Single-cell mass cytometric analysis of peripheral immunity and multiplex plasma marker profiling of non-small cell lung cancer patients receiving PD-1 targeting immune checkpoint inhibitors in comparison with platinum-based chemotherapy

INTRODUCTION: The effect of platinum-based chemotherapy (Chem.) and second- or multiple- line immune checkpoint PD-1 blocking therapy by Nivolumab or Pembrolizumab (ICI) was assayed in the peripheral blood of non-small cell lung cancer (NSCLC) patients. METHODS: Flow cytometry was used to detect NSCLC-related antigen binding IgG antibodies. The Luminex MagPix multiplex bead-based cytokine/chemokine detecting system was used to quantitatively measure 17 soluble markers in the plasma samples. Single-cell mass cytometry was applied for the immunophenotyping of peripheral leukocytes. RESULTS: The incubation of patient derived plasma with human NSCLC tumor cell lines, such as A549, H1975, and H1650, detected NSCLC-specific antibodies reaching a maximum of up to 32% reactive IgG-positive NSCLC cells. The following markers were detected in significantly higher concentration in the plasma of Chem. group versus healthy non-smoker and smoker controls: BTLA, CD27, CD28, CD40, CD80, CD86, GITRL, ICOS, LAG-3, PD-1, PD-L1, and TLR-2. The following markers were detected in significantly higher concentration in the plasma of ICI group versus healthy non-smoker and smoker controls: CD27, CD28, CD40, GITRL, LAG-3, PD-1, PD-L1, and TLR-2. We showed the induction of CD69 and IL-2R on CD4+ CD25+ T-cells upon chemotherapy; the exhaustion of one CD8+ T-cell population was detected by the loss of CD127 and a decrease in CD27. CD19+CD20+, CD79B+, or activated B-cell subtypes showed CD69 increase and downregulation of BTLA, CD27, and IL-2R in NSCLC patients following chemotherapy or ICI. DISCUSSION: Peripheral immunophenotype caused by chemotherapy or PD-1 blocking was shown in the context of advanced NSCLC.