Multifunctional Analysis of CD4⁺T-Cell Response as Immune-Based Model for Tuberculosis Detection

Mono- and multifunctional specific CD4(+) and CD8(+) T-cell responses were evaluated to improve the immune-based detection of active tuberculosis (TB) and latent infection (LTBI). We applied flow cytometry to investigate cytokines profile (IFN-γ, TNF-α, and IL-2) of T cells after stimulation with TB antigens in 28 TB-infected subjects (18 active TB and 10 LTBI) and 10 uninfected controls. Cytokines production by CD4(+) T cells at single-cell levels was higher in TB-infected subjects than uninfected controls (P < 0.0001). Assigning to activated CD4(+) T cells, producing any of the three cytokines, a cut-off >0.45%, it was possible to differentiate TB-infected (>0.45%) by uninfected subjects (<0.45%). Among TB-infected subjects, the frequencies of multifunctional CD4(+) T cells, simultaneously producing all 3 cytokines, are lower in active TB than LTBI subjects (P = 0.003). Thus, assigning to triple-positive CD4(+) T cells a cut-off <0.182%, TB-infected individuals could be classified as active TB subjects (<0.182%) or LTBI subjects (>0.182%). The magnitude of CD8(+) T-cell responses showed no differences between active TB and LTBI. Multifunctional CD4(+) T-cell responses could have the potential to identify at single time point subjects without TB infection and patients having active or latent TB.