Killer Cells In Peripheral Blood Research Articles

Dynamic changes of phenotype and function of natural killer cells in peripheral blood before and after thermal ablation of hepatitis B associated hepatocellular carcinoma and their correlation with tumor recurrence

BackgroundThermal therapy induces an immune response in patients with hepatocellular carcinoma (HCC), but the dynamic characteristics of the natural killer (NK) cell immune response post-thermal ablation remain unclear. We conducted a prospective longitudinal cohort study to observe the dynamic changes of phenotype and function of NK cells in peripheral blood before and after thermal ablation of hepatitis B-associated HCC and their correlation with tumor recurrence.MethodsFifty-six patients clinically and pathologically confirmed with hepatitis B-associated HCC were selected for thermal ablation. Peripheral blood was collected on day 0, day 7, and month 1. NK cell subsets, receptors, and killing function were detected by flow cytometry, and the LDH levels were examined. Overall recurrence and associated variables were estimated using Kaplan–Meier, log-rank, and Cox proportional-hazards analyses.ResultsThe frequency of CD3−CD56+ cells was increased on day 7 (P < 0.01) without significant differences between D0 and M1. NKG2D, NKp44, NKp30, CD159a, and CD158a expression was increased on M1 (all P < 0.05). The granzyme B and IFN-γ expression in NK cells were higher on M1 vs. D0 (P < 0.05). On day 7, the NK cell lysis activity of the target K562 cells was increased (P < 0.01) but decreased on M1 (P < 0.05). Survival analysis showed that CD158a expression and IFN-γ and perforin release on day 0 were associated with the risk of HCC recurrence. Cox regression analysis showed that the expression changes in CD56, NKp46, granzyme B, and perforin (D7-D0) induced by thermal ablation were associated with recurrence-free survival (RFS) of patients with HCC.ConclusionThermal ablation increased the frequency and function of CD3−CD56+ NK cells in the peripheral blood of patients with HCC. These cells tended to be more differentiated and activated. Notably, expression levels of NK cell receptors NKp46, perforin, and granzyme B were associated with RFS.

Natural killer cells in peripheral blood at diagnosis predict response to immunosuppressive therapy in severe aplastic anemia.

Immunosuppressive therapy (IST) consisting of antihuman thymocyte globulin and cyclosporine A is the first-line therapy for patients with severe aplastic anemia (AA) who are ineligible for undergoing bone marrow transplantation. The aim of the study was to evaluate the correlation between natural killer (NK) cells and response to IST in SAA patients. We retrospectively included 93 AA patients and detected NK cells in peripheral blood by flow cytometry. Both the proportion and absolute number of NK cells in newly diagnosed SAA patients were significantly lower than in controls, while the proportion and absolute number of NK cells in complete remission patients treated with IST were remarkably increased compared with treatment-naïve SAA patients. Additionally, the absolute number of NK cells at diagnosis was positively correlated with initial blood counts. For SAA patients receiving IST, the proportion of NK cells at baseline and 6months was significantly higher in responders than in non-responders. Unexpectedly, we found that the increase in the proportion of NK cells at 6months after IST was closely related to the recovery of hematopoiesis. ROC curve identified 7.3% of NK cells proportion at diagnosis as the cutoff value to predict response to IST. The response rate was higher in NK proportion high group than in NK proportion low group. Multivariate logistic regression analysis further confirmed the independent predictive value of NK cells proportion in assessing IST response. The proportion of NK cells at diagnosis may serve as a promising predictor of response to IST in patients with SAA.

Effects of fucoidan on gut flora and tumor prevention in 1,2-dimethylhydrazine-induced colorectal carcinogenesis

