CD56 Natural Killer Research Articles

Expression of antigen-presenting cells in lung of postmortem SARS-CoV-2 cases.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a deadly pulmonary disease with impaired immunological response that causes significant tissue damage and organ failure. Postmortem examination of the lung is a useful tool for understanding the immunopathogenesis of this virus. Lung autopsy samples from seven dead SARS-CoV-2 patients were obtained and evaluated using hematoxylin and eosin stain to analyze the histopathological changes in those samples, on the other hand, Immunohistochemical (IHC) staining was used for detection of CD21, CD1a, CR1 (CD35), CD68, Myeloperoxidase (MPO), CD15, CD56, CD3, CD20, CD4, and CD8 cells markers. Histopathological examination revealed diffuse alveolar damage with extensive parenchymal architecture distortion, intravascular fibrin clot, deposition of collagen fibers, vascular congestions and blood vessels containing thrombi, pneumocyte type II with inflammatory cell infiltration. The IHC staining for the innate immune cells such as antigen-presenting cells (APCs) including dendritic cells, Macrophages, and neutrophils showed a strong positive staining, while CD56 Natural killer (NK) cells showed negative staining. On the other hand, the specific immune cells including; CD20 B cells, CD3 T cells, and CD4 helper T cells, showed positive staining while CD8 Cytotoxic T cells showed negative staining. The lung autopsy samples from patients with COVID-19 confirmed the presence of APCs through the positive staining of CD21, CD1a, CD35, CD68, MPO, and CD15 expressed the virus recognition, proinflammatory cytokine production, and adaptive immune cells activation through CD3, CD4, and CD20 positive staining and the role of APCs in the severity of pulmonary infection and pathogenesis of SARS-CoV-2 infection however the absence of the CD56 NK and CD8 cytotoxic T explains the worse infection status for the patients.

Diagnostic significance of blood lymphocyte activation markers in pre-eclampsia.

The adaptive and innate immune system is important in both initiating and preventing functional disorders during pregnancy, one of which is pre-eclampsia. The research aims to conduct the comparative quantification of selected subpopulations of peripheral blood immunoregulatory cells in pregnant women with pre-eclampsia in the third trimester. The marker receptors CD4, CD8, CD95, CD25, and CD27 and the marker antigen HLA-DR were considered. The screening was performed by flow cytometry with dual phenotyping using phycoerythrin- and fluorescein-isothiocyanate-labeled monoclonal antibodies. Data processing consisted in calculating a likelihood value to assess the statistical significance of the difference between the samples. A statistically significant decrease in the subpopulation titer of T and B lymphocytes with marker receptors CD4, CD8, and CD19 was found in pre-eclampsia patients. In the CD4 carrier T-lymphocyte population, there was an increased expression of the CD25/CD95 activation and apoptosis markers. In the CD8 T-killer population, a decreased representation of the CD27/CD25/CD95 markers of differentiation, activation, and apoptosis was deterministic. The expression pattern of the major histocompatibility complex antigen HLA-DR did not change significantly in normality and pathology. The titer of peripheral natural killer cells carrying the CD56 marker increased in patients with various degrees of disease severity, while the number of CD16 natural killer remained at the level of the control group. The research results suggest that a change in the ratio of the above receptors is a diagnostic indicator for pre-eclampsia.

Screening and identification of genes related to ferroptosis in keratoconus

Corneal keratoconus (KC) is a dilated (ectatic) corneal disease characterized by a central thinning of the cornea, which causes protrusion into a conical shape that seriously affects vision. However, due to the complex etiology of keratoconus, its entire mechanism remains unclear and there is no mechanism-directed treatment method. Ferroptosis is a novel programmed cell death mechanism related to lipid peroxidation, stress, and amino acid metabolism, which plays a crucial role in various diseases. This study aimed to explore the relationship between keratoconus and ferroptosis, to provide new insights into the mechanism of keratoconus development, and potential treatment options based on further elucidation of this mechanism. The corresponding mRNA microarray expression matrix data of KC patients were obtained from GEO database (GSE204791). Weighted co-expression network analysis (WGCNA) and support vector machine recursive feature elimination (SVM-RFE) were selected to screen hub genes, which were overlapped with ferroptosis genes (FRGs) from FerrDb. GO and GSEA were performed to analyze differential pathways, ssGSEA was used to determine immune status, and then, feasible drugs were predicted by gene-drug network. Additionally, we predicted the miRNA and IncRNA of hub genes to identify the underlying mechanism of disease so as to predict treatment for the disease. The epithelial transcriptome from keratoconus tissue mRNA microarray data (GSE204791) was extracted for the main analysis, including eight epithelial cells and eight epithelial control cells. The differential genes that were overlapped by WGCAN, SVM-RFE and FRGs were mainly related to oxidative stress, immune regulation, cellular inflammation, and metal ion transport. Through further analysis, aldo–keto reductase family 1 member C3 (AKR1C3) was selected, and negatively correlated with mature CD56 natural killer (NK) cells and macrophages. Then, gene-drug interaction network analysis and miRNA prediction were performed through the website. It was concluded that four immune-related drugs (INDOMETHACIN, DAUNORUBICIN, DOXORUBICIN, DOCETAXEL) and a miRNA (has-miR-184) were screened to predict potential drugs and targets for disease treatment. To our knowledge, this was the first report of KC being associated with ferroptosis and prompted search for differential genes to predict drug targets of gene immunotherapy. Our findings provided insight and a solid basis for the analysis and treatment of KC.

