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Abstract
Background Head and neck squamous cell carcinomas (HNSCCs) represent the sixth most common cancer worldwide with an estimated 65,630 cases and 14,500 deaths in the United States last year. Fortuitously, by virtue of their high mutational burden and robust neoantigenome, HNSCCs harbor an abundance of tumor-specific antigen (TSA)-T cells; and, thus, represent an ideal target for autologous adoptive therapy. However, the lack of a reliable biomarker to accurately identify bona fide TSA-T cells among a heterogenous population of tumor-infiltrating lymphocytes (TILs) has precluded the complete translation of this otherwise promising therapeutic strategy.
Methods We developed an interaction-based chemoenzymatic labeling method to rapidly and efficiently identify TSA-T cells: the α-(1,3)-fucosyltransferase–FucoID–strategy. Using the FucoID method, we profiled TSA-T cells identified by proximity-based chemoenzymatic labeling (figure 1).
Results We find that FucoID labeled T cells in both the tumor and tumor-draining lymph node compartments feature a population of TSA-T cells with exhausted-stem cell like phenotypes and robust antitumoral cytotoxic activity. Additionally, when subjected to conventional ex vivo expansion protocols, these FucoID-labeled T cells are resistant to differentiation and anergy following adoptive transfer in vivo. Through tandem T cell receptor (TCR) and transcriptomic sequencing at the single-cell level, FucoID-labeled T cells from the tumor constitute a defined population with potent effector function (figure 2). An analysis of the FucoID-labeled T cells from the draining lymph node that share a TCR repertoire with those from the tumor reveals a unique population with high TCF-7 expression and stem-like features (figure 3).
Conclusions The FucoID proximity-based labeling strategy represents a translatable, antigen-agnostic method to exclusively and expediently identify TSA-reactive T cells with phenotypes optimal for ex vivo expansion, in vivo persistence, and antitumor cytotoxicity. This work represents a paradigm shift in the approach to adoptive T cell therapies, which can immediately inform the design of next-generation immune oncology trials for HNSCC.
FucoID Identification Strategy. The a-(1,3)-fucosyltransferase (FT) identification strategy - FucolD employs (A) GDP-FT that is chemoenzymatically conjugated to the DC surface via a short PEG linker. (B) FT-conjugated DCs (FT-DCs) subsequently transfer GDP-Fucose-Biotin labels to T cells via proximity-based DC:T cell interactions
Tumor-antigen specific T cell precursors in draining lymph nodes. TCR sequencing reveals tumor-antigen-specific T cell precursors in tumor-draining lymph nodes. (A) In vitro killing of 4MOSC1 tumor cells by expanded CD8+ TIL subsets; Biotin- non-TSA T cell, Biotin+= TSA-T cell (E:T 2:1, n=3). (B) Right -TCR-β sequencing from biotin+ CD8+ TILs and dLNs. Left - No overlap biotin and biotin- T cells in dLNs. (red = Overlapping CDR3s)
Tandem TCR & scRNA-seq of FucolD-labeled T cells from tumor and LN. A. Cartoon schema of the experimental approach - (middle) CD4 (red) and CD8 (blue) T cells are isolated from tumors or draining lymph nodes of 4MOSC1-tumor bearing animals and subjected to (1,3)-fucosyltransferase (FT) labelling - FucolD - via proximity-based interactions with GDP- FT chemoenzymatically conjugated autologous DCs that have been previously exposed to tumor homogenate; (right) FucolD-labelled and unlabelled T cells are then processed for tandem scRNA-sequencing and TCR-sequencing. B. UMAP of resultant T cell populations after scRNA-sequencing with (right) total number of T cells in each identified population. C. UMAP showing distribution of FucolD-labelled and unlabelled T cells. D. UMAP showing the distribution of T cells derived from either tumor or tumor draining lymph nodes. E. UMAP showing the distribution of CD4 and CD8 T cells. F. (left) FucolD-labelled CD4 and CD8 T cells from tumor and lymph node with (right) overlapping TCR clonotypes from (B). G. (left) Subsampled FucolD-labelled CD4 T cells with overlapping clonotypes from (F). H. (left) Subsampled FucolD-labelled CD8 T cells with overlapping clonotypes from (F). I. Dot plot highlighting key gene expression signatures of subsampled FucolD-labelled CD8 T cell populations with overalapping TCR clonotypes from (H). J. Selected differential gene expression of subsampled FucolD-labelled CD8 T cell populations from (H)
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