Characterization of innate lymphoid cell subsets infiltrating melanoma and epithelial ovarian tumors.

The innate lymphoid cell (ILC) family is composed of heterogeneous innate effector and helper immune cells that preferentially reside in tissues where they promote tissue homeostasis. In cancer, they have been implicated in driving both pro- and anti-tumor responses. This apparent dichotomy highlights the need to better understand differences in the ILC composition and phenotype within different tumor types that could drive seemingly opposite anti-tumor responses. Here, we characterized the frequency and phenotype of various ILC subsets in melanoma metastases and primary epithelial ovarian tumors. We observed high PD-1 expression on ILC subsets isolated from epithelial ovarian tumor samples, while ILC populations in melanoma samples express higher levels of LAG-3. In addition, we found that the frequency of cytotoxic ILCs and NKp46+ILC3 in tumors positively correlates with monocytic cells and conventional type 2 dendritic cells, revealing potentially new interconnected immune cell subsets in the tumor microenvironment. Consequently, these observations may have direct relevance to tumor microenvironment composition and how ILC subset may influence anti-tumor immunity.

Interleukin-2 is required for NKp30-dependent NK cell cytotoxicity by preferentially regulating NKp30 expression.

Natural killer (NK) cells are key effectors in cancer immunosurveillance, eliminating a broad spectrum of cancer cells without major histocompatibility complex (MHC) specificity and graft-versus-host diseases (GvHD) risk. The use of allogeneic NK cell therapies from healthy donors has demonstrated favorable clinical efficacies in treating diverse cancers, particularly hematologic malignancies, but it requires cytokines such as IL-2 to primarily support NK cell persistence and expansion. However, the role of IL-2 in the regulation of activating receptors and the function of NK cells expanded for clinical trials is poorly understood and needs clarification for the full engagement of NK cells in cancer immunotherapy. Here, we demonstrated that IL-2 deprivation significantly impaired the cytotoxicity of primary expanded NK cells by preferentially downregulating NKp30 but not NKp46 despite their common adaptor requirement for expression and function. Using NK92 and IL-2-producing NK92MI cells, we observed that NKp30-mediated cytotoxicity against myeloid leukemia cells such as K562 and THP-1 cells expressing B7-H6, a ligand for NKp30, was severely impaired by IL-2 deprivation. Furthermore, IL-2 deficiency-mediated NK cell dysfunction was overcome by the ectopic overexpression of an immunostimulatory NKp30 isoform such as NKp30a or NKp30b. In particular, NKp30a overexpression in NK92 cells improved the clearance of THP-1 cells in vivo without IL-2 supplementation. Collectively, our results highlight the distinct role of IL-2 in the regulation of NKp30 compared to that of NKp46 and suggest NKp30 upregulation, as shown here by ectopic overexpression, as a viable modality to harness NK cells in cancer immunotherapy, possibly in combination with IL-2 immunocytokines.

Conformational changes in the Niemann-Pick type C1 protein NCR1 drive sterol translocation.

The membrane protein Niemann-Pick type C1 (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. Saccharomyces cerevisiae NCR1 is localized to the vacuolar membrane, where it is suggested to carry sterols across the protective glycocalyx and deposit them into the vacuolar membrane. However, documentation of a vacuolar glycocalyx in fungi is lacking, and the mechanism for sterol translocation has remained unclear. Here, we provide evidence supporting the presence of a glycocalyx in isolated S. cerevisiae vacuoles and report four cryo-EM structures of NCR1 in two distinct conformations, named tense and relaxed. These two conformations illustrate the movement of sterols through a tunnel formed by the luminal domains, thus bypassing the barrier presented by the glycocalyx. Based on these structures and on comparison with other members of the Resistance-Nodulation-Division (RND) superfamily, we propose a transport model that links changes in the luminal domains with a cycle of protonation and deprotonation within the transmembrane region of the protein. Our model suggests that NPC proteins work by a generalized RND mechanism where the proton motive force drives conformational changes in the transmembrane domains that are allosterically coupled to luminal/extracellular domains to promote sterol transport.

