目的 探讨细胞因子IL-23与IL-12对NK细胞功能的影响及可能的机制.方法 密度梯度离心法分离人外周血单个核细胞(PBMCs)或磁珠纯化NK细胞,不刺激或用IL-23或IL-12刺激,用流式细胞术和ELISA法检测NK细胞产生IFN-γ的情况;以K562或Jurkat细胞作为靶细胞,用流式细胞术检测NK细胞的杀伤功能并分析NK细胞在不同的刺激条件下杀伤相关分子的表达情况及pSTAT的表达情况.结果 与未刺激组相比,IL-23和IL-12均可以诱导NK细胞呈剂量和时间依赖方式产生IFN-γ;但IL-12而非IL-23可以增强NK细胞对靶细胞K562或Jurkat细胞的杀伤功能.进一步研究表明,IL-12而非IL-23可以诱导杀伤相关分子TRAIL及CD107a/b的表达.此外,IL-12诱导NK细胞表达更高水平的pSTAT4,而IL-23诱导NK细胞表达更高水平的pSTAT3.结论 与IL-12相比,IL-23亦可以诱导NK细胞产生细胞因子但不能增强NK细胞的杀伤功能,IL-23不能诱导杀伤相关分子TRAIL及CD107a/b的表达,IL-23可以诱导低水平的pSTAT4但高水平的pSTAT3的表达.

目的 鉴定1例非肌性肌球蛋白重链9(MYH9)基因相关疾病家系的致病突变.方法 调查收集先证者和家系成员病史资料,观察其临床特征和实验室检查指标.采用芯片捕获高通量测序方法检测先证者MYH9基因,确定突变位点后,对先证者和家系成员进行Sanger验证.结果 该家系三代4名患者均有鼻衄、瘀斑紫癜、外伤血肿或月经量增多病史,长期存在镜下血尿和蛋白尿.血涂片镜检均有血小板减少、巨大血小板和粒细胞异常包涵体"三联征".所有患者MYH9基因第40内含子供体剪接位点存在错义突变c.5765+2T>A(p.R1922Rfs43),且该基因突变与疾病表型共分离.结论 该家系存在MYH9基因c.5765+2T>A(p.R1922Rfs43)剪接突变,是MYH9基因相关疾病的致病突变,为国内首次报道.

目的 探讨结核性胸液细胞(pleural fluid cells,PFCs)中NK细胞的活化标志、记忆相关表型、NK细胞表面受体表达以及分泌细胞因子IFN-γ的情况.方法 以正常人外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)作对照,应用多色流式细胞术分析结核性胸液中NK细胞活化及记忆相关表面分子的表达情况,同时分析在细胞因子IL-12的作用下结核性胸液中NK细胞分泌IFN-γ的情况.结果 与PBMCs中的NK细胞相比,PFCs中的NK细胞表达高水平早期活化标志CD69,并表达T细胞记忆相关分子CD45RO.进一步分析NK细胞的记忆相关表型,约50％的CD45RO+NK细胞同时表达CD27,而几乎99％的CD45RO+NK细胞不表达CD62L.PFCs中约7.3％的NK细胞同时表达CD45RO和NKG2D,约2.4％的NK细胞同时表达CD45RO和NKG2A.NK细胞功能实验提示在细胞因子IL-12的作用下,CD45RO+NK细胞分泌更高水平的IFN-γ.结论 结核性胸液中NK细胞具有记忆细胞的表型特征,表现出CD45RO+CD27+CD62L-效应型记忆细胞特征,在IL-12因子刺激下,CD45RO+NK细胞产生更高水平的IFN-γ,可能在预防结核感染中发挥重要作用.

