Allogeneic hematopoietic stem cell transplantation for children with aggressive natural killer cell leukemia: one case report with a literature review / 中华器官移植杂志

Objective:To summarize the clinical features, treatments and prognoses of aggressive natural killer cell leukemia (ANKL) in children.Methods:Clinical data and follow-up results were retrospectively reviewed for one hospitalized case of ANKL in June 2019.Through a literature search, the relevant items were retrieved from the databases of China National Knowledge Infrastructure, WanFang and PubMed using the Chinese and English keywords of "aggressive natural killer cell leukemia" and "children" up to December 2021.Results:This 8-year-old girl was diagnosed with ANKL by flow cytometric immunophenotype and immunohistochemical stain.Fever was the initial manifestation accompanied by sallow complexion, fatigue, enlargement of liver, spleen and lymph node and hematopenia of three lines.Allogeneic hematopoietic stem cell transplantation (allo-HSCT) was performed after chemotherapy.As of April 2022, the child stayed in a disease-free survival state after follow-ups for over 2 years.The literature search finally yielded 7 eligible Chinese and 10 English reports with a total of 17 pediatric ANKLs.In this group, there were fever (n=15), rash (n=1), perineal mass (n=1) and diarrhea, vomiting and other digestive tract symptoms (n=1). Six cases were misdiagnosed during an early stage of disease.4 cases received chemotherapy alone, 3 cases received chemotherapy regimen for acute lymphoblastic leukemia, 1 child died and one death occurred after received chemotherapy regimen of "cisplatin + vincristine + doxorubicin + ifosfamide". Allo-HSCT was performed in 5 patients after remission with chemotherapy and one child died from multiple organ failure at 9 months after allo-HSCT.Nine cases gave up treatment.Conclusions:ANKL has a rapid disease progression, diverse clinical manifestations, easy misdiagnosis and poor prognosis.For suspected ANKL cases, clinicians perform multiple bone perforations at multiple sites and immunophenotype by flow cytometry as soon as possible to confirm the diagnosis.Currently allo-HSCT offers a long-term survival of ANKL patients.

A biomimetic liver cancer on-a-chip reveals a critical role of LIPOCALIN-2 in promoting hepatocellular carcinoma progression

Hepatic stellate cells (HSCs) represent a significant component of hepatocellular carcinoma (HCC) microenvironments which play a critical role in tumor progression and drug resistance. Tumor-on-a-chip technology has provided a powerful in vitro platform to investigate the crosstalk between activated HSCs and HCC cells by mimicking physiological architecture with precise spatiotemporal control. Here we developed a tri-cell culture microfluidic chip to evaluate the impact of HSCs on HCC progression. On-chip analysis revealed activated HSCs contributed to endothelial invasion, HCC drug resistance and natural killer (NK) cell exhaustion. Cytokine array and RNA sequencing analysis were combined to indicate the iron-binding protein LIPOCALIN-2 (LCN-2) as a key factor in remodeling tumor microenvironments in the HCC-on-a-chip. LCN-2 targeted therapy demonstrated robust anti-tumor effects both in vitro 3D biomimetic chip and in vivo mouse model, including angiogenesis inhibition, sorafenib sensitivity promotion and NK-cell cytotoxicity enhancement. Taken together, the microfluidic platform exhibited obvious advantages in mimicking functional characteristics of tumor microenvironments and developing targeted therapies.

The effects of yogurt consumption on immune function in university male track and field athletes -A randomized, double-blind, placebo-controlled, parallel-group study- / 体力科学

This study aimed to evaluate the effects of ingesting yogurt fermented with Lactobacillus delbrueckii subsp. bulgaricus (OLL1073R-1) on the immune function of healthy university men track and field athletes. Study design Randomized, double-blind, placebo-controlled, parallel-group study. A total of 37 track and field athletes aged ≥18 years were randomly assigned into two groups. For 2 weeks, two bottles of yogurt fermented with OLL1073R-1 and Streptococcus thermophilus OLS3059 or placebo sour milk were ingested daily to the participants. During the intake period, a 1-week training camp was held and participants were subjected to strenuous exercise. Natural killer (NK) cell activity, which is the primary endpoint, was significantly lower in the placebo group after ingestion than that at baseline; however, it remained unchanged during the pre-exercise level of the yogurt group. The two-way repeated measures analysis of variance showed an interaction effect in the NK cell activity change (P=0.018) and a significant difference between the groups after the 2-week ingestion (P=0.015). Among the secondary endpoints, cytokines and chemokines levels involved in activating innate immunity maintained or enhanced only in the yogurt group. ALT, LDH, and CK significantly elevated only in the placebo group. Furthermore, amino acid levels were significantly lower in the placebo group after ingestion than that at baseline; however, it remained unchanged during the pre-exercise level in the yogurt group. Consuming yogurt fermented with OLL1073R-1 prevents the decline in immune function associated with strenuous exercise. Additionally, the yogurt may contribute to stable physical condition.

