IL-12/15/18-induced cell death and mitochondrial dynamics of human NK cells.

Natural killer (NK) cells are lymphocytes with potent antitumor functions and, consequently, several NK cell-based strategies have been developed for cancer immunotherapy. A remarkable therapeutic approach is the adoptive transfer of NK cells stimulated with IL-12, IL-15 and IL-18. This cytokine stimulation endows NK cells with properties that resemble immunological memory and, for this reason, they are known as cytokine-induced memory-like (CIML) NK cells. Very promising results have been reported in clinical trials and yet, there are still unknown aspects of CIML NK cells. Here, we have conducted a preliminary study of their mitochondrial dynamics. Our results show that upon IL-12/15/18 stimulation the viability of NK cells decreased and an increment in mitochondrial superoxide levels was observed. In addition, we found that mitochondria appeared slightly elongated and their cristae density decreased following IL-12/15/18 stimulation, possibly in a process mediated by the low levels of optic atrophy type 1 (OPA1) protein. Interestingly, although mitophagy was slightly impaired, an increase in autophagic flux was observed, which might explain the reduced viability and the accumulation of unfit mitochondria. Our findings could be of relevance in order to design new strategies intended to improve the mitochondrial fitness of IL-12/15/18-stimulated NK cells with the aim of improving their therapeutic efficacy.

P815-based redirected degranulation assay to study human NK cell effector functions.

Natural killer (NK) cells are part of the innate immune system, the classic cytotoxic population of innate lymphoid cells (ILCs). They can directly kill virus-infected or tumor cells through different mechanisms without prior sensitization using their lytic functions in response to different signals (target cell ligands and/or inflammatory cytokines) and secreting cytokines, such as interferon gamma (IFNγ) and tumor necrosis factor (TNF). NK cells use antibody-dependent cell-mediated cytotoxicity (ADCC) to recognize and kill cells expressing target antigens when they are antibody coated. Redirected cytotoxicity is a technique used to target cells that do not per se activate NK cells. Here, we use redirected degranulation, a surrogate technique that correlates with redirected lysis. The P815 cell line (mouse mastocytoma) express fragment crystallizable gamma receptor II (FcγRII) and therefore could bind the Fc portion of mouse IgG antibodies, which through their fragment antigen-binding (Fab) may recognize NK cells activating receptors leading to target cell lysis. This technique could be used to determine the inhibitory or activating capacity of different receptors or isoforms and in immunotherapy using T cell and NK cell activators.

In vivo expansion of a CD9+ decidual-like NK cell subset following autologous hematopoietic stem cell transplantation.

Autologous hematopoietic stem cell transplantation (autoHSCT) is a treatment option for hematological disorders and pediatric solid tumors. After an autoHSCT, natural killer (NK) cells are the first lymphocyte subset returning to normal levels. To uncover global changes during NK cell reconstitution after autoHSCT, we performed RNA-sequencing on NK cells before and after autoHSCT. Results showed profound changes in the gene expression profile of NK cells immediately after autoHSCT. Several biological processes including cell cycle, DNA replication and the mevalonate pathway were enriched. Significantly, we observed that following autoHSCT, NK cells acquired a decidual-like gene expression profile, including the expression of CD9. By using multiparametric flow cytometry, we confirmed the expansion of NK cells expressing CD9 immediately after autoHSCT, which exhibited higher granzyme B and perforin expression levels than CD9- NK cells. These results provide insights into the physiopathology of NK cells during their reconstitution after autoHSCT.

Cytokine-Induced Memory-Like NK Cells: From the Basics to Clinical Applications.

Natural killer (NK) cells are lymphocytes with a key role in the defense against viral infections and tumor cells. Although NK cells are classified as innate lymphoid cells (ILCs), under certain circumstances they exhibit adaptive and memory-like features. The latter may be achieved, among others, by a brief stimulation with interleukin (IL)-12, IL-15 and IL-18. These cytokine-induced memory-like (CIML) NK cells resemble the trained immunity observed in myeloid cells. CIML NK cells undergo transcriptional, epigenetic and metabolic reprogramming that, along with changes in the expression of cell surface receptors and components of cytotoxic granules, are responsible for their enhanced effector functions after a resting period. In addition, these memory-like NK cells persist for a long time, which make them a good candidate for cancer immunotherapy. Currently, several clinical trials are testing CIML NK cells infusions to treat tumors, mostly hematological malignancies. In relapse/refractory acute myeloid leukemia (AML), the adoptive transfer of CIML NK cells is safe and complete clinical remissions have been observed. In our review, we sought to summarize the current knowledge about the generation and molecular basis of NK cell memory-like responses and the up-to-date results from clinical trials with CIML NK cells.