Adoptive T-cell therapy represents a promising therapeutic approach for the treatment of cancer. Successful adoptive immunotherapy depends on the ex vivo priming and expansion of antigen-specific T ...cells. However, the in vitro generation of adequate numbers of functional antigen-specific T cell remains a major obstacle. It is important to develop efficient and reproducible methods to generate high numbers of antigen-specific T cells for adoptive T-cell transfer. We have developed a new artificial antigen-presenting cell (aAPC) by transfection of major histocompatibility (MHC) class I negative Daudi cells with a peptide-beta2-microglobulin-MHC fusion construct (single-chain aAPC) ensuring presentation of the peptide-MHC complex of interest. Using this artificial antigen-presenting cell, we could generate up to 9.2 x 10(8) antigen-specific cytotoxic CD8(+) T cells from 10 ml blood. In vitro generated T cells lysed endogenously presented antigens. Direct comparison of the single-chain aAPC with autologous monocyte-derived dendritic cells demonstrated that these cells were equally efficient in stimulation of T cells. Finally, we were able to generate antigen-specific T cell lines from perpheral blood mononuclear cells of patients receiving cytotoxic chemotherapy. The use of single-chain aAPC represent a promising option for the generation of antigen-specific CD8(+) T cells, which could be used for adoptive T-cell therapy.
NF-κB is a key transcriptional regulator of inflammatory responses, but also controls expression of prosurvival genes, whose products protect tissues from damage and may thus act indirectly in an ...antiinflammatory fashion. The variable importance of these two distinct NF-κB-controlled responses impacts the potential utility of NF-κB inhibition as a treatment strategy for intractable inflammatory conditions, such as inflammatory bowel disease. Here, we show in murine models that inhibition of IKKβ-dependent NF-κB activation exacerbates acute inflammation, but attenuates chronic inflammatory disease in the intestinal tract. Acute ulcerating inflammation is aggravated because of diminished NF-κB-mediated protection against epithelial cell apoptosis and delayed mucosal regeneration secondary to reduced NF-κB-dependent recruitment of inflammatory cells that secrete cytoprotective factors. In contrast, in IL-10-deficient mice, which serve as a model of chronic T cell-dependent colitis, ablation of IKKβ in the intestinal epithelium has no impact, yet IKKβ deficiency in myeloid cells attenuates inflammation and prolongs survival. These results highlight the striking context and tissue dependence of the proinflammatory and antiapoptotic functions of NF-κB. Our findings caution against the therapeutic use of IKKβ/NF-κB inhibitors in acute inflammatory settings dominated by cell loss and ulceration.
Myeloid-derived suppressor cells (MDSC) represent a heterogeneous population of immature myeloid cells that accumulate in blood, liver, spleen and tumors upon chronic inflammation and tumor ...development in patients and mice. Acute hepatitis is characterized by a fast infiltration of inflammatory cells in the liver and increased enzymatic activity at this organ that could lead into liver fibrosis and chirrosis. We have studied the biology of hepatic MDSC in acute hepatitis. Unexpectedly, hepatic MDSC, which accumulate in the liver of mice bearing subcutaneous tumors, failed to suppress inflammatory responses upon Con A injection, but instead were responsible for exacerbating acute liver damage. Phenotypic, genetic and functional studies demonstrated rapid changes of hepatic MDSC from a suppressor phenotype into a pro-inflammatory subset as early as 3 hours after Con A injection. An increase in the expression of pro-inflammatory cytokines, costimulatory molecules such as CD80, CD86 and CD40 along with a loss of suppressor function was noticed in mice upon Con A treatment. These changes were CD40-dependent and not found in CD40-/- MDSC. Interestingly, CD40 ligation of human MDSC in vitro resulted in down-regulation of arginase I expression and suppressor function. Finally, blockade of ROS production in hepatic MDSC ameliorated hepatocyte damage suggesting that MDSC mediated toxicity was ROS dependent. We believe that these findings reflect how MDSC plasticity can be modulated to promote inflammation, opening a new path for therapies targeting innate suppressive cells in cancer patients.
A link between inflammation and cancer has long been suspected, but its molecular nature remained ill defined. A key player in inflammation is transcription factor NF-kappaB whose activity is ...triggered in response to infectious agents and proinflammatory cytokines via the IkappaB kinase (IKK) complex. Using a colitis-associated cancer model, we show that although deletion of IKKbeta in intestinal epithelial cells does not decrease inflammation, it leads to a dramatic decrease in tumor incidence without affecting tumor size. This is linked to increased epithelial apoptosis during tumor promotion. Deleting IKKbeta in myeloid cells, however, results in a significant decrease in tumor size. This deletion diminishes expression of proinflammatory cytokines that may serve as tumor growth factors, without affecting apoptosis. Thus, specific inactivation of the IKK/NF-kappaB pathway in two different cell types can attenuate formation of inflammation-associated tumors. In addition to suppressing apoptosis in advanced tumors, IKKbeta may link inflammation to cancer.