Colorectal cancer (CRC) is one of the major malignancies in humans. This study was designed to evaluate the effects of fucoidan on gut flora and tumor prevention in 1,2-dimethylhydrazine-induced colorectal carcinogenesis in rats. We found that dietary fucoidan treatment decreased the tumor incidence and mean tumor weight and increased cell apoptosis. Fucoidan treatment decreased the expression of β-catenin C-Myc, CyclinD1 and Survivin, while the Hippo pathway was activated with increased phosphorylation levels of mammalian sterile 20-like kinase 1 and 2, large tumor suppressor 1 and 2, and Yes-associated protein. Compared with the model group, the levels of interleukin (IL)-17 and IL-23 were decreased, but the levels of interferon-γ, IL-4 and IL-10 were increased, in the fucoidan group. Fucoidan treatment increased natural killer cells in peripheral blood and the proportion of CD4+ T cells. Immunofluorescence detection of colorectal tumor tissues showed decreased expression of Foxp3 and up-regulated expression of CD68 in the fucoidan group. Moreover, fucoidan treatment decreased the levels of diamine oxidase and lipopolysaccharides and up-regulated the levels of tight junction proteins. 16S rDNA high-throughput sequencing revealed that fucoidan treatment decreased the abundance of Prevotella and increased the abundance of Alloprevotella. Fucoidan increased the levels of butyric acid and valeric acid compared to the model group. This study provides experimental evidence that dietary fucoidan may prevent colorectal tumorigenesis by regulating gut microecology and body immunity. Meanwhile, fucoidan activated the Hippo pathway and down-regulated the β-catenin pathway to induce tumor cell apoptosis and suppress tumor growth.

Effect of perioperative allogeneic red blood cell infusion on T cell subsets and natural killer cells in peripheral blood of patients with gastric cancer

Objective To investigate the effect of allogeneic red blood cell transfusion on T lymphocyte subsets and natural killer (NK) cells in peripheral blood of patients with gastric cancer during perioperative period. Methods 50 patients with gastric cancer in our hospital from January 2017 to December 2017 were randomly divided into control group (n=25) and observation group (n=25). Perioperative allogeneic erythrocyte transfusion was performed in the observation group, while no allogeneic erythrocyte transfusion was performed in the control group. Blood samples were taken from two groups before and after operation 3 d, and serum T lymphocyte subsets (CD3+, CD4+, CD8+, CD4+ /CD8+ ) and NK cell levels were measured. T cell subsets and NK cell levels of patients in observation group, control group and observation group at different clinical stages before and after operation were counted. According to blood transfusion volume, the patients in the observation group were divided into <4 U group and <4 U group. T cell subsets and NK cell levels before and after operation were compared between the two groups. Results There was no significant difference in CD3+, CD4+, CD8+, CD4+ /CD8+, NK cells between the two groups before operation (P>0.05). The levels of CD3+, CD4+, CD4+ /CD8+, NK cells in the two groups after operation were lower than those before operation and CD8+ levels were higher than those before operation. The levels of CD3+, CD4+, CD4+ /CD8+, NK cells in the observation group were lower than those in the control group, and the levels of CD8+ in the observation group were higher than those in the control group (P 0.05). After operation, the levels of CD3+, CD4+, CD4+ /CD8+, NK cells in the two groups were lower and CD8+ levels were higher than those before operation. The levels of CD3+, CD4+, CD4+ /CD8+, NK cells in stage Ⅰ-Ⅱ patients were higher than those in stage Ⅲ-Ⅳ patients, and CD8+ levels were lower than those in stage Ⅲ-Ⅳ patients (P 0.05). After operation, the levels of CD8+ in the two groups were higher than those before operation, while the levels of CD3+, CD4+, CD4+ /CD8+, NK cells were lower than those before operation; The levels of CD3+, CD4+, CD4+ /CD8+, NK cells in the 4 U group, and CD8+ levels were lower than those in >4 U group (P<0.05). Conclusions Perioperative allogeneic red blood cell transfusion can affect T cell subsets and NK cell content in peripheral blood of patients with gastric cancer, and there are some differences in the effects of different clinical stages of gastric cancer patients, which can cause the suppression of cellular immune function. Key words: Stomach neoplasms; Perioperative period; Erythrocyte transfusion; T-lymphocyte subsets; Killer cells, natural

Normal Values of T, B and NK Lymphocyte Subpopulations in Peripheral Blood of Healthy Cuban Adults.

