重组人IL-15促人脐血CD34+造血干细胞定向分化为NK细胞

目的 探讨IL－15促CD34^ 造血干细胞定向分化为NK细胞的作用。方法 采用MACS法分离脐血中CD34^ 细胞，分别用IL－15、SCF（造血干细胞因子）和IL－15 SCF体外培养，流式细胞仪测定CD3、CD16、CD56分子，MTT法检测NK细胞杀伤活性。结果 IL－15可以促进CD34^ 细胞分化为以CD56^ CD16^-为主的NK细胞，SCF具有较强的协同作用。结论 IL－15具有促使NK细胞定向分化的作用。     

CD34+ cells cultured in stem cell factor and interleukin-2 generate CD56+ cells with antiproliferative effects on tumor cell lines

In vitro stimulation of CD34+ cells with IL-2 induces NK cell differentiation. In order to define the stages of NK cell development, which influence their generation from CD34 cells, we cultured G-CSF mobilized peripheral blood CD34+ cells in the presence of stem cell factor and IL-2. After three weeks culture we found a diversity of CD56+ subsets which possessed granzyme A, but lacked the cytotoxic apparatus required for classical NK-like cytotoxicity. However, these CD56+ cells had the unusual property of inhibiting proliferation of K562 and P815 cell lines in a cell-contact dependent fashion.     

NK cells differentiated from bone marrow,cord blood and peripheral blood stem cells exhibit similar phenotype and functions

In the present study, we investigated the differentiation of human NK cells from bone marrow, cord blood and mobilized peripheral blood purified CD34⁺ stem cells using a potent culture system. Elutriated CD34⁺ stem cells were grown for several weeks in medium supplemented with stem cell factor (SCF) and IL-15 in the presence or absence of a murine stromal cell line (MS-5). Our data indicate that IL-15 induced the proliferation and maturation of highly positive CD56⁺ NK cells in both types of culture, although murine stromal cells slightly increased the proliferation of NK cells. NK cells differentiated in the presence of MS-5 were mostly CD56⁺ CD7⁻ and a small subset expressed CD16. These in vitro differentiated CD56⁺ NK cells displayed cytolytic activity against the HLA class I ⁻ target K562. The CD56⁺ CD16⁺ subset also lysed NK-resistant Daudi cells. Neither of these NK subsets were shown to express Fas ligand. Total CD56⁺ cells expressed high amounts of transforming growth factor-β and granulocyte-macrophage colony-stimulating factor, but no IFN-γ. Investigation of NK receptor expression showed that most CD56⁺ cells expressed membrane CD94 and NKG2-A mRNA. PCR analysis revealed that p58 was also expressed in these cells. The role of CD94 in NK cell-mediated cytotoxicity was assessed on human HLA-B7-transfected murine L cells. While a low cytotoxic activity towards HLA-B7 cells was observed, the HLA-DR4 control cells were killed with high efficiency. These studies demonstrate that cytolytic and cytokine-producing NK cells may be derived from adult and fetal precursors by IL-15 and that these cells express a CD94 receptor which may influence their lytic potential.     

Interleukin-15 Promotes the Commitment of Cord Blood CD34^＋ Stem Cells into NK Cells

Naturalkiller(NK)cellsweresubgroupoflymphocytes thatplayedanessentialroleinthecellularbasedimmune defenseagainstvirus infectedandtransfectedcells.His torically,inteleukin 2(IL 2)wasregardedasthenatural activatorforNKcells.Recently,itwasdiscoveredthat IL …     

Accumulation of CD16-CD56+ natural killer cells with high affinity interleukin 2 receptors in human early pregnancy decidua.

