Percentage Of Natural Killer Cells Research Articles

Regulatory natural killer cell expression in atopic childhood asthma

Introduction: Different subsets of natural killer (NK) cells were found to play a role in pathogenesis of allergy. We sought to investigate the expression of regulatory NK cells (CD56+CD16+CD158+) in atopic children with bronchial asthma in order to outline the value of these cells as biomarkers of disease severity and/or control. Methods: A cross sectional controlled study was carried out in the Pediatric Allergy and Immunology Unit, Ain Shams University. The study included 45 atopic children [mean age(SD)= (2.9) years] with bronchial asthma (BA) and/or allergic rhinitis (AR)as well as 40 healthy matched controls. Enrolled subjects underwent complete blood counting and flow cytometric measurement of NK cell (CD16+ CD56+) and regulatory NK cells (CD16+CD56+CD158+). Results: Patients had significantly higher regulatory NK cell percentages [mean (SD)= 41 (52) %] than controls [mean (SD)=15 (7.1)]; p ≤0.001. Regulatory NK cell counts and percentages did not vary with the concomitant presence of AR or the degree of asthma control. Regulatory NK cell counts tended to be higher in children with moderate/severe BA compared to those with mild asthma but the difference did not reach statistical significance (U= -1.8, p=0.06). NK cell counts [mean (SD)= 159 (164) cells/μl] and percentages [mean (SD)= 3.7 (3.2) %] were comparable among patients and controls and did not vary with the presence of AR ( p = 0.51, 0.95) or with the degree of asthma control. NK cells absolute counts and percentages tended to be higher among patients with moderate/severe compared to mild asthma but the difference did not reach statistical significance. Conclusions: Regulatory NK cells seem to be increased in childhood asthma. We recommend wider scale prospective studies on steroid-naive subjects involving measurement of cytokines that are secreted by different types of NK cells. Keywords: Natural killer, regulatory, asthma, children, allergy

Measurement of uterine natural killer cell percentage in the periimplantation endometrium from fertile women and women with recurrent reproductive failure: establishment of a reference range

Uterine natural killer cells are the major leukocytes present in the periimplantation endometrium. Previous studies have found controversial differences in uterine natural killer cell percentage in women with recurrent reproductive failure compared with fertile controls. We sought to compare the uterine natural killer cell percentage in women with recurrent reproductive failure and fertile controls. This was a retrospective study carried out in university hospitals. A total of 215 women from 3 university centers participated in the study, including 97 women with recurrent miscarriage, 34 women with recurrent implantation failure, and 84 fertile controls. Endometrial biopsy samples were obtained precisely 7 days after luteinization hormone surge in a natural cycle. Endometrial sections were immunostained for CD56 and cell counting was performed by a standardized protocol. Results were expressed as percentage of positive uterine natural killer cell/total stromal cells. The median uterine natural killer cell percentage in Chinese ovulatory fertile controls in natural cycles was 2.5% (range 0.9-5.3%). Using 5th and 95th percentile to define the lower and upper limits of uterine natural killer cell percentage, the reference range was 1.2-4.5%. Overall, the groups with recurrent reproductive failure had significantly higher uterine natural killer cell percentage than the controls (recurrent miscarriage: median 3.2%, range 0.6-8.8%; recurrent implantation failure: median 3.1%, range 0.8-8.3%). However, there was a subset of both groups (recurrent miscarriage: 16/97; recurrent implantation failure: 6/34) that had lower uterine natural killer cell percentage compared to fertile controls. A reference range for uterine natural killer cell percentage in fertile women was established. Women with recurrent reproductive failure had uterine natural killer cell percentages both above and below the reference range.

Profile of differentially expressed Toll-like receptor signaling genes in the natural killer cells of patients with Sézary syndrome.

