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Objective:To investigate the expression of programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway in patients with acute myeloid leukemia (AML) and its relationship with clinical features and prognosis, and to examine its effect on PD-1-positive natural killer (NK) cells against AML cells in vitro.Methods:The bone marrow samples of 65 AML patients and the peripheral blood of 32 AML patients diagnosed in Affiliated Cancer Hospital of Zhengzhou University from July 2019 to December 2020 were prospectively collected, and the peripheral blood of 24 healthy people was taken as healthy control. The expression level of PD-L1 in bone marrow tumor cells and expression level of PD-1 in peripheral blood NK cells were detected by flow cytometry. The correlations of PD-1 expression in bone marrow tumor cells and PD-1 expression in NK cells with the clinicopathological features, curative effect and prognosis of patients were analyzed. Flow cytometry was used to detect the expression level of PD-L1 in AML cell line THP-1 (target cells) and the expression level of PD-L1 in NK cell line NKL (effector cells). THP-1 cells treated with and without 25 μmol/L of PD-L1 inhibitor fraxinellone were used as experimental group and control group, and co-cultured with NKL cells at different effector-to-target ratios. The apoptosis of THP-1 cells and the expression of NKG2D in NKL cells were detected by flow cytometry, the cell proliferation status was detected by CCK-8 and the cell proliferation inhibition rate was calculated; the levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the supernatant of co-culture system were detected by enzyme-linked immunosorbent assay (ELISA).Results:The proportion of AML patients with PD-L1-positive expression in bone marrow tumor cells was higher than that in the healthy control group [38.5% (25/65) vs. 8.3% (2/24), P = 0.029]. The proportion of AML patients with PD-1-positive expression in peripheral blood NK cells was higher than that in the healthy control group [40.6% (13/32) vs. 12.5% (3/24), P = 0.035]. There were no statistical differences in sex, age, hemogram, proportion of primordial cells, risk stratification, chromosomal karyotype, gene mutation (except NPM1 gene), fusion gene and French-American-British cooperative group (FAB) typing between patients with PD-L1 positive and negative in bone marrow tumor cells and between patients with PD-1 positive and negative in peripheral blood NK cells (all P > 0.05). In relapsed/refractory patients, the proportion of patients with PD-L1-positive expression in bone marrow tumor cells was higher than that in newly treated patients [58.8% (10/17) vs. 31.2% (15/48), P = 0.045]. There was no significant difference in the proportion of patients with PD-1-positive expression in peripheral blood NK cells between relapsed/refractory patients and newly treated patients [(38.5% (5/13) vs. 42.1% (8/19), P = 0.837]. There was no statistical difference in complete remission (CR) rate between PD-L1 positive and negative patients [69.6% (16/23) vs. 74.3% (26/35), P > 0.05]. There was no statistical difference in CR rate between PD-1 positive and negative patients [66.7% (8/12) vs. 70.6% (12/17), P > 0.05]. There was no statistical difference in recurrence rate after CR between PD-L1 positive and negative patients [12.5% (2/16) vs. 19.2% (5/26), P > 0.05]. There was no statistical difference in recurrence rate after CR between PD-1 positive and negative patients [25.0% (2/8) vs. 16.7% (2/12), P > 0.05]. Flow cytometry showed that the positive rate of PD-1 in NKL cells was (67±6)% and the positive rate of PD-L1 in THP-1 cells was (85±5)%. After co-culture with NKL cells, the apoptotic rate and proliferation inhibition rate of THP-1 cells were higher in the experimental group compared with the control group, the expression of NKG2D on the surface of NKL cells was elevated, and the levels of IFN-γ and TNF-α in the co-culture supernatant were increased. Conclusions:In AML patients, the expression of PD-L1 in bone marrow tumor cells is high, and the expression of PD-1 in peripheral blood NK cells is also high. The expression of PD-L1 in bone marrow tumor cells of relapsed/refractory AML patients is higher than that of newly treated patients. Inhibition of PD-L1 expression in THP-1 cells can enhance the tumor killing activity of NKL cells in vitro. The mechanism may be that inhibition of PD-L1 expression in THP-1 cells up-regulates the expression of NKL cell activated receptor NKG2D and promotes the secretion of IFN- γ and TNF- α.
