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

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

Research output: Contribution to journalArticle

Abstract

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 patients, or the status of autoimmune diseases. Two HLA-A2 restricted CTL clones expressing BV17 were isolated from a tumor infiltrating lymphocytes (TIL) 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 culture 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 specific competitor or competitors specific for the MART-1((27- 35)) reactive CTL clone (CO-41) and the gp100((209-217)) reactive CTL clone (CO-4). Each competitor contained a segment of the TCR BC region that serve as an internal reference standard. Using the BV17 competitor we were able to accurately and reproducibly measure cDNA templates at a frequency as low as 1/100,000 using cDNA samples of known TCRBV subfamily composition. This competitor was used to monitor the frequency of BV17 expressing T cell in the TIL 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 predominantly to the expansion of the CO-4 clone. This method can be used to meaningfully quantify the precursor frequency of T cell mRNA in prepared samples via TCR subfamily or TCR sequence specific primers.

Original languageEnglish (US)
Pages (from-to)93-102
Number of pages10
JournalJournal of Immunotherapy
Volume22
Issue number2
StatePublished - 1999
Externally publishedYes

Fingerprint

T-Cell Antigen Receptor
Clone Cells
Polymerase Chain Reaction
Tumor-Infiltrating Lymphocytes
Carbon Monoxide
Complementary DNA
Melanoma
T-Lymphoid Precursor Cells
T-Lymphocytes
HLA-A2 Antigen
T-Cell Receptor Genes
Superantigens
Peptides
Autoantigens
Neoplasm Antigens
Immunotherapy
Autoimmune Diseases
Allografts
Organism Cloning
Vaccines

Keywords

  • Competitive PCR
  • CTL clone
  • T cell receptor
  • Tumor infiltrating lymphocytes

ASJC Scopus subject areas

  • Cancer Research
  • Pharmacology
  • Immunology

Cite this

Quantitation of T-cell receptor frequencies by competitive PCR : Generation and evaluation of novel TCR subfamily and clone specific competitors. / McKee, Mark D.; Clay, Timothy M.; Rosenberg, Steven A.; Nishimura, Michael I.

In: Journal of Immunotherapy, Vol. 22, No. 2, 1999, p. 93-102.

Research output: Contribution to journalArticle

McKee, Mark D. ; Clay, Timothy M. ; Rosenberg, Steven A. ; Nishimura, Michael I. / Quantitation of T-cell receptor frequencies by competitive PCR : Generation and evaluation of novel TCR subfamily and clone specific competitors. In: Journal of Immunotherapy. 1999 ; Vol. 22, No. 2. pp. 93-102.
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