Effector granules in human T lymphocytes: the luminal proteome of secretory lysosomes from human T cells

Abstract

BackgroundCytotoxic cells of the immune system have evolved a lysosomal compartment to store and mobilize effector molecules. In T lymphocytes and NK cells, the death factor FasL is one of the characteristic marker proteins of these so-called secretory lysosomes, which combine properties of conventional lysosomes and exocytotic vesicles. Although these vesicles are crucial for immune effector function, their protein content in T cells has so far not been investigated in detail.ResultsIn the present study, intact membranous vesicles were enriched from homogenates of polyclonally activated T cells and initially characterized by Western blotting and electron microscopic inspection. The vesicular fraction that contained the marker proteins of secretory lysosomes was subsequently analyzed by 2D electrophoresis and mass spectrometry. The proteome analysis and data evaluation revealed that 70% of the 397 annotated proteins had been associated with different lysosome-related organelles in previous proteome studies.ConclusionWe provide the first comprehensive proteome map of T cell-derived secretory lysosomes with only minor contaminations by cytosolic, nuclear or other proteins. This information will be useful to more precisely address the activation-dependent maturation and the specific distribution of effector organelles and proteins in individual T or NK cell populations in future studies.

An Extended Proteome Map of the Lysosomal Membrane Reveals Novel Potential Transporters

“…8). Many subunits of these complexes have been identified in previous proteomic studies of lysosomes (15-17, 19, 72), phagosomes (54 -56, 59, 60, 73, 74), or lysosome-related organelles (57,58,(75)(76)(77)(78). However, these complexes were most often not as extensively documented as in this work.…”

“…To our knowledge, the MbL2385 list is the most extensive published to date for lysosomes (15-17, 19, 72), phagosomes (54 -56, 59, 60, 73, 74), or lysosome-related organelles (57,58,(75)(76)(77)(78). Its IMP content (32%) is much higher than that commonly obtained if no specific subfractionation treatment is performed (5-15% IMPs (34)), but it is very similar to that obtained in a study of placental lysosomal membranes that also used an organic solvent treatment (16).…”

Preparing the lethal hit: interplay between exo- and endocytic pathways in cytotoxic T lymphocytes

“…Interestingly, the above-mentioned CLEM on CTLs derived from synaptobrevin2-knockin mice revealed two surprising pieces of data. First, the synaptobrevin2-positive CGs were very homogeneous in diameter (about 350 nm), indicating that, in contrast to the postulate by Schmidt and coworkers [63, 64], only one class of mature CGs exists. This finding was supported by recent combined total internal reflection fluorescence (TIRF) microscopy and membrane capacitance measurements that determined a homogeneous diameter of fusing CGs of 312 nm [66].…”

“…Adding to the complexity, however, is the possibility that more than one population of mature CGs exist. Schmidt and coworkers [63, 64] demonstrated, by combining density gradient centrifugation, proteomics and electron microscopy that two subpopulations of CGs exist. Proteomic profiling of T cell organelles separated by density gradient centrifugation revealed the presence of two species of lysosome-related organelles: a larger, electron-light clear fraction with a diameter between 300 and 700 nm and a smaller, electron-dense dark fraction with a diameter of less than 300 nm.…”

Protein networks and activation of lymphocytes

“…Already, the membrane proteomes of primary B cells, NK cells, and T cells have been characterized utilizing this technique on plasma membrane enriched samples [14–16]. Studies have also performed proteomic characterizations of enriched secretory lysosomes from cytotoxic T cells and NK cells [17–19]. …”