HLA-II Expression Marks Activated and Clonally Expanded CD8+ T Cells and Improves Tumor-Reactivity Prediction Across Human Cancers.

Tumor-infiltrating CD8+ T cells are transcriptionally heterogeneous, and robust features linking their clonal dynamics to experimentally defined tumor reactivity remain incompletely characterized. We integrated single-cell RNA sequencing (scRNA-seq) and paired T-cell receptor sequencing (scTCR-seq) data from 31 single-cell immune datasets to characterize CD8+ T-cell states associated with clonal expansion and tumor reactivity. Across cancers, HLA-II-expressing CD8+ T cells were enriched in activated, cytotoxic, proliferative, exhausted, and terminally differentiated states and were preferentially associated with expanded TCR clonotypes. Pseudotime analysis indicated that HLA-II expression occurs along late differentiation states. HLA-DR blockade reduced CD25 upregulation in activated mixed PBMC and purified CD8+ T-cell cultures and attenuated CellTrace CFSE dilution in purified CD8+ T cells. An HLA-II-associated CD8+ T-cell signature showed cancer-type-dependent survival associations, including longer overall survival in several cohorts. To evaluate its contribution to tumor-reactivity prediction, an expansion-pretrained DNN was adapted using experimentally annotated tumor-reactive and non-tumor-reactive clonotypes from six pancreatic ductal adenocarcinoma samples and evaluated in 2225 functionally annotated CD8+ T cells from three held-out samples. Adding six HLA-II genes improved ROC-AUC across three feature panels from 0.540 to 0.781, 0.626 to 0.732, and 0.838 to 0.878, respectively, and improved PR-AUC from 0.570 to 0.739, 0.640 to 0.718, and 0.799 to 0.869. These findings identify HLA-II expression as a feature of activated and clonally expanded CD8+ T-cell states and support the complementary value of HLA-II-associated transcriptional features for prioritizing candidate tumor-reactive TCRs.