Natural Killer Cell Markers Research Articles

Identification and Validation of a Novel Signature Based on NK Cell Marker Genes to Predict Prognosis and Immunotherapy Response in Lung Adenocarcinoma by Integrated Analysis of Single-Cell and Bulk RNA-Sequencing.

Natural killer (NK) cells, the effectors of the innate immune system, have a remarkable influence on cancer prognosis and immunotherapy. In this study, a total of 1,816 samples from nine independent cohorts in public datasets were enrolled. We first conducted a comprehensive analysis of single-cell RNA-sequencing data of lung adenocarcinoma (LUAD) from the Gene Expression Omnibus (GEO) database and determined 189 NK cell marker genes. Subsequently, we developed a seven-gene prognostic signature based on NK cell marker genes in the TCGA LUAD cohort, which stratified patients into high-risk and low-risk groups. The predictive power of the signature was well verified in different clinical subgroups and GEO cohorts. With a multivariate analysis, the signature was identified as an independent prognostic factor. Low-risk patients had higher immune cell infiltration states, especially CD8+ T cells and follicular helper T cells. There existed a negative association between inflammatory activities and risk score, and the richness and diversity of the T-cell receptor (TCR) repertoire was higher in the low-risk groups. Importantly, analysis of an independent immunotherapy cohort (IMvigor210) revealed that low-risk patients had better immunotherapy responses and prognosis than high-risk patients. Collectively, our study developed a novel signature based on NK cell marker genes, which had a potent capability to predict the prognosis and immunotherapy response of LUAD patients.

Eight Weeks of Daily Cannabidiol Ingestion Does Not Alter Peripheral Blood Mononuclear Cell Profile or Natural Killer Cell Cytotoxicity

ObjectiveThe purpose of this randomized placebo‐controlled trial was to determine the effects of an 8‐week cannabidiol (CBD) intervention on peripheral blood mononuclear cells (PBMCs) profile and natural killer cell (NKC) cytotoxicity.HypothesisFollowing an 8‐week CBD intervention, participants consuming CBD will have an increased percentage of NKC in peripheral blood, as well as decreased NKC cytotoxicity determined by a measure of K562 cell viability.MethodsPhysically active men and women (18‐45 y), currently meeting the American College of Sports Medicine (ACSM) guidelines for physical activity were randomly assigned to placebo (CN, n=5) or CBD (CB, n=9) groups. Participants consumed capsulized control; coconut derived medium chain triglycerides (MCT; 225mg/day) or CBD (50mg/day with 175mg of MCT) daily for 8 weeks. Participants completed measures of body size, body composition (BodPod) and fitness (VO2max), and peripheral blood was drawn before and after the intervention. Ficoll‐isolated PBMCs were co‐incubated for 4h with K562 human leukemia cells before cytotoxicity analysis by flow cytometry. Before co‐culture, PBMCs were stained with T cell marker CD3, and NKC marker, CD56, for immune profiling. K562 cells were pre‐stained with cell‐specific CD71 (transferrin receptor) and the cell viability dye Calcein AM. Data are presented as mean ± standard deviation with significance set at a=0.05. At the pre intervention time point, data was not normally distributed, so a Welch’s t‐test was preformed to determine group differences. A 2 (group) x 2 (time) analysis of variance (ANOVA) was used to identify any interactions or main effects.ResultsAt the pre‐intervention time points, there were no significant differences between groups with respect to participant characteristics (age: 27.1 ± 6.1 y; height: 169 ± 9.7 cm; weight: 73.8 ± 15.5 kg; lean body mass: 57.5 ± 12.0 kg; body fat: 21.5 ± 8.3%; VO2Peak: 45.2 ± 7.6 ml/kg/min). There were no significant differences in percentage of NKC (Pre: 8.8 ± 5.8%; Post:7.4 ± 5.7%), natural killer T‐cells (NKT; Pre: 1.7 ±1.1; Post: 1.5 ±1.1%), or T‐cells (Pre: 47.37±13.3; Post: 46.29±10.17%) when CN was compared to CB following the intervention. Furthermore, there were no differences in K562 cell mean or median fluorescence intensity of Calcein AM when CN was compared to CB following the intervention.ConclusionEight weeks of CBD (50mg/day) did not alter NKC, NKT, or T‐cell distribution in PBMCs. In line with these findings, NKC showed similar cytolytic function through no change in mean or median fluorescence intensity of Calcein AM expression of K562 human leukemia cells, indicating no change in cell viability following CBD supplementation. This preliminary study suggests that CBD does not alter the distribution of circulating mononuclear immune cells or NKC cytolytic function.

