Editors' ChoiceImmunology

A basic strategy for detecting CD8 T cell specificity

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Science Translational Medicine  05 Oct 2016:
Vol. 8, Issue 359, pp. 359ec161
DOI: 10.1126/scitranslmed.aai8748

Recognition of short peptide sequences presented on major histocompatibility complex (MHC) class I by the T cell receptors (TCRs) of adaptive immune CD8 T cells is essential for defense against pathogens and malignant cells and informs several autoimmune diseases. Accurately determining the molecular targets of CD8 T cells could thus accelerate the development of novel therapeutics; yet, to date, characterization of their specificities has been limited by the use of fluorescently or heavy-metal–tagged peptide-MHC (pMHC) multimers. Fundamentally, these approaches are restricted to screening approximately 100 of the millions of potential pMHCs at once due to spectral overlap or the scarcity of unique heavy metals, respectively. Now, Bentzen and colleagues have overcome this experimental hurdle by leveraging DNA barcoding, which theoretically allows for billions of unique pMHCs to be examined simultaneously.

Using a straightforward combinatorial construction scheme, the authors generated over a thousand unique, sequenceable oligonucleotides coupled to pMHCs. To determine the linearity and sensitivity of their approach, they mixed peripheral blood mononuclear cells (PBMCs) from donors with different CD8 T cell specificities. They showed detection of pMHC-specific CD8 T cells down to 0.008% of total, although it is worth noting that their sensitivity depends on absolute, not relative, cellular numbers. Expanding their analysis to PBMCs from several donors, the authors observed good quantitative agreement with a matched set of fluorescently labeled pMHCs, supporting the robustness of their technique. Next, by generating additional cancer-specific multimers, they identified pMHC-specific T cells within both tumor-infiltrating lymphocytes and PBMCs with greater sensitivity than is achievable using fluorescently labeled analogs. Last, they demonstrated that their method is compatible with intracellular cytokine staining, a commonly used functional assay.

While several obstacles remain—including realizing rapid and cost-effective pipelines for pMHC production, counteracting potential interference effects between pMHCs, and developing strategies to control for confounding differences in TCR affinity—Bentzen et al. provide a solid foundation for future endeavors. Collectively, their results hint at exciting possibilities, such as diagnosing cancers and monitoring their treatments from blood samples or directly linking specificity to T cell phenotype and TCR sequence at the single-cell level, that could one day help transform clinical and basic immunology.

A. K. Bentzen et al., Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes. Nat. Biotechnol. 10.1038/nbt.3662 (2016). [Abstract]

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