Tumors evade immune cytotoxicity by altering the surface topology of NK cells.

The highly variable response rates to immunotherapies underscore our limited knowledge about how tumors can manipulate immune cells. Here the membrane topology of natural killer (NK) cells from patients with liver cancer showed that intratumoral NK cells have fewer membrane protrusions compared with liver NK cells outside tumors and with peripheral NK cells. Dysregulation of these protrusions prevented intratumoral NK cells from recognizing tumor cells, from forming lytic immunological synapses and from killing tumor cells. The membranes of intratumoral NK cells have altered sphingomyelin (SM) content and dysregulated serine metabolism in tumors contributed to the decrease in SM levels of intratumoral NK cells. Inhibition of SM biosynthesis in peripheral NK cells phenocopied the disrupted membrane topology and cytotoxicity of the intratumoral NK cells. Targeting sphingomyelinase confers powerful antitumor efficacy, both as a monotherapy and as a combination therapy with checkpoint blockade.

Mitochondrial fragmentation limits NK cell-based tumor immunosurveillance.

Natural killer (NK) cells have crucial roles in tumor surveillance. We found that tumor-infiltrating NK cells in human liver cancers had small, fragmented mitochondria in their cytoplasm, whereas liver NK cells outside tumors, as well as peripheral NK cells, had normal large, tubular mitochondria. This fragmentation was correlated with reduced cytotoxicity and NK cell loss, resulting in tumor evasion of NK cell-mediated surveillance, which predicted poor survival in patients with liver cancer. The hypoxic tumor microenvironment drove the sustained activation of mechanistic target of rapamycin-GTPase dynamin-related protein 1 (mTOR-Drp1) in NK cells, resulting in excessive mitochondrial fission into fragments. Inhibition of mitochondrial fragmentation improved mitochondrial metabolism, survival and the antitumor capacity of NK cells. These data reveal a mechanism of immune escape that might be targetable and could invigorate NK cell-based cancer treatments.

Natural Killer Cells Promote Fetal Development through the Secretion of Growth-Promoting Factors.

Natural killer (NK) cells are present in large populations at the maternal-fetal interface during early pregnancy. However, the role of NK cells in fetal growth is unclear. Here, we have identified a CD49a+Eomes+ subset of NK cells that secreted growth-promoting factors (GPFs), including pleiotrophin and osteoglycin, in both humans and mice. The crosstalk between HLA-G and ILT2 served as a stimulus for GPF-secreting function of this NK cell subset. Decreases in this GPF-secreting NK cell subset impaired fetal development, resulting in fetal growth restriction. The transcription factor Nfil3, but not T-bet, affected the function and the number of this decidual NK cell subset. Adoptive transfer of induced CD49a+Eomes+ NK cells reversed impaired fetal growth and rebuilt an appropriate local microenvironment. These findings reveal properties of NK cells in promoting fetal growth. In addition, this research proposes approaches for therapeutic administration of NK cells in order to reverse restricted nourishments within the uterine microenvironment during early pregnancy.

Reconstituted CD74+ NK cells trigger chronic graft versus host disease after allogeneic bone marrow transplantation.

Chronic graft-versus-host disease (cGVHD) is the most common long-term complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The patients with pulmonary cGVHD in particular have a very poor prognosis. NK cells are the first reconstituted lymphocyte subset after allo-HSCT; however, the impact of reconstituted NK cells on cGVHD is unclear. Here, we found allogeneic recipients showed obvious pulmonary cGVHD. Surprisingly, deletion of reconstituted NK cells resulted in maximal relief of pulmonary cGVHD. Mechanistically, reconstituted NK cells with donor profiles modulated the pulmonary inflammatory microenvironment to trigger cGVHD. Reconstituted NK cells secreted IFN-γ and TNF-α to induce CXCL10 production by epithelial cells, which recruited macrophages and CD4+ T cells to the lungs. Then macrophages and CD4+ T cells were activated by the inflammatory microenvironment, thereby mediating lung injury. Through assessment of differences in cellular energy, we found that CD74+ NK cells with high mitochondrial potential and pro-inflammatory activity triggered pulmonary cGVHD. Furthermore, targeted elimination of CD74+ NK cells using the anti-CD74 antibody significantly alleviated pulmonary cGVHD but preserved the CD74- NK cells to exert graft-versus-leukemia (GVL) effects. Data from human samples corroborated our findings in mouse models. Collectively, our results reveal that reconstituted CD74+ NK cells trigger pulmonary cGVHD and suggest that administration of CD74 antibody was a potential therapeutic for patients with cGVHD.