Immunohistochemical Evaluation of NKP46 Receptor Expression and the Number of NK Cells in the Endometrium of Patients with Endometriosis.

Background: Endometriosis is a medical condition that can cause infertility in women. Women with endometriosis experience a decrease in NK cell cytotoxic activity against endometrial cells, ultimately contributing to the spread of these cells. Objective: To assess the frequency of NK cells and the expression of the NKP46 receptor in endometrial tissue from patients with endometriosis using immunohistochemistry. Methods: 30 endometrial tissue specimens were collected from three groups of cases with mild (n=11), moderate (n=10), and severe endometriosis (n=9), respectively. Additionally, 20 normal endometrial tissue specimens were collected as the control group. Immunohistochemical staining was carried out using specific human monoclonal antibodies against CD56 and NKP46 molecules. Results: Cases with severe endometriosis had a significantly higher number of CD56+ uterine NK cells (26.19±2.50) compared to fertile women (15.02±0.622) and women with mild to moderate endometriosis (p<0.001). However, there was no significant difference between the mild to moderate patients compared with the healthy women (p>0.05). Endometrial NKp46 expression was lower in women with severe endometriosis (0.447±0.0829) compared to fertile women (0.987±0.115, p=0.03). The NKp46+/CD56+ cell ratio was also lower in women with severe endometriosis (0.019±0.003) compared to fertile women (0.072±0.011, p=0.01). Conclusion: Women with severe endometriosis demonstrated an increased rate of infiltrated uterine NK cells and a significant decrease in NKP46 expression compared to fertile women. Therefore, NK cells and the NKp46 receptor may be involved in the development of endometriosis.

Largely preserved functionality after the combined loss of NKG2D, NCR1 and CD16 demonstrates the remarkable plasticity of NK cell responsiveness.

Natural killer (NK) cells play an important role in the early defense against tumors and virally infected cells. Their function is thought to be controlled by the balance between activating and inhibitory receptors, which often compete for the same ligands. Several activating receptors expressed on virtually all NK cells lack an inhibitory partner, most notably CD16, NCR1 and NKG2D. We therefore hypothesized that a signal through at least one of these receptors is always required for full NK cell activation. We generated animals lacking all three receptors (TKO) and analyzed their NK cells. In vitro, TKO NK cells did not show reduced ability to kill tumor targets but displayed hyperresponsiveness to NK1.1 stimulation. In vivo, TKO animals had a minor reduction in their ability to control non-hematopoietic tumors and cytomegalovirus infection, which was the result of reduced NK cell activity. Together, our findings show that activating NK cell receptors without an inhibitory partner do not provide a 'master' signal but are integrated in the cumulative balance of activating and inhibitory signals. Their activity is controlled through regulation of the responsiveness and expression of other activating receptors. Our findings may be important for future development of NK cell-based cancer immunotherapy.

Identification and characterization of protein interactions with the major Niemann-Pick type C disease protein in yeast reveals pathways of therapeutic potential.

Niemann-Pick type C (NP-C) disease is a rare lysosomal storage disease caused by mutations in NPC1 (95% cases) or NPC2 (5% cases). These proteins function together in cholesterol egress from the lysosome, whereby upon mutation, cholesterol and other lipids accumulate causing major pathologies. However, it is not fully understood how cholesterol is transported from NPC1 residing at the lysosomal membrane to the endoplasmic reticulum (ER) and plasma membrane. The yeast ortholog of NPC1, Niemann-Pick type C-related protein-1 (Ncr1), functions similarly to NPC1; when transfected into a mammalian cell lacking NPC1, Ncr1 rescues the diagnostic hallmarks of cholesterol and sphingolipid accumulation. Here, we aimed to identify and characterize protein-protein interactions (PPIs) with the yeast Ncr1 protein. A genome-wide split-ubiquitin membrane yeast two-hybrid (MYTH) protein interaction screen identified 11 ER membrane-localized, full-length proteins interacting with Ncr1 at the lysosomal/vacuolar membrane. These highlight the importance of ER-vacuole membrane interface and include PPIs with the Cyb5/Cbr1 electron transfer system, the ceramide synthase complex, and the Sec61/Sbh1 protein translocation complex. These PPIs were not detected in a sterol auxotrophy condition and thus depend on normal sterol metabolism. To provide biological context for the Ncr1-Cyb5 PPI, a yeast strain lacking this PPI (via gene deletions) exhibited altered levels of sterols and sphingolipids including increased levels of glucosylceramide that mimic NP-C disease. Overall, the results herein provide new physical and genetic interaction models to further use the yeast model of NP-C disease to better understand human NP-C disease.