Tim-3, CD9 Molecule Expressions on Natural Killer (NK) and T-Lymphocytes with NK Functions (NKT) of the Peripheral Blood at Different Trimesters of Physiological Pregnancy

Natural killer cells (NK) and T-lymphocytes with NK functions (NKT) are the leading effectors of the mother’s immune tolerance to a semi-allogeneic fetus and have a fetal trophic effect during physiological pregnancy. Tim-3 (T-cell Ig and mucin domain-containing protein 3) and CD9 molecules play a critical role in the immunoregulatory and fetal trophic functions of NK and NKT, but their expression in peripheral blood cells has not been studied. The aim of this work was to study the expression of Tim-3 and CD9 in peripheral blood NK and NKT during physiological pregnancy. The object of the study was the peripheral blood of healthy women in I and III trimesters of a physiological pregnancy. The control group consisted of healthy non-pregnant women in the first phase of the menstrual cycle. Expression of Tim-3, CD9 molecules was analyzed by flow cytometry on regulatory NK (CD16–CD56bright) and NKT (CD16–CD56+), cytotoxic NK (CD16+CD56dim/–) and NKT (CD16+CD56+). It was found that in the first trimester of pregnancy, the total number and subpopulation composition of NK and NKT cells did not change. Tim-3 expression increased in all NK and NKT subpopulations, except for cytotoxic CD16+CD56dimNK. CD9 expression increased in all NK subpopulations, but in NKT did not differ from non-pregnant. At the same time, a direct correlation between CD9 and Tim-3 expressions was revealed in regulatory NK and NKT in the first trimester of pregnancy. In the third trimester, the regulatory CD16–CD56brightNK number increased, while cytotoxic CD16+CD56dimNK and regulatory CD16–CD56+NKT decreased compared to non-pregnant women. The number of CD16+CD56–NK did not change in I and III trimesters of a physiological pregnancy. Tim-3 expression was upregulated in all NK and cytotoxic NKT subpopulations, while CD9 was upregulated only in regulatory NKs. Thus, Tim-3 and CD9 molecule expressions of s on different NK and NKT subpopulations changed during I and III trimesters of a physiological pregnancy, which plays an important role in the regulation of their phenotype and functions.

Generation Natural Killer Cell-Mimic Nanoparticles for Active Targeting of Acute Myeloid Leukemia

Natural killer (NK) cells play a crucial role in recognizing and killing emerging tumor cells. Via an array of activating and inhibitory receptors, NK cells can target and kill abnormal cells, including tumor cells, without prior antigen sensitization. However, fully-established tumors employ numerous mechanisms to inactivate NK cells or hide from them, for example by shedding NK cell-activating ligands, like NK2GD (Chiossone et al., 2018). Here, we describe the engineering of nanoparticles (NPs) functionalized with tumor-recognizing and cytotoxic components of NK cells to address this problem and develop a nanoparticle-based treatment strategy simulating NK cell functionality. Liposomes were synthesized using the thin film hydration method followed by extrusion and functionalized them with the death ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), immunoglobulin Fc-binding peptide (FCP, replicating the function of NK CD16 receptor) and the therapeutic antibody, anti-CD38 (daratumumab, Panel A) to target acute myeloid leukemia (AML). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to monitor morphology and size of liposomes. The potential of NK cell-mimic NPs (CD38-NK.NPs) to target and kill AML cells was evaluated on primary AML blasts and a disseminated AML xenograft for which the CD38-positive AML cell line, OCI-AML2 was engrafted into 8-9 week-old female NSG mice. Upon establishment of AML, the mice were injected intraperitoneally (i.p) with 5 doses of either naked liposomes (as vehicle control), soluble TRAIL protein (sTRAIL), LP/TRAIL or CD38-NK.NPs every two days. Tumor burden was monitored by determining the frequency of live human CD45+ cells in the peripheral blood, spleen and bone marrow with flow cytometry. Liposome was chosen as the base of the NP due to its high biocompatibility, biodegradability, cell-like characteristics and capability to penetrate through vessel walls (Pattni, Chupin and Torchilin, 2015). TEM and DLS measurements showed the generated NK.NPs were monodisperse with an average diameter of 145 nm, a size enabling vessel transmigration and avoiding kidney filtration (Gaumet et al., 2008). Immunofluorescent labelling of TRAIL, FCP and antibody verified CD38-NK.NPs functionalization. In vitro, both TRAIL-conjugated liposomes and CD38-NK.NPs were more effective in killing AML cell lines (ML-1, ML-2, Kasumi-1, and OCI-AML2) than sTRAIL, indicating that TRAIL-mediated cytotoxicity can be enhanced by presenting TRAIL in a conformation close to its membrane-bound structure. No significant difference could be detected between LP/TRAIL vs. CD38-NK.NP cytotoxicity in the in vitro assays. On the other hand, CD38-NK.NPs were more effective in killing primary AML blasts than non-targeted, NP-conjugated TRAIL (LP/TRAIL, 13±17% vs 4±15% killing, respectively). Moreover, CD38-NK.NPs showed considerably higher activity relative to the NK cell line, KHYG-1, against CD38-positive AML cell lines. Notably, CD38-NK.NPs showed no significant cytotoxic effect on non-malignant peripheral blood leukocytes. In vivo, NK cell-mimic NPs were more efficient in targeting and eradicating CD38-positive AML cells that either sTRAIL or LP/TRAIL, shown by the reduced percentage of circulating hCD45+ AML cells in treated mice (Panel B). CD38-NK.NPs were also more efficient in inhibiting accumulation of AML cells in the bone and spleen than sTRAIL and LP/TRAIL (Panel B), confirming the ability of CD38-NK.NPs to actively target AML cells expressing the targeted biomarker. Of note, CD38-NK.NPs showed no detectable systemic toxicity or damage to kidneys, liver and pancreas in healthy NSG mice. Overall, we have developed an NK cell-mimic nanoparticle able to actively target AML based on AML surface marker expression without any detectable systematic toxicity. Importantly, due to the modular built of the NK.NPs, they can be functionalized by any therapeutic tumor-targeting antibody via the immunoglobulin-binding peptide (FCP), thus enabling selective targeting tumor cells based on expression of tumor-specific markers and enabling personalized therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

