Interferon-α mediating the functional damage of CD56dimCD57+natural killer cells in peripheral blood of systemic lupus erythematosuss / 北京大学学报(医学版)

OBJECTIVE@#To investigate the regulatory effect of interferon-α (IFN-α) on the apoptosis and killing function of CD56dimCD57+ natural killer (NK) cells in systemic lupus erythematosus (SLE) patients, and to explore the specific mechanism.@*METHODS@#A total of sixty-four newly treated SLE patients and sixteen healthy controls (HC) enrolled in the Second Hospital of Dalian Medical University were selected as the research subjects. And the gene expression levels of molecules related to NK cell-killing function were detected by real-time quantitative polymerase chain reaction. CD56dimCD57+ NK cells were co-cultured with the K562 cells, and the apoptotic K562 cells were labeled with Annexin-Ⅴ and 7-amino-actinomycin D. Peripheral blood mononuclear cells were treated with 20, 40, and 80 μmol/L hydrogen peroxide (H2O2), and treated without H2O2 as control, the expression level of perforin (PRF) was detected by flow cytometry. The concentration of IFN-α in serum was determined by enzyme linked immunosorbent assay. The expression levels of IFN-α receptors (IFNAR) on the surface of CD56dimCD57+ NK cells were detected by flow cytometry, and were represented by mean fluorescence intensity (MFI). CD56dimCD57+ NK cells were treated with 1 000 U/mL IFN-α for 24, 48 and 72 h, and no IFN-α treatment was used as the control, the apoptosis and the expression levels of mitochondrial reactive oxygen species (mtROS) were measured by flow cytometry and represented by MFI.@*RESULTS@#Compared with HC(n=3), the expression levels of PRF1 gene in peripheral blood NK cells of the SLE patients (n=3) were decreased (1.24±0.41 vs. 0.57±0.12, P=0.05). Compared with HC(n=5), the ability of peripheral blood CD56dimCD57+ NK cells in the SLE patients (n=5) to kill K562 cells was significantly decreased (58.61%±10.60% vs. 36.74%±6.27%, P < 0.01). Compared with the control (n=5, 97.51%±1.67%), different concentrations of H2O2 treatment significantly down-regulated the PRF expression levels of CD56dimCD57+ NK cells in a dose-dependent manner, the 20 μmol/L H2O2 PRF was 83.23%±8.48% (n=5, P < 0.05), the 40 μmol/L H2O2 PRF was 79.53%±8.56% (n=5, P < 0.01), the 80 μmol/L H2O2 PRF was 76.67%±7.16% (n=5, P < 0.01). Compared to HC (n=16), the serum IFN-α levels were significantly increased in the SLE patients (n=45) with moderate to high systemic lupus erythematosus disease activity index (SLEDAI≥10) [(55.07±50.36) ng/L vs. (328.2±276.3) ng/L, P < 0.001]. Meanwhile, compared with HC (n=6), IFNAR1 expression in peripheral blood CD56dimCD57+ NK cells of the SLE patients (n=6) were increased (MFI: 292.7±91.9 vs. 483.2±160.3, P < 0.05), and compared with HC (n=6), IFNAR2 expression in peripheral blood CD56dimCD57+ NK cells of the SLE patients (n=7) were increased (MFI: 643.5±113.7 vs. 919.0±246.9, P < 0.05). Compared with control (n=6), the stimulation of IFN-α (n=6) significantly promoted the apoptosis of CD56dimCD57+ NK cells (20.48%±7.01% vs. 37.82%±5.84%, P < 0.05). In addition, compared with the control (n=4, MFI: 1 049±174.5), stimulation of CD56dimCD57+ NK cells with IFN-α at different times significantly promoted the production of mtROS in a time-dependent manner, 48 h MFI was 3 437±1 472 (n=4, P < 0.05), 72 h MFI was 6 495±1 089 (n=4, P < 0.000 1), but there was no significant difference at 24 h of stimulation.@*CONCLUSION@#High serum IFN-α level in SLE patients may induce apoptosis by promoting mtROS production and inhibit perforin expression, which can down-regulate CD56dimCD57+ NK killing function.

Construction of NKG2D CAR-NK92 cells and its killing effect on multiple myeloma cells / 细胞与分子免疫学杂志

Objective This study aims to construct and identify the chimeric antigen receptor NK92 (CAR-NK92) cells targeting NKG2D ligand (NKG2DL) (secreting IL-15Ra-IL-15) and verify the killing activity of NKG2D CAR-NK92 cells against multiple myeloma cells. Methods The extracellular segment of NKG2D was employed to connect 4-1BB and CD3Z, as well as IL-15Ra-IL-15 sequence to obtain a CAR expression framework. The lentivirus was packaged and transduced into NK92 cells to obtain NKG2D CAR-NK92 cells. The proliferation of NKG2D CAR-NK92 cells was detected by CCK-8 assay, IL-15Ra secretion was detected by ELISA and killing efficiency was detected by lactate dehydrogenase (LDH) assay. The molecular markers of NKp30, NKp44, NKp46, the ratio of apoptotic cell population, CD107a, and the secretion level of granzyme B and perforin were detected using flow cytometry. In addition, the cytotoxic mechanism of NKG2D CAR-NK92 cells on the tumor was verified by measuring the degranulation ability. Moreover, after NKG2D antibody inhibited effector cells and histamine inhibited tumor cells, LDH assay was utilized to detect the effect on cell-killing efficiency. Finally, the multiple myeloma tumor xenograft model was constructed to verify its anti-tumor activity in vivo. Results Lentiviral transduction significantly increased NKG2D expression in NK92 cells. Compared with NK92 cells, the proliferation ability of NKG2D CAR-NK92 cells was weaker. The early apoptotic cell population of NKG2D CAR-NK92 cells was less, and NKG2D CAR-NK92 cells had stronger cytotoxicity to multiple myeloma cells. Additionally, IL-15Ra secretion could be detected in its culture supernatant. NKp44 protein expression in NKG2D CAR-NK92 cells was clearly increased, demonstrating an enhanced activation level. Inhibition test revealed that the cytotoxicity of CAR-NK92 cells to MHC-I chain-related protein A (MICA) and MICB-positive tumor cells was more dependent on the interaction between NKG2D CAR and NKG2DL. After stimulating NKG2D CAR-NK92 cells with tumor cells, granzyme B and perforin expression increased, and NK cells obviously upregulated CD107α. Furthermore, multiple myeloma tumor xenograft model revealed that the tumors of mice treated with NKG2D CAR-NK92 cells were significantly reduced, and the cell therapy did not sensibly affect the weight of the mice. Conclusion A type of CAR-NK92 cell targeting NKG2DL (secreting IL-15Ra-IL-15) is successfully constructed, indicating the effective killing of multiple myeloid cells.

Gene Polymorphisms of Patients with Lymphoma-Associated Hemophagocytic Syndrome in Longyan area, Fujian Province / 中国实验血液学杂志

OBJECTIVE@#To analyze the gene polymorphisms of patients with lymphoma-associated hemophagocytic syndrome in Longyan area, Fujian province.@*METHODS@#A total of 125 patients with lymphoma-associated hemophagocytic syndrome in Longyan, Fujian province, admitted to Longyan First Hospital from May 2017 to November 2020 were selected. Peripheral venous blood was collected from all the patients, and the genotypes of perforin 1 (PRF1) and interleukin-10 (IL-10) gene loci were detected by PCR-fluorescence probe method, and the correlation between PRF1 and IL-10 gene polymorphisms and lymphoma-associated hemophagocytic syndrome was analyzed.@*RESULTS@#The mutation frequencies of PRF1 gene loci rs885821 (C>T), rs885822 (C>T), rs1889490 (G>A) in patients with lymphoma-associated hemophagocytic syndrome were 10.40%, 78.8% and 64.4%, respectively. The mutation frequencies of rs1800872 (A>C), rs1800871 (C>T) and rs1800896 (G>A) of IL-10 loci were 56.0%, 45.2% and 77.6%, respectively.@*CONCLUSION@#PRF1 and IL-10 gene loci were polymorphic in patients with lymphoma-associated hemophagocytic syndrome in Longyan area, Fujian province. Alleles C and G of PRF1 and IL-10 were risk factors, and alleles T and A were protective factors.