INTRODUCTION Quantification of lymphocyte subpopulations is useful for evaluating immune response in states of health and disease, including immunodeficiencies, autoimmunity, infections and cancer. Studies have found that concentrations and proportions of different cell subpopulations vary with geographic location, age, sex and ethnicity. Knowing the normal values of these cells and their variation in healthy populations will contribute to improved clinical practice and scientific research. OBJECTIVE Estimate normal absolute concentrations and percentages of the most abundant lymphocyte subpopulations in peripheral blood and their relation to sex and age. METHODS A cross-sectional analysis was conducted in 129 healthy adults, 61 men and 68 women aged 18-80 years; 89 aged <50 years and 40 ≥50 years. We included individuals who agreed to participate by written informed consent. Exclusion criteria were chronic disease, or use of tobacco, alcohol or medications that can alter immune system cell numbers and functions. Through dual platform flow cytometry, we determined absolute and percentage values for T lymphocyte subsets CD3+, CD3+/CD4+T, CD3+/CD8+T, CD19+ B cells and CD3-/CD56+ natural killer cells in peripheral blood, using an 8-color flow cytometer. We estimated medians and the 2.5 and 97.5 percentiles and calculated the Pearson correlation coefficient to evaluate associations. Significance tests were also used to compare groups. The significance threshold was p = 0.05 in all cases. RESULTS Ranges of absolute values and percentages (%) were: total lymphocytes: 1200-3475 cells/μL (20.2-49.3); CD3+ T cells: 880-2623 cells/μL (56.5-84.7); CD3+/CD4+ T cells: 479-1792 cells/μL (30.3-55.7); CD3+/CD8+ T cells: 248-1101 cells/μL (13.2-42.9); CD19+ B cells: 114-1491 cells/μL (5.4-49.5); CD3-/CD56+ natural killer cells: 70-652 cells/μL (3.7-28.0); and the CD4+:CD8+ index: 0.80-3.92. Absolute numbers--but not percentages--of lymphocytes and CD3+ T cells were higher in those <50 years (p = 0.025 and 0.020, respectively). Absolute values and relative percentages of CD3+/CD8+ and relative values of CD3+/ CD4+ T cells were significantly higher in the younger subgroup (p = 0.004 and p = 0.047). Age was not associated significantly with B lymphocytes or natural killer cells. Absolute and relative values ​​of CD3+/CD4+ T lymphocytes were significantly higher in women (p = 0.009 and 0.036, respectively). CONCLUSIONS. Absolute numbers of total lymphocytes and T and CD3+/CD8+ T lymphocytes are higher in younger individuals. In percentage values, CD3+/CD4+ T lymphocytes are lower in older persons. Absolute and percentage values ​​of CD3+/CD4+ T phenotype are higher in women. These differences justify adjusting clinical analyses to different values ​​by age and sex. KEYWORDS T lymphocytes, B lymphocytes, normal values, flow cytometry, age, sex, Cuba.

Propofol Promotes Activity and Tumor-Killing Ability of Natural Killer Cells in Peripheral Blood of Patients with Colon Cancer.

BackgroundWe investigated the effect of propofol on activities and tumor-killing ability of natural killer (NK) cells in patients with colon cancer.Material/MethodsTwenty colon cancer patients and 20 healthy subjects were included. Peripheral blood (5 ml) was collected from all patients and healthy subjects. NK cells in peripheral blood were separated by negative screening using immunomagnetic beads. Flow cytometry was used to determine expression of activated receptors, inhibitory receptors, killing effector molecules, and proliferation-associated markers on NK cell surfaces. After in vitro treatment with propofol for 24 h, expression of activated receptors, inhibitory receptors, killing effector molecules, and proliferation-associated markers on NK cell surfaces was examined again. In addition, the tumor-killing effect of NK cells was studied by co-culture with K562 cells or colon cancer SW620 cells at a ratio of 1: 1.ResultsThe number of NK cells in peripheral blood from colon cancer patients was increased compared with healthy subjects, but activities and proliferation ability of the NK cells were decreased. The tumor-killing effect of NK cells isolated from colon cancer patients was decreased. Of note, propofol promoted activation of NK cells from colon cancer patients. In addition, propofol increased expression of tumor-killing effector molecules by NK cells and the proliferation ability of NK cells. Propofol also enhanced the killing effect of NK cells on colon cancer cells.ConclusionsThe present study demonstrates that propofol promotes the activity and tumor-killing ability of NK cells in peripheral blood of patients with colon cancer.

