Validation of differentially expressed genes
About 3 ml of whole blood from 10 initially treated pulmonary TB patients and 15 healthy people controls (HC) was collected in a heparin sodium anticoagulant tube. The inclusion criteria of the TB group was sputum MTB-positive, and/or interferon-gamma release assay (IGRA)-positive pulmonary TB patients without anti-TB treatment or with anti-TB treatment for less than 1 month. The inclusion criteria of HC were IGRA-negative and without abnormal findings in lung computed tomography (CT) scan. All samples were respectively stimulated with MTB CFP-10-ESAT-6 fusion protein for 24h(35). Total RNA was extracted using Trizol reagent (Invitrogen, USA), and cDNA was reverse transcribed from 1μg of purified RNA using PrimeScriptTM RT Reagent Kit (Takara, Japan). The primer sequences for amplification of the DE genes were obtained from PrimerBank and synthesized by Sangon Biotech (Shanghai, China) and shown in Table 1. RT-qPCR amplification of the genes was performed using the RT-qPCR kit with the SYBRGreen Ⅰ method (KAPA SYBR® FAST, South Africa). The quantification of mRNA was examined using RT-qPCR on Roche 480 (Roche, Switzerland) with the following program: pre-denatured at 95℃ for 3min; 40 cycles of denaturing at 95℃ for 10s, annealing at 60℃ for the 20s; extension at 72℃ for 1s. RT-qPCR of each cDNA sample was repeated twice, and the final Ct value was the mean of the two times. The relative expression amount of Amy2a, Retn, and Sftpd was calculated by the 2−△△Ct method using the GAPDH gene as an internal reference.