A General Functional Response of Cytotoxic T Lymphocyte-Mediated Killing of Target Cells

Cytotoxic T lymphocytes (CTLs) kill virus-infected cells and tumor cells, and play a critical role in immune protection. Our knowledge of how the CTL killing efficiency varies with CTL and target cell numbers is limited. Here, we simulate a region of lymphoid tissue using a cellular Potts model to characterize the functional response of CTL killing of target cells, and find that the total killing rate saturates both with the CTL and the target cell densities. The relative saturation in CTL and target cell densities is determined by whether a CTL can kill multiple target cells at the same time, and whether a target cell can be killed by many CTLs together. We find that all the studied regimes can be well described by a double-saturation (DS) function with two different saturation constants. We show that this DS model can be mechanistically derived for the cases where target cells are killed by a single CTL. For the other cases, a biological interpretation of the parameters is still possible. Our results imply that this DS function can be used as a tool to predict the cellular interactions in cytotoxicity data.     

A General Functional Response of Cytotoxic T Lymphocyte-Mediated Killing of Target Cells

Cytotoxic T lymphocytes (CTLs) kill virus-infected cells and tumor cells, and play a critical role in immune protection. Our knowledge of how the CTL killing efficiency varies with CTL and target cell numbers is limited. Here, we simulate a region of lymphoid tissue using a cellular Potts model to characterize the functional response of CTL killing of target cells, and find that the total killing rate saturates both with the CTL and the target cell densities. The relative saturation in CTL and target cell densities is determined by whether a CTL can kill multiple target cells at the same time, and whether a target cell can be killed by many CTLs together. We find that all the studied regimes can be well described by a double-saturation (DS) function with two different saturation constants. We show that this DS model can be mechanistically derived for the cases where target cells are killed by a single CTL. For the other cases, a biological interpretation of the parameters is still possible. Our results imply that this DS function can be used as a tool to predict the cellular interactions in cytotoxicity data.     

Vpr Stimulates Viral Expression and Induces Cell Killing in Human Immunodeficiency Virus Type 1-Infected Dividing Jurkat T Cells

In this study we investigated the effects of Vpr during human immunodeficiency virus (HIV) infection of proliferating Jurkat T cells by using a vesicular stomatitis virus envelope G glycoprotein pseudotyped HIV superinfection system. We observe that the expression of Vpr results in a severe reduction in the life span of HIV type 1 (HIV-1)-infected dividing T cells in culture. In agreement with a recent report (S. A. Stewart, B. Poon, J. B. M. Jowett, and I. S. Chen, J. Virol. 71:5579–5592, 1997), we show that events characteristic of apoptotic cell death are involved in the Vpr-mediated cytopathic effects. Our results also show that infection with viruses expressing the wild-type vpr gene results in an increase in viral gene expression and production. Interestingly, the effects of Vpr on cell viability and on viral gene expression both correlate with the ability of the protein to induce a cell cycle arrest in the G₂/M phase. Mutagenesis analyses show that the C terminus of Vpr is essential for these biological activities. Although the role of Vpr is currently associated with the infection of nondividing cells, our results suggest that Vpr can also directly increase viral replication in vivo in infected dividing T cells. Furthermore, these in vitro observations suggest that Vpr-mediated cytotoxic effects could contribute to the CD4⁺ depletion associated with AIDS progression.     