ACTIVITY OF NATURAL KILLER CELLS IN BIOLOGICAL FLUIDS FROM PATIENTS WITH COLORECTAL AND OVARIAN CANCERS

Objective. To compare the functional activity of natural killer cells in peripheral blood and ascites from patients with different stages of colorectal and ovarian cancers and benign ovarian tumors. Material and methods. The study included 10 patients with stage IIIC ovarian cancer (FIGO, 2009), 5 patients with benign ovarian tumors (BOTs), and 15 patients with colorectal cancer (T 2–4 N 0–2 M 0 ). The control group consisted of 5 healthy donors. To evaluate the number and functional activity of NK-cells in peripheral blood and ascites, the FACS Canto II Flow Cytometer was used. Results. In peripheral blood of patients with ovarian and colorectal cancers, the relative number of activated NK-cells capable of secreting granzyme B (GB) (CD56 + CD107a + GB + PF-) was significantly lower and the proportion of degranulated NK-cells (CD56 + CD107a + GB- PF-) was higher than those of healthy donors. Low total NK-cell counts in peripheral blood were a distinctive feature of ovarian cancer patients (p < 0.05). The proportion of activated peripheral blood NK-cells, containing granules of cytolytic enzymes GB and perforin (PF) increased with tumor growth. However, lymph node metastasis in patients with colorectal cancer did not affect the level and activation of NK-cells. The comparative analysis of NK-populations in patients with benign and malignant ovarian tumors revealed that the level of CD56 + cells was significantly higher in tumor ascites compared to peripheral blood. In patients with BTs, the levels of CD56 + CD107a + and activated CD56 + CD107a + GB-PF-degranulated cells was higher in ascites than in blood. In patients with ovarian cancer, the level of degranulated cells was higher in peripheral blood than in malignant ascites. Conclusion. The tumor cells and tumor microenvironment were found to affect the number and the functional activity of NK-cells. The accumulation of free fluid within the peritoneal cavity in patients with both benign and malignant ovarian tumors resulted in significantly decreased levels of NK-cells in peripheral blood and increased levels in ascites. The differences in the functional activity of NK-cells in ascites and peripheral blood of patients with ovarian cancer require further investigation of the lymphocyte receptor status and possibly cytokine-producing function of NK-cells.

Expression of regulatory T cells and natural killer cells in peripheral blood of patients in different stages of chronic hepatitis B virus infection

Expression of regulatory T cells and natural killer cells in peripheral blood of patients in different stages of chronic hepatitis B virus infection

Abstract B56: An immuno-gene therapy clinical trial evaluating in situ vaccination of malignant pleural mesothelioma with intrapleural delivery of adenovirus-interferon-alpha-2b in combination with chemotherapy

Abstract Background: “In situ vaccination” immune-gene therapy has the ability to induce broad, polyclonal anti-tumor responses directed by the patient's own immune system using standard “off the shelf” agents. In this trial, we activated endogenous tumor immunity by injection of an adenovirus expressing a Type I interferon into the pleural space of patients with malignant pleural mesothelioma (MM). Based on preclinical data showing synergy with chemotherapy, all patients then received standard systemic cytotoxic therapy. Methods: Two doses of intrapleural administration of a replication-defective recombinant adenoviral vector containing the human interferon-alpha (hIFN-α2b) gene at a dose of 3x1011 viral particles were given concomitant with a 14-day course of high-dose cyclo-oxygenase-2 (COX-2) inhibitor (Celecoxib) to reduce side effects and to modify the tumor microenvironment by decreasing PGE- 2 levels. This was followed by standard first-line or second-line chemotherapy agents. Primary outcome measures were safety, overall best response rate, and survival. Bio-correlates were measured. Results: Forty patients were treated in this study: 18 patients received first-line Pemetrexed-based chemotherapy; 7 patients who had previously received front-line Pemetrexed-based chemotherapy &amp;gt;6 months prior to enrollment received Pemetrexed-based second-line chemotherapy. Additionally, in the second-line chemotherapy arm, fifteen patients (n=15) received gemcitabine-based chemotherapy. Treatment was well tolerated and adverse events were comparable to historical controls. Follow-up chest CT scans demonstrated an overall response rate of 20% by Modified RECIST criteria and disease control rate (DCR) of 85% (partial and complete responses plus stable disease) at initial follow-up scan after the first two cycles of chemotherapy. Encouragingly, median survival for all patients with epithelial histology (including both first and second line) was 26 months (95% CI: 15-ND); median overall survival (MOS) for patients with non-epithelial histology (both first and second line) was 6.5 months (95% CI: 5.50 – ND). [See figure] Historical MOS with first line chemotherapy alone is 13.3 months. No clear predictors for response were identified including: baseline immunologic parameters (i.e. activated T cells or number of regulatory T cells); the presence of the immune-gene signature in their biopsies; peak levels of interferon in blood or pleural fluid; or induction of anti-tumor antibodies, activated T cells, or natural killer cells in peripheral blood. Conclusions: The combination of intrapleural Ad.IFN-α2b vector, Celecoxib, and systemic chemotherapy proved safe in patients with unresectable malignant pleural mesothelioma. Overall survival rates were significantly higher than historical controls, particularly in the second-line groups. The results of this study support proceeding with a multi-center randomized clinical trial of chemo-immunogene therapy versus standard chemotherapy alone. Citation Format: D. H. Sterman, E. Alley, J. Friedberg, S. Metzger, J. Stevenson, E. Moon, A. R. Haas, A. Vachani, S. I. Katz, G. Cheng, J. Sun, D. F. Heitjan, L. Litzky, K. Cengel, C. B. Simone, II, M. Culligan, M. Culligan, S. M. Albelda. An immuno-gene therapy clinical trial evaluating in situ vaccination of malignant pleural mesothelioma with intrapleural delivery of adenovirus-interferon-alpha-2b in combination with chemotherapy. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B56.