Sézary syndrome (SS), an aggressive and leukemic form of cutaneous T-cell lymphoma, usually results in shortened survival. Improving innate immunity in SS by targeting natural killer (NK) cells with Toll-like receptor (TLR) agonists could be an interesting modulatory strategy. We evaluated the NK cell populations in SS patients assessing activating and inhibitory receptors expression and profiled the differential expression of TLR signaling pathway genes in unstimulated NK cells and after TLR7/8 stimulation. We observed preserved CD56bright NK cells and a low percentage of CD56dim NK cells in the peripheral blood of SS patients compared to those in the healthy control group. Both NK cell populations showed down-modulation of NKG2C and NKG2D expression, which was associated with high serum levels of the soluble form of NKG2D ligands. In contrast, an expansion of “memory” CD57+ NKG2C+ NK cells and high cytomegalovirus antibody titers were detected in SS patients. Profiling of the TLR signaling genes in NK cells from SS patients showed an abundance of differentially expressed genes (DEGs) in NK cells in the unstimulated condition, with mostly up-regulation of NFκB/JNK p38 pathway genes, but there was down-regulation of type I (IFN-α/β) and II (IFN-γ) interferon and IL-12A. After activation of NK cells with TLR7/8 agonist, the down-regulated genes correlated with the IFN response, and IL-12 became up-regulated, together with other antitumor factors. NK cell activation with a dual agonist for TLR7 and TLR8 is able to induce the expression of IFN-γ and type I IFN, which can improve immunity in SS patients.

Experiment research of natural killer cells amplification in vitro and the killing effect on ovarian cancer cells

Objective: To amplify natural killer (NK) cells in vitro and explore its killing effect on ovarian cancer cells. Methods: (1) The separation of NK cells and identification. A total of 20 ml peripheral blood of one healthy volunteer was collected in Nov. 2015, Peking University People's Hospital. The peripheral blood mononuclear cells of normal volunteers were isolated, cultured in vitro and amplificated cultivation for 14 days with K562 cells transfected and expressing interleukin 21 (IL-21-K562) as nourish cells. The number and dynamic state of the growth cells were monitored during the cultured process. Cells were harvested and counted after 14 days cultured. The NK cells phenotypes were detected by flow cytometry. (2) The killing effect of NK cells on ovarian cancer cells: the ratio of effector cells (NK cells) and target cells (ovarian cancer cells and its control) was 50∶1, 20∶1, 10∶1, 5∶1 or 1∶1, NK cells killing effect on ovarian cancer cells was detected by the lactate dehydrogenase (LDH) release experiments. Results: (1) The results of NK cells establishment and phenotypic characterization: the cells were induced in vitro for 14 days by amplification culture. With the extension of incubation time, the number of NK cells increased constantly, from 2.0×10(7) on day 0 to 5.1×10(9) on day 14. Obvious amplification of the total number of cells were detected for 255 times. Living cells unstained by trypan blue eventually reached 95% above. Before and after the induction and amplification in vitro, the percentage of NK cells(CD(3)(-)CD(5)(6+)cells) in CD(3)- cells were 2.33% and 85.32%, respectively (P<0.01), which covered the whole lymphocytes 1.06% and 69.42%, respectively (P<0.01), which showed that NK was the main cell type in the amplificated lymphocytes. (2) The killing rate of NK cells on ovarian cancer cells in vitro: the results detected by LDH release experiments showed that NK cells could performed strong nonspecific killing effect on ovarian cancer cell lines SKOV3, HOC1A, 3AO and CAOV3, as well the normal ovarian cell line T29 and NK sensitive cell line K562, and the killing effect increased significantly along with the increase of effector cells and target cells ratio (P<0.01). When the ratio was 1∶1, the killing rate was 37% for K562, while the rate of killing of other cells was around 10% (P<0.05). When the effect-target ratio was 20∶1 and 50∶1, in addition to CAOV3 cells (more than 70%), NK cells had a kill rate of more than 80% for other ovarian cancer cells lines and their control cell K562 and T29 cells (P>0.05). Conclusion: NK cells could be established in vitro and have a good non-specific killing effect on ovarian cancer cells.

Peripheral Lymphocyte Subsets as a Marker of Parkinson's Disease in a Chinese Population.

In this study, we conducted a clinical analysis of lymphocytesubtypes in 268 patients with Parkinson's disease (PD) to assess their clinical impact as a potential marker of advanced PD in Chinese patients. The participants comprised 268 sporadic PD patients and 268 healthy controls. The numbers of natural killer (NK) cells and CD3+, CD3+CD4+, CD3+CD8+, and CD19+ lymphocytes from peripheral blood were determined by immunostaining and flow cytometric analysis and the percentages of these CD+ T cells were calculated. The ratio of regulatory T (Treg)/helper T 17 (Th17) lymphocytes from 64 PD patients and 46 controls was determined by flow cytometric analysis. The results showed that the percentage of NK cells was higher in advanced PD patients than in controls (22.92%±10.08% versus 19.76%±10.09%, P=0.006), while CD3+ T cells are decreased (62.93%±9.27% versus 65.75%±9.13%, P=0.005). The percentage of CD19+B cells in male patients was lower (P=0.021) than in female patients, whereas NK cells were increased (P<0.0001). The scores on the Unified Parkinson's Disease Rating Scale (UPDRS) and the Non-Motor Symptoms Scale in late-onset PD patients were significantly higher than those in early-onset patients (P=0.024 and P=0.007, respectively). The percentage of CD19+ B cells in patients with UPDRS scores >24 was lower than in those with scores <24 (10.17%±4.19% versus 12.22%±5.39%, P=0.009). In addition, the Treg/Th17 ratio in female patients was higher than that in female controls (13.88±6.32 versus 9.94±4.06, P=0.042). These results suggest that the percentages of NK cells, CD3+ T cells, and CD19+ B cells along with the Treg/Th17 ratio in peripheral blood may be used to predict the risk of PD in Chinese individuals and provide fresh avenues for novel diagnostic biomarkers and therapeutic designs.