Human pancreatic adenocarcinoma has an overall poor prognosis. Therapeutic efforts are often ineffective because of late diagnosis and a high degree of chemoresistance. Overexpression of transforming ...growth factor alpha in the pancreas of transgenic mice causes the formation of premalignant ductal lesions and the development of invasive ductal adenocarcinoma. The aim of the present study was to explore regulation of proapoptotic and antiapoptotic signals during pancreatic tumor development in mice.
EL-TGFalpha-hGH transgenic mice crossbred to p53-deficient mice develop ductal pancreatic adenocarcinoma resembling the human disease. During the multistep carcinogenesis up-regulation of Bcl-x(L) is evident early and persists throughout tumorigenesis as detected by Real Time PCR, Western blot analysis, and immunofluorescence.
Up-regulation of Bcl-x(L) is evident early in tumor development and persists throughout tumorigenesis. The transcription factors Stat3 and NF-kappaB induce increased Bcl-x(L) expression in the premalignant lesions and tumor cells. Inhibition of either transcription factor alone leads to Bcl-x(L) down-regulation in transient transfection assays. Functional analysis shows that blocking of both Stat3 and NF-kappaB together induces programmed cell death in murine pancreatic tumor cells.
These findings indicate that apoptosis resistance precedes formation of invasive pancreatic cancer. Therefore, combined inhibition of Stat3 and NF-kappaB might represent a novel strategy for tumor prevention and therapy.
To determine the effects of rolipram, a specific type IV phosphodiesterase inhibitor, on tumor necrosis factor (TNF)-alpha production and HIV-1 replication.
TNF-alpha enhances HIV-1 replication in ...vitro; blocking TNF-alpha and thereby inhibiting HIV-1 replication may therefore potentially delay progression of HIV disease. Pentoxifylline is a non-specific phosphodiesterase inhibitor that blocks TNF-alpha synthesis and HIV-1 replication in vitro and has been shown in preliminary clinical studies to decrease viral replication in HIV-1-infected patients. Rolipram, which selectively inhibits the predominant phosphodiesterase isoenzyme of monocytes, inhibits lipopolysaccharide (LPS)-induced TNF-alpha with 500-fold greater potency than pentoxifylline. We, therefore, hypothesized that rolipram would be a powerful inhibitor of HIV-1 replication.
The effects of rolipram and pentoxifylline on TNF-alpha production and HIV-1 replication were determined in infected and uninfected peripheral blood mononuclear cells (PBMC), in a chronically infected promonocytic cell line (U1) and in an acutely infected monocytic cell line (BT4A3.5). TNF-alpha was determined by specific radioimmunoassay and HIV-1 replication was measured by p24 antigen and HIV-1 mRNA production.
Rolipram inhibited TNF-alpha production in LPS- and phorbol myristate acetate (PMA)-stimulated PBMC and in PMA-stimulated U1 cells. Rolipram also inhibited HIV-1 replication in the U1 cell line, as well as in acutely infected PBMC and BT4A3.5 cells. Depending on the experimental conditions, rolipram was 10-600 times more potent, on a molar basis, than pentoxifylline.
Rolipram is a potent inhibitor HIV-1 replication and therefore deserves further investigation as a potential therapeutic agent in the treatment of HIV-1-infected patients.
To discern the T cell subtype associated with T cell differentiation, the expression of CD45RA and CD27 was measured from total CD8(high) cells and from human T cell lymphotropic virus type I ...(HTLV-I) Tax11-19 peptide-specific CD8(+) cells in peripheral blood lymphocytes of patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Phenotypically defined memory and/or effector cells (CD45RA(-)CD27(+), CD45RA(+)CD27(-), and CD45RA(-)CD27(-)) were increased in HAM/TSP CD8(+) cells, compared with those of HTLV-I-seronegative healthy control subjects. The percentage of human leukocyte antigen (HLA)-DR-positive cells was also increased in CD8(+) cells of HAM/TSP, compared with those in HLA-DR(+)CD8(+) cells of healthy control subjects. HTLV-I provirus load correlated with the frequency of Tax11-19-specific CD8(+) cells. The high frequency of memory and/or effector type HTLV-I Tax11-19-specific CD8(+) cells suggests that continuous restimulation driven by HTLV-I antigens in vivo may be associated with the pathogenesis of HAM/TSP.
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