Single-cell immunophenotyping revealed the association of CD4+ central and CD4+ effector memory T cells linking exacerbating chronic obstructive pulmonary disease and NSCLC

Abstract

IntroductionTobacco smoking generates airway inflammation in chronic obstructive pulmonary disease (COPD), and its involvement in the development of lung cancer is still among the leading causes of early death. Therefore, we aimed to have a better understanding of the disbalance in immunoregulation in chronic inflammatory conditions in smoker subjects with stable COPD (stCOPD), exacerbating COPD (exCOPD), or non-small cell lung cancer (NSCLC).MethodsSmoker controls without chronic illness were recruited as controls. Through extensive mapping of single cells, surface receptor quantification was achieved by single-cell mass cytometry (CyTOF) with 29 antibodies. The CyTOF characterized 14 main immune subsets such as CD4+, CD8+, CD4+/CD8+, CD4−/CD8−, and γ/δ T cells and other subsets such as CD4+ or CD8+ NKT cells, NK cells, B cells, plasmablasts, monocytes, CD11cdim, mDCs, and pDCs. The CD4+ central memory (CM) T cells (CD4+/CD45RA−/CD45RO+/CD197+) and CD4+ effector memory (EM) T cells (CD4+/CD45RA−/CD45RO+/CD197−) were FACS-sorted for RNA-Seq analysis. Plasma samples were assayed by Luminex MAGPIX® for the quantitative measurement of 17 soluble immuno-oncology mediators (BTLA, CD28, CD80, CD27, CD40, CD86, CTLA-4, GITR, GITRL, HVEM, ICOS, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, TLR-2) in the four studied groups.ResultsOur focus was on T-cell-dependent differences in COPD and NSCLC, where peripheral CD4+ central memory and CD4+ effector memory cells showed a significant reduction in exCOPD and CD4+ CM showed elevation in NSCLC. The transcriptome analysis delineated a perfect correlation of differentially expressed genes between exacerbating COPD and NSCLC-derived peripheral CD4+ CM or CD4+ EM cells. The measurement of 17 immuno-oncology soluble mediators revealed a disease-associated phenotype in the peripheral blood of stCOPD, exCOPD, and NSCLC patients.DiscussionThe applied single-cell mass cytometry, the whole transcriptome profiling of peripheral CD4+ memory cells, and the quantification of 17 plasma mediators provided complex data that may contribute to the understanding of the disbalance in immune homeostasis generated or sustained by tobacco smoking in COPD and NSCLC.

Identification of immune subsets with distinct lectin binding signatures using multi-parameter flow cytometry: correlations with disease activity in systemic lupus erythematosus

“…PBMCs were isolated as described previously by our group ( 42 , 43 ). Briefly, after the collection of 20 mL peripheral blood into an EDTA vacutainer (Becton Dickinson, Franklin-Lakes, USA), PBMCs were isolated by standard density gradient centrifugation using Leucosep tubes (Greiner Bio-One, Kremsmünster, Austria).…”

Comparative single-cell multiplex immunophenotyping of therapy-naive patients with rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus shed light on disease-specific composition of the peripheral immune system

“…Cells were processed for CyTOF as described previously by our group with minor modifications ( 37 ). Briefly, cryotubes were thawed in a 37°C water bath for 2 min, and cells were transferred into 14 ml of cRPMI at 37°C and centrifuged at 350 g for 6 min at room temperature (RT).…”

Closing Editorial: Immunophenotyping in Autoimmune Diseases and Cancer 3.0

“…Immunophenotyping using fluorochrome- or metal-labeled antibodies has become a widely used technology to reveal the functional heterogeneity of leukocytes and to monitor protein expression detected by single-cell flow cytometry [ 1 ]. Recent progress in fluorescence flow cytometry and mass cytometry has enabled high-dimensional resolution of the complex immunophenotype in both autoimmune diseases and cancer [ 2 , 3 , 4 ]. Therefore, we invited authors to publish their latest achievements related to the perturbation of the regulation of immune activation and the discovery of rare subpopulations of innate and adaptive immune players in autoimmune diseases or cancer.…”