Background: The success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK) cells. Here, we targeted an intracellular inhibiting protein ‘cytokine inducible SH2-containing protein’ (CISH) in NK cells to evaluate the impact on their functions and antitumor properties. Methods: To further understand CISH functions in NK cells, we developed a conditional Cish-deficient mouse model in NK cells (Cish^fl/flNcr1^Ki/+ ). NK cells cytokine expression, signaling and cytotoxicity has been evaluated in vitro. Using intravenous injection of B16F10 melanoma cell line and EO711 triple negative breast cancer cell line, metastasis evaluation was performed. Then, orthotopic implantation of breast tumors was performed and tumor growth was followed using bioluminescence. Infiltration and phenotype of NK cells in the tumor was evaluated. Finally, we targeted CISH in human NK-92 or primary NK cells, using a technology combining the CRISPR(i)-dCas9 tool with a new lentiviral pseudotype. We then tested human NK cells functions. Results: In Cish^fl/flNcr1^Ki/+ mice, we detected no developmental or homeostatic difference in NK cells. Global gene expression of Cish^fl/flNcr1^Ki/+ NK cells compared with Cish^+/+Ncr1^Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways, triggering CISH protein expression. Primed Cish^fl/flNcr1^Ki/+ NK cells display increased activation upon NCR stimulation. Cish^fl/flNcr1^Ki/+ NK cells display lower activation thresholds and Cish^fl/flNcr1^Ki/+ mice are more resistant to tumor metastasis and to primary breast cancer growth. CISH deletion favors NK cell accumulation to the primary tumor, optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression, limiting NK cell exhaustion. Finally, using CRISPRi, we then targeted CISH in human NK-92 or primary NK cells. In human NK cells, CISH deletion also favors NCR signaling and antitumor functions. Conclusion: This study represents a crucial step in the mechanistic understanding and safety of Cish targeting to unleash NK cell antitumor function in solid tumors. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy.

Natural killer (NK) cells are a vital part of the innate immune system capable of rapidly clearing mutated or infected cells from the body and promoting an immune response. Here, we find that NK cells activated by viral infection or tumor challenge increase uptake of fatty acids and their expression of carnitine palmitoyltransferase I (CPT1A), a critical enzyme for long-chain fatty acid oxidation. Using a mouse model with an NK cell–specific deletion of CPT1A, combined with stable ¹³ C isotope tracing, we observe reduced mitochondrial function and fatty acid–derived aspartate production in CPT1A-deficient NK cells. Furthermore, CPT1A-deficient NK cells show reduced proliferation after viral infection and diminished protection against cancer due to impaired actin cytoskeleton rearrangement. Together, our findings highlight that fatty acid oxidation promotes NK cell metabolic resilience, processes that can be optimized in NK cell–based immunotherapies.

Natural killer (NK) cells are innate lymphocytes responsible for the elimination of infected or transformed cells. The activation or inhibition of NK cells is determined by the balance of target cell ligand recognition by stimulatory and inhibitory receptors on their surface. Previous reports have suggested that the glycosaminoglycan heparin is a ligand for the natural cytotoxicity receptors NKp30, NKp44 (human), and NKp46 (both human and mouse). However, the effects of heparin on NK cell homeostasis and function remain unclear. Here, we show that heparin does not enhance NK cell proliferation or killing through NK cell activation. Alternatively, in mice models, heparin promoted NK cell survival in vitro and controlled B16-F10 melanoma metastasis development in vivo. In human NK cells, heparin promisingly increased interferon (IFN)-γ production in synergy with IL-12, although the mechanism remains elusive. Our data showed that heparin is not able to increase NK cell cytotoxicity.

Understanding the pathways and regulation of human haematopoiesis, in particular, lymphopoiesis, is vital to manipulation of these processes for therapeutic purposes. However, although haematopoiesis has been extensively characterised in mice, translation of these findings to human biology remains rudimentary. Here, we describe the isolation of three progenitor subsets from human foetal bone marrow that represent differential stages of commitment to the natural killer (NK) cell lineage based on IL-15 responsiveness. We identify CD7 as a marker of IL-15 responsive progenitors in human bone marrow and find that this expression is maintained throughout commitment and maturation. Within the CD7⁺ fraction, we focussed on the lineage potential of three subsets based on CD127 and CD117 expression and observed restricted lymphoid and biased NK cell potential amongst subsets. We further demonstrate the presence of subsets similar in both phenotype and function in umbilical cord blood and the bone marrow of humanised mice, validating these as appropriate sources of progenitors for the investigation of human haematopoiesis. Overall, we describe several stages in the process of lymphopoiesis that will form the basis of investigating the regulators of this process in humans.