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We analysed the promoter methylation status of five genes, involved in adhesion (EPB41L3, TSLC-1), apoptosis (RASSF1, RASSF2) or angiogenesis (TSP-1), in intraoperative sentinel lymph node (SLN) biopsy samples from patients with breast cancer, that had been processed by the one-step nucleic acid amplification (OSNA) technique. SLN resection is performed to estimate the risk of tumour cells in the clinically negative axilla, to avoid unnecessary axillary lymph node dissection. OSNA is currently one of the eligible molecular methods for detecting tumour cells in SLNs. It is based on the quantitative evaluation of cytokeratin 19 mRNA which allows distinguishing between macrometastasis, micrometastasis and isolated tumour cells, on the basis of the quantity of tumour cells present. There have been no prior studies on the question whether or not samples processed by OSNA can be used for further molecular studies, including epigenetic abnormalities which are some of the most important molecular alterations in breast cancer. Genomic DNA was extracted from samples obtained from 50 patients diagnosed with primary breast cancer. The content of tumour cells in SLNs was evaluated by OSNA, and the promoter methylation status of the selected genes was analysed by methylation-specific PCR. All were found to be hypermethylated to a variable degree, and RASSF1 hypermethylation was significantly associated with macrometastasis, micrometastasis and isolated tumour cells (p = 0.002). We show that samples used for OSNA are suitable for molecular studies, including gene promoter methylation. These samples provide a new source of material for the identification of additional biomarkers. [ABSTRACT FROM AUTHOR]

The release of cytokines by T cells strongly defines their functional activity in vivo. The ability to produce multiple cytokines has been associated with beneficial immune responses in cancer and infectious diseases, while their progressive loss is associated with T-cell exhaustion, senescence and anergy. Consequently, strategies that enhance the multifunctional status of T cells are a key for immunotherapy. Dendritic cells (DCs) are professional antigen presenting cells that regulate T-cell functions by providing positive and negative co-stimulatory signals. A key negative regulator of T-cell activity is provided by binding of programmed death-1 (PD-1) receptor on activated T cells, to its ligand PD-L1, expressed on DCs. We investigated the impact of interfering with PD-L1/PD-1 co-stimulation on the multifunctionality of T cells, by expression of the soluble extracellular part of PD-1 (sPD-1) or PD-L1 (sPD-L1) in human monocyte-derived DCs during antigen presentation. Expression, secretion and binding of these soluble molecules after mRNA electroporation were demonstrated. Modification of DCs with sPD-1 or sPD-L1 mRNA resulted in increased levels of the co-stimulatory molecule CD80 and a distinct cytokine profile, characterized by the secretion of IL-10 and TNF-α, respectively. Co-expression in DCs of sPD-1 and sPD-L1 with influenza virus nuclear protein 1 (Flu NP1) stimulated Flu NP1 memory T cells, with a significantly higher number of multifunctional T cells and increased cytokine secretion, while it did not induce regulatory T cells. These data provide a rationale for the inclusion of interfering sPD-1 or sPD-L1 in DC-based immunotherapeutic strategies. [ABSTRACT FROM AUTHOR]

T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNγ and TNFα in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells. [ABSTRACT FROM AUTHOR]