树突状细胞体外诱导抗肝癌免疫

目的应用树突状细胞（ＤＣ）在体外诱导高效而特异抗肝癌免疫．方法自肝癌患者外周血中分离ＤＣ;以粒／巨噬细胞集落刺激因子（ＧＭＣＳＦ）及白介素４（ＩＬ４）联合刺激ＤＣ;以人肝癌细胞系ＨｅｐＧ２肿瘤细胞的肿瘤相关抗原（ＴＡＡ）激活ＤＣ;ＤＣ诱导自体Ｔ淋巴细胞增殖、分化为细胞毒性Ｔ细胞（ＣＴＬ）;检测ＣＴＬ及其上清液对ＨｅｐＧ２肿瘤细胞、ＬＯＶＯ肿瘤细胞及ＨＯＳ８６０３肿瘤细胞的细胞毒作用．结果经人肝癌细胞系ＨｅｐＧ２肿瘤细胞的ＴＡＡ激活并经ＧＭＣＳＦ及ＩＬ４联合刺激后,肝癌患者外周血ＤＣ能够诱导自体Ｔ淋巴细胞增殖分化为ＣＴＬ,该ＣＴＬ及其上清液对ＨｅｐＧ２肿瘤细胞均有高效而特异性的杀伤作用（杀伤率分别为９２％±１０５％和４１％±８９％）．结论肝癌患者外周血ＤＣ体外能够诱导高效而特异抗肝癌免疫．提示ＤＣ作为一新概念上的抗肿瘤疫苗可能在肿瘤治疗及预防中发挥重要作用．     

Induction of tumor immunity and cytotoxic t lymphocyte responses using dendritic cells transduced by adenoviral vectors encoding HBsAg: comparison to protein immunization

Dendritic cells (DC) are specialized antigen-presenting cells with powerful immunostimulatory properties. Their use for induction of anti-tumor immunity has been limited by several factors, including identification of appropriate tumor-associated antigens, delivery of antigens to DC, and maintaining DC in a highly activated state. Here, DC propagated in vitro were transduced with an adenoviral (Ad) vector to express hepatitis B surface antigen (HBsAg), an antigen present in hepatocellular carcinoma (HCC). Many patients with HCC demonstrate evidence of prior HBV exposure, suggesting that the presence of the virus in a quiescent state may promote tumorigenesis. Ad-HBsAg-transduced DC stimulated strong cytotoxic T lymphocyte (CTL) responses to HBsAg-expressing tumor cells, and protected mice from lethal tumor challenge. Immunity was antigen-specific, as wild-type tumor (HBsAg -) grew normally. Furthermore, DC transduced with an irrelevant vector had no effect. Vaccination with HBsAg protein, a clinically utilized preparation that confers immunity to HBV infection, did not protect against tumor challenge even though it induced a strong antibody response. These studies describe for the first time the contributions of humoral and cellular immune responses to tumor immunity induced by Ad-transduced DC compared to protein vaccination.     

Selective transduction of HIV-1-infected cells by the combination of HIV and MMLV vectors

Human immunodeficiency virus 1 (HIV-1)-infected cells are important targets of gene therapy for acquired immune deficiency syndrome. We have developed a novel strategy for targeted gene transfer into HIV-1-infected cells based on 2-step gene transfer. The first step involves the stable introduction of the HIV vector containing the ecotropic Moloney murine leukemia virus (MMLV) receptor gene (EcoRec) into human CD4+ T cells as a molecular switch. Because the HIV-long terminal repeat (HIV-LTR) is Tat inducible, it is expected that EcoRec is expressed only after HIV-1 infection. Northern blot analysis and a retrovirus binding assay confirmed that the HIV-LTR of the integrated vector was silent in transduced cells but strongly transactivated in HIV-1 infection. High levels of EcoRec expression were observed only in HIV-1-infected cells. These cells became highly susceptible to ecotropic MMLV infection and, therefore, in the second step, HIV-1-infected cells were selectively transduced with ecotropic MMLV vectors. More than 70% of HIV-1-infected cells were transduced by this strategy. These findings indicate that this 2-step method can be used for selective and stable gene transfer into HIV-1-infected cells.     

Inhibition of HIV-1 replication by an antiviral xanthate compound in vitro

The antiviral xanthate compound tricyclodecan-9-yl-xanthogenate (code name D609) is capable of inhibiting DNA and RNA viruses in vitro. It can also inhibit the shedding of infectious HIV into the tissue culture medium from chronically infected lymphoma cells (KE37-III) as shown by infectivity assays and Western blots of the supernatant. HIV-specific proteins, however, were accumulated intracellularly. The initiation of a de novo HIV replication after infection of permissive KE37-1 cells was completely inhibited at concentrations of D609 which still permitted mitotic divisions of the cells. Furthermore, the selective antiviral activity of the xanthate compound was evidenced by the absence of HIV replicative intermediate DNA. The expression of cellular genes, such as c-myc, remained unimpaired within these cells.     

