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

Abstract

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.

Keywords: CD4+ T cells; Lung cancer; multiplex immunohistochemistry; neoadjuvant immunotherapy; spatial interaction.

Figure 1

Identification and characterization of tumor microenvironment in NSCLC. (A) Cohorts and samples. (B) mIHC staining was used to characterize immune indicators and select the ROIs and proceed with tissue segmentation, cell segmentation, and Phenotype and identification, and the boxes represent ROIs. (C) Overview of the analysis variables. CAF, cancer-associated fibroblast; HIF-1α, hypoxia-inducible factor 1α; ICI, immune checkpoint inhibitor; mIHC, multiplex immunohistochemistry; MVD, microvessel density; NND, nearest neighbor distance; NSCLC, non-small cell lung cancer; ROI, region of interest.

Figure 2

mIHC characterization of the impact of CD4⁺ T cells, CD8⁺ T cells, and CD68⁺ macrophage densities in different regions of NSCLC patients on the efficacy of neoadjuvant immunotherapy. (A) Representative mIHC images from panel 1 and tissue segmentation images, where CK⁺ indicates the epithelial compartment, and CK⁻ indicates the stromal compartment. Epi, epithelial compartment. Str, stromal compartment, and the boxes represent enlarged immune indicators. (B) Representative images of CD4⁺ T cells, CD8⁺ T cells, and CD68⁺ macrophages, and the boxes represent enlarged immune indicators. (C) Differences in CD8⁺ T cell density between responders and non-responders in the stromal compartment of all patients (left), and the corresponding ROC curve (right). (D) Differences in CD8⁺ T cell density between responders and non-responders in the total compartment of PD-L1 low-expressing patients (left), and the corresponding ROC curve (right). (E) Differences in CD8⁺ T cell density between responders and non-responders in the stromal compartment of PD-L1 low-expressing patients (left), and the corresponding ROC curve (right). AUC, area under the curve; DAPI, 4',6-diamidino-2-phenylindole; mIHC, multiplex immunohistochemistry; N, non-response; NSCLC, non-small cell lung cancer; PD-L1, programmed cell death-ligand 1; R, response; ROC, receiver operating characteristic; Se, sensitivity; Sp, specificity; TPS, tumor proportion score.

Figure 3

The impact of spatial interactions between CD4⁺ T cells and CD8⁺ T cells, as well as CD68⁺ macrophages, on the efficacy of neoadjuvant immunotherapy in PD-L1 low-expressing NSCLC patients. (A) mIHC staining was used to analyze differences in the NND between CD4⁺ T cells and CD8⁺ T cells in different compartments and representative images (left). (B) mIHC staining was used to analyze differences in the NND between CD4⁺ T cells and CD68⁺ macrophages in different compartments and representative images (left). (C) ROC curve of the NND between CD4⁺ T cells and CD8⁺ T cells in the total compartment in relation to treatment efficacy. (D) ROC curve of the NND between CD4⁺ T cells and CD8⁺ T cells in the stromal compartment in relation to treatment efficacy. (E) Radar chart showing the relationship between the proximity (10/20/30 μm) of CD4⁺ T cells and CD8⁺ T cells and the efficacy of neoadjuvant immunotherapy in NSCLC patients. AUC, area under the curve; DAPI, 4',6-diamidino-2-phenylindole; Epi, epithelial compartment; mIHC, multiplex immunohistochemistry; N, non-response; NND, nearest neighbor distance; NSCLC, non-small cell lung cancer; PD-L1, programmed cell death-ligand 1; R, response; ROC, receiver operating characteristic; Se, sensitivity; Sp, specificity; Str, stromal compartment, and the boxes represent enlarged immune indicators.

Figure 4

A shorter NND between CD4⁺ T cells and CD8⁺ T cells is positively correlated with MVD. (A) A multispectral mixed image of panel 2 from the mIHC showing representative illustrations of tumor cells, CD4⁺ T cells, HIF-1α-expressing cells, MVD, and CAFs, and the boxes represent enlarged immune indicators. (B) Correlation heatmap of various immune markers within different compartments in PD-L1 low-expressing responders. (C) Correlation heatmap of various immune markers within different compartments in PD-L1 low-expressing non-responders. CAF, cancer-associated fibroblast; DAPI, 4',6-diamidino-2-phenylindole; HIF-1α, hypoxia-inducible factor-1α; mIHC, multiplex immunohistochemistry; MVD, microvascular density; NND, nearest neighbor distance; PD-L1, programmed cell death-ligand 1; SMA, smooth muscle actin; TPS, tumor proportion score.