A one step PCR procedure for analysis of tumor specific T lymphocyte responses

In an effort to develop optimal conditions for analysis of tumor specific T lymphocyte responses, we have studied the effect of changes in the concentration of oligonucleotide primers on the synthesis of TCR cDNAs in a one step PCR procedure using V beta 10 gene subfamily as a model. It was found that synthesis of the TCR cDNAs increases in a roughly linear fashion at primer concentrations between 0.005-0.05 mu M Evaluation of the use of low concentration (0.005 mu M) Of primers showed these conditions to be adequate for the analysis of TCR V beta subfamilies in the spleen of BALB/c mice, but not in the peritoneal exudate cells (PECs), the latter requiring ten-fold higher concentrations of the variable region primers to compensate for the overall low frequency of T lymphocytes in the PECs in comparison to the spleen. Use of these optimal conditions to detect L1210 tumor specific T lymphocyte responses showed that, in the immunized mice, L1210 specific T lymphocyte responses are detectable in the PECs, but not in the spleen cells from these mice, Thus, upon i.p. immunization of DBA/2 mice with irradiated L1210 lymphoma cells, followed by analysis of the PECs by RT/PCR, three TCR V beta subfamilies, including V beta 8.2, V beta 15 and V beta 16 were found to contain specific major TCR cDNA bands. The approach P-32 isotope (0.5 mu Ci) followed by direct analysis of the PCR described here is very efficient as it uses a small amount of the products on a denaturing acrylamide/urea gel. Furthermore, data is also presented that shows that quantitative differences in the levels of individual TCR cDNAs in a particular V beta subfamily are preserved during PCR amplification.