The Expression and Function of NK Cells in Patients with Acute Myeloid Leukemia

To explore the expression characteristics of antigens and functional markers of natural killer (NK) cells in patients with acute myeloid leukemia (AML). Multi-parameter flow cytometry was used to detect NK cell surface markers and their functional indicators in 56 newly diagnosed AML patients and 24 healthy controls, including activating receptors NKG2D, NKP46, DNAM-1, and killing indicators granzyme B, perforin. Referring to the WHO hematopoiesis and lymph tissue tumor classification criteria, 56 cases were roughly divided into three types: AML M1, M2, and M4/M5. However, there was no differences about NK cells among the three types, so it was no longer subdivided. NK cells were divided into two groups: CD3-CD56hiCD16- (CD56hiNK) and CD3-CD56dimCD16+ (CD56dimNK). Compared with CD56dimNK cell population, except for NKP46, the positive expression levels of NKG2D and other receptors of CD56hiNK cells in AML patients decreased (P<0.001). Compared with healthy controls, the proportion of CD56hiNK cells in AML patients increased, while the number and proportion of NK cells and proportion of CD56dimNK cells significantly decreased (P<0.05). The proportion of perforin in CD56hiNK cells significantly increased (P<0.05). The expression of DNAM-1 in CD56hiNK cells, NKG2D, DNAM-1, and perforin in CD56dimNK cells decreased significantly (P<0.05). There was no statistically significant difference in expression of other functional indexes in AML patients compared with corresponding indexes of healthy controls. In addition, the proportion of CD56hiNK cells was positively correlated with the expression of CD34+ in AML (r=0.303). Compared with CD56dimNK, the ratio of CD56hiNK and the expression of functional markers in AML patients are lower. Compared with healthy controls, the number and expression ratio of NK cells in AML patients decrease and the expression of functional markers is abnormal, indicating that its function is impaired.

The Critical and Diverse Roles of CD4-CD8- Double Negative T Cells in Nonalcoholic Fatty Liver Disease.

Background & AimsHepatic inflammation is a hallmark of nonalcoholic fatty liver disease (NAFLD). Double negative T (DNT) cells are a unique subset of T lymphocytes that do not express CD4, CD8, or natural killer cell markers, and studies have suggested that DNT cells play critical and diverse roles in the immune system. However, the role of intrahepatic DNT cells in NAFLD is largely unknown.MethodsThe proportions and RNA transcription profiling of intrahepatic DNT cells were compared between C57BL/6 mice fed with control diet or methionine-choline–deficient diet for 5 weeks. The functions of DNT cells were tested in vitro and in vivo.ResultsThe proportion of intrahepatic DNT cells was significantly increased in mice with diet-induced NAFLD. In NAFLD mice, the proportion of intrahepatic TCRγδ+ DNT cells was increased along with elevated interleukin (IL) 17A; in contrast, the percentage of TCRαβ+ DNT cells was decreased, accompanied by reduced granzyme B (GZMB). TCRγδ+ DNT cell depletion resulted in lowered liver IL17A levels and significantly alleviated NAFLD. Adoptive transfer of intrahepatic TCRαβ+ DNT cells from control mice increased intrahepatic CD4 and CD8 T cell apoptosis and inhibited NAFLD progression. Furthermore, we revealed that adrenic acid and arachidonic acid, harmful fatty acids that were enriched in the liver of the mice with NAFLD, could induce apoptosis of TCRαβ+ DNT cells and inhibit their immunosuppressive function and nuclear factor kappa B (NF-κB) or AKT signaling pathway activity. However, arachidonic acid facilitated IL17A secretion by TCRγδ+ DNT cells, and the NF-κB signaling pathway was involved. Finally, we also confirmed the variation of intrahepatic TCRαβ+ DNT cells and TCRγδ+ DNT cells in humans.ConclusionsDuring NAFLD progression, TCRγδ+ DNT cells enhance IL17A secretion and aggravate liver inflammation, whereas TCRαβ+ DNT cells decrease GZMB production and lead to weakened immunoregulatory function. Shifting of balance from TCRγδ+ DNT cell response to one that favors TCRαβ+ DNT regulation would be beneficial for the prevention and treatment of NAFLD.

Isolation of Innate Lymphoid Cells from Murine Intestinal Lamina Propria.

Natural killer (NK) cells are innate cytotoxic immune cells essential for mediating first-line defense against various environmental antigens. With the discoveries of other subsets of innate lymphocytes over the last decade, NK cells are categorized as innate lymphoid cells (ILC) and as the innate counterparts of cytotoxic T cells. Besides NK cells, ILCs are classified into three groups distinguished by their dependence on distinct transcription factors for development and unique effector functions. Subsets of ILCs share many surface proteins that, however, have initially been identified as NK cell markers, making them hard to be distinguished for detailed investigations. Here, we describe a method to identify and individually isolate subsets of innate lymphoid cells from gut lamina propria using cell surface markers.

Liver Regeneration in Chronic Liver Injuries: Basic and Clinical Applications Focusing on Macrophages and Natural Killer Cells.

Background & AimsLiver regeneration is a necessary but complex process involving multiple cell types besides hepatocytes. Mechanisms underlying liver regeneration after partial hepatectomy and acute liver injury have been well-described. However, in patients with chronic and severe liver injury, the remnant liver cannot completely restore the liver mass and function, thereby involving liver progenitor-like cells (LPLCs) and various immune cells.ResultsMacrophages are beneficial to LPLCs proliferation and the differentiation of LPLCs to hepatocytes. Also, cells expressing natural killer (NK) cell markers have been studied in promoting both liver injury and liver regeneration. NK cells can promote LPLC-induced liver regeneration, but the excessive activation of hepatic NK cells may lead to high serum levels of interferon-γ, thus inhibiting liver regeneration.ConclusionsThis review summarizes the recent research on 2 important innate immune cells, macrophages and NK cells, in LPLC-induced liver regeneration and the mechanisms of liver regeneration during chronic liver injury, as well as the latest macrophage- and NK cell-based therapies for chronic liver injury. These novel findings can further help identify new treatments for chronic liver injury, saving patients from the pain of liver transplantations.

Korean Red Ginseng Enhances Immunotherapeutic Effects of NK Cells via Eosinophils in Metastatic Liver Cancer Model.

