A Novel ZAP-70 Dependent FRET Based Biosensor Reveals Kinase Activity at both the Immunological Synapse and the Antisynapse

Many hypotheses attempting to explain the speed and sensitivity with which a T-cell discriminates the antigens it encounters include a notion of relative spatial and temporal control of particular biochemical steps involved in the process. An essential step in T-cell receptor (TCR) mediated signalling is the activation of the protein tyrosine kinase ZAP-70. ZAP-70 is recruited to the TCR upon receptor engagement and, once activated, is responsible for the phosphorylation of the protein adaptor, Linker for Activation of T-cells, or LAT. LAT phosphorylation results in the recruitment of a signalosome including PLCγ1, Grb2/SOS, GADS and SLP-76. In order to examine the real time spatial and temporal evolution of ZAP-70 activity following TCR engagement in the immune synapse, we have developed ROZA, a novel FRET-based biosensor whose function is dependent upon ZAP-70 activity. This new probe not only provides a measurement of the kinetics of ZAP-70 activity, but also reveals the subcellular localization of the activity as well. Unexpectedly, ZAP-70 dependent FRET was observed not only at the T-cell -APC interface, but also at the opposite pole of the cell or “antisynapse”

Granzyme B-td TOMATO, un nouvel outil fluorescent pour le suivi de la cytolyse chez la souris

La fonction de cytolyse est un mécanisme majeur des effecteurs du système immunitaire pour éliminer les cellules infectées ou tumorales. Cette fonction associe l'activité de la perforine, qui forme des pores dans la membrane d'une cellule cible, à la sécrétion de protéases: les granzymes. Ces dernières sont des molécules pro-apoptotiques qui induisent la mort de la cellule cible. Les granzymes et en particulier granzyme B ciblent plusieurs voies intracellulaires complémentaires pour assurer l'efficacité de la cytolyse. Or il est difficile d'observer directement la fonction de cytolyse au cours de réponse immunitaire in vivo dans des conditions physiologiques. Dans les travaux présentés dans cette thèse, nous avons développé un nouveau modèle qui permet de suivre la fonction de cytolyse en temps réel par l'expression d'une protéine de fusion fluorescente GZMB-tdTomato. Les résultats obtenus par expression rétrovirale ont montré que la protéine de fusion est correctement exprimée dans les vésicules cytolytiques qui deviennent fluorescentes. Dans un second temps, nous avons réalisé un nouveau modèle murin qui exprime GZMB-tdTomato de manière substituée au GZMB natif par recombinaison homologue (Knock In). Nous avons mis en évidence que la protéine de fusion conserve l'activité catalytique de la protéine native et ses caractéristiques (conditions d'expression, de maturation, de sécrétion et demeure active après le passage dans la cellule cible lors de la cytolyse). En utilisant un modèle murin exprimant un TCR transgénique nous avons pu suivre le déroulement de la fonction de cytolyse de lymphocytes cytotoxiques en temps réel par video microscopies.Cytolysis is a major function used by the immune system's effectors to kill infected or tumor cells. Cytolysis depends on the pore forming protein perforin and the secretion of proteases of the granzyme family. Granzymes, including granzyme B (GZMB) have pro-apoptotic features and induce target cell death. Several complementary pathways are triggered by granzymes to ensure efficient cytolysis. It remains difficult to directly observe cytolysis during in vivo immune responses under physiological conditions. In this PhD we developed a new model to visualize cytolytic function in real time by expression of a fusion protein: GZMB-tdTomato. Results obtained from retroviral transduction showed that the fusion protein is correctly expressed in cytolytic vesicles, which became fluorescent. We then constructed a new mouse model by homologous recombination (Knock In) that express GZMB-tdTomato substituted for the native GZMB. The fusion protein conserves the catalytic activity of GZMB and its features (expression, maturation, secretion conditions) and remains active after its passage into target cells. Using TCR transgenic OTI cells, we followed the sequence of events of cytolysis from lymphocytes in real time by videomicroscopy. We also observed the cytolytic vesicles relocalization towards the cell contact zone and the death of target cell by cytolysis. Finally, we studied in vivo differentiation of naïve lymphocyte to cytolytic effector cells (the acquisition of cytolysis) and target cell death after bacterial infection

Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

<div><p>To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells’ plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granules to the synaptic contact zone. A perforin-dependent step was next visualized by the fluorescence signal from the non-permeant dye TO-PRO-3 at the synaptic cleft, minutes before the labeling of the target cell nucleus, characterizing a previously undescribed synaptic event in CTL cytolysis. Transferred OVA-specific GZMB-Tom-expressing CD8 T cells acquired GZMB-Tom expression in Listeria monocytogenes-OVA infected mice as soon as 48h after infection. These GZMB-Tom positive CD8 T cells localized in the splenic T-zone where they interacted with CD11c positive dendritic cells (DC), as shown by GZMB-Tom granule redistribution to the T/DC contact zone. GZMB-Tom-KI mice thus also provide tools to visualize acquisition and activation of cytolytic function in vivo.</p></div

In vivo acquisition of GZMB-Tom expression by GZMB-Tom-KI/KI-KI-OT1 CD8 T cells in response to infection.

<p>Naive CD45.1 B6 mice were used as recipients of 3.10⁶ naive CD8 T cells from GZMB-Tom-KI/KI-OT1 CD45.2 mice. Mice were i.v. injected 11h later with 10.000 U Listeria-OVA. Immunohistology (see Materials and Methods) on a spleen section from mice collected 48h after infection shows staining of transferred GZMB-Tom-KI CD8 T cells (CD45.2, yellow) with GZMB-Tom (red) and either staining for B cells (B220, blue) (A) or for CD11c (cyan) and F4.80 (purple) (B). Magnifications highlight the location of the GZMB-Tom containing granules.</p

Comparison of T and NK cell composition in the lymphoid organs of C57BL/6 and GZMB-Tom-KI mice.

<p>FACS analysis of CD4 and CD8 T lymphocytes in spleen, lymph nodes (LN) and thymus (A, B) and of NK cells in spleen (B) from C57BL/6 (WT) and from GZMB-Tom-KI/KI mice. In B, level of GZMB-Tom expression is shown on CD4 and CD8 T lymphocytes and on NK cells (spleen) or CD4CD8 double positive (DP) cells (thymus) from WT (filled in grey) and GZMB-Tom-KI (black line) mice. In C, splenocytes were cultured with 1000U/ml IL-2 and analyzed before culture (D0) and 3 (D3) and 4 (D4) days later for GZMB-Tom expression on NK cells, defined as NK1.1+ CD3- cells. One representative experiment of at least two experiments is shown.</p

Visualization of the kinetics of CTL activation, granule relocalization and target cell death.

<p>OT1 CTL from GZMB-Tom-KI/KI (A), WT (B) or Perf-KO-GZMB-Tom-KI/KI (C) prepared as in Fig. 7 were labeled with 2.5 µM Fluo-4. For video microscopy analysis, RMA-S target cells pre-incubated with 1 µM OVA peptide and loaded with 1 µM Calcein AM were deposited onto poly-lysine activated Labtek wells, before the addition of the Fluo-4 labeled CTL as described in Materials and Methods. TO-PRO-3 was present in the medium (see Materials and Methods). Fluorescence signals from GZMB-Tom (A, C), Fluo-4 (reported as rainbow RGB false color) and calcein (cyan) as well as brightfield (A, B, C) were recorded every 12.5 sec for around 90 min. Image J or ZEN software was used for image analysis. Images reporting all 4 signals (upper A; upper C), GZMB-Tom (red), TO-PRO-3 (green) and brightfield (lower A; lower C) or 3 signals (upper B) and TO-PRO-3 (green) and brightfield (lower B) are shown for selected times. Videos are provided as Supporting Information. Statistical analysis of the different events observed in those videos are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067239#pone.0067239.s005" target="_blank">Fig.S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067239#pone.0067239.s006" target="_blank">S6</a>.</p

Analysis of GZMB-Tom-KI distribution in CTL by confocal microscopy.