Synergistic Mechanism of Interferon alpha-1b, Interleukin-2 and Thalidomide for Immune Regulation in Patients with Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explore the synergistic immunomodulatory mechanism of interferon alpha-1b, interleukin-2 and thalidomide (ITI) regimen on patients with acute myeloid leukemia (AML).@*METHODS@#Sixty eight untreated de novo or relapsed or refractory or maintenance therapy patients with AML admitted in the Affiliated Cancer Hospital of Zhengzhou University and the other 11 medical units from March 2016 to May 2019 were treated with ITI regimen. Peripheral blood specimen per patient was collected into EDTA-K3 anticoagulation vacuum tube before the administration of ITI and 3 months after the treatment; peripheral blood lymphocyte subsets and perforin and Granzyme B expression were analyzed by using flow cytometry; the levels of VEGF, IFN-γ, TNF-α and IL-6 in the plasma were detected by using a cytometric bead array. Thirty-five healthy subjects from the hospital physical examination centre were selected as normal controls.@*RESULTS@#The ratio of CD4@*CONCLUSION@#The ITI regimen can raise the ratio of CD4

Síndrome Hemofagocítico en Niños Reporte de Caso / Hemophagocytic Syndromein children: a case report

Introducción:El síndrome hemofagocítico (SHF) es reconocido como un conjunto de signos clínicos y hallazgos laboratoriales que tienen un grave compromiso en la salud y vitalidad de los niños con una incidencia de 1.2 casos/millón/año. Puede pasar subdiagnosticado y confundido con sepsis de foco inespecífico Caso clínico:Niño de 4 años de edad, sin antecedentes de importancia. Ingresado desde el servicio de emergencia por presentar 20 días de fiebre y dolor abdominal. Requirió intubación por franca falla respiratoria y el ingreso a la Unidad de Cuidados Intensivos Pediátricos. Con hipotensión e insuficiencia hepática, pancitopeniay esplenomegalia. Evolución: Se descartaron infecciones bacterianas con policultivos, SARS-Cov 2negativo,se descartaron inmunodeficiencias congénitas y adquiridas.TORCHnegativo, VDRL no reactivo.La prueba de Epstein Barr fue positivo para IgM.Se determinó endocarditis con derrame pericárdico global. Estudio de biopsia medular normocromía, normocitosis, pancitopenia y blastos <5%, sin infiltración tumoral. Se estableció el Diagnóstico de SHFse inicióciclosporina y corticoterapia.Requirió ventilación mecánica por 20 días con período de pronación de 36 horas. Fue dado de alta a pediatríay posteriormente a domicilio, para control por consulta externa. Conclusión: El diagnóstico del SHF es inusual y subestimado al momento de la evaluación clínica. En el presente reporte se asocia a la presencia del Virus Epstein Barr

Introduction: Hemophagocytic syndrome (HPS) is recognized as a set of clinical signs and laboratory findings that have a serious compromise on the health and vitality of children with an incidence of 1.2 cases / million / year. It can be underdiagnosed and confused with sepsis with a non-specific focus. Clinical case: 4-year-old boy, with no significant history. Admitted from the emergency service due to 20 days of fever and abdominal pain. She required intubation due to frank respiratory failureand admission to the Pediatric Intensive Care Unit. With hypotension and liver failure, pancytopenia and splenomegaly. Evolution: Bacterial infections were ruled out with polycultures, SARS-Cov 2 negative, congenital and acquired immunodeficiencies were ruled out. Negative TORCH, non-reactive VDRL. The Epstein Barr test was positive for IgM. Endocarditis with global pericardial effusion was determined. Medullary biopsy study normochromia, normocytosis, pancytopenia, and blasts <5%, without tumor infiltration. The diagnosis of SHF was established, cyclosporine and corticosteroid therapy were started. He required mechanical ventilation for 20 days with a 36-hour pronation period. He was discharged to pediatrics and later at home, for outpatient control. Conclusion: The diagnosis of HHS is unusual and underestimated at the time of clinical evaluation. In this report it is associated with the presence of the Epstein Barr Virus

Fatal familial hemophagocytic lymphohistiocytosis with perforin gene (PRF1) mutation and EBV-associated T-cell lymphoproliferative disorder of the thyroid

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare fatal autosomal recessive disorder of immune dysregulation. The disease presents most commonly in the first year of life; however, symptomatic presentation throughout childhood and adulthood has also been identified. Biallelic mutation in the perforin gene is present in 20%­50% of all cases of FHL. Secondary hemophagocytic lymphohistiocytosis (HLH) in association with hematological malignancies is known; however, whether mutations in HLH-associated genes can be associated with FHL and hematolymphoid neoplasms is not well documented. Also, Epstein­Barr-virus- (EBV) positive systemic T-cell lymphoproliferative disease (SE-LPD) in the setting of FHL is not clearly understood. Here, we present the case of a young boy who presented with typical features of childhood FHL harboring the perforin gene (PRF1) mutation, and had SE-LPD diagnosed on autopsy, along with evidence of recent EBV infection. The patient expired due to progressive disease. Five siblings died in the second or third decade of life with undiagnosed disease. Genetic counseling was provided to the two surviving siblings and parents, but they could not afford genetic testing. One surviving sibling has intermittent fever and is on close follow-up for possible bone marrow transplantation.