Detection and quantification of HIV-1 specific CD4 helper and CD8 cytotoxic cells: their role in HIV-1-infected individuals and vaccine recipients

There is a strong link between virus specific CD8 T-cell function and the efficiency of regulatory CD4 helper T cells. Controlling viraemia in HIV-1-infected individuals requires the maintenance of strong CD4 and CD8 T-cell responses. These responses should be elicited by prophylactic vaccination and by postexposure immunotherapy. This review will examine the methods that are available for the detection and quantification of HIV-1 specific CD4 and CD8 T-cell responses. We will also discuss the methods that should be used to identify these responses in HIV-1-infected individuals, seropositive recipients of immunotherapy and seronegative vaccinees. Finally, we will give examples of how responses observed in vitro relate to those known to occur in vivo .     

Molecular modeling and in vitro activity of an HIV-1-encoded glutathione peroxidase

Based on theoretical evidence, it has been proposed that HIV-1 may encode several selenoprotein modules, one of which (overlapping the env gp41-coding region) has highly significant sequence similarity to the mammalian selenoprotein glutathione peroxidase (GPx; EC ). The similarity score of the putative HIV-1 viral GPx homolog relative to an aligned set of known GPx is 6.3 SD higher than expected for random sequences of similar composition. Based on that alignment, a molecular model of the HIV-1 GPx was constructed by homology modeling from the bovine GPx crystal structure. Despite extensive truncation relative to the cellular GPx gene, the structural core and the geometry of the catalytic triad of selenocysteine, glutamine, and tryptophan are well conserved in the viral GPx. All of the insertions and deletions predicted by the alignment proved to be structurally feasible. The model is energetically favorable, with a computed molecular mechanics strain energy close to that of the bovine GPx structure, when normalized on a per-residue basis. However, considering the remote homology, this model is intended only to provide a working hypothesis allowing for a similar active site and structural core. To validate the theoretical predictions, we cloned the hypothetical HIV-1 gene and found it to encode functional GPx activity when expressed as a selenoprotein in mammalian cells. In transfected canine kidney cells, the increase in GPx activity ranged from 21% to 43% relative to controls (average 30%, n = 9, P < 0.0001), whereas, in transfected MCF7 cells, which have low endogenous GPx activity, a near 100% increase was observed (average 99%, n = 3, P < 0.05).     

Sustained multilineage gene persistence and expression in dogs transplanted with CD34(+) marrow cells transduced by RD114-pseudotype oncoretrovirus vectors

Previous studies have shown that the choice of envelope protein (pseudotype) can have a significant effect on the efficiency of retroviral gene transfer into hematopoietic stem cells. This study used a competitive repopulation assay in the dog model to evaluate oncoretroviral vectors carrying the envelope protein of the endogenous feline virus, RD114. CD34-enriched marrow cells were divided into equal aliquots and transduced with vectors produced by the RD114-pseudotype packaging cells FLYRD (LgGLSN and LNX) or by the gibbon ape leukemia virus (GALV)-pseudotype packaging cells PG13 (LNY). A total of 5 dogs were studied. One dog died because of infection before sustained engraftment could be achieved, and monitoring was discontinued after 9 months in another animal that had very low overall gene-marking levels. The 3 remaining animals are alive with follow-ups at 11, 22, and 23 months. Analyses of gene marking frequencies in peripheral blood and marrow by polymerase chain reaction revealed no significant differences between the RD114 and GALV-pseudotype vectors. The LgGLSN vector also contained the enhanced green fluorescent protein (GFP), enabling us to monitor proviral expression by flow cytometry. Up to 10% of peripheral blood cells expressed GFP shortly after transplantation and approximately 6% after the longest follow-up of 23 months. Flow cytometric analysis of hematopoietic subpopulations showed that most of the GFP-expressing cells were granulocytes, although GFP-positive lymphocytes and monocytes were also detected. In summary, these results show that RD114-pseudotype oncoretroviral vectors are able to transduce hematopoietic long-term repopulating cells and, thus, may be useful for human stem cell gene therapy.     

Myeloma cells exhibit an increase in proteasome activity and an enhanced response to proteasome inhibition in the bone marrow microenvironment in vivo

The proteasome inhibitor bortezomib has a striking clinical benefit in patients with multiple myeloma. It is unknown whether the bone marrow microenvironment directly contributes to the dramatic response of myeloma cells to proteasome inhibition in vivo. We have used the well‐characterized 5TGM1 murine model of myeloma to investigate myeloma growth within bone and response to the proteasome inhibitor bortezomib in vivo. Myeloma cells freshly isolated from the bone marrow of myeloma‐bearing mice were found to have an increase in proteasome activity and an enhanced response to in vitro proteasome inhibition, as compared with pre‐inoculation myeloma cells. Treatment of myeloma‐bearing mice with bortezomib resulted in a greater reduction in tumor burden when the myeloma cells were located within the bone marrow when compared with extra‐osseous sites. Our results demonstrate that myeloma cells exhibit an increase in proteasome activity and an enhanced response to bortezomib treatment when located within the bone marrow microenvironment in vivo. Am. J. Hematol., 2009. © 2009 Wiley‐Liss, Inc.     