5-脱氧杂氮胞苷抑制小鼠附植前的胚胎发育

DNA甲基化在哺乳动物发育过程中有关键作用．在小鼠附植前胚胎发育过程中,DNA甲基化一直处于动态变化过程中．通过将体外受精胚在5-AZA-CdR中持续培养,研究5-AZA-CdR对小鼠附植前胚胎发育的影响,为附植前胚胎发育机理的研究及5-AZA-CdR的毒副作用研究提供试验基础．从原核期加入不同浓度的5-AZA-CdR时,胚胎不能发育到桑椹胚(0.2 和1.0 μmol/L)和4-细胞胚(5.0 μmol/L);从2-细胞期加入时,胚胎阻滞于未致密化的8-细胞(0.2 和1.0 μmol/L)和3/4-细胞期(5.0 μmol/L);而当从4-细胞加入时,虽然胚胎能够发育到早期桑椹胚,但发育比例同对照相比显著降低(P < 0.05)．进一步检测凋亡、基因组DNA甲基化和整体转录活性,结果显示,高浓度的5-AZA-CdR导致8-细胞和早期桑椹胚发生早期凋亡,而低浓度的5-AZA-CdR引起8-细胞和早期桑椹胚基因组DNA甲基化的降低和转录活性的降低,并且这种降低呈浓度依赖性．所以加入低浓度的5-AZA-CdR时,胚胎的DNA甲基化降低,引起转录活性的降低,进而导致胚胎发育的停滞．     

B7-H1 and a Mathematical Model for Cytotoxic T Cell and Tumor Cell Interaction

The surface protein B7-H1, also called PD-L1 and CD274, is found on carcinomas of the lung, ovary, colon, and melanomas but not on most normal tissues. B7-H1 has been experimentally determined to be an antiapoptotic receptor on cancer cells, where B7-H1-positive cancer cells have been shown to be immune resistant, and in vitro experiments and mouse models have shown that B7-H1-negative tumor cells are significantly more susceptible to being repressed by the immune system. We derive a new mathematical model for studying the interaction between cytotoxic T cells and tumor cells as affected by B7-H1. By integrating experimental data into the model, we isolate the parameters that control the dynamics and obtain insights on the mechanisms that control apoptosis.     

Template Properties of 5-Methyl-2'-Deoxycytidine and 5-Hydroxymethyl-2'-Deoxycytidine in Reactions with Human Translesion and Reparative DNA Polymerases

Molecular Biology - 5-Methyl-2'-deoxycytidine (mC) and the product of its controlled oxidation, 5-hydroxymethyl-2'-cytidine (hmC), play a key role in the epigenetic regulation of gene...     

Pan-Bcl-2 Inhibitor AT-101 Enhances Tumor Cell Killing by EGFR Targeted T Cells

Pancreatic cancer is a deadly disease and has the worst prognosis among almost all cancers and is in dire need of new and improved therapeutic strategies. Conditioning of tumor cells with chemotherapeutic drug has been shown to enhance the anti-tumor effects of cancer vaccines and adoptive cell therapy. In this study, we investigated the immunomodulatory effects of pan-Bcl-2 inhibitor AT-101 on pancreatic cancer (PC) cell cytotoxicity by activated T cells (ATC). The effects of AT-101 on cytotoxicity, early apoptosis, and Granzyme B (GrzB) and IFN-γ signaling pathways were evaluated during EGFR bispecific antibody armed ATC (aATC)-mediated killing of L3.6pl and MiaPaCa-2 PC cells pre-sensitized with AT-101. We found that pretreatment of tumor cells with AT-101 enhanced susceptibility of L3.6pl and MiaPaCa-2 tumor cells to ATC and aATC-mediated cytotoxicity, which was in part mediated via enhanced release of cytolytic granule GrzB from ATC and aATC. AT-101-sensitized L3.6pl cells showed up-regulation of IFN-γ-mediated induction in the phosphorylation of Ser⁷²⁷-Stat1 (pS⁷²⁷-Stat1), and IFN-γ induced dephosphorylation of phospho-Tyr⁷⁰⁵-Stat3 (pY⁷⁰⁵-Stat3). Priming (conditioning) of PC cells with AT-101 can significantly enhance the anti-tumor activity of EGFRBi armed ATC through increased IFN-γ induced activation of pS⁷²⁷-Stat1 and inhibition of pY⁷⁰⁵-Stat3 phosphorylation, and resulting in increased ratio of pro-apoptotic to anti-apoptotic proteins. Our results verify enhanced cytotoxicity after a novel chemotherapy conditioning strategy against PC that warrants further in vivo and clinical investigations.     

