Granzyme B-tdTomato, a new probe to visualize cytolytic effector cell activation

Authors


An important function of the immune system consists in eliminating infected or transformed cells. Naive CD8+ T lymphocytes differentiate in peripheral lymphoid organs following a first antigen contact. There they acquire the different constituents of the cytolytic machinery and become cytolytic T lymphocytes (CTLs), before migration to the tissues where they meet their specific target. Target cell killing is mediated by the release of granules expressing the Lamp-1 marker 1 and containing effector proteins including perforin 2, 3 and granzymes (granzyme A (GZMA) and B (GZMB) being the main proteases). Effective target cell lysis depends on many factors; so deciphering the mechanisms involved is important, in particular to palliate the failings of the immune system during tumor development.

Transient labeling of acidic granules with Lysotracker has elegantly been used to analyze kinetics of granule polarization in CTL/target conjugates. Intracellular staining of fixed and permeabilized cells has allowed elucidation of important steps of CTL granule movements, fusion and degranulation 4–6. In order to develop a fluorescent probe that would stably label the contents of cytolytic granules in living cells, we designed a construct encoding a fusion protein composed of an N-terminal GZMB, a 12 amino-acid linker and a C-terminal tdTomato (tdTom) (excitation: 554 nM, emission: 581 nm, stable at the acidic pH of the granules (pKa 4.7) 7, GZMB-tdTom). This was inserted in the retroviral expression vector MSCV-IRES-HuCD2t (Supporting Information Fig. 1).

We first transduced a T-cell hybridoma (HybT) and obtained stable expression of GZMB-tdTom in granules co-expressing GZMB and Lamp-1 (Supporting Information Fig. 2–5). Immunoblots revealed the fusion protein GZMB-tdTom at 85 kDa and tdTom at 55 kDa MW, as expected (Supporting Information Fig. 4). GZMB enzymatic activity could be detected in GZMB-tdTom-HybT cells, albeit at a low level as compared with that in CTLs (Supporting Information Fig. 5D). Whether this results from incomplete processing of the protein in HybT cells requires further investigation (Supporting Information Fig. 5D).

To address more physiological conditions, we transduced normal CD8+ CTLs with the GZMB-tdTom construct (Supporting Information Fig. 6). As observed by confocal microscopy, the GZMB-tdTom fusion protein was localized in granules (Fig. 1A). Co-localization between GZMB-tdTom, Lamp-1 and GZMB was observed in granules of CTLs alone (Fig. 1B-i) in CTL/antigenic target conjugates (Fig. 1B-ii) that had re-localized the red granules to the cell–cell contact zone, and in conjugates of CTLs with targets presenting control peptide (Fig. 1B-iii). Redistribution of granules to the CTL/target contact zone was observed in 80% of antigen-specific versus 5% of non-specific conjugates (Supporting Information Fig. 7B). TdTom-transduced cells expressed red tdTom protein spread throughout the cytoplasm (Fig. 1B-iv) and similarly to untransduced CTLs (Supporting Information Fig. 7A) relocalized GZMB-containing granules expressing Lamp-1 to the CTL/target contact zone (Fig. 1B-iv). Mathematical analyses showed that GZMB-tdTom colocalized with Lamp-1 and GZMB (Pearson's Rr coefficient around 0.55) whereas tdTom did not show any colocalization (Rr 0.1) (Supporting Information Fig. 7C).

Figure 1.

GZMB-tdTom-transduced P14-TCR CTLs express the fusion protein in vesicles/granules that redistribute in CTL/target cell conjugates. Purified Hu-CD2+ GZMB-tdTom-transduced P14-TCR CTLs were used as described in Supporting Information. Targets were RMA-S cells loaded with antigenic (gp33) or control (HY) peptide (10–6 M). (A and B) CTL-target conjugates were analyzed after 30 min at 37°C. Bars = 5 μm. (A) Confocal analysis on fixed GZMB-tdTom-transduced CTLs, alone (left) or conjugated to an antigenic target (t, right). (B) Confocal analysis on fixed and permeabilized cells: GZMB-tdTom-transduced CTLs (i) alone, (ii) conjugated with antigenic or (iii) control targets (t, shown in brightfield); and (iv) TdTom-transduced CTLs conjugated with antigenic targets. Fluorescence is shown for tdTom (red), a-Lamp-1 (green) and a-GZMB (blue). For statistics on colocalization between GZMB-tdTom, anti-Lamp-1 and anti-GZMB fluorescence see Supporting Information Fig. 7A and B. (C) i and ii Antigenic (gp33) or (iii and iv) control (HY) targets were deposited on slides. GZMB-tdTom-transduced TCR-P14 CTLs loaded with Fluo-4 (see Supporting Information) were then deposited. Signals for (i and iii) green Fluo-4 (Ca++ flux) and (ii and iv) red fluorescence (GZMB-tdTom) were recorded for 30–40 min by time lapse video microscopy (Zeiss Meta 560). Images at the indicated time points are shown; for the complete videos see the Supporting Information. For quantification of the images, see Supporting Information Fig. 7D. Analysis and relative quantification of all the images were performed with Image J software. The data are representative of three experiments.