HTLV type 1 Tax transduction in microglial cells and astrocytes by lentiviral vectors

Infection with human T cell leukemia virus type 1 (HTLV-1) can result in the development of HAM/TSP, a nonfatal, chronic inflammatory disease involving neuronal degeneration and demyelination of the central nervous system. Elevated levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and IL-1 observed in the cerebrospinal fluid of HAM-TSP patients suggest that cytokine dysregulation within the CNS is involved in neuropathogenesis. HTLV-1 infection and enhanced expression of TNF-alpha by microglial cells, astrocytes, and macrophages has been hypothesized to lead to the destruction of myelin and oligodendrocytes in the CNS. Although the association of HTLV-2 infection and development of neurological disease is more tenuous, HTLV-2 has also been found to be associated with peripheral neuropathies. To investigate the roles of HTLV Tax(1) and Tax(2) in the induction of cytokine disregulation in these cell types, we are currently developing gene delivery vectors based on human immunodeficiency virus type-1 (HIV-1) capable of stably coexpressing the HTLV-1 or -2 tax and eGFP reporter genes in primary human cells. Transduction frequencies of up to 50%, as assessed by eGFP expression, can be achieved in human monocyte-derived macrophages and in explanted cultures of human microglia. Preliminary data suggest that Tax(1) expression is sufficient to up-regulate the proinflammatory cytokine profile in explanted human microglial cells. Future experiments will compare and evaluate the effect of tax(1) and tax(2) gene expression on the cellular proinflammatory cytokine expression profile, as well as demonstrate the effects of transducing human fetal astrocytes and PBMC-derived macrophages.     

Migration of V delta 1 and V delta 2 T cells in response to CXCR3 and CXCR4 ligands in healthy donors and HIV-1-infected patients: competition by HIV-1 Tat

We show that HIV-1-infected patients have increased concentrations of circulating V delta 1 T cells (2.2%-9.0% of T lymphocytes; healthy donors, 1.0%-2%) and, in some instances, V delta 2 T cells (3.5%-4.8% vs 2.0%-3.3%). In these patients, both V delta 1 and V delta 2 T cells are CXCR3+CXCR4+, whereas in healthy donors CXCR4 was preferentially expressed on V delta 1 T lymphocytes. gamma delta T cells transmigrated across endothelial monolayers, in response to interferon-gamma-inducing protein-10 (IP-10/CXCL10), stromal cell-derived factor-1 (SDF-1/CXCL12), or both, according to the expression of the specific receptors CXCR3 and CXCR4. Interestingly, 6Ckine/SLC/CCL21 was more effective than IP-10/CXCL10 on V delta 1 CXCR3+ cells, whereas V delta 2 CXCR3+ cells were driven more efficiently by IP-10/CXCL10. IP-10/CXCL10- and SDF-1/CXCL12-induced transmigration was dependent on phosphoinositide-3 kinase (PI-3K), as demonstrated by the use of the specific blockers wortmannin and LY294002 and by the activation of the downstream serine kinase Akt/PKB on ligation of CXCR3 and CXCR4. Occupancy of CXCR3, but not of CXCR4, led to CAMKII activation; accordingly, the CAMKII inhibitors KN62 and KN93 decreased IP-10/CXCL10- but not SDF-1/CXCL12-driven transmigration. Finally, HIV-1 Tat, which is present in the serum of HIV-1-infected patients, interferes with the chemotactic activity of these chemokines because of the cysteine-rich domain of the protein, which contains CXC and CC chemokine-like sequences.     

The frequency of HIV-specific interferon- gamma -producing CD8 T cells is associated with both age and level of antigenic stimulation in HIV-1-infected children

Ex vivo interferon (IFN)- gamma -producing CD8 T cells specific for human immunodeficiency virus (HIV) Env, Gag, and Pol antigens were measured in the peripheral blood of 55 children not receiving highly active antiretroviral therapy (HAART) and 70 children receiving HAART. In children not receiving HAART, the frequency of HIV-specific IFN- gamma -producing CD8 T cells was positively correlated with age and was not associated with plasma viral load or CD4 T cell levels. In children receiving HAART, the frequency of HIV-specific IFN- gamma -producing CD8 T cells was directly correlated with plasma viral load, and its association with age remained significant. In conclusion, the frequency of HIV-specific IFN- gamma -producing CD8 T cells in children is primarily determined by both age and plasma viral load.     

