BCL2A1 marks an early, less-exhausted CD8+ tumor-infiltrating lymphocyte state associated with PD-1 blockade response in NSCLC
10 affiliations
- 1International Ph.D. Program in Medicine, College of Medicine, Taipei Medical University
- 2Department of Oncology, Faculty of Medicine, Can Tho University of Medicine and Pharmacy
- 3Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University
- 4School of Respiratory Therapy, College of Medicine, Taipei Medical University
- 5Division of Pulmonary Medicine, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University
- 6Division of Pulmonary Medicine, Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University
- 7Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University
- 8TMU Research Center of Thoracic Medicine, Taipei Medical University
- 9Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University
- 10Core Laboratory of Immune Monitoring, Office of Research & Development, Taipei Medical University
Abstract
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.
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BibTeX
@article{pham2026bcl2a1,
title = {{BCL2A1 marks an early, less-exhausted CD8+ tumor-infiltrating lymphocyte state associated with PD-1 blockade response in NSCLC}},
author = {Hoang Minh Quan Pham and Chia-Ling Chen and Po-Hao Feng and Kang-Yun Lee and Yu-Ping Hung and Chiou-Feng Lin},
journal = {Frontiers in Immunology},
year = {2026},
month = oct,
doi = {10.3389/fimmu.2026.1908216},
url = {https://www.frontiersin.org/articles/10.3389/fimmu.2026.1908216/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - BCL2A1 marks an early, less-exhausted CD8+ tumor-infiltrating lymphocyte state associated with PD-1 blockade response in NSCLC
AU - Hoang Minh Quan Pham
AU - Chia-Ling Chen
AU - Po-Hao Feng
AU - Kang-Yun Lee
AU - Yu-Ping Hung
AU - Chiou-Feng Lin
PY - 2026
DA - 2026/10/07
JO - Frontiers in Immunology
DO - 10.3389/fimmu.2026.1908216
UR - https://www.frontiersin.org/articles/10.3389/fimmu.2026.1908216/full
AB - 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.
SN - 1664-3224
ER -