目的 应用E6流式细胞仪建立检测CD34+细胞百分比和绝对计数的方法并进行验证.方法 收集2019年1月至2019年12月在深圳市儿童医院血液肿瘤科因重型β-地中海贫血行异基因造血干细胞移植的患儿89例,以移植患儿供的骨髓或外周血为研究对象,应用E6流式细胞仪建立ISHAGE法检测CD34+细胞百分比,应用"流式直接体积法"计算CD34+细胞的绝对计数,并结合白细胞手工计数对CD34+细胞绝对计数的结果进行验证.结果 结合白细胞手工计数对CD34+细胞的绝对计数结果的正确性进行验证,Pearson相关分析结果显示:供者外周血干细胞或骨髓干细胞CD34+细胞绝对计数流式结果与CD34+细胞绝对计数计算结果高度相关(r=0.93,R2=0.87,P<0.0001);两次供者外周血干细胞或骨髓干细胞白细胞计数和单个核百分比结果高度一致;同样,两次供者外周血干细胞或骨髓干细胞CD34+细胞流式计数百分比和绝对计数结果亦高度一致.结论应用E6流式细胞仪的"流式直接体积法"计算CD34+细胞绝对计数结果准确,可以作为造血干细胞移植术前计算供者外周血干细胞或骨髓干细胞CD34+细胞数目的可靠依据.

目的 探讨急性髓系白血病自然缓解的诱因和可能的发生机制.方法 回顾分析1例急性单核细胞白血病发生自然缓解患儿的临床资料,并结合相关文献探讨自然缓解的可能机制.结果 1岁4月龄男性患儿,确诊为急性髓系单核细胞白血病,未经化疗后发生骨髓自然缓解,约1年后出现髓系肉瘤复发,予行HLA半相合造血干细胞移植术,目前预后良好.回顾既往文献发生自然缓解患者大部分缓解之前有发热史,通常由感染导致,也可由药物撤除或输血导致.结论 髓系白血病发生自然缓解很少见,未来或许可以通过免疫疗法达到治疗白血病的目的.

目的 动态监测重型β-地中海贫血患儿异基因造血干细胞移植(allo-HSCT)术前及术后第1、2、3、6和12个月淋巴细胞亚群比例的变化,探讨淋巴细胞的恢复情况及重建特点.方法 选取行allo-HSCT的27例重型β-地中海贫血患儿,其中人类白细胞抗原(HLA)全相合移植术20例,HLA不全相合移植术7例.采用流式细胞术(FCM)动态监测患者移植前及术后1年内CD3+T细胞、CD3+CD4+T细胞、CD3+CD8+T细胞、CD4+/CD8+比值、CD19+B细胞及CD16+CD56+自然杀伤(NK)细胞比例的变化.结果 与allo-HSCT术前比较,重型β-地中海贫血患儿术后CD16+CD56+NK细胞比例恢复最快,术后第1、2、3、6个月均高于术前(P<0.05),第12个月恢复至术前水平(P>0.05).CD3+T细胞比例术后第1个月与术前比较差异无统计学意义(P>0.05),第2个月起降低且低于术前(P<0.01),第12个月仍低于术前(P<0.01).CD3+CD8+T细胞比例术后第1个月明显高于术前(P<0.01),随后逐渐下降,至术后第12个月时仍高于术前(P<0.05).CD3+CD4+T细胞比例术后明显低于术前(P<0.01),且呈缓慢回升趋势,至术后第12个月仍明显低于术前(P<0.01).CD4+/CD8+比值长期倒置,虽然呈缓慢上升趋势,但仍明显低于术前(P<0.01).CD19+B细胞比例术后第1个月明显低于术前(P<0.01),术后第2个月恢复至术前水平(P>0.05),术后第6个月和第12个月明显高于术前(P<0.01、P<0.05).术前及术后同一时间点男性、女性患儿之间及HLA全相合移植与HLA不全相合移植之间各淋巴细胞亚群比例差异均无统计学意义(P>0.05).结论 重型β-地中海贫血患儿allo-HSCT术后淋巴细胞亚群的免疫重建恢复顺序依次为CD16+CD56+NK细胞、CD3+CD8+T细胞、CD19+B细胞、CD3+T细胞、CD3+CD4+T细胞.