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Objective:To investigate the clinical efficacy of lenalidomide combined with bortezomib and dexamethasone (RVd) regimen in treatment of newly diagnosed multiple myeloma (NDMM) patients and its effect on the levels of regulatory T cells (Treg cells) and natural killer (NK) cells.Methods:Thirty-eight NDMM patients who were admitted to the Second Affiliated Hospital of Bengbu Medical College from September 2019 to May 2022 were selected for a prospective study, and were divided into control group (18 cases) and observation group (20 cases) according to random number table method. The control group was treated with bortezomib+epirubicin+dexamethasone (VAd) regimen, and the observation group was treated with RVd regimen. The efficacy and safety were compared between the two groups. The levels of Treg cells (CD4 + CD25 + FOXP3 +) and NK cells (CD3 - CD56 + CD16 +) before and after treatment in the two groups were detected by flow cytometry, and the results were compared. Results:After 4 courses of treatment, the objective response rate (ORR) of the observation group was 95.0% (19/20), which was higher than that of the control group [77.8% (14/18)], and the difference was statistically significant ( P = 0.016). Before treatment, there was no statistical difference in the levels of Treg cells and NK cells between the two groups ( P values were 0.381 and 0.650). After treatment, the level of Treg cells in the control group increased from (1.5±0.5)% before treatment to (4.7±1.3)% ( P = 0.008), while the level of Treg cells in the observation group increased from (1.4±0.5)% before treatment to (6.8±1.5)% ( P = 0.001), and the level in the observation group was higher than that in the control group ( P = 0.027); the level of NK cells in the control group increased from (16±6)% before treatment to (20±5)% ( P = 0.004), while the level of NK cells in the observation group increased from (16±6)% before treatment to (24±6)% ( P = 0.006), and the level in the observation group was higher than that in the control group ( P = 0.032). The incidence rates of thrombocytopenia and neutropenia in the observation group were higher than those in the control group, and the differences were statistically significant ( P values were 0.012 and 0.027), which was reversible after active treatment. There was no statistical difference in the incidence rates of other adverse reactions (all P>0.05). Conclusions:RVd regimen for NDMM is clinically effective, safe and reliable, and the patients' levels of Treg cells and NK cells elevate after treatment.
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Objective:To analyze the value of cytotoxic T lymphocyte and natural killer cell levels in prognosis evaluation of patients with ST-elevation myocardial infarction (STEMI).Methods:A total of 158 patients with STEMI who underwent percutaneous coronary intervention in The Second People's Hospital of Liaocheng from September 2020 to August 2021 were included in this study. The ratio of cytotoxic T lymphocytes to natural killer cells was measured immediately after admission and 48 hours after surgery. These patients were followed up for 1 month after treatment. They were divided into the adverse cardiovascular event group (occurrence group) and no adverse cardiovascular event group (non-occurrence group) according to the occurrence of cardiovascular adverse events. The influential factors of the prognosis of STEMI and the correlation between the influential factors and STEMI were analyzed.Results:Among 158 patients with STEMI, 27 patients had adverse cardiovascular events, accounting for 17.09%. There were significant differences in systolic blood pressure, left ventricular ejection fraction, and low-density lipoprotein levels between the occurrence and non-occurrence groups ( t = 2.82, 4.27, 2.32, all P < 0.05). At 48 hours after surgery, the levels of cytotoxic T lymphocytes [(22.75 ± 8.39)%, (29.23 ± 4.61)%] and natural killer cells [(13.73 ± 4.64)%, (20.64 ± 4.52)%] in the peripheral blood in the occurrence and non-occurrence groups were significantly decreased compared with before surgery [ t = -5.05, -83.68, -142.71, -7 084.80, all P < 0.001]. Before and 48 hours after surgery, the levels of cytotoxic T lymphocytes [(27.47 ± 3.35)%, (22.75 ± 8.39)%] and natural killer cells [(21.42 ± 4.36)%, (13.73 ± 4.64)%] in the peripheral blood in the occurrence group were significantly lower than those in the non-occurrence group ( t = 7.68, 13.10, 4.16, 5.76, all P < 0.001). Univariate analysis showed that preoperative cytotoxic T lymphocytes < 27.47%, preoperative natural killer cells < 21.42%, left ventricular ejection fraction, and low-density lipoprotein may be the risk factors that affect the prognosis of patients with STEMI ( P < 0.000, 0.012, 0.019, 0.033). Cox regression analysis showed that preoperative cytotoxic T lymphocytes < 27.47% and preoperative natural killer cells < 21.42% were independent risk factors affecting the prognosis of patients with STEMI (both P < 0.001). Conclusion:Reduced levels of baseline cytotoxic T lymphocytes and natural killer cells in patients with STEMI suggest an increased risk of poor prognosis.