Most human peripheral blood natural killer (NK) cells express the phenotype CD16+CD56+. However, a very minor subset of NK cells express CD16-CD56+, and these NK cells bear both interleukin 2 receptor (IL-2R)alpha (p55) and IL-2R beta (p75) (high affinity IL-2 receptors). In this report, we demonstrate that in human early pregnancy decidua--an interface between maternal immunocompetent cells and fetus (placenta)--abundant (approximately 83%) CD16-CD56+ NK cells with high affinity IL-2 receptors were present, and these cells responded to low amounts of IL-2 (4.5 pM). These CD16-CD56+ NK cells significantly expressed an early activation antigen, CD69, in vivo, whereas peripheral CD16-CD56+ NK cells did not express CD69. These findings suggest that CD16-CD56+ NK cells in early pregnancy decidua may be activated in vivo, and may play an important role in immunoregulation during early pregnancy. Also, decidual lymphocytes may be useful materials to study the mechanism of MHC-unrestricted cytotoxicity of this type of NK cells.     

转染IL-21的CD34~+脐血造血干细胞抗裸鼠卵巢癌作用研究

目的 探讨IL-21转染的脐血造血干细胞(CD34~+UBSC·IL-21)对荷卵巢癌裸鼠的治疗作用.方法 从脐血分离CD34~+造血干细胞,体外培养扩增后用于重组体pIRES2-IL-21-EGFP转染.以肿瘤大小、荷瘤鼠生存期判断CD34~+UBSC-IL-21对荷瘤裸鼠的治疗效应.以RT-PCR、免疫荧光、ELISA、Western blot、脾细胞增殖试验及免疫组化法分别鉴定CD34~+UBSC和肿瘤组织中IL-21的表达及活性.裸鼠脾细胞中NK细胞含量及脾细胞的杀伤效应、血清中IFN-γ和TNF-α水平分别用FCM与ELISA检测.结果 pIRES2-IL-21-EGFP成功转染CD34~+UBSC.CD34~+UBSC-IL-21能抑制肿瘤生长,延长荷瘤裸鼠生存期,治疗鼠肿瘤局部能表达IL-21、血清IFN-γ和TNF-α水平升高,NK细胞含量及NK细胞杀伤活性明显增强,与其他组相比,差异有统计学意义(P<0.01).结论 转染IL-21的CD34~+UBSC有良好的抗裸鼠卵巢癌作用,该结果为临床使用UBSC为载体的基因治疗卵巢癌研究奠定了基础.     

G-CSF-mobilized CD34+ cells cultured in interleukin-2 and stem cell factor generate a phenotypically novel monocyte

To study the early stages of development from stem cells of the CD56+ cell population [which includes natural killer (NK) cells], granulocyte-colony stimulating factor-mobilized peripheral blood CD34+ cells from healthy donors were sorted to >99% purity and cultured in the presence of stem cell factor and interleukin (IL)-2. After 3 weeks in culture, the majority of cells acquired CD33, with or without human leukocyte antigen-DR and CD14. In 20 stem cell donors tested, 8.7 +/- 8.8% of cells were CD56+. Two major CD56+ subsets were identified: CD56(bright), mainly CD33- cells (7+/-10%, n=11) with large, granular lymphocyte morphology, and CD56dim, mainly CD33+ (2.5+/-2, n=11) cells with macrophage morphology. The CD56bright population had cytoplasmic granzyme A but lacked killer inhibitory receptor, suggesting they were immature NK cells. The CD56dim, CD33+, population lacked NK markers. They may represent a minor subset of normal monocytes at a developmental stage comparable with the rare CD56+ CD33+ hybrid myeloid/NK cell leukemia. Consistent with a monocyte nature, CD56dimCD33+ proliferated and produced a variety of cytokines upon lipopolysaccharide stimulation, including IL-8, IL-6, monocyte chemoattractant protein-1, and macrophage-derived chemokine but not interferon-gamma. In a short-term cytotoxicity assay, they failed to kill but powerfully inhibited the proliferation of the NK-resistant cell line P815. The generation of CD56+ cells was negatively regulated by hyaluronic acid and IL-4, indicating that extracellular matrix may play an important role in the commitment of CD34+ cells into CD56 myeloid and lymphoid lineages.     