目的 比较腺病毒(adeno virus,ADV)与呼吸道合胞病毒(respiratory syncytial virus,RSV)肺炎患儿及健康儿童外周血淋巴细胞亚群的百分比例并探讨其临床意义.方法 收集2014年1月～2015年12月期间在深圳市儿童医院住院的腺病毒肺炎患儿(ADV组)30例,呼吸道合胞病毒肺炎患儿(RSV组)30例(入选依据:60例病毒感染患儿肺部听诊均可闻及中小湿啰音,肺部X线片有点状或片状阴影,临床诊断为肺炎,直接免疫荧光法检测相关病毒抗原结果为阳性且无其他病原感染的临床及实验室证据)以及健康体检儿童(健康组)35例,应用流式细胞术检测上述研究对象外周血中CD3+T细胞,CD3+CD4+T细胞,CD3+CD8+T细胞,CD3-CD56+NK细胞及CD3-CD19+B细胞的百分比并比较各组间的差异.结果 与健康组相比较,ADV组[(55.04±8.45)％vs(69.07±9.30)％,t=3.32,P=0.002]与RSV组[(60.90±11.12)％vs(69.07±9.30)％,t=2.43,P=0.004]的CD3+T细胞比例均下降,差异具有统计学意义(P<0.01),特别是ADV组[(21.26±6.16)％vs(26.97±7.63)％,t=1.87,P=0.04]与RSV组[(21.56±7.06)％vs(26.97±7.63)％,t=1.68,P=0.04]的CD3+CD8+T细胞比例明显下降,差异具有统计学意义(P<0.05);与此同时,与健康组相比,ADV组[(28.22±5.16)％vs(17.06±6.42)％,t=3.25,P=0.001]和RSV组[(29.14±10.96)％vs(17.06±6.42)％,t=2.79,P=0.009]的CD3-CD9+B细胞比例升高,差异具有统计学意义(P<0.05).与健康组[(13.54±5.95)％,t=2.01,P=0.03]及ADV组[(16.26±8.34)％,t=2.89,P=0.005]相比,RSV组的CD3-CD56+NK细胞亚群比例[(9.59±5.54)％]下降,差异具有统计学意义(P<0.05).结论 腺病毒肺炎和呼吸道合胞病毒肺炎患儿外周血淋巴细胞亚群比例存在统计学差异,病毒性肺炎患儿体内可能存在免疫应答紊乱.

Our previous result indicated that memory-like human natural killer (NK) cells from TB pleural fluid cells (PFCs) produced large amounts of IFN-γ in response to Bacille Calmette Guerin (BCG). Furthermore, recent studies have shown that human lymphoid tissues harbored a unique NK cell subset that specialized in production of interleukin (IL)-22, a proinflammatory cytokine that mediates host defense against pathogens. Yet little information was available with regard to the properties of IL-22 production by memory-like human NK cells. In the present study, we found that cytokines IL-15 induced and IL-12 enhanced the levels of IL-22 by NK cells from TB PFCs. In addition, IL-22 but not IL-17 was produced by NK cells from PFCs in response to BCG and M.tb-related Ags. More importantly, the subset of specific IL-22-producing NK cells were distinct from IFN-γ-producing NK cells in PFCs. CD45RO+ or CD45RO- NK cells were sorted, co-cultured with autologous monocytes and stimulated with BCG for the production of IL-22. The result demonstrated that CD45RO+ but not CD45RO- NK cells produced significantly higher level of IL-22. Anti-IL-12Rβ1 mAbs (2B10) partially inhibit the expression of IL-22 by NK cells under the culture with BCG. Consistently, BCG specific IL-22-producing NK cells from PFCs expressed CD45ROhighNKG2Dhighgranzyme Bhigh. In conclusion, our data demonstrated that memory-like antigen-specific CD45RO+ NK cells might participate in the recall immune response for M. tb infection via producing IL-22, which display a critical role to fight against M. tb.

