Liu et al. develop PRECISE-seq, a single-cell platform linking TCR specificity and avidity to T cell phenotypes in vivo. They reveal that high-potency antiviral T cells become exhausted, while tumor-reactive CD8+ T cells acquire a regulatory Ly49+ state that is restrained by PD-1 blockade, resulting in effector revival within tumors. Linking T cell phenotypes with antigen specificity and functional avidity is critical for understanding in vivo immune responses in infection and cancer. Here, we develop PRECISE-seq, a method that integrates multi-omics T cell analysis with contact-dependent proximity labeling for rapid screening of disease-relevant T cell repertoires, and for linking the relative TCR avidity with T cell phenotypes at single-cell resolution. PRECISE-seq accurately retrieves CMV-specific clonotypes from human peripheral blood and quantitatively measures functional avidity in physiological contexts. We find that high-potency CMV-specific T cells preferentially acquire an exhausted phenotype. In tumors, polyclonal tumor-reactive CD8+ T cells predominantly differentiate into a protumor Ly49+ regulatory state (TLy49), characterized by inhibitory killer cell lectin-like receptor expression and originating from effector memory T cells along a trajectory distinct from exhaustion. Notably, PD-1 blockade reduces TLy49 formation and promotes effector revival, which correlates with responsiveness to immunotherapy. Together, PRECISE-seq enables high-resolution mapping of TCR potency and T cell phenotype, revealing a regulatory axis shaping T cell fate in tumors. Graphical Abstract A multi-part diagram shows workflow linking T-cell antigen specificity, phenotype, PD-1 response mechanisms, and prediction of clinical outcomes.Panel A: An illustration shows the identification of antigen specificity. It includes a depiction of CD 8 cells interacting with antigens and labeled with biotin. Single-cell RNA sequencing (scRNA-seq) and single-cell TCR sequencing (scTCR-seq) are illustrated with colored bars representing different gene segments. Panel B: A UMAP plot shows the phenotype and TCR potency. The plot is labeled with UMAP_1 and UMAP_2 axes and shows clusters of cells with different TCR potency and exhaustion scores. Panel C: A diagram illustrates the Ly49 plus regulatory state. It shows immune checkpoints, NK receptors, SPP1, granzyme C, and Ly49 genes interacting with a cell. Panel D: A diagram depicting the mechanism of discovery of alpha PD-1 therapy. It shows different types of T cells (T subscript EM, T subscript EFF, T subscript EX, T subscript Ly49) and their responses to alpha PD-1 therapy. Panel E: A diagram predicts clinical response. It shows two figures representing responders and non-responders based on the levels of T subscript EM, T subscript EFF, and T subscript Ly49/KIR cells.

INTRODUCTION: Triple-negative breast cancer (TNBC) is characterized as the most unfavorable prognosis of the breast cancer subtype. Chemotherapy is currently the primary treatment owing to a persistent lack of effective alternative medicine. To fulfill this unaddressed clinical requirement, we utilized the PGLU-Fc-III-4C MsAb platform to develop a nanobioconjugate trispecific antibody (TsAb; CD3×CD137×CD276) that targeted CD3, CD137, and CD276, aiming to restrict the growth of TNBC tumors (4T1 model) and provide a novel therapeutic strategy. METHODS: The capacity for binding to target cells and anti-tumor effects of TsAb in vitro were evaluated. In vivo antitumor efficacy and biosafety were further assessed in the 4T1 subcutaneous tumor model. Anti-tumor immune responses induced by TsAb on tumor-infiltrating CD8+ T cells were monitored. RESULTS: The TsAb effectively bound and facilitated interactions between 4T1 tumor cells and T cells, significantly boosting the anti-tumor effect. TsAb promoted the expansion of tumor and splenic T lymphocytes and facilitated the recruitment of splenic and blood T lymphocytes to tumor tissues. Compared with the PGLU-Fc-III-4C-IgG treatment group, the TsAb treatment group had varying degrees of increase in CD8+ tissue-resident memory T cells (TRM), central memory T cells (TCM), and terminal effector memory T cells (TEM) in tumor tissues. The TsAb treatment group exhibited a significant increase of PD-1+ CD8+ T cells and TCF1-Tim-3+ CD8+ terminally exhausted T cells in tumor tissues. The safety profile demonstrated no obvious systemic toxicity. DISCUSSION: Briefly, TsAb mediated CD8+ T cell activation, proliferation, and terminal differentiation, accompanied by increasing cytokine production to eliminate the tumor. Meanwhile, no obvious systemic toxicity was observed. In general, the CD3×CD137×CD276 nanobioconjugate trispecific antibody provides a promising immunotherapeutic approach via regulating CD8+ T cell immune response for the treatment of triple-negative breast cancer.

UNLABELLED: Neoadjuvant immunotherapy improves outcomes in esophageal squamous cell carcinoma (ESCC), yet ∼70% of patients fail to respond. Pretreatment biopsies and plasma provide critical opportunities for biomarker discovery. In this study, we performed plasma proteomic profiling and identified soluble glycoprotein nonmetastatic melanoma protein B (sGPNMB) as the most elevated circulating protein in nonresponders. Mechanistically, tumor cell-derived sGPNMB suppressed CD8+ T-cell receptor signaling via the SDC4-CD148 axis to induce functional exhaustion, with secretion being required for its immunosuppressive activity. Cancer-associated fibroblast-epithelial (CAF-Epi) niches promoted SOX2 upregulation in tumor cells, transcriptionally activating GPNMB expression. In humanized patient-derived xenograft models, circulating GPNMB levels predicted response to PD-1 blockade, and GPNMB inhibition synergized with therapy. Across retrospective cohorts and a prospective clinical trial, a multimodal model combining plasma GPNMB levels, CAF-Epi niche detection, and clinical-pathologic features achieved robust predictive accuracy for immunotherapy response and survival. These findings establish a spatial-circulating biomarker framework for precision ESCC immunotherapy. SIGNIFICANCE: Tumor-derived soluble GPNMB, transcriptionally activated by SOX2 within CAF-Epi niches, drives CD8+ T-cell exhaustion and resistance to PD-1 blockade in ESCC. Integrating circulating GPNMB levels with CAF-Epi niche features and clinical-pathologic factors, we develop and validate a clinically scalable multimodal model for predicting immunotherapy response.