A New Format of Single Chain Tri-specific Antibody with Diminished Molecular Size Efficiently Induces Ovarian Tumor Cell Killing

A combination of bi-specific antibodies (BsAb), anti-tumor×anti-CD3 and anti-tumor×anti-CD28, is effective in vitro and in vivo, whereas production of two kinds of bi-specific antibodies is labor intensive and administration is complicated. Accordingly, we previously developed a new model of single chain tri-specific antibody (scTsAb), sTRI, which linked both the CD3 and CD28 signals for T-cell activation in one molecule, and demonstrated its capacity for triggering T-cells to kill ovary tumor cells. To improve the pharmacokinetics further and decrease the immunogenicity of scTsAb, we have now generated a new format of scTsAb, TR3H, whose molecular size is smaller than sTRI. Here we describe the construction, purification and characterization of TR3H. TR3H scTsAb bound to effector cells and tumor target cells specifically and induced redirected lyses of ovary tumor cells through freshly isolated, unstimulated human peripheral blood lymphocytes (PBLs). This new format of scTsAb possesses properties that support its potential as a new tumor immunotherapeutic agent. Revisions requested August 2005; Revisions received 14 September 2005     

A Cancer Vaccine Induces Expansion of NY-ESO-1-Specific Regulatory T Cells in Patients with Advanced Melanoma

Cancer vaccines are designed to expand tumor antigen-specific T cells with effector function. However, they may also inadvertently expand regulatory T cells (Treg), which could seriously hamper clinical efficacy. To address this possibility, we developed a novel assay to detect antigen-specific Treg based on down-regulation of surface CD3 following TCR engagement, and used this approach to screen for Treg specific to the NY-ESO-1 tumor antigen in melanoma patients treated with the NY-ESO-1/ISCOMATRIX^TM cancer vaccine. All patients tested had Treg (CD25^bright FoxP3⁺ CD127^neg) specific for at least one NY-ESO-1 epitope in the blood. Strikingly, comparison with pre-treatment samples revealed that many of these responses were induced or boosted by vaccination. The most frequently detected response was toward the HLA-DP4-restricted NY-ESO-1_157–170 epitope, which is also recognized by effector T cells. Notably, functional Treg specific for an HLA-DR-restricted epitope within the NY-ESO-1_115–132 peptide were also identified at high frequency in tumor tissue, suggesting that NY-ESO-1-specific Treg may suppress local anti-tumor immune responses. Together, our data provide compelling evidence for the ability of a cancer vaccine to expand tumor antigen-specific Treg in the setting of advanced cancer, a finding which should be given serious consideration in the design of future cancer vaccine clinical trials.     

5-Aza-CdR对人胃癌细胞株生物学行为及RASSFlA mRNA表达的影响

观察不同浓度的5-氮-2′-脱氧胞苷(5-Aza-CdR)对人胃癌细胞株BGC-823、SGC-7901、MKN-28生长及RASSF1A mRNA表达的影响。方法:分别以0．4μmol/L、1．6μmol/L、6．4μmol/L、25．6μmol/L、102．4μmol/L浓度的5-Aza-CdR处理人胃癌细胞株BGC-823、SGC-7901、MKN-28,MTT比色法测定72h时间段的吸光度值、计算抑制率,流式细胞仪检测5-Aza-CdR对胃癌细胞株生长周期及凋亡的影响,RT-PCR检测5-Aza-CdR处理前、后抑癌基因RASSF1A mRNA的表达。结果:5-Aza-CdR抑制体外培养人胃癌细胞株BGC-823、SGC-7901、MKN-28生长,呈浓度依赖性;5-Aza-CdR能有效诱导BGC-823、SGC-7901、MKN-28细胞凋亡;RT-PCR检测人胃癌细胞株SGC-7901、MKN-28无RASSF1A mRNA表达,经5-Aza-CdR处理后基因重新表达, BGC-823处理前后RASSF1A mRNA均有表达。结论:新型抑癌基因RASSF1A与胃癌的发生相关,5-Aza-CdR能抑制胃癌细胞株的增殖,并促进凋亡,其机制可能与RASSF1A基因的重新表达有关。     