Natural killer T (NKT) cells from mouse and human play an important role in the immune responses against Mycobacterium tuberculosis. However, the function of CD3(+)TCRvβ11(+) NKT cells at the local site of M. tuberculosis infection remains poorly defined. In the present study, we found that after stimulation with M. tuberculosis antigens, NKT cells isolated from tuberculosis (TB) pleural fluid mononuclear cells (PFMCs) produced IL-21 and other cytokines including IFN-γ, TNF-α, IL-2 and IL-17. IL-21-expressing NKT cells in PFMCs displayed effector memory phenotype, expressing CD45RO(high)CD62L(low)CCR7(low). Moreover, NKT cells expressed high levels of CXCR5 and all of IL-21-expressing NKT cells co-expressed CXCR5. The frequency of BCL-6-expression was higher in IL-21-expressing but not in non-IL-21-expressing CD3(+)TCRvβ11(+) NKT cells. Sorted CD3(+)TCRvβ11(+) NKT cells from PFMCs produced IFN-γ and IL-21 after stimulation, which expressed CD40L. Importantly, CD3(+)TCRvβ11(+) NKT cells provided help to B cells for the production of IgG and IgA. Taken together, our data demonstrate that CD3(+)TCRvβ11(+) NKT cells from a local site of M. tuberculosis infection produce IL-21, express CXCR5 and CD40L, help B cells to secrete IgG and IgA, and may participate in local immune responses against M. tuberculosis infection.

We have previously shown that human memory-like NK cells were persistent in tuberculous pleurisy but it was unclear how NK cells migrated into the pleural fluids. At present, we found that NK cells from TB pleural fluid cells (PFCs) expressed significantly higher levels of CXCR3 and CXCR4 than NK cells from PBMCs. Migration assay demonstrated that IP-10 and SDF-1 induced more migration of NK cells from PFCs than PBMCs. CD45RO+ or CD45RO− NK cells from PFCs were co-cultured with autologous monocytes and stimulated with BCG. The results showed CD45RO+ but not CD45RO− NK cells produced significantly higher levels of IFN-γ, which was IL-12-dependent since anti-IL-12Rβ1 mAbs could significantly inhibit the IFN-γ by NK cells. Collectively, our data demonstrated that human Mycobacterium tuberculosis-specific NK cells were migrated into the local site of TB infection mainly via IP-10/CXCR3 and SDF-1/CXCR4 axis, memory-like NK cells might display an important role against M. tuberculosis infection.

In schistosomiasis, limited information is available about the role of interleukin-17 (IL-17) in lung, despite the fact that this cytokine plays a crucial role during pro-inflammatory immune responses. In our study, we observed CD4+T cells changed after the infection. Furthermore, ELISA and FACS results revealed that Schistosoma japonicum infection could induce a large amount of IL-17 in mouse pulmonary lymphocytes. IL-17-producing cells, including Th17 cells, CD8+T (Tc) cells, γδT cells and natural killer T cells, was also associated with the development of lung inflammatory diseases. FACS results indicated that Th17 cell was the main source of IL-17 in the infected pulmonary lymphocytes after phorbol-12-myristate-13-acetate (PMA) and Ionomycin stimulation. Moreover, FACS results revealed that the percentage of Th17 cells continued to increase as over the course of S. japonicum infection. Additionally, cytokines co-expression results demonstrated that Th17 cells could express more IL-4 and IL-5 than IFN-γ. Reducing IL-17 activity by using anti-IL-17 ameliorated the damage and decreased infiltration of inflammatory cells in infected C57BL/6 mouse lungs. Collectively, these results suggest Th17 cells is the major IL-17-producing cells population and IL-17 contributes to pulmonary granulomatous inflammatory during the S. japonicum infection.

