Abstract 1654: Immune repertoire amongst subpopulations of tumor infiltrating lymphocytes (TILs) in colorectal cancer: Oligoclonality is predominantly observed in cytotoxic CD8+ TILs
Authors: Koo Si Lin , Ten Rachel Rui Xian , Aung Thin Zar , Koh Dennis , Wang Who Whong , Leow Wei Qiang , Lim Kiat Hon , Chew Suk Peng , Yuan Ju , Lim Bing , Albani Salvatore , Tan Iain Beehuat 
Published: May 2015

Abstract
Introduction: The immunophenotype and density of immune cell infiltrates in colorectal cancer (CRC) has consistently been shown to correlate with patient prognosis suggesting that the immune microenvironment plays an important role in regulating tumor progression. However, the immune repertoire of these tumor infiltrating lymphocytes has not been well characterized.
Methods: In this pilot study, we characterized the immue repertoire through TCR sequencing of T lymphocytes in the CRC tumor microenvironment and normal adjacent colonic mucosa. We collected fresh tumor and adjacent normal colonic mucosal tissue specimens from 2 patients who underwent surgery for CRC. A part of the tumor specimen was dissociated and sorted for collection of CD4+ and CD8+ T lymphocytes using FACSAria III cell sorter. 4 samples consisting of fresh frozen tumor, sorted CD4+ and CD8+ TILs and fresh frozen adjacent normal colonic mucosal tissue of each of the 2 patients were then sent for T-cell receptor β (TCRβ) complementarity-determining region 3 (CDR3) amplification and high-throughput next generation sequencing using the ImmunoSEQ platform at Adaptive Biotechnologies. The TCRβ CDR3 regions were then determined based on the definitions established by the ImMunoGeneTics Collaboration.
Results: The median total number of unique TCRβ CDR3 sequences identified in each of the 8 specimens evaluated was 5726 (range 475 to 54699), with an average of 78.3% of each specimen being productive sequences (range 75.1% to 81.9%). We next evaluated the diversity of T cell repertoire in terms of abundance and distribution of the T cell populations. There were more unique T cell clones present in the adjacent normal colonic mucosa (54699 and 24255) than in the tumor (7026 and 20887). Amongst the sorted TILs, there were less unique CD8+ T cell clones (561 and 475) compared to unique CD4+ T cell clones (2913 and 4426). There were fewer numbers of dominant clones making up 50% of total CDR3 reads in the CD8+ sorted TILs (15 and 29) compared to sorted CD4+ TILs (150 and 179), tumor tissue (390 and 668) and adjacent normal mucosa (1125 and 185).
Conclusion: Preliminary data from this pilot study suggests an oligoclonal population of cytotoxic T cells within the tumor microenvironment. We hypothesize that clonal expansion of these cytotoxic T cells were in response to specific tumor antigen stimulation. We have expanded the T cells and generated patient derived CRC cell lines. Further analysis of this population of expanded tumor infiltrating lymphocytes against their specific tumor cell lines are ongoing to evaluate T cell recognition and cytotoxic activity.
Citation Format: Si Lin Koo, Rachel Rui Xian Ten, Thin Zar Aung, Dennis Koh, Who Whong Wang, Wei Qiang Leow, Kiat Hon Lim, Suk Peng Chew, Ju Yuan, Bing Lim, Salvatore Albani, Iain Beehuat Tan. Immune repertoire amongst subpopulations of tumor infiltrating lymphocytes (TILs) in colorectal cancer: Oligoclonality is predominantly observed in cytotoxic CD8+ TILs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1654. doi:10.1158/1538-7445.AM2014-1654