Phenotypic and functional heterogeneity of natural killer cells from umbilical cord blood mononuclear cells

Natural killer cells (NK) from umbilical cord blood (CB) play an important role in allogeneic stem cell transplantation and defending infections of newborn. Based on the surface expression of CD56 and CD16 or inhibitory and activatory receptors, NK cells could be subdivided into various subsets with distinct functions. To investigate the biological characterization of NK subsets, the phenotypes and intracellular proteins in freshly isolated CB NK subsets were analyzed at the single cell level by flow cytometry in current study. The production of IFN-gamma and cytotoxicity against K562 target cells were also evaluated after stimulation with IL-12. The results showed that NK cells from CB could be divided into four subsets on the basis of CD56 and CD16 expression. Interestingly, CB NK cells expressed CD45RA but not CD45RO molecules that is similar to the na?ve T cells. Moreover, CD27, a memory T cell marker, highly expressed on CD56(hi)CD16- NK cells. The killing-associated molecules, NKG2A, NKG2D, CD95 and the intracellular granzyme B and perforin were heterogeneously expressed among the 4 subsets. Addition of IL-12 into cultures resulted in the induction of IFN-gamma expression by CD56(hi)CD16- and CD56(lo)CD16- subsets and the enhancement of NK cytolytic activity. Taken together, this study elucidated the heterogeneity in phenotypes and biological functions of CB NK cells.     

Activation of human NK cells by plasmacytoid dendritic cells and its modulation by CD4+ T helper cells and CD4+ CD25hi T regulatory cells

Plasmacytoid dendritic cells (pDC) represent a specialized cell population that produce type I interferon (IFN) in response to virus. Although type I IFN is a natural killer (NK) cell modulator, a direct role for pDC in coordinating NK cell functions has not yet been elucidated in detail, especially in humans. Here we report that human pDC, following engagement of Toll-like receptor (TLR) 9, not only activate autologous NK cells, as indicated by the induction of CD69 expression, but also enhance their effector functions, especially cytotoxicity. Moreover, they can induce selective proliferation of CD56bright CD16- NK cells. This activity can be strongly augmented by the addition of autologous CD4+ CD25- T helper cells in an IL-2-dependent fashion. Strikingly, CD4+ CD25hi T regulatory (Treg) cells completely abrogate this IL-2-dependent proliferation of NK cells, while they are not able to influence NK cell activation or proliferation solely induced by pDC. Our data show that TLR9-engaged pDC represent a critical stimulus for human NK cells and that CD4+ Th cells and CD4+ CD25hi Treg cells play an important role in modulating human NK cell responses.     

Altered Coexpression of Lectin-Like Receptors CD94 and CD161 on NK and T Cells in HIV Patients

HIV patients either on highly active antiviral therapy (HAART) or therapy naive were analyzed for their CD56 phenotype and cytokine production in comparison to healthy controls (HC). Both NK cell populations (CD56(dim) and CD56(bright)) are found to be present in all groups with selectively decreased CD56(dim) NK cells in HAART naive patients. Patients on HAART exhibited significantly diminished numbers of CD161+CD56(dim) NK cells. CD56(dim) were equally potent in producing IFNgamma in all three groups. The number of TNFalpha+CD56(bright) NK cells from patients on HAART and TNFalpha+CD56(dim) NK cells from HAART naive patients was significantly reduced as compared to healthy controls. In summary our data revealed that functional capacities and coexpression patterns of lectin-like receptors on lymphocytes are differentially affected in HIV patients depending on the state of therapy (under HAART or HAART naive) or the cell type (NK or T cells), respectively.     