AIM:To study the mechanism underlying the IL-12-induced cytotoxic function of NK cells to Jurkat cells.METHODS:NK cells from peripheral blood mononuclear cells (PBMCs) were purified by magnetic sorting and stimulated with or without IL-12. The expression of genes on IL-12-treated and non-IL-12-treated NK cells was analyzed by gene chips and the expression of cytolytic molecules was evaluated by flow cytometry.RESULTS:Seventeen genes were up- (5/17) or down-regulated (12/17) on IL-12-treated NK cells compared with non-IL-12-treated NK cells (fold change≥10). IL-12-induced expression of TRAIL on NK cells mediated the cytotoxicity to Jurkat cells. The expression of TRAIL on subsets of CD56(+);CD16(+); and CD56(-);CD16(+); NK cells significantly increased after the stimulation with IL-12 and Jurkat cells expressed high level of TRAIL receptor 2 (TRAIL-R2). Importantly, the neutralizing mAbs against TRAIL (RIK-2) significantly inhibited the cytotoxicity of NK cells induced by IL-12.CONCLUSION:The expression of TRAIL on human NK cells induced by IL-12 was one of the major mechanisms of cytotoxicity to Jurkat cells.

This paper aimed to analyze the roles of IL-12 and SCF on the cell divisions and IL-12Rβ expression of CD34+ cells.The mononuclear cells were isolated from neonatal umbilical cord blood(CBMC) and adult peripheral blood(PBMC).Then the frequency of CD34+ cells from CBMCs and PBMCs were detected by FACS.Finally,we analyzed the effects of IL-12 and SCF on CD34+ cells.About 0.13% of CD34+ cells in PBMCs and 1.53% of CD34+ cells in CBMCs were detected.The frequency of CD34+ cells from CBMCs was higher than that from PBMCs.Furthermore,SCF could increase the expression of CD25 on CD34+ cells.In conclusion,IL-12 and SCF could increase cell divisions and expression of IL-12Rβ1 on CD34+ cells.

This paper aimed to characterize the phenotypic and biological properties of NK cells in human pleural fluid cells(PFCs).Firstly,we isolated PFCs and PBMCs from pleural fluids and normal individuals,respectively.Then,surface markers and intracellular cytotoxic molecules of PFCs and PBMCs were stained with monoclonal antibodies and analyzed at a single cell level by flow cytometry.Our results showed that three distinct subpopulations(CD56+CD16-,CD56+CD16+ and CD56-CD16+) of NK cells from PFCs could be identified based on the expression of CD56 and CD16 molecules.The total number of NK cells was decreased and the subset of CD56+CD16-NK cells in pleural fluid from tuberculosis patients was increased compared with PBMCs.The expression of granzyme B was significantly lower on subsets of CD56+CD16+ and CD56-CD16+ NK cells from PFCs as compared with PBMCs,which was consistent with the higher level of CD107a/b on subsets of CD56+CD16-,CD56+CD16+ and CD56-CD16+ NK cells from PFCs(P < 0.05).The expressions of NKG2A,NKG2D,CD69 and CD25 were markedly higher on subsets of CD56+CD16+ and CD56-CD16+ NK cells from PFCs as compared with PBMCs(P < 0.05).In conclusion,the percentage of NK cells from PFCs is decreased compared with PBMCs;lower expression of granzyme B and higher expression of CD69 indicate that NK cells might fight against the tuberculosis infection.

Th1 cell-mediated immune responses at the site of active infection are important to restrict the growth of M. tuberculosis (MTB) and for the spontaneous resolution of patients with tuberculous pleurisy (TBP). In the present study, we found that without any stimulation, CD4(+) T cells in pleural fluid cells (PFCs) from patients with TBP expressed significantly higher levels of CD69 than PBMCs from patients with tuberculosis (TB) or healthy donors. CD4(+)CD69(+) T cells expressed T-bet and IL-12Rβ2. After stimulation with MTB-specific antigens, CD4(+)CD69(+) T cells expressed significantly higher levels of IFN-γ, IL-2 and TNF-α than CD4(+)CD69(-) T cells, demonstrating that CD4(+)CD69(+) T cells were MTB-specific Th1 cells. In addition, CD4(+)CD69(+) T cells were mostly polyfunctional Th1 cells that simultaneously produced IFN-γ, IL-2, TNF-α and displayed an effector or effector memory phenotype (CD45RA(-)CCR7(-)CD62L(-)CD27(-)). Moreover, the percentages of CD4(+)CD69(+) T cells were significantly and positively correlated with polyfunctional T cells. Interestingly, sorted CD4(+)CD69(+) but not CD4(+)CD69(-) fractions by flow cytometry produced IFN-γ, IL-2 and TNF-α that were significantly regulated by CD4(+)CD25(+) Treg cells. Taken together, based on the expression of CD69, we found a direct quantitative and qualitative method to detect and evaluate the in vivo generated MTB-specific polyfunctional CD4(+) T cells in PFCs from patients with TBP. This method can be used for the potential diagnosis and enrichment or isolation of MTB-specific Th1 cells in the investigations.