Abstract P5-04-14: Decreased functions of natural killer cells in peripheral blood of advanced triple negative breast cancer patients

Abstract Background : Natural killer (NK) cells are a major player in innate immune response. Two distinct NK cell subsets with different functions as the strongest cytotoxic activity (CD56dimCD16+) or cytokine production (CD56brightCD16+/-) have been known. In this study, we evaluated the phenotypic and functional difference of NK cells in peripheral blood (pB) from the patients who had advanced triple negative (TN) breast cancer compared to healthy controls. Methods : We enrolled advanced triple negative (TN) breast cancer patients treated at the National Cancer Center, Korea between March 2012 and March 2014. Healthy controls who visited for health screening were recruited at the same time period and peripheral blood (pB) samples were collected. In patient groups, pB was sampled either prior to the initiation or during the courses of chemotherapy. Then, patients were retrospectively classified into two groups as follows; non-responders (the group A) and responders (the group B) to the treatment. NK cells were isolated from peripheral blood mononuclear cells (PBMC) and analyzed based on their expression of CD56 and CD16. We looked at cytotoxic activity of pB NK cells against K562 cell line and measured CD107a expression as a parameter of NK cell activation. Results : The absolute numbers of lymphocytes and NK cells per cubic millimeter of pB were similar among healthy controls (N=24), the group A (N=16), and the group B (N=21). There was no significant difference in the proportion of pB NK cell subsets among three groups except CD56brightCD16+/- subset which was significantly higher in the group B compared to healthy controls (P=0.0014). Even in the presence of similar proportions of NK subsets, the activities of CD56dimCD16+ NK cells measured by CD107a expression were significantly lower both in the group A (P=0.0023) and in the group B (P=0.0127) compared with those of control group. The activities of CD56dimCD16+ NK cells were not different between group A and B. In the case of CD56brightCD16+/- NK cells, CD107a positivity was significantly lower in the group A compared with controls (P=0.0068) and group B (P=0.0465). Conclusions : Our data suggested that cytolytic functions of NK cells were significantly decreased in advanced TN breast cancer patients compared with those of healthy controls despite a similar proportion of NK cell subsets in pB. Citation Format: In Hae Park, Sun-Young Kong, Joo Hyun Kang, Hye Jin Mo, Tae Sik Kim, Keun Seok Lee, Jungsil Ro. Decreased functions of natural killer cells in peripheral blood of advanced triple negative breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-14.

The decrease in NKG2D+ Natural Killer cells in peripheral blood of patients with metastatic colorectal cancer.

Natural killer(NK)cellsplay importantrolesin the immune defenseagainst tumors such as colorectal cancer. In humans, NKG2D is an activating immune receptor constitutively expressed in most cytotoxic lymphocytes including NK and CD8+ T cells. In this study, the expression of NKG2D molecule was investigated in peripheral blood NK cells from colorectal cancer patients and compared with healthy subjects. We studied 21 non-metastatic (low-grade), 17 non-metastatic (high-grade), 16 metastatic colorectal cancer patients, and 24 healthy controls. Peripheral blood samples were obtained to isolate peripheral blood mononuclear cells (PBMCs) and the percentage of peripheral blood NKG2D+CD3-CD56+ NK cells was analyzed by flow cytometry. The expression of NKG2D at mRNA level was also measured by real-time PCR in both, patients and control subjects. The results showed a significant reduction in the percentage of NKG2D+NK cells as well as NKG2D mRNA expression in peripheral blood of metastatic colon cancer patients. This result suggests that decreased expression of activatingNKG2Dreceptor in metastatic colorectal cancer might compromise NK cell function and allow tumor to evade immunity (Tab. 3, Fig. 4, Ref. 33).

