Homeostatic Dysregulation of Systemic CD8+ T Cell Compartment in Lung Cancer Patients
Description
Supporting data values
Files
Steps to reproduce
Flow cytometry and sorting Thawed PBMCs were washed twice with complete media (RPMI-1640 (Welgene) supplemented with 10% FBS, Penicillin-Streptomycin (Welgene), 2-mercaptoethanol (Welgene), non-essential amino acid (Welgene), glutamine (Welgene), and HEPES (Welgene)), and used for further experiments. Surface molecules were stained in staining media (PBS (Welgene) supplemented with 3% FBS and EDTA (Bioneer)) for 30 min. For intracellular molecules, samples were fixed and permeabilized with BD Cytofix/Cytoperm (BD) or FOXP3/Transcription Factor Staining Buffer Set (eBioscience) for 20 min, then stained for intracellular molecules for 30 min. All processes were done on ice. Prepared samples were run using CytoFLEX S (Beckman Coulter), or CytoFLEX LX (Beckman Coulter). Patient grouping strategies To group patients according to perforin (prfn) fold change (FC) and granzyme B (gnzB) FC, FCs were acquired by dividing prfn+ (and gnzB+) frequencies after ICI therapy with baseline prfn+ (and gnzB+) frequencies of the respective patient. Patients with high FC (FC≥1.1; ~30% of total patients) were considered FChi and the rest as FClo. To group patients according to baseline prfn, gznB, DN-Tem, or DP-Tem, frequencies of these cells in total Tem were accessed. Top ~30% were considered patients with high frequency of these cells. To investigate biomarker efficiency of DN-Tem and DP-Temra, DN-Tem and DP-Temra frequencies were assessed in total Tem and total Temra, respectively, in peripheral blood of lung cancer patients and healthy individuals. Patients with DN-Tem and DP-Temra frequencies higher than that of most (>~80%) healthy individuals who were analyzed under the same experiment settings (DN-Tem≥36% and DP-Temra≥17.2% for NSCLC patients and DN-Tem≥42% and DP-Temra≥11% for SCLC patients) were considered DN-Temhi and DP-Temrahi. The threshold for CXCR3+.DN-Tem (≥1.25%) was determined with a similar approach. Clinical analysis During ICI treatment, a follow-up CT scan for the response assessment was performed every 2 to 3 cycles (Cohort #1, #2, #3) or 4 to 6 cycles (Cohort #4) of ICI treatment. The clinical response to the treatment was defined by the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. The best response was defined as complete response (CR) if cancer was no longer detectable, partial response (PR) if the size of the cancer decreased, stable disease (SD) if there was no progression, and progressive disease (PD) if the size of the cancer increased on CT up to 3 months after starting the treatment. In addition, PR or SD by 6 months was defined as durable clinical benefit (DCB). Forrest plot was analyzed using the indicated variables against PR. Hazard ratios were calculated against one factor (labeled as reference) in each variable.