KIR- Ligand Interactions in Hypertensive Disorders in Pregnancy.

HypothesisThe activity of natural killer (NK) cells is considered an important factor for the tolerance of the fetus during pregnancy. The complications of pregnancy, such as hypertensive disorders (HDP), may be therefore associated with this immune compartment.MethodsThe current study included 41 pregnant women diagnosed with HDPs (Gestational Hypertension; GH or Preeclampsia; PE) and 21 healthy women. All the patients were under continuous obstetric care during the pregnancy and labour. The number of mother-child mismatches within killer immunoglobulin-like receptors (KIRs), their ligands [MM], and missing KIR ligands [MSLs] was assessed. KIRs and their ligands were assessed with Next Generation Sequencing (NGS) and Polymerase Chain Reaction Sequence-Specific Oligonucleotide (PCR-SSO) typing. The subsets of NK cells were assessed with multicolor flow cytometry and correlated to the number of MSLs.ResultsThe number of MSLs was significantly higher in HDP patients when compared to healthy non-complicated pregnancy patients. Some MSLs, such as those with 2DS2 activating KIR, were present only in HDP patients. The percentage of CD56+CD16-CD94+ NK cells and CD56+CD16-CD279+ NK cells correlated with the number of MSLs with inhibiting KIRs only in healthy patients. In HDP patients, there was a correlation between the percentage of CD56-CD16+CD69+ NK cells and the number of MSLs with inhibiting and activating KIRs. As compared to the healthy group, the percentage of CD56+CD16-CD279+ NK cells and CD56-CD16+CD279+ NK cells were lower in HDP patients. HDP patients were also characterized by a higher percentage of CD56+CD16+perforin+ NK cells than their healthy counterparts.ConclusionsPatients with HDP were characterized by a higher number of MSLs within the KIRs receptors. It seemed that the number of MSLs in the healthy group was balanced by various receptors, such as CD94 or inhibitory CD279, expressed on NK cells. Conversely, in HDP patients the number of MSLs was associated with the activation detected as the increased level of CD69+ NK cells.

Study the effect of natural killer cells in pationts with blood cancer disease from other when the injury-infected corona emerging

Lymphocytes and subgroups of natural killer cells (CD16 and CD56) play an important role in maintaining the function of the immune system. After infection with COVID-19, the total number of lymphocytes and subgroups changes, indicating a possible association between lymphocyte subgroup change and causative mechanisms. For viral diseases, the study showed a clear decrease in peripheral lymphocytes in patients with coronavirus-19. The reason may be attachment to the virus or indirectly due to immune injuries from inflammatory mediators. It is possible that the secretion of lymphocytes circulating in inflammatory lung tissues also leads to lymphocytopenia. And the viral infection can cause an imbalance in the levels of lymphocyte subgroups, as the cell surface molecules of CD16 and CD56 natural killer cells participate in the humoral and cytotoxic immunity against viral infection, where a decrease in the level of these cells was observed less in severe cases compared to mild cases. Patients with lymphocytic leukemia often suffer from weak immune status When they are exposed to risk factors for contracting COVID-19.

Tissue Trafficking Kinetics of Rhesus Macaque Natural Killer Cells Measured by Serial Intravascular Staining.

