Exocytic responses of single leukaemic human cytotoxic T lymphocytes stimulated by agents that bypass the T cell receptor

Abstract

Cytotoxic T lymphocytes kill targets via secretion of lytic agents including perforin and granzymes. Recently, new methods have been developed to monitor cytotoxic T lymphocyte degranulation. These include detecting the appearance of lysosome-associated membrane protein on the cell's surface, and monitoring decreases in cellular perforin content. We have combined these methods with microscopy and flow cytometry to provide the first analysis of how single cytotoxic T cells degranulate. We used TALL-104 human leukaemic cytotoxic T cells as a model system, and stimulated them with thapsigargin and PMA, soluble agents that mimic the two major signalling pathways activated by T cell receptor cross-linking. Our results indicate that essentially every TALL-104 cell responds to maximal stimulation by releasing about half of its lytic granule complement. This reflects complete release of the contents of half the cell's granules, rather than partial release of the contents of all of the granules. Sub-maximal stimulation reduces both the fraction of cells that respond and the magnitude of single cell responses. We find that individual cells respond to maximal stimulation with a variable latency, and provide evidence that, once it starts, degranulation is a slow process taking tens of minutes.

An initial and rapid step of lytic granule secretion precedes microtubule organizing center polarization at the cytotoxic T lymphocyte/target cell synapse

“…Lytic granule secretion happens, for instance, when CTLs are triggered by soluble stimuli such as phorbol-12-myristate-13-acetate and ionomycin (22). Our results are in line with these observations and provide further support to the notion that docking and fusion of lytic granules to the CTL plasma membrane can occur independently of microtubule polarization events.…”

Calcineurin Activation Is Only One Calcium-dependent Step in Cytotoxic T Lymphocyte Granule Exocytosis

“…Ca 2ϩ -free Ringer's solution contained 145 NaCl, 4.5 KCl, 1 MgCl 2 , 1 mM EGTA, 5 HEPES, and 10 glucose (pH 7.4 with NaOH). N ␣ -Benzyloxycarbonyl-L-lysine Thiobenzyl ester (BLT) Esterase Assays-Granyzme B released from TALL-104 cells was assayed by measuring hydrolysis of BLT essentially as described previously (13,20,22). Absorbance measurements were made with a Bio-Tek Synergy HT-I plate reader (Bio-Tek Instruments, Winooski, VT) read at 410 nm after subtraction of an appropriate blank.…”

“…We first expressed a truncated constitutively active form of calcineurin A fused to GFP (CnAmut-GFP) and measured exocytosis in cells stimulated with TG ϩ PMA using LAMP externalization. We used reduced extracellular Ca 2ϩ (0.5 mM) because our previous work indicates that exocytosis is maximal in the presence of higher Ca 2ϩ (20); this would preclude detection of enhancement of exocytosis resulting from calcineurin expression. Fig.…”

“…1 To whom correspondence should be addressed: Dept. To gather positive evidence supporting a role of calcineurin, we exploited a new assay of exocytosis that detects exposure of lysosome-associated membrane protein-1 (LAMP-1) to the extracellular solution as a consequence of exocytosis (17)(18)(19)(20)(21) to examine the effects of expressing calcineurin-inhibitory peptides and wild-type or mutant constitutively active Ca 2ϩ -independent calcineurin A on exocytosis. Taken together, our results provide strong support for the idea that calcineurin activation is required for lytic granule exocytosis.…”

Ganglioside Inhibition of CD8+ T Cell Cytotoxicity: Interference with Lytic Granule Trafficking and Exocytosis

“…5A), from 76 6 4% polarized granules in untreated CD8 + CTL to 43 6 7% after GD1a exposure, and to 19 6 2% by exposure to the WT tumor-derived gangliosides. These results suggested that gan- To answer this question, we quantified degranulation of CD8 + CTL in response to the combination of phorbol ester and ionophore (PMA/ionomycin), a stimulus that induces release of lytic granules by CD8 + CTL in a nonvectorial manner, that is, independent of the ability of lytic granules to accumulate at the immunological synapse (26). In these experiments, exogenous gangliosides also inhibited lytic granule release by CD8 + CTL in response to PMA/ionomycin stimulation (Fig.…”