Evaluation of cytotoxic function and apoptosis in interleukin (IL)-12/IL-15-treated umbilical cord or adult peripheral blood natural killer cells by a propidium-iodide based flow cytometry

Both deficient natural killer (NK) cell effector function and increased propensity to apoptosis of neonatal NK cells contribute to the increased susceptibility to infection in the neonates. Interleukin (IL)-12 and IL-15 are two immunoregulatory cytokines known to enhance cytolytic function of neonatal NK cells. The present study aims to simultaneously investigate the effect of IL-12/IL-15 on K562 cytotoxicity as well as NK cells apoptosis of enriched umbilical cord blood (CB) and adult peripheral blood (APB) NK cells, using flow cytometric cytotoxicity assays. The results indicated that (i) prior to cytotoxicity assays, CB NK cells underwent greater degree of spontaneous apoptosis than did APB NK cells; (ii) both IL-12 and IL-15 inhibited the spontaneous apoptosis of CB NK cells, while IL-15 promoted the apoptosis in APB NK cells; (iii) the deficient K562 cytotoxicity of CB NK cells could be enhanced to levels comparable with that of APB NK cells by IL-15; (iv) IL-15 increased the percentages of apoptosis in NK–K562 conjugates in a dose-dependant manner in both CB and APB with a greater effect seen with APB NK cells; (v) target-induced apoptosis was observed with APB NK cells which were further enhanced with IL-15. However, CB NK cells, unstimulated or IL-15-activated, were resistant to K562-induced apoptosis. Thus, the multi-parameter flow cytometry analysis not only demonstrates better for the deficient CB NK function but also provides greater details for cytotoxic mechanisms of NK cells.     

Human congenital infection with Trypanosoma cruzi induces phenotypic and functional modifications of cord blood NK cells

We studied the phenotype and activity of cord blood natural killer (NK) cells in newborns congenitally infected with Trypanosoma cruzi. We found that the proportion of CD56(bright) NK cells was significantly decreased in cord blood from these newborns, suggesting they may have been recruited to secondary lymphoid organs. The remaining CD56(bright) NK cells exhibited a defective ability in the production of interferon (IFN)-gamma following in vitro activation with interleukin (IL)-12 + IL-2 or IL-12 + IL-15 cytokines, as compared with NK cells from uninfected newborns. In addition, cord blood NK cells from congenitally infected newborns stimulated with cytokines have a decreased release of granzyme B (GrB) when incubated with K562 target cells. This defect in cytotoxic effector function is associated with a reduced surface expression of activating NK receptors (NKp30, NKp46, and NKG2D) on CD56(dim) NK cells compared with uninfected newborns. These alterations of fetal NK cells from congenitally infected newborns may reflect a down-regulation of the NK cell response after an initial peak of activation and could also be the result of T. cruzi modulating the immune response.     

Effector Mechanisms against Neuroblastoma Cells and Their Modulation by Lymphokines

The susceptibility of four neuroblastoma (NB) cell lines, SKN-DZ, GOTO, IMR-32, and NB-1 to natural killer (NK) cells and cytotoxic T cells (CTL) and its modulation by lymphokines were studied using a ⁵¹ Cr-release assay in vitro. The cytotoxic activity against 4 NB cell lines differed very much from person to person. However, the activity from every donor was significantly enhanced by culturing of peripheral blood lymphocytes (PBL) with lymphokines, interleukin-2 (IL2) or interferon (IFN), for 12 hours. Cold target inhibition assay revealed that the cytotoxic activity of IL2– or IFN-treated cells against NB cell lines was nonspecific and was inhibited by NK-sensitive K562. In addition, the CTL were not induced by culturing of PBL with mitomycin C-treated NB cell lines for five days, even in the presence of IL2. These results suggest that it is not cytotoxic T cells but NK cells that are important as effector cells to NBs and that the increase in NK cell activity due to lymphokines may be of profound biological significance in immune surveillance of NBs.     