This paper aimed to compare the characteristics of IL-22-producing T cells in human normal membrane of intestinal tract and peripheral blood mononuclear cells(PBMC).We isolated the mononuclear cells from the membrane of intestinal tract and peripheral blood,respectively.The production of IL-22 by T cells and the correlation of IL-22 with IFN-γ and IL-17 were detected by FACS after stimulation with anti-CD3 plus anti-CD28.Finally,the surface markers CD45RO,CD62L,CCR7,CCR6,CCR10 and CCR4 were analyzed.Compared with the lower secretion of IL-22 by CD4+ and CD8+ T cells from PBMCs(0.6%;0.57%),3.15% of CD4+ IL-22+ and 4% CD8+ IL-22+ could be detected in membrane of intestinal tract after stimulation with anti-CD3 plus anti-CD28.A certain subset of CD4+ IL-22+ and CD8+ IL-22+ T cells was existed and they were distinct from Th1,Th17,Tc1,Tc17 cells.IL-22+ T cells in the membrane of intestinal tract expressed higher levels of CD45RO,part of these cells expressed CCR7 and fewer expressed CD62L.Furthermore,CD4+IL-22+ and CD8+IL-22+ T cells expressed higher levels of CCR10(55.3%;73.9%) and part of CCR6 and CCR4 in the membrane of intestinal tract.In conclusion,IL-22 is mainly produced by the effector or central memory T cells from the membrane of intestinal tract.In addition,a certain subset of IL-22-producing T cells is distinct from Th1,Th17,Tc1,Tc17 cells.

AIM To evaluate cytokine production and subsets in PBMCs from PPD+ normal donors after stimulation with BCG. METHODS PBMCs were isolated from PPD+ normal individuals, and cytokine production and BCG-specific T cell subsets were assessed by eight-color flow cytometry. RESULTS Following stimulation with BCG, CD4+ but not CD8+ T cells expressed IFN-γ, IL-2 and TNF-α. Phenotypic analysis indicated that cytokine-producing cells were CD4+CD45RO+CD62L-CD27- and CD4+CD45RO+CD62L-CD27+. CONCLUSION BCG predominantly induced CD4+ T cells to produce cytokines following stimulation with BCG. Further analysis indicated that these cells are CD4+CD45RO+CD62L- effector memory cells, suggesting that these cells probably played essential role in preventing TB infection.

Immunosuppressive mediators in tuberculosis pleurisy (pleural fluid (PF)) are associated with the course of disease, but they remain poorly defined. To study the local immune status of patients with tuberculosis pleurisy, we examined the effect of PF on the functions of T cells and the differentiation of Th1 cells. PF could inhibit the ability of T cells to produce cytokines. However, tumor-necrosis factor (TNF)-α derived from non-T cells was not impaired. Further analysis indicated that cell activation and cell cycle progression were also suppressed. Moreover, PF could inhibit Th1 cell differentiation. Importantly, we found that inhibitors of indoleamine 2,3-dioxygenase (IDO) and adenosine and neutralizing antibodies against IL-10 and transforming growth factor (TGF)-β could reverse cytokine production, suggesting that IDO, adenosine, IL-10 and Transforming growth factor–β1 in PF might take part in impairing T-cell functions. Taken together, our data demonstrate for the first time that several immunopathological factors participate in the downregulation of T-cell functions in local PF.