The in vivo tissue distribution and trafficking patterns of natural killer (NK) cells remain understudied. Animal models can help bridge the gap, and rhesus macaque (RM) primates faithfully recapitulate key elements of human NK cell biology. Here, we profiled the tissue distribution and localization patterns of three NK cell subsets across various RM tissues. We utilized serial intravascular staining (SIVS) to investigate the tissue trafficking kinetics at steady state and during recovery from CD16 depletion. We found that at steady state, CD16+ NK cells were selectively retained in the vasculature while CD56+ NK cells had a shorter residence time in peripheral blood. We also found that different subsets of NK cells had distinct trafficking kinetics to and from the lymph node as well as other lymphoid and non-lymphoid tissues. Lastly, we found that following administration of CD16-depleting antibody, CD16+ NK cells and their putative precursors retained a high proportion of continuously circulating cells, suggesting that regeneration of the CD16 NK compartment may take place in peripheral blood or the perivascular compartments of tissues.

­Association of Macrophage Activation Syndrome, Natural Killer (NK) Cell Function and Genetic Polymorphisms With Dengue Disease Severity

Background: Severe dengue is characterized by shock, organ dysfunction, and coagulopathy, driven by an excessive host inflammatory response. We investigated the role of hyperinflammation/macrophage activation syndrome (MAS) in dengue pathogenesis including MAS biomarkers, Natural Killer (NK) cell function and genetic polymorphisms. Methods: Samples were included from patients recruited into an observational study in Vietnam and divided into three severity grades (plasma leak severity grades 0,1 and 2). MAS biomarkers, were measured at 4 time-points. The frequencies, activation status and cytotoxic potential of NK cells were analysed by flow cytometry. RNA was extracted from sorted CD56+ NK cells and libraries prepared using SMART-Seq Stranded Kit and sequenced using HiSeq3000 (Illumina). Results: 70 hospitalized dengue patients were included. Patients with severe plasma leakage (grade 2 leakage) had higher MAS biomarkers than patients without plasma leakage, including a higher proportion had ferritin levels of >2000 ng/ml (83.3% vs 45.2%) and H scores (148.5 vs.105.5). Activated CD69+ total NK and CD56 dim cells were decreased in patients with severe leakage compared to grade 1. Perforin expression was reduced in NK CD56 dim and CD56 bright cells in grade 2 patients compared to grade 1, and granzyme B MFI levels were reduced in total NK cells and CD56 dim NK cells. RNA sequencing analysis revealed three previously unreported SNPs in the genes involved in NK cell function to be associated with more severe disease; two in the NK cell lectin-like receptor K1 gene (KLRK1) and one in the PRF1 gene, encoding perforin 1. Conclusions: Features of MAS/hyperinflammation are associated with dengue disease severity, including higher MAS biomarkers, impaired NK cell activation/cytotoxicity and specific SNPs in genes involved in NK cell cytolytic function (KLRK1 and PRF-1). Clinical trials of targeted therapeutic immunomodulation for patients with dengue-associated MAS are warranted. Funding Information: Young Investigator Research Grant: NMRC/ OFYIRG17may066 awarded to S Yacoub; Open Fund - Individual Research Grant, National Medical Research Council, Singapore NMRC/OFIRG/0049/2017 awarded to L.Rivino. OM Sessions was supported by the Singapore Ministry of Education (https://www.moe.gov.sg) with a Startup grant. Declaration of Interests: The authors report no COI. Ethics Approval Statement: Ethical approval was obtained from the Oxford Tropical Research Ethics Committee and the Ethics review Committee at NHTD. Written informed consent was obtained from all participants. Experimental work on PBMCs was performed at Duke-NUS Medical School after approval by the Singapore-NUS Institutional Review Board.

Increased Peripheral NKG2A-NKG2D+CD3-CD16+CD56dim NK Cell Subset Was Positively Correlated with Antiphospholipid Antibodies in Patients of Obstetric Antiphospholipid Syndrome

ABSTRACT Obstetric antiphospholipid syndrome (OAPS) is an autoimmune disorder with severe life-threatening complications shown during pregnancy. It has been reported that the increase in CD16+CD56dim natural killer (NK) cells in peripheral blood are risk factors for recurrent miscarriages, but this expression of CD16+CD56dim NK cells in OAPS patients has not been reported, and the mechanism is not clearly illustrated. In this study, we compared the distributional profiles of different NK cell subsets and the expressions of NK cell-activating receptors in peripheral blood of patients with OAPS and healthy women. Our results showed significantly increased NKG2A−NKG2D+ subset and decreased NKG2A+NKG2D− subset in CD3− CD16+CD56dim NK cells, CD3−CD16−CD56bright NK cells and CD56+T cells in OAPS patients compared with those in healthy control women. The CD27−CD11b+ subset significantly increased in CD3−CD16+CD56dim NK cells in OAPS patients compared with those in healthy control women. In addition, the NKG2A−NKG2D+ subset in CD3−CD16 + CD56dim NK subset in triple positivity was higher than single positivity OAPS patients. At the optimal diagnostic threshold established by ROC analysis, using the cut-off of NKG2A−NKG2D+ and CD27−CD11b+ subset in CD3−CD16+CD56dim NK cells is 10.10% and 92.75%, the sensitivity of NKG2A−NKG2D+ and CD27−CD11b+ to detect patients with OAPS compared with healthy control results was 94.1% and 60.8%, and specificity was 84.2% and 89.5%, respectively, with an area under the curve (AUC) of 0.903 and 0.829, respectively. The NKG2A−NKG2D+ subset in CD3−CD16+CD56dim NK cells was positively correlated with the antiphospholipid antibodies lg anti-aCL IgG, lg anti-aCL IgM, lg anti-aCL IgA, lg anti-β2GP1 IgM and Complement 4(C4), while the CD27+CD11b+ subset in CD3−CD16 + CD56dim NK cells was correlated with lg anti-β2GP1 IgG and lg anti-β2GP1 IgA. These results suggested that the NK cytotoxic function enhanced in OAPS patients and unbalanced of NK activating receptors and inhibiting receptors may contribute to the immune pathogenesis of OAPS.