Metastasis decreases the survival rate of patients with liver cancer. Therefore, novel anti-metastatic strategies are needed. Korean Red Ginseng (KRG) is often ingested as a functional food with an immune-boosting effect. We investigated a combination of KRG and natural killer (NK) cells as a novel immunotherapy approach. SK-Hep1 cells were injected into the tail vein of NRGA mice to establish an experimental metastasis model. KRG, NK cells, or a combination of KRG and NK cells were administered. Tumor growth was observed using an in vivo imaging system, and metastatic lesions were evaluated by histological analysis and immunohistochemistry. Bioluminescence intensity was lower in the KRG and NK cell combination group than in the other groups, indicating that the combination treatment suppressed the progression of metastasis. CD56 expression was used as a NK cell marker and hematological analysis was performed. The combination treatment also decreased the expression of matrix metalloproteinases and the area of metastatic lesions in liver and bone tissues, as well as increased the eosinophil count. Expression of cytokines-related eosinophils and NK cells was determined by Western blotting analysis. The expression of interleukin 33 (IL33) was induced by the combination of KRG and NK cells. High IL33 expression was associated with prolonged overall survival in the Kaplan–Meier plotter. Our results suggest that KRG enhances the immune activity of NK cells by IL-33 through eosinophils and suppresses metastatic liver cancer progression.

Liver X receptors regulate natural killer T cell population and antitumor activity in the liver of mice

The nuclear receptors liver X receptor α (LXRα) and LXRβ are lipid sensors that regulate lipid metabolism and immunity. Natural killer T (NKT) cells, a T cell subset expressing surface markers of both natural killer cells and T lymphocytes and involved in antitumor immunity, are another abundant immune cell type in the liver. The potential function of the metabolic regulators LXRα/β in hepatic NKT cells remains unknown. In this study, we examined the role of LXRα and LXRβ in NKT cells using mice deficient for LXRα and/or LXRβ, and found that hepatic invariant NKT (iNKT) cells are drastically decreased in LXRα/β-KO mice. Cytokine production stimulated by the iNKT cell activator α-galactosylceramide was impaired in LXRα/β-KO hepatic mononuclear cells and in LXRα/β-KO mice. iNKT cell-mediated antitumor effect was also disturbed in LXRα/β-KO mice. LXRα/β-KO mice transplanted with wild-type bone marrow showed decreased iNKT cells in the liver and spleen. The thymus of LXRα/β-KO mice showed a decreased population of iNKT cells. In conclusion, LXRα and LXRβ are essential for NKT cell-mediated immunity, such as cytokine production and hepatic antitumor activity, and are involved in NKT cell development in immune tissues, such as the thymus.

High Density of CD16+ Tumor-Infiltrating Immune Cells in Recurrent Ovarian Cancer Is Associated with Enhanced Responsiveness to Chemotherapy and Prolonged Overall Survival.

Simple SummaryThe late—and in most cases at an advanced stage—diagnosis of patients with ovarian cancer (OC) and the high recurrence rate make this malignant disease the most lethal among gynecological cancers. With a mortality-to-incidence ratio of 0.74, OC is a tumor with the fifth most frequent progression after esophageal cancer, liver cancer, pancreatic cancer, and brain tumors. The updated FIGO staging system is the gold standard in the clinic and includes surgical, radiologic, and pathologic elements to describe the extent of OC. This system is used to describe tumor extent, plan further therapy, and predict prognosis. However, it is consistently observed that patients with identical stages and treatments have a completely different outcome in terms of survival and recurrence. This fact indicates that this classification alone is not sufficient for the prognosis of OC in the vast majority of cases. Over the last two decades, many studies have demonstrated the critical role of the tumor microenvironment in tumorigenesis, progression, prognosis, and response to chemotherapy. In the current study, we investigate the role of CD16 expression in OC.Background: Ovarian cancer (OC) is the most aggressive and fatal malignancy of the female reproductive system. Debulking surgery with adjuvant chemotherapy represents the standard treatment, but recurrence rates are particularly high. Over the past decades, the association between the immune system and cancer progression has been extensively investigated. However, the interaction between chemotherapy and cancer immune infiltration is still unclear. In this study, we examined the prognostic role of CD16 expression in OC, as related to the effectiveness of standard adjuvant chemotherapy treatment. Methods: We analyzed the infiltration by immune cells expressing CD16, a well-characterized natural killer (NK) and myeloid cell marker, in a tissue microarray (TMA) of 47 patient specimens of primary OCs and their matching recurrences by immunohistochemistry (IHC). We analyzed our data first in the whole cohort, then in the primary tumors, and finally in recurrences. We focused on recurrence-free survival (RFS), overall survival (OS), and chemosensitivity. Chemosensitivity was defined as RFS of more than 6 months. Results: There was no significant correlation between CD16 expression and prognosis in primary carcinomas. However, interestingly, a high density of CD16-expressing tumor-infiltrating immune cells (TICs) in recurrent carcinoma was associated with better RFS (p = 0.008) and OS (p = 0.029). Moreover, high CD16 cell density in recurrent ovarian carcinoma showed a significant association with chemosensitivity (p = 0.034). Univariate Cox regression analysis revealed that the high expression of CD16+ TIC in recurrent cancer biopsies is significantly associated with an increased RFS (HR = 0.49; 95% CI 0.24–0.99; p = 0.047) and OS (HR = 0.28; 95% CI 0.10–0.77; p = 0.013). However, this was not independent of known prognostic factors such as age, FIGO stage, resection status, and the number of chemotherapy cycles. Conclusions: The high density of CD16-expressing TICs in recurrent ovarian cancer is associated with a better RFS and OS, thereby suggesting a previously unsuspected interaction between standard OC chemotherapy and immune cell infiltration.

