[Influence and Mechanism of Immunophenotyping on the Efficacy 
of Neoadjuvant Therapy in Non-small Cell Lung Cancer]

The low response rate to immunotherapy can be partially attributed to tumor immune escape mechanisms arising from the heterogeneous tumor microenvironment. This study aims to determine the impact of inflammatory non-small cell lung cancer (NSCLC) on the efficacy of neoadjuvant immunotherapy combined with chemotherapy at the histological level, and to investigate the predictive value of specific CD8+ and CD4+ T cell numbers, as well as spatial interactions, in treatment response.

A retrospective study included 43 patients with NSCLC who underwent neoadjuvant immunotherapy combined with chemotherapy at Shandong Cancer Hospital from January 2021 to June 2023. Preoperative biopsy specimens were collected and subjected to multiplex immunofluorescence staining [CD8/programmed cell death protein 1 (PD-1)/T cell immunoglobulin and mucin-domain containing protein 3 (TIM-3)/CD4/ forkhead box protein 3 (FoxP3)/cytokeratin (CK)/4',6-diamidino-2-phenylindole (DAPI)]. InForm software was used to perform tissue segmentation (epithelial and stromal regions) and quantify the density and spatial proximity of tumor cells, CD8+ T cells and their subsets (cytotoxic, pre-exhausted and exhausted), as well as CD4+ T cells and their subsets (conventional and regulatory). NSCLC was classified into three subtypes based on the relative infiltration levels of CD8+ T cells in both the epithelial and stromal compartments: inflamed (both compartments>10/1000), excluded (epithelial compartment<10/1000 and stromal compartment>10/1000), and desert (both compartments<10/1000). The Kolmogorov-Smirnov test, Fisher's exact test, Mann-Whitney U test and Logistic regression were used to identify factors associated with major pathological response (MPR).

Inflamed, excluded, and desert NSCLC accounted for 65.1%, 27.9% and 7.0%, respectively. Compared with patients with non-inflamed NSCLC, those with inflamed NSCLC exhibit a higher MPR rate (71.4% vs 33.3%, P=0.016). Both univariate and multivariate Logistic regression analyses confirmed that the inflamed subtype is an independent protective factor against the acquisition of MPR in NSCLC patients (OR=0.20, 95%CI: 0.05-0.74, P=0.020; adjusted OR=0.17, 95%CI: 0.03-0.80, P=0.030). Analysis of the spatial distance between CD8+ and CD4+ T cells within the epithelial regions of inflamed NSCLC revealed that the effective density of cytotoxic CD8+ T cells within a 30 μm radius of regulatory CD4+ T cells was lower in the MPR group than in the non-MPR group (0.00 vs 0.33, P=0.037).

Patients with inflamed NSCLC demonstrate superior efficacy when receiving neoadjuvant immunotherapy combined with chemotherapy. This may be due to reduced proximity between regulatory CD4+ T cells and cytotoxic CD8+ T cells.