Iron Limitation Restores Autophagy and Increases Lifespan in the Yeast Model of Niemann-Pick Type C1.

Niemann-Pick type C1 (NPC1) is an endolysosomal transmembrane protein involved in the export of cholesterol and sphingolipids to other cellular compartments such as the endoplasmic reticulum and plasma membrane. NPC1 loss of function is the major cause of NPC disease, a rare lysosomal storage disorder characterized by an abnormal accumulation of lipids in the late endosomal/lysosomal network, mitochondrial dysfunction, and impaired autophagy. NPC phenotypes are conserved in yeast lacking Ncr1, an orthologue of human NPC1, leading to premature aging. Herein, we performed a phosphoproteomic analysis to investigate the effect of Ncr1 loss on cellular functions mediated by the yeast lysosome-like vacuoles. Our results revealed changes in vacuolar membrane proteins that are associated mostly with vesicle biology (fusion, transport, organization), autophagy, and ion homeostasis, including iron, manganese, and calcium. Consistently, the cytoplasm to vacuole targeting (Cvt) pathway was increased in ncr1∆ cells and autophagy was compromised despite TORC1 inhibition. Moreover, ncr1∆ cells exhibited iron overload mediated by the low-iron sensing transcription factor Aft1. Iron deprivation restored the autophagic flux of ncr1∆ cells and increased its chronological lifespan and oxidative stress resistance. These results implicate iron overload on autophagy impairment, oxidative stress sensitivity, and cell death in the yeast model of NPC1.

The NK cell receptor NKp46 recognizes ecto-calreticulin on ER-stressed cells.

Natural killer (NK) cell kill infected, transformed and stressed cells when an activating NK cell receptor is triggered1. Most NK cells and some innate lymphoid cells express the activating receptor NKp46, encoded by NCR1, the most evolutionarily ancient NK cell receptor2,3. Blockage of NKp46 inhibits NK killing of many cancer targets4. Although a few infectious NKp46 ligands have been identified, the endogenous NKp46 cell surface ligand is unknown. Here we show that NKp46 recognizes externalized calreticulin (ecto-CRT), which translocates from the endoplasmic reticulum (ER) to the cell membrane during ER stress. ER stress and ecto-CRT are hallmarks of chemotherapy-induced immunogenic cell death5,6, flavivirus infection and senescence. NKp46 recognition of the P domain of ecto-CRT triggers NK cell signalling and NKp46 caps with ecto-CRT in NK immune synapses. NKp46-mediated killing is inhibited by knockout or knockdown of CALR, the gene encoding CRT, or CRT antibodies, and is enhanced by ectopic expression of glycosylphosphatidylinositol-anchored CRT. NCR1)-deficient human (and Nrc1-deficient mouse) NK cells are impaired in the killing of ZIKV-infected, ER-stressed and senescent cells and ecto-CRT-expressing cancer cells. Importantly, NKp46 recognition of ecto-CRT controls mouse B16 melanoma and RAS-driven lung cancers and enhances tumour-infiltrating NK cell degranulation and cytokine secretion. Thus, NKp46 recognition of ecto-CRT as a danger-associated molecular pattern eliminates ER-stressed cells.

Tumor-associated NK cells facilitate tumor growth via NKp46 in immunocompetent murine hepatocellular carcinoma.

