Endocytosis of Cytotoxic Granules Is Essential for Multiple Killing of Target Cells by T Lymphocytes

Abstract

CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner. Following acidification, endocytosed CGs are recycled through early and late, but not recycling endosomes. CGs are refilled with granzyme B at the late endosome stage and polarize to subsequent synapses formed between the CTL and new target cells. Importantly, inhibiting CG endocytosis in CTLs results in a significant reduction of their cytotoxic activity. Thus, our data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process.

Various Stages of Immune Synapse Formation Are Differently Dependent on the Strength of the TCR Stimulus

“…Biotinylated anti-CD3 on supported planar bilayers have been used to reliably report downstream signaling events in T cells with clear segregation of the IS into SMACs comparable to that observed with a peptide MHC stimulation [19]. We also have evidence that CTLs stimulated with anti-CD3 vs. cognate peptide-MHC complexes behave similarly in our functional readouts (see materials and methods in [13]. Physical parameters such as mechanical forces, however, do influence agonist TCR-pMHC interactions occurring during T cell priming processes [25].…”

“…In neurons there are reports of fast and slow modes of synaptic vesicle membrane fusion and retrieval depending on the strength of the stimulus, temperature, and synaptic maturation [22]. We could previously demonstrate that Synaptobrevin2 is the major v-SNARE in cytotoxic granules of murine CTLs and that recycling of cytotoxic granule membrane components is essential for multiple killing of target cells [13,23]. Chang et al speculated there may be differences in the requirement of granule recycling during acute and chronic infections due to differences in the viral load [13].…”

“…We could previously demonstrate that Synaptobrevin2 is the major v-SNARE in cytotoxic granules of murine CTLs and that recycling of cytotoxic granule membrane components is essential for multiple killing of target cells [13,23]. Chang et al speculated there may be differences in the requirement of granule recycling during acute and chronic infections due to differences in the viral load [13]. Therefore, we investigated the temporal dynamics of CG membrane proteins at the IS upon fusion in CTLs stimulated with differing stimulatory anti-CD3 concentrations.…”

“…Naïve CD8 + T cells were isolated from splenocytes using a FlowComp Dynabeads Mouse CD8 + positive isolation kit (ThermoFisher Scientific, Karlsruhe, Germany) as described previously [13]. The cells were cultured at a concentration of 1 × 10 6 cells per mL in AIMV Medium (ThermoFisher Scientific) supplemented with 10% FCS, 0.5% Pen/Strep and 40 µM BME (Carl Roth, Karlsruhe, Germany).…”

“…It has been suggested in T helper and naïve T cells that differing TCR strengths induce different modes of immune synapses and that stronger TCR stimuli induce longer lasting or more sustained synapses [11]. CTLs form short term synapses because they can rapidly induce granule induced killing of target cells [12][13][14]. Therefore, we studied how different TCR stimuli influences different parameters of IS formation within this timeframe.…”