Clinical Characteristics of Aggressive NK-Cell Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explore and summarize the clinical characteristics and treatment of aggressive NK-cell leukemia (ANKL), and provide new insights for clinical diagnosis and treatment of this disease.@*METHODS@#The clinical data of 7 patients with ANKL admitted to the First Affiliated Hospital of Wannan Medical College from March 2014 to July 2021 were retrospectively analyzed, and their clinical characteristics, laboratory and imaging results, treatment and outcomes were analyzed.@*RESULTS@#Among the 7 patients, 5 were males and 2 were females, with a median age of 47 (33-69) years old. The morphology of bone marrow cells in 7 patients showed similar large granular lymphocytes. Immunophenotyping revealed abnormal NK cells in 5 cases. By the end of follow-up, 6 cases died and 1 case survived, with a median survival time of 76.9 (4-347) days.@*CONCLUSION@#ANKL is a rare disease with short course and poor prognosis. If combined with hemophagocytic syndrome (HPS), the prognosis is even worse. There is no unified treatment method at present, and the use of PD-1 inhibitors may prolong the survival in some patients.

Research Progress of Chimeric Antigen Receptor Modified NK Cells in the Treatment of Acute Myeloid Leukemia --Review / 中国实验血液学杂志

Chimeric Antigen Receptor (CAR) is a research hotspot in the field of cellular immunotherapy in recent years, and CAR-T cells therapy are developing rapidly in hematological malignant tumors, but their clinical application is still limited by related risks. It is particularly important to find more optimized immunoreactive cells. Natural killer (NK) cells, as key effector cells of innate immunity, can kill tumor or infected cells quickly without prior sensitization. Autologous or allogeneic NK cell infusion has become an effective cell therapy for acute myeloid leukemia (AML). CAR-NK cells combine the advantages of CAR targeting tumor specific antigens and enhancing immune cells activity with the innate antitumor function of NK cells to enhance the targeting and lytic activity of NK cells to AML primordial cells. At present, most of the CAR-NK treatments for AML are still in the stage of specific target screening and verification. This article reviews the latest research progress of CAR-NK cell therapy in the field of AML therapy.

Screening of immune cell activators from Astragali Radix using a comprehensive two-dimensional NK-92MI cell membrane chromatography/C18 column/time-of-flight mass spectrometry system / 药物分析学报

Astragali Radix(AR)is a clinically used herbal medicine with multiple immunomodulatory activities that can strengthen the activity and cytotoxicity of natural killer(NK)cells.However,owing to the complexity of its composition,the specific active ingredients in AR that act on NK cells are not clear yet.Cell membrane chromatography(CMC)is mainly used to screen the active ingredients in a complex system of herbal medicines.In this study,a new comprehensive two-dimensional(2D)NK-92MI CMC/C18 column/time-of-flight mass spectrometry(TOFMS)system was established to screen for potential NK cell acti-vators.To obtain a higher column efficiency,3-mercaptopropyltrimethoxysilane-modified silica was synthesized to prepare the NK-92MI CMC column.In total,nine components in AR were screened from this system,which could be washed out from the NK-92MI/CMC column after 10 min,and they showed good affinity for NK-92MI/CMC column.Two representative active compounds of AR,isoastragaloside Ⅰ and astragaloside Ⅳ,promoted the killing effect of NK cells on K562 cells in a dose-dependent manner.It can thus suggest that isoastragaloside Ⅰ and astragaloside Ⅳ are the main immunomodulatory compo-nents of AR.This comprehensive 2D NK-92MI CMC analytical system is a practical method for screening immune cell activators from other herbal medicines with immunomodulatory effects.