Analysis of PRF1gene variant in a child with late-onset familial hemophagocytic lymphohistiocytosis type 2 and severe central nervous system disease / 中华医学遗传学杂志

OBJECTIVE@#To detect genetic mutations in a child with late-onset hemophagocytic lymphohistiocytosis (HLH).@*METHODS@#Clinical data of an 8-year-5-month-old girl with recurrent HLH and severe central nervous system disease was analyzed. Next-generation sequencing was used to detect mutation in the exons and adjacent introns of 17 genes associated with HLH. Suspected mutations were confirmed by Sanger sequencing. Influence of mutations on protein function was predicted with SIFT and PolyPhen-2 software.@*RESULTS@#The child was found to carry compound heterozygous mutations of the PRF1 gene. Among these, the c.1349C>T (p.Thr450Met) mutation, with a SIFT predictive value of -4.921 (Deleterious variant) and a PolyPhen-2 predictive value of 1.000 (Probably damaging), was inherited from her father and known to be pathogenic. The c.1273dupT (p.Trp425fsX457) mutation was inherited from her mother and previously unreported, which resulted in the deletion of almost the entire C2 domain (amino acid residues 413 to 540) and carboxyl terminal of perforin, which seriously affected the function of the protein.@*CONCLUSION@#The c.1349C>T (p.Thr450Met) and c.1273 dupT (p.Trp425fsX457) compound heterozygous mutations of the PRF1 gene probably underlie the disease in this patient.

A preliminary study on SLAMF6 expression in patients with severe aplastic anemia / 中华血液学杂志

Objective: To explore the expression of SLAMF6 on CD8(+) T cells in patients with severe aplastic anemia (SAA) and its correlation with disease immune status. Methods: By flow cytometry (FCM), SLAMF6 expression level in peripheral blood CD8(+) T cells was detected in 21 patients with SAA and 15 normal controls respectively from February 2017 to April 2018. The correlation between SLAMF6 expression level and hematopoietic functions, including HGB, PLT, the neutrophil granulocyte and reticulocyte absolute value in peripheral blood, hyperplasia degree (percentage of granulocytes, erythrocytes, lymphocytes and megakaryocytes in bone marrow) and perforin, granzyme B, IFN-γ expression level in CD8(+) T cells were evaluated. To further confirm the effect of SLAMF6 on CD8(+) T cells, anti-SLAMF6 Ab was used to block SLAMF6 pathway (IgG as control), and FCM was used to detect the perforin, granzyme B, and IFN-γ production of CD8(+) T cells. Results: The expression of SLAMF6 on CD8(+) T cells in untreated SAA patients[(56.29±12.97)%]was significantly lower than that of normal controls[(80.96±7.36)%](t=-7.672, P<0.001). The expression of SLAMF6 on CD8(+) T cells in SAA patients were positively correlated with the HGB, PLT, the neutrophil granulocyte and reticulocyte absolute value in peripheral blood, percentage of granulocytes, erythrocytes in bone marrow (all P<0.05), but they were negatively correlated with the percentage of lymphocytes in bone marrow, and the expression of perforin, granzyme B, and IFN-γ of CD8(+) T cells (all P<0.05). After blocking SLAMF6 pathway by anti-SLAMF6 Ab, the expression levels of perforin, granzyme B and IFN-γ in SAA patients were significantly higher than those in the untreated group, and the differences were statistically significant (all P<0.05). Conclusions: SLAMF6 is significantly down-regulated on CD8(+) T cells in SAA patients, which may act as a negative immunoregulatory molecule participating in the mechanism of SAA by affecting the functional molecules secretion on CD8(+) T cells.

Expressions and clinical significance of GAS1, IL-1RAP and PRF1 in patients with ALK positive anaplastic large cell lymphoma / 中华血液学杂志

