Quantitation of T-cell receptor frequencies by competitive polymerase chain reaction: Dynamics of T-cell clonotype frequencies in an expanding tumor-infiltrating lymphocyte culture

Mark D. McKee, Timothy M. Clay, Rochelle A. Diamond, Steven A. Rosenberg, Michael I. Nishimura

Research output: Contribution to journalArticle


The use of T-cell receptor (TCR) genes as markers for antigen-reactive T cells is dependent on the ability of the TCR genes to rapidly identify antigen-reactive T-cell clonotypes in patient samples. We recently reported a competitive reverse-transcriptase polymerase chain reaction (cRT-PCR) method that can measure the frequency of individual TCRBV subfamilies and clonotypes in mixed lymphocyte populations more accurately than other semiquantitative PCR assays. However, it is impractical to measure changes in the absolute frequency of each TCRBV subfamily to identify those T cells with increasing frequency after antigen stimulation in vivo or in vitro. Therefore, we have modified our cRT-PCR method to more rapidly identify expanding T-cell populations by combining all of the TCRBV subfamily-specific competitors into a single sample to determine the relative abundance of each TCRBV subfamily. Using an expanding TIL 620 culture, we identified four TCRBV (BV2, BV12, BV17, and BV23) subfamilies that expanded over a 23-day period. These subfamilies accounted for 23% of the T cells in the day 35 culture and increased to 57%, 92%, and 80% of the days 44, 51, and 58 cultures respectively. Analysis of DNA sequences demonstrated that the observed expansion was caused primarily by a single clonotype within each subfamily. T cells expressing BV17 and BV23 recognized gp100 and MART-1 respectively. Therefore. this cRT-PCR method can detect expanding T-cell populations based solely on their TCRBV subfamily expression. Furthermore, T-cell expansion in a mixed TIL population was a good predictor of antigen reactivity.

Original languageEnglish (US)
Pages (from-to)419-429
Number of pages11
JournalJournal of Immunotherapy
Issue number4
StatePublished - Jul 1 2000



  • Competitive PCR
  • Melanoma
  • T-cell receptor
  • TIL

ASJC Scopus subject areas

  • Immunology
  • Pharmacology
  • Cancer Research

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