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.
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📋 Methods
Live cell imaging of CTL motility and NFAT-GFP nucleo-cytoplasmic shuttling
To examine CTL motility and NFAT activation at varying [Ca 2+ ] i , HA-CTL were transferred into medium with either low (5 mM) or high (100 mM) K + concentration and allowed to adhere to murine ICAM-1-Fc-coated (12.5 μg/ml, R&D Systems) plastic. High [K + ] e depolarizes the cell membrane and reduces the driving force for cytosolic Ca 2+ -influx upon cell activation. Cells were either left untreated or activated with 2 μM thapsigargin, cultured for 30 min to allow for stabilization of [Ca 2+ ] i at different concentrations, and then recorded by multiphoton live cell imaging for 10 minutes at 30 sec. intervals.
Calculation of NFAT memory time
HA-CTL were prestimulated for 30 min. with 1 μg/ml ionomycin and 50 ng/ml PMA or with splenocytes pulsed with 10 −9 M HA 515-523 peptide (1:4 ratio). Alternatively, cells were prestimulated with Concanavalin A (10μg/ml) for 15 min. Subsequent stimulation was carried out for 60 minutes in presence of BFA, along with 100μg/ml cycloheximide (CHX), 1 μM CsA, 100mM αMM or 50 μg/ml ActD. ActD was added 15, 30, 45, or 60 min. after BFA. Cells were then stained for intracellular IFN-γ and Egr2, and analyzed by flow cytometry. MP-IVM recordings of T CM and CTL in LNs and tumors All experiments were in accordance with NIH guidelines and were approved by the Institutional Animal Committees of Massachusetts General Hospital. Analysis of T-B cell interactions in LNs was carried out as previously described ( Mempel et al., 2006 ). Dorsal skinfold chamber (DSFC) tumors were grown with modifications from previously published techniques ( Fukumura et al., 1998 ). Two aliquots of 10 6 H2B-Cerulean-expressing tumor cells were subcutaneously injected in the backs of mice, the first proximal to the neck (to ensure continued robust lymphatic drainage of tumor tissue after installation of DSFCs over the second tumor), the second ~ 1.5 cm left of the dorsal midline approximately halfway from the neck to the tail base. 5–7 days later, 5 × 10 6 NFAT-GFP- and H2B-mRFP-expressing HA-CTL were injected i.v.. The following day, DSFCs were installed in a way that the distal tumor was centered in the imaging chamber and accessible to longitudinal investigation by MP-IVM, which was performed under general anesthesia with Ketamine and Xylazine on days 2, 5, and 7 after CTL transfer. Blood plasma was visualized by i.v. injection of 2 μl (4 pmol) of QTracker 655 quantum dot suspension (Invitrogen). Some mice were injected i.v. with 10 6 CD4 + CD25 + HA-T reg cells purified from pgk-HAxTCR-HA mice by immunomagnetic selection (Miltenyi Biotech) one day before tumor implantation.
Show full methods section
Live cell imaging of CTL motility and NFAT-GFP nucleo-cytoplasmic shuttling
To examine CTL motility and NFAT activation at varying [Ca 2+ ] i , HA-CTL were transferred into medium with either low (5 mM) or high (100 mM) K + concentration and allowed to adhere to murine ICAM-1-Fc-coated (12.5 μg/ml, R&D Systems) plastic. High [K + ] e depolarizes the cell membrane and reduces the driving force for cytosolic Ca 2+ -influx upon cell activation. Cells were either left untreated or activated with 2 μM thapsigargin, cultured for 30 min to allow for stabilization of [Ca 2+ ] i at different concentrations, and then recorded by multiphoton live cell imaging for 10 minutes at 30 sec. intervals.
