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Diel vertical migration (DVM) is a common predator avoidance strategy of zooplankton. Migration to deeper water layers during the day to escape visually hunting predators is most likely to require physiological adaptations to periodically changing temperatures. Polyunsaturated fatty acids (PUFA) play crucial roles in membrane temperature acclimation. Exposure to cold temperatures typically results in an increase in the relative abundance of PUFA in cell membranes and PUFA requirements of Daphnia increase with decreasing temperatures. To assess the role of dietary PUFA in coping with temperature fluctuations experienced during DVM, we reared Daphnia magna at either constantly warm or fluctuating temperatures, simulating DVM both with and without dietary PUFA supplementation. We show that the well‐known positive effect of dietary eicosapentaenoic acid supplementation on offspring production and population growth rates of D. magna is more pronounced at alternating temperatures than at constantly warm temperatures. Exposure to alternating temperatures caused modification in body PUFA concentrations and, consequently, increased lipid peroxidation. However, detrimental effects of lipid peroxidation were not evident. Our data demonstrate that the capacity to cope with the distinct temperature fluctuations experienced during DVM increases with dietary eicosapentaenoic acid supplementation, suggesting that an adequate dietary PUFA supply is crucial especially for migrating Daphnia populations. A dietary deficiency in long‐chain PUFA may thus severely constrain predator evasion, potentially resulting in increased mortality and cascading effects on lower trophic levels.

Dipeptidyl peptidase 4 (DPP4, CD26) is a serine protease detected on several immune cells and on epithelial cells of various organs. Besides the membrane-bound enzyme, a catalytically active soluble form (sCD26/DPP4) is detected in several body fluids. Both variants cleave off dipeptides from the N-termini of various chemokines, neuropeptides, and hormones. CD26/DPP4 plays a fundamental role in the regulation of blood glucose levels by inactivating insulinotropic incretins and CD26/DPP4 inhibitors are thus routinely used in diabetes mellitus type 2 therapy to improve glucose tolerance. Such inhibitors might also prevent the CD26/DPP4-mediated inactivation of the T-cell chemoattractant CXCL10 released by certain tumors and thus improve anti-tumor immunity and immunotherapy. Despite its implication in the regulation of many (patho-)physiological processes and its consideration as a biomarker and therapeutic target, the cellular source of sCD26/DPP4 remains highly debated and mechanisms of its release are so far unknown. In line with recent reports that activated T lymphocytes could be a major source of sCD26/DPP4, we now demonstrate that CD26/DPP4 is stored in secretory granules of several major human cytotoxic lymphocyte populations and co-localizes with effector proteins such as granzymes, perforin, and granulysin. Upon stimulation, vesicular CD26/DPP4 is rapidly translocated to the cell surface in a Ca2+-dependent manner. Importantly, activation-induced degranulation leads to a massive release of proteolytically active sCD26/DPP4. Since activated effector lymphocytes serve as a major source of sCD26/DPP4, these results might explain the observed disease-associated alterations of sCD26/DPP4 serum levels and also indicate a so far unknown role of CD26/DPP4 in lymphocyte-mediated cytotoxicity.

Granulysin (GNLY) is a cationic antimicrobial, proinflammatory, and cytotoxic effector protein primarily expressed in human cytotoxic T and NK cells. Its two variants, the 15 kDa precursor and the mature 9 kDa protein processed by proteolysis, act on different microbes or infected and transformed target cells and utilize mechanistically different effector activities. In human peripheral blood lymphocytes of healthy individuals, both forms of GNLY are detected in TCR αβ+ (CD4+ and CD8+) T cells, TCR γδ+ T cells, and CD3−CD56+ NK cells. In general, classical cytotoxic cells (i.e. CD8+ TCR αβ+ T cells, TCR γδ+ T cells, and NK cells) contain effector proteins in higher abundance in more cells of the subset as compared to TCR αβ+ CD4+ T cells. Imaging flow cytometry analyses demonstrate that the subcellular localization and internal pools of 9 kDa and 15 kDa GNLY are virtually non-overlapping. The 9 kDa form is enriched in dense granules that also contain granzymes (Grz) and carry CD107a, whereas 15 kDa GNLY is associated with CD107a-negative lysosome-related effector vesicles. We further demonstrate that 15 kDa GNLY serves as an additional indicator for non-classical, PKC-dependent degranulation while the liberation of granules containing 9 kDa GNLY requires calcium mobilization. Our studies provide a deeper insight into the subcellular localization and release mechanisms of the individual GNLY species. This information will not only be useful for the interpretation of GNLY-related pathophysiologies, but also for the development of therapeutic interventions employing distinct GNLY effector functions for microbial targeting or immunoregulation.

It is widely accepted that cytotoxic T and NK cells store effector proteins including granzymes, perforin and Fas ligand (FasL) in intracellular granules, often referred to as secretory lysosomes. Upon target cell encounter, these organelles are transported to the cytotoxic immunological synapse, where they fuse with the plasma membrane to release the soluble effector molecules and to expose transmembrane proteins including FasL on the cell surface. We previously described two distinct species of secretory vesicles in T and NK cells that differ in size, morphology and protein loading, most strikingly regarding FasL and granzyme B. We now show that the signal requirements for the mobilization of one or the other granule also differ substantially. We report that prestored FasL can be mobilized independent of extracellular Ca2+, whereas the surface exposure of lysosome-associated membrane proteins (Lamps; CD107a and CD63) and the release of granzyme B are calcium-dependent. The use of selective inhibitors of actin dynamics unequivocally points to different transport mechanisms for individual vesicles. While inhibitors of actin polymerization/dynamics inhibit the surface appearance of prestored FasL, they increase the activation-induced mobilization of CD107a, CD63 and granzyme B. In contrast, inhibition of the actin-based motor protein myosin 2a facilitates FasL-, but impairs CD107a-, CD63- and granzyme B mobilization. From our data, we conclude that distinct cytotoxic effector granules are differentially regulated with respect to signaling requirements and transport mechanisms. We suggest that a T cell might ‘sense’ which effector proteins it needs to mobilize in a given context, thereby increasing efficacy while minimizing collateral damage.