6-Mercaptopurine modifies cerebrospinal fluid T cell abnormalities in paediatric opsoclonus-myoclonus as steroid sparer.

The purpose of this study was to evaluate the capacity of 6-mercaptopurine (6-MP), a known immunosuppressant, to normalize cerebrospinal fluid (CSF) lymphocyte frequencies in opsoclonus-myoclonus syndrome (OMS) and function as a steroid sparer. CSF and blood lymphocytes were immunophenotyped in 11 children with OMS (without CSF B cell expansion) using a comprehensive panel of cell surface adhesion, activation and maturation markers by flow cytometry, and referenced to 18 paediatric controls. Drug metabolites, lymphocyte counts and liver function tests were used clinically to monitoring therapeutic range and toxicity. In CSF, adjunctive oral 6-MP was associated with a 21% increase in the low percentage of CD4+ T cells in OMS, restoring the CD4/CD8 ratio. The percentage of CD4+ T cells that were interferon (IFN)-γ+ was reduced by 66%, shifting the cytokine balance away from T helper type 1 (Th1) (proinflammatory) predominance. The percentage of natural killer (NK) cells decreased significantly in CSF (-32%) and blood (-67 to -82%). Low blood absolute lymphocyte count was more predictive of improvement in CSF lymphocyte proportions (correlated with % CD4+ T cells) than the 6-thioguanine level (no correlation). 6-MP was difficult to titrate: 50% achieved the target absolute lymphocyte count (<1·5 K/mm); 20%, the 'therapeutic' 6-thioguanine level; and 40% the non-toxic 6-methylmercaptopurine level. Side effects and transaminase elevation were mild and reversible. Clinical steroid-sparing properties and lowered relapse frequency were demonstrated. 6-MP displayed unique pharmacodynamic properties that may be useful in OMS and other autoimmune disorders. Its steroid sparer capacity is limited to children in whom the therapeutic window can be reached without limiting pharmacokinetic factors or side effects.

Spleen contributes to restraint stress induced changes in blood leukocytes distribution

Psychological stress has great impacts on the immune system, particularly the leukocytes distribution. Although the impacts of acute stress on blood leukocytes distribution are well studied, however, it remains unclear how chronic stress affects leukocytes distribution in peripheral circulation. Furthermore, there is no report about the role of spleen in the blood leukocytes distribution induced by stress. Here we show that spleen contributes to the alteration of restraint stress induced blood leukocytes distribution. Our data confirmed that restraint stress induced anxiety-like behavior in mice. Furthermore, we found that restraint stress decreased the CD4/CD8 ratio and elevated the percentages of natural killer cells, monocytes and polymorphonuclear myeloid-derived suppressor cell. We demonstrated that activation of hypothalamic-pituitary-adrenal axis (HPA) and sympathetic nervous system (SNS) contributes to restraint stress induced alteration of blood leukocyte distribution. Interestingly, we found that splenectomy could reverse the change of CD4/CD8 ratio induced by restraint stress. Together, our findings suggest that activation of HPA axis and SNS was responsible for the blood leukocyte subsets changes induced by restraint stress. Spleen, at least in part, contributed to the alteration in peripheral circulation induced by restraint stress.

Role of natural killer cells in liver transplantation treatment of liver cancer.

