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).
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).
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.