Natural killer(NK) cells comprise one subset of the innate lymphoid cells family. Despite reported anti-tumor activity of NK cells, their tangible contribution to tumor control remains controversial. This is due to the incomplete understanding of NK alterations within tumor microenvironment(TME). Here we showed, using murine hepatocellular carcinoma(HCC) model, that early NK cells deletion markedly attenuated tumor growth in a CD8+T cells dependent manner. This effect was accompanied by an enhanced CD8+T cells effector function in tumor rather than circulating blood. Then, we demonstrated that abundant NKp46+ NK subset, but not NKp46- NK, were recruited towards tumor microenvironment during tumor progression. Frequency of intratumor NKP46+ NK cells were inversely related to CD8+T cells activation, and positively correlated with tumor growth. Intratumor NKp46+ NK cells exhibited dysfunction and increased expression of inhibitory receptors, when compared with NKp46- NK cells. Blockade of NK cells-associated NKp46 effectively attenuated HCC growth. Infusion of tumor-derived NKp46+ NK cells markedly enhanced HCC growth in vivo, in contrast to tumor cells inoculation alone. The further mechanistic investigations unveiled that NK cells boosted tumor growth by NKp46-mediated impairment of CD8+T cells effector function. Overall, this work supported a previously unappreciated regulatory property of tumor-associated NK cells in HCC, and NKp46 as a potential target against HCC in clinical setting.

Overexpression of lipocalin 2 in PBX1-deficient decidual NK cells promotes inflammation at the maternal-fetal interface.

PROBLEM: Impairment of PBX1 expression in decidual natural killer (dNK) cells is associated with the pathogenesis of unexplained recurrent spontaneous abortion, which results in fetal growth restriction (FGR) by affecting the secretion of downstream growth factors. However, whether other mechanisms limit embryo growth in decidua containing PBX1-deficient natural killer (NK) cells is unknown. METHOD OF STUDY: Pbx1f/f ; Ncr1Cre mice were employed to explore the underlying mechanisms by which PBX1- NK cells affect embryonic development. To simulate the clinical testing of pregnant women, Doppler ultrasound imaging was used to detect embryo implantation and development. Differentially expressed genes (DEGs) in PBX1- NK cells that may affect normal pregnancy were screened using RNA-sequencing and real-time PCR. Immune cell changes caused by DEGs were detected by flow cytometry. Finally, the mechanism of FGR was explored by injecting the protein LCN2, corresponding to the selected DEG, into mice. RESULTS: We verified the embryonic dysplasia in pregnant Pbx1f/f ; Ncr1Cre mice by Doppler ultrasound imaging and found that LCN2 was upregulated in dNK cells. We also observed higher infiltration of neutrophils and macrophages in the decidua of Pbx1f/f ; Ncr1Cre mice. Finally, we found an increase in the number and activation of neutrophils at the maternal-fetal interface after injecting LCN2 into pregnant mice and observed that these mice showed signs of FGR. CONCLUSION: Excessive LCN2 secreted by PBX1- dNK cells at the maternal-fetal interface recruit neutrophils and causes an inflammatory response, which is related to FGR.

A blinded multicenter investigation: Accentuated NK lymphocyte CD335 (NKp46) expression predicts reproductive failures after IVF.

