Mycobacterium tuberculosis, the leading cause of tuberculosis (TB), remains a significant global health challenge. According to the World Health Organization, approximately one-third of individuals worldwide has latent TB infection (LTBI) and are at a risk of developing active TB ⁽¹⁾. Approximately 5%–10% of cases with LTBI may progress to active TB, especially in individuals with a compromised immune system ^(2-6). Additionally, at least one-third of individuals with HIV worldwide are co-infected with M. tuberculosis, with 8%–10% developing active TB annually ⁽⁷⁾. Therefore, rapid diagnostic tools and effective LTBI treatments are crucial for reducing and managing TB burden. Accurately identifying individuals with LTBI at the highest risk of progression to active disease is essential for effective control efforts.

Tuberculin skin test (TST) has been considered the gold standard for LTBI diagnosis ⁽⁸⁾. However, TST results are often influenced by cross-reactivity between the bacille Calmette–Guérin (BCG) vaccine and various environmental nontuberculous mycobacteria because the purified protein derivative used in TST contains antigens common to BCG and certain nontuberculous mycobacterial strains ⁽²⁾. Interferon-gamma release assay (IGRA), an alternative diagnostic method for LTBI, detects M. tuberculosis infection by measuring the release of interferon-gamma (IFN-γ) from immune cells stimulated with M. tuberculosis-specific antigens, such as early secretory antigen target 6 and culture filtrate protein 10 ⁽⁹⁾. These antigens are highly specific to M. tuberculosis because they are absent in all BCG strains and most major nontuberculous mycobacteria ⁽¹⁰⁾. These antigens serve as strong T-cell targets during M. tuberculosis infection. IGRAs have been promising in reducing false positives, especially among low-risk individuals vaccinated with BCG. However, their sensitivity is limited to immunocompromised patients and young children. Additionally, IGRAs cannot distinguish between active pulmonary TB and LTBI cases ⁽¹¹⁾.

Various cytokines and regulatory factors are crucial in the pathogenesis and control of M. tuberculosis infections ^(12-16). Research indicates that IFN-γ alone is insufficient to prevent disease progression, highlighting the importance of tumor necrosis factor-alpha (TNF-α) in controlling M. tuberculosis infection ^(17,18). Additionally, interleukin-2 (IL-2) has been found to correlate with pathogen load, whereas elevated levels of IL-4 and IL-10 are strongly associated with active TB ⁽¹⁹⁾. Using a diluted whole blood intracellular cytokine staining (ICS) assay, we measured the frequency of purified protein derivatives (PPD)-specific CD4+ T cells that produce IFN-γ, TNF-α, and IL-2 in 25 participants and investigated their clinical relevance. This study seeks to investigate immune responses to assess the diagnostic potential of ICS assays for identifying LTBI.

Healthy adults aged >20 years were recruited between January 2016 and September 2017 from Severance Hospital in Seoul, South Korea. This study excluded individuals with acute or chronic illnesses, history of active TB, or any TB-related symptoms. The participants’ BCG vaccination status was evaluated through a survey and physical inspection of BCG scars on either upper arm. The TB infection status was determined using the QuantiFERON-TB Gold In-Tube assay (QFT-GIT). None of the participants had an HIV infection or chronic conditions, such as diabetes mellitus, chronic renal failure, cancer, or chronic liver disease, nor did they have any immunosuppressive conditions or acute infections. This study was approved by The Institutional Review Board of Severance Hospital (IRB No. 4-2014-1108). All participants provided written informed consent to allow the use of their clinical data and specimens for TB biomarker research. Peripheral blood samples were collected from all participants in sodium heparin tubes (Becton) for QFT and ICS assays ⁽²⁰⁾.

Statistical analyses were performed using GraphPad Prism 6 software, as outlined in the figure legends. The Mann–Whitney U-test was used to compare the study groups. All P-values were two-sided, with P < 0.05 considered statistically significant.

We recruited 63 healthy Korean adults aged >20 years with normal chest radiographs and no history of TB. Among those with known TB exposure, 13 tested positive in the QFT and were classified as having LTBI. Contrastingly, 12 participants without any known TB exposure tested negative for QFT and were designated as healthy controls (HCs) (Fig. 1). Additional demographic information about the participants, including sex and presence of a BCG scar, are presented in Table 1. We used a diluted whole blood ICS assay to assess the frequency of PPD-specific CD4+ T cells that produce IFN-γ, TNF-α, and IL-2 in 25 participants (Fig. 2A). The populations of CD4+ T cells producing IFN-γ, TNF-α, and IL-2 were significantly more prevalent in the LTBI group compared to the HC group (P = 0.0023, P = 0.0002, P = 0.0473; Fig. 2B). Additionally, the frequency of PPD-specific polyfunctional CD4+ T cells that produced IFN-γ, IL-2, and TNF-α was higher in the LTBI group (P < 0.0001; Fig. 2C). These findings indicate that infection with M. tuberculosis triggered an antigen-specific, polyfunctional Th-1 response in the participants.