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Objective:To evaluate the effect of ozone preconditioning on splenic natural killer (NK) cells in septic mice.Methods:Twenty-four SPF-grade male C57BL/6 mice, aged 6-8 weeks, weighing 18-22 g, were divided into 4 groups ( n=6 each) according to the random number table method: control group (group C), lipopolysaccharide (LPS)group, ozone+ LPS group (O 3+ LPS) and air+ LPS group (Air+ LPS). The sepsis was induced by intraperitoneal injection of LPS 10 mg/kg. Ozone preconditioning was started at 5 days before developing the model: ozone 1 mg/kg was intraperitoneally injected once a day for 5 consecutive days, the equal volume of air was injected in Air+ LPS group. The survival was observed within 72 h after LPS injection, and sepsis score and ear temperature (once every 2 h, an average was calculated) were recorded. The posterior orbital venous blood samples were taken at 6 and 24 h after LPS injection for determination of serum interferon-γ (IFN-γ) and interleukin-10 (IL-10) concentrations using enzyme-linked immunosorbent assay. The spleen was then taken, and a single cell suspension of the spleen was prepared for measurement of the percentage of NK cells in the spleen by flow cytometry. Results:Compared with C group, the ear temperature, sepsis score and 72-h survival rate were significantly decreased, serum IFN-γ and IL-10 concentrations were increased at each time point after LPS injection, and the percentage of splenic NK cells was increased at 6 h after LPS injection and decreased at 24 h after LPS injection in LPS, Air+ LPS and O 3+ LPS groups ( P<0.05). Compared with LPS group, the ear temperature, sepsis score and 72-h survival rate were significantly increased, serum IFN-γ concentrations were decreased at each time point after LPS injection, serum IL-10 concentrations were increased at each time point after LPS injection, and the percentage of splenic NK cells was decreased at 6 h after LPS injection and increased at 24 h after LPS injection in O 3+ LPS group ( P<0.05), and no significant change was found in the parameters mentioned above in Air+ LPS group ( P>0.05). Conclusions:The mechanism by which ozone preconditioning reduces sepsis may be related to reduction of inflammatory responses and regulation of splenic NK cell levels in septic mice.
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ABSTRACT Objective To determine the role of the AKT pathway in the regulating of natural Killer-induced apoptosis of acute myeloid leukemia cells and to characterize the associated molecular mechanisms. Methods BALB/c nude mice were injected with HL60 cells to induce a xenogenic model of subcutaneous leukemic tumors. Mice were treated with perifosine, and their spleens were analyzed using biometry, histopathology, and immunohistochemistry. Gene expression analysis in leukemia cells was performed by real-time PCR. Protein analysis of leukemia and natural Killer cells was performed by flow cytometry. AKT inhibition in HL60 cells, followed by co-culture with natural Killer cells was performed to assess cytotoxicity. Apoptosis rate was quantified using flow cytometry. Results Perifosine treatment caused a reduction in leukemic infiltration in the spleens of BALB/c nude mice. In vitro , AKT inhibition reduced HL60 resistance to natural Killer-induced apoptosis. AKT inhibition suppressed the immune checkpoint proteins PD-L1, galectin-9, and CD122 in HL60 cells, but did not change the expression of their co-receptors PD1, Tim3, and CD96 on the natural Killer cell surface. In addition, the death receptors DR4, TNFR1, and FAS were overexpressed by AKT inhibition, thus increasing the susceptibility of HL60 cells to the extrinsic pathway of apoptosis. Conclusion The AKT pathway is involved in resistance to natural Killer-induced apoptosis in HL60 cells by regulating the expression of immune suppressor receptors. These findings highlight the importance of AKT in contributing to immune evasion mechanisms in acute myeloid leukemia and suggests the potential of AKT inhibition as an adjunct to immunotherapy.