GARP-mediated active TGF-ß1 induces bone marrow NK cell dysfunction in AML patients with early relapse post-allo-HSCT.

Relapse is a leading cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute myeloid leukemia (AML). However, the underlying mechanisms remain poorly understood. Natural killer (NK) cells play a crucial role in tumor surveillance and cancer immunotherapy, and NK cell dysfunction has been observed in various tumors. Here, we performed ex vivo experiments to systematically characterize the mechanisms underlying the dysfunction of bone marrow-derived NK (BMNK) cells isolated from AML patients experiencing early relapse after allo-HSCT. We demonstrated that higher levels of active transforming growth factor ß1 (TGF-ß1) were associated with impaired effector function of BMNK cells in these AML patients. TGF-ß1 activation was induced by the overexpression of glycoprotein A repetitions predominant on the surface of CD4+ T cells. Active TGF-ß1 significantly suppressed mTORC1 activity, mitochondrial oxidative phosphorylation, the proliferation, and cytotoxicity of BMNK cells. Furthermore, pretreatment with the clinical stage TGF-ß1 pathway inhibitor, galunisertib, significantly restored mTORC1 activity, mitochondrial homeostasis, and cytotoxicity. Importantly, the blockade of the TGF-ß1 signaling improved the antitumor activity of NK cells in a leukemia xenograft mouse model. Thus, our findings reveal a mechanism explaining BMNK cell dysfunction and suggest that targeted inhibition of TGF-ß1 signaling may represent a potential therapeutic intervention to improve outcomes in AML patients undergoing allo-HSCT or NK cell-based immunotherapy.

Interactions between driver genes shape the signaling pathway landscape and direct hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies, whose initiation and development are driven by alterations in driver genes. In this study, we identified four driver genes (TP53, PTEN, CTNNB1, and KRAS) that show a high frequency of somatic mutations or copy number variations (CNVs) in patients with HCC. Four different spontaneous HCC mouse models were constructed to screen for changes in various kinase signaling pathways. The sgTrp53 + sgPten tumor upregulated mTOR and noncanonical nuclear factor-κB signaling, which was shown to be strongly inhibited by rapamycin (an mTOR inhibitor) in vitro and in vivo. The JAK-signal transducer and activator of transcription (STAT) signaling was activated in Ctnnb1mut + sgPten tumor, the proliferation of which was strongly inhibited by napabucasin (a STAT3 inhibitor). Additionally, mTOR, cytoskeleton, and AMPK signaling were upregulated while rapamycin and ezrin inhibitors exerted potent antiproliferative effects in sgPten + KrasG12D tumor. We found that JAK-STAT, MAPK, and cytoskeleton signaling were activated in sgTrp53 + KrasG12D tumor and the combination of sorafenib and napabucasin led to the complete inhibition of tumor growth in vivo. In patients with HCC who had the same molecular classification as our mouse models, the downstream signaling pathway landscapes associated with genomic alterations were identical. Our research provides novel targeted therapeutic options for the clinical treatment of HCC, based on the presence of specific genetic alterations within the tumor.

Effective treatment of severe COVID-19 patients with tocilizumab.