<p>GZMB-Tom-KI CTL were produced from LN T cells by a-CD3/CD28 stimulation (as in Fig. 3) followed by IL-2 expansion for analysis at day 8 of culture and treated as described in Materials and Methods. A: Cells were labeled with anti-CD3 (17A2-A647) before fixation and single fluorescence of tdTom (red) or anti-CD3 (green), as well as merged fluorescence images are shown. B-D: Cells were fixed and permeabilized for labeling with antibodies diluted in Saponin buffer. Labeling with a-CD107a (Lamp-1, 1D4B-A488, green) and a-GZMB (MHGB05-APC, blue) (B, D) or a-GZMA (3GB8.5-FITC, green) and a-GZMB (blue) (C) is shown as well as tdTom fluorescence (red) (B-D). In (D) CTL were derived from GZMB-Tom-KI/KI-OT1 mice (see Methods) and incubated for 25 min at 37°C with RMA-S target cells loaded with the OVA peptide (10⁻⁶ M). Histograms of an RGB line profile show colocalization of tdTom (red), a-Lamp-1 (green) and a-GZMB (blue). A-D: For evaluation of colocalization, variations of two fluorescence intensities were compared and Rr Pearson's coefficients were calculated (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067239#pone.0067239-Mouchacca1" target="_blank">[39]</a>, and see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067239#pone.0067239.s003" target="_blank">Fig.S3</a>). Rr = 0 (no co-localization) for CD3/tdTom (A); Rr around 0.65 (good colocalization) for tdTom/Lamp-1, Lamp-1/GZMB and tdTom/GZMB (B); Rr = 0.45 for both tdTom/GZMA and GZMA/GZMB and 0.60 for tdTom/GZMB (C); Rr around 0.7 for all 3 couples tdTom/Lamp-1, Lamp-1/GZMB and tdTom/GZMB in the CTL/target conjugates (D).</p

Protease activity of GZMB-Tom degranulated from GZMB-Tom-KI/KI CTL.

<p><b>A</b>: OT1 CTL from WT, GZMB-Tom-KI/KI (GZMB-Tom), GZMB-KO, Perf-KO and Perf-KO-GZMB-Tom-KI/KI (Perf-KO/GZMB-Tom) mice were harvested 6 days after stimulation as in Fig. 5. 210⁶ cells in 100 µl medium were incubated in medium (M) or were activated with Ionomycin 2 µg/ml and PMA 50 ng/ml (I+P) or with coated a-CD3 (CD3) for 4h to induce CTL degranulation. Supernatants were assayed for GZMB protease activity after GZMB capture on a-GZMB-coated plates (kit QuickZyme Biosciences) according to the <i>manufacturer’s protocol</i>. GZMB activity is reported as optical density at 405 nm for sample minus background. One experiment representative of three with similar results is presented. Statistics are shown for values of GZMB activity measured after activation with I+P or with a-CD3 versus medium for each CTL, as well as for genetically-modified CTL versus WT CTL for either I+P (green lines) or a-CD3 (blue lines) activation. P values as in Fig. 5. <b>B and C</b>: GZMB-Tom activity measured in EG7-DEVD target cells during incubation with CTL. OT1 CTL from WT, GZMB-Tom-KI/KI (GZMB-Tom), Perf-KO-GZMB-Tom-KI/KI (Perf-KO/GZMB-Tom) and GZMB-KO mice were prepared from LN CD8 T cells by antigenic stimulation in culture and expansion with IL-2 (see Materials and Methods). Targets cells are EG7-DEVD, EL4 cells expressing OVA as well as the FRET-based fluorescent probe CFP-DEVD-YFP (see Materials and Methods). EG7-DEVD cells were incubated alone or in the presence of the various types of CTL (+ CTL) at 1/1 ratio (10⁵ cells each) for 1h at 37°C. FACS analysis represents the FRET fluorescence of the probe versus CFP fluorescence with discrimination of two separate zones on the diagonal (see Materials and Methods). The upper zone represents the FRET signal of the un-cleaved probe, while the lower one corresponds to FRET disruption after cleavage of the probe. The % of cells in the deFRET zones is indicated on the graphs (B). Statistical analyses from 2 experiments at effector to target ratios from 0.3–3/1 are shown in (C) as % deFRET with deFRET for the WT CTL set at 100%. Results are from more than 3 experiments for WT and GZMB-Tom CTL, and two experiments for GZMB-KO and Perf-KO-GZMB-Tom CTL. Statistics are shown for % deFRET comparing WT, GZMB-KO and Perf-KO/GZMB-Tom CTL with GZMB-Tom CTL. P values as in Fig. 5.</p

