We have developed immunological techniques for monitoring of tumour antigen specific CD4+ T cell responses. We set an in vitro system able to detect fona fide in vivo primed CD4+ T cells. Purified CD4+ T cells are first stimulated in vitro once in the presence peptides, previously identified as containing naturally processed epitopes on tumour-associated antigens. After two weeks, proliferation and cytokine secretion (IFN-?, IL-5, IL-4, IL-2, TNF-?, IL-10, TGF-?, IL-17, granzyme and perforin) are tested. To estimate the frequency of antigen specific CD4+ T cell we us...

Real time and immunohistochemistry analysis was performed in a study to evaluate whether iNOS can serve as a prognostic factor in stage III melanoma survival. The analysis showed that both iNOS and COX-2 alone significantly predicted OS. The BRAF/NRAS mutation status did not significantly differ between patient groups, although iNOS significantly correlated with BRAF mutation frequency. Furthermore, iNOS and COX-2 were significantly linked to a number of metastatic lymph nodes. However, iNOS maintained its statistical significance together with a number of lymph node...

The method is about transfecting CD4(+) and CD8(+) T cells with mRNA encoding recombinant immunoreceptors for use in the adoptive immunotherapy of cancer. CD4(+) and CD8(+) T cells were efficiently transfected with immunoreceptors specific for ErbB2 and CEA. The immunoreceptor expression was transient with half-maximal expression at day 2 and no detectable immunoreceptor expression at day 9 after electroporation. Opposed to standard procedures using retroviral gene transduction to constitutively express immunoreceptors in T cells, transient immunoreceptor expressi...

Using MAGE-A3 as a model antigen we established a functional read-out system by transfection of CD4 T cells with RNA encoding T cell receptors recognizing MHC DP4 restricted MAGE-A3 epitopes.

We have expertise in western blotting. Western blot was used, for instance, for the demonstration of protein kinase R phosphorylation on transduction.

We have knowhow on ELISPOT analysis of long peptide vaccines.

Our lab has expertise about ELISPOT analyses from the long peptide trial in end stage cervical carnoma, resected cervical carcinoma and VIN III patients.

An Elispot assay was applied in the establishment of immunomonitoring vaccination trials with RNA transfected DC by using overlapping peptides.

We have know-how about performing LDA assays. We have, for instance, monitored vaccine induced T cell responses for the two clinical trials. We found CD8+ and CD4+ T cell responses against the immunological tracers KLH and TK, and against the NTPs and MAGE-3, respectively.

We have experience about using Elispot assays for monitoring vaccine induced specific anti-tumor T cell responses.

Our lab has expertise in performing ELISA assays. For example, we have monitored vaccine induced T cell responses for two clinical trials. We found CD8+ and CD4+ T cell responses against the immunological tracers KLH and TK, and against the NTPs and MAGE-3, respectively.

In our laboratory we have know-how on Elispot analysis. For instance, an IFN-ELISPOT assay was used to determine that co-cultures of DC and T cells led to antigen specific, IFN-? secreting T cells after 1 restimulation at a DC:T cell ratio of 1:20.

Knowledge on ELISPOT analyses is available in our lab for immunomonitoring of patients treated with Apo-DC +/- GM-CSF.

We have experience with ELISA assay for IL-17 from e-Bioscience.

We have experience with ELISA for IL-12p70 from R&D System.

An ELISA assay using the kit from Biosource was performed according to the manufacturer’s instructions for the measurement of different cytokines.

Our lab has expertise with Elispot assays. We evaluated the capacity of autologous DC and HEK293 cells transfected with relevant HLA alleles to function as T cell targets in Elispot assays upon transfection with tumor antigen encoding plasmids.

We have know-how on assays to measure the proportions of Treg cells. For instance, we set up an assay to measure the proportions of Treg cells in tissues or cells that is based on the demethylation of a region of the FOXP3 promoter, which is observed in human Treg cells but not in the other T cells that can express FOXP3 transiently after activation. Starting from DNA, two quantitative PCR are used, which amplify the methylated and unmethylated FOXP3 promoter sequences. The ratio between the expression levels is, in our hands, a faithful Treg marker. We will pursue...

Using this peptide library technique and ELISPOT analysis patients´ PBMC are pre-screened for CD4- and CD8- T cell responses and individual sets of 4 to 8 peptides chosen for further analysis.

In our lab we have experience in performing real time PCR analyses.