Research advances on chimeric antigen receptor cellular therapy for hepatocellular carcinoma / 中华肝胆外科杂志

Hepatocellular carcinoma is one of the most common cancers and causes of cancer-related death in the world, the insidious onset, rapid progression and poor prognosis make the treatment more difficult. At present, the current therapeutic options, include surgical resection, ablation, postoperative recurrenceare still with disadvantages. The efficacy of targeted drug therapy is also unsatisfactory. Immunotherapy is a promising research direction. Immunosuppressants at the molecular level have shown initial success, while adoptive immunocell therapy at the cellular level has also shown promising results, the typical example is chimeric antigen receptor cell therapy. The purpose of this review is to summarize the recent research progress on chimeric antigen receptor cellular therapy in liver cancer.

Jinfukang Inhibits Lung Cancer Metastasis by Regulating Immune Senescence / 肿瘤防治研究

Objective To explore the effect of immune senescence on lung cancer metastasis and reveal the mechanism of Fuzheng traditional Chinese medicine Jinfukang in the prevention and treatment of the metastasis. Methods A lung metastasis model of Lewis lung cancer cells was established in C57BL/6 mice with different ages (15 months, 6 months, and 2 months). Mice in the 6-month-old group were given Jinfukang intragastrically for 42 days. Pulmonary metastasis was analyzed by in vivo imaging, anatomical microscopic observation, and HE staining. The proportion of memory T cells and NK cells in peripheral blood was detected by flow cytometry. Results The lung metastatic tumor formation rate of 15-month-old and 6-month-old mice was significantly higher than that of 2-month-old mice (all P < 0.05). Abundance of CD4+T cells in peripheral blood was positively correlated with age (2-month-old vs. 6-month-old, P=0.041; 2-month-old vs.15-month-old, P=0.041; 6-month-old vs.15-month-old, P=0.953). The abundance of NK cells was negatively correlated with age (2-month-old vs. 6-month-old, P=0.009; 2-month-old vs.15-month-old, P=0.009; 6-month-old vs. 15-month-old, P=0.574). However, the survival time of mice in the Jinfukang group was longer (P > 0.05) and the level of NK cells in peripheral blood was significantly higher (P=0.029) than those in the normal saline group. Conclusion Immune senescence can promote the metastasis of lung cancer. The prolongation of the survival time of mice administered with Jinfukang may be related to delaying immune senescence and increasing the number of NK cells.

Progress of immune cell reconstitution after allogeneic hematopoietic stem cell transplantation / 国际儿科学杂志

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective cure for many hematologic and non-hematologic diseases.However, infection morbidity and mortality remain high after allo-HSCT, which is closely related to the recovery of immune cell subsets.The detailed information on the recovery of natural killer(NK), T and B cell subsets can better predict and regulate the occurrence of adverse events.This article will review the current rules, influencing factors and the relationship between the outcome and the immune reconstitution of NK cells, T cells and B cells after hematopoietic stem cell transplantation.

EBER + extranodal NK/T lymphoma in patient with idiopathic CD4 lymphocytopenia / Linfoma T/NK EBER positivo en paciente con linfocitopenia CD4 idiopática

Abstract Idiopathic CD4 lymphocytopenia (ICL) not related to HIV is an infrequent and severe condition with no etiology defined until now. The concomitant presence of an underlying disease, especially an oncohematological process, could be related to the immune physiopathology and the development of the im munosuppressive state. On the other hand, Epstein Barr virus is a well-known oncogenic pathogen described in the development of several types of lymphoma which might be reactivated in the ICL. There is still no specific treatment for this syndrome, so the therapeutic scope for these patients is the treatment of opportunistic diseases and the administration of specific antimicrobials as prophylaxis. We present a patient with an uncommon asso ciation of an ICL and an extranodal T/NK lymphoma with detection of VEB nuclear RNA by in situ hybridization (EBER). Diagnosis was challenging which led the health team to carry out many studies over several months

Resumen La linfocitopenia CD4 idiopática (ICL) no relacionada al HIV es una condición grave e infrecuente sin una etiología aún definida. La presencia de una enfermedad subyacente, especialmente un proceso oncohematológico, podría tener relación en la fisiopatología del proceso inmunológico. Por otro lado, el virus Epstein Barr (VEB) es bien conocido por ser un patógeno oncogénico descrito en el desarrollo de diversos tipos de linfomas, el cual podría ser reactivado en estados de inmunosupresión severa. No existe aún un tratamiento específico para este síndro me, por lo que el objetivo terapéutico en estos pacientes radica en el manejo profiláctico y activo de las distintas enfermedades oportunistas ante las cuales son susceptibles. Se presenta un paciente con un déficit grave de linfocitos CD4 de causa idiopática, y un diagnóstico posterior de linfoma T/NK extraganglionar con detección de RNA nuclear de VEB por hibridización in situ (EBER), una asociación poco descrita en la literatura médica.