Liver cancer caused by diet or life style is a significant public health problem. Liver transplantation (LT) is a commonly used method of treatment for the liver cancer. The present study aimed to determine whether assessing the net state of natural killer (NK) cell function following LT distinguishes patients at risk for transplantation rejection. A total of 53 patients were involved; all underwent LT for hepatocellular carcinoma with (n=13) or without (n=40) transplantation rejection. The density of interferon-γ (IFN-γ) in blood serum was examined and patients were divided into two groups: Higher (H) and lower (L), on the basis of IFN-γ density. The percentage of NK cells and their producing cytokines was detected using fluorescence-activated cell sorting in peripheral blood and liver samples. As evaluation indexes of liver function, aspartate transaminase (AST) and alanine transaminase (ALT) were detected in blood serum. NK cell activation of the H-group was observed to be higher than the L-group, specifically the expression of NK group 2D, cluster of differentiation 69 and IFN-γ were higher than the L-group. The H-group exhibited a higher level of AST and ALT, which indicates the potential for acute transplantation rejection. The results of the present study indicate that NK cells and NK-derived IFN-γ serve an important function in regulating the rejection of LT and tumor metastasis in response to LT.

Sexual Dimorphic Responses in Lymphocytes of Healthy Individuals after Carica papaya Consumption.

Sexual dimorphism in immune response is widely recognized, but few human studies have observed this distinction. Food with endo-immunomodulatory potential may reveal novel sex-biased in vivo interactions. Immunomodulatory effects of Carica papaya were compared between healthy male and female individuals. Volunteers were given fixed meals supplemented with papaya for 2 days. Changes in blood immune profiles and hormone levels were determined. In females, total natural killer (NK) cell percentages decreased (12.7 ± 4.4 vs 14.6 ± 5.8%, p = 0.018, n = 18) while B cells increased (15.2 ± 5.5 vs 14.5 ± 5.0, p = 0.037, n = 18) after papaya consumption. Increased 17β-estradiol (511.1 ± 579.7 vs 282.7 ± 165.0 pmol/l, p = 0.036, n = 9) observed in females may be crucial to this change. Differentiation markers (CD45RA, CD69, CD25) analyzed on lymphocytes showed naïve (CD45RA+) non-CD4+ lymphocytes were reduced in females (40.7 ± 8.1 vs 46.8 ± 5.4%, p = 0.012, n = 8) but not males. A general suppressive effect of papaya on CD69+ cells, and higher percentage of CD69+ populations in females and non-CD4 lymphocytes, may be relevant. CD107a+ NK cells were significantly increased in males (16.8 ± 7.0 vs 14.7 ± 4.8, p = 0.038, n = 9) but not females. Effect in females may be disrupted by the action of progesterone, which was significantly correlated with this population (R = 0.771, p = 0.025, n = 8) after papaya consumption. In males, total T helper cells were increased (33.4 ± 6.4 vs 32.4 ± 6.1%, p = 0.040, n = 15). Strong significant negative correlation between testosterone and CD25+CD4+ lymphocytes, may play a role in the lower total CD4+ T cells reported in males. Thus, dissimilar immune profiles were elicited in the sexes after papaya consumption and may have sex hormone influence.

Changes in natural killer cell subsets defined by CD11b and CD27 in spleen of septic mice

Objective To evaluate the changes in natural killer(NK)cell subsets defined by CD11b and CD27 in the spleen of septic mice. Methods A total of 168 pathogen-free healthy male C57BL/6 mice, weighing 20-30 g, aged 8-10 weeks, were divided into 2 groups(n=84 each)using a random number table: sham operation group(Sham group)and sepsis group(Sep group). Sepsis was induced by cecal ligation and puncture in chloral hydrate-anesthetized mice.Thirty mice in each group were selected to assess the survival within 4 days after operation, and the survival rate was calculated.At 2, 4, 6, 24, 48 and 72 h after operation(T1-6), 6 mice were randomly selected, and blood samples were taken from the eyeballs for determination of serum tumor necrosis factor-alpha and interleukin-10 concentrations by enzyme-linked immunosorbent assay.Six mice were randomly selected at T4-6 and sacrificed, and the spleens were removed for measurement of the percentage of NK cells and their subsets by flow cytometry. Results Compared with Sham group, the survival rate was significantly decreased at different time points, the serum necrosis factor-alpha concentration at T1-3, 6 and serum interleukin-10 concentration at T3-6 were increased, the percentage of NK cells was decreased at T4-6, the percentage of CD27-CD11b+ NK cells was decreased and the percentage of CD27+ CD11b+ and CD27+ CD11b-NK cells was increased at T5, the percentage of CD27-CD11b-NK cells was decreased at T4, 5, and the percentage of CD27-CD11b-NK cells was increased at T6 in Sep group(P<0.05). Conclusion Changes in NK cell subsets defined by CD11b and CD27 in the spleen play an important role in the development of sepsis in mice. Key words: Sepsis; Killer cells, natural; Antigens, CD27; Antigens, CD11b