In vitro functions of natural killer cells in peripheral blood of patients with severe aplastic anemia

To observe the in vitro killing functions of natural killer (NK) cells for K562 cells in peripheral blood of severe aplastic anemia (SAA) patients and explore the role of NK cells in the immunological pathogenesis of SAA. NK cells (CD3(-)CD56(+)CD16(+)) as effector cells were sorted from peripheral blood mononuclear cells (PBMNC) of 12 SAA patients and 10 normal controls by magnetic activated cell sorting system (MACS)from June 2013 to August 2014. K562 cells as target cells were co-cultured with the effector cells (1: 1, 1: 5, 1: 10). Using Annexin V and PI double staining markers, the apoptotic rate of K562 cells were measured by flow cytometry after culturing. The apoptotic rate of K562 cells in SAA patients was (10.30% ± 6.08%) according to the ratio of effector-target (1: 1). And it was significantly higher than that in normal controls (6.30% ± 3.46%) (P < 0.05). The apoptotic rate of K562 cells in SAA patients were 10.30% ± 6.08%, 16.47% ± 8.29% and 25.45% ± 9.88% respectively according to the ratio of effector-target(1: 1, 5: 1, 10: 1). The higher ratio of effector-target, the higher apoptotic rate of K562 cells. There were statistic differences among three groups (P < 0.05). There are enhanced in vitro functions of NK cells in peripheral blood of SAA patients NK cells may play an important role in abnormal immune tolerance mechanisms of SAA patients.

Expansion of natural killer cells in peripheral blood in a Japanese elderly with human T-cell lymphotropic virus type 1-related skin lesions.

Natural killer (NK) cells were proposed to play an important role in the pathogenesis of human T-cell lymphotropic virus type 1- (HTLV-1-) associated neurologic disease. Our patient was a 77-year-old Japanese man, who had been treated for infective dermatitis associated with HTLV-1 for nearly 10 years. When referred to us, he had facial eczema/edema as well as extensive dermatitis at the neck/upper chest and nuchal area/upper back regions. Dermal lesions had CD3+CD4+ cells, but no NK cells. Flow cytometry of his peripheral blood showed a phenotype of CD2+ (97%), CD3+ (17%), CD4+ (12%), CD7+ (94%), CD8+ (6%), CD11c+ (70%), CD16+ (82%), CD19+ (0%), CD20+ (0%), CD56+ (67%), HLA-DR+ (68%), and NKp46+ (36%). Absolute numbers of CD56+NK cells in the peripheral blood were in a range of 986/μL–1,270/μL. The expanded NK cells in the peripheral blood are considered to be reactive, to maintain the confinement of the HTLV-1-positive CD4+ cells in the skin, and to prevent the progression of the disease.

Patients With Endometriosis of the Rectosigmoid Have a Higher Percentage of Natural Killer Cells in Peripheral Blood

Study ObjectiveTo estimate the concentration of natural killer (NK) cells in the peripheral blood in patients with and without endometriosis. DesignCase-control study (Canadian Task Force classification II-2). SettingTertiary referral hospital. PatientsOne hundred fifty-five patients who had undergone videolaparoscopy were divided into 2 groups: those with endometriosis (n = 100) and those without endometriosis (n = 55). InterventionsThe percentage of NK cells relative to peripheral lymphocytes was quantified at flow cytometry in 155 patients who had undergone laparoscopy. In addition to verifying the presence of endometriosis, stage of disease and the sites affected were also evaluated. Measurements and Main ResultsThe mean (SD) percentage of NK cells was higher (15.3% [9.8%]) in patients with endometriosis than in the group without the disease (10.6% [5.8%]) (p < .001). The percentage of NK cells was highest (19.8 [10.3%]) in patients with advanced stages of endometriosis and in those in whom the rectosigmoid colon was affected. In a statistical model of probability, the association of this marker (NK cells ≥11%) with the presence of symptoms such as pain and intestinal bleeding during menstruation and the absence of previous pregnancy yielded a 78% likelihood of the rectosigmoid colon being affected. ConclusionCompared with patients without endometriosis, those with endometriosis demonstrate a higher concentration of peripheral NK cells. The percentage of NK cells is greater, primarily in patients with advanced stages of endometriosis involving the rectosigmoid colon. Therefore, it may serve as a diagnostic marker for this type of severe endometriosis, in particular if considered in conjunction with the symptoms.