The peripheral blood NK cell diversity is highly complex. Recent studies have described more than a thousand phenotypes sharing NK cell receptors (NKRs), across the leukocyte lineages. Previously, we have found that accentuated NK p46 phenotype has prognostic value for NK cytotoxicity status, and is characteristic for patients with recurrent implantation failure (RIF). In a blinded investigation we studied blood samples from IVF women before embryo transfer (pre-implantation genetic tested [PGT] embryos n = 116; not tested embryos n = 219). We studied NKp46 expression by flow cytometry and anti-cardiolipin antibody (aCL) levels. aCL results were transmitted to the clinic but NKp46 expression was blinded (for us and for the clinic) and not analyzed before termination of the study (end of last pregnancy). Association of NKp46 phenotype with clinical pregnancy rate (CPR), pregnancy failure (PF) rate and life birth rate (LBR) were analyzed. aCL positive and IvIg treated cases were excluded. IVF success was dependent on p46 NK phenotype in patients with PGT embryos. Elevated p46 expression on NK (>93%) as well as decreased (<66%) significantly reduce CPR (OR 12.7 and 3.8) without affecting pregnancy failure frequency. Both accentuations (taken together) resulted in a significant reduction of LBR (OR 3.9 p = 0.019) compared with non-accentuated phenotypes (p46 levels 66-93%). Elevated NK cell levels (>14.5% weakly) were associated with PF (OR 3.1 p = 0.069), but not significantly with reduced LBR. In contrast, numbers of NKCD335+ lymphocytes (>11.5%) were a significant predictor of PF (OR-4.0 p<0.05) and decreased LBR (OR 2.1 p = 0.06). At the same time, accentuated numbers of NKCD335neg lymphocytes (<0.7 and >4%) were also associated with decreased LBR (OR 2,65 p = 0.05). In patients with NKCD335++ numbers (<5 and >21%), we found a weakly association with IVF failure. We found similar associations in IVF patients without PGT -A but at lower significance levels regardless the higher number of patients. Impact of NKp46 phenotype for IVF success was significant in patients with donor's ET and almost imperceptible in patients > 35y.o. with own embryo transfer. Accentuated increased or decreased CD335 expression on NK was associated with embryo implantation failure. Balanced CD335 levels form a condition favorable for implantation. Elevated numbers of p46+NK (CD3-CD56+CD335+) predicts pregnancy failures at higher significance levels than elevated NK cell numbers. Elevated numbers of p46negNK (CD3-CD56+CD335-) indicate reduced LBR. Accentuation of p46 expression on NK cells is associated with reproductive failures. In combination with PGD it provides a powerful prediction algorithm and treatment option.

Targeting CISH enhances natural cytotoxicity receptor signaling and reduces NK cell exhaustion to improve solid tumor immunity.

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 (Cishfl/flNcr1Ki/+ ). 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 Cishfl/flNcr1Ki/+ mice, we detected no developmental or homeostatic difference in NK cells. Global gene expression of Cishfl/flNcr1Ki/+ NK cells compared with Cish+/+Ncr1Ki/+ 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 Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ 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.

Reduced Expression of Natural Killer Cell-Related Activating Receptors by Peripheral Blood Mononuclear Cells from Patients with Breast Cancer and Their Improvement by Zoledronic Acid.

BACKGROUND/AIM: Natural killer (NK) cell receptors affect the NK cell-mediated elimination of malignant cells. In this experimental study the effect of Zoledronic acid (ZOL) was investigated on the expression of NK activating- (NKP46 and NKG2D) and inhibitory (KIR2DL1) receptors by Phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) from breast cancer (BC) patients. MATERIALS AND METHODS: Peripheral blood mononuclear cell-extracted RNA from thirty breast cancer women and twenty-five healthy subjects was analyzed for gene expression of NKP46, NKG2D and KIR2DL1 using real time-PCR. Then, the PBMCs from BC patients were cultured in the presence of PHA with 5 µg/ml, 10 or 20 µg/ml of ZOL for 32 hours and expression of the aforementioned receptors was determined. RESULTS: Expression of NKP46, NKG2D and NKP46/KIR2DL1 ratio in BC women were lower than healthy group (P<0.01, P<0.04 and P<0.05, respectively). NKP46 expression was up-regulated by PHA-stimulated PBMCs treated with 10 µg/ml and 20 µg/ml of ZOL compared with PHA-stimulated cultures (P<0.01 and P<0.05, respectively). NKG2D expression remarkably increased by PHA-stimulated cultures treated with 5 µg/ml, 10 µg/ml and 20 µg/ml of ZOL compared with PHA-stimulated cultures (P<0.05 and P<0.02 and P<0.04, respectively). CONCLUSION: Expression of NK cell-related activating receptors decreased in BC patients. ZOL can improve the expression of NK activating receptors.

Porcine Plasmacytoid Dendritic Cells Are Unique in Their Expression of a Functional NKp46 Receptor.

