A Subset of Circulating Blood Mycobacteria-Specific CD4 T Cells Can Predict the Time to Mycobacterium tuberculosis Sputum Culture Conversion Catherine Riou 1. , Clive M. Gray 1 * . , Masixole Lugongolo 2 , Thabisile Gwala 2 , Agano Kiravu 1 , Pamela Deniso 3 , Lynsey Stewart-Isherwood 3 , Shaheed Vally Omar 2 , Martin P. Grobusch 4 , Gerrit Coetzee 2 , Francesca Conradie 3 , Nazir Ismail 2 , Gilla Kaplan 5 , Dorothy Fallows 5 1 Division of Immunology, Institute of Infectious Disease and Molecular Medicine and National Health Laboratory Services, University of Cape Town, Cape Town, South Africa, 2 National Institute for Communicable Diseases, National Health Laboratory Services, Johannesburg, South Africa, 3 Right to Care and the Clinical HIV Research Unit, University of the Witwatersrand, Johannesburg, South Africa, 4 Center of Tropical Medicine and Travel Medicine, Department of Infectious Diseases, Division of Internal Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 5 Laboratory of Mycobacterial Immunity and Pathogenesis, Public Health Research Institute at the International Center for Public Health, Newark, New Jersey, United States of America Abstract We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR + Ki67 + on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67 + HLA-DR + Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67 + HLA-DR + T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNc + IL2 + TNFa + CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial- specific CD4 T cells (Ki67 + HLA-DR + ) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment. Citation: Riou C, Gray CM, Lugongolo M, Gwala T, Kiravu A, et al. (2014) A Subset of Circulating Blood Mycobacteria-Specific CD4 T Cells Can Predict the Time to Mycobacterium tuberculosis Sputum Culture Conversion. PLoS ONE 9(7): e102178. doi:10.1371/journal.pone.0102178 Editor: Delia Goletti, National Institute for Infectious Diseases (L. Spallanzani), Italy Received February 16, 2014; Accepted June 16, 2014; Published July 21, 2014 Copyright: ß 2014 Riou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by an National Institute of Allergy and Infectious Diseases grant (RO1 AI80737) and a Fogarty Global Infectious Diseases fellowship (D43TW008264). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: clive.gray@uct.ac.za . These authors contributed equally to this work. Introduction The tuberculosis (TB) epidemic in many parts of the world has been greatly exacerbated in recent years, not only by the HIV co- epidemic, but also by the rise in multidrug resistant (MDR) strains of M. tuberculosis (Mtb). MDR-TB is defined by resistance to rifampicin (RIF) and isoniazid (INH), the two most effective drugs against TB and the backbone of standard short-course therapy [1,2]. The quest for new pharmaceuticals to combat both drug susceptible and resistant TB and expand treatment options for patients with MDR-TB is a major challenge. At present, the success of TB therapy is established by the risk of relapse within the first 2 years after treatment, which necessitates long clinical trials and extended follow-up of patients. Thus, to support clinical trials and improve case management, early predictors of clinical outcome that can serve as interim indicators of treatment response are needed. The only currently accepted interim indicators are sputum culture conversion after 2 months of standard therapy and time to culture positivity at the start of treatment, which provides an indicator of bacillary load [3–5]. However, culture-based methods require 6–8 weeks for a result and are only appropriate for patients who are sputum culture positive at baseline. To address this clinical need, as well as to shorten the time required for clinical trials of new TB drugs in the pipeline, extensive efforts to discover early biomarkers of response to TB treatment are currently underway [6–9]. The evaluation of candidate host immune biomarkers is a particularly active area of research, which additionally can contribute to our general understanding of the pathogenesis of TB disease [10]. A number of investigators have examined serodiagnostic markers in patients before and during TB treatment, including inflammatory molecules, cytokines and chemokines, as well as antibodies against Mtb proteins [10–12] and the host blood transcriptome [13,14]. Others have assessed PLOS ONE | www.plosone.org 1 July 2014 | Volume 9 | Issue 7 | e102178