Antibody-Dependent Cellular Cytotoxicity and Natural Killer Activity Against HTLV-1 Infected Cells

Antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) ativity were examined using MT-2 cells persistently infected by HTLV-1 as target cells, and mononuclear cells as effector cells, from helathy one-week-old newborn babies, infants, children and adults. More than 10% of ADCC was observed in 17 newborn babies out of 22 (77.3%) and in all 67 healthy one-month-old babies to adults, by adding serum from anti-HTLV-1 positive carriers. When anti-HTLV-1 negative serum was added, less than 10% of ADCC was observed. If infants without anti-HTLV-1 antibodies were breast-fed they had the possibility of HTLV-1 vertical transmission. There was no significant decrease in NK activity between 90 healthy newborn babies, infants, children, or adults. These results suggest that ADCC and NK activity protect against the transmission of HTLV-1 from mother to child.     

Genotype, phenotype, and outcomes of nine patients with T-B+NK+ SCID

There are few reports of clinical presentation, genotype, and HCT outcomes for patients with T-B+NK+ SCID. Between 1981 and 2007, eight of 84 patients with SCID who received and/or were followed after HCT at UCSF had the T-B+NK+ phenotype. One additional patient with T-B+NK+ SCID was identified as the sibling of a patient treated at UCSF. Chart reviews were performed. Molecular analyses of IL7R, IL2RG, JAK3, and the genes encoding the CD3 T-cell receptor components δ (CD3D), ε (CD3E), and ζ (CD3Z) were carried out. IL7R mutations were documented in four patients and CD3D mutations in two others. Three patients had no defects found. Only two of nine patients had an HLA-matched related HCT donor. Both survived, and neither developed GVHD. Five of seven recipients of haploidentical grafts survived. Although the majority of reported cases of T-B+NK+ SCID are caused by defects in IL7R, CD3 complex defects were also found in this series and should be considered when evaluating patients with T-B+NK+ SCID. Additional genes, mutations in which account for T-B+NK+ SCID, remain to be found. Better approaches to early diagnosis and HCT treatment are needed for patients lacking an HLA-matched related donor.     

Activation of NKT Cells by IL 15 Plus GM CSF Stimulation and Its Cytotoxicity to Neuroblastoma Cells

Ｎｅｕｒｏｂｌａｓｔｏｍａ (ＮＢ)ｉｓｔｈｅｍｏｓｔｃｏｍｍｏｎｍａｌｉｇ ｎａｎｔｔｕｍｏｒｏｆｔｈｅｓｙｍｐａｔｈｅｔｉｃｎｅｒｖｏｕｓｓｙｓｔｅｍｉｎｃｈｉｌ ｄｒｅｎ .Ｔｈｅｌｏｎｇ ｔｅｒｍｓｕｒｖｉｖａｌｉｓｌｏｗｗｉｔｈｆｒｅｑｕｅｎｔｒｅ ｃｕ     

Role of interleukin‐15 in the development of mouse olfactory nerve

Interleukin (IL)‐15 interacts with components of the IL‐2 receptor (R) and exhibits T cell‐stimulating activity similar to that of IL‐2. In addition, IL‐15 is widely expressed in many cell types and tissues, including the central nervous system. We provide evidence of a novel role of IL‐15 in olfactory neurogenesis. Both IL‐15 and IL‐15Rα were expressed in neuronal precursor cells of the developing olfactory epithelium in mice. Adult IL‐15Rα knockout mice had fewer mature olfactory neurons and proliferating cells than wild‐type. Our results suggest that IL‐15 plays an important role in regulating cell proliferation in olfactory neurogenesis.     

Ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children

BACKGROUND: Allogeneic natural killer (NK) cells are known to show medium to high cytotoxic activity against HLA-nonidentical leukemia or tumor cells. For a possible benefit of post transplant treatment with NK cells after haploidentical stem cell transplantation (haplo-SCT) we developed a clinical scale procedure for NK cell processing observing Good Manufacturing Practice (GMP). METHODS: Allogeneic donor NK cells were selected from 15 unstimulated leukaphereses using two rounds of immunomagnetic T cell depletion, followed by an NK cell enrichment step. CD56 (+)CD3 (-) NK cells were stimulated and expanded in vitro according to GMP. Quality control of NK cell purity, residual T cells and cytotoxic activity was done by multi-coloured flow cytometric analyses. RESULTS: Purification led to an absolute number of 234-1 237 x 10 (6) CD56 (+)CD3 (-) NK cells from leukapheresis harvests with a median purity of 95 % and a 4 to 6(1/2) log depletion of T cells. After two weeks stimulation with IL-2 a five-fold expansion of NK cells with a T cell contamination below 0.1 % was reached. Median cell viability was 95 % after purification and 99 % after expansion. The IL-2 stimulated NK cells showed a highly increased lytic activity against the MHC-I deficient K562 cells compared to freshly isolated NK cells and a medium cytotoxicity against patients' leukemic cells. CONCLUSIONS: Clinical scale enrichment and activation of allogeneic donor NK cells is feasible. High dose NK cell application may be a new treatment option for pediatric patients with leukemia or solid tumors in case of minimal residual disease or unbalanced chimerism post haplo-SCT as we could show for the first three patients .     

Impaired natural killer activity in lymphohistiocytosis syndrome

In six patients with well-documented lymphohistiocytosis syndrome, natural killer activity was found to be profoundly impaired and could not be increased by incubation in vitro with interferon. This abnormality was not found in parents of the affected children. A clear correlation with the activity of the disease was observed, although a delay of a few weeks (possibly reflecting the life span of NK cells in blood) was seen in the disappearance of NK activity after the onset of the disease and its reappearance after remission. No absolute correlation was observed between NK activity and percentage of leukocytes detected by the Leu-7 monoclonal antibody. Our findings indicate that testing NK activity is useful for the diagnosis of lymphohistiocytosis syndrome and can be used as an index of activity of the disease, among other major clinical and biologic signs of this syndrome. Reversal of the NK activity defect (rather than detection of Leu-7 positive cells) appears to be a good criterion of complete remission.     

Cytotoxic Function of Umbilical Cord Blood Natural Killer Cells: Relevance to Adoptive Immunotherapy

Decreased graft-versus-host disease (GVHD), ease of accessibility, and sustained engraftment encourage the use of umbilical cord blood (UCB) as an alternative source to bone marrow for immune reconstitution in children with leukemia. Natural killer (NK) cells rapidly expand after stem cell transplantation and are important for regulating GVHD and providing graft-versus-leukemia (GVL) effects. This review highlights the phenotypic and functional differences between UCB NK cells and adult peripheral blood (APB) NK cells, and discusses the possible therapeutic benefit of using UCB NK cells for adoptive immunotherapy in leukemia. Alloreactive NK cells show potent cytotoxic activities against human leukocyte antigen (HLA)-nonidentical leukemic cells and reduce leukemia relapses. The higher numbers of NK progenitors in UCB makes it a convenient source for ex vivo expansion of UCB NK cells for posttransplant treatment. UCB NK cells readily respond to interleukin-15, which may greatly enhance their antitumor effect. Activation and expansion protocols for UCB NK cells are currently being developed.     

Cytotoxic function of umbilical cord blood natural killer cells: relevance to adoptive immunotherapy

Decreased graft-versus-host disease (GVHD), ease of accessibility, and sustained engraftment encourage the use of umbilical cord blood (UCB) as an alternative source to bone marrow for immune reconstitution in children with leukemia. Natural killer (NK) cells rapidly expand after stem cell transplantation and are important for regulating GVHD and providing graft-versus-leukemia (GVL) effects. This review highlights the phenotypic and functional differences between UCB NK cells and adult peripheral blood (APB) NK cells, and discusses the possible therapeutic benefit of using UCB NK cells for adoptive immunotherapy in leukemia. Alloreactive NK cells show potent cytotoxic activities against human leukocyte antigen (HLA)-nonidentical leukemic cells and reduce leukemia relapses. The higher numbers of NK progenitors in UCB makes it a convenient source for ex vivo expansion of UCB NK cells for posttransplant treatment. UCB NK cells readily respond to interleukin-15, which may greatly enhance their antitumor effect. Activation and expansion protocols for UCB NK cells are currently being developed.     