Objective: To investigate the expressions of growth arrest-specific protein (GAS1), IL-1 receptor accessory protein (IL-1RAP) and perforin (PRF1) in patients with anaplastic lymphoma kinase positive, anaplastic large cell lymphoma (ALK+ ALCL) and their relationships with clinical significances and the prognoses of ALK+ ALCL. Methods: Twenty-six formalin-fixed paraffin-embedded (FFPE) samples of ALK+ ALCL patients who were diagnosed from January 2011 to September 2016 were collected. Twelve FFPE samples of patients with ALK+ALCL, 13 FFPE samples of patients with peripheral T cell lymphoma (not otherwise specified) (PTCL-NOS) and 8 FFPE samples of patients with angioimmunoblastic T-cell lymphoma (AITL) were used as control groups. RQ-PCR and immunohisto-chemical staining were used to detect the mRNA and protein expressions of GAS1, IL-1RAP and PRF1. The clinical data were analyzed. Results: ①The expression levels of GAS1, IL-1RAP and PRF1 gene and protein in ALK+ ALCL group were higher than those of the control groups (P<0.05), but the expression levels had no statistically significant differences between the control groups (P>0.05). ②Patients with elevated lactate dehydrogenase (LDH) (0.77 vs 1.38, z=-3.292, P=0.001) or International prognostic index (IPI)≥3(0.62 vs 1.29, z=-2.495, P=0.013) had lower expression level of GAS1. Patients with stage Ⅲ/Ⅳ disease (0.89 vs 1.18, z=-2.212, P=0.027) or IPI≥3 (0.48 vs 1.13, z=-2.008, P=0.045) had lower expression level of PRF1. IL-1RAP expression level was not associated with clinical features. ③ALK+ ALCL patients in complete remission (CR) group had higher expression levels of GAS1 and PRF1 than patients in non-remission (NR) group (P values were 0.016 and 0.009). ④Kaplan-Meier survival analysis showed that patients with high expression levels of GAS1 and PRF1 had longer median overall survival and progression-free survival than patients with low expression levels of GAS1 and PRF1. Conclusion: GAS1, IL-1RAP and PRF1 could be molecular markers for ALK+ ALCL patients. They have potential diagnostic value and can be used for differential diagnosis in some difficult cases. ALK+ ALCL patients with high expression levels of GAS1 or PRF1 have better curative effects and prognoses.

Non-invasive messenger RNA transcriptional evaluation in human kidney allograft dysfunction

The aim of the present study was to evaluate messenger RNA expression in kidney allograft recipients. Forty-four kidney transplant recipients were evaluated up to three months after grafting. After transplantation, peripheral blood samples were drawn sequentially for real-time polymerase chain reaction analyses of perforin and TIM-3 genes. Biopsies were obtained to evaluate acute graft dysfunction and interpreted according to the Banff classification. Eight patients presented episodes of acute rejection. Recipients with rejection had significantly higher levels of TIM-3 mRNA transcripts compared to those without rejection (median gene expression 191.2 and 36.9 mRNA relative units, respectively; P<0.0001). Also, perforin gene expression was higher in patients with rejection (median gene expression 362.0 and 52.8 mRNA relative units; P<0.001). Receiver operating characteristic curves showed that the area under the curve (AUC) for the TIM-3 gene was 0.749 (95%CI: 0.670-0.827). Perforin gene mRNA expression provided an AUC of 0.699 (95%CI: 0.599 to 0.799). Overall accuracy of gene expression was 67.9% for the TIM-3 gene and 63.6% for the perforin gene. Combined accuracy was 76.8%. Negative predictive values were 95.3% for the TIM-3 gene, 95.5% for the perforin gene, and 95.4% in the combined analyses. Gene expression was significantly modulated by rejection treatment decreasing 64.1% (TIM-3) and 90.9% (perforin) compared to the median of pre-rejection samples. In conclusion, the longitudinal approach showed that gene profiling evaluation might be useful in ruling out the diagnosis of acute rejection and perhaps evaluating the efficacy of treatment.

Familial Hemophagocytic Lymphohistiocytosis Type 2 in a Korean Infant With Compound Heterozygous PRF1 Defects Involving a PRF1 Mutation, c.1091T>G

No abstract available.

