Analysis of Cytotoxic Granules and Constitutively Produced Extracellular Vesicles from Large Granular Lymphocytic...

Large granular lymphocyte leukemias (LGLLs) are rare lymphoproliferative malignancies caused by clonal expansion of granular lymphocytes. T-cell LGLL and natural killer (NK) cell LGLL are defined based on their cellular origin. Their clinical manifestation and pathophysiology vary depending on the subtype and include, e.g., neutropenia, anemia, recurrent infections, and autoimmunity. A limited number of available patient-derived cell lines are considered valuable tools to study the biology of these malignancies. They differ in the expression of lineage-specific surface markers, but generally contain cytotoxic effector molecules in characteristic granules.

We investigated the presence and release of lysosome-associated effector proteins in patient-derived LGLL cell lines by flow and imaging cytometry, by Western blotting and by bottom-up proteomics profiling.

The tested cell lines did not express FasL (CD178), but did express CD26/DPP4⁺. Intracellularly, we detected major differences in the abundance and subcellular distribution of granzymes, perforin, and granulysin. Similar differences were seen in enriched lysosome-related effector vesicles (LREVs). The proteomics profiling of enriched EVs from an NK-LGLL line (NKL) and a T-LGLL line (MOTN-1), confirmed individual profiles of effector molecules.

Our analyses underscore the individual distribution of effector proteins but also open new routes to define the role of intra- and extracellular granules in the disease manifestation or pathology of LGLLs.