High-throughput and high-dimensional single-cell analysis of antigen-specific CD8+ T cells.

Although critical to T cell function, antigen specificity is often omitted in high-throughput multiomics-based T cell profiling due to technical challenges. We describe a high-dimensional, tetramer-associated T cell antigen receptor (TCR) sequencing (TetTCR-SeqHD) method to simultaneously profile cognate antigen specificities, TCR sequences, targeted gene expression and surface-protein expression from tens of thousands of single cells. Using human polyclonal CD8+ T cells with known antigen specificity and TCR sequences, we demonstrate over 98% precision for detecting the correct antigen specificity. We also evaluate gene expression and phenotypic differences among antigen-specific CD8+ T cells and characterize phenotype signatures of influenza- and Epstein-Barr virus-specific CD8+ T cells that are unique to their pathogen targets. Moreover, with the high-throughput capacity of profiling hundreds of antigens simultaneously, we apply TetTCR-SeqHD to identify antigens that preferentially enrich cognate CD8+ T cells in patients with type 1 diabetes compared to healthy controls and discover a TCR that cross-reacts with diabetes-related and microbiome antigens. TetTCR-SeqHD is a powerful approach for profiling T cell responses in humans and mice.

Ushering in Integrated T Cell Repertoire Profiling in Cancer.

Advances in immune profiling techniques have dramatically changed the cancer immunotherapy and monitoring landscape. High-throughput protein and gene expression technologies have paved the way for the discovery of therapeutic targets and biomarkers, and have made monitoring therapeutic response possible through the ability to independently assay the phenotype, specificity, exhaustion status, and lineage of single T cells. Although valuable insights into response profiling have been gained with current technologies, it has become evident that single-method profiling is insufficient to accurately capture an antitumor T cell response. We discuss and propose new methods that combine multiple axes of analysis to provide a comprehensive analysis of T cell repertoire in the fight against cancer.

Single-cell RNA sequencing of lung adenocarcinoma reveals heterogeneity of immune response-related genes.

Immunotherapy has emerged as a promising approach to treat cancer. However, partial responses across multiple clinical trials support the significance of characterizing intertumor and intratumor heterogeneity to achieve better clinical results and as potential tools in selecting patients for different types of cancer immunotherapies. Yet, the type of heterogeneity that informs clinical outcome and patient selection has not been fully explored. In particular, the lack of characterization of immune response-related genes in cancer cells hinders the further development of metrics to select and optimize immunotherapy. Therefore, we analyzed single-cell RNA-Seq data from lung adenocarcinoma patients and cell lines to characterize the intratumor heterogeneity of immune response-related genes and demonstrated their potential impact on the efficacy of immunotherapy. We discovered that IFN-γ signaling pathway genes are heterogeneously expressed and coregulated with other genes in single cancer cells, including MHC class II (MHCII) genes. The downregulation of genes in IFN-γ signaling pathways in cell lines corresponds to an acquired resistance phenotype. Moreover, analysis of 2 groups of tumor-restricted antigens, namely neoantigens and cancer testis antigens, revealed heterogeneity in their expression in single cells. These analyses provide a rationale for applying multiantigen combinatorial therapies to prevent tumor escape and establish a basis for future development of prognostic metrics based on intratumor heterogeneity.

High-throughput determination of the antigen specificities of T cell receptors in single cells.

We present tetramer-associated T-cell receptor sequencing (TetTCR-seq) to link T cell receptor (TCR) sequences to their cognate antigens in single cells at high throughput. Binding is determined using a library of DNA-barcoded antigen tetramers that is rapidly generated by in vitro transcription and translation. We applied TetTCR-seq to identify patterns in TCR cross-reactivity with cancer neoantigens and to rapidly isolate neoantigen-specific TCRs with no cross-reactivity to the wild-type antigen.

Normalized Synergy Predicts That CD8 Co-Receptor Contribution to T Cell Receptor (TCR) and pMHC Binding Decreases As TCR Affinity Increases in Human Viral-Specific T Cells.

The discovery of naturally occurring T cell receptors (TCRs) that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC) has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D) system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds) rather than synergy (total CD8 cooperation) alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D) analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our previously established TCR discovery platform using 2D TCR affinity and sequence test would allow for selection of TCRs specific to any given antigen with the desirable attributes of high TCR affinity, CD8 co-receptor independence and functional superiority. Utilizing TCRs with less CD8 contribution could be beneficial for adoptive cell transfer immunotherapies using naturally occurring or genetically engineered T cells against viral or cancer-associated antigens.