A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools

Meytal Dror Levinsky, Baruch Brenner, Michal Yalon, Zohar Levi, Zvi Livneh, Zoya Cohen, Tamar Paz-Elizur, Rachel Grossman, Zvi Ram*, Ilan Volovitz*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Personalized vaccines against patient-unique tumor-associated antigens represent a promising new approach for cancer immunotherapy. Vaccine efficacy is assessed by quantification of changes in the frequency and/or the activity of antigen-specific T cells. Enzyme-linked immunosorbent spot (ELISpot) and flow cytometry (FCM) are methodologies frequently used for assessing vaccine efficacy. We tested these methodologies and found that both ELISpot and standard FCM [monitoring CD3/CD4/CD8/IFNγ/Viability+CD14+CD19 (dump)] demonstrate background IFNγ secretion, which, in many cases, was higher than the antigen-specific signal measured by the respective methodology (frequently ranging around 0.05–0.2%). To detect such weak T-cell responses, we developed an FCM panel that included two early activation markers, 4-1BB (CD137) and CD40L (CD154), in addition to the above-cited markers. These two activation markers have a close to zero background expression and are rapidly upregulated following antigen-specific activation. They enabled the quantification of rare T cells responding to antigens within the assay well. Background IFNγ-positive CD4 T cell frequencies decreased to 0.019% ± 0.028% and CD8 T cells to 0.009% ± 0.013%, which are 19 and 13 times lower, respectively, than without the use of these markers. The presented methodology enables highly sensitive monitoring of T-cell responses to tumor-associated antigens in the very low, but clinically relevant, frequencies.

Original languageEnglish
Article number574
JournalCancers
Volume15
Issue number3
DOIs
StatePublished - Feb 2023

Keywords

  • ELISpot
  • RNA vaccine
  • T cells
  • cancer
  • cancer testis antigens
  • flow cytometry
  • neoantigens
  • peptide vaccine
  • personalized cancer vaccine
  • self-antigens

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