Phenotypic and Functional Heterogeneity of Natural Killer Cells from Umbilical Cord Blood Mononuclear Cells

Natural killer cells (NK) from umbilical cord blood (CB) play an important role in allogeneic stem cell transplantation and defending infections of newborn. Based on the surface expression of CD56 and CD16 or inhibitory and activatory receptors, NK cells could be subdivided into various subsets with distinct functions. To investigate the biological characterization of NK subsets, the phenotypes and intracellular proteins in freshly isolated CB NK subsets were analyzed at the single cell level by flow cytometry in current study. The production of IFN-γ and cytotoxicity against K562 target cells were also evaluated after stimulation with IL-12. The results showed that NK cells from CB could be divided into four subsets on the basis of CD56 and CD16 expression. Interestingly, CB NK cells expressed CD45RA but not CD45RO molecules that is similar to the naïve T cells. Moreover, CD27, a memory T cell marker, highly expressed on CD56^hiCD16^? NK cells. The killing-associated molecules, NKG2A, NKG2D, CD95 and the intracellular granzyme B and perforin were heterogeneously expressed among the 4 subsets. Addition of IL-12 into cultures resulted in the induction of IFN-γ expression by CD56^hiCD16^? and CD56^loCD16^? subsets and the enhancement of NK cytolytic activity. Taken together, this study elucidated the heterogeneity in phenotypes and biological functions of CB NK cells.     

Methylprednisolone induces preferential and rapid differentiation of CD34+ cord blood precursors toward NK cells

Previous studies showed that methylprednisolone (MePDN) down-regulates the surface expression of activating NK receptors and sharply inhibits the NK cytotoxicity both in vitro and in vivo. Since MePDN is administered to patients undergoing hemopoietic stem cell transplant to treat acute graft versus host disease (GvHD), we analyzed whether it could also inhibit the NK cell differentiation from CD34(+) hemopoietic cell precursors, thus interfering with the development of effector cells with anti-leukemic potential. We show that MePDN promotes the in vitro differentiation of CD161+CD56+/- immature NK cells by inducing a rapid expression of NKp46, NKG2D, DNAX-accessory molecule 1 (DNAM-1), leukocyte function-associated antigen-1 and NKG2A and an efficient cytolytic activity. This phenotypic and functional NK cell maturation occurred more rapidly than in parallel control cultures performed in the absence of MePDN. In addition, MePDN induced CD33+CD161-CD56- myeloid precursors to switch toward NK cells. It is also of note that immature NK cells when cultured in the absence (but not in the presence) of MePDN produced high amounts of IL-8. These data indicate that MePDN can accelerate the in vitro NK cell differentiation, thus revealing a dichotomous effect on immature versus mature NK cells; in addition, interference with the in vitro development of myeloid cells occurred. These effects should be further investigated in hemopoietic stem cell transplanted patients receiving steroids to treat GvHD.     

绿脓杆菌制剂与IL-12在诱导人NK细胞IFN-γ产生中的协同作用

探讨绿脓杆菌制剂(piliated Pseudomonas Aeruginosa,PPA)与IL-12协同诱导人PBMC和NK细胞IFN-γ的产生。分离健康人PBMC和纯化NK细胞分别与培养液、PPA、IL-12或PPA+IL-12共同培养,利用酶联免疫吸附法(ELISA)检测无细胞培养上清中IFN-γ的水平。同时采用流式细胞仪在单个细胞水平上分析PPA和IL-12诱导IFN-γ产生的淋巴细胞亚群。结果显示,单独应用亚适剂量的IL-12或PPA刺激人PBMC或纯化NK细胞,不能诱导或只能诱导低水平IFN-γ的产生。当PPA和IL-12共同与人PBMC或纯化NK细胞孵育后,PPA呈时间和剂量依赖性与IL-12协同诱导PBMC和纯化NK细胞产生大量IFN-γ。细胞亚群分析的结果表明,PPA和IL-12协同诱导CD56+NK细胞产生IFN-γ,但对CD4+T和CD8+T细胞无明显作用。PPA与IL-12协同促进NK细胞IFN-γ的产生,提示PPA和IL-12能直接刺激NK细胞发生免疫应答。     

IL-21 induces both rapid maturation of human CD34+ cell precursors towards NK cells and acquisition of surface killer Ig-like receptors

