PD-1/PD-L1 immune-checkpoint blockade induces immune effector cell modulation in metastatic non-small cell lung cancer patients: A single-cell flow cytometry approach

Abstract

Peripheral immune-checkpoint blockade with mAbs to programmed cell death receptor-1 (PD-1) (either nivolumab or pembrolizumab) or PD-Ligand-1 (PD-L1) (atezolizumab, durvalumab, or avelumab) alone or in combination with doublet chemotherapy represents an expanding treatment strategy for metastatic non-small cell lung cancer (mNSCLC) patients. This strategy lays on the capability of these mAbs to rescue tumor-specific cytotoxic T lymphocytes (CTLs) inactivated throughout PD-1 binding to PD-L1/2 in the tumor sites. This inhibitory interactive pathway is a physiological mechanism of prevention against dangerous overreactions and autoimmunity in case of prolonged and/or repeated CTL response to the same antigen peptides. Therefore, we have carried out a retrospective bioinformatics analysis by single-cell flow cytometry to evaluate if PD-1/PD-L1-blocking mAbs modulate the expression of specific peripheral immune cell subsets, potentially correlated with autoimmunity triggering in 28 mNSCLC patients. We recorded a treatment-related decline in CD4⁺ T-cell and B-cell subsets and in the neutrophil-to-lymphocyte ratio coupled with an increase in natural killer T (NKT), CD8⁺PD1⁺ T cells, and eosinophils. Treatment-related increase in autoantibodies [mainly antinuclear antibodies (ANAs) and extractable nuclear antigen (ENA) antibodies] as well as the frequency of immune-related adverse events were associated with the deregulation of specific immune subpopulations (e.g., NKT cells). Correlative biological/clinical studies with deep immune monitoring are badly needed for a better characterization of the effects produced by PD-1/PD-L1 immune-checkpoint blockade.

Keywords: NKT; NSCL; bioinformatics; flow cytometry; immune checkpoint inhibitors.

Figure 1

Cell clustering and phenotype identification of PBMCs of mNSCLC patients. Previously compensated flow cytometry standard (FCS) files have been used for large-scale immune monitoring using FlowCT. (A) Cell number per time point (basal (BAS) vs. post-treatment (POST) after pre-processing, quality control, and normalization. (B) Uniform manifold approximation and projection (UMAP) dimensionality reduction techniques for full visualization of 19 independent clusters. PBMC, Peripheral blood mononuclear cells; mNSCLC, metastatic non-small cell lung cancer.

Figure 2

Correlation of different cell clustering and mNSCLC patient survival. Post-treatment fold change of previously identified cells is calculated and used for downstream analyses. (A) Values of seven fundamental cell clusters before and after therapy are shown as grouped dot plot, paired Student’s t-test is used for the analysis of each cluster in the different treatment conditions, and clusters with p-value < 0.05 were considered as significant. y-Axis represents % of cells on total lymphocytes for B/T/NKT cells, total cellularity for monocytes and neutrophils, and an absolute number for NLR. (B) NLR calculation in two different study centers to demonstrate baseline differences. (C) Survival analysis based on population fold changes; p-value < 0.1 considered as trend, and p-value < 0.05 considered as statistically significant. mNSCLC, metastatic non-small cell lung cancer; NLR, neutrophil-to-lymphocyte ratio. *= pvalue<0.05.

Figure 3

CD8⁺ T cells and NKT cell frequency changes in response to treatment in groups with different inflammatory and autoimmunity markers. (A) Boxplot representing changes on peripheral blood T_regs and PD-1⁺CD8⁺ T lymphocytes as effect of treatment. (B) Total percentage of different cell subsets before and after therapy is reported for patients positive and negative for antinuclear antibodies (ANA, AAbs); only significant results are indicated. (C) Abundance of different cell subsets before and after therapy is reported for patients experiencing or not irAEs. Cell subsets with p-value < 0.1 only are represented. NKT, natural killer T; irAEs, immuno-related adverse events. *= pvalue<0.05. , **= p value<0.01 ns, not significant.