Following TCR/antigen engagement, calcium flux and PKC activation are important signals for gene activation and granule migration to the CTL/target contact zone preceding degranulation 4, 8. CTLs preloaded with Fluo-4 were used to monitor by video microscopy the Ca++ fluxes and the redistribution of GZMB-tdTom-containing granules. When GZMB-tdTom-transduced P14-TCR CTLs faced a specific target, an attachment signal preceded a rapid Ca++ flux (10–20 s) and granule translocation to the contact zone occurring at various times (20–480 s) (Fig. 1C-i and ii, Supporting Information Fig. 7D, Video 1). No significant signal was observed when the CTLs were facing control targets (Fig. 1C-iii and iv, Video 2). These kinetics are in agreement with published studies using CTL clones 6, 9.

We used the Lamp-1 exposure method to assess CTL degranulation in response to antigenic stimulation and to observe the fate of GZMB-tdTom during that process. GZMB-tdTom-transduced P14-TCR CTLs exposed Lamp-1 in response to gp33-loaded RMA-S, the extent of degranulation being dependent on peptide concentration (Fig. 2A). The percent of GZMB-tdTom fluorescent CTLs markedly decreased (from 20% for non-stimulated or control-peptide stimulated CTLs to 13% for CTLs activated with 10−6 M gp33-loaded RMA-S), with a level of GZMB-tdTom fluorescence much lower in Lamp-1–positive (MRFI 422 (MRFI, mean relative fluorescence intensity)) as compared to Lamp-1–negative (607) CTLs. GZMB expression as measured on fixed and permeabilized cells were also reduced (about 50%) in the antigen-activated CTLs (data not shown). These results suggest that the whole GZMB-tdTom fusion protein was released during degranulation. Similarly, analysis of GZMB-tdTom-transduced OT1-TCR-Gzmb-KO (Gzmb, GZMB-encoding gene) CTLs, in which the only source of GZMB is GZMB-tdTom, showed that expression of GZMB-tdTom as well as GZMB was markedly decreased upon CTL activation with OVA-expressing cells (Supporting Information Fig. 8). We also found that the capacity of GZMB-tdTom-transducted P14-TCR CTLs to kill specific targets was not affected as compared to that of untransduced CTLs (Fig. 2B).

Figure 2.

Functional characteristics of GZMB-tdTom-transduced CTLs. (A) Release of GZMB-tdTom during CTL/target interaction. GZMB-tdTom-transduced P14-TCR CTLs were analyzed for antigen-induced Lamp-1 exposure when stimulated with RMA-S targets loaded with 10–6, 10–8,10–10 and 10–12 M gp33 antigenic peptide or 10–6 M HY control peptide for 1 h (1/1 CTL/target ratio). FACS analysis was performed on live cells gated on P14-TCR(Vα2)-CD8+ T cells and red fluorescence versus exposed Lamp-1 are shown. Data are representative of five experiments. (B) CTL activity of untransduced or GZMB-tdTom-transduced P14-TCR CTLs. CTL activity was tested in a classical 4 h 51Cr-release assay on RMA-S target cells loaded with 10–6, 10–8 and 10–10 M peptide gp33, as indicated. CTL/target ratio=2.5. Non-specific lysis (on HY-loaded RMA-S) was subtracted. Data are replicates from one experiment.

To our knowledge, two attempts at expressing fluorescent GZMB fusion proteins have been reported, but they were not expressed in CTLs 10, 11. Here, we described a new probe that allows visualization of cytolytic granules in living cells during their migration to the CTL/target cell contact zone. It also permits monitoring of GZMB release during antigen-induced degranulation and should be useful to further decipher the various steps leading to CTL activation and cytolytic effector function.

Acknowledgements

This work was supported by institutional funding from «Institut National de la Santé et de la Recherche Médicale» and «Centre National de la Recherche Scientifique», and by grants from «National du Cancer», EC Integrated Project “Cancer Immunotherapy” and CARS Explorer (to A.-M.S.-V.). P.M. and V.G. were supported, respectively, by doctoral fellowships from “Association pour la Recherche sur le Cancer” and “Ministère de la Recherche et de la Technologie”. We thank Bernard Malissen, for his support, Lee Leserman and Stephane Méresse for suggestions and critical reading of the manuscript, Mathieu Fallet and M. Bajénoff for help with video imaging and the personnel of the CIML Imaging and animal facilities for assistance.

Conflict of interest: The authors declare no financial or commercial conflict of interest.