A ONE-STEP PCR PROCEDURE FOR ANALYSIS OF TUMOR-SPECIFIC T-LYMPHOCYTE RESPONSES

In an effort to develop optimal conditions for analysis of tumor speci fic T lymphocyte responses, we have studied the effect of changes in t he concentration of oligonucleotide primers on the synthesis of TCR cD NAs in a one step PCR procedure using V beta 10 gene subfamily as a mo del. It was found that synthesis of the TCR cDNAs increases in a rough ly linear fashion at primer concentrations between 0.005-0.05 mu M Eva luation 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 subfam ilies in the spleen of BALB/c mice, but not in the peritoneal exudate cells (PECs), the latter requiring ten-fold higher concentrations of t he variable region primers to compensate for the overall low frequency of T lymphocytes in the PECs in comparison to the spleen. Use of thes e optimal conditions to detect L1210 tumor specific T lymphocyte respo nses showed that, in the immunized mice, L1210 specific T lymphocyte r esponses 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, thr ee 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 describ ed 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 am plification.