The CD95 (APO-1/Fas) system is an important mediator of T-cell cytotoxicity. We investigated this system in 22 hepatocellular carcinomas (HCCs) from patients. All HCCs had partially or completely lost the expression of the CD95 receptor constitutively expressed by normal liver cells and might thus evade CD95-mediated killing. We also considered a new mechanism of immune evasion, namely, the active destruction of T-lymphocytes by tumor cells expressing CD95 ligand (CD95L). CD95L messenger RNA and protein could be detected in the HCCs. In coculture experiments, HepG2 hepatoblastoma cells, expressing CD95L mRNA after treatment with cytostatic drugs, killed CD95+ Jurkat lymphocytes. Our data suggest that tumor cells can evade immune attack by down-regulation of the CD95 receptor and killing of lymphocytes through expression of CD95L.

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Azathioprine and its metabolite 6-mercaptopurine (6-MP) are immunosuppressive drugs that are used in organ transplantation and autoimmune and chronic inflammatory diseases such as Crohn disease. However, their molecular mechanism of action is unknown. In the present study, we have identified a unique and unexpected role for azathioprine and its metabolites in the control of T cell apoptosis by modulation of Rac1 activation upon CD28 costimulation. We found that azathioprine and its metabolites induced apoptosis of T cells from patients with Crohn disease and control patients. Apoptosis induction required costimulation with CD28 and was mediated by specific blockade of Rac1 activation through binding of azathioprine-generated 6-thioguanine triphosphate (6-Thio-GTP) to Rac1 instead of GTP. The activation of Rac1 target genes such as mitogen-activated protein kinase kinase (MEK), NF-κB, and bcl-x L was suppressed by azathioprine, leading to a mitochondrial pathway of apoptosis. Azathioprine thus converts a costimulatory signal into an apoptotic signal by modulating Rac1 activity. These findings explain the immunosuppressive effects of azathioprine and suggest that 6-Thio-GTP derivates may be useful as potent immunosuppressive agents in autoimmune diseases and organ transplantation.

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Azathioprine and its metabolite 6-mercaptopurine (6-MP) are immunosuppressive drugs that are used in organ transplantation and autoimmune and chronic inflammatory diseases such as Crohn disease. However, their molecular mechanism of action is unknown. In the present study, we have identified a unique and unexpected role for azathioprine and its metabolites in the control of T cell apoptosis by modulation of Rac1 activation upon CD28 costimulation. We found that azathioprine and its metabolites induced apoptosis of T cells from patients with Crohn disease and control patients. Apoptosis induction required costimulation with CD28 and was mediated by specific blockade of Rac1 activation through binding of azathioprine-generated 6-thioguanine triphosphate (6-Thio-GTP) to Rac1 instead of GTP. The activation of Rac1 target genes such as mitogen-activated protein kinase kinase (MEK), NF-κB, and bcl-x L was suppressed by azathioprine, leading to a mitochondrial pathway of apoptosis. Azathioprine thus converts a costimulatory signal into an apoptotic signal by modulating Rac1 activity. These findings explain the immunosuppressive effects of azathioprine and suggest that 6-Thio-GTP derivates may be useful as potent immunosuppressive agents in autoimmune diseases and organ transplantation.

Alterations of TGF-beta signaling have been described in colorectal cancer, although the molecular consequences are largely unknown. By using transgenic mice overexpressing TGF-beta or a dominant-negative TGF-betaRII, we demonstrate that TGF-beta signaling in tumor infiltrating T lymphocytes controls the growth of dysplastic epithelial cells in experimental colorectal cancer, as determined by histology and a novel system for high-resolution chromoendoscopy. At the molecular level, TGF-beta signaling in T cells regulated STAT-3 activation in tumor cells via IL-6. IL-6 signaling required tumor cell-derived soluble IL-6R rather than membrane bound IL-6R and suppression of such TGF-beta-dependent IL-6 trans-signaling prevented tumor progression in vivo. Taken together, our data provide novel insights into TGF-beta signaling in colorectal cancer and suggest novel therapeutic approaches for colorectal cancer based on inhibition of TGF-beta-dependent IL-6 trans-signaling.