Natural killer cells as a predictive biomarker for response to anti-PD-1 therapy in patients with advanced solid tumors.

e21055 Background: Assessment of PD-L1 expression in tumor biopsy has been widely used for predicting anti-PD-1 therapy. Several factors, including tumor heterogeneity, false negative staining and unavailability of biopsy samples limit the application. Natural killer (NK) cells exert cytotoxicity against cancer cells. Therefore, we explored using NK cells percentage in peripheral blood (PB) as a biomarker to identify pts who are more likely to benefit from anti-PD-1 therapy. Methods: Pts treated with anti-PD-1 antibody (JS001) 1 mg/kg, 3mg/kg or 10mg/kg every 2 weeks in a Phase I clinical trial (ClinicalTrials.gov Identifier: NCT02836795) had PB collected at baseline and at time of radiographic evaluation performed every 8 wks. Disease status (shrinking stable disease, growing stable disease, partial response, complete response, or progressive disease) was characterized using RECIST v1.1 and immune-related response criteria. Frequencies of NK cells (CD3-CD16+CD56+) were measured by flow cytometry. Results: Thirty-six patients were enrolled in this trial, including 22 melanoma, 9 urothelial carcinoma and 5 renal cell carcinoma. As of 1/27/2017, median treatment duration: 3.92 months. Radiographic tumor evaluation has been performed in 32 pts: 1/32 CR, 6/32 PR, 6/ 32 shrinking SD, 4/32 growing SD and 15/32 PD. Pts with high NK cells at baseline observed 60% (6/10) Clinical Benefit Rate (CR/PR/ shrinking SD) versus 40% (13/32) in overall pts. Patients with clinical benefit had higher frequency of NK cells at baseline compared to pts with radiographic growing SD and PD (mean 24.3% vs. 17.9%, P = 0.076). Furthermore, pts with NK cell level consistently above UN or increasing above UN during treatment are more likely to get clinical benefit than those under UN (7/10 vs 3/22, P = 0.013). Conclusions: Baseline high NK cells frequency may predict a favorable response to anti-PD-1 therapy, which provides a non-invasive way to monitor response to PD-1 blockade. These results are being validated in a phase II trial. Clinical trial information: NCT02836795.

Atopy in children with juvenile systemic lupus erythematosus is associated with severe disease.

The influence of co-existing atopy on the prognosis of juvenile systemic lupus erythematosus (JSLE) was assessed in this study. Patients diagnosed with JSLE between October 2005 and April 2016 were enrolled in a prospective study and followed up for 2 years. Management of patients was evaluated using the systemic lupus erythematosus disease activity index 2000 (SLEDAI-2K) score and laboratory variables. Eighty JSLE patients were enrolled at diagnosis and divided into those with (n = 35) and without (n = 45) atopy. When compared with the non-atopic group, atopic patients showed higher SLEDAI-2K score at disease onset (16.09 vs. 11.18), higher erythrocyte sedimentation rate (52.89 vs. 38.27 mm/h), higher percentage of total B-cells (25.85 vs. 19.51%), lower percentage (7.26 vs. 9.03%) and cytotoxicity (9.92 vs. 11.32%) of natural killer cells, and lower complement C3 (0.51 vs. 0.69 g/L) (all p<0.05). At 1, 3, 6, 12, 18, and 24 months, JSLE patients with atopy reached higher SLEDAI-2K score and lower ΔSLEDAI-2K improvement rate (at 1 month, 8.34 vs. 4.71 and 43.63 vs. 57.95%, respectively; at 3 months, 8.57 vs. 2.62 and 48.39 vs. 75.10%, respectively; at 6 months, 6.91 vs. 2.38 and 53.59 vs. 77.26%, respectively; at 12 months, 4.71 vs. 1.80 and 69.54 vs. 84.10%, respectively; at 18 months, 4.66 vs. 2.02 and 68.14 vs. 82.93%, respectively; at 24 months, 8.57 vs. 2.62 and 70.00 vs. 81.88%, respectively; all p<0.05). During the 24 months of follow-up, the total number of disease flares was higher in JSLE patients with co-existing atopy (3.77 vs. 1.51, p<0.05), and the atopic group needed much more time to reach the stable condition of the disease (6.88 vs. 4.65 months, p<0.05). JSLE patients combined with co-existing atopy had more severe disease at diagnosis and poorer outcomes than JSLE patients without atopy.

