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 Romidepsin 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 BTK inhibitor manufacturer degranulation being dependent on peptide concentration (Fig. 2A). The percent of GZMB-tdTom fluorescent ifenprodil 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.