Effective Melanoma Immunotherapy with Interleukin-2 Delivered by a Novel Polymeric Nanoparticle

Interleukin-2 (IL-2) has been shown to possess antitumor activity in numerous preclinical and clinical studies. However, the short half-life of recombinant IL-2 protein in serum requires repeated high-dose injections, resulting in severe side effects. Although adenovirus-mediated IL-2 gene therapy has shown antitumor efficacy, the host antibody response to adenoviral particles and potential biosafety concerns still obstruct its clinical applications. Here we report a novel nanopolymer for IL-2 delivery, consisting of low molecular weight polyethylenimine (600 Da) linked by β-cyclodextrin and conjugated with folate (named H1). H1 was mixed with IL-2 plasmid to form H1/pIL-2 polyplexes of around 100 nm in diameter. Peritumoral injection of these polyplexes suppressed the tumor growth and prolonged the survival of C57/BL6 mice bearing B16-F1 melanoma grafts. Importantly, the antitumor effects of H1/pIL-2 (50 μg DNA) were similar to those of recombinant adenoviruses expressing IL-2 (rAdv-IL-2; 2 × 10(8) pfu). Furthermore, we showed that H1/pIL-2 stimulated the activation and proliferation of CD8+, CD4+ T cell, and natural killer cells in peripheral blood and increased the infiltration of CD8+, CD4+ Tcells, and natural killer cells into the tumor environment. In conclusion, these results show that H1/pIL-2 is an effective and safe melanoma therapeutic with an efficacy comparable to that of rAdv-IL-2. This treatment represents an alternative gene therapy strategy for melanoma.

Foxp3high and Foxp3low Treg cells differentially correlate with T helper 1 and natural killer cells in peripheral blood

Regulatory T (Treg) cells interact with B, natural killer (NK), and dendritic cells in addition to other T cells. In this study, we aimed at determining whether Foxp3+ T cells and subpopulations have any correlation with other lymphocyte subsets and their functions in a systemic immune environment. Peripheral blood was drawn from 22 nonpregnant healthy women. T, B, and NK cell subpopulations were measured by immunophenotype analysis. Intracellular Foxp3, cytokine expression (tumor necrosis factor-α [TNF-α], interferon-γ [IFN-γ], and interleukin-10 [IL]-10), and NK-cell cytotoxicity were analyzed by flow cytometric analysis. Correlations between Foxp3+ T cells and other immune variables were analyzed under control of age and menstrual phases. Foxp3+, Foxp3low, and CD4+Foxp3+ cells significantly correlated with CD4+CD25+, CD4+CD25dim, and CD4+CD25bright cells. Foxp3+, Foxp3low, and CD4+Foxp3+ cells positively correlated with CD3+ and CD3+CD4+ T cells, but negatively correlated with CD3−CD56+ and CD3−CD56dim NK cells. CD4+Foxp3high Treg cells were positively correlated with CD3+CD4+TNF-α+ (p = 0.014) and negatively correlated with CD3+CD8+IL-10+ T cells (p = 0.001). The ratio of type 1/2 cytokine-producing CD3+CD8+ cells demonstrated a positive correlation with CD4+Foxp3high cells (p ≤ 0.01). CD8+Foxp3+ cells were positively correlated with CD3+CD4+IL-10+ cells (p = 0.007) and negatively correlated with CD3+CD8+TNF-α+ cells (p = 0.008). In conclusion, each Foxp3+ Treg cell subpopulation has unique immune interaction, which controls particular subsets of lymphocytes.

