Sánchez, Pedro Arias

Abstract Introduction Screening individuals with latent tuberculosis infection (LTBI) from regions with varying TB endemicity is critical for global TB control. Immune responses to TB differ due to variations in exposure, immune adaptations, and environmental factors. This study evaluated antigen-specific immune responses in untreated LTBI individuals from low- and high-TB-endemic settings. Methods Peripheral blood mononuclear cells from untreated LTBI individuals from low-endemic (USA) and high-TB-endemic settings (Mexico) were stimulated with PPD, MTB300, ESAT-6/CFP-10 (RD1) peptides, mycobacterial glycopeptidolipids (GPL), and controls. We compare flow cytometric immune responses of T-cell intracellular cytokines and activation markers using non-parametric statistical methods. Results We studied age-matched, untreated LTBI individuals who tested IGRA(+)/TST(+) from low-TB-endemic (n = 18) and 18 healthcare workers from a high-TB-endemic settings. IGRA results showed no statistical differences across groups. The frequency of CD3+CD8+IFN-γ+HLA-DR+ and total CD3+TNF-α+ T cells after RD1 stimulation was significantly higher in the high-TB-endemic group (P < 0.05). Additionally, we observed significant increased frequencies in CD3/CD4+TNF-α+HLA-DR+ and CD8+TNF-α+HLA-DR+ T cells stimulated by GPL in the same group, probably associated with nontuberculous mycobacteria (NTM) exposures. Our findings suggest possible recent TB and NTM exposures in healthcare workers from a high-TB-endemic setting, resulted in increased antigen-specific adaptive TNF-α and IFN-γ producing T cells, which are important for pathogen control and immunity. Conclusion We found significant differences in antigen-specific cell immunity, including HLA-DR activated T-cells, a marker of recent antigen encounter, in individuals from a high-TB-endemic settings. These findings underscore the importance of settings-specific immune biomarkers for improving LTBI diagnostics, clinical management, and vaccination trials. Funding Source National Institute of Allergy and Infectious Diseases at the National Institutes of Health Topic Categories Immune Mechanisms of Human Disease (HUM)

Introduction Asymptomatic individuals with negative Interferon-γ release assay (IGRA) and tuberculin skin test (TST) results are often labeled as tuberculosis-unexposed individuals. We hypothesize that flow cytometric (FC) detection of antigen-stimulated T-cells in peripheral blood can differentiate IGRA(-)/TST(-) individuals with and without prior potential M. tuberculosis (Mtb) exposure. Methods We used FC multiparameter profiling of T-cell activation markers in TST/IGRA-negative individuals at risk for Mtb exposure and in unexposed controls. Results We studied 54 IGRA(-)/TST(-) subjects, including 27 individuals at risk for prior Mtb exposure. While quantitative IGRA results were negative in all patients, FC profiling of CD3/CD4 + IFN-γ + HLA-DR + T-cells after stimulation with a pool of Mtb-specific region of difference-1 (RD1) peptides, mycobacterial (PPD and MTB300), or Candida antigens, statistically differentiated between unexposed and potentially exposed individuals (P<0.05). ROC analysis showed PPD and RD1 peptides responses had AUCs >0.7 (sensitivity of 55.5–74% and specificity of 66.6–85.1%). The presence of a polyfunctional RD1-reactive CD3/CD8 + IFN-γ + TNF-α + T-cell subset also significantly differentiated these groups (AUC 0.66). Other T-cell markers demonstrated no significant differences. Conclusions FC detection of antigen-specific IFN-γ + HLA-DR + CD4+T-cells and IFN-γ + TNF-α + CD8 + T-cells can differentiate IGRA(-)/TST(-) individuals at risk of Mtb exposure from unexposed controls. Further validation is needed to determine whether this immunoprofiling approach can improve detection of Mtb infections and identification of unexposed individuals for future biomarker studies and vaccine trails.