Lysosome-related intra- and extracellular effector vesicles from human T and NK cells

• Applicant: Professor Dr. Ottmar Janßen
• Subject Area: Immunology
• Term: from 2015 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 271889415

Cytotoxic T and NK cells eliminate virus-infected or transformed cells by local exposure and release of cytotoxic effector proteins. The initial concept that “secretory lysosomes” store granzymes and perforin and release them into the cytotoxic immunological synapse (IS), and - at the same time - transport the membrane-associated death FasLigand to the cell surface does not seem to be compatible with more recent findings. We described two distinct types of lysosome-related effector vesicles (LREV), which display stable and homogeneous protein contents in healthy and leukemic T cell subpopulations and NK-cells, but are mobilized in either a PKC-dependent (non-classical degranulation) or calcium-dependent (classical degranulation) fashion. In the first funding period, we could verify these observations based on several marker proteins (e.g. FasL, LAMP-1, CD63, granzymes, granulysins) and characterize the release mechanisms in more detail. So far, it was not known that the exopeptidase CD26/DPP4 is also stored in LREV of cytotoxic lymphocytes. We showed that CD26/DPP4 co-localizes with markers for dense granules and is released upon TCR-stimulation or target cell exposure in a calcium-dependent manner. This indicates that cytotoxic effectors and immunoregulatory or metabolically active proteins are mobilized via similar vesicular routes. Based on the identified marker proteins, we plan to further characterize the mobilization of LREV under physiological conditions and in the context of target cell lysis. In terms of translation, we aim at identifying peculiarities of vesicles from leukemia patients, especially with LGL leukemias, where LREV of neoplastic cells might be causative for the often associated autoimmune phenomena, and we will employ an innovative in vitro approach to decipher the cell biological/immunological impact of gene defects that result in lysosomal storage diseases.In the context of intracellular vesicles, one has to keep in mind that T and NK cells do not only express effector molecules locally or release them into the IS, but they also constitutively - or more pronounced after activation - release various extracellular vesicles. Exosomes or microvesicles might thus serve for intracellular communication and effector function also at distant sites. We already know that intracellular LREV and exosomes/microvesicles display similar protein contents. Aim of the proposed work is an in-depth analysis of either spontaneously released or inducible extracellular vesicles regarding their protein content and surface decoration. A direct comparison with intracellular LREV will clarify the origin and signal requirements for the release of these extracellular vesicles.

• DFG Programme: Research Grants