Effects of Iron Overload on the Apoptosis and Function of Splenic CD8+ T Cells in Mice / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of iron overload on apoptosis and function of splenic CD8+ T cells in mice.</p><p><b>METHODS</b>Forty C57BL/6 mice were randomly divided into control groups, Iron overload (IO), IO+NAC and IO+DFX groups. The iron overload model was established by intraperitoneal injection of iron dextran, and saline was injected as the control. The levels of intracellular reactive oxygen species (ROS) and labile iron pool (LIP) were analyzed by measuring the mean fluorescence intensity (MFI) of 2-7 dichlorofluorescein (DCF) or calcein. The ratio of CD8+ T cells and the levels of IFN-γ, TNF-α, Granzyme-B, and perforin in CD8+ T cells were detected by flow cytometry. The CD8+ T cell apoptosis was determined by flow cytometry with Annexin V/PI double staining. Real-time PCR was used to detect the expression of IFN-γ, TNF-α, Granzyme-B, perforin, BCL-2, and bax at mRNA level in CD8+ T cells.</p><p><b>RESULTS</b>Iron overload was found by spleen iron staining and flow cytometry. The level of intracellular ROS in iron overload (IO) groups was higher than that of the control groups (P<0.01). The percentage of CD8+ T cells in spleen from mice with IO was lower than that in control groups (P<0.05). The expression of IFN-γ and Granzyme-B in CD8+ T cells in IO group were lower than that in control group, the expression of IFN-γ and Granzyme-B at mRNA level in CD8+ T cells was lower than that of control group (P<0.05). CD8+ T cell apoptosis in iron overload group was significantly higher than that in control groups (P<0.01); the expression of BCL-2 at mRNA level was lower than that in control group, but the expression of BAX at mRNA level was higher than that in control group (P<0.05). These effects could be reversed after treating iron-overloaded mice with DFX or NAC.</p><p><b>CONCLUSION</b>Iron overload can inhibit the ratio of CD8+ T cells of splenic cells in mice, decrease the expression of IFN-γ, Granzyme-B, increase the apoptosis of CD3+ CD8+/CD8-. These effects may be regulated through increasing the intracellular ROS level, and can be partially reversed after treating iron-overloaded mice with DFX or NAC.</p>

Expression of perforin and granzyme-B in peripheral blood lymphocyte in patients with prostate cancer and the clinical significance / 中南大学学报(医学版)

OBJECTIVE@#To explore the expression of perforin and granzyme-B in peripheral blood lymphocyte (PBL) in patients with prostate cancer (PCa) and the clinical significance.@*METHODS@#The expressions of perforin and granzyme-B in PBL were detected by fluorescence quantitative reverse transcription polymerase chain reaction. The results of perforin and granzyme-B expression were compared among patients with PCa (n=60), patients with BPH (benign prostatic hyperplasia, n=40) and healthy controls (n=20).@*RESULTS@#Th e expressions of perforin and granzyme-B in patients with PCa were significantly lower than that in patients with BPH or that in the healthy controls (P<0.05), respectively. Furthermore, in PCa patients with low pathological grade, the expressions of perforin and granzyme-B in PBL was statistically higher than that in patients with high pathological grade (P<0.05). The expressions of perforin and granzyme-B in PCa patients at high clinical stage was statistically lower than that in PCa patients at low clinical stage (P<0.05).@*CONCLUSION@#The results of this study suggest that development and progression of PCa might be associated with poor immune status of patients.

Attenuation of Peripheral Regulatory T-Cell Suppression of Skin-Homing CD8+T Cells in Atopic Dermatitis

PURPOSE: Cutaneous lymphocyte-associated antigen (CLA)-expressing CD8+T cells have been known to play an important role in the pathogenesis of atopic dermatitis (AD). However, the mechanisms underlying the loss of self-tolerance remain unclear. Regulatory T cells (Tregs) play a key role in the development of homeostasis in the immune system. We, therefore, hypothesized that a reduced ability of Tregs to inhibit autologous CD8+CLA+T cells might be underlying mechanism in AD. MATERIALS AND METHODS: CD8+CLA+T cells and Tregs were obtained from the peripheral blood of AD patients and control volunteers. The frequencies of CD8+CLA+T cells were evaluated. The proliferative responses of CD8+CLA+T cells were assessed by flow cytometry, and the levels of transforming growth factor-beta1 (TGF-beta1) and interleukin-10 (IL-10) in culture supernatants were detected by enzyme-linked immunosorbent assay. RESULTS: Our results revealed higher frequency and increased expression of perforin and granzyme-B in peripheral CD8+CLA+T cells in AD, and lower inhibitory ability of Tregs on proliferation of CD8+CLA+T cells in AD. Meanwhile, the levels of TGF-beta1 produced by Tregs were significantly lower in AD, and anti-TGF-beta1 abolished such suppression. CONCLUSION: The attenuated inhibitory ability of Tregs on hyper-activated autologous CD8+CLA+T cells, mediated by TGF-beta1, plays an important role in the pathogenesis of AD.