Human natural killer cell expansion in vitro using mitomycin-C treatment as an alternative to irradiation of modified K562 feeder cells.

Abstract Introduction Current methods for in vitro expansion of Natural Killer (NK) cells require feeder cell irradiation, which is not readily available in many biomedical research or clinical facilities. Here we evaluated mitomycin-C (MMC) treatment of feeder cells as an alternative to irradiation for the purpose of expanding NK cells in human peripheral blood mononuclear cell (PBMC) cultures. Methods Using PBMCs from 4 healthy blood donors and genetically modified K562 feeder cells (K562-mb21-41BBL cells) that preferentially stimulate NK cells, we evaluated the efficacy of MMC treatment in limiting the proliferation of the feeder cells at a range of doses (0 ug/mL, 2 ug/mL, 10 ug/mL, 20 ug/mL, 30 ug/mL, and 200 ug/mL) with 3-hour incubation. We used 7AAD dye and CFSE proliferation dye to measure viability and cell proliferation, respectively. We then compared the percentages of NK cells (CD3neg, CD56pos) in PBMCs cultured for 14 days in the presence of MMC-treated feeder cells or irradiated feeder cells using multiparametric flow cytometry. Results We found that 10 ug/mL dose of MMC for 3-hrs achieved a significantly higher percentage of viable cells compared to higher doses (p&amp;lt;0.05, ANOVA), and also significantly limited proliferation compared to untreated feeder cells. Furthermore we found no significant difference in NK cell expansion by 10 ug/mL MMC treated feeder cells and 100 grays irradiated feeder cells. Discussion In conclusion, we found that 10 ug/mL mitomycin-C treatment of modified K562 feeder cells was optimal for limiting their proliferation for NK cell expansion that was equivalent to using irradiated feeder cells.

Role of Natural Killer Cells in Sonic Hedgehog driven Medulloblastoma

Abstract Medulloblastoma (MB) is the most common paediatric neoplasm of the CNS. Standard therapy is mainly curative; however, the severe side effects make immunotherapy an attractive therapeutic option. Natural Killer (NK) cells are innate lymphoid cells that can be used for adoptive cell therapy. Herein, we studied the role of NK cells in MB growth control, focusing on Sonic Hedgehog (SHH) subgroup, using the MB cell line DAOY. The contribution of SHH pathway on NK cell recruitment to MB was analysed performing an orthotopic xenograft of DAOY in nude mice treated with a pharmacological inhibitor of the SHH pathway. FACS analysis revealed higher percentage of NK cells in cerebella of treated mice compared to control group. Based on this in vivo evidence, we characterized the mechanism underlying NK cell recruitment. Pharmacological inhibition of the SHH pathway in DAOY raised the expression and secretion of chemokines involved in NK cell migration. Chemotaxis assay confirmed that these secreted molecules were functional. Successively, we assessed the ability of NK cells to recognize SHH-MB. By FACS analysis we determined the tumor line expression of ligands for the NKG2D and DNAM1 activating receptor. In particular, ULBP3 and ULBP2 were expressed at high levels. Moreover, we found that NK cells co-culture with DAOY induced degranulation that was reduced in the presence of anti-NKG2D and anti-DNAM1 blocking antibodies, suggesting an involvement of these receptors in triggering degranulation. Besides, DAOY increased the production of IFNγ in NK cells pre-activated of with suboptimal doses of IL12 and IL15. Our data provide evidence for a role of NK cells in the control of SHH-MB growth.