Expansion of Phenotypically Senescent CD57+ CD8 T Cells Is Associated with Impaired Immunocompetence after Allogeneic Hematopoietic Stem Cell Transplantation

Background: T cell senescence is a physiological process typically associated with aging. In addition, lymphopenia and chronic immune activation can result in T cell senescence. CD57, a member of the N-CAM family initially described as a natural killer cell marker, has been reported as a marker of human senescent CD8 T cells. Percentages of CD57+ CD8 T cells increase during aging as well as during chronic viral infections. Early studies have reported increased proportions of CD57-expressing CD8 T cells after autologous and allogeneic hematopoietic stem cell transplantation (HSCT). Whether these cells can be considered a counterpart of the senescent population found during aging is at present unknown. More importantly, whether the expansion of CD57+ CD8 T cells is associated with impaired immunocompetence after allogeneic HSCT remains to be investigated.Materials and Methods: With written informed consent, peripheral blood mononuclear cells were collected from healthy controls (HC, n=21) and patients undergoing allogeneic HSCT (n=115) at Geneva University Hospitals. Proportions of CD57+ CD8 T cells as well as phenotypic and functional characteristics of CD57+ CD8 T cells were assessed by flow cytometry. Virus-specific CD8 T cells were identified by flow cytometry based on IFNg and/or TNFa intra-cytoplasmic expression after 6h in vitro stimulation with peptides derived from CMV, EBV, HHV6, BKV and Adenovirus. Torque Teno Virus (TTV) replication was quantified by quantitative PCR as previously described (Pradier et al., Front Immunol 2020; doi: 10.3389/fimmu.2020.00998).Results: CD8 T cells recovered from allogeneic HSCT displayed significantly higher proportions of CD57+ cells compared with healthy controls (HC, median 23% [range 6%-67%]; HSCT 58% [11%-97%]; p= 8.1e−06; Figure 1A-B). Such a difference was detected at early time points after transplantation and further increased at later time points (Figure 1B). After taking into account T cell subsets heterogeneity, we observed higher proportions of CD57+ cells in CD45RA- CCR7+ CD27+ central memory (CM; p= 0.00057), CD45RA- CCR7- CD27+ transitional memory (TM; p=1.1e-05) and CD45RA- CCR7- CD27- effector memory (EM; p=2.6e−06) CD8 T cells from allogeneic HSCT recipients compared with healthy controls. We did not observe any significant differences in CD57 expression in naïve nor in TEMRA CD8 T cells. Phenotypically, CD57+ CD8 T cells from allogeneic HSCT recipients displayed a senescent immunophenotype characterized by the low surface expression of CD127 that was further downregulated in CD57+ CD8 T cells from allogeneic HSCT recipients compared with healthy controls (p=0.00067). Functionally, CD57+ CD8 T cells from allogeneic HSCT recipients displayed a similar capacity to produce IFN-g, TNF-a, granzyme B and perforin when compared to CD57+ CD8 T cells from healthy controls. Virus-specific CD8 T cells identified upon stimulation with CMV, EBV, HHV6, BKV and Adenovirus peptides mainly displayed a CD27- effector memory phenotype in HSCT recipients (Figure 1C) and expressed higher levels of CD57 in HSCT recipients compared with healthy controls for CMV (p=0.017(; EBV p=0.018; Figure 1C-D). with a trend not reaching significance was for adenovirus, HHV6 and BK-virus-specific CD8 T cells. Using the replication of the non-pathogenic anellovirus TTV as a measure of impaired immunocompetence, we observed that the proportion of CD57-expressing cells among effector memory CD8 T cells positively correlated with TTV titers in allogeneic HSCT recipients (R=0.32, p=0.019; Figure 1E).Conclusion: These results show that the proportion of phenotypically senescent CD57+ CD8 T cells increases after allogeneic HSCT as a result of an increased expression at the surface of memory CD8 T cells, including virus-specific cells. Moreover, CD57 expression at the surface of EM CD8 T cells, a highly enriched population in allogeneic HSCT recipients, correlated with higher replication of TTV, reflecting a status of impaired immunocompetence after allogeneic HSCT. Studies are ongoing to determine the utility of CD57 expression on T cells as a biomarker to predict infectious complications after allogeneic HSCT. [Display omitted] DisclosuresChalandon: Incyte, BMS, Pfizer, Abbie, MSD, Roche, Novartis, Gilead, Amgen, Jazz, Astra Zenec: Other: Travel EXpenses, Accomodation; Incyte: Speakers Bureau; Incyte, BMS, Pfizer, Abbie, MSD, Roche, Novartis, Amgen: Other: Advisory Board. Simonetta: BMS Cellgene: Other: Ad-Hoc Advisory Board.

CD16 and CD56 mRNA Expression in Decidua of Patients with Missed and Spontaneous Abortions

