Spatiotemporal multi-omics reveal ISG15⁺ T cells as a predictive transitional state in cancer immunotherapy

Technical Summary: Spatiotemporal multi-omics reveal ISG15⁺ T cells as a predictive transitional state in cancer immunotherapy

Problem Statement
The clinical efficacy of immune checkpoint blockade (ICB) is dictated by the plasticity of tumor-infiltrating T cells. While the PD-1/PD-L1 axis has revolutionized oncology, durable responses remain limited to a subset of patients. Current paradigms often view T cell exhaustion as a hierarchical trajectory from progenitor exhausted (Tpex) to terminally exhausted (Tex-term) states. However, the existence, biological fate, and therapeutic utility of intermediate, stress-adapted T cell states—specifically those imprinted by type I interferons (IFN-I)—remain controversial. It is unclear whether these populations act as terminally dysfunctional "sinks" or as mobilizable precursors that can be reprogrammed into active effectors. Furthermore, existing pan-cancer atlases often lack the scale and resolution to confidently define rare, transient, or plastic cellular states across diverse malignancies.

Methodology
To address these gaps, the authors constructed the largest pan-cancer single-cell T cell atlas to date, integrating 119 scRNA-seq datasets comprising 2.35 million T cells from 1,758 samples across 39 malignancies. The study employed a multi-modal, orthogonal validation strategy:

• Pan-Cancer Atlas Construction: Rigorous batch-effect correction (BBKNN, Harmony) and unsupervised clustering were used to delineate T cell subpopulations.
• Longitudinal HNSCC Modeling: A high-resolution scRNA-seq cohort (232,665 cells) was generated from 28 tissue samples spanning normal mucosa, precancerous lesions, and invasive head and neck squamous cell carcinoma (HNSCC).
• Multi-Omic Integration: The study integrated single-cell ATAC-seq (scATAC-seq) to map chromatin accessibility, spatial transcriptomics (Visium HD and Xenium 5K) to resolve spatial niches at subcellular resolution, and bulk RNA-seq for clinical correlation.
• In Vivo Validation: Mouse models (4NQO-induced spontaneous HNSCC and 4MOSC1 orthotopic tongue tumors) were used to track disease progression and response to anti-PD-1 therapy.
• Functional Assays: Adoptive transfer experiments utilized IFN-β-conditioned OT-I CD8⁺ T cells to model the ISG15⁺ state and assess antitumor capacity.
• Clonal Tracking: scTCR-seq data were integrated to reconstruct clonal trajectories and lineage diversion following ICB treatment.

Key Contributions and Results

1. Identification of ISG15⁺ T Cells as a Distinct Subset:
   The pan-cancer atlas revealed a previously underrecognized, interferon-responsive population of ISG15⁺ T cells within both CD4⁺ and CD8⁺ compartments. These cells are broadly conserved across malignancies but are strikingly enriched in HNSCC. Unlike canonical subsets, ISG15⁺ CD8⁺ T cells exhibit a hybrid phenotype, co-expressing potent cytotoxic effectors (GZMA, PRF1, GNLY) and exhaustion markers (PDCD1, CTLA4, LAG3).

2. Spatiotemporal Dynamics and Malignancy Specificity:
   ISG15⁺ T cells show a stepwise accumulation from normal tissue to precancerous lesions and finally to invasive carcinoma. This accumulation is specific to the malignant tumor microenvironment (TME), as these cells remain scarce in benign inflammatory conditions (e.g., oral lichen planus, periodontitis). Spatial transcriptomics (Visium HD and Xenium) confirmed that ISG15⁺ T cells are physically sequestered within the hypoxic, antigen-dense tumor core, specifically juxtaposed with proliferating (PCNA⁺) tumor epithelial cells.

3. Epigenetic Plasticity and Regulatory Mechanisms:
   scATAC-seq analysis demonstrated that ISG15⁺ T cells are not terminally fixed. They maintain open chromatin at both cytotoxic loci (e.g., GNLY) and exhaustion/suppressive loci (e.g., LAYN, ENTPD1), positioning them at a multipotent epigenetic crossroads. The state is driven by a shared IFN-I signaling axis, with master transcription factors IRF7 and STAT1 robustly co-activated.

   • Interaction Mechanism: Spatial interactome modeling identified the LGALS9-CD44 axis as the primary communication channel between ISG15⁺ T cells and proliferating tumor cells, potentially acting as a "tether" that enforces this stalled, stress-adapted state.

4. Lineage Diversion and Therapeutic Mobilization:
   Crucially, the study demonstrates that ISG15⁺ T cells are a mobilizable reservoir. Clonal tracking via scTCR-seq revealed that upon ICB treatment, clones previously stalled in the ISG15⁺ state undergo massive lineage diversion, differentiating into functional effector (Teff) and tissue-resident memory (Trm) compartments. In vivo, anti-PD-1 treatment led to a significant reduction in intratumoral ISG15⁺ T cell abundance, correlating with their conversion into effectors. Adoptive transfer of IFN-β-conditioned ISG15⁺-like CD8⁺ T cells confirmed their latent, mobilizable antitumor capacity.

5. Predictive Biomarker Potential:
   The study establishes a bidirectional predictive model:

   • Baseline: High pre-treatment abundance of ISG15⁺ T cells correlates with favorable clinical response to ICB, suggesting a sufficient "spring-loaded" precursor pool.
   • Post-Treatment: Persistent retention of ISG15⁺ T cells post-therapy is associated with non-response, indicating a failure of state transition and therapeutic resistance.

Significance
The paper redefines ISG15⁺ T cells not as terminally exhausted sinks, but as epigenetically poised, transitional reservoirs that dictate immunotherapeutic outcomes. By integrating spatiotemporal multi-omics, the authors provide a mechanistic framework showing how chronic IFN-I signaling and spatial tethering (via LGALS9-CD44) maintain these cells in a plastic, stress-adapted state. The findings suggest that the baseline accumulation and subsequent treatment-induced clearance of ISG15⁺ T cells serve as robust biomarkers for ICB success. Furthermore, the identification of these cells as a mobilizable intermediate state highlights them as potential therapeutic targets; disrupting the spatial tethers or modulating IFN-I signaling could unlock these stalled reservoirs to enhance the efficacy of immune checkpoint blockade and other immunotherapies. The authors have made the data and an interactive portal available to facilitate community exploration of these states.