Adult suspected primary hemophagocytic syndrome complicated with aggressive natural killer cell leukemia: report of one case and review of literature / 白血病·淋巴瘤

Objective:To improve the understanding of adult primary hemophagocytic syndrome (HPS) with aggressive natural killer cell leukemia (ANKL).Methods:The clinicopathological data of one adult patient with suspected primary HPS complicated with ANKL in Huiqiao Medical Center, Nanfang Hospital of Southern Medical University in October 2017 were retrospectively analyzed, and literatures were reviewed.Results:A 21-year-old male patient presented with persistent fever, hemocytopenia, splenomegaly, low fibrinogen, a significant increase in ferritin, hemophagocytes in bone marrow, decreased natural killer (NK) cell activity, and increased soluble CD25. Flow cytometry detection showed that the expression of NK cells was abnormal, and there were familial lysosomal trafficking regulator (LYST) and UNC13D gene defects. He was suspected of primary HPS complicated with ANKL. The patient was given 4 courses of EPOCH+PEG-Asp (etoposide, dexamethasone, vindesine, cyclophosphamide, doxorubicin hydrochloride liposome, pegaspargase) regimen chemotherapy, 20 mg of citalopidine twice a week maintenance therapy and matched unrelated hematopoietic stem cell transplantation. After 35 months of follow-up, he got sustained remission.Conclusions:Even if there are secondary causes of adult HPS, it is necessary to screen out related genes to avoid misdiagnosis. HPS patients with ANKL progress rapidly, and the early mortality is high. EPOCH+ PEG-Asp regimen induction therapy and allogeneic hematopoietic stem cell transplantation should be used as early as possible after diagnosis.

Chimeric antigen receptor-modified immune cells and their application in tumor immunotherapy / 中国肿瘤生物治疗杂志

@# [Abstract] Chimeric antigen receptor-modified T cells (CAR-T cells) refers to the products after transferring genetic material with specific antigen recognition domain and T cell activation signal domains into T cells by gene modification technology, thus rendering the modified T cells activated by binding directly to specific antigens on the surface of tumor cells in the CAR-T cell therapy. In recent years, CAR-T cells have achieved remarkable results in the treatment of hematological diseases, bringing new hope to patients with hematological tumors. CAR-T cell therapy has become one of the most promising tumor immunotherapies, and CAR-T cells have become a hot spot for research and development by major companies. However, due to the side effects such as cytokine storm and poor treatment effect on solid tumors, the clinical application of CAR-T cells still faces challenges. In addition to traditional T cells, other immune cells are being explored for the application of CAR, for example, modifying immune cells such as NK cells, γδT cells, NKT cells, and macrophages to improve the effectiveness of these immune cells in killing tumors, and simultaneously reduce the adverse reactions caused by CAR-T cell immunotherapy. This review compares the advantages and disadvantages of different CAR-modified immune cells in tumor therapy and provides new ideas and enlightenments for the clinical development and application of CAR-modified immune cells in tumor immunotherapy.

Application of human IL-15 transgenic NCG mouse in preclinical evaluation of CAR-NK cell therapy for tumor treatment / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To explore the application value of human IL-15 transgenic NCG mice (NCG-hIL-15 mice) in preclinical evaluation of chimeric antigen receptor modified NK (CAR-NK) cell therapy for tumor treatment. Methods: qPCR and WB were performed to detect the expression of human IL-15 in the bone marrow and main organs (spleen, liver, lung, kidney and pancreas) of transgenic mice. After being transfused with human PBMC-derived NK (PB-NK) cells, the NCG-hIL-15 mice and control NCG mice were continuously monitored for the in vivo amplification of NK cells and the changes in body weight and survival time. Flow cytometry was used to detect the differential expressions of activated receptors and inhibitory receptors in amplified NK cells. WB was used to detect the expressions of perforin and granzyme-B. NCG-hIL-15 mice or NCG mice bearing MIAPaca-2 cell transplanted tumor were treated with anti-MUC1-CAR-NK cell reinfusion; then, the CAR-NK cell survival in different groups of mice was detected by Flow cytometry, and the survival time of tumor bearing mice was recorded and tumor growth was detected by in vivo imaging. Results: The results indicated that PB-NK cells could proliferate stably within 10 weeks in NCG-hIL-15 mice without obvious graft versus host diseases (GVHD) during the observation period. The in vivo-expanded human NK cells maintained the original expression patterns of various surface molecules, including KARs and KIRs. Compared with the NK cells in NCG mice, the NK cells in NCG-hIL-15 mice contained significantly higher amounts of granzyme-B and perforin (all P<0.05). CAR-NK cells showed significantly increased survival rate and stronger tumor-inhibitory effect in NCG-hIL-15 mice as compared with those in control NCG mice, resulting in significantly prolonged survival in NCG-hIL-15 mice (all P<0.01). Conclusion: NCG-hIL-15 mouse model has potential application value in preclinical trial and biological evaluation of NK cell-based immunotherapy.

