Abstract
Despite advances in diagnosing latent Mycobacterium tuberculosis infection (LTBI), we still lack a diagnostic test that differentiates LTBI from active tuberculosis (TB) or predicts the risk of progression to active disease. One reason for the absence of such a test may be the failure of current assays to capture the dynamic complexities of the immune responses associated with various stages of TB, since these assays measure only a single parameter (release of IFN-γ) and rely on prolonged (overnight) T cell stimulation. We describe a novel, semi-automated RNA flow cytometry assay to determine whether immunological differences can be identified between LTBI and active TB. We analyzed antigen-induced expression of Th1 cytokine mRNA after short (2- and 6-h) stimulation with antigen, in the context of memory T cell immunophenotyping. IFNG and TNFA mRNA induction was detectable in CD4+ T cells after only 2 h of ex vivo stimulation. Moreover, IFNG- and TNFA-expressing CD4+ T cells (Th1 cells) were more frequent in active TB than in LTBI, a difference that is undetectable with conventional, protein-based cytokine assays. We also found that active TB was associated with higher ratios of effector memory to central memory Th1 cells than LTBI. This effector memory phenotype of active TB was associated with increased T cell differentiation, as defined by loss of the CD27 marker, but not with T cell exhaustion, as determined by PD-1 abundance. These results indicate that single-cell-based, mRNA measurements may help identify time-dependent, quantitative differences in T cell functional status between latent infection and active tuberculosis.
Highlights
Human infection with the intracellular pathogen, Mycobacterium tuberculosis, manifests itself in two forms
We showed that production of IFNG and TNFA mRNA, in response to ex vivo antigen stimulation associated with latent Mycobacterium tuberculosis infection (LTBI), was mostly found in CD4+ T cells (>90%), with negligible contribution by other lymphocyte subsets [26]
The present study showed that RNA flow cytometry affords rapid detection of cytokine mRNA induction in ex vivo stimulated CD4+ T cells
Summary
Human infection with the intracellular pathogen, Mycobacterium tuberculosis, manifests itself in two forms. Even the most accurate ex vivo LTBI assays, which measure IFN-γ release by antigen-stimulated peripheral T cells (Interferon gamma release assays-IGRA), do not distinguish between LTBI and active TB, nor do they provide information on the risk of reactivation and progression to disease [6,7,8]. Multi-parameter, T-cell-based assays have addressed either production of multiple cytokines [10,11,12] or memory phenotypes and expression of activation markers [13,14,15,16,17,18,19,20,21,22] Some of these studies have generated potentially promising results [for example, [22]], supporting the possibility that host signatures of infection stage or immunological protection can be identified. We utilized a semi-automated RNA flow cytometry platform [24] to determine whether a multi-parametric (mRNA and protein) assay for T cell memory phenotypes and cytokine production identifies differences between LTBI and active TB
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