BCL2A1 marks an early, less-exhausted CD8+ tumor-infiltrating lymphocyte state associated with PD-1 blockade response in NSCLC

Background The tumor microenvironment exerts intense survival pressure on CD8 + tumor-infiltrating lymphocytes (TILs), yet the cellular basis for an earlier, less-exhausted state associated with response to immune checkpoint blockade (ICB) in non-small-cell lung cancer (NSCLC) remains unclear. We previously identified BCL2A1 as a CD8 + TIL-derived marker predictive of a response to PD-1 blockade. Here we combine deep single-cell discovery with experimental validation in primary human T cells to characterise the BCL2A1-associated CD8 + TIL state and its candidate upstream signal. Methods We integrated four transcriptomic cohorts—pre-treatment bulk RNA-seq with CD8 + TIL deconvolution (n=60), on-treatment scRNA-seq/TCR-seq (n=11), longitudinal scRNA-seq/TCR-seq (n=18), and an independent post-treatment cohort (n=59)—with in silico perturbation, pathway, and metabolic-flux analyses, and experimentally tested a key prediction of this state by flow cytometry in primary human CD8 + T cells. Results BCL2A1 high CD8 + TILs showed elevated BCL2A1 expression without coordinated upregulation of anti-apoptotic BCL2-family genes, maintained minimal exhaustion, and occupied an early, BCL6 -, TCF7 -, and NFKB1 -regulated pseudotime state. Across longitudinal and post-treatment cohorts, they formed diverse, low-expansion repertoires rather than dominant expanded clones, with reduced biosynthetic flux (branched-chain amino acid and purine metabolism) consistent with a non-proliferative state. NF-κB signaling was elevated, and TNF-α was nominated as the leading candidate upstream ligand of BCL2A1 . We then experimentally tested this prediction: in primary human CD8 + T cells, TNF-α induced BCL2A1 protein ≈13-fold (44.5% vs. 3.4% BCL2A1 + ; MFI 2.93-fold; P < 0.0001), whereas CD3/CD28 activation alone did not, establishing a TNF-α-specific, protein-level response. Conclusions Combining integrative single-cell analysis with experimental validation in primary human T cells, we define BCL2A1 as a marker of an early, less-exhausted CD8 + TIL state associated with improved ICB outcomes in NSCLC; a survival-related role is supported by these associations but was not functionally tested.