Lamp-1 externalization and GZMB-Tom degranulation during CTL activation.

<p>OT1 CTL from B6 (WT) and from GZMB-Tom-KI/KI mice were prepared from LN CD8 T cells by antigenic triggering and expanded with IL-2 as described in Materials and Methods. To measure Lamp-1 externalization, 10⁵ CTL were stimulated for 2h at 37°C with RMA-S target cells pre-loaded with the relevant OVA peptide (Rel P) at different concentrations (10⁻⁶–10⁻¹² M) or Irrelevant peptide (Irr P) at 10⁻⁶ M. Effector to target ratio was 3/1. a-Lamp-1 Ab was added to the activation medium (see Materials and Methods). FACS analysis was performed on cells gated as CD8 positive (CTL). One experiment with concordant duplicates is shown, and is representative of at least 3 experiments. A: Overlays of the analysis are represented for Lamp-1 versus GZMB-Tom for WT and GZMB-Tom-KI/KI OT1 CTL. <b>B</b> and <b>C</b>: Quantification of the experiments as % CTL positive for lamp-1 mAb uptake (B) and CTL tdTom Mean Fluorescence Intensity (MFI) (C). D: Overlays of the analysis are represented for all cells including CTL and RMA-S cells and are plotted as CD8 versus GZMB-Tom fluorescence. Quantification of the experiments gated on the RMA-S target cells as % cells positive for tdTomato (E) and tdTom MFI (F). Statistics are shown for values of Lamp-1 externalization for CTL incubated with RMA-S targets pre-loaded with different concentrations of relevant peptide versus irrelevant peptide (C), as well as for acquisition of tdTomato fluorescence as % (E) and as MFI (F) by RMA-S targets pre-loaded with different concentrations of relevant peptide versus irrelevant peptide. P<0.01 (**); P<0.05 (*); P>0.05 (NS) (see Materials and Methods).</p

GZMB-Tom and GZMB expression as a function of T cell division in culture.

<p>T cells purified from LN of C57Bl/6 (WT) or GZMB-Tom-KI/KI mice pre-labeled with 2.5 µM CTV were cultured (0.3 10⁶/well) on costar plates (24-well) pre-coated with anti-CD3 10 µg/ml with additional soluble anti-CD28 (2 µg/ml final) (a-CD3/28) or not (a-CD3). <b>A</b>: GZMB-Tom expression (tdTom) is shown in CD4 and CD8-gated T cells before (Unactivated) and after 20h, 40h or 72h in culture as a function of cell division (CTV). <b>B</b>: In parallel, GZMB expression as detected with anti-GZMB mAb (GZMB) on fixed and permeabilized cells is shown before (Unactivated) and 72h after culture with anti-CD3 or anti-CD3/CD28 as in (A). One experiment representative of 3 independent experiments is shown.</p