Abstract A60: Transcriptional profile of CD56negCD16pos Natural Killer cells within endemic Burkitt lymphoma

Abstract Endemic Burkitt lymphoma (eBL), the most common pediatric cancer in equatorial Africa, has long been associated with two etiologic agents: Epstein-Barr virus (EBV) and Plasmodium falciparum malaria. On the other hand, Natural Killer (NK) cells are essential in the clearance of infected cells as well as play a critical role in tumor immunosurveillance. Despite chronic viral infections as HIV, HCV, which have been found to promote the expansion of a CD56 (negative) NK cell subset with impaired effector function, previous cancer studies have been limited to investigations of CD56dimCD16pos NK cells. We previously published that eBL children had significantly more CD56negCD16pos NK cells compared to age-matched healthy children from a low and high malaria transmission area. Here, we characterized this CD56negCD16pos NK subset using histology staining, multicolor flow cytometry, and RNA-sequencing. We found CD56negCD16pos NK cells to be morphologically similar to CD56dimCD16pos NK cells but had higher expression of FCGR3B (CD16b) (p=0.000008) and MPEG1 (Perforin 2) (p=0.0000007), and lower expression of KLRF1 (NKp80), IL18RAP and IL18R1 (IL18 receptor) (p=0.000003, p=0.000003 and p=0.00005, respectively). Phenotypically, CD56negCD16pos NK cells share characteristics of adaptive NK cells; however, they differ from “memory-like” NK cells, with consistent expression of NKG2C/CD57, lower expression of NKp46 and CD160, and higher expression of KIR3DL1 compared to CD56dimCD16pos NK cells. Finally, higher plasma levels of IL-12p70 and IL-2 (known to help NK cell activation and proliferation) and IL-6 and MIP1a were found in healthy compared to eBL children. Together, these findings suggest that the expansion of CD56negCD16pos NK cells could help explain diminished tumor immunosurveillance and might be a mechanism by which EBV-infected cells evades NK-cell mediated immunity. Citation Format: Catherine S. Forconi, Cliff Oduor, John M. Ong’echa, Jeff Bailey, Ann M. Moormann. Transcriptional profile of CD56negCD16pos Natural Killer cells within endemic Burkitt lymphoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A60.

Reduced levels of circulating natural killer cells in children with celiac disease

Background Celiac disease (CD) is an autoimmune disease characterized by malabsorption. Serologic testing for CD consists of Ig A type of antitissue transglutaminase (tTG), antiendomysium (EMA). These tests are helpful in monitoring adherence to the gluten-free diet (GFD). Natural killer (NK) cell count alterations have been reported in various diseases, such as cancer, Crohn’s disease, malnutrition, and autoimmune disorders.
 Objective To compare peripheral blood NK cell counts in children with celiac disease (CD) to healthy controls. The second aim was to analyze for possible correlations between NK cells (CD3-/CD16+, CD56+) and tissue transglutaminase (tTG)-IgA and tTG-IgG, as well as endomysial antibody EMA-IgA indicating gluten sensitivity.
 Methods Fifty children with CD were compared to 48 healthy children as controls, with similar age and sex distribution. Peripheral blood NK cell counts were measured by flow cytometry.
 Results The median (P25-P75) ages of the 50 celiac patients (23 male; 46%) and 48 controls (21 male; 44%) were 10 (2-17) years and 9 (3-17) years, respectively. Mean follow-up duration was 3 years, ranging from 1-10 years. All CD patients had positive tTG-IgA and EMA-IgA tests while it was negative in all (100 %) control patients. The absolute number of circulating CD16+ NK cells (259.52 vs. 1404.36 μ/L) and CD56+ NK cells (366.24 vs. 2440.46 μ/L) were significantly lower in the celiac group than the control group (P<0.05 for both). The absolute numbers of circulating white blood cells (7785 vs. 8165 μ/L) and lymphocytes (3106 vs. 3173 μ/L) were not significantly different between the celiac and control groups (P>0.05 for both). Correlation analysis between the absolute number of circulating NK cells and tTG-IgA, tTG-IgG, and EMA-IgA levels in CD patients revealed no significant relationships (P>0.05 for all).
 Conclusions Peripheral blood NK cell count were significantly lower in celiac patients than controls, hence, decreased NK cell counts may be an abnormal feature seen in autoimmune diseases. NK cell count in celiac patients had no significant correlations to tTG-IgA, tTG-IgG, or EMA-IgA levels. Therefore, NK cell count may be inappropriate marker for monitoring compliance to a gluten free diet.