Quantities and function of NK cells in patients with immune thrombocytopenia

Objective: To investigate natural killer (NK) cell quantities and function in patients with immune thrombocytopenia (ITP) . Methods: A total of 66 ITP patients (34 newly diagnosed and 32 in complete remission) were collected from September 2015 to May 2016 in Tianjin Medical University General Hospital, and 30 healthy volunteers were recruited as controls. The percentages of NK cells and their subsets in peripheral blood, the expression of activating receptor (NKp44), inhibitory receptor (NKG2A) and CD16, perforin and granzyme β were detected by flow cytometry. The correlation between the above parameters and patients' immune status and platelet level were evaluated. Results: (1)The percentage of CD3(-)CD56(+) NK cells in newly diagnosed patients (10.99%±4.89%)and patients in complete remission (9.73%±6.75%) were significantly lower than that in healthy controls (14.67%±7.24%)(P=0.023, 0.003). The percentage of NK cells Bright subset was significantly lower in the newly diagnosed patients(0.48%±0.23%)and those in complete remission (0.41%±0.33%) than in healthy controls(0.64%±0.32%)(P=0.037, 0.002); the percentage of Dim subset was also significantly lower in the newly diagnosed (10.16%±5.02%) and patients in complete remission (8.07%±5.74%) than in healthy controls(14.16%±7.19%) (P=0.009, 0.007). (2)The proportion of Bright subset in total NK cells in new diagnosed ITP patients (6.48%±4.33%) was significantly higher than that in healthy controls (4.21%±2.70%)(P=0.020); the proportion of Dim NK cells subset in new diagnosed ITP patients (93.51%±4.33%) was significantly lower than that in healthy controls(95.79%±2.70%) (P=0.020). (3)The expression of activating receptor NKp44 in new diagnosed ITP patients was significantly lower than that in complete remission group and healthy controls[0.28%(0.95%)vs 0.61%(2.05%), 0.92%(0.90%); P=0.047, 0.048]; the expression of inhibitory receptor NKG2A in new diagnosed ITP patients was significantly higher than that in healthy controls(42.34%±23.86% vs 29.25%±12.83%, P=0.009). The proportion of CD16 was significantly lower in the newly diagnosed patients than in healthy controls(93.51%±4.33%95.79%±2.70%, P=0.020). (4)The expression of perforin in the newly diagnosed ITP patients was significantly lower than that in healthy controls [87.52%(25.29%)vs 91.55%(8.29%), P=0.025]; the expression of granzyme β in ITP patients and controls showed no statistically significant difference. (5)The level of NK cells in ITP patients was negatively correlated with CD3(+) CD8(+) T cells (r=-0.387, P=0.012) and CD5(+) CD19(+) B cells in peripheral blood (r=-0.273, P=0.028), positively correlated with the ratio of CD3(+) CD4(+) /CD3(+) CD8(+) (r=0.358, P=0.028) and peripheral platelet count (r=0.314, P=0.011). Conclusion: Deceased quantities and impaired total NK function, insufficient suppression of autoreactive T and B cells might play a role in the pathogenesis of ITP.

Cytotoxicity of Natural Killer Cells Activated Through NKG2D Contributes to the Development of Bronchiolitis Obliterans in a Murine Heterotopic Tracheal Transplant Model.

Bronchiolitis obliterans after lung transplantation is a major cause of postoperative mortality in which T cell-mediated immunity is known to play an important role. However, the exact contribution of natural killer (NK) cells, which have functions similar to CD8+ T cells, has not been defined. Here, we assessed the role of NK cells in murine bronchiolitis obliterans through heterotopic tracheal transplantations and found a greater percentage of NK cells in allografts than in isografts. Depletion of NK cells using an anti-NK1.1 antibody attenuated bronchiolitis obliterans in transplant recipients compared with controls. In terms of NK cell effector functions, an improvement in bronchiolitis obliterans was observed in perforin-KO recipient mice compared to wild type (WT). Furthermore, we found upregulation of NKG2D-ligand in allografts and demonstrated the significance of this using grafts expressing Rae-1, a murine NKG2D-ligand, which induced severe bronchiolitis obliterans in WT and Rag-1 KO recipients. This effect was ameliorated by injection of anti-NKG2D blocking antibody. Together, these results suggest that cytotoxicity resulting from activation of NK cells through NKG2D leads to the development of murine bronchiolitis obliterans.

Distinct Effects of Dexamethasone on Human Natural Killer Cell Responses Dependent on Cytokines.

Glucocorticoids (GCs) have long been known to be immune suppressive and synthetic variants are widely used in the treatment of inflammatory disorders. Here, we report that, while suppressing the initial production of interferon-γ (IFN-γ), the synthetic GC dexamethasone (Dex) enhances the proliferation and survival of natural killer (NK) cells stimulated with interleukin-2 (IL-2) + interleukin-12 (IL-12). Inhibition of mTOR complex 1 by rapamycin revealed the immunosuppressive activity of Dex was independent from the effect of enhancing NK cell proliferation. In the presence of IL-2 + IL-12, Dex also increased the percentage of NK cells that were CD16+ and DNAM1bright, increased the level of expression of CD94 or NKG2A, and improved mitochondrial function of NK cells. Moreover, NK cells treated with cytokines IL-2 and IL-12 + Dex, followed by a 7-day rest, displayed an increased IFN-γ response upon restimulation. Thus, there is a dichotomic effect of GCs on NK cell function dependent on the local cytokine milieu; the NK cell effector response is initially suppressed, but, dependent on the cytokines present, Dex can also augment the proliferation, survival, and reactivity of human NK cells in a secondary recall response.