Evaluation of the Effect of Lentinula Edodes Mycelia Extract on Immune Function in Healthy Subjects Placebo-controlled, Parallel-group Study / 日本補完代替医療学会誌

In this study, we examined the effects of Lentinula edodes mycelia (LEM) on fatigue and immune function in healthy subjects. 22 people were randomly assigned in a 1:1 ratio to the LEM ingestion group and the placebo group. Daily oral ingestion of LEM for 4 weeks (600 mg/day) significantly improved the VAS fatigue score compared with before treatment (p<0.01). Additionally, NK cell activity was increased by ingestion of LEM (p<0.01) and was higher level compared to the placebo group (p<0.05). These results indicate that oral ingestion of LEM may improve the feeling of fatigue and immune function.

Nanocarrier-mediated PiggyBac transposon system for preparation of CAR-NK cells / 中国肿瘤生物治疗杂志

@#Objective: To explore the gene transduction method of chimeric antigen receptor (CAR) mediated by novel cationic polymer nanocarrier mPEG-P (Asp-AED-g-HFB) (PAEF) and PigyBac transposon system to modify natural killer (NK) cells, providing a new strategy for immunotherapy of cancer cells. Methods: PAEF/DNA (transposase+transposon) complex were prepared. The particle size distribution and surface potential of PAEF/DNA complexes were measured with Nano-ZSE Dynamic Light Scattering System (Malvern Instruments). The DNA encapsulation rate, release and stability of PAEF were evaluated by DNA gel electrophoresis, and then by combiningwithparticlesizeandsurfacepotentialtodeterminethepreferentialN/PratiotoenterNKcells.Thecell cytotoxicity of PAEF/DNA complexes under different N/P ratios was analyzed by CCK-8 cytotoxicity test. Transduction efficiency of NK cells was evaluated by Fluorescence microscopy and Flow cytometry, and the feasibility of PAEF gene transfection vectors was assessed. Results: PAEF could encapsulate DNA to form nano-complexes with the diameter of 100-150 nm, which was suitable to mediate DNA entering into cells. PAEF could completely encapsulate DNA with N/P ratio of 20. In the presence of reducing agent dithiothreitol (DTT), PAEF had a good ability to release DNA. NK-92 cells transfected with PAEF/DNA complex, which was formed at the N/P ratio of 80, attained a significantly higher cell viability than cells of lipofectamine transfection group [(72.50±3.9)% vs (64.03±1.8)%, P<0.05]; Fluorescence microscopic observation showed more fluorescence and higher fluorescence intensity in cells of PAEF/DNA group; Flow cytometry showed the highest transfection efficiency of 83.4%. Conclusions: Nanocarrier PAEF can encapsulate DNA well by electrostatic adsorption, and has good biocompatibility and high efficiency for gene transduction. It provides a good experimental basis for adoptive immunotherapy.

Expression and clinical significance of the NK cell-activating receptor NKG2D and its ligand in liver tissue of patients with primary biliary cholangitis / 临床肝胆病杂志