Reduced levels of circulating natural killer cells in children with celiac disease

Background Celiac disease (CD) is an autoimmune disease characterized by malabsorption. Serologic testing for CD consists of Ig A type of antitissue transglutaminase (tTG), antiendomysium (EMA). These tests are helpful in monitoring adherence to the gluten-free diet (GFD). Natural killer (NK) cell count alterations have been reported in various diseases, such as cancer, Crohn’s disease, malnutrition, and autoimmune disorders.
 Objective To compare peripheral blood NK cell counts in children with celiac disease (CD) to healthy controls. The second aim was to analyze for possible correlations between NK cells (CD3-/CD16+, CD56+) and tissue transglutaminase (tTG)-IgA and tTG-IgG, as well as endomysial antibody EMA-IgA indicating gluten sensitivity.
 Methods Fifty children with CD were compared to 48 healthy children as controls, with similar age and sex distribution. Peripheral blood NK cell counts were measured by flow cytometry.
 Results The median (P25-P75) ages of the 50 celiac patients (23 male; 46%) and 48 controls (21 male; 44%) were 10 (2-17) years and 9 (3-17) years, respectively. Mean follow-up duration was 3 years, ranging from 1-10 years. All CD patients had positive tTG-IgA and EMA-IgA tests while it was negative in all (100 %) control patients. The absolute number of circulating CD16+ NK cells (259.52 vs. 1404.36 μ/L) and CD56+ NK cells (366.24 vs. 2440.46 μ/L) were significantly lower in the celiac group than the control group (P<0.05 for both). The absolute numbers of circulating white blood cells (7785 vs. 8165 μ/L) and lymphocytes (3106 vs. 3173 μ/L) were not significantly different between the celiac and control groups (P>0.05 for both). Correlation analysis between the absolute number of circulating NK cells and tTG-IgA, tTG-IgG, and EMA-IgA levels in CD patients revealed no significant relationships (P>0.05 for all).
 Conclusions Peripheral blood NK cell count were significantly lower in celiac patients than controls, hence, decreased NK cell counts may be an abnormal feature seen in autoimmune diseases. NK cell count in celiac patients had no significant correlations to tTG-IgA, tTG-IgG, or EMA-IgA levels. Therefore, NK cell count may be inappropriate marker for monitoring compliance to a gluten free diet.

Abstract A67: Augmented antitumor activity of NK-92 MI cells expressing a chimeric antigen receptor targeting human mesothelin

Abstract Adoptive natural killer (NK) cell immunotherapy using primary NK cells has been challenging, to date. The use of an ”off-the -shelf” NK cell line where multiple genetic changes can be introduced has advantages. In this study, we explored the use of the NK-92MI cell line expressing a chimeric antigen receptor (CAR) targeting human mesothelin, the current target in our human CAR-T cell therapy trials. The NK-92MI cell line (which makes its own IL-2) was purchased from ATCC and transduced with a lentivirus encoding GFP (vector control) or an anti-human mesothelin scFv-41BB-CD3 ζ second-generation CAR (MesoCAR). Addition of IL-21 enhanced transduction. The transduced cells (MesoCar-NK-92MI) were sorted by flow cytometry to achieve an expression level of >98%. The NK markers NKp30, NKp44, NKG2D, 2B4, and CD56 were highly expressed on the Car-NK-92MI cell line (from 75% to 99.9%). To test the antitumor effects in vitro, three types of human tumor lines transduced to express human mesothelin were tested. Lung cancer (A549Meso), mesothelioma (EMMeso), and head and neck cancer (SSC15Meso) cells were exposed to vector control NK-92MI or MesoCar-NK-92MI cells for 22 hours at varying E:T ratios. The supernatants were collected for detection of cytokines and granzyme B. We also determined the killing function of MesoCar-NK-92MI cells after irradiation of the cells at 10 Gy. To test in vivo activity, NSG mice were injected in the flank with 5 million EMMeso cells. After tumors were established, each mouse was intravenously injected with 10 million vector control NK-92MI cells or MesoCar-NK-92MI cells with a boost injection of 5 million effectors on days 4, 8, and 11. Tumor volumes were assessed over time and mice were sacrificed on day 18. In vitro killing results showed augmented antitumor effect in MesoCar-NK-92MI compared to vector control NK-92MI. Irradiated MesoCar-NK-92MI cells still possessed good antitumor activity with only 32% loss of activity on average. Increased cytokine and granzyme B secretion was also seen with the MesoCar-NK-92MI cell line (with or without irradiation) compared to NK-92MI cells. In animal studies, irradiated MesoCar-NK-92MI cells significantly inhibited tumor growth by an average of 59% with no obvious toxicity or tumor growth in other organs. The non-CAR-expressing NK-92MI cells had significantly less antitumor activity. In conclusion, use of an irradiated NK-92MI cell line expressing a CAR targeting human mesothelin could become a safe and possible “off-the-shelf” adoptive cell therapy that could be used alone or in combination with CAR-T cells or other treatments. Citation Format: Jing Sun, Patrick Woodruff, Soyeon Kim, Estela Noguera-Ortega, Evguenia Arguiri, Maria Liousia, Steven Maceko, Edmund Moon, Steven Albelda. Augmented antitumor activity of NK-92 MI cells expressing a chimeric antigen receptor targeting human mesothelin [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A67.