B- and T-cell subpopulations in patients with severe idiopathic membranous nephropathy may predict an early response to rituximab

Primary membranous nephropathy (PMN) is characterized by antibodies to the podocyte, but little is known about B- and T-cell populations and their response to rituximab is controversial. To help resolve this we compared 33 lymphocyte subpopulations and 27 cytokines/chemokines in 25 patients with severe PMN and 27 age-matched healthy individuals. At baseline, patients had a significantly increased percentage of naive B-cells with significantly decreased switched and non-switched memory B-cells. There was a significantly decreased percentage of natural killer (NK) cells with an increase in the CD56brightCD16-/lo NK subset. There were a significantly decreased percentage of regulatory T cells, together with an increased plasma concentration of TNF-alpha, IL-5 and IL-2RA. We then investigated 16 patients at eight days and three and six months after treatment with rituximab added to supportive therapy compared to nine patients with supportive therapy alone. After rituximab, B-cell recovery was still incomplete at six months, with persistent alterations of B-cell subsets, a significant increase of both T-regulatory (Treg) cells and NK cells, and a significant decrease of both the CD56brightCD16-/lo NK subset and TNF-alpha levels. The patients who clinically responded to rituximab had a significantly lower percentage of Tregs at baseline compared to non-responders and a significantly increased percentage at day eight. Tregs remained unchanged in non-responders and in patients treated with supportive therapy alone. Thus, evaluation of Tregs might be useful for predicting early response to rituximab.

Chronic Mental Stress Induces Reversible Reduction of Natural Killer Cells and CD56&lt;sup&gt;dim &lt;/sup&gt;Subpopulation

Generally, studying to be a health care provider is a stressful and demanding field and the students have to face many stressors that may affect their general health status including the immune system. This work aimed at studying the effect of prolonged naturalistic life-stress exposure on the percentage of peripheral blood T-lymphocytes and Natural Killer (NK) cells in female laboratory medicine students. 52 peripheral blood samples in the last week of the final written exam (stress time point) and 27 samples after 12 weeks rest (control) were withdrawn and analyzed by flow cytometry. At the stress time point, there was a significant high T helper cells percentage with elevation of T helper/T cytotoxic ratio, P value <0.001. Also, significant low percentages of NK cells and CD56dim together with high CD56bright subpopoulations were detected, P value <0.001. Lymphocyte analysis of the subgroup that had an attach of common cold (34.6%) revealed significant reduction in the number of T cytotoxic and NK cells, P values 0.042 and 0.001 respectively. This study concluded that in humans, naturalistic chronic stress as expressed in academic exams has the potential to negatively affect the immune system, but normality is regained after sufficient stress relieve measures. Replication in larger and more diverse sample populations with inclusion of males for comparison is required, Also assessments of NK cell cytotoxicity and T helper cell subsets especially T regulatory cells are required for future studies.

Frequency and phenotype of natural killer cells and natural killer cell subsets in bovine lymphoid compartments and blood.

SummaryNatural killer (NK) cells are widely distributed in lymphoid and non‐lymphoid tissues, but little is known about the recirculation of NK cells between blood and tissues. This is relevant to understanding recirculation in the steady‐state and also for determining the roles for NK cells in vaccine‐induced immunity and responses to infection. Therefore, the percentage of NK cells and their phenotype across peripheral blood, afferent lymph and lymph nodes in steady‐state conditions was investigated in cattle using the pseudo‐afferent lymphatic cannulation model. CD2+ CD25lo NK cells were the predominant subset of NK cells within the blood. In contrast, CD2− CD25hi NK cells were the main subset present within the skin‐draining afferent lymphatic vessels and lymph nodes, indicating that CD2− NK cells are the principal NK cell subset trafficking to lymph nodes via the afferent lymphatic vessel. Furthermore, a low percentage of NK cells were present in efferent lymph, which were predominantly of the CD2− subset, indicating that NK cells can egress from lymph nodes and return to circulation in steady‐state conditions. These compartmentalization data indicate that NK cells represent a population of recirculating lymphocytes in steady‐state conditions and therefore may be important during immune responses to vaccination or infection.