Purpose: In glioma-in contrast to various other cancers-the impact of T-lymphocytes on clinical outcome is not clear. We investigated the clinical relevance and regulation of T-cell infiltration in glioma.Experimental Design: T-cell subpopulations from entire sections of 93 WHO II-IV gliomas were computationally identified using markers CD3, CD8, and Foxp3; survival analysis was then done on primary glioblastomas (pGBM). Endothelial cells expressing cellular adhesion molecules (CAM) were similarly computationally quantified from the same glioma tissues. Influence of prominent cytokines (as measured by ELISA from 53 WHO II-IV glioma lysates) on CAM-expression in GBM-isolated endothelial cells was determined using flow cytometry. The functional relevance of the cytokine-mediated CAM regulation was tested in a transmigration assay using GBM-derived endothelial cells and autologous T-cells.Results: Infiltration of all T-cell subsets increased in high-grade tumors. Most strikingly, within pGBM, elevated numbers of intratumoral effector T cells (T eff , cytotoxic and helper) significantly correlated with a better survival; regulatory T cells were infrequently present and not associated with GBM patient outcome. Interestingly, increased infiltration of T eff cells was related to the expression of ICAM-1 on the vessel surface. Transmigration of autologous T cells in vitro was markedly reduced in the presence of CAM-blocking antibodies. We found that TGF-b molecules impeded transmigration and downregulated CAM-expression on GBM-isolated endothelial cells; blocking TGF-b receptor signaling increased transmigration.Conclusions: This study provides comprehensive and novel insights into occurrence and regulation of T-cell infiltration in glioma. Specifically, targeting TGF-b1 and TGF-b2 might improve intratumoral T-cell infiltration and thus enhance effectiveness of immunotherapeutic approaches. Clin Cancer Res; 17(13); 4296-308. Ó2011 AACR.
DNA methylation patterns of mammalian genomes are generated in gametogenesis and early embryonic development. Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the process. Both enzymes contain a long N-terminal regulatory region linked to a conserved C-terminal domain responsible for the catalytic activity. Although a PWWP domain in the N-terminal region has been shown to bind DNA in vitro, it is unclear how the DNA methyltransferases access their substrate in chromatin in vivo. We show here that the two proteins are associated with chromatin including mitotic chromosomes in mammalian cells, and the PWWP domain is essential for the chromatin targeting of the enzymes. The functional significance of PWWPmediated chromatin targeting is suggested by the fact that a missense mutation in this domain of human DNMT3B causes immunodeficiency, centromeric heterochromatin instability, facial anomalies (ICF) syndrome, which is characterized by loss of methylation in satellite DNA, pericentromeric instability, and immunodeficiency. We demonstrate that the mutant protein completely loses its chromatin targeting capacity. Our data establish the PWWP domain as a novel chromatin/ chromosome-targeting module and suggest that the PWWP-mediated chromatin association is essential for the function of the de novo methyltransferases during development.
Severe immune suppression is frequent in late-stage tumor patients and promotes tumor immune evasion and subsequent tumor progression. Regulatory T cells (Treg) are major suppressors of anti-tumor immune responses. Therefore, targeting of Treg has become a key goal of anti-tumor therapy. Several preclinical and clinical observations suggest that Treg can be depleted by cyclophosphamide. Over a period of 3 months, we investigated the effect of metronomic low-dose cyclophosphamide on Treg numbers, suppressive capacity and proliferation on endogenous anti-tumor T-cell responses and on their correlation to clinical outcome in 12 patients with treatment-refractory metastasized breast cancer who received single-agent 50 mg cyclophosphamide p.o. daily. Cyclophosphamide treatment initially caused a significant reduction in circulating Treg by more than 40% (P = 0.002). However, Treg numbers completely recovered during the treatment due to increased proliferative activity and maintained their suppressive capacity. Treg depletion coincided with a strong increase in breast tumor-reactive T cells (P = 0.03) that remained at high levels during the whole period. Numbers of tumor-reactive T cells but not of Treg correlated with disease stabilization (P = 0.03) and overall survival (P = 0.027). We conclude that metronomic low-dose cyclophosphamide only transiently reduces Treg but induces stable tumor-specific T-cell responses, which correlate with improved clinical outcome in advanced-stage breast cancer patients.
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