Skip to main content

Volume 3 Supplement 2

30th Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2015)

  • Poster presentation
  • Open access
  • Published:

Tetramer based approach for efficient identification and isolation of neo-antigen specific CD8 T cells from peripheral blood (PBL) of patients with metastatic cancers


Adoptive cell therapy with T cells bearing mutation specific T cell receptors (TCR) can be an effective method for treating metastatic cancers. The objective of this study was to identify mutation reactive T cells in the circulation of patients with different types of metastatic cancer.


The strategy utilized whole exome sequencing data to identify somatic non-synonymous mutations and then in-silico algorithms to predict minimal epitopes encoding mutated amino acids for each patient specific HLA-allele. CD8-enriched PBL from each patient were stained with tetramers generated in house by a UV-exchangeable technique as previously described for A*02:01, A*03:01, A*11:01, B*07:02, and a commercial tetramer was acquired for B*57:01. Based on the initial staining frequency (+tetramer+ T cells recognizing 7 unique neo-antigens from the PBL of 4 patients (ranging from 1 to 4 per patient). We enriched the frequencies of CD8+tetramer+ cells from 0.5 to >85%, 0.3 to >65% and 0.01 to 3% from the PBL of patients with colorectal (3971-A*02:01), lung (4014-B*57:01), and ovarian (4067-B*07:02) cancers respectively, using individual tetramers. Populations reactive with three HLA-A*11:0-restricted and one HLA-A*03:01-restricted neoantigens were also isolated from the PBL of lung cancer patients 4014 and 4037, respectively, using a pooled tetramer approach.


Overall, the isolated T cells recognized mutated epitopes when co-cultured with autologous CD14+ monocytes pulsed with mutated peptides in the context of appropriate MHC-I alleles including HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, HLA-B*07:02, and HLA-B*57:01, with reactivity detected using IFN-γ ELISA. Using single cell PCR, we could clone the TCRs reactive with an HLA-*02:01-presented colon cancer neoantigen and an HLA-B*57:01-presented lung cancer neoantigen. Evaluation of PBL retrovirally-transduced with these TCRs demonstrated that they bound to tetramers and secreted IFN-γ when co-cultured with CD14+ monocytes pulsed with appropriate mutated peptides.


To conclude, tetramers offer a sensitive, fast and reliable methodology to isolate mutation specific tumor reactive T cells from PBL of cancer patients. Furthermore, this method facilitates the identification, and cloning of mutation reactive TCR with which to construct receptor-engineered T cells for adoptive T cell therapy.

Author information

Authors and Affiliations


Rights and permissions

Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.

The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this licence, visit

The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bharathan, M., Trebska-McGowan, K., Anna, P. et al. Tetramer based approach for efficient identification and isolation of neo-antigen specific CD8 T cells from peripheral blood (PBL) of patients with metastatic cancers. j. immunotherapy cancer 3 (Suppl 2), P47 (2015).

Download citation

  • Published:

  • DOI: