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 one step PCR procedure using Vbeta10 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 muM. Evaluation of the use of low concentration (0.005 muM) of primers showed these conditions to be adequate for the analysis of TCR Vbeta 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 Vbeta subfamilies, including Vbeta8.2, Vbeta15 and Vbeta16 were found to contain specific major TCR cDNA bands. The approach described here is very efficient, as it uses a small amount of the 32P isotope (0.5 muCi) followed by direct analysis of the PCR 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 Vbeta subfamily are preserved during PCR amplification.
Abu-hadid
M M
MM
Department of Molecular Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Fuji
H
H
Hsu
S
S
Sood
A K
AK
D000818:Animals; D001483:Base Sequence; D002999:Clone Cells; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D015329:Gene Rearrangement, T-Lymphocyte; D007939:Leukemia L1210 / Q000276:immunology*; D051379:Mice; D008811:Mice, Inbred DBA; D008969:Molecular Sequence Data; D010529:Peritoneal Cavity / Q000166:cytology; D016133:Polymerase Chain Reaction / Q000379:methods*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D013154:Spleen / Q000166:cytology
D017931:DNA Primers; D018076:DNA, Complementary; D016693:Receptors, Antigen, T-Cell, alpha-beta