Multi-omics analysis identifies altered EOMES-associated differentiation programs of precursor effector-memory CD8⁺ T cells in transplant-associated cutaneous squamous cell carcinoma

BACKGROUND: Transplant-associated cutaneous squamous cell carcinoma (TSCC) is one of the most common malignancies in solid organ transplant recipients and is strongly associated with long-term immunosuppression. However, the transcriptional programs and molecular mechanisms through which chronic immunosuppression reshapes intratumoral CD8⁺ T-cell differentiation remain poorly understood. METHODS: We integrated single-cell transcriptomic and T-cell receptor repertoire analyses of TSCC and immunocompetent cutaneous squamous cell carcinoma (SCC). An independent SCC cohort containing paired tumors and adjacent tissues, together with matched spatial transcriptomic data, was used to evaluate the association between EOMES and antitumor CD8⁺ T-cell differentiation. RESULTS: Single-cell transcriptomic analysis revealed marked accumulation of precursor effector-memory CD8⁺ T cells (Pre-Tem) in TSCC, accompanied by reduced cytotoxic programs and clonal expansion. Trajectory and regulatory-network analyses revealed retention of precursor-like differentiation states associated with reduced EOMES expression and regulon activity. In silico perturbation modeling predicted EOMES as a candidate transcriptional regulator linked to cytotoxic gene expression and effector differentiation. An independent SCC cohort further supported the biological relevance of EOMES-associated programs in antitumor CD8⁺ T-cell states. Cell-cell communication analysis identified CXCL10-CXCR3 signaling as a dominant pathway targeting Pre-Tem cells and related with diminished EOMES-associated differentiation programs. Spatial transcriptomics further identified spatial co-localization among CXCL10 signaling, EOMES-associated states, and Pre-Tem cells within a tumor-margin niche. CONCLUSIONS: Pre-Tem accumulation and altered differentiation states represent a previously unrecognized feature of immune dysfunction in TSCC. EOMES-associated programs were closely linked to precursor-to-effector CD8⁺ T-cell differentiation, while CXCL10-CXCR3 signaling may contribute to impaired precursor-to-effector differentiation. These findings provide a conceptual framework for understanding immune dysfunction in transplant-associated malignancies and establish a rationale for future mechanistic and translational studies.