Altered CD4 T cell response in oligometatastic non-small cell lung cancer brain metastasis

Abstract

Lung cancer is the leading cause of cancer mortality globally, with brain metastasis (BM) in 40% of advanced non-small-cell lung cancer (NSCLC) cases. In 15% of these cases, the brain is the sole affected organ (oligometastasis), which correlates with a better prognosis compared to widespread disease. It remains unclear if brain-only metastasis without systemic spread is due to the immune system's ability to control systemic tumor progression. We studied the immune cell compositions in NSCLC patients with BM, identifying novel patterns associated with BM, and specifcally with either oligo- or polymetastatic spread. Multi-parametric immune phenotyping of peripheral blood primarily showed alterations in the CD4⁺ T cell compartment, with increased CD4⁺ T_H17 cells, and higher IL-17 levels in NSCLC BM patients compared to healthy individuals. Furthermore, CD4⁺ T cells in BM patients exhibited lower CD73 expression and reduced effector memory differentiation. There was also decreased intratumoral infiltration and a distinct CD4⁺ T cell profile in oligo-synchronous BM, both in the tumor microenvironment and peripheral blood, compared to polymetastatic BM patients. Additionally, CD73 was significantly upregulated in CD4⁺ and T regulatory cells of oligo-synchronous (BM simulantously with primary-tumor diagnosis) BM. These findings suggest that CD4⁺ T cells play a crucial role in the biology of NSCLC BM and potentially contribute to differences in metastatic patterns, as oligo-synchronous BM shows a more significant alteration in the CD4⁺ T cell immune profile, both locally at the tumor site and systemically.

Keywords: Brain metastasis; CD4; Immunophenotyping; NSCLC; T cells.

Fig. 1

(a.) Immunohistochemical detection and estimation of positive staining for CD3, CD8, and CD4 on TILs in brain metastatic tissues. Representative stainings (high and low infiltration)are shown for both intra-tumoral (IT) and peri-tumoral (PT) regions. Magnification: x20. (b.) Comparison of the staining scores for CD3, CD8, and CD4 in all groups combined, and between oligo-synchronous BM (Sync), oligo-metachronous BM (Meta), and poly BM (Poly) individually in both IT and PT regions. BM: brain metastasis, Sync: oligo-synchronous BM, Meta: oligo-metachronous BM, and poly: poly metastasis, negative: no stained cells, low: <10%, moderate: 10–40%, and high: >40%

Fig. 2

(a) Flow cytometric immunophenotyping of T cell activation and exhaustion marker expression of peripheral blood from NSCLC BM patients and age-matched healthy donors (HD). (b) Spider plots of CD4⁺ and CD8⁺ differentiation phenotype differences detected in peripheral blood of BM patients and HD. (c) Box plots depicting expression of T regs in HD and BM patients with no significant differences found. (d) A significantly higher frequency of pro-inflammatory T_H17 T cells among cancer patients compared to HD was observed, (e) whereas a reduced HLA-DR expression did not support and activated phenotype in CD4⁺ T cells. (f) Box plots depicting expression of different cytokines IFNγ, TNFα, IL-2, IL-4, IL-10, and IL-17 on CD4⁺ and CD8⁺ cells. (g) Box plots for PD-1, KLRG1 and CD73 expression on CD8⁺ T cells, and TGIT expression on T regs. Mean ± SEM, t test. P values are defined as * < 0.05; ** < 0.01; and *** < 0.001

Fig. 3

Flow cytometric immunophenotyping of T cell differentiation and CD73 expression in the different BM groups. (a) Spider plots of CD4 + and CD8 + differentiation phenotypes of peripheral blood in BM groups. Populations are defined by expressions of CD45RA, CD27, CD28, and CCR7 markers. (b) Box plots depicting expression of CD4 + naïve, TeffRA+, central memory, and TeffRA- (T_EM) expressions between BM groups. (c) Box plots of CD73 expressions in CD8+, CD4+, and Treg cells between BM groups

Fig. 4

Phonograph clustering of different markers in the three BM groups combined, and individual UMAPs display CD73 expression in synchronous, metachronous, and polymetastatic BM. Heatmap and boxplot of markers expression in cluster 5: CD4+, CD27+, CD28+, CD127+, CD73 + and CD45RA-, CD25- T cells. Values describe population size as percentages. Sync: oligo-synchronous BM, Meta: oligo-metachronous BM, and poly: poly metastasis)