Functional status and spatial interaction of T cell subsets driven by specific tumor microenvironment correlate with recurrence of non-small cell lung cancer

BACKGROUND: The anti-tumoral or pro-tumoral roles of CD4(+) and CD8(+) T cells typify the complexity of T cell subsets function in cancer. In the non-small cell lung cancer (NSCLC), the density and topology of distinct T cell phenotypes at the tumor center (TC) versus the invasive margin (IM) are largely unknown. Here, we investigated T cell subsets density and distribution within TC and IM regions in NSCLC and its impact on the prognosis. METHODS: We performed multiplex immunofluorescence using a tissue microarray of samples from 99 patients with locally advanced NSCLC to elucidate the distributions of tumor cell, T cell subpopulations (CD4/conventional CD4/regulatory CD4/CD8/cytotoxic CD8/pre-dysfunctional CD8/dysfunctional CD8), microvessel density (MVD), cancer-associated fibroblasts (CAFs) and hypoxia-inducible factor-1α (HIF-1α) in TC and IM tissues. Cell-to-cell nearest neighbor distances and interactions were analyzed using the phenoptrreports R package. Cox regression was used to evaluate the associations between T cell subsets density and proximity to tumor cells and recurrence-free survival (RFS). Correlations between different cell subsets were examined by Spearman’s or Kruskal-Wallis tests. RESULTS: In the locally advanced NSCLC, the proportion of tumor cells and CAFs in IM is lower than in the TC, while MVD, CD4(+), and CD8(+) T lymphocytes were increased, and tumor cells were closer to T lymphocytes and their subsets. The density and proximity of CD4(+) and CD8(+) T cells in the TC and IM regions were not associated with RFS, but in the IM area, increased density of dysfunctional CD8 and closer regulatory CD4 to tumor cells were independent risk factors for recurrence (HR were 3.536 and 2.884, respectively), and were positively correlated with HIF-1α(+)CD8 (r = 0.41, P = 0.000) and CAFs (P = 0.017), respectively.s CONCLUSIONS: In locally advanced NSCLC, the functional status of T cells in the IM region is closely related to recurrence. The density of dysfunctional CD8 and the proximity of regulatory CD4 to tumor cells were independent risk factors for recurrence, and are positively correlated with the hypoxia response of CD8(+) T cells and CAFs. Targeting hypoxia or CAFs is expected to further sensitize therapy.