Study Objective to estimate relative mRNA expression of receptors CD16 and CD56 in decidual tissue of the patients with missed and spontaneous abortions. Design Prospective cohort study. Setting Academic affiliated tertiary care center. Patients or Participants Patients with spontaneous abortions (n=22), missed abortions (n=22) and women with normal pregnancy, admitted for artificial abortions (control group, n=57) were examined at 6-10 weeks of gestation. Patients with severe extragenital diseases, antiphospholipid syndrome, endocrine disorders, uterine malformations, and fetal chromosomal abnormalities were excluded from the research. Interventions Uterine abrasion. Measurements and Main Results Decidual tissue was obtained by uterine abrasion. Relative mRNA expression of CD16 and CD56 was defined by reverse transcription-quantitative polymerase chain reaction (RT-PCR). Peptidylprolyl isomerase А and beta-actin were used as housekeeping genes. Expression of mRNA was counted in relative units by ΔΔCq method. Data were performed as the median and interquartile range and compared using Kruskal-Wallis and Mann-Whitney tests (GraphPad Prism 9.0.2). Relative mRNA expression of CD56 (a marker of natural killer (NK) cells) in decidual tissue of the patients with spontaneous and missed abortions didn't have significant differences with the control group, which confirms the data of meta-analysis (Seshadri S., Sunkara S.K., 2013). Relative mRNA expression of CD16 in decidua of patients with missed abortions (837,53 (313,00; 1082,39)) was lower compared to women with spontaneous abortions (1077 (510,23; 1541,37)) and the control group (1692,57 (1243,34; 2759,13)) (p<0,0001 and p<0,00001, respectively). Expression of CD16 in decidua of the patients with spontaneous abortions was lower, than in the control group (p<0,001). CD16 (FcγRIII) is involved in antibody-dependent cellular cytotoxicity. Uterine NK cells have CD56 + CD16- phenotype (Tang A.W., 2011). The main cells in the decidua expressing CD16 are monocytes, macrophages (Ning F. et al., 2016), and neutrophils (Presicce P. et al., 2018). Conclusion CD16 can play an important role in the pathogenesis of early miscarriages. to estimate relative mRNA expression of receptors CD16 and CD56 in decidual tissue of the patients with missed and spontaneous abortions. Prospective cohort study. Academic affiliated tertiary care center. Patients with spontaneous abortions (n=22), missed abortions (n=22) and women with normal pregnancy, admitted for artificial abortions (control group, n=57) were examined at 6-10 weeks of gestation. Patients with severe extragenital diseases, antiphospholipid syndrome, endocrine disorders, uterine malformations, and fetal chromosomal abnormalities were excluded from the research. Uterine abrasion. Decidual tissue was obtained by uterine abrasion. Relative mRNA expression of CD16 and CD56 was defined by reverse transcription-quantitative polymerase chain reaction (RT-PCR). Peptidylprolyl isomerase А and beta-actin were used as housekeeping genes. Expression of mRNA was counted in relative units by ΔΔCq method. Data were performed as the median and interquartile range and compared using Kruskal-Wallis and Mann-Whitney tests (GraphPad Prism 9.0.2). Relative mRNA expression of CD56 (a marker of natural killer (NK) cells) in decidual tissue of the patients with spontaneous and missed abortions didn't have significant differences with the control group, which confirms the data of meta-analysis (Seshadri S., Sunkara S.K., 2013). Relative mRNA expression of CD16 in decidua of patients with missed abortions (837,53 (313,00; 1082,39)) was lower compared to women with spontaneous abortions (1077 (510,23; 1541,37)) and the control group (1692,57 (1243,34; 2759,13)) (p<0,0001 and p<0,00001, respectively). Expression of CD16 in decidua of the patients with spontaneous abortions was lower, than in the control group (p<0,001). CD16 (FcγRIII) is involved in antibody-dependent cellular cytotoxicity. Uterine NK cells have CD56 + CD16- phenotype (Tang A.W., 2011). The main cells in the decidua expressing CD16 are monocytes, macrophages (Ning F. et al., 2016), and neutrophils (Presicce P. et al., 2018). CD16 can play an important role in the pathogenesis of early miscarriages.

EZH2 identifies the precursors of human natural killer cells with trained immunity.

Objective:Trained immunity of natural killer (NK) cells has shown great potential in the treatment of cancers by eliciting enhanced effector responses to restimulation by cytokines or cancer cells for long time periods after preactivation. However, the human NK cells responsible for the generation and maintenance of trained immunity are largely unknown. We hypothesized that heterogeneous human NK cells would respond differentially to stimulation with a combination of IL-12, IL-15, and IL-18, and that an NK cell subset might exist that is mainly responsible for the induction of trained immunity. On the basis of our hypothesis, we aimed to identify the subset from which cytokine-trained human NK cells originate and to explore possible regulatory targets for drug intervention.Methods:Flow cytometry assays were performed to analyze the functions of cytokine-trained NK cells and examine cell division and protein expression in NK cell subsets. Single-cell RNA sequencing (scRNA-seq) plus TotalSeq™ technology was used to track the heterogeneity of NK cells during the induction of trained immunity.Results:Traditional developmental markers for peripheral NK cells were unable to identify the precursors of human NK cells with trained immunity. Therefore, we used scRNA-seq plus TotalSeq™ technology to track the heterogeneity of NK cells during the induction of trained immunity and identified a unique cluster of CD57−NKG2A+EZH2+IFNG+MKI67+IL12R+IL15R+IL18R+ NK cells. Enrichment and pseudotime trajectory analyses suggested that this cluster of NK cells contained the precursor of trained NK cells. We then used flow cytometry to further investigate the role of EZH2 in trained NK precursors and found that CD57−NKG2A+EZH2+ NK cells had faster cell cycles and an enhanced trained phenotype, and EZH2 inhibition significantly impaired the induction of trained immunity in NK cells. These results suggested that EZH2 is a unique epigenetic marker of precursors of human NK cells with trained immunity.Conclusions:Our work revealed human NK heterogeneity in the induction of trained immunity, identified the precursor subset for trained NK cells, and demonstrated the critical role of EZH2 in the induction of trained immunity in human NK cells.

Serum HBV pregenomic RNA exhibited opposite associations with NKdim and NKbright cell immunity in treatment-naïve chronic hepatitis B patients.

