Editors' ChoiceImmunology

Viewing T Cell Biology Through a Genomic Lens

Science Translational Medicine  09 Jul 2014:
Vol. 6, Issue 244, pp. 244ec119
DOI: 10.1126/scitranslmed.3009627

The human body faces a constant onslaught of potential pathogenic organisms. To combat this blitz, the immune system has evolved an adaptive response to specifically address the vast array of attackers. This adaptive response includes T cells, which express T cell receptors that are rearranged at the genetic level during T cell development, allowing for this diversity of antigen specificity. After encounter with antigen, these specific T cells proliferate and generate diverse functional subsets with various phenotypes. Immunologists often rely on flow cytometry to characterize the phenotype of T cells at a single-cell level, but functional assays to measure proliferation or cytokine release are performed with populations of T cells. Similarly, Sanger sequencing of T cell receptors relies on in vitro clonal expansion of T cells, which alters their phenotype. Thus, until now, there has been a Heisenberg-esque block: It has not been possible to identify and characterize single T cells without a priori knowledge of their specificity.

In this paper, Han and colleagues developed a deep-sequencing–based method to integrate the phenotypic and functional characteristics of single T cells along with their identity and clonal ancestry. First, they sorted single T cells into wells using flow cytometry; then, they combined reverse transcription polymerase chain reaction (RT-PCR) for the T cell receptor (with 76 primers) and RT-PCR for a panel of transcription factors and cytokines that define functional T cell subsets (34 primers). An aliquot of the amplified cDNA was used in a second nested PCR reaction, and then a third PCR reaction added an individual barcode for deep sequencing. Using this platform, they demonstrated that tumor-infiltrating T cells in colon cancer have undergone clonal expansion and that multiple T cell clones with similar specificity are enriched in tumor tissue compared with adjacent tissue or peripheral blood. Both of these findings suggest that tumor-infiltrating T cells are indeed reacting to tumor antigens. The sequence-based phenotype of the tumor-infiltrating T cells was also different than T cells from adjacent tissue or from peripheral blood. Last, by integrating the sequence and functional information at the single-cell level, the authors were able to determine that a tumor-infiltrating T cell had clonally expanded and evolved to lose FOXP3 expression and gain interleukin-17 production. This platform will aid in identifying targetable T cell antigens and in understanding the events that guide different T cell responses in health and disease.

A. Han et al., Linking T-cell receptor sequence to functional phenotype at the single-cell level. Nat. Biotechnol., published online 22 June 2014 (10.1038/nbt.2938). [PubMed]

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