Natural Killer Cells in Peripheral Blood and Lung Tissue Are Associated With Chronic Rejection After Lung Transplantation

Natural killer (NK) cells have the capacity to recognize and respond to alloantigen, yet their role in lung transplant rejection is not well defined. The aim of this study was to correlate NK cell numbers and immunophenotype in peripheral blood and tissue with graft function after lung transplantation. NK cell subsets were immunophenotyped in peripheral blood (n = 41). Lung tissue was stained for NK cells via CD16 and morphologic assessment (n = 30). Peripheral blood NK cells were activated in patients with chronic rejection, but the overall number of cells was lower in these patients when compared with stable patients. Furthermore, there was significantly more CD16(+) NK cells in the lung compartment of patients with bronchiolitis obliterans syndrome compared with stable patients (p = 0.001). In patients with chronic rejection, peripheral blood NK cells are activated but their numbers decrease, while the number of NK cells in the lungs increases. This suggests NK cells systemically activate and migrate to the lung during the progression of chronic rejection after lung transplantation.

Severe combined immunodeficiency caused by a splicing abnormality of the CD3? gene

CD3delta deficiency is a recently identified rare form of severe combined immunodeficiency. We analysed the CD3delta gene in a Japanese family with severe combined immunodeficiency. The patients lacked T-cells with normal numbers of B-cells and natural killer cells in peripheral blood. We found a novel homozygous mutation in the splicing acceptor site of intron 2 (IVS2-2A --> G) in these patients. Analysis of patients' mononuclear cells revealed the CD3delta splicing abnormality. Chest X-ray films and computed tomography revealed small sized thymuses in these patients. The CD3delta gene should be analysed in patients with severe combined immunodeficiency lacking T-cells with normal B- and natural killer cells irrespective of the thymus size.

Immune responses to HTLV-I(ACH) during acute infection of pig-tailed macaques.

Human T cell lymphotropic virus type 1 (HTLV-I) is causally linked to adult T cell leukemia/lymphoma (ATL) and a chronic progressive neurological disease, HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A nonhuman primate model that reproduces disease symptoms seen in HTLV-I-infected humans might facilitate identification of initial immune responses to the virus and an understanding of pathogenic mechanisms in HTLV-I-related disease. Previously, we showed that infection of pig-tailed macaques with HTLV-I(ACH) is associated with multiple signs of disease characteristic of both HAM/TSP and ATL. We report here that within the first few weeks after HTLV-I(ACH) infection of pig-tailed macaques, serum concentrations of interferon (IFN)-alpha increased and interleukin-12 decreased transiently, levels of nitric oxide were elevated, and activation of CD4(+) and CD8(+) lymphocytes and CD16(+) natural killer cells in peripheral blood were observed. HTLV-I(ACH) infection elicited virus-specific antibodies in all four animals within 4 to 6 weeks; however, Tax-specific lymphoproliferative responses were not detected until 25-29 weeks after infection in all four macaques. IFN-gamma production by peripheral blood cells stimulated with a Tax or Gag peptide was detected to varying degrees in all four animals by ELISPOT assay. Peripheral blood lymphocytes from one animal that developed only a marginal antigen-specific cellular response were unresponsive to mitogen stimulation during the last few weeks preceding its death from a rapidly progressive disease syndrome associated with HTLV-I(ACH) infection of pig-tailed macaques. The results show that during the first few months after HTLV-I(ACH) infection, activation of both innate and adaptive immunity, limited virus-specific cellular responses, sustained immune system activation, and, in some cases, immunodeficiency were evident. Thus, this animal model might be valuable for understanding early stages of infection and causes of immune system dysregulation in HTLV-I-infected humans.

Natural killer cells in human peripheral blood and primary cutaneous natural killer cell lymphomas may express cutaneous lymphocyte antigen.

In normal human peripheral blood, cutaneous lymphocyte antigen is expressed by memory T cells, suggesting a specific tissue-homing population of T cells. In this study it is demonstrated that 6% of CD56 + natural killer cells in peripheral blood also express cutaneous lymphocyte antigen (CLA). It was also detected that most of the tumor cells in primary cutaneous nasal-type natural killer cell lymphomas were CLA-positive, whereas primary nasal natural killer cell lymphomas were CLA-negative. Although natural killer cells traditionally are known to be non-specific immune cells without antigen specificity and little is known about the role of natural killer cells in skin diseases, the results of this study suggest the existence of a subset of skin-associated CLA+ CD56+ natural killer cells. These natural killer cells may be related to the pathogenesis of primary cutaneous natural killer cell lymphomas.