Mitochondrial transfer from MSCs to T cells induces Treg differentiation and restricts inflammatory response.

Mesenchymal stem cells (MSCs) have fueled ample translation for the treatment of immune-mediated diseases. They exert immunoregulatory and tissue-restoring effects. MSC-mediated transfer of mitochondria (MitoT) has been demonstrated to rescue target organs from tissue damage, yet the mechanism remains to be fully resolved. Therefore, we explored the effect of MitoT on lymphoid cells. Here, we describe dose-dependent MitoT from mitochondria-labeled MSCs mainly to CD4+ T cells, rather than CD8+ T cells or CD19+ B cells. Artificial transfer of isolated MSC-derived mitochondria increases the expression of mRNA transcripts involved in T-cell activation and T regulatory cell differentiation including FOXP3, IL2RA, CTLA4, and TGFβ1, leading to an increase in a highly suppressive CD25+ FoxP3+ population. In a GVHD mouse model, transplantation of MitoT-induced human T cells leads to significant improvement in survival and reduction in tissue damage and organ T CD4+ , CD8+ , and IFN-γ+ expressing cell infiltration. These findings point to a unique CD4+ T-cell reprogramming mechanism with pre-clinical proof-of-concept data that pave the way for the exploration of organelle-based therapies in immune diseases.

Immune status of splenectomized patients following vaccination against encapsulated bacteria

Aim: The current study aimed to observe the effectiveness of a pneumococcal vaccine and a Haemophilus influenzae vaccine in splenectomized patients by quantification of serum antibody and complement levels, analysis of T and B lymphocytes with flow-cytometry and determination of the percentage of Natural Killer cells and the CD4+ to the CD8+ lymphocyte ratio.Material and Methods: The study included 22 adult patients who had undergone splenectomy between May 1999 and December 2006. The Pneumo-23 vaccine was administered for protection against Streptococcus pneumonia to all patients, whereas the Act-HIB vaccine was administered for protection against Haemophilus influenzae type B to 12 patients after January 2002.Hemogram, IgG and IgM antibody concentrations, serum levels of Complement protein 3 and Complement Protein 4 were obtained and lymphocytes were analyzed whether they were CD3+ and CD19+ by flow cytometry. At the second time point, in 2009, CD4+/CD8+ T-cell ratio and the percentage of CD3/CD16+CD56 Natural Killer cells were evaluated in addition to aforementioned parameters.Results: All of the study patients had normal IgG levels. Three patients had low levels of IgM. C3 and C4 values were normal. Ten patients showed a low CD3+ T-cell distribution at 5 years. The percentage of CD19+ B-lymphocytes returned to normal. Sustained suppression of CD19 level was observed only in one patient with immune thrombocytopenic purpura. CD8 distribution was normal in all patients. At 5 years, reduced CD4 percentage was detected in 5 patients with immune thrombocytopenic purpura, 3 of whom had suppressed levels of Natural Killer cells concomitantly. Normal levels of IgG, C3, C4 and CD19+ B-cell percentages were found at the final laboratory assessment for all patients.Conclusion: Our findings indicate that immunological functions are restored following administration of the Pneumo-23 and Act-HIB vaccines. Further studies are needed to obtain a prognostic immune profile index that could help predict patients at high risk for post-splenectomy sepsis.

Increased degranulation of immune cells is associated with higher cervical viral load in HIV-infected women.

The activation of effector immune cells at the cervicovaginal mucosa (CVM) might influence the cervical HIV load and thus the secondary transmission; however, limited information is available about the innate effector cells at CVM during HIV infection. In this study, we quantified and assessed the activation of the effector immune cells at the CVM of HIV-infected women with different disease outcomes: nonprogressive HIV disease (LTNPs) and chronic HIV-infected (CHI) and their relationship with cervical viral shedding. The phenotype and frequency of cytobrush-derived effector immune cells like natural killer cells, T cells, and dendritic cells and their degranulation status (CD107a expression as a surrogate marker of activation) was determined using flow cytometry in age-matched HIV- infected and uninfected women and their association with cervical HIV load was determined. The frequencies of dendritic cells, CD56, CD56 natural killer cell subsets were similar in both the study groups and also within the HIV-infected women with and without progressive disease. The frequencies of CD56CD16 natural killer cells (P = 0.04) and degranulating CD56 natural killer cells were significantly higher among HIV-infected women (P < 0.05). Among HIV-infected women, LTNP women showed reduced degranulation of natural killer and CD8 T cells than seen in the CHI women, which was also associated with lower cervical viral load (P < 0.05). The present study showed that increased degranulation of natural killer and T cells is associated with higher HIV shedding at the CVM of HIV-infected women. Hence reduction of the local immune activation at CVM could be an effective strategy to reduce the cervical viral load.

IL-25 promotes Th2 bias by upregulating IL-4 and IL-10 expression of decidual γδT cells in early pregnancy.