After analyzing the immune characteristics of patients with severe coronavirus disease 2019 (COVID-19), we have identified that pathogenic T cells and inflammatory monocytes with large amount of interleukin 6 secreting may incite the inflammatory storm, which may potentially be curbed through monoclonal antibody that targets the IL-6 pathways. Here, we aimed to assess the efficacy of tocilizumab in severe patients with COVID-19 and seek a therapeutic strategy. The patients diagnosed as severe or critical COVID-19 in The First Affiliated Hospital of University of Science and Technology of China (Anhui Provincial Hospital) and Anhui Fuyang Second People's Hospital were given tocilizumab in addition to routine therapy between 5 and 14 February 2020. The changes of clinical manifestations, computerized tomography (CT) scan image, and laboratory examinations were retrospectively analyzed. Fever returned to normal on the first day, and other symptoms improved remarkably within a few days. Within 5 d after tocilizumab, 15 of the 20 patients (75.0%) had lowered their oxygen intake, and 1 patient needed no oxygen therapy. CT scans manifested that the lung lesion opacity absorbed in 19 patients (90.5%). The percentage of lymphocytes in peripheral blood, which decreased in 85.0% of patients (17/20) before treatment (mean, 15.52 ± 8.89%), returned to normal in 52.6% of patients (10/19) on the fifth day after treatment. Abnormally elevated C-reactive protein decreased significantly in 84.2% of patients (16/19). No obvious adverse reactions were observed. All patients have been discharged on average 15.1 d after giving tocilizumab. Preliminary data show that tocilizumab, which improved the clinical outcome immediately in severe and critical COVID-19 patients, is an effective treatment to reduce mortality.

Natural killer cells promote intra-cellular-infected trophoblasts survival via APOD-LRP1 axis.

Natural killer (NK) cells are known for their potent ability to kill stressed cells, whereas host cells infected with intra-cellular bacteria may also be benefit from the selective killing function of NK cells and survive. The mechanism of how NK cells protect host cells infected with intra-cellular bacteria is still unclear. Here, we discovered that decidual NK (dNK) cells cannot only eliminate intra-cellular bacteria which infected trophoblasts, but can also synthesize more lipids and transport lipids to trophoblasts to avoid their apoptosis. Mechanically, NK cells synthesize more lipids accompanied by increasing expression of apolipoprotein APOD. Lipids in NK cells can be delivered to trophoblast cells through APOD, maintaining adequate lipid droplet content and lipid metabolism homeostasis in trophoblasts. Blocking the APOD receptor LRP1 abolished lipid transport from NK cells to trophoblasts, and the reduction of lipid droplets caused by bacterial infection in trophoblast cells could not be restored, culminating in cell apoptosis. Our study provides new evidence for the immune surveillance and protective effect of NK cells on embryos during early pregnancy.

Immunogenic senescence sensitizes lung cancer to LUNX-targeting therapy.

The higher immunogenicity of tumors usually predicts favorable therapeutic responses. Tumor antigens dominate the immunogenic character within tumors. We investigated if there was a targetable tumor antigen during immunogenic chemotherapy within lung cancer. Chemotherapy-induced immunogenic senescence was demonstrated using a multi-marker, three-step workflow, and RNA-sequencing data. The ability of anti-lung-specific X protein (LUNX) antibody to suppress the survival of senescent lung cancer cells was evaluated in vitro and in vivo using real-time cytotoxicity analysis and xenograft mouse models, respectively. The induction of cellular senescence by immunogenic chemotherapy boosted cell-surface shuttling of LUNX and enhanced the immunogenic features of senescent tumor cells, which sensitized lung cancer cells to anti-LUNX antibody-mediated therapy and contributed to tumor suppression. The immunogenic senescence-mediated anti-tumor response was triggered by the direct action of antibody on tumor cells, strengthened by natural-killer cells through an antibody-dependent cell-mediated cytotoxicity response, and ultimately, led to tumor control. Our findings suggest that LUNX is a lung cancer targetable-immunogenic antigen. The proportion of lung cancers responding to LUNX-targeting therapy could be expanded substantially by immunogenic chemotherapy that induces senescence-associated translocation of LUNX to the plasma membrane.

Uterine NK cell functions at maternal-fetal interface†.

During pregnancy, maternal decidual tissue interacts with fetal trophoblasts. They constitute the maternal-fetal interface responsible for supplying nutrition to the fetus. Uterine natural killer (uNK) cells are the most abundant immune cells at the maternal-fetal interface during early pregnancy and play critical roles throughout pregnancy. This review provides current knowledge about the functions of uNK cells. uNK cells have been shown to facilitate remodeling of the spiral artery, control the invasion of extravillous trophoblast (EVT) cells, contribute to the induction and maintenance of immune tolerance, protect against pathogen infection, and promote fetal development. Pregnancy-trained memory of uNK cells improves subsequent pregnancy outcomes. In addition, this review describes the distinct functions of three uNK cell subsets: CD27-CD11b-, CD27+, and CD27-CD11b+ uNK cells.