Helicobacter pylori infection is associated with chronic gastritis, peptic ulceration, and gastric carcinoma. The potential role of CD95-mediated apoptosis was investigated in a panel of gastric biopsies obtained from patients with H . pylori -associated chronic gastritis ( n ϭ 29) and with noninfected normal mucosa ( n ϭ 10). Immunohistochemistry revealed increased CD95 receptor expression in epithelial and lamina propria cells in chronic gastritis. By in situ hybridization, CD95 ligand mRNA was absent or low in normal mucosa but expressed at high levels in lamina propria lymphocytes and, unexpectedly, in epithelial cells in chronic gastritis. Apoptotic cells were rare in normal mucosa but were observed regularly in chronic gastritis in close proximity to CD95 ligand mRNA expression throughout the epithelial and lamina propria cells.In a functional analysis gastric epithelial cell lines were incubated with supernatants of H .

Major limitations of currently investigated␣␤T cells redirected against cancer by transfer of tumor-specific ␣␤TCR arise from their low affinity, MHC restriction, and risk to mediate self-reactivity after pairing with endogenous ␣ or ␤TCR chains. Therefore, the ability of a defined ␥9␦2TCR to redirect ␣␤T cells selectively against tumor cells was tested and its molecular interaction with a variety of targets investigated. Functional analysis revealed that a ␥9␦2TCR efficiently reprograms both CD4 ؉ and CD8 ؉ IntroductionThe major challenge in the field of adoptive immunotherapy is the generation of tumor-reactive ␣␤T cells which can be applied to a broad variety of cancer patients. To facilitate the rapid generation of tumor-reactive ␣␤T cells, it has been proposed that ␣␤T cells can be reprogrammed with genes encoding for a tumor-specific ␣␤TCR or a chimeric receptor. 1 Several such receptors are already being used to redirect ␣␤T cells in phase 1 clinical trials. 1,2 However, reprogramming ␣␤T cells with defined ␣␤TCRs is substantially hampered by their restriction to HLA types, thus limiting the number of patients who can be treated with one ␣␤TCR. In addition, pairing of introduced with endogenous ␣␤TCR chains can induce life-threatening autoreactivity. 3,4 One attractive alternative to mediate a selective antitumor reactivity with a high-affinity TCR might arise from the ability of ␥␦T cells to mediate antitumor reactivity while ignoring a healthy environment. [5][6][7] Isolated ␥9␦2T cells efficiently kill tumor cells of hematologic malignancies and from solid tumors. 7 However, the function and proliferation capacity of ␥␦T cells is frequently heavily impaired in cancer patients 8 making autologous ␥␦T cells less attractive for immune interventions. On the other hand, as end-stage cancer patients can easily elicit ␣␤T-cell immune responses against, for example, viral Ags, 9,10 ␣␤T cells might serve as carriers for broadly tumor-reactive ␥␦TCRs.The recognition of mevalonate metabolites (phosphoantigens) 11 which are overexpressed in a broad range of tumor cells has been suggested as an important mechanism by which multiple ␥9␦2TCR can sense malignant transformation as the recognition involves TCR domains which are conserved in most ␥9␦2TCRs. [12][13][14] In addition, ␥9␦2TCR G115 has been also suggested to bind to a complex of Apolipoprotein AI (ApoAI) and F1-ATPase, 15 a complex mitochondrial enzyme found on the surface of many malignant cells. 16 This knowledge might allow a rational design of ␥␦T cell-based immunotherapies. Therefore, we investigated whether a defined ␥9␦2TCR can be efficiently expressed in ␣␤T cells, mediate tumor-specific proliferation of ␣␤T cells, and redirect both effector CD8 ϩ and helper CD4 ϩ ␣␤T-cell subsets against a broad panel of tumor cell lines while ignoring normal cells in vitro and in vivo. MethodsCell lines, Abs, the retroviral transduction and expansion of ␣␤T cells, functional T-cell assays 11,[17][18][19][20] as well as the animal model used are described in supp...