Research Article Multifunctional Analysis of CD4 + T-Cell Response as Immune-Based Model for Tuberculosis Detection Miriam Lichtner, 1,2 Claudia Mascia, 1 Ilaria Sauzullo, 1 Fabio Mengoni, 1 Serena Vita, 1 Raffaella Marocco, 2 Valeria Belvisi, 2 Gianluca Russo, 1 Vincenzo Vullo, 1 and Claudio M. Mastroianni 1,2 1 Department of Public Health and Infectious Diseases, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University, Piazzale Aldo Moro 5, 00185 Rome, Italy 2 Infectious Diseases Unit, Sapienza University, Corso della Repubblica 79, 04100 Latina, Italy Correspondence should be addressed to Claudio M. Mastroianni; claudio.mastroianni@uniroma1.it Received 17 September 2014; Revised 30 December 2014; Accepted 30 December 2014 Academic Editor: Vishwanath Venketaraman Copyright © 2015 Miriam Lichtner et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mono- and multifunctional specifc CD4 + and CD8 + T-cell responses were evaluated to improve the immune-based detection of active tuberculosis (TB) and latent infection (LTBI). We applied fow cytometry to investigate cytokines profle (IFN-, TNF- , and IL-2) of T cells afer 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 ( < 0.0001). Assigning to activated CD4 + T cells, producing any of the three cytokines, a cut-of >0.45%, it was possible to diferentiate 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 ( = 0.003). Tus, assigning to triple- positive CD4 + T cells a cut-of <0.182%, TB-infected individuals could be classifed as active TB subjects (<0.182%) or LTBI subjects (>0.182%). Te magnitude of CD8 + T-cell responses showed no diferences 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. 1. Introduction Mycobacterium tuberculosis (Mtb) infects more than 2 billion people worldwide; 90% of Mtb-infected individuals are able to resist overt tuberculosis (TB) disease determining the state of latency of infection (LTBI) [1]. Although latent and active TB disease are likely part of a dynamic spectrum [2, 3], individuals with LTBI are classically considered to be asymptomatic and not infectious; thus, the accurate classif- cation of TB status is essential since treatment and prevention approaches are entirely diferent. All existing tests for LTBI diagnosis, the tuberculin skin test (TST) and the newer interferon-gamma release assays (IGRAs), are acceptable but remain imperfect tests [4]. Tey represent indirect markers of Mtb exposure and provide immunological evidence of host sensitization to TB antigens. Both tests depend on cell-mediated immunity, and neither test can accurately diferentiate between active TB and LTBI, distinguish reactivation from reinfection, or discriminate the various stages within the spectrum of Mtb infection [5, 6]. Tus, there is a need for newer biomarkers to classify patients at a single time point as having active TB, LTBI, or no infection. Alternative immunological methods have been investi- gated in recent years [7–11]. In particular, multifunctional T cells, defned by their ability to coexpress two or more cytokines, have showed a better diagnostic yield than IGRA to detect TB infection [12–14] and have improved discrimi- nation between active TB and LTBI [7–11, 15]. However, currently there is no consensus whether multi- functional T cells represent a marker of protective immunity or disease activity. Studies in animal models revealed a poten- tial association of multifunctional T1 cells with protective Hindawi Publishing Corporation Journal of Immunology Research Volume 2015, Article ID 217287, 10 pages http://dx.doi.org/10.1155/2015/217287