Quantitation of T-cell receptor frequencies by competitive PCR: Generationand evaluation of novel TCR subfamily and clone specific competitors

T cell receptor (TCR) V gene usage has been used to characterize the immune response to bacteria, viruses, allografts, self antigens, tumor antigens, and superantigens. Sensitive methods to detect changes in the frequency of TCR subfamilies or clonotypes might be useful in evaluating the efficacy of vaccines against infectious agents, immunotherapy treatments for cancer pa tients, or the status of autoimmune diseases. Two HLA-A2 restricted CTL clo nes expressing BV17 were isolated from a tumor infiltrating lymphocytes (TI L) culture of a patient with metastatic melanoma. One clone recognized the MART-1((27-35)) peptide and the other clone recognized the gp100((209-217)) peptide. The frequency of each of these CTL clones in an expanding TIL cul ture was measured using a novel competitive RT-PCR (cRT-PCR) strategy. cRT- PCR uses a single primer pair to amplify template cDNA simultaneously with a modified DNA competitor molecule. A rapid two-step PCR technique followed by a single cloning step was used to generate a TCR BV17 subfamily specifi c competitor or competitors specific for the MART-1((27-35)) reactive CTL c lone (CO-41) and the gp100((209-217)) reactive CTL clone (CO-4). Each compe titor contained a segment of the TCR BC region that served as an internal r eference standard. Using the BV17 competitor we were able to accurately and reproducibly measure cDNA templates at a frequency as low as 1/100,000 usi ng cDNA samples of known TCRBV subfamily composition. This competitor was u sed to monitor the frequency of BV17 expressing T cells in the TTL and PMBC of a patient with metastatic melanoma. We determined that the frequency of BV17 expressing T cells increased from 4.5% of the culture on day 35 to 60 .7% of the culture on day 58. Expansion of the BV17 subfamily was due predo minantly to the expansion of the CO-4 clone. This method can be used to mea ningfully quantify the precursor frequency of T cell mRNA in prepared sampl es via TCR subfamily or TCR sequence specific primers.