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SUMMARY OBJECTIVES: The aim of this study was to evaluate CD56 immunostaining in the stroma of benign and malignant ovarian epithelial neoplasms and associate the CD56 immunostaining with prognostic factors and survival in ovarian cancer. METHODS: Patients with ovarian epithelial neoplasia (n=77) were studied with a prospective cohort. The CD56 immunostaining was evaluated in the peritumoral stroma. Two groups were evaluated: benign ovarian neoplasms (n=40) and malignant ovarian neoplasms (n=37). Data were recorded for histological type and grade, International Federation of Gynecology and Obstetrics staging, molecular subtype, and lymph node metastases. Fisher's exact test and Kaplan-Meier survival curves were used, with a significance level of ≤0.05. RESULTS: We found greater CD56 stromal immunostaining in malignant neoplasms when compared to the group of benign neoplasms (p=0.00001). There was no significant difference in relation to the prognostic factors and survival. CONCLUSION: Malignant ovarian neoplasms showed higher stromal CD56 immunostaining. As the prognostic value of natural killer in ovarian cancer is controversial, knowing the specific function of each cell present both in the tumor tissue and systemically may help guide successful immunotherapies in the near future.
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Liver-resident natural killer (LrNK) cells, as a type of newly discovered tissue-resident natural killer cells, have a strong immune killing function. During the development and progression of hepatocellular carcinoma (HCC), the function of LrNK cells is impaired and such cells may promote the progression of HCC by upregulating the expression of related immune checkpoints. Based on the latest research, this article reviews the immune function of LrNK cells and their role in the development and progression of HCC, in order to explore the application prospect of these cells in HCC immunotherapy.
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Autoantibody-related congenital heart block (ACHB) is a passively acquired autoimmune disease developing in fetuses after exposuring to maternal anti-Ro/Sj?gren's syndrome type A (SSA) antibody and/or anti-La/SSB antibody transported across the placenta, which contributes to fetal heart conduction system damage and signal conduction block at the atrioventricular node. However, fetal atrioventricular block does not necessarily occur with the presence of maternal autoantibodies, indicating its complex pathogenesis. This review focuses on the theories of calcium channels and apoptosis, the influence of other maternal factors and environmental changes on ACHB and the roles of natural killer cells and human leukocyte antigen in ACHB, aiming to provide reference for further study on the pathogenesis.
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Abstract | Introduction: Immunological markers have been described during COVID-19 and persist after recovery. These immune markers are associated with clinical features among SARS- CoV-2 infected individuals. Nevertheless, studies reporting a comprehensive analysis of the immune changes occurring during SARS-CoV-2 infection are still limited. Objective: To evaluate the production of proinflammatory cytokines, the antibody response, and the phenotype and function of NK cells and T cells in a Colombian family cluster with SARS-CoV-2 infection. Materials and methods: Proinflammatory cytokines were evaluated by RT-PCR and ELISA. The frequency, phenotype, and function of NK cells (cocultures with K562 cells) and T-cells (stimulated with spike/RdRp peptides) were assessed by flow cytometry. Anti-SARS-CoV-2 antibodies were determined using indirect immunofluorescence and plaque reduction neutralization assay. Results: During COVID-19, we observed a high proinflammatory-cytokine production and a reduced CD56bright-NK cell and cytotoxic response. Compared with healthy controls, infected individuals had a higher frequency of dysfunctional CD8+ T cells CD38+HLA-DR-. During the acute phase, CD8+ T cells stimulated with viral peptides exhibited a monofunctional response characterized by high IL-10 production. However, during recovery, we observed a bifunctional response characterized by the co-expression of CD107a and granzyme B or perforin. Conclusion: Although the proinflammatory response is a hallmark of SARS-CoV-2 infection, other phenotypic and functional alterations in NK cells and CD8+ T cells could be associated with the outcome of COVID-19. However, additional studies are required to understand these alterations and to guide future immunotherapy strategies.