Decidual immune cells (DICs), consisting of both innate and adaptive immune cells, have a pivotal role in maintaining immune tolerance for normal pregnancy. Our previous study demonstrated that interleukin (IL)-25 stimulates the proliferation of decidual stromal cells (DSCs) in an autocrine manner. However, the role of IL-25 in functional regulation of DICs is largely unknown. Flow cytometry was used to analyze the expression of IL-25 and its receptor (IL-17RB) in DICs, and the effect of IL-25 on the expression of Ki-67, IL-4, IL-10, interferon (IFN)-γ and transforming growth factor (TGF)-β in decidual γδT cells. In addition, ELISA assays were performed to detect the secretion of IL-10 and TGF-β in decidual γδT cells. The present findings indicated that decidual CD56 bright CD16-natural killer (NK) cells, natural killer T (NKT) cells, regulatory T (Treg) cells, CD3+ T cells, macrophages and γδT cells co-expressed IL-25 and IL-17RB, particularly γδT cells. Recombinant human (rh) IL-25 protein upregulated the expression of Ki-67, IL-4, and IL-10, but downregulated the expression of IFN-γ in γδT cells; however, anti-human IL-25 or IL-17RB neutralizing antibody reversed these effects. These data suggest that IL-25 may promote IL-10 production by γδT cells as well as the proliferation of γδT cells, and possibly forms a positive feedback loop to maintain a T helper 2 cell bias at the maternal-fetal interface and further contributes to the maintenance of successful pregnancy.

Ex vivo culture of human atherosclerotic plaques: A model to study immune cells in atherogenesis

Background and aimsThe mechanisms that drive atherosclerotic plaque progression and destabilization in humans remain largely unknown. Laboratory models are needed to study these mechanisms under controlled conditions. The aim of this study was to establish a new ex vivo model of human atherosclerotic plaques that preserves the main cell types in plaques and the extracellular components in the context of native cytoarchitecture. MethodsAtherosclerotic plaques from carotid arteries of 28 patients undergoing carotid endarterectomy were dissected and cultured. At various time-points, samples were collected and analysed histologically. After enzymatic digestion, single cells were analysed with flow cytometry. Moreover, tissue cytokine production was evaluated. ResultsWe optimised the plaque dissection protocol by cutting plaques into circular segments that we cultured on collagen rafts at the medium–air interface, thus keeping them well oxygenated. With this technique, the relative presence of T and B lymphocytes did not change significantly during culture, and the sizes of lymphocyte subsets remained stable after day 4 of culture. Macrophages, smooth muscle cells, and fibroblasts with collagen fibres, as well as T and B lymphocyte subsets and CD16 natural killer cells, remained largely preserved for 19 days of culture, with a continuous production of inflammatory cytokines and chemokines. ConclusionsOur new model of ex vivo human atherosclerotic plaques, which preserves the main subsets of immune cells in the context of tissue cytoarchitecture, may be used to investigate important aspects of atherogenesis, in particular, the functions of immune cells under controlled laboratory conditions.

Hepatitis C virus-induced NK cell activation causes metzincin-mediated CD16 cleavage and impaired antibody-dependent cytotoxicity

The Fc receptor family for immunoglobulin (Ig)G type III (FcγRIII, CD16) is an activating receptor on natural killer (NK) cells and an essential mediator of antibody-dependent cellular cytotoxicity (ADCC). There is only limited information on its role during chronic hepatitis C virus (HCV) infection. We studied CD16 expression in relation to NK cell functional activity in HCV-infected patients and sought mechanistic insights into virus-induced modulation. NK cell CD16 expression and activation status were evaluated ex vivo by flow cytometry in HCV-infected patients and healthy controls (HC) as well as in vitro after co-culture with HCV-infected HuH7.5 cells. Rituximab-mediated ADCC was assessed in HC and HCV-infected patients using Daudi cells as a target. The role of metzincins in CD16 down-modulation was assessed using specific inhibitory molecules and by evaluating intracellular mRNA levels. HCV-infected patients exhibited increased frequencies of ex vivo activated NK cells and a concomitantly decreased NK CD16 expression, which resulted in impaired ADCC activity. Moreover, exposure of NK cells to culture-derived HCV recapitulated the ex vivo findings of decreased CD16 expression and increased NK cell activation. Importantly, blockade of metzincin-mediated shedding activity, including selective a disintegrin and metalloproteinase 17 (ADAM-17) inhibition, restored NK CD16 expression. Successful treatment with direct-acting antivirals partially improved NK ADCC function despite delayed CD16 reconstitution. Chronic HCV infection induces NK cell activation resulting in ADAM-17-dependent CD16 shedding and consequent impaired ADCC function. Altered ADCC may contribute to failure to eradicate HCV-infected hepatocytes. We show here that hepatitis C virus (HCV) activates natural killer (NK) lymphocytes which, as a consequence, loose their Fc receptor for IgG (CD16), an essential molecule for antibody binding. We show that this occurs through the action of enzymes named metzincins, resulting in altered NK-mediated antibody-dependent killing (ADCC) of target cells. This mechanism may contribute to HCV persistence and may represent a general phenomenon whereby some viruses can escape host's immune responses.