Table 1

Baseline characteristics of the participants

	QFT-GIT (cut off 0.35 IU/mL)
	Positive	Negative
Age	42.5 ± 13.8	30.0 ± 13.1
Sex, female	16 (69.2)	11 (91.7)
BCG scar	3 (23.1)	10 (83.3)

Values are presented as mean ± standard deviation or n (%).

QFT-GIT, the QuantiFERON-TB Gold In-Tube assay.

Fig. 1. Flow chart of enrolled participants. The study included healthy adults aged >20 years with normal chest radiography results and no history of tuberculosis (TB). Among participants with known TB exposure, 13 are QuantiFERON-TB Gold In-Tube assay (QFT-GIT)-positive and identified as having latent TB infection (LTBI). Conversely, 12 participants without known TB exposure are QFT-negative and considered as healthy controls (HCs).

Fig. 2. Intracellular cytokine staining analysis in latent tuberculosis infection (LTBI) and healthy control (HC) groups. (A) Gating strategy for identifying polyfunctional CD4+ T cells. For each sample, singlets are selected, followed by excluding CD14+ and CD19+ cells. Lymphoid and CD3+CD4+ T cells are gated sequentially, and cytokine gates were applied. (B) Data show single-cytokine expression levels, including interferon-gamma (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-α), for LTBI individuals and HCs. (C) Polyfunctionality of CD4+ T cells is compared between LTBI individuals (red plots) and HCs (blue plots). *P < 0.05, **P < 0.001, ***P < 0.0001, ****P < 0.00001.

The optimal cut-off level for CD4+ T cells producing IFN-γ, TNF-α, IL-2, and polyfunctional CD4+ T cells was determined using receiver operating characteristic curve analysis (Fig. 3). The results indicate that CD4+ T cells producing IFN-γ, TNF-α, IL-2, and polyfunctional CD4+ T cells may serve as effective diagnostic markers, as there were statistically significant differences between the LTBI group and HCs. The appropriate cutoff points for the ICS assay were found to be significant for each target. Table 2 presents the overall sensitivity and specificity of CD4+ T cells producing IFN-γ, TNF-α, IL-2, and polyfunctional CD4+ T cells. Notably, polyfunctional CD4+ T cells exhibited high sensitivity (92.31%) and specificity (100.00%).

Table 2

The diagnostic utility of CD4+ T cell producing IFN-γ, IL-2, TNF-α, and polyfunctional CD4+ T cells for LTBI

ICS marker	AUC	Cut-off	Sensitivity	Specificity	P-value
IFN-γ	0.8462	>0.1315	76.92%	91.67%	0.0033
IL-2	0.9131	>0.1435	76.92%	91.67%	0.0005
TNF-α	0.7430	>0.1750	61.54%	91.67%	0.0471
Polyfunctional	0.9776	>0.0470	92.31%	100.00%	0.0001

IFN-γ, interferon-gamma; IL-2, interleukin-2; TNF-α, tumor necrosis factor-alpha; LTBI, latent tuberculosis infection; ICS, intracellular cytokine staining; AUC, area under the curve.

Fig. 3. Comparison of receiver operating characteristic (ROC) curve of (A) interferon-gamma (IFN-γ), (B) interleukin-2 (IL-2), (C) tumor necrosis factor-alpha (TNF-α), and (D) combination (INF-γ+, IL-2+, and TNF-α+).

Previous studies have assessed the cytokine profiles of CD4+ T cells and shown that cytokine responses are predominantly triggered by latency-associated M. tuberculosis antigens ^(21,22). However, the diagnostic potential of CD4+ T cell cytokines, particularly polyfunctional CD4+ T cells, for identifying LTBI remains unclear. This study has some limitations. The sample size was limited, and further research involving a larger cohort from multiple centers is required to better understand the cytokine profiles of CD4+ T cells. Further studies involving active TB patients are required to assess whether polyfunctional CD4+ T cells can effectively differentiate LTBI from active TB.

Therefore, polyfunctional CD4+ T cells are potential biomarkers for LTBI diagnosis.

None.

No potential conflict of interest relevant to this article was reported.

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning (RS-2023-00211685), BB21plus funded by Busan Metropolitan City, Busan Techno Park, and the support program for university development at the Catholic University of Pusan in 2023.