Resumen | Introducción. Se han descrito diferentes marcadores inmunológicos durante la COVID-19, los cuales persisten incluso después de la convalecencia y se asocian con los estadios clínicos de la infección. Sin embargo, aún son pocos los estudios orientados al análisis exhaustivo de las alteraciones del sistema inmunológico en el curso de la infección. Objetivo. Evaluar la producción de citocinas proinflamatorias, la reacción de anticuerpos, y el fenotipo y la función de las células NK y los linfocitos T en una familia colombiana con infección por SARS-CoV-2. Materiales y métodos. Se evaluaron las citocinas proinflamatorias mediante RT-PCR y ELISA; la frecuencia, el fenotipo y la función de las células NK (en cocultivos con células K562) y linfocitos T CD8+ (estimulados con péptidos spike/RdRp) mediante citometría de flujo, y los anticuerpos anti-SARS-CoV-2, mediante inmunofluorescencia indirecta y prueba de neutralización por reducción de placa. Resultados. Durante la COVID-19 hubo una producción elevada de citocinas proinflamatorias, con disminución de las células NK CD56 bright y reacción citotóxica. Comparados con los controles sanos, los individuos infectados presentaron con gran frecuencia linfocitos T CD8+ disfuncionales CD38+HLA-DR-. Además, en los linfocitos T CD8+ estimulados con péptidos virales, predominó una reacción monofuncional con gran producción de IL-10 durante la fase aguda y una reacción bifuncional caracterizada por la coexpresión de CD107a y granzima B o perforina durante la convalecencia. Conclusión. Aunque la reacción inflamatoria caracteriza la infección por SARS-CoV-2, hay otras alteraciones fenotípicas y funcionales en células NK y linfocitos T CD8+ que podrían asociarse con la progresión de la infección. Se requieren estudios adicionales para entender estas alteraciones y guiar futuras estrategias de inmunoterapia.
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Infecciones por Coronavirus , Células Asesinas Naturales , Linfocitos T , Anticuerpos Neutralizantes , InflamaciónRESUMEN
Objective:To investigate the effects of upregulation of cAMP response element binding protein (CREB) on proliferation, invasion and natural killer (NK) cell killing activity of esophageal adenocarcinoma.Methods:23 patients with esophageal cancer treated in Shaanxi Provincial People′s Hospital from March 2017 to December 2019 were selected. The protein expression of CREB in esophageal adenocarcinoma and adjacent normal tissues was detected by immunohistochemistry; Esophageal adenocarcinoma cell line TE-10 was cultured in vitro. TE-10 cells transfected with si-NC were set as the control group and TE-10 cells transfected with si-CREB were set as the si-CREB group. Western blot was used to detect the protein level of CREB, MHC class Ⅰ chain-related A (MICA) and MHC class Ⅰ chain-related B (MICB); Flow cytometry was employed to detect the expression of MICA and MICB; clone formation experiment and Transwell were used to detect the proliferative and invasive ability of TE-10 cells; lactate dehydrogenase (LDH) releasing assay was used to detect cytotoxicity of NK cells against TE-10 cells. Results:The expression of CREB in esophageal adenocarcinoma tissue was higher than that in normal tissues adjacent to cancer; the protein level of CREB in esophageal cancer cell TE-10 was higher than that of human normal esophageal epithelial cells HEEC ( P<0.05). The proliferative and invasive ability of TE-10 cells in the si-CREB group was significant lower than that in the control group ( P<0.05), while the expression of MICA and MICB, the killing rate of NK cells on TE-10 cells was significant higher than that in the control group ( P<0.05). Conclusions:CREB was highly expressed in esophageal adenocarcinoma tissues and cells. Silencing CREB could inhibit the proliferation and invasion of esophageal adenocarcinoma cells, and enhance the killing sensitivity of NK cells to esophageal adenocarcinoma cells by up-regulating the expression of MICA and MICB.