The activating receptor NKp46 shows a unique expression pattern on porcine leukocytes. We showed already that in swine not all NK cells express NKp46 and that CD3+NKp46+ lymphocytes form a T-cell subset with unique functional properties. Here we demonstrate the expression of NKp46 on CD4highCD14-CD172a+ porcine plasmacytoid dendritic cells (pDCs). Multicolor flow cytometry analyses revealed that the vast majority of porcine pDCs (94.2% ± 4) express NKp46 ex vivo and have an increased expression on the single-cell level compared to NK cells. FSC/SSChighCD4highNKp46+ cells produced high levels of IFN-α after CpG ODN 2216 stimulation, a hallmark of pDC function. Following receptor triggering with plate-bound monoclonal antibodies against NKp46, phosphorylation of signaling molecules downstream of NKp46 was analyzed in pDCs and NK cells. Comparable to NK cells, NKp46 triggering led to an upregulation of the phosphorylated ribosomal protein S6 (pS6) in pDCs, indicating an active signaling pathway of NKp46 in porcine pDCs. Nevertheless, a defined effector function of the NK-associated receptor on porcine pDCs could not be demonstrated yet. NKp46-mediated cytotoxicity, as shown for NK cells, does not seem to occur, as NKp46+ pDCs did not express perforin. Yet, NKp46 triggering seems to contribute to cytokine production in porcine pDCs, as induction of TNF-α was observed in a small pDC subset after NKp46 cross-linking. To our knowledge, this is the first report on NKp46 expression on pDCs in a mammalian species, showing that this receptor contributes to pDC activation and function.

The Natural Cytotoxicity Receptor NKp44 (NCR2, CD336) Is Expressed on the Majority of Porcine NK Cells Ex Vivo Without Stimulation.

Natural killer (NK) cells have been studied extensively in humans and mice for their vital role in the vertebrate innate immune system. They are known to rapidly eliminate tumors or virus infected cells in an immune response utilizing their lytic properties. The natural cytotoxicity receptors (NCRs) NKp30 (NCR3), NKp44 (NCR2), and NKp46 (NCR1) are important mediators of NK-cell cytotoxicity. NKp44 expression was reported for NK cells in humans as well as in some non-human primates and found exclusively on activated NK cells. Previously, no information was available on NKp44 protein expression and its role in porcine lymphocytes due to the lack of species-specific monoclonal antibodies (mAbs). For this study, porcine-specific anti-NKp44 mAbs were generated and their reactivity was tested on blood and tissue derived NK cells in pigs of different age classes. Interestingly, NKp44 expression was detected ex vivo already on resting NK cells; moreover, the frequency of NKp44+ NK cells was higher than that of NKp46+ NK cells in most animals analyzed. Upon in vitro stimulation with IL-2 or IL-15, the frequency of NKp44+ NK cells, as well as the intensity of NKp44 expression at the single cell level, were increased. Since little is known about swine NK cells, the generation of a mAb (clone 54-1) against NKp44 will greatly aid in elucidating the mechanisms underlying the differentiation, functionality, and activation of porcine NK cells.

The transcription factor HIF-1α mediates plasticity of NKp46+ innate lymphoid cells in the gut.

Gut innate lymphoid cells (ILCs) show remarkable phenotypic diversity, yet microenvironmental factors that drive this plasticity are incompletely understood. The balance between NKp46+, IL-22-producing, group 3 ILCs (ILC3s) and interferon (IFN)-γ-producing group 1 ILCs (ILC1s) contributes to gut homeostasis. The gut mucosa is characterized by physiological hypoxia, and adaptation to low oxygen is mediated by hypoxia-inducible transcription factors (HIFs). However, the impact of HIFs on ILC phenotype and gut homeostasis is not well understood. Mice lacking the HIF-1α isoform in NKp46+ ILCs show a decrease in IFN-γ-expressing, T-bet+, NKp46+ ILC1s and a concomitant increase in IL-22-expressing, RORγt+, NKp46+ ILC3s in the gut mucosa. Single-cell RNA sequencing revealed HIF-1α as a driver of ILC phenotypes, where HIF-1α promotes the ILC1 phenotype by direct up-regulation of T-bet. Loss of HIF-1α in NKp46+ cells prevents ILC3-to-ILC1 conversion, increases the expression of IL-22-inducible genes, and confers protection against intestinal damage. Taken together, our results suggest that HIF-1α shapes the ILC phenotype in the gut.