The NK cell maturation from CD34(+) Lin(-) hematopoietic cell precursors is a complex process that requires the direct contact with stromal cells and/or the synergistic effect of different cytokines. In this study we show that IL-21 is capable of inducing an accelerated NK cell maturation when added to cultures of CD34(+) Lin(-) cells isolated from human cord blood supplemented with IL-15, Flt3-L and SCF. After 25 days of culture, 50% of CD56(+) cells expressed various NK cell markers including the NKp46 and NKp30 triggering receptors, the CD94/NKG2A inhibitory receptor and CD16. At day 35, substantial fractions of NK cells expressed KIR, CD8 and CD2, i.e. surface markers expressed by mature NK cells, that are virtually undetectable in developing NK cells cultured in the absence of IL-21. Remarkably, similar to mature NK cells all these markers were included in the CD56(dim) cell fraction, while the CD56(bright) population was only composed of CD94/NKG2A(-) and CD94/NKG2A(+) cells. Thus, IL-21 allows the induction of a full NK cell maturation in vitro and offers an important tool for dissecting the molecular mechanisms involved in different steps of NK cell maturation and in the acquisition of a mature KIR repertoire.     

Characterization of cord blood natural killer and lymphokine activated killer lymphocytes following ex vivo cellular engineering.

Cord blood (CB) natural killer (NK) and lymphokine-activated killer (LAK) cytotoxic cells are poorly characterized but might be used to treat minimal residual and/or recurrent malignant disease. Currently, there is no mechanism to use CB for adoptive cancer cellular immunotherapy after CB transplantation (CBT). Recognizing this as a deficiency, we hypothesized that CB aliquots could be engineered ex vivo for potential donor lymphocyte infusion after CBT. Cryopreserved CB aliquots were thawed, depleted of monocytes, and cultured in serum-free medium alone or serum-free medium with anti-CD3 and interleukins 2, 7, and 12 combined with antibody/cytokines for 48 hours. Immunophenotyping, cytotoxicity, and proliferation were evaluated. A significant expansion of CD3+ was seen, in addition to increases in lymphocyte subsets of CD8+, CD8+/CD25+, and CD3+/45RO+ versus medium alone. A significant enhancement of CD3 proliferation (P<.001), NK cytotoxicity, NK subset expansion, LAK cytotoxicity, and T-helper 1 subset expansion was also demonstrated. Significant enrichment was seen in NK CD16+/CD56+bright, CD16+/CD56+dim, CD56+bright and CD56+dim/KIR3DL1+, CD56+bright and CD56+dim/KIR2DL1+, CD56+bright and CD56+dim/KIR2DL2+ and CD94+/NKG2a+ subsets. These increases in CB NK subsets were in part secondary to augmentation of cell survival. Further, survival of NOD-SCID mice xenografted with human K562 cells and treated with CB cells expanded with antibody/cytokines was significantly higher than that in animals that received no treatment (phosphate buffered saline) and those that were treated with CB ex vivo expanded in medium alone (P<.005, respectively). These data suggest that cryopreserved CB cells could be ex vivo engineered for potential use as adoptive cancer cellular immunotherapy for donor lymphocyte infusion after CBT.     

CIK/NK细胞4周培养过程中的CD27、CD28的表达及其意义

目的:了解细胞因子诱导杀伤（CIK）细胞和自然杀伤（NK）细胞在培养过程中CD27和CD28的表达变化规律。方法:通过流式细胞仪检测CD3+CD56+ CIK细胞以及CD3-CD56+ NK细胞在4周培养过程中其上CD27和CD28的表达变化。结果:CD27在CIK和NK细胞上均于培养的第2周达到最高峰,分别为（44.57±4.07）％和（51.02±5.20）％,但之后逐步下降,在培养第4周降至30%左右的水平。CD28在CIK和NK细胞上分别是在第3周和第2周时达到表达的最高峰,为（4.40±0.66）％和（45.14±3.58）％,之后迅速下降,在培养第4周已经几乎消失。结论:根据CD27和CD28在CIK/NK细胞上的表达变化规律,CIK/NK细胞的收获时机以培养第3周为宜。     

