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

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.

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.

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.

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.