A brief in vitro stimulation of natural killer (NK) cells with interleukin (IL)-12, IL-15, and IL-18 endow them a memory-like behavior, characterized by higher effector responses when they are restimulated after a resting period of time. These preactivated NK cells, also known as cytokine-induced memory-like (CIML) NK cells, have several properties that make them a promising tool in cancer immunotherapy. In the present study, we have described the effect that different combinations of IL-12, IL-15, and IL-18 have on the generation of human CIML NK cells. Our data points to a major contribution of IL-15 to CIML NK cell-mediated cytotoxicity against target cells. However, the synergistic effect of the three cytokines grant them the best polyfunctional profile, that is, cells that simultaneously degranulate (CD107a) and produce multiple cytokines and chemokines such as interferon γ, tumor necrosis factor α, and C-C motif chemokine ligand 3. We have also analyzed the involvement of each cytokine and their combinations in the expression of homing receptors CXCR4 and CD62L, as well as the expression of CD25 and IL-2-induced proliferation. Furthermore, we have tested the effects of the Jak1/2 inhibitor ruxolitinib in the generation of CIML NK cells. We found that ruxolitinib-treated CIML NK cells expressed lower levels of CD25 than non-treated CIML NK cells, but exhibited similar proliferation in response to IL-2. In addition, we have also found that ruxolitinib-treated NK cells displayed reduced effector functions after the preactivation, which can be recovered after a 4 days expansion phase in the presence of low doses of IL-2. Altogether, our results describe the impact that each cytokine and the Jak1/2 pathway have in the phenotype, IL-2-induced proliferation, and effector functions of human CIML NK cells.

Reproductive longevity is critical for fertility and impacts healthy ageing in women, yet insights into the underlying biological mechanisms and treatments to preserve it are limited. Here, we identify 290 genetic determinants of ovarian ageing, assessed using normal variation in age at natural menopause (ANM) in ∼200,000 women of European ancestry. These common alleles influence clinical extremes of ANM; women in the top 1% of genetic susceptibility have an equivalent risk of premature ovarian insufficiency to those carrying monogenic FMR1 premutations. Identified loci implicate a broad range of DNA damage response (DDR) processes and include loss-of-function variants in key DDR genes. Integration with experimental models demonstrates that these DDR processes act across the life-course to shape the ovarian reserve and its rate of depletion. Furthermore, we demonstrate that experimental manipulation of DDR pathways highlighted by human genetics increase fertility and extend reproductive life in mice. Causal inference analyses using the identified genetic variants indicates that extending reproductive life in women improves bone health and reduces risk of type 2 diabetes, but increases risks of hormone-sensitive cancers. These findings provide insight into the mechanisms governing ovarian ageing, when they act across the life-course, and how they might be targeted by therapeutic approaches to extend fertility and prevent disease.

Unrepaired DNA damage during embryonic development can be potentially inherited by a large population of cells. However, the quality control mechanisms that minimize the contribution of damaged cells to developing embryos remain poorly understood.Here, we uncovered an ATR-and CHK1-mediated transcriptional response to replication stress (RS) in ESCs that induces genes expressed in totipotent two-cell (2C) stage embryos and 2C-like cells. This response is mediated by Dux, a multicopy retrogene defining the cleavage-specific transcriptional program in placental mammals. In response to RS, DUX triggers the transcription of 2C-like markers such as murine endogenous retrovirus-like elements (MERVL) and Zscan4. This response can also be elicited by ETAA1-mediated ATR activation in the absence of RS. ATR-mediated activation of DUX requires GSRF1 dependent post-transcriptional regulation of Dux mRNA. Strikingly, activation of ATR expands ESCs fate potential by extending their contribution to both embryonic and extraembryonic tissues. These findings define a novel ATR dependent pathway involved in maintaining genome stability in developing embryos by controlling ESCs fate in response to RS. BackgroundESCs are characterized by self-renewal and the ability to propagate for several cycles in vitro and in vivo 1 . Even if ESCs exhibit several markers of RS 2 , they are able to maintain genome integrity more efficiently than differentiated cells 1 . The mechanisms underlying such distinctive feature are largely unknown. ESC colonies harbor a small transient subpopulation of cells (2C-like cells) with functional andtranscriptional features of totipotent 2C-stage embryos 3-5 . Transition to 2C-like cells has been shown to promote maintenance of genome integrity and survival of ESCs in long-term culture 6-8 . In addition, several studies have demonstrated that transition to the 2C-like state confers expanded developmental potential to ESCs, making them capable of contributing to both embryonic and extra-embryonic tissues (also referred to as bidirectional cell fate potential) 3-5 . However, the molecular players underlying the transition to the 2C-like state in ESC culture and its possible physiological relevance in vivo in maintaining genome integrity and expanding cell fate potential in a developing embryo are not fully understood.Here we provide several lines of evidence that ATR and CHK1-mediated response to RS triggers the activation of 2C-specific genes in ESCs and mouse embryos. This transition is hampered in ESCs derived from ATR-deficient Seckel and CHK1 haploinsufficient mouse models and following ATR or CHK1 inhibition. Significantly, we show that ETAA1-mediated ATR activation is sufficient to trigger the formation of 2C-like cells in the absence of RS.Mechanistically, ATR-induced transition to 2C-like state is mediated by post-transcriptional regulation of the Dux gene, which shapes the transcriptional signature of 2C-like cells and totipotent 2C-stage embryos in placental mammals 9-11 . ATR-dependent regulation of Du...

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this protocol describes the procedure of in vitro fertilisation of mouse oocytes. The procedure is performed at the transgenic core facility by a specialist according to the protocols found here: https://www.infrafrontier.eu/knowledgebase/protocols/cryopreservation-protocols