Abstract
Cell death plays a fundamental role in mounting protective and pathogenic immunity. Etosis is a cell death mechanism defined by the release of extracellular traps (ETs), which can foster inflammation and exert microbicidal activity. While etosis is often associated with innate cells, recent studies showed that B cells and CD4+ T cells can release ETs. Here we investigate whether CD8+ T cells can also release ETs, which might be related to cytotoxicity and tissue pathology. To these ends, we first employed an in vitro system stimulating human CD8+ T cells isolated from healthy volunteers with anti-CD3/anti-CD28. Using time-frame video, confocal and electron microscopy, we demonstrate that human CD8+ T cells release ETs upon stimulation (herein LETs – lymphocyte extracellular traps), which display unique morphology and functional characteristics. CD8+ T cell-derived LETs form long strands that co-localize with CD107a, a marker of vesicles containing cytotoxic granules. In addition, these structures connect the LET-releasing cell to other neighboring cells, often resulting in cell death. After demonstrating the release of LETs by human CD8+ T cells in vitro, we went on to study the occurrence of CD8-derived LETs in a human disease setting. Thus, we evaluated the occurrence of CD8-derived LETs in lesions from patients with human tegumentary leishmaniasis, where CD8+ T cells play a key role in mediating pathology. In addition, we evaluated the association of these structures with the intensity of the inflammatory infiltrate in early and late cutaneous, as well as in mucosal leishmaniasis lesions. We demonstrated that progression and severity of debilitating and mutilating forms of human tegumentary leishmaniasis are associated with the frequency of CD8+ T cells in etosis, as well as the occurrence of CD8-derived LETs carrying CD107a+ vesicles in the lesions. We propose that CD8+ T cell derived LETs may serve as a tool for delivering cytotoxic vesicles to distant target cells, providing insights into mechanisms of CD8+ T cell mediated pathology.
Highlights
T cell activation and function is dependent on mechanisms that involve direct cell-cell interaction, as well as autocrine, paracrine and endocrine responses to soluble factors produced by a variety of cells
Our study demonstrates for the first time that human CD8+ T lymphocytes release extracellular traps (LETs) upon stimulation, and that these LETs are morphologically distinct from the ones released by CD4+ T cells
ETs derived from CD8+ T cells form long DNA filaments connecting one cell to another, contain CD107a+ vesicles, and induce death in cells that enter in contact with it
Summary
T cell activation and function is dependent on mechanisms that involve direct cell-cell interaction, as well as autocrine, paracrine and endocrine responses to soluble factors produced by a variety of cells. LEF were incubated with anti-CD8 and anti-CD4 monoclonal antibodies for 30 min at 4°C, washed twice with PBS, and resuspended at the concentration of 1 × 106 cells/ml. For some assays (as indicated in the text), purified lymphocytes were labeled with 5 mM carboxyfluorescein succinimidyl ester (CFSE, Sigma, St. Louis, MO, USA) by incubation for 10 min at room temperature, washed with PBS, plated in coverslips and stimulated or not with anti-CD3 and anti-CD28, as described above, for 24 h. Cells were washed twice, permeabilized by incubation for 15 min with 0.5% saponin solution, washed, and subjected to intracellular staining with anti-TNF-APC, anti-CD107a-FITC monoclonal antibodies for 20 min at room temperature.
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