Profiling of the immune repertoire in COVID-19 patients with mild, severe, convalescent, or retesting-positive status.

Forty-seven samples of peripheral blood mononuclear cells from four groups of coronavirus disease (COVID)-19 patients (mild, severe, convalescent, retesting-positive) and healthy controls were applied to profile the immune repertoire of COVID-19 patients in acute infection or convalescence by transcriptome sequencing and immune-receptor repertoire (IRR) sequencing. Transcriptome analyses showed that genes within principal component group 1 (PC1) were associated with infection and disease severity whereas genes within PC2 were associated with recovery from COVID-19. A "dual-injury mechanism" of COVID-19 severity was related to an increased number of proinflammatory pathways and activated hypercoagulable pathways. A machine-learning model based on the genes associated with inflammatory and hypercoagulable pathways had the potential to be employed to monitor COVID-19 severity. Signature analyses of B-cell receptors (BCRs) and T-cell receptors (TCRs) revealed the dominant selection of longer V-J pairs (e.g., IGHV3-9-IGHJ6 and IGHV3-23-IGHJ6) and continuous tyrosine motifs in BCRs and lower diversity of TCRs. These findings provide potential predictors for COVID-19 outcomes, and new potential targets for COVID-19 treatment.

PBX1 promotes development of natural killer cells by binding directly to the Nfil3 promoter.

The transcription factor nuclear factor interleukin-3-regulated protein (NFIL3, also called E4BP4) is crucial for commitment of natural killer (NK) cells from common lymphoid progenitors (CLPs). However, the identity of the factor that can regulate NFIL3 directly during the NK-cell development is not known. Here, we reveal that pre-B-cell leukemia transcription factor 1 (PBX1) can upregulate the NFIL3 expression directly. We used conditional knockout mice in which PBX1 in hematopoietic cells was specifically absent. The number of NK-committed progenitor pre-NKP cells and rNKP cells was reduced significantly in the absence of PBX1, which was consistent with NFIL3 deficiency. Also, the NFIL3 expression in NK cells was decreased if PBX1 was absent. We demonstrated that PBX1 was bound directly to the promoter of Nfil3 and facilitated transcription. Upon knockout of the binding site of PBX1 in the Nfil3 promoter, mice showed fewer NK-precursor cells and NK cells, just like that observed in Nfil3 knockout mice. Furthermore, asparagine N286 in the homeodomain of PBX1 controlled the binding of PBX1 to the Nfil3 promoter. Collectively, these findings demonstrate that the transcription factor PBX1 promotes the early development of NK cells by upregulating the Nfil3 expression directly.

Why tocilizumab could be an effective treatment for severe COVID-19?

A severe pneumonia-associated respiratory syndrome caused by a new coronavirus was identified in December 2019 (COVID-19), spread rapidly and has become a world-wide public health challenge. About 25% of COVID-19 patients experienced severe complications including acute respiratory distress syndrome (ARDS), and even progressed into an intensive care unit (ICU) admission and died. The exploration for the mortality causes and advancing novel therapeutic development of severe COVID-19 is crucial at the moment. The biopsy samples analysis at autopsy suggested that increased alveolar exudate caused by aberrant host immune response and inflammatory cytokine storm probably impedes alveolar gas exchange and contributes to the high mortality of severe COVID-19 patients. Our research has identified that pathogenic T cells and inflammatory monocytes incite inflammatory storm with large amount of interleukin 6, therefore monoclonal antibody that targets the IL-6 pathways may potentially curb inflammatory storm. Moreover, Tocilizumab treatment that blocking IL-6 receptors showed inspiring clinical results including temperature returned to normal quickly and respiratory function improved. Therefore, we suggest that Tocilizumab is an effective treatment in severe patients of COVID-19 to calm the inflammatory storm and reduce mortality.

Natural killer cells promote immune tolerance by regulating inflammatory TH17 cells at the human maternal-fetal interface.