ObjectiveTo investigate the association of the expression of the NK cell-activating receptor NKG2D, its ligand major histocompatibility complex class I chain-related gene A (MICA), and related cytokines ［interferon-γ (IFN-γ), interleukin-10 (IL-10), and interleukin-15 (IL-15)］ with intrahepatic inflammation in primary biliary cholangitis (PBC). MethodsLiver biopsy specimens were collected from 30 patients with PBC (PBC group), 15 patients with chronic hepatitis B (CHB group), and 10 patients with nonalcoholic fatty liver disease (NAFLD group), who were hospitalized in The Second Affiliated Hospital of Kunming Medical University from August 2014 to June 2015. The degree of liver inflammation (G) and fibrosis degree (S) of the liver specimens were determined, and immunohistochemistry was used to measure the expression of NKG2D, MICA, IFN-γ, IL-10, and IL-15 in liver tissue (the scores were determined based on the number of cells stained and the degree of staining to evaluate the expression of each marker). A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the t-test was used for comparison between two groups; a Spearman correlation analysis was used to investigate correlation. ResultsIn the PBC group, the expression of NKG2D increased with the degree of inflammation, and the patients with G3-4 inflammation had significantly higher expression than those with G1-2 inflammation (G1 vs G2 vs G3 vs G4: 1.4±0.05 vs 1.56±0.05 vs 1.86±0.11 vs 2.60±0.17, F=150.8, P＜0.05); the expression of NKG2D decreased with fibrosis degree (S3 vs S4: 2.30±0.17 vs 1.56±0.05, t=-1.52, P＜0.05). In the PBC group, there was no significant difference in MICA between G3 and G4 (0.11±0.01 vs 0.20±0.03, t=-2.20, P＞0.05) and between S3 and S4 (0.12±0.02 vs 0.18±0.03, t=-2.64, P＞0.05). In the PBC group, there was a significant difference in the expression of IL-15 between the patients with different degrees of inflammation (G1 vs G2 vs G3 vs G4: 0.70±0.10 vs 1.50±0.10 vs 1.93±0.11 vs 2.60±0.17, F=251.3, P＜0.05), while there was no significant difference between the patients with different fibrosis degrees (S3 vs S4: 2.00±0.05 vs 2.40±0.30, t=-1.62, P＞0.05). In the CHB group, there was a significant difference in the expression of IL-15 between the patients with different degrees of inflammation (G1 vs G2 vs G3: 0.73±0.15 vs 1.96±0.15 vs 2.50±0.17, F=150, P＜0.05) and between the patients with different fibrosis degrees (S1 vs S2 vs S3: 0.70±0.10 vs 21.96±0.15 vs 2.50±0.17, F=158.7, P＜0.05). In the PBC group, the expression of IL-10 was only observed in the patients with G1 inflammation (0.16±0.01), and in the CHB group, the expression of IL-10 was observed in the patients with G1 and G2 inflammation, with no significant difference (G1 vs G2: 0.19±0.01 vs 0.13±0.01, t=-1.522, P＞0.05). In the patients with PBC, the expression of IL-15 in liver tissue was positively correlated with the levels of alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (GGT) (r=0.241 and 0.407, P=0.014 and 0.045). ConclusionThe NK cell-activating receptor NKG2D affects the degree of intrahepatic inflammation in PBC, and the NKG2D ligand MICA is expressed in the advanced stage of PBC and can downregulate NKG2D. The expression of IL-15 increases with the degree of inflammation in PBC and is positively correlated with the levels of ALP and GGT, suggesting that the activation of NK cells and abnormal secretion of cytokines are involved in the development and progression of PBC and IL-15 may be used as an auxiliary index for the diagnosis of PBC.

Evaluation of immunological parameters in pregnant women: low levels of B and NK cells / Avaliação dos parâmetros imunológicos em mulheres gestantes: baixos níveis de células B e NK

Abstract Objective To describe the immunological and hematological reference intervals of low-risk pregnant women. Methods A cross-sectional retrospective database analysis of a basic and translational study analyzing the hematological evaluation blood counts and immunophenotyping of TCD3 + , TCD4 + , TCD8 + , B, and natural killer (NK) cells of the peripheral blood in 79 low-risk pregnant women and of 30 control women from the state of Pernambuco, Brazil, was performed. Results No significant differences were detected between the hematological profiles of the 2nd and 3rd trimesters. Nevertheless, the median level of B cells decreased significantly in the 2nd (174 x 103 μL; p < 0.002) and 3rd trimesters (160 x 103 μL; p < 0.001), compared with the control group (296 x 103 μL). Similarly, the median level of NK cells was lower in the 2nd (134 x 103 μL; p < 0.0004) and 3rd trimesters (100 x 103 μL, p < 0.0004), compared with the control group (183 x 103 μL). In contrast, relative TCD4+ and TCD8+ levels increased in the 2nd and 3rd trimesters compared with the controls (TCD4 + : 2nd trimester = 59%; p < 0.001; 3rd trimester = 57%; p < 0.01; control = 50%; and TCD8 + : 2nd trimester = 31%; p < 0.001; 3rd trimester = 36%; p < 0.01; control = 24%). Conclusion Low-risk pregnant women have ~ 40% less B and NK cells in the peripheral blood, compared with non-pregnant women. These parameters may improve health assistance for mothers and contribute to define reference values for normal pregnancies.

