The G Protein-Coupled Receptor GPR56 Is an Inhibitory Checkpoint for NK Cell Migration.

G protein-coupled receptors (GPCRs) represent the largest family of surface receptors and are responsible for key physiological functions, including cell growth, neurotransmission, hormone release, and cell migration. The GPCR 56 (GPR56), encoded by ADGRG1, is an adhesion GPCR found on diverse cell types, including neural progenitor cells, melanoma cells, and lymphocytes, such as effector memory T cells, γδ T cells, and NK cells. Using RNA-sequencing and high-resolution flow cytometry, we found that GPR56 mRNA and protein expression increased with NK cell differentiation, reaching its peak in adaptive NK cells. Small interfering RNA silencing of GPR56 led to increased spontaneous and chemokine-induced migration, suggesting that GPR56 functions as an upstream checkpoint for migration of highly differentiated NK cells. Increased NK cell migration could also be induced by agonistic stimulation of GPR56 leading to rapid internalization and deactivation of the receptor. Mechanistically, GPR56 ligation and downregulation were associated with transcriptional coactivator with PDZ-binding motif translocation to the nucleus and increased actin polymerization. Together, these data provide insights into the role of GPR56 in the migratory behavior of human NK cell subsets and may open possibilities to improve NK cell infiltration into cancer tissues by releasing a migratory checkpoint.

The Lysosomal Calcium Channel TRPML1 Maintains Mitochondrial Fitness in NK Cells through Interorganelle Cross-Talk.

Cytotoxic lymphocytes eliminate cancer cells through the release of lytic granules, a specialized form of secretory lysosomes. This compartment is part of the pleomorphic endolysosomal system and is distinguished by its highly dynamic Ca2+ signaling machinery. Several transient receptor potential (TRP) calcium channels play essential roles in endolysosomal Ca2+ signaling and ensure the proper function of these organelles. In this study, we examined the role of TRPML1 (TRP cation channel, mucolipin subfamily, member 1) in regulating the homeostasis of secretory lysosomes and their cross-talk with mitochondria in human NK cells. We found that genetic deletion of TRPML1, which localizes to lysosomes in NK cells, led to mitochondrial fragmentation with evidence of collapsed mitochondrial cristae. Consequently, TRPML1-/- NK92 (NK92ML1-/-) displayed loss of mitochondrial membrane potential, increased reactive oxygen species stress, reduced ATP production, and compromised respiratory capacity. Using sensitive organelle-specific probes, we observed that mitochondria in NK92ML1-/- cells exhibited evidence of Ca2+ overload. Moreover, pharmacological activation of the TRPML1 channel in primary NK cells resulted in upregulation of LC3-II, whereas genetic deletion impeded autophagic flux and increased accumulation of dysfunctional mitochondria. Thus, TRPML1 impacts autophagy and clearance of damaged mitochondria. Taken together, these results suggest that an intimate interorganelle communication in NK cells is orchestrated by the lysosomal Ca2+ channel TRPML1.

Adaptive single-KIR+NKG2C+ NK cells expanded from select superdonors show potent missing-self reactivity and efficiently control HLA-mismatched acute myeloid leukemia.

BACKGROUND: Natural 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. METHODS: We 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. RESULTS: ADAPT-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 CD45dim blast subtypes. CONCLUSIONS: These 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.

Prospects for NK Cell Therapy of Sarcoma.

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.

Sex-Specific Cut-Offs for High-Sensitivity Cardiac Troponin: Is Less More?

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.