Combined TLR2/4-Activated Dendritic/Tumor Cell Fusions Induce Augmented Cytotoxic T Lymphocytes

Induction of antitumor immunity by dendritic cell (DC)-tumor fusion cells (DC/tumor) can be modulated by their activation status. In this study, to address optimal status of DC/tumor to induce efficient antigen-specific cytotoxic T lymphocytes (CTLs), we have created various types of DC/tumor: 1) un-activated DC/tumor; 2) penicillin-killed Streptococcus pyogenes (OK-432; TLR4 agonist)-activated DC/tumor; 3) protein-bound polysaccharides isolated from Coriolus versicolor (PSK; TLR2 agonist)-activated DC/tumor; and 4) Combined OK-432- and PSK-activated DC/tumor. Moreover, we assessed the effects of TGF-β1 derived from DC/tumor on the induction of MUC1-specific CTLs. Combined TLR2- and TLR4-activated DC/tumor overcame immune-suppressive effect of TGF-β1 in comparison to those single activated or un-activated DC/tumor as demonstrated by: 1) up-regulation of MHC class II and CD86 expression on DC/tumor; 2) increased fusion efficiency; 3) increased production of fusions derived IL-12p70; 4) activation of CD4⁺ and CD8⁺ T cells that produce high levels of IFN-γ; 5) augmented induction of CTL activity specific for MUC1; and 6) superior efficacy in inhibiting CD4⁺CD25⁺Foxp3⁺ T cell generation. However, DC/tumor-derived TGF-β1 reduced the efficacy of DC/tumor vaccine in vitro. Incorporating combined TLRs-activation and TGF-β1-blockade of DC/tumor may enhance the effectiveness of DC/tumor-based cancer vaccines and have the potential applicability to the field of adoptive immunotherapy.     

PD-L1 Expression on Retrovirus-Infected Cells Mediates Immune Escape from CD8+ T Cell Killing

Cytotoxic CD8+ T Lymphocytes (CTL) efficiently control acute virus infections but can become exhausted when a chronic infection develops. Signaling of the inhibitory receptor PD-1 is an important mechanism for the development of virus-specific CD8+ T cell dysfunction. However, it has recently been shown that during the initial phase of infection virus-specific CD8+ T cells express high levels of PD-1, but are fully competent in producing cytokines and killing virus-infected target cells. To better understand the role of the PD-1 signaling pathway in CD8+ T cell cytotoxicity during acute viral infections we analyzed the expression of the ligand on retrovirus-infected cells targeted by CTLs. We observed increased levels of PD-L1 expression after infection of cells with the murine Friend retrovirus (FV) or with HIV. In FV infected mice, virus-specific CTLs efficiently eliminated infected target cells that expressed low levels of PD-L1 or that were deficient for PD-L1 but the population of PD-L1high cells escaped elimination and formed a reservoir for chronic FV replication. Infected cells with high PD-L1 expression mediated a negative feedback on CD8+ T cells and inhibited their expansion and cytotoxic functions. These findings provide evidence for a novel immune escape mechanism during acute retroviral infection based on PD-L1 expression levels on virus infected target cells.     

hnRNPK与Nef相互作用并有利于细胞表面CD4的表达

目的:前期不同的研究分别证明HIV蛋白Nef下调宿主细胞表面受体CD4的表达,以及Nef与宿主细胞蛋白heterogeneous nuclear ribonucleoprotein K(hnRNPK)存在相互作用。因此提出了两个值得研究探讨的重要问题:(1)hnRNPK是否参与调节细胞表面CD4的表达?(2)Nef是否通过hnRNPK调节细胞表面CD4的表达?方法:利用半体外GST-pulldown技术验证Nef与hnRNPK存在相互作用。通过瞬时转染的方式将HIV-1Nef表达在He La-CD4细胞里,同时利用siRNA干扰技术敲低hnRNPK,最后运用流式细胞技术检测细胞表面CD4的表达水平。结果:(1)GST-pulldown结果验证了Nef与hnRNPK存在相互作用;(2)Nef的表达使细胞表面CD4水平下降约75%;(3)不管是否有Nef,hnRNPK的敲低都使细胞表面CD4表达水平明显下降(50%);同样的,Nef下调CD4的作用也不受hnRNPK敲低的影响。结论:(1)hnRNPK与Nef相互作用;(2)hnRNPK有利于细胞表面CD4的表达,其与Nef的下调作用的关系尚不明确,Nef对CD4的下调作用可能涉及有其他因素参与的复杂调控。     