Resumo Objetivo Descrever o intervalo de referência imunológico e hematológico de gestantes de baixo risco. Métodos Realizou-se uma análise retrospectiva, de um estudo básico e translacional, analisando o perfil hematológico e a imunofenotipagem das células TCD3 + , TCD4 + , TCD8 + , B e natural killer (NK) do sangue periférico de 79 gestantes de baixo risco e de 30 mulheres (controles) do estado de Pernambuco, Brasil. Resultados Não observamos diferenças significativas entre os perfis hematológicos do 2° e 3° trimestres. No entanto, houve redução das células B no 2° (média = 174 x 103 μL; p < 0,002) e no 3° trimestres (160 x 103 μL; p < 0,001), comparado como grupo controle (296 x 103 μL). A mediana das células NK foi menor no 2° (134 x 103 μL; p < 0,0004) e no 3° trimestres (100 x 103 μL; p < 0,0004), comparado com o grupo controle (183 x 103 μL). Porém, o percentual de TCD4+ e de TCD8+ aumentou no 2° e 3° trimestres em relação aos controles (TCD4 + : 2° trimestre = 59%; p < 0,001; 3° trimestre = 57%; p < 0,01; controle = 50%; e TCD8 + : 2° trimestre = 31%; p < 0,001; 3° trimestre = 36%; p < 0,01; controle = 24%). Conclusão Mulheres grávidas de baixo risco têm ~ 40% menos células B e NK no sangue periférico em comparação com mulheres não grávidas. Estes parâmetros podem melhorar a assistência à saúde das mães e contribuir para a definição de valores de referência para gestações normais.

Changes in natural killer cell subsets marked with CD27 and CD11b for HBV carrier mice / 中华肝脏病杂志

Objective@#To evaluate the changes in natural killer cell subsets marked with CD27 and CD11b for HBV carrier mice.@*Methods@#The pAAV-HBVl.2 plasmid was injected into the tail vein of C57BL/6 mice by hydrodynamic injection method to construct HBV-carrier model group and empty vector as the control group. Liver function and virological examination at different time points were used to judge the construction of HBV- plasmid carrier animal model. Flow cytometry was used to detect the frequency of NK cells and CD11b combined with CD27 NK cell subsets in spleen and liver. GraphPad Prism software was used for statistical analysis.@*Results@#HBV-carrier mouse model was successfully constructed. There were no statistically significant difference in NK cell frequencies between spleen and liver of HBV carrier mice (P> 0.05), compared to control group. NK cells were divided into four subsets with in combination to CD27 and CD11b: CD11b+CD27-(CD11b+SP), CD11b+CD27+(DP), CD11b-CD27+(CD27+SP) and CD11b-CD27-(DN). Furthermore, the spleen of HBV-carrier mice had no statistically significant difference (P> 0.05) with the frequency of the four NK-cell subsets. The frequency of DN NK cell subsets was significantly increased in the liver of HBV carrier mice than control group (P< 0.001); however, the frequency of CD11b+SP cell subsets was significantly decreased (P < 0.05).There were no statistical significance in the frequency comparison between NK subgroups of DP and CD27+SP NK cell subsets (P> 0.05).@*Conclusion@#HBV-carrier mice with abnormal distribution of hepatic NK cell subsets significantly increased and decreased the frequency of DN NK cell subsets and CD11b+SP cell subsets.