Hepatitis B virus (HBV) pregenomic RNA (pgRNA) is a new biomarker that reflects HBV replication, but its relationship with natural killer (NK) cell immunity in chronic hepatitis B (CHB) is unknown. We assessed serum HBV pgRNA levels in 323 CHB patients by reverse transcription-polymerase chain reaction, assessed cytokine production and activation and inhibitory markers of NK cells by flow cytometry, and measured serum cytokines by enzyme-linked immunosorbent assays (ELISAs). Among the different CHB phases, the serum HBV pgRNA level was highest in the immune-tolerant (IT) and immune-active (IA) phases. Regarding NK and NKdim cells, HBV pgRNA was negatively associated with frequencies, but positively associated with NKp44 and NKp46 expression (activation markers). Regarding NKbright cells, serum HBV pgRNA was positively associated with frequency and programmed cell death protein 1 (PD1) expression (inhibitory marker), but negatively associated with NKp44 and NKp46. Serum HBV pgRNA was not associated with NKp30 (activation marker) on NK cells or subsets. Lastly, serum HBV pgRNA was positively correlated with the levels of serum IL-7 and IL-12P40 (NK cell-promoting cytokines) and negatively correlated with serum prostaglandin E2 (PGE2) level (which negatively regulates NK cells). In conclusion, we found varied relationships between serum HBV pgRNA and NK cells and subsets, indicating that HBV pgRNA may play a complicated role in NK cell-related immunity, providing new information on HBV and host immunity.

Low Expression of T/NK-Cell Signature Predicts Poor Survival in Patients with Primary Testicular and Diffuse Large B-Cell Lymphoma

Primary testicular lymphoma (PTL) is a rare and aggressive disease accounting for 1% of malignant lymphomas. Majority of the PTLs are diffuse large B-cell lymphomas (DLBCL). Recently, the impact of tumor microenvironment (TME) and limited immune surveillance on the lymphoma pathogenesis and survival has been recognized. Here, we aimed to characterize cellular and molecular immunological profiles in PTL, and associate the findings with outcome.We used NanoString digital gene expression profiling with the PanCancer Immune panel to assess the expression of 770 immune response genes in tumor tissue of 60 patients with PTL. RNA-sequencing data from a cohort of 96 patients with primary DLBCL was used to validate the findings.Unsupervised hierarchical clustering revealed clusters of genes with differential expression between the PTL patients. The clusters were enriched for genes related to T-cell and natural killer (NK) cell markers and signaling (e.g. CD3, CD4, CD8, ITGB2, PRF1, GZMA/B/H/M/K), as well as cytokine signaling (e.g. IL4, IL9, IL17A/B, TNFSF11, CXCR1, CCL28). Interestingly, the group of patients with low expression of T/NK-cell signature genes had a significantly worse progression-free survival (5-year PFS 33% vs. 65%, p=0.012) and overall survival (5-year OS 33% vs. 67%, p=0.004), as compared to the other patients. In Cox multivariate analysis with international prognostic index (IPI), T/NK-cell signature retained independence for prediction of better PFS and OS (RR=0.356, p=0.014 and RR=0.306, p=0.006, respectively). The signature was identified also in a cohort of 96 patients with primary DLBCL, but not in Hodgkin lymphoma. Multiplex immunohistochemistry confirmed lower percentage of CD3+, CD3+CD4+, and CD3+CD8+ lymphocytes in the tumor tissue of the group of PTL patients with poor prognosis (11.8% vs. 24.1%, 5.0% vs. 15.6%, and 4.6% vs. 12.3%, respectively), and association of their higher amount with favorable PFS and OS. Moreover, the RNA level expression of MHC class I and II genes was significantly lower in the poor prognosis group.In conclusion, our results demonstrate that loss of T/NK cell signature and MHC class I and II gene expression are associated with high risk of recurrence and death in patients with PTL and DLBCL. They further highlight the importance of the immune escape as a mechanism of treatment resistance. DisclosuresPollari:Takeda: Other: Conference attendance fees and travel expences; BMS: Other: Conference attendance fees and travel expences; Novartis: Other: Conference attendance fees and travel expences; Pierre Fabre: Other: Conference attendance fees and travel expences; Roche: Consultancy. Mannisto:Takeda: Honoraria, Other: Travel expence; Roche: Honoraria, Other: Travel expence; Amgen: Other: Travel expence; Novartis: Other: Travel expence; Celgene: Other: Travel expence; Gilead: Other: Travel expence; Pfizer: Honoraria; SOBI: Honoraria. Mustjoki:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Celgene: Honoraria. Leppa:Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Research Funding; Merck: Consultancy, Honoraria; Janssen Cilag: Consultancy, Research Funding.

