TIL Transcriptomic States Enable Successful TCR Prediction

Engineered cell-based immunotherapies have proven effective; however, targeting tumor-specific antigens without destroying normal cells represents a major challenge to their use in solid tumors. To evaluate the transcriptomic profiles of neoantigen-reactive tumor-infiltrating lymphocytes (TIL) in archival metastatic patient tumor samples, Lowery, Krishna, and colleagues performed single-cell RNA sequencing as well as T-cell receptor (TCR) sequencing on both CD8⁺ and CD4⁺ T cells from the metastatic cancer of 10 patients across multiple solid tumor types. Twelve unique transcriptional clusters were defined and ranged from activated TIL to resident-memory TIL and CD4⁺ regulatory TIL. Integration of TCR clonotypes within these 12 TIL phenotypic clusters showed that a majority of TIL clones were unexpanded singletons, while most of the oligoclonal TIL expansion occurred in the differentiated dysfunctional CD8⁺ TIL cluster, the resident-memory CD8⁺ TIL cluster, and the effector-memory CD8⁺ TIL cluster. Analysis of TIL clonotypes enriched in the peripheral blood or the tumor showed that the cellular states of tumor-expanded clones were distinct from blood-emigrant TILs within metastatic tumors. Moreover, 31 experimentally verified neoantigen TCR clonotypes (NeoTCR) were expressed on 325 single T cells and targeted driver neoantigens as well as nonmutated viral/tumor-associated antigens, with the vast majority distributed between the CD4⁺ and CD8⁺ differentiated dysfunctional states. This was irrespective of patient tumor histology and suggests a shared neoantigen-specific TIL exhaustion program within metastatic cancers. The genes differentially expressed by these NeoTCR T cells were then used to derive NeoTCR signatures, which could then be applied to score T cells in independent samples. In four validation samples (metastatic colon cancer), these NeoTCR signatures successfully identified tumor-relevant TCRs, showing that approximately half of the predicted TCRs were either tumor-, neoantigen-, or tumor-associated antigen–reactive, and indicating the shared existence of dysfunctional states regardless of antigen class. Therefore, this study identifies a shared signature of neoantigen-specific CD4⁺ and CD8⁺ T cells in metastatic solid tumors, ultimately contributing to the identification of tumor-specific TCRs with the use of limited TIL material.

Lowery FJ, Krishna S, Yossef R, Parikh NB, Chatani PD, Zacharakis N, et al. Molecular signatures of antitumor neoantigen-reactive T cells from metastatic human cancers. Science 2022;375:877–84.

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