Costunolide and Dehydrocostus Lactone as Inhibitors of Killing Function of Cytotoxic T Lymphocytes

Costunolide and dehydrocostus lactone were isolated from an extract of mokko (Saussurea lappa Clarke) as inhibitors of killing activity of cytotoxic T lymphocytes (CTL). Mokko lactone was also isolated as an inactive compound from the extract. The structure-activity relationship indicated that α-methylenel- γ-butyrolactone is required for the inhibitory effect. Costunolide markedly inhibited the granule exocytosis and the production of inositol phosphates in response to anti-CD3 monoclonal antibody (rnAb) stimulation at a concentration that did not affect the binding of anti-CD3 mAb. Tyrosine phosphorylation induced by crosslinking of CD3 molecules was significantly inhibited by costunolide in a dose-dependent manner. These results suggest that costunolide inhibits the killing activity of CTL through preventing the increase in tyrosine phosphorylation in response to the crosslinking of T-cell receptors.     

Tag7 (PGLYRP1) in Complex with Hsp70 Induces Alternative Cytotoxic Processes in Tumor Cells via TNFR1 Receptor

Tag7 (also known as peptidoglycan recognition protein PGRP-S, PGLYRP1), an innate immunity protein, interacts with Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against some tumor cell lines. In this study, we have analyzed the programmed cell death mechanisms that are induced when cells interact with the Tag7-Hsp70 complex, which was previously shown to be released by human lymphocytes and is cytotoxic to cancer cells. We show that this complex induces both apoptotic and necroptotic processes in the cells. Apoptosis follows the classic caspase-8 and caspase-3 activation pathway. Inhibition of apoptosis leads to a switch to the RIP1-dependent necroptosis. Both of these cytotoxic processes are initiated by the involvement of TNFR1, a receptor for TNF-α. Our results suggest that the Tag7-Hsp70 complex is a novel ligand for this receptor. One of its components, the innate immunity protein Tag7, can bind to the TNFR1 receptor, thereby inhibiting the cytotoxic actions of the Tag7-Hsp70 complex and TNF-α, an acquired immunity cytokine.     

A Biased Competition Theory of Cytotoxic T Lymphocyte Interaction with Tumor Nodules

The dynamics of the interaction between Cytotoxic T Lymphocytes (CTL) and tumor cells has been addressed in depth, in particular using numerical simulations. However, stochastic mathematical models that take into account the competitive interaction between CTL and tumors undergoing immunoediting, a process of tumor cell escape from immunesurveillance, are presently missing. Here, we introduce a stochastic dynamical particle interaction model based on experimentally measured parameters that allows to describe CTL function during immunoediting. The model describes the competitive interaction between CTL and melanoma cell nodules and allows temporal and two-dimensional spatial progression. The model is designed to provide probabilistic estimates of tumor eradication through numerical simulations in which tunable parameters influencing CTL efficacy against a tumor nodule undergoing immunoediting are tested. Our model shows that the rate of CTL/tumor nodule productive collisions during the initial time of interaction determines the success of CTL in tumor eradication. It allows efficient cytotoxic function before the tumor cells acquire a substantial resistance to CTL attack, due to mutations stochastically occurring during cell division. Interestingly, a bias in CTL motility inducing a progressive attraction towards a few scout CTL, which have detected the nodule enhances early productive collisions and tumor eradication. Taken together, our results are compatible with a biased competition theory of CTL function in which CTL efficacy against a tumor nodule undergoing immunoediting is strongly dependent on guidance of CTL trajectories by scout siblings. They highlight unprecedented aspects of immune cell behavior that might inspire new CTL-based therapeutic strategies against tumors.