We analyzed changes in intratumoral CD8+ and CD4+ T-cell subpopulations following neoadjuvant chemoimmunotherapy in non-small cell lung cancer. We then assessed whether these alterations favored better outcomes and explored their association with the tumor microenvironment.Paired pre- and post-treatment samples from 32 patients with non-small cell lung cancer who underwent neoadjuvant chemoimmunotherapy at Shandong Cancer Hospital (January 2021-June 2023) were analyzed retrospectively. A quantitative analysis of tumor cells and their microenvironment was performed using a tissue microarray and a multiplex immunofluorescence technique. The analysis was based on the number of cells per thousand nucleated cells. Patients exhibiting a major pathologic response were classified as responders. The delta parameter (post-treatment minus pre-treatment) was utilized to assess changes in these indicators, and associations with treatment response were identified using the Wilcoxon Signed-Rank test and logistic regression analyses.Of the 32 patients, 59.38% were classified as responders. Across all patients, neoadjuvant chemoimmunotherapy significantly reduced the densities of dysfunctional CD8+ resident memory T cells and cytotoxic and dysfunctional CD8+ bystander T cells, while conventional CD4+ T cells increased significantly. Similar trends were observed in the response group. In the non-response group, only cytotoxic CD8+ bystander T cells were reduced in number. Logistic regression analysis revealed that a high delta conventional CD4+ T cells is more favorable for MPR (OR = 0.13, p = 0.038), exhibiting a similar trend to changes in HIF-1α (p = 0.049).Alterations in specific CD8+ and CD4+ T-cell subpopulations during neoadjuvant chemoimmunotherapy may favor better outcomes and are potentially associated with tumor hypoxia. These findings provide a new perspective on developing strategies to improve treatment sensitivity in non-small cell lung cancer.Copyright © 2025 Wu, Yang, Sun, Zhao, Geng, Cao, Chen, Yan, Yang, Sun and Xing.