High PD-1 expression on regulatory and effector T-cells in lung cancer draining lymph nodes

Abstract

The treatment of advanced nonsmall cell lung cancer (NSCLC) with PD-1/PD-L1 immune checkpoint inhibitors has improved clinical outcome for a proportion of patients. The current challenge is to find better biomarkers than PD-L1 immunohistochemistry (IHC) that will identify patients likely to benefit from this therapy. In this exploratory study we assessed the differences in T-cell subsets and PD-1 expression levels on T-cells in tumour-draining lymph nodes (TDLNs) and peripheral blood mononuclear cells (PBMCs). To evaluate this, flow cytometric analyses were performed on endobronchial ultrasound-guided (EBUS) fine-needle aspirates (FNA) from TDLNs of patients with NSCLC, and the results were compared to paired PBMC samples. For a select number of patients, we were also able to obtain cells from a non-TDLN (NTDLN) sample. Our data show that the frequency of PD-1⁺ CD4⁺ and CD8⁺ T-cells, as well as the PD-1 expression level on activated regulatory T (aT_reg) and CD4⁺ and CD8⁺ T-cells, are higher in TDLNs than in PBMCs and, in a small sub-analysis, NTDLNs. These elevated PD-1 expression levels in TDLNs may reflect tumour-specific T-cell priming and conditioning, and may serve as a predictive or early-response biomarker during PD-1 checkpoint blockade.

FIGURE 1

PD-1 expression on activated regulatory T-cells (aT_reg) and CD4⁺ T-cells. a) Frequencies of aT_reg and PD-1⁺ aT_reg in peripheral blood mononuclear cells (PBMCs) and tumour-draining lymph nodes (TDLNs) (p<0.01). b) Representative dot plot showing PD-1^hi gating on activated (act)CD4⁺ T-cells in TDLNs, and frequencies of PD-1^hi actCD4 T-cells among PBMCs and TDLNs (p<0.001). c) PD-1 mean fluorescence intensity levels on aT_reg, actCD4⁺ and cytokine-secreting (cyt)CD4⁺ T-cells. Paired t-tests were performed to determine statistical significance between matched PBMCs and TDLNs: **: p<0.01.

FIGURE 2

PD-1 expression on CD8⁺ T-cells. a) Representative examples of PD-1 expression on CD8⁺ T-cells in peripheral blood mononuclear cells (PBMCs) and tumour-draining lymph nodes (TDLNs). b) Percentages of PD-1 (p<0.0001) and PD-1 mean fluorescence intensity levels on CD8⁺ T-cells present in PBMCs and TDLNs (p<0.05). c) Percentages of proliferating Ki67⁺ CD8⁺ T-cells in PBMCs and TDLNs (p<0.05). d) Pearson correlations between total percentages of PD-1⁺ cells on activated regulatory T-cells (aT_reg) and CD8⁺ T-cells, activated (act)CD4⁺ T-cells and cytokine-secreting (cyt)CD4⁺ T-cells for the six TDLN samples in which we could evaluate aT_reg frequencies.

FIGURE 3

PD-1 expression on CD4⁺ and CD8⁺ T-cells in matched tumour-draining lymph nodes (TDLN) and non-tumour draining lymph node (NTDLN) samples. a) Percentages of PD-1^hiactivated (act)CD4⁺ T-cells in matched TDLNs and NTDLNs (n=2). b) Percentages of PD-1⁺ and PD-1 mean fluorescence intensity levels on CD8⁺ T-cells in matched TDLNs and NTDLNs (n=3).