A human CD34(+) subset resides in lymph nodes and differentiates into CD56bright natural killer cells

In humans, T cells differentiate in thymus and B cells develop in bone marrow (BM), but the natural killer (NK) precursor cell(s) and site(s) of NK development are unclear. The CD56bright NK subset predominates in lymph nodes (LN) and produces abundant cytokines compared to the cytolytic CD56dim NK cell that predominates in blood. Here, we identify a novel CD34dimCD45RA(+) hematopoietic precursor cell (HPC) that is integrin alpha4beta7bright. CD34dimCD45RA(+)beta7bright HPCs constitute <1% of BM CD34(+) HPCs and approximately 6% of blood CD34(+) HPCs, but >95% of LN CD34(+) HPCs. They reside in the parafollicular T cell regions of LN with CD56bright NK cells, and when stimulated by IL-15, IL-2, or activated LN T cells, they become CD56bright NK cells. The data identify a new NK precursor and support a model of human NK development in which BM-derived CD34dimCD45RA(+)beta7bright HPCs reside in LN where endogenous cytokines drive their differentiation to CD56bright NK cells in vivo.     

Role of chemokines in the biology of natural killer cells

Natural killer (NK) cells participate in innate and adaptive immune responses to obligate intracellular pathogens and malignant tumors. Two major NK cell subsets have been identified in humans: CD56(dim) CD16+ and CD56(bright) CD16-. Resting CD56(dim) CD16+ NK cells express CXCR1, CXCR2, CXCR3, CXCR4, and CX3CR1 but no detectable levels of CC chemokine receptors on the cell surface. They migrate vigorously in response to CXCL12 and CXC3L1. In contrast, resting CD56(bright) CD16- NK cells express little CXCR1, CXCR2, and CXC3R1 but high levels of CCR5 and CCR7. Chemotaxis of CD56(bright) CD16- NK cells is stimulated most potently by CCL19, CCL21, CXCL10, CXCL11, and CXCL12. Following activation, NK cells can migrate in response to additional CC and CXC chemokines. Cytolytic activity of NK cells is augmented by CCL2, CCL3, CCL4, CCL5, CCL10, and CXC3L1. Moreover, proliferation of CD56(dim) CD16+ NK cells is costimulated by CCL19 and CCL21. Activated NK cells produce XCL1, CCL1, CCL3, CCL4, CCL5, CCL22, and CXCL8. Chemokines secreted by NK cells may recruit other effector cells during immune responses. Furthermore, CCL3, CCL4, and CCL5 produced by NK cells can inhibit in vitro replication of HIV. CCL3 and CXL10 expression appear to be required for protective NK cell responses in vivo to murine cytomegalovirus or Leishmania major, respectively. Moreover, NK cells participate in the in vivo rejection of transduced tumor cells that produce CCL19 or CCL21. Thus, chemokines appear to play an important role in afferent and efferent NK cell responses to infected and neoplastic cells.     

自体外周血造血干细胞移植后造血重建中CD226分子在NK细胞上表达的变化

本文应用双重免疫荧光染色和流式细胞术分析,观察自体外周血造血干细胞移植患者造血重建中,CD226分子在CD56bright和CD56dimNK细胞亚群表达的变化.结果显示,在自体外周血造血干细胞移植造血重建中,于干细胞回输第12天,CD56 NK细胞占外周血淋巴细胞百分率为26.6%,其中CD56bright亚群,占CD56 NK细胞87.3%;CD56 CD226 细胞占CD56 NK细胞92.1%,CD56brightCD226 细胞占CD56 CD226 细胞89.9%.在自体外周血造血干细胞移植造血重建中,CD56bright亚群是NK细胞造血重建最早出现并占优势的一个亚群, CD226分子可能作为一种分化标记主要表达在CD56bright亚群上.