMM: α-methyl mannoside. (E) Intracellular IFN-γ after treatment with CHX, CsA, αMM, or with ActD starting at different time-points during the measurement phase. (F) Quantification of “NFAT memory time” for Ifng based on expression above background as shown in E. (G, H) Intracellular Egr2 and “NFAT memory time” for Egr2 in the same experimental setting as shown for IFN-γ in E and F. MFI* is the product of percentage and MFI of IFN-γ or Egr2 in cells. Mean and SD of triplicates from one representative experiment out of 3 is shown. P-value is derived through Student’s t test. n.s.=not significant; * = p<0.05.
Fig. 5
Fig. 5
Rapid activation and NFAT memory in CTL during tumor rejection. (A) Experimental design. (B) Intravital micrograph from the stroma-parenchyma border in a CT26HA tumor 5 days after injection of HA-CTL expressing NFAT-GFP (green) and H2B-mRFP (red). Tumor cells express H2B-Cerulean (blue). Blood plasma was visualized through i.v.-injected quantum dots (white). Scale bar=50 μm. See also movies S4 and S6. (C and D) Image sequences from similar recordings as shown in B. (C) Rapid nuclear translocation of NFAT in an HA-CTL upon contact with an HA-expressing tumor cell. See also movie S5. (D) Robust migration of an HA-CTL with nuclear NFAT. Time in min:sec. Scale bar=10 μm in both C and D. (E) Correlation of NFAT activation (color-coded NFAT SI) and cell motility in CTL in the tumor parenchyma. Traces are selected from 269 (15 and 254 from CT26 and CT26HA tumors, respectively) recorded in 2–8 movies per condition in 2 independent experiments. (F) Population analysis of CTL instantaneous velocity and NFAT activation in the tumor parenchyma. Dot plots show data from one representative movie per condition, recorded 5 days after CTL transfer. Numbers in grids represent percentages in sectors.
Fig. 6
Fig. 6
Rising intracellular [Ca2+] leads to CTL deceleration before inducing NFAT nuclear translocation. (A) NFAT-GFP-expressing CTL were cultured in medium containing low (5 mM) or high (100 mM) concentrations of K+ and treated (or not) with Thapsigargin (TG) for 30 min. Then, with TG present, their migration on ICAM-1-coated coverslips as well as NFAT activation was recorded for 10 min. by live cell imaging. (B) Representative micrographs of all conditions tested. (C) CTL migratory tracks. Color-code represents NFAT signaling index. Circles highlight tracks of immobilized CTL without NFAT activation. (D) Correlation of cell motility and NFAT signaling index. One representative experiment out of 2 is shown.
Fig. 7
Fig. 7
Comparable NFAT activation through stable and unstable CTL-APC contacts in tumor tissue. (A) Experimental design: Prior to tumor implantation, mice were seeded with HA-T reg cells. (B) Image sequence showing HA-CTL expressing NFAT-GFP (green) and mCherry (red) sequentially interacting with at least three different tumor cells (marked 1, 2, 3). Note that NFAT stays nuclear between cell contacts, evident in insets showing magnified views of the CTL. See also movie S9. Time in min:sec. Scale bar=20 μm. (C) Correlation of NFAT activation (color-coded NFAT SI) and motility in HA-CTL in the tumor parenchyma in presence or absence of HA-T reg cells. Traces are selected from 420 (254 and 166 in absence and presence of HA-T reg cells, respectively) recorded in 8–9 movies per condition, from 2 independent experiments. (D) Distribution of instantaneous NFAT activation states in HA-CTL infiltrating the tumor parenchyma in presence (red lines) or absence (black lines) of HA-T reg cells at the indicated time-points after CTL transfer. Numbers indicate percentage of events with NFAT SI>0.75. Data are pooled for each time-point and condition. (E) Arrest coefficient (AC) analysis of CTL track segments defined by continuous NFAT activation (SI>0.75 for >2 min) at the indicated time-points in absence (back symbols) or presence (red symbols) of HA-T reg cells. Each symbol represents an individual track segment, bars represent medians. The shaded area highlights data points representing non-arrested cells (AC<0.7). P values were determined by Mann-Whitney test. Pooled data represent all ‘NFATon segments’ in two to three movies per condition and time-point, collected in two independent experiments. (F) Graphic summary of data in E. (G) NFAT Signaling x Motility Index analysis in HA-CTL infiltrating the tumor parenchyma.

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