The interpretation of the results obtained from immunomonitoring of clinical trials is a diYcult task due to the variety of methods and protocols available to detect vaccine-speciWc T-cell responses. This heterogeneity as well as the lack of standards has led to signiWcant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer ("CIMT") in order to compare techniques and protocols applied for the enumeration of antigen-speciWc T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the "CIMT monitoring panel". A total of 13 centers from six European countries participated in the study in which pretested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-speciWc T-cells in each sample using tetramer staining and one functional assay. The results of the Wrst testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-speciWc CD8 + T-cells by tetramer staining. Analysis by ELISPOT was inXuenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the Wrst phase. 123The recommendations improved the number of antigenspeciWc T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identiWcation of distinct variables that inXuence the sensitivity of diVerent T-cell assays and to formally show that a deWned correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such "two-step" inter-laboratory projects could deWne rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.

We studied global gene expression in three melanoma cell lines with the most common and potent V600E mutation in the B-RAF gene-four cell lines with a common Q61R mutation in the N-RAS gene and three cell lines with no mutations using human HG-U133A 2.0 micro-arrays with 22 277 transcripts. Data analysis using stringent criteria revealed several upregulated and downregulated genes in cell lines with B-RAF and N-RAS mutations compared with cell lines without mutations. We found 29 genes specifically upregulated and 32 genes downregulated in cell lines with B-RAF mutations, whereas 70 genes were upregulated and 39 downregulated in cell lines with N-RAS mutations; 11 genes showed overlapping upregulation and 45 downregulation. The micro-array data for nine selected genes were validated by the real-time PCR technique. Expression of a large number of genes, that encode members or regulators of the RAS/RAF/MEK/ERK pathways or are involved in metastasis or invasion, was affected in cell lines with mutations in B-RAF and N-RAS. Upregulated genes in cell lines with mutations included dual-specificity phosphatase 6 (DUSP6), sprouty 2 (SPRY2), v-akt murine thymoma viral oncogene homolog 3 (AKT3) and matrix metalloproteinase 14 (MMP14); downregulated genes included interleukin 18 (IL18), Krüppel-like factor 5 (KLF5) and inhibitor of DNA binding 2 (ID2). Our results, though carried on cell lines, provide a novel insight into the effect of mutations in the B-RAF and N-RAS genes on global gene expression in melanoma and highlight the complexity of mechanisms involved in tumour initiation and maintenance.

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Interleukin-12 (IL-12), a heterodimeric cytokine, is important in the generation of a Th1-biased immune response. Several polymorphisms have been described in IL12B, the gene encoding the p40 subunit of IL-12. A bi-allelic polymorphism within the IL12B promoter region has been reported to show association with diseases as diverse as severe childhood asthma and fatal cerebral malaria. In order to define the molecular basis for these disease associations, we investigated the secretion of IL-12 by human monocyte-derived dendritic cells. Homozygotes for the IL12B promoter polymorphism showed a 10-fold difference in median p70 secretion in response to CD40 ligation. Remarkably, this difference resulted from the inability of most allele 1 homozygotes to secrete heterodimeric IL-12. In contrast, most of the donors homozygous for allele 2 had detectable secretion. These findings are important for the understanding of the highly complex regulation of IL-12 secretion, and its consequent impact on disease susceptibility, in humans.