Closer proximity of pre-treatment CD4+ T cells to CD8+ T cells favor response to neoadjuvant immunotherapy in patients with PD-L1 low-expressing non-small cell lung cancer.

Background: Neoadjuvant chemo-immunotherapy improves non-small cell lung cancer (NSCLC) outcomes, but remission rates vary, emphasizing the need for biomarkers. This study aimed to investigate the impact of the baseline intratumoral CD4⁺ T-cell-adjacent microenvironment on the efficacy of neoadjuvant immunotherapy in NSCLC and its correlation with hypoxia-inducible factor-1α (HIF-1α), microvessel density (MVD), and cancer-associated fibroblasts (CAFs).

Methods: Tumor samples from 49 NSCLC patients before neoadjuvant immunotherapy were retrospectively collected and subjected to multiplex immunohistochemistry staining (panel 1: DAPI/CK/CD4/CD8/CD68; Panel 2: DAPI/CK/CD4/HIF-1α/CD31/α-SMA) to characterize CD4⁺ T cells, CD8⁺ T cells CD68⁺ macrophages, HIF-1α⁺ cells, HIF-1α⁺CD4⁺ cells, MVD, and CAF. Mann-Whitney U test and receiver operating characteristic (ROC) curve were used to assess the relationship between the number and spatial distribution of each metric and the efficacy of the treatment, and Spearman's rank correlation was used to assess the correlation of each metric.

Results: In 49 NSCLC patients, responders (54.2%) and non-responders (45.8%). Single-indicator analysis revealed a positive correlation between high infiltration of CD8⁺ T cells in the stromal area and response to treatment in overall and programmed cell death-ligand 1 (PD-L1) low-expressing patients [CD8⁺ T (str) density: overall patient, 38 vs. 16, P=0.03; tumor proportion score (TPS) 1-49% subgroup, 37 vs. 14, P=0.04], with an area under curve (AUC) 0.684 and 0.746, respectively. CD4⁺ T cells combined with CD8⁺ T cells or CD68⁺ macrophages were analyzed and found to be more efficacious than CD4⁺T^hiCD8⁺T^hi compared to CD4⁺T^loCD8⁺T^lo in patients with low expression of PD-L1 (P=0.03). Assessment of the nearest neighbor distance (NND) of CD4⁺ T cells and their adjacent cells revealed that the closer the CD4⁺ T cells and CD8⁺ T cells in the overall compartment, the better the efficacy in NSCLC patients, especially in patients with low PD-L1 expression [CD4⁺ T to CD8⁺ T (all) NND: overall patients, 34 vs. 47 μm, P=0.03; TPS 1-49% subgroup, 34 vs. 69 μm, P=0.006], and the AUC was 0.670 and 0.830, respectively. Notably, this favorable spatial interaction may not be dependent on direct contact between CD4⁺ T cells and CD8⁺ T cells within 10/20/30 μm (P>0.05). Furthermore, in overall and PD-L1 low-expressing patients, the closer the distance between CD4⁺ T cells and CD8⁺ T cells, the higher the MVD (overall patients, r=-0.39, P=0.008; TPS 1-49% subgroup, r=-0.49, P=0.01).

Conclusions: The baseline intratumoral CD4⁺ T-cell-adjacent microenvironment in NSCLC is associated with the efficacy of neoadjuvant immunotherapy for NSCLC, with the closer proximity of pre-treatment CD4⁺ T cells and CD8⁺ T cells, the better the treatment efficacy in NSCLC patients (even especially in the low-expressing PD-L1 population), and is associated with high MVD.