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Standardized method for the immuno-monitoring of DC vaccination trials

Post-clinical protocol

We are progressing steadily towards the delivery of a standardized method for the immuno-monitoring of DC vaccination trials using a finite number of defined tumor antigens. Our starting point was the method that we had developed previously to estimate blood frequencies of CD8 T lymphocytes against one defined antigenic peptide. It was based on an in vitro restimulation of blood lymphocytes with the peptide and growth factors over two weeks followed by detection with the appropriate HLA class I tetramer. In order to estimate frequencies of responding cells, these mixed lymphocyte peptide cultures (MLPC) are conducted in limiting dilution condition. We have validated two important modifications to this protocol.

i) MLPC/tetramer with multiple peptides presented by HLA class I molecules. We now have a stimulation method that can be used for up to 8 antigenic peptides simultaneously, all presented by HLA class I molecules. To avoid competition between the peptides for HLA binding, the initial stimulation of the PBMC is carried out by dividing the initial population into 8 fractions, each of which is pulsed with one peptide, and pooling these individually pulsed fractions before the distribution into the limiting dilution microcultures. For tetramer detection, we obtain similar results with either of two methods. The first is simply to mix all tetramers, a procedure that, if each reagent is carefully titrated, does not generate a disturbing non specific labeling. A positive microculture has then to be retested individually with each tetramer. The advantage of this procedure is that the initial screening has to be carried out only once. This procedure is applicable when the proportion of positive microcultures is low. The second method is to carry out the screening with mixes of three tetramers with an overlap of one. For tetramers 1-8 this gives 1,2,3 and 3,4,5 and 5,6,7 and 7,8,1. Here the initial screening uses four labelings, but one can immediately determine which antigen is recognized in which microculture.

ii) MLPC/tetramer including a peptide presented by HLA class II molecules. With the availability of a new type of HLA class II tetramer, consisting of recombinant HLA-DP4 molecules folded with the MAGE-A3 peptide presented by DP4, we could test whether the MLPC/tetramer method could be used also for a vaccine peptide recognized by CD4 T cells. We found out that the addition of the MAGE-3.DP4 peptide to the mix of 8 peptides described above allowed for the stimulation, in the same microcultures, of both CD4 and CD8 peptide-specific responder cells. Tetramer detection was as sensitive as for the class I reagents and could be performed simultaneously. This improvement could be very useful for all the trials that combine class I and class II antigenic peptides.

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