Development of gene expression signatures for latent and clinical tuberculosis (TB)

For further identify the differentiation between latent and clinical tuberculosis (TB), we employed whole genome microarray expression profiling to study genes with significant expression change in peripheral CD4+T cells between healthy control, latent tuberculosis (LTB) and clinical tuberculosis (TB). Our experiment included 4 groups: healthy donor (HD), latent TB1 (LTB1) with low IFN-gamma release level, latent TB2 (LTB2) with high IFN-gamma release level, and tuberculosis (TB) with high IFN-gamma release level. Human peripheral blood mononuclear cells were collected, from which CD4+T cells were isolated. Total RNA of each individuals of each group was extracted from peripheral CD4+T cells. One μg of RNA mixture, pooled equivalently by each individual total RNA of each group, was administrated microarray test. Compared with HD, through analyzing enriched-Gene Ontology (GO) terms and KEGG pathways of each group, we found peripheral CD4+T cells might had different ability for mycobacterium tuberculosis infection in LTB1, LTB2 and TB. Finally we detected that TNFSF13/APRIL and TNFSF13B/BAFF was significant up-regulation in both CD4+T cells and serum of TB by real time PCR and ELISA, respectively. Peripheral CD4+ T cells were purified by positive selection using magnetic beads (BD IMagTM anti-human CD4 Particles-DM, BD Biosciences). Total RNA was extracted from CD4+ T cells (CD4-RNA) of LTB1, LTB2 and TB patients and healthy controls by RNeasy Mini Kit (QIAGEN).Then, RNA quality was checked and mixed with an equal quantity of each individual. The number of included-individuals in each group was 11 for HD, 11 for LTB1, 12 for LTB2 and 11 for TB.