Natural killer (NK) cells accumulate at the maternal-fetal interface in large numbers, but their exact roles in successful pregnancy remain poorly defined. Here, we provide evidence that T(H)17 cells and local inflammation can occur at the maternal-fetal interface during natural allogenic pregnancies. We found that decidual NK cells promote immune tolerance and successful pregnancy by dampening inflammatory T(H)17 cells via IFN-γ secreted by the CD56(bright)CD27(+) NK subset. This NK-cell-mediated regulatory response is lost in patients who experience recurrent spontaneous abortions, which results in a prominent T(H)17 response and extensive local inflammation. This local inflammatory response further affects the regulatory function of NK cells, leading to the eventual loss of maternal-fetal tolerance. Thus, our data identify NK cells as key regulatory cells at the maternal-fetal interface by suppressing T(H)17-mediated local inflammation.

Molecular signatures and transcriptional regulatory networks of human immature decidual NK and mature peripheral NK cells.

Many differences exist between human immature and mature natural killer (NK) cells, but their respective molecular signatures and transcriptional regulators are relatively unknown. To gain new insights into the diversity and developmental regulation of human NK cells, we used data from high-resolution microarrays with independent verification to describe a comprehensive comparative analysis between immature decidual NK (idNK) cells with a CD56(bright) CD16(-) T-bet(-) phenotype and mature peripheral NK (mpNK) cells with a CD56(dim) CD16(+) T-bet(+) phenotype. This study shows that many novel growth factors, cytokines, and chemokines are expressed by NK cells, and they may regulate NK-cell development or function in an autocrine manner. Notably, we present that idNK and mpNK cells are enriched for homeobox and zinc-finger transcription factors (TFs), respectively. Additionally, many novel candidate transcriptional regulators are common to both idNK and mpNK cells. We further describe the transcriptional regulatory networks of NK cells and show that the endogenous growth factors, cytokines, and TFs enriched in idNK cells regulate each other and may contribute to idNK-cell immaturity. Together, these findings provide novel molecular signatures for immature and mature NK cells, and the novel candidate regulators identified here can be used to describe and further understand NK-cell differentiation and function.

Subsets of human natural killer cells and their regulatory effects.

Human natural killer (NK) cells have distinct functions as NK(tolerant) , NK(cytotoxic) and NK(regulatory) cells and can be divided into different subsets based on the relative expression of the surface markers CD27 and CD11b. CD27⁺ NK cells, which are abundant cytokine producers, are numerically in the minority in human peripheral blood but constitute the large population of NK cells in cord blood, spleen, tonsil and decidua tissues. Recent data suggest that these NK cells may have immunoregulatory properties under certain conditions. In this review, we will focus on these new NK cell subsets and discuss how regulatory NK cells may serve as rheostats or sentinels in controlling inflammation and maintaining immune homeostasis in various organs.

TGF-ß1 down-regulation of NKG2D/DAP10 and 2B4/SAP expression on human NK cells contributes to HBV persistence.

The mechanism underlying persistent hepatitis B virus (HBV) infection remains unclear. We investigated the role of innate immune responses to persistent HBV infection in 154 HBV-infected patients and 95 healthy controls. The expression of NKG2D- and 2B4-activating receptors on NK cells was significantly decreased, and moreover, the expression of DAP10 and SAP, the intracellular adaptor proteins of NKG2D and 2B4 (respectively), were lower, which then impaired NK cell-mediated cytotoxic capacity and interferon-γ production. Higher concentrations of transforming growth factor-beta 1 (TGF-ß1) were found in sera from persistently infected HBV patients. TGF-ß1 down-regulated the expression of NKG2D and 2B4 on NK cells in our in vitro study, leading to an impairment of their effector functions. Anti-TGF-ß1 antibodies could restore the expression of NKG2D and 2B4 on NK cells in vitro. Furthermore, TGF-ß1 induced cell-cycle arrest in NK cells by up-regulating the expression of p15 and p21 in NK cells from immunotolerant (IT) patients. We conclude that TGF-ß1 may reduce the expression of NKG2D/DAP10 and 2B4/SAP, and those IT patients who are deficient in these double-activating signals have impaired NK cell function, which is correlated with persistent HBV infection.