Expansion and function of MHC restricted killer T cells derived from umbilical cord blood / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was to expand the cytotoxic T lymphocytes (CTL) through inducing the differentiation of umbilical blood monomuclear cells (UBMNC) by using various combination of cytokines, and to investigate the functions of expanded CTL.</p><p><b>METHODS</b>The MNC were isolated by ficoll density gradient centrifugation. Then, the PHA-P, IFN-γ combined with IL-2, IL-15 and other cytokines were used for induction and expansion of the cord blood-derived CTL. The biological function of CTL was examined by phenotype analysis, cytotoxic tests and real-time fluorescence quantitative PCR.</p><p><b>RESULTS</b>After expansion for 15 days, the cell number increased by 1522% ± 137%. The content of CD3(-)CD8(-) cells in uncultured cord blood MNC was 95%, and the CD3(+)CD8(+) CTL cells reached 82.77% in cultured cord blood MNC after expansion for 15 days. The expanded CTL cell showed the cytotoxic activity against K562 and HeLa cell line. The killing rate of MNC was 61.88 ± 1.08%. After expansion, the killing rate could reach to 90% with the average value of 90.33 ± 2.02%. The expanded CTL cells highly expressed some key cytokines, such as granzyme A, granzyme B, GM-CSF, granulysin, IFN-γ, TGF-β, TNF-α and perforin. Compared with the control group, the expression of IFN-γ and TGF-β significantly increased (P < 0.05), and the other factors dramatically increased (P < 0.01).</p><p><b>CONCLUSION</b>The cord blood-derived CTL can be expanded by different combinations of cytokines. These protocols may provide alternative choices for CTL cell expansion in tumor adoptive immunotherapy.</p>

Association between gene polymorphisms of Perforin 1 and hemophagocytic lymphohistiocytosis / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate frequency distribution of gene polymorphisms of PRF1 gene in children with hemophagocytic lymphohistiocytosis (HLH), and to explore whether the possible gene polymorphisms of PRF1 gene confer an increased risk of susceptibility to HLH.</p><p><b>METHODS</b>Forty-eight children who were diagnosed with HLH between January 2009 and December 2013 (HLH group) and 100 healthy children (control group) were enrolled in this study. The gene polymorphisms in the coding region of PRF1 gene, which consists of three exons and two introns, were genotyped by PCR, followed by direct sequencing.</p><p><b>RESULTS</b>Three single nucleotide polymorphisms (SNPs) were revealed in the coding sequence of PRF1 in the 48 children with HLH. Seven SNPs were detected in the noncoding sequence. Other two SNPs in the noncoding sequence including rs10999426 and rs10999427 were detected only in 5 healthy children (5%). There was no significant difference in allelic frequencies of all the SNPs above between the HLH and control groups (P>0.05). Haplotype analysis showed there was a pair-wise linkage disequilibrium between rs10999426 and rs10999427 (D=1, r2=1), but there was no significant difference in the distribution of A-T haplotype between the HLH and control groups (P>0.05).</p><p><b>CONCLUSIONS</b>There is no association between gene polymorphisms of PRF1 gene and the susceptibility to HLH. There is a pair-wise linkage disequilibrium between rs10999426 and rs10999427, but a low detection rate of A-T haplotype in healthy children indicates that it might not play a protective role in the development of HLH.</p>

A novel PRF1 gene mutation in a fatal neonate case with type 2 familial hemophagocytic lymphohistiocytosis / 소아과

Hemophagocytic lymphohistiocytosis (HLH) occurs in the primary form (genetic or familial) or secondary form (acquired). The familial form of HLH (FHL) is a potentially fatal autosomal recessive disorder that occurs because of constitutional defects in cell-mediated cytotoxicity. Here, we report a fatal neonatal case of type 2 FHL (FHL2) that involved a novel frameshift mutation. Clinically, the newborn presented with severe sepsis-like features and required mechanical ventilation and continuous venovenous hemodiafiltration. Flow cytometry analysis showed marked HLH and complete absence of intracytoplasmic perforin expression in cytotoxic cells; therefore, we performed molecular genetic analyses for PRF1 mutations, which showed that the patient had a compound heterozygous mutation in PRF1, that is, c.65delC (p.Pro22Argfs*2) and c.1090_1091delCT (p.Leu364Glufs*93). Clinical and genetic assessments for FHL are required for neonates with refractory fever and progressive multiple organ failure, particularly when there is no evidence of microbiological or metabolic cause.