Polymorphonuclear leucocyte and natural killer cell functions in mature and premature newborns

In this study the phagocytic and natural killer cell (NK) functions in 17 premature and 30 mature newborns are compared. The ability of polymorphonuclear phagocytes (PMNs) to ingest, digest and lyse (antibody-dependent cell-mediated cytotoxicity, ADCC) opsonized sheep red blood cells and NK activity were tested. Examinations were performed in cord and venous blood within 6 h or 3-4 days after delivery. Results of examinations were compared with normal values for the group of healthy 4- to 15-year-old children. To assess the influence of the newborn's maturity and age on the tested PMNs and NK functions, the following comparisons were made. (1) Cord vs. peripheral venous blood: only ADCC was higher in peripheral than in cord blood. (2) Mature newborn cells obtained either 6 h or 3-4 days after delivery: ingestion and ADCC were lower and NK activity was higher 3-4 days after delivery. (3) Premature vs. mature newborn cells tested 3-4 days after delivery: ingestion and ADCC were higher while NK activity was lower in premature newborns. (4) Premature newborns tested at 3-4 days vs. mature newborns tested within 6 h after birth: ingestion was lower in the prematures while digestion, ADCC and NK activity were similar. (5) Cells from all newborns tested vs. those of healthy older children: results depend on the interval after birth when newborns were tested. Thus, within the first 6 h after delivery, mature newborns had higher ingestion and ADCC capacity but lower digestion and NK activity. Later, 3-4 days after birth, ingestion, ADCC and NK activity were lower in mature newborns. In the prematures at that interval NK activity was lower. (6) There was a positive correlation between gestational age and NK activity of newborns.     

Immunological aspects of progeria (Hutchinson-Gilford syndrome) in a 15-month-old child

A thorough analysis of the immunological status was conducted in a 15-month-old child with progeria (Hutchinson-Gilford syndrome). Total leukocyte and neutrophil counts were slightly increased, and monocytes were decreased. Percentage and number of CD4+ cells in the blood were mildly decreased as well as the CD4/CD8 cell ratio. CD20 (B-cell marker) bearing cells and cells bearing Ia-antigens were increased, as well as CD16 and CD56 marker-bearing cells (natural-killer cells, NK). Kymphocyte proliferation upon stimulation with phytohaemagglutinin and purified protein derivative were decreased, and with pokeweed mitogen increased. NK cell activity appeared increased, particularly at lower effector: target cell ratios.     

特应质的肺炎支原体肺炎患儿自然杀伤细胞亚型的变化

目的 探讨肺炎支原体肺炎(MPP)患儿NK细胞亚型的变化及其与特应质之间的关系.方法 收集2010年7月-2011年2月于本院住院或门诊就诊的MPP患儿33例,其中MPP伴特应质患儿18例,MPP不伴特应质组15例.同时收集非MPP(NMPP)患儿20例,其中NMPP伴特应质患儿9例,NMPP不伴特应质患儿11例.另收集15例健康儿童作为健康对照组.收集所有研究对象外周静脉血标本,采用密度梯度离心法分离获得外周血单个核细胞,分别用标准刺激剂[佛波酯(PMA)+离子霉素(Ion)]孵育4 h,或特异性肺炎支原体(MP)抗原孵育育24h,或不予刺激剂孵育,应用流式细胞术测定NK细胞内细胞因子IL-4和IFN-γ水平.结果 1.PMA+ Ion刺激后,MPP组、NMPP组和健康对照组NK细胞内IL-4、IFN-γ表达的差异均无统计学意义(Pa＞0.05).2.在MP抗原不同水平刺激下(0.1μg·L-1、0.2μg·L-1),MPP组NK细胞内IL-4、IFN-γ水平高于NMPP组及健康对照组(Pa＜0.05),NMPP组与健康对照组无统计学差异(Pa＞0.05).MPP伴特应质组IL-4水平高于MPP不伴有特应质组(Pa＜0.05),而IFN-γ表达水平在2组间则无明显差异(Pa＞0.05):IL-4和IFN-γ表达水平在NMPP伴特应质组和NMPP不伴特应质组患儿之间均无明显差异(Pa＞0.05).3.MPP组提高MP抗原刺激水平,IL-4表达水平的增幅随之上升,而IFN-γ表达水平的增幅则逐渐下降.结论 在MPP患儿中,标准刺激剂不能改变NK细胞来源的IL4、IFN-γ水平,用MP抗原刺激能明显提升NK细胞来源的IL-4及IFN-γ水平,且以IL-4升高为主.这一现象在MPP伴特应质的患儿中更为突出,且与MP抗原水平有关.MP感染促使NK细胞来源的IL-4水平明显升高,与特应质相互协同,从而加重过敏症状.     