Mechanism of CD226-dependant NK cell regulation in mouse obesity / 中华微生物学和免疫学杂志

Objective To investigate the effects of CD226 knockout ( KO) on obese mice fed with high fat diet and to analyze the composition of immune cells in CD226KO obese mice for further elucidating the immunological mechanism of CD226 involved in high fat diet-induced obesity. Methods Both wild-type ( WT) and CD226KO mice were randomly divided into two groups, high-fat and normal diet groups, and fed for 14 weeks to establish the type 2 diabetes model. Immune cells in mouse spleen and peripheral blood were analyzed by flow cytometry. In in vitro experiments, NK92-MI cells were infected with pshRNA-CD226 lenti-virus to silence CD226 expression, and then qPCR was performed to detect the expression of Foxp3, TNF-αand IFN-γ at mRNA level. Results In the high-fat diet groups, CD226KO mice had lower blood glucose, serum insulin and HOMA-IR than WT mice, but higher HOMA-IS and HOMA-β. CD226KO could reduce compensatory hyperplasia of islet tissue, and significantly down-regulate the proportion of spleen NK cells in mice. The proportion of CD3-CD49b+CD25+Foxp3+regulatory NK cells (NKreg) increased significantly in CD226KO mice. CD226KO could significantly increase Foxp3 expression in NK92-MI cells and decrease the expression of TNF-α and IFN-γ. Conclusions CD226KO can alleviate insulin resistance, increase the number of islet β-cell and improve islet β-cell function in obese mice. The mechanism might be related to the up-regulation of Foxp3+ NKreg ratio.

Regulatory effect of CLEC2D-CD161 interaction on killing capacity of decidual natural killer cells / 中华围产医学杂志

Objective@#To investigate the regulatory effect of CLEC2D-CD161 interaction on killing capacity of decidual natural killer (dNK) cells during early pregnancy and its association with the incidence of recurrent spontaneous abortion (RSA).@*Methods@#Decidua tissues were collected from normal pregnancies (n=16) and RSA cases (n=6) at 6-10 gestational weeks in the Department of Obstetrics and Gynecology of Peking University Third Hospital from October 2018 to May 2019. (1) Expressions of CLEC2D and CD161 in decidua from early pregnancy were detected using immunofluorescence. (2) Primary dNK cells were isolated from decidua from early pregnancy. dNK cells pre-treated with CD161 antibody (blocking CD161, B-CD161) were co-cultured with JEG-3 cells which were knocked-down by CLEC2D small interfering RNA (siCLEC2D), followed by killing capacity assessment of dNK cells by cytotoxicity assay and determination of expressions of related molecules by quantitive real-time polymerase chain reaction. (3) Western blot and flow cytometry were used to detect the expression of CLEC2D and CD161 in decidua tissues. Cytotoxicity assay was performed to analyze the killing capacity of dNK cells. T test was used for statistical analysis between normal and RSA cases.@*Results@#(1) CLEC2D was mainly expressed in extravillous trophoblast (EVT) cells and CD161 was mainly detected in dNK cells. CD161-positive dNK cells and CLEC2D-positive EVT cells were adjacently located in decidua tissues allowing their interaction. (2) Cytotoxicity assay suggested that CD161 blocking in dNK cells or CLEC2D knockdown in JEG-3 cells could enhance the cytotoxicity of dNK cells. The target cell lysis rates at the effector-target ratios of 40∶1, 20∶1, 10∶1 and 5∶1 in B-CD161 group were (59.12±4.56)%, (25.96±5.44)%, (13.60±8.94)% and (12.53±8.94)%, and in IgG control group were (20.01±1.96)%, (8.51±1.32)%, (3.24±0.75)% and (3.82±1.92)%, respectively. There were significant differences between the two groups at the effector-target ratios of 40∶1 (t=13.922, P<0.01) and 20∶1 (t=5.403 P<0.05), but not at 10∶1 or 5∶1 (P>0.05). The target cell lysis rates at the effector-target ratios of 40∶1, 20∶1, 10∶1 and 5∶1 in si-CLEC2D group were (43.37±2.01)%, (32.99±2.08)%, (23.47±1.36)% and (11.48±0.37)%, and in the negative control (NC) group were (15.54±1.46)%, (13.84±1.68)%, (9.94±3.01) and (5.50±0.99)%, respectively. Differences between the two groups at all effector-target ratios were statistically significant (t=19.402, 12.400, 7.093 and 9.842, all P<0.01). Moreover, the expression of dNK killing-related factor granzyme B in the siCLEC2D group was higher than that in the NC group. (3) Compared with the normal pregnancy group, the RSA group showed decreased CD161 expression and increased killing capacity of dNK cells, but no significant difference in CLEC2D expression.@*Conclusions@#At early pregnancy, CLEC2D on EVT cells can interact with CD161 on dNK cells, which inhibits the cytotoxicity of dNK cells and induces immune tolerance at the fetal-maternal interface. Decreased expression of CD161 in decidua results in increased cytotoxicity of dNK cells, which may be one of the causes of immune rejection in RSA.