Calculation of NFAT memory time
HA-CTL were prestimulated for 30 min. with 1 μg/ml ionomycin and 50 ng/ml PMA or with splenocytes pulsed with 10 −9 M HA 515-523 peptide (1:4 ratio). Alternatively, cells were prestimulated with Concanavalin A (10μg/ml) for 15 min. Subsequent stimulation was carried out for 60 minutes in presence of BFA, along with 100μg/ml cycloheximide (CHX), 1 μM CsA, 100mM αMM or 50 μg/ml ActD. ActD was added 15, 30, 45, or 60 min. after BFA. Cells were then stained for intracellular IFN-γ and Egr2, and analyzed by flow cytometry. MP-IVM recordings of T CM and CTL in LNs and tumors All experiments were in accordance with NIH guidelines and were approved by the Institutional Animal Committees of Massachusetts General Hospital. Analysis of T-B cell interactions in LNs was carried out as previously described ( Mempel et al., 2006 ). Dorsal skinfold chamber (DSFC) tumors were grown with modifications from previously published techniques ( Fukumura et al., 1998 ). Two aliquots of 10 6 H2B-Cerulean-expressing tumor cells were subcutaneously injected in the backs of mice, the first proximal to the neck (to ensure continued robust lymphatic drainage of tumor tissue after installation of DSFCs over the second tumor), the second ~ 1.5 cm left of the dorsal midline approximately halfway from the neck to the tail base. 5–7 days later, 5 × 10 6 NFAT-GFP- and H2B-mRFP-expressing HA-CTL were injected i.v.. The following day, DSFCs were installed in a way that the distal tumor was centered in the imaging chamber and accessible to longitudinal investigation by MP-IVM, which was performed under general anesthesia with Ketamine and Xylazine on days 2, 5, and 7 after CTL transfer. Blood plasma was visualized by i.v. injection of 2 μl (4 pmol) of QTracker 655 quantum dot suspension (Invitrogen). Some mice were injected i.v. with 10 6 CD4 + CD25 + HA-T reg cells purified from pgk-HAxTCR-HA mice by immunomagnetic selection (Miltenyi Biotech) one day before tumor implantation.
Analysis of NFAT nucleocytoplasmic shuttling
The spatial overlap between NFAT-GFP and H2B-mRFP signals was used to calculate the NFAT signaling index (SI) as a measure of NFAT nuclear accumulation (See also Figure S2 ). To this end, centroids of H2B-mRFP-stained cell nuclei were measured in Imaris (Bitplane) and used to define the nuclear center. Normalized 2D-radial profile integrated fluorescence intensity histograms were then generated for EGFP and mRFP signals, using ImageJ and the radial profile plug-in ( http://rsbweb.nih.gov/ij/plugins/radial-profile.html ), to assess the distribution of each reporter as a function of distance from nuclear centers. In individual recordings we found that in non-signaling cells with visually determined nuclear exclusion of NFAT-GFP, this overlap was 30–40% (due to deviation from perfect sphericity of most nuclei as well as the 3D geometry of cells), while full nuclear accumulation yielded 100% overlap. To adjust for slight variability between different experiments we arbitrarily defined an SI of 0 to represent the 1st percentile of the distribution of overlap values obtained from an individual recording, while 100% overlap was defined as an SI of 1. All annotation of NFAT signaling status was automated through ImageJ scripting and validated visually.
Statistical analysis
For normally distributed data, experimental groups were compared through Student’s t test. Otherwise, Mann-Whitney test was used.
Supplementary Material 01 02 03 04 05 06 07 08 09 10 11
📊 Figures
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 (u201cNFAT-GFPu201d). TAD: N-terminal transactivation d...
Fig. 2
Kinetics of NFAT nucleocytoplasmic shuttling in T cells in vivo. ( A ) Experimental setup: HA-T CM expressing NFAT-GFP (green) and H2B-mRFP (red) were transferred i.v. into mice with 7-day-old CT26 tu...
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...
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 ...
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 ...
Fig. 6
Rising intracellular [Ca 2+ ] 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) concent...
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 ...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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