Clinical characteristics and mutation analysis of X-linked severe combined immunodeficiency in China

Abstract

X-linked severe combined immunodeficiency (X-SCID) is a rare, life-threatening immune disorder, caused by mutations of the gene for the γ-chain (γc) of the interleukin-2 receptor, IL2RG. We analyzed the clinical, immunologic, and molecular characteristics of children with X-SCID, attempting to improve the diagnosis and treatment of X-SCID in China.

Methods

X-SCID was suspected in male infants with recurrent or persistent infections. Eleven male infants from ten unrelated Chinese families were included. The IL2RG gene was amplified and sequenced, followed by mutation analysis in these children and their female relatives. X-linked short tandem repeat (X-STR) typing was done to define the maternal lymphocyte engraftment.

Results

The 11 children exhibited recurrent infections and 10 of them had lymphopenia. B cells were present in all patients, T cells were markedly reduced in 10, and NK cells were markedly reduced in 9. Nine IL2RG gene mutations were identified in the 11 children, with 5 novel mutations. One patient was found to have the maternal lymphocyte engraftment.

Conclusion

The clinical presentations and immunologic characteristics of the X-SCID patients were accordingly quite uniform despite the heterogeneity of mutations locating almost in the entire γc gene.     

神经母细胞瘤脂类提取物对NKT细胞激活作用的实验研究

目的 研究神经母细胞瘤脂类提取物对健康儿童自然杀伤T（NKT）细胞增殖程度和对NKT细胞介导的对自身肿瘤细胞的细胞毒作用的影响。方法 甲醇／氯仿法提取神经母细胞瘤脂类提取物作为刺激物，采集健康儿童外周血单核细胞，培养出成熟的树突细胞，采集并分离同一个体外周血T细胞与树突细胞共同培养，分别加入脂类提取物或（和）白细胞介素15（IL－15）和粒－单系细胞集落刺激因子（GM－CSF）。测定各组T细胞的增殖程度的变化，流式细胞仪测定NKT细胞占T细胞的比例（NKT／T）并分选出NKT细胞对神经母细胞瘤细胞毒作用。结果 肿瘤脂类提取物刺激组T细胞增殖程度、NKT／T比例、NKT细胞毒作用高于对照组（P＝0．043；P＝0．020；P＝0．032）。共刺激组（肿瘤脂类提取物＋IL－15＋GM－CSF）T细胞增殖程度、NKT／T比例、NKT细胞毒作用高于对照组（P＝0．001；P＝0．001；P＝0．020），肿瘤脂类提取物刺激组（P＝0．004；P＝0．030；P＝0．010）和细胞因子刺激组（P＝0．044；P＝0．049；P＝0．048）。结论 神经母细胞瘤脂类提取物可以有效激活NKT细胞，发挥对自身肿瘤细胞的细胞毒作用。细胞因子IL－15和GM－CSF可以增强脂类提取物的激活作用。     

NK cell immunotherapy for high-risk neuroblastoma relapse after haploidentical HSCT

Little is known on strategies to prevent or to treat relapses occurring after haploidentical stem cell transplantation (haplo-HSCT) performed for the high-risk neuroblastoma (NB). We describe a 6-year-old male with refractory NB who relapsed 22 months after haplo-HSCT. A complete remission was obtained with a combination of immuno-chemotherapy based on donor NK cells transplants, IL2 infusions and temozolomide/topotecan. This case is an incentive to explore both the immediate therapeutic effect of haplo-graft provided via haplo-NK cells and the immunogenic platform that haplo-HSCT offers for future treatment. Our post-relapse strategy shows that chemo- and bio-treatment should be viewed as complementary therapeutic options.     