NK Cell Homing and Persistence in Bone Marrow after Adoptive Immunotherapy Correlates with Therapy Response

Background: Natural killer (NK) cells are innate lymphoid cells capable of killing leukemic cells without prior sensitization. Adoptive immunotherapy with allogeneic NK cells has been used at the University of Minnesota and was shown to be effective in a subset of patients with acute myeloid leukemia refractory to therapy. The factors determining clinical success of adoptive NK cell immunotherapy are largely unknown. We hypothesized that NK cell homing and persistence in the bone marrow niche may be predictive for the clinical outcomes.Methods: We retrospectively analyzed bone marrow biopsy findings 14 days after NK cell infusion in 109 patients treated at our institution between 2003-2014. To evaluate the homing and persistence of NK cell in the bone marrow, multiple methods were used. Immunohistochemical staining for the NK cell marker CD56 was performed on the core biopsy in available cases. Flow cytometry data performed on aspirate material at the time of the biopsy was re-evaluated to enumerate the NK cell percentage. Finally the percentage of Large Granular Lymphocytes was determined in a semi-quantitative manner from Wright-Giemsa stained aspirate smears. CD56 immunohistochemical stain was quantified by counting the number of CD56 positive cells per microscopic field of view (High Power Field, HPF) (400 x magnifications) and averaging over the 10 microscopic fields with the highest density of CD56 positive cells. Cases with obvious CD56 staining of blasts on the trephine core were excluded from further analysis.Results: 95 bone marrow biopsy cases could be stained for CD56, but 6 cases were excluded from analysis due to CD56 staining leukemic blast population. For the whole study group the average CD56+ cell density per High Power Field (HPF) was 10.1 (SD=17). A control group composed of 10 patients with bone marrow biopsy shortly after chemotherapy, but who did not receive NK cell infusion had an average of 3.8 CD56+ cells per HPF (SD=3.4).Patients who had a higher density of NK cells in the bone marrow compared to the control group (more than 3.8 cells/HPF) were less likely to have persistence of acute leukemia as defined by ≥5% blasts, than patients with an NK cell density similar to the control group (Figure 1, Chi-square test, p=0.037). As well, patients with <5% leukemic blasts had more CD56 positive cells per HPF (Median 5.6, range 0.1-95.8 Versus median 1.8, range 0.1-28.1, p=0.009, Mann-Whitney test) and had a greater proportion of NK cells by flow cytometry (p=0.004, Mann-Whitney test).The clinical factors which may affect NK cell homing and persistence were also evaluated. Total Body Irradiation (TBI) at a dose 200 cGy for 2 days was applied as part of pre-infusion conditioning in a subset of the patients. Patients who received TBI (32 with TBI Vs 55 without) had a significantly higher density of CD56 positive cells (Figure 2, Median 4.75Vs 2.8, p=0.04, Mann-Whitney test). Among those patients who did not receive TBI conditioning, a subset received an anti-CD25 immunotoxin agent to reduce regulatory T cells (Denileukin Difitox, Ontak). The density of NK cells was significantly higher in the patients who received anti-CD25 immunotoxin (Figure 3, Median 9.8 Vs 2.5, p=0.02, Mann-Whitney test).Conclusions: Homing and survival/expansion of adoptively transferred NK cells in the bone marrow 14 days after infusion correlates with morphologic remission of acute leukemia. While the factors influencing NK cell homing and persistence in the BM niche remain to be elucidated, our results suggest a role for TBI as part of the conditioning and anti-CD25 immunotoxin in promoting the persistence of adoptively transferred NK cells in the bone marrow niche. [Display omitted] DisclosuresMiller:Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding; Celegene: Consultancy.

Follicular lymphoma of the ocular adnexa: Clinicopathological findings with flow cytometry analysis of eight cases.

Flow cytometry (FCM) is used to evaluate cell surface markers of various leukocyte populations quantitatively. However, little is known about the usefulness of FCM in follicular lymphoma (FL) of the ocular adnexa. The aim of this study was to evaluate the clinicopathological features and FCM results in ocular adnexal FL. This is a retrospective multicenter case study on clinical and immunohistochemical features. All tumors, surgically excised, were diagnosed based on histopathology, immunoglobulin heavy chain gene rearrangement, and FCM. The percentages (%) of B-cell markers, T-cell markers, a natural killer cell marker, and cell surface kappa/lambda measured by FCM analysis in tumor tissues were searched based on medical records. This study enrolled nine tumors in eight FL patients (three men and five women). The median age at the time of initial presentation was 74 years. All the tumors surgically excised histologically exhibited cluster of differentiation (CD)10, CD20, and BCL2-positive cells. At the time of ophthalmic diagnosis, lymphomas were already disseminated throughout the body in five cases. FCM demonstrated high percentage of B-cell markers including CD10, CD19, CD20, and CD23 in all nine tumors. CD10 population was 73.5% ± 11.9% in seven out of nine tumors, while that in the other two tumors was particularly low being 11.7% ± 1.13%, which showed the relatively high T-cell lineages compared to the other seven tumors. For ophthalmologists involving managements of ocular adnexal tumors, FCM can provide useful information for complementing the diagnosis and understanding pathophysiology of FL.

Prognostic significance of tumor-infiltrating lymphocytes and macrophages in nasopharyngeal carcinoma: a systematic review and meta-analysis.

Many studies have investigated the prognostic value of tumor-infiltrating lymphocytes (TILs) and tumor-infiltrating macrophages (TIMs) in patients with nasopharyngeal carcinoma (NPC), but the results remain controversial. Here, we performed a meta-analysis to evaluate the prognostic significance of TILs/TIMs in patients with NPC METHODS: The study was registered with PROSPERO (CRD42021234078). PubMed, Embase, and Web of Science databases were searched up to Dec 30, 2020. We reviewed studies that evaluated the relationship between TILs/TIMs and overall survival (OS), disease-free survival (DFS), or progression-free survival (PFS) in NPC. For TILs, CD3, CD4, CD8, and FOXP3 were searched as T-cell markers, CD19 and CD20 as B-cell markers, and CD56 as a natural killer cell marker. For TIMs, CD68 and CD163 were searched as total and M2 macrophage markers, respectively. In total, 19 studies with 3708 NPC were included in this meta-analysis. We found that high total numbers of TILs were significantly associated with favorable OS [hazard ratio (HR) 0.46, 95% confidence interval (CI) 0.38-0.57 and PFS (HR 0.48, 95% CI 0.38-0.62)]. In contrast, tumor infiltration by CD3+ T cells (HR 0.55, 95% CI 0.39-0.76), CD4+ T cells (HR 0.40, 95% CI 0.18-0.85), and CD8+ T cells (HR 0.56, 95% CI 0.34-0.93) correlated positively with OS. No significant relationship was found between survival and tumor infiltration by FOXP3+ T cells, CD68+ macrophages, or CD163+ macrophages. Our findings revealed that tumor infiltration by CD3+ , CD4+ , and CD8+ T cells could be prognostic biomarkers in NPC.

