Management of patients presenting to the Emergency Department with chest pain is continuously evolving. In the setting of acute coronary syndrome, the availability of high-sensitivity cardiac troponin assays (hs-cTn) has allowed for the development of algorithms aimed at rapidly assessing the risk of an ongoing myocardial infarction. However, concerns were raised about the massive application of such a simplified approach to heterogeneous real-world populations. As a result, there is a potential risk of underdiagnosis in several clusters of patients, including women, for whom a lower threshold for hs-cTn was suggested to be more appropriate. Implementation in clinical practice of sex-tailored cut-off values for hs-cTn represents a hot topic due to the need to reduce inequality and improve diagnostic performance in females. The aim of this review is to summarize current evidence on sex-specific cut-off values of hs-cTn and their application and usefulness in clinical practice. We also offer an extensive overview of thresholds reported in literature and of the mechanisms underlying such differences among sexes, suggesting possible explanations about debated issues.

BackgroundNatural killer (NK) cells hold great promise as a source for allogeneic cell therapy against hematological malignancies, including acute myeloid leukemia (AML). Current treatments are hampered by variability in NK cell subset responses, a limitation which could be circumvented by specific expansion of highly potent single killer immunoglobulin-like receptor (KIR)+NKG2C+adaptive NK cells to maximize missing-self reactivity.MethodsWe developed a GMP-compliant protocol to expand adaptive NK cells from cryopreserved cells derived from select third-party superdonors, that is, donors harboring large adaptive NK cell subsets with desired KIR specificities at baseline. We studied the adaptive state of the cell product (ADAPT-NK) by flow cytometry and mass cytometry as well as cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq). We investigated the functional responses of ADAPT-NK cells against a wide range of tumor target cell lines and primary AML samples using flow cytometry and IncuCyte as well as in a mouse model of AML.ResultsADAPT-NK cells were >90% pure with a homogeneous expression of a single self-HLA specific KIR and expanded a median of 470-fold. The ADAPT-NK cells largely retained their adaptive transcriptional signature with activation of effector programs without signs of exhaustion. ADAPT-NK cells showed high degranulation capacity and efficient killing of HLA-C/KIR mismatched tumor cell lines as well as primary leukemic blasts from AML patients. Finally, the expanded adaptive NK cells had preserved robust antibody-dependent cellular cytotoxicity potential and combination of ADAPT-NK cells with an anti-CD16/IL-15/anti-CD33 tri-specific engager led to near-complete killing of resistant CD45dimblast subtypes.ConclusionsThese preclinical data demonstrate the feasibility of off-the-shelf therapy with a non-engineered, yet highly specific, NK cell population with full missing-self recognition capability.

Natural killer (NK) cells are innate lymphoid cells with potent antitumor activity. One of the most NK cell cytotoxicity-sensitive tumor types is sarcoma, an aggressive mesenchyme-derived neoplasm. While a combination of radical surgery and radio- and chemotherapy can successfully control local disease, patients with advanced sarcomas remain refractory to current treatment regimens, calling for novel therapeutic strategies. There is accumulating evidence for NK cell-mediated immunosurveillance of sarcoma cells during all stages of the disease, highlighting the potential of using NK cells as a therapeutic tool. However, sarcomas display multiple immunoevasion mechanisms that can suppress NK cell function leading to an uncontrolled tumor outgrowth. Here, we review the current evidence for NK cells’ role in immune surveillance of sarcoma during disease initiation, promotion, progression, and metastasis, as well as the molecular mechanisms behind sarcoma-mediated NK cell suppression. Further, we apply this basic understanding of NK–sarcoma crosstalk in order to identify and summarize the most promising candidates for NK cell-based sarcoma immunotherapy.

Induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells offer a promising platform for off-the-shelf immunotherapy against cancer. A unique benefit of iPSC-derived immune effector cells is the possibility to perform multiple precision editing steps at the single cell level to achieve a homogenous effector cell population tailored to target a desired cancer type and equipped with selected functional properties. These functional edits are superimposed on the innate reactivity of NK cells to stress ligands and MHC downregulation (missing self). The ability of NK cells to sense missing self is based on a functional calibration to self MHC during a process termed NK cell education, the latter being critically dependent on signaling through inhibitory receptors, including CD94/NKG2A and killer cell immunoglobulin-like receptors (KIR). Whereas the process of NK cell differentiation into mature effector cells from iPSCs has been well characterized, the role of natural variation in inhibitory receptor expression and NK cell education remains poorly defined in iNK cells. We used mass cytometry to map the receptor repertoire in series of iNK cell lines and genetic edits thereof during differentiation and in vitro expansion (Figure 1A and B). Similar to peripheral blood NK cells, the receptor repertoire was diversified but genetically hardwired showing consistent patterns within each iNK cell line but with slight variation between genetically distinct lines. NKG2A was the dominantly expressed inhibitory receptor ranging from 13% to 87% with the highest expression in multi-edited iNK cell lines engineered to express a chimeric antigen receptor against CD19, a high affinity, non-cleavable FcγRIIIa receptor (CD16) and a recombinant IL15 signaling complex (CAR19-iNK cells). KIR expression was generally low in all tested iNK cell lines but increased gradually during culture and was further increased by genetic silencing of NKG2A receptors. Interestingly, silencing of NKG2A lead to increased levels of the activating receptor NKG2C. We monitored degranulation by iNK cell variants against K562 engineered to express varying levels of HLA-E as well as CD19+ Nalm-6 cells. Genetic silencing of ß2microglobulin (ß2m), associated with reduced levels of HLA-class I and HLA-E, led to dampened global functional responses in iNK cells, suggesting a positive impact of education during iNK cell differentiation and expansion (Figure 1C). Subset stratification revealed that NKG2A+ iNK cells showed superior functionality compared to NKG2A- iNK cells across all iNK cell lines tested, albeit less striking in CAR19-iNK cells that showed the highest overall natural cytotoxicity (Figure 1D). Knockdown of NKG2A led to a general reduction in functional capacity of NK92 cells (Figure 1E-F) and CAR19-iNK cells (Figure 1H), supporting a critical role for NKG2A-driven education in iNK cells. Given the superior functionality of NKG2A+ iNK cells, we next addressed whether this advantage was countered by expression of the check point ligand HLA-E during target cell interactions. Although we noted a slight inhibitory impact on natural cytotoxicity in NK cells isolated and expanded from peripheral blood (PB-NK) against K562 cells expressing physiological levels of HLA-E, this effect was completely overridden in iNK cells and did not interfere with NKG2A+ CAR-iNK cell recognition of HLA-E expressing CD19+ target cells (Figure 1G-H). Indeed, NKG2A+ CAR19-iNK showed superior degranulation against HLA-E expressing CD19+ Nalm-6 targets compared to CRISPR-edited NKG2A-/- CAR19-iNK cells (Figure 1I). Our results shed light on the regulatory gene circuits and cellular programs that determine functional potential in iPSC-derived NK cells products. Specifically, our results point to a crucial role for NKG2A-driven acquisition of a mature effector cell phenotype in combination with functional education through cognate ligands. Importantly, iNK cell education is operational during iNK cell differentiation and expansion without interfering with recognition of tumor targets expressing HLA-E. Figure 1 Disclosures Cichocki: Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding. Mahmood:Fate Therapeutics, Inc: Current Employment. Gaidarova:Fate Therapeutics, Inc: Current Employment. Bjordahl:Fate Therapeutics: Current Employment. Chu:Fate Therapeutics, Inc: Current Employment. Groff:Fate Therapeutics, Inc: Current Employment. Denholtz:Fate Therapeutics, Inc: Current Employment. Miller:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Vycellix: Consultancy; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; GT Biopharma: Consultancy, Patents & Royalties, Research Funding. Lee:Fate Therapeutics, Inc.: Current Employment. Kaufman:Fate Therapeutics: Consultancy. Goodridge:Fate Therapeutics, Inc: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Malmberg:Fate Therapeutics: Consultancy, Patents & Royalties; Vycellix: Membership on an entity's Board of Directors or advisory committees.