Interleukin-15 enhances CD4+ CD45RA+ expression on umbilical cord blood mononuclear cells

The reduced incidence of graft‐vs.‐host disease following umbilical cord blood (CB) transplantation may be related to the functional immaturity of newborn T cells expressing mainly the naive CD45RA phenotype. Expansion of CD4⁺ CD45RA⁺ T cells using cytokines may benefit neonates and infants with human immunodeficiency virus (HIV) infection, as a preferential decline in CD4⁺ CD45RA⁺ cells has been noted as HIV disease progresses. The aim of the study was to investigate the effect of interleukin (IL)‐15, a novel cytokine similar to IL‐2 in biological activities, on CD45RA/RO expression and apoptosis in umbilical cord blood (CB) and adult peripheral blood (APB) mononuclear cells (MNCs). Prior to culture, CB MNCs contained a greater number of CD4⁺ CD45RA⁺ cells and fewer CD4⁺ CD45RO⁺ cells than did APB MNCs. When incubated with RPMI‐1640 containing 10% fetal calf serum for 7 days, the percentage of CD45RA⁺ cells within CD4⁺ T cells (%CD45RA⁺/CD4⁺) significantly decreased compared to that of fresh CB MNCs. IL‐15 exerted a dose‐dependent increase of %CD45RA⁺/CD4⁺ and a corresponding decrease of %CD45RO⁺/CD4⁺ in CB MNCs, an effect not observed with APB MNCs treated with IL‐15. The percentages of CD45RA⁺ and CD45RO⁺ expression within CD8⁺ cells, however, were not influenced by IL‐15, in either CB or APB MNCs. A greater number of CB MNCs underwent apoptosis than did APB MNCs after 7 days of culture in RPMI‐1640 containing 10% fetal calf serum. IL‐15 did not inhibit apoptosis but induced proliferation comparable to that achieved in APB MNCs. The ability of IL‐15 to preferentially enhance the proliferation of CD4⁺ CD45RA⁺ cells in CB MNCs suggests a role for immunomodulative therapy in HIV‐infected newborns and infants.     

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目的了解布地奈德联合沙丁胺醇雾化吸入治疗儿童哮喘急性发作前后其气道炎性细胞、白细胞介素-6(IL-6)、IL-8、肿瘤坏死因子-α(TNF-α)的变化,探讨其影响机制。方法对上海市第五人民医院2002-09—2003-12收治的急性哮喘发作患儿采用上述联合治疗1周,共对34例急性发作期、24例缓解期患儿和15名正常儿童的诱导痰液进行炎性细胞计数和分类,测定其中IL-6、IL-8、TNF-α水平。结果急性期哮喘患儿的总细胞数、嗜酸性细胞、中性粒细胞和单核细胞比例及IL-8、IL-6、TNF-α水平均高于正常对照组。缓解期哮喘患儿除嗜酸细胞及淋巴细胞比例明显高于正常对照组外,其余上述炎性细胞比例及细胞因子水平均降至正常。结论布地奈德联合沙丁胺醇雾化吸入可显著降低急性哮喘发作患儿气道分泌物内嗜酸性细胞、中性粒细胞和单核细胞数量及IL-8、IL-6、TNF-α水平。     

Natural killer cell function in children with malignant solid neoplasias

Natural killer (NK) cell numbers and lytic activity were determined in 40 children with various types of solid malignant neoplasias and in 25 control children by NKH-1 monoclonal antibody and cytotoxicity against K562 target cells, respectively. Patients were analyzed at the time of diagnosis before initiation of therapy and followed over a median time of 15.8 months. Mean NK cell numbers and lytic activity were similar among different types of tumor analyzed. Patients with localized disease (stages I, II; n = 25) also showed values not statistically different from those of patients in advanced disease (stages III, IV; n = 15). According to their response to therapy, patients were divided into three groups: group 1 (complete remission; n = 28), group 2 (partial remission; n = 5), and group 3 (progression of disease; n = 6). Patients in group 3 showed at the time of diagnosis a mean NK activity significantly lower than that of patients in groups 1 and 2 and control children (P = 0.007). The defect in NK cell lytic capacity in vitro observed in patients with progressive disease suggests that NK cells play a role in the control of neoplastic growth in vivo and may imply that some children with refractory progressive disease can benefit from immunomodulation destined to improve the lytic potential of NK cells.