A subset of cytotoxic effector memory T cells enhances CAR T cell efficacy in a model of pancreatic ductal adenocarcinoma.

In humans, the natural killer (NK) cell marker CD161 identifies several subsets of T cells, including a polyclonal CD8 αβ T cell receptor-expressing subset with characteristic specificity for tissue-localized viruses. This subset also displays enhanced cytotoxic and memory phenotypes. Here, we characterized this unique T cell subset and determined its potential suitability for use in chimeric antigen receptor (CAR) T cell therapy. In mice, gene expression profiling among the CD161-equivalent CD8+ T cell populations (CD8+NK1.1+) revealed substantial up-regulation of granzymes, perforin, killer lectin-like receptors, and innate signaling molecules in comparison to CD8+NK1.1- T cells. Adoptive transfer of CD8+NK1.1+ cells from previously exposed animals offered substantially enhanced protection and improved survival against melanoma tumors and influenza infection compared to CD8+NK1.1- cells. Freshly isolated human CD8+CD61+ T cells exhibited heightened allogeneic killing activity in comparison to CD8+CD61- T cells or total peripheral blood mononuclear cells (PBMCs). To determine whether this subset might improve the antitumor efficacy of CAR T cell therapy against solid tumors, we compared bulk PBMCs, CD8+CD161-, and CD8+CD161+ T cells transduced with a human epidermal growth factor receptor-2 (HER2)-specific CAR construct. In vitro, CD8+CD161+ CAR-transduced T cells killed HER2+ targets faster and with greater efficiency. Similarly, these cells mediated enhanced in vivo antitumor efficacy in xenograft models of HER2+ pancreatic ductal adenocarcinoma, exhibiting elevated expression of granzymes and reduced expression of exhaustion markers. These data suggest that this T cell subset presents an opportunity to improve CAR T cell therapy for the treatment of solid tumors.

CRISPR-Cas9–mediated TIM3 knockout in human Natural Killer Cells Enhances Growth Inhibitory Effects on Human Glioma Cells

Abstract Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based-immunotherapy might be a promising target in GBM. T cell immunoglobulin mucin family member 3 (TIM3), a receptor expressed on NK cells, has been suggested as a marker of dysfunctional NK cells. We established TIM3 knockout in NK cells, using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating of TIM3 exon 2- or exon 5-targeting guide RNA-Cas9 protein complexes (RNPs) inhibited TIM3 expression on NK cells with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted the intended genome sites. The expression of other checkpoint receptors, i.e., programmed cell death 1 (PD1), Lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and TACTILE (CD96) were unchanged on the TIM3 knockout NK cells. Real time cell growth assays revealed that TIM3 knockout enhanced NK cell–mediated growth inhibition of GBM cells. These results demonstrated that TIM3 knockout enhanced human NK cell mediated cytotoxicity on GBM cells. Future, CRISPR-Cas9 mediated TIM3 knockout in NK cells may prove to be a promising immunotherapeutic alternative in patient with GBM.

Factors Predicting the Presence of Maternal Cells in Cord Blood and Associated Changes in Immune Cell Composition.

BackgroundCord blood (CB) samples are increasingly used as a source of hematopoietic stem cells in transplantation settings. Maternal cells have been detected in CB samples and their presence is associated with a better graft outcome. However, we still do not know what influences the presence of maternal microchimerism (MMc) in CB samples and whether their presence influences CB hematopoietic cell composition.Patients and MethodsHere we test whether genetic, biological, anthropometric and/or obstetrical parameters influence the frequency and/or quantity of maternal Mc in CB samples from 55 healthy primigravid women. Mc was evaluated by targeting non-shared, non-inherited Human Leukocyte Antigen (HLA)-specific real-time quantitative PCR in whole blood and four cell subsets (T, B lymphocytes, granulocytes and/or hematopoietic progenitor cells). Furthermore CB samples were analyzed for their cell composition by flow cytometry and categorized according to their microchimeric status.ResultsMMc was present in 55% of CB samples in at least one cell subset or whole blood, with levels reaching up to 0.3% of hematopoietic progenitor cells. Two factors were predictive of the presence of MMc in CB samples: high concentrations of maternal serological Pregnancy-Associated-Protein-A at first trimester of pregnancy (p=0.018) and feto-maternal HLA-A and/or –DR compatibility (p=0.009 and p=0.01 respectively). Finally, CB samples positive for MMc were significantly enriched in CD56+ cells compared to CB negative for MMc.ConclusionsWe have identified two factors, measurable at early pregnancy, predicting the presence of maternal cells in CB samples at delivery. We have shown that MMc in CB samples could have an influence on the hematopoietic composition of fetal cells. CD56 is the phenotypic marker of natural killer cells (NK) and NK cells are known to be the main effector for graft versus leukemia reactions early after hematopoietic stem cell transplantation. These results emphasize the importance of MMc investigation for CB banking strategies.