Tcf1 Sustains the Expression of Multiple Regulators in Promoting Early Natural Killer Cell Development.

T cell factor 1 (Tcf1) is known as a critical mediator for natural killer (NK) cell development and terminal maturation. However, its essential targets and precise mechanisms involved in early NK progenitors (NKP) are not well clarified. To investigate the role of Tcf1 in NK cells at distinct developmental phases, we employed three kinds of genetic mouse models, namely, Tcf7fl/flVavCre/+, Tcf7fl/flCD122Cre/+ and Tcf7fl/flNcr1Cre/+ mice, respectively. Similar to Tcf1 germline knockout mice, we found notably diminished cell number and defective development in BM NK cells from all strains. In contrast, Tcf7fl/flNcr1Cre/+ mice exhibited modest defects in splenic NK cells compared with those in the other two strains. By analyzing the published ATAC-seq and ChIP-seq data, we found that Tcf1 directly targeted 110 NK cell-related genes which displayed differential accessibility in the absence of Tcf1. Along with this clue, we further confirmed that a series of essential regulators were expressed aberrantly in distinct BM NK subsets with conditional ablating Tcf1 at NKP stage. Eomes, Ets1, Gata3, Ikzf1, Ikzf2, Nfil3, Runx3, Sh2d1a, Slamf6, Tbx21, Tox, and Zeb2 were downregulated, whereas Spi1 and Gzmb were upregulated in distinct NK subsets due to Tcf1 deficiency. The dysregulation of these genes jointly caused severe defects in NK cells lacking Tcf1. Thus, our study identified essential targets of Tcf1 in NK cells, providing new insights into Tcf1-dependent regulatory programs in step-wise governing NK cell development.

Immunological Profiling of COVID-19 Patients with Pulmonary Sequelae.

Cellular immunity may be involved in organ damage and rehabilitation in patients with coronavirus disease 2019 (COVID-19). We aimed to delineate immunological features of COVID-19 patients with pulmonary sequelae (PS) 1 year after discharge. Fifty COVID-19 survivors were recruited and classified according to radiological characteristics, including 24 patients with PS and 26 patients without PS. Phenotypic and functional characteristics of immune cells were evaluated by multiparametric flow cytometry. Patients with PS had an increased proportion of natural killer (NK) cells and a lower percentage of B cells than patients without PS. Phenotypic and functional features of T cells in patients with PS were predominated by the accumulation of CD4-positive (CD4+) T cells secreting interleukin 17A (IL-17A), short-lived effector-like CD8+ T cells (CD27-negative [CD27-] CD62L-), and senescent T cells with excessive secretion of granzyme B/perforin/interferon gamma (IFN-γ). NK cells were characterized by the excessive secretion of granzyme B and perforin and the downregulation of NKP30 and NKP46; highly activated NKT and γδ T cells exhibited NKP30 and TIM-3 upregulation and NKB1 downregulation in patients with PS. However, immunosuppressive cells were comparable between the two groups. The interrelationship of immune cells in COVID-19 was intrinsically identified, whereby T cells secreting IL-2, IL-4, and IL-17A were enriched among CD28+ and CD57- cells and cells secreting perforin/granzyme B/IFN-γ/tumor necrosis factor alpha (TNF-α)-expressed markers of terminal differentiation. CD57+ NK cells, CD4+Perforin+ T cells, and CD8+ CD27+ CD62L+ T cells were identified as the independent predictors for residual lesions. Overall, our findings unveil the profound imbalance of immune landscape that may correlate with organ damage and rehabilitation in COVID-19. IMPORTANCE A considerable proportion of COVID-19 survivors have residual lung lesions such as ground-glass opacity and fiber streak shadow. To determine the relationship between host immunity and residual lung lesions, we performed an extensive analysis of immune responses in convalescent patients with COVID-19 1 year after discharge. We found significant differences in immunological characteristics between patients with pulmonary sequelae and patients without pulmonary sequelae 1 year after discharge. Our study highlights the profound imbalance of immune landscape in the COVID-19 patients with pulmonary sequelae, characterized by the robust activation of cytotoxic T cells, NK cells, and γδ T cells, as well as the deficiencies of immunosuppressive cells. Importantly, CD57+ NK cells, CD4+Perforin+ T cells, and CD8+ CD27+ CD62L+ T cells were identified as the independent predictors for residual lesions.