Development of NK cell expansion methods using feeder cells from human myelogenous leukemia cell line

BACKGROUND: Natural killer (NK) cells constantly survey surrounding tissues and remove newly generated cancer cells, independent of cancer antigen recognition. Although there have been a number of attempts to apply NK cells for cancer therapy, clinical application has been somewhat limited because of the difficulty in preparing a sufficient number of NK cells. Therefore, ex vivo NK cell expansion is one of the important steps for developing NK cell therapeutics. METHODS: CD3+ depleted lymphocytes were cocultured with IL-2 and with feeder cells (peripheral blood mononuclear cells [PBMCs], K562, and Jurkat) for 15 days. Expanded NK cells were tested for cytotoxicity against cancer cell lines. RESULTS: We compared feeder activities of three different cells-PBMC, K562, and Jurkat. K562 expanded NK cells by almost 20 fold and also showed powerful cytotoxic activity against cancer cells. K562-NK cells remarkably expressed the NK cell activation receptors, NKG2D, and DNAM-1. K562-NK cells exhibited more than two-fold production of cytotoxic granules compared with Jurkat-NK cells, producing more perforin and granzyme B than naive NK cells. CONCLUSION: Our findings suggest that K562 are more efficient feeder cells than Jurkat or PBMCs. K562 feeder cells expanded NK cells by almost 20 fold and showed powerful cytotoxic activity against cancer cells. We herein propose an intriguing approach for a design of NK cell expansion.

Analysis of clinical phenotype and genetic mutations of a pedigree of familial hemophagocytic lymphohistiocytosis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze mutations in a pedigree of familial hemophagocytic lymphohistiocytosis (FHLH) from Sichuan and provide genetic counseling for the family.</p><p><b>METHODS</b>Clinical data of a case with FHLH diagnosed at West China Second Hospital was retrospectively analyzed. Genomic DNA was extracted from peripheral blood samples of the proband and his family members. Eight candidate genes for primary HLH were amplified with PCR and analyzed by direct sequencing.</p><p><b>RESULTS</b>The proband was diagnosed as HLH based on clinical manifestations of recurrent fever for 2 months, hepatosplenomegaly, lymphadenopathy, pancytopenia, hyperferritinemia, and decreased fibrinogen and hemophagocytosis in bone marrow. Genetic testing for primary HLH was carried out considering the relapse of illness after hormone therapy for 8 weeks and the family history. The results of gene sequencing showed that the proband has carried compound heterozygous mutations in PRF1 gene (c.1349C> T in exon 3 and c.445G> A in exon 2). His father has carried a heterozygous mutation (c.445G> A in exon 2) and nonsense mutation (c.900C> T in exon 3), and his mother carried a heterozygous mutation (c.1349C> T in exon 3). Both c.1349C> T and c.445G> A have been previously reported as pathogenic mutations.</p><p><b>CONCLUSION</b>The family has been diagnosed as familial HLH type 2 based on clinical and laboratory examinations and molecular genetic testing. Gene sequencing has indicated that is was a recessive type familial HLH.</p>

Clinicopathologic features of intestinal natural killer/T-cell lymphoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the clinicopathologic features, diagnosis and differential diagnosis of intestinal natural killer (NK)/T-cell lymphoma.</p><p><b>METHODS</b>The clinical features, histopathology, immunohistochemical findings and follow-up data of 14 cases of intestinal NK/T-cell lymphoma were retrospectively reviewed.</p><p><b>RESULTS</b>The male-to-female ratio was 9:5. The medium age of patients was 45 years. The sites of involvement included small intestine (6 cases), colon (6 cases) or both (2 cases). The main clinical manifestations were an abdominal mass, other gastrointestinal symptoms such as abdominal pain, as well as systemic symptoms such as fever and cachexia. Intestinal perforation complicated by acute peritonitis might occur in advanced disease. Histologically, the intestinal wall showed full-thickness infiltration by medium-sized atypical lymphoid cells with pleomorphic nuclei, prominent inflammatory background, angiocentric/angiodestructive growth pattern and coagulative necrosis. Immunohistochemical study showed that the tumor cells were positive for CD3ε, CD43, CD56, granzyme B and perforin. They were negative for CD20, CD79α and MPO. In-situ hybridization for Epstein-Barr virus encoded RNA (EBER) showed negative signals. A high proliferative index was demonstrated by Ki-67 immunostaining. Follow-up data of 8 cases were available, with duration of follow up ranging from 0.5 to 36 months. Five patients died within 20 months.</p><p><b>CONCLUSIONS</b>Extranodal NK/T-cell lymphoma, nasal-type primarily involving intestine is rare and tends to carry an aggressive clinical course. The relatively non-specific clinical manifestations of intestinal NK/T-cell lymphoma may result in misdiagnosis in some cases. A comprehensive evaluation of clinical manifestations, pathologic features and immunohistochemical findings is essential for definitive diagnosis.</p>