Perforin expression in T cells and virological response to PEG-interferon alpha2b in HIV-1 infection

OBJECTIVE AND DESIGN: Interferon alpha (IFNalpha), which is known to directly inhibit the HIV-1 replicative cycle and to increase the activity of cytotoxic T lymphocytes (CTL), is being tested as an anti-HIV agent. As CTL play a major role in immune defence against HIV, we wanted to further characterize CTL activity and the effect of IFNalpha on it. METHODS: We followed by flow cytometry the intracellular expression of the key mediator of cytotoxicity, perforin, in peripheral blood T cells of patients treated with IFNalpha. RESULTS: We observed that the percentage of T cells harbouring perforin was higher in infected subjects than in non-infected controls. Administration of IFNalpha2b attached to polyethylene glycol increased this perforin expression further and reduced viral load (P = 0.010). The increase in the percentage of T cells expressing perforin correlated with IFNalpha-induced decrease in viral load (r, 0.753; P = 0.003). In addition, the level of perforin expression before IFNalpha administration was inversely correlated with viral load remaining after IFNalpha administration (r, -0.647; P= 0.017). CONCLUSION: The pre-therapeutic percentage of perforin-positive T cells might be a predictive marker of the virological response to IFNalpha in HIV-1-infected patients.     

胰腺癌Capan-2细胞与树突状细胞融合诱导特异性抗癌免疫应答的体外研究

目的观察人胰腺癌Capan-2细胞与树突状细胞(DC)融合后诱导的特异性抗肿瘤免疫反应。方法收集2013年4月-2015年3月于沈阳军区总医院消化科就诊的人类白细胞抗原(HLA)-A2~+胰腺癌患者6例,从患者外周血单个核细胞中分离并培养DC。所获DC分为3组:(1)使用聚乙二醇-二甲基亚砜诱导法,将DC与Capan-2细胞融合以负载肿瘤抗原;(2)DC与Capan-2细胞共培养;(3)单独DC培养。通过流式细胞术检测PE-MUC4/FITC-CD86抗体双标细胞评估融合率;应用四甲基偶氮唑盐法检测各组DC的存活率变化。使用干扰素(IFN)γ酶联免疫法检测DC诱导的细胞毒T淋巴细胞(CTL)活化反应。采用51Cr标准细胞毒实验检测DC诱导的抗原特异性CTL对体外胰腺癌细胞的杀伤作用。多组间比较采用方差分析,进一步两两比较采用LSD-t检验。结果DC与Capan-2融合细胞同时表达DC表型(CD86)和MUC4分子,CD86与MUC4双阳性表达率为(38.30±7.30)%,明显高于共培养组(7.21±1.06)%;融合细胞组DC存活率呈时间依赖性下降,转染后96 h的存活率降低至62.81%,而与Capan-2细胞共培养组DC存活率稳定在80%以上,两组间差异有统计学意义(P0.05)。DC-Capan-2融合细胞诱导的CTL 24 h IFNγ释放量为(85.34±2.97)U/ml,DC、Capan-2共培养组DC诱导的IFNγ释放量为(19.07±4.25)U/ml,两转染组的差异有统计学意义(P0.05)。DC-Capan-2融合细胞诱导的特异性CTL能够有效识别和杀伤HLA-A2~+/MUC4~+的Capan-2细胞及HLAA2~+/MUC4-的PANC-1细胞,但不能有效识别和杀伤HLA-A2-/MUC4-的Mia Pa Ca-2细胞和HLA-A2-/MUC4~+的As PC-1细胞。结论胰腺癌细胞与DC融合后可诱导出显著的CTL抗肿瘤免疫反应,此过程受主要组织相容性复合体Ⅰ类抗原呈递的限制。     

In vitro cytotoxic response to human myeloma plasma cells by peripheral blood leukocytes from patients with multiple myeloma and benign monoclonal gammopathy.

Peripheral blood leukocytes (PBL) from myeloma patients were studied for their capacity to lyse plasma cells from myeloma patients, benign monoclonal gammopathy (BMG) patients, and nonneoplastic disease patients. Plasma cells were isolated from bone marrow, labeled with 51Cr, and cultured with PBL isolated from patients with myeloma, BMG, or nonneoplastic disease, as well as normal individuals. PBL from patients with multiple myeloma demonstrated responses to autologous or allogeneic myeloma plasma cells. Optimum conditions for cytotoxic response included a responder-to-stimulator ratio of 1:1 and an effector-to-target ratio of 20:1. PBL from normal individuals or patients with BMG failed to demonstrate this response. However, PBL from BMG patients, but not normal individuals, could be induced to kill myeloma plasma cells (but not nonmyeloma plasma cells) by simultaneous stimulation with allogeneic lymphocytes and myeloma plasma cells.