Bladder tumor ILC1s undergo Th17-like differentiation in human bladder cancer.

PURPOSE: Human innate lymphoid cells (hILCs) are lineage-negative immune cells that do not express rearranged adaptive antigen receptors. Natural killer (NK) cells are hILCs that contribute to cancer defense. The role of non-NK hILCs in cancer is unclear. Our study aimed to characterize non-NK hILCs in bladder cancer. EXPERIMENTAL DESIGN: Mass cytometry was used to characterize intratumoral non-NK hILCs based on 35 parameters, including receptors, cytokines, and transcription factors from 21 muscle-invasive bladder tumors. Model-based clustering was performed on t-distributed stochastic neighbor embedding (t-SNE) coordinates of hILCs, and the association of hILCs with tumor stage was analyzed. RESULTS: Most frequent among intratumoral non-NK hILCs were hILC1s, which were increased in higher compared with lower stage tumors. Intratumoral hILC1s were marked by Th17-like phenotype with high RORγt, IL-17, and IL-22 compared to Th1 differentiation markers, including Tbet, perforin, and IFN-γ. Compared with intratumoral hILC2s and hILC3s, hILC1s also had lower expression of activation markers (NKp30, NKp46, and CD69) and increased expression of exhaustion molecules (PD-1 and Tim3). Unsupervised clustering identified nine clusters of bladder hILCs, which were not defined by the primary hILC subtypes 1-3. hILC1s featured in all the nine clusters indicating that intratumoral hILC1s displayed the highest phenotypic heterogeneity among all hILCs. CONCLUSIONS: hILC1s are increased in higher stage tumors among patients with muscle-invasive bladder cancer. These intratumoral hILC1s exhibit an exhausted phenotype and Th17-like differentiation, identifying them as potential targets for immunotherapy.

Ex Vivo Expanded and Activated Natural Killer Cells Prolong the Overall Survival of Mice with Glioblastoma-like Cell-Derived Tumors.

Glioblastoma (GBM) is the leading malignant intracranial tumor and is associated with a poor prognosis. Highly purified, activated natural killer (NK) cells, designated as genuine induced NK cells (GiNKs), represent a promising immunotherapy for GBM. We evaluated the anti-tumor effect of GiNKs in association with the programmed death 1(PD-1)/PD-ligand 1 (PD-L1) immune checkpoint pathway. We determined the level of PD-1 expression, a receptor known to down-regulate the immune response against malignancy, on GiNKs. PD-L1 expression on glioma cell lines (GBM-like cell line U87MG, and GBM cell line T98G) was also determined. To evaluate the anti-tumor activity of GiNKs in vivo, we used a xenograft model of subcutaneously implanted U87MG cells in immunocompromised NOG mice. The GiNKs expressed very low levels of PD-1. Although PD-L1 was expressed on U87MG and T98G cells, the expression levels were highly variable. Our xenograft model revealed that the retro-orbital administration of GiNKs and interleukin-2 (IL-2) prolonged the survival of NOG mice bearing subcutaneous U87MG-derived tumors. PD-1 blocking antibodies did not have an additive effect with GiNKs for prolonging survival. GiNKs may represent a promising cell-based immunotherapy for patients with GBM and are minimally affected by the PD-1/PD-L1 immune evasion axis in GBM.