The incidence of nontuberculous mycobacteria lung disease (NTM-LD) has increased over the last two decades and become an important clinical issue (1, 2). However, the etiology remains unclear (3, 4). Among NTM-LD, Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are the most predominant pathogens in North America and East Asia, and they are the two most frequently isolated species responsible for NTM-LD (4, 5). Since MAC and MAB exist ubiquitously in the environment, airway colonization with MAC or MAB is not uncommon. In fact, less than half of patients with positive sputum cultures for MAC and MAB clinically have the disease (6, 7), indicating the importance of host vulnerability to NTM pulmonary infection (8).

Patients with NTM-LD become immunocompromised through complex host–pathogen interactions. Attenuated immune response of peripheral blood mononuclear cells (PBMCs) has been reported by MAC stimulation (9, 10). However, the pathogenesis of the immune attenuation in NTM-LD has yet to be understood. Our previous report showed that programmed death-1 (PD-1), a suppressive co-receptor for T-cell activation (11), was increased on lymphocytes in patients with MAC-LD and might play an essential role in attenuating host immunity (10). However, the decreased cytokine production has not been totally reverted by in vitro PD-1 blockade (10). In regard to other immune regulators, T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), a negative regulator of T helper 1 immunity, is similar to other T-cell inhibitory receptors, such as PD-1 (12, 13). Increasing expression of TIM3 on T cells may mediate the decrease of secretion of TNF-α and IFN-γ in viral infections, mycobacterial infection, or tumors (14–16). The prognosis of double positivity of TIM3 (+) and PD-1 (+) seems worse (14, 17). However, the role of the TIM3 pathway in modulating T-cell immune responses has been scarcely studied in NTM-LD in terms of clinical manifestation and cellular function. It is important to understand the pathogenesis and immune modulation. Therefore, we conducted this study to investigate TIM3 expression in NTM-LD patients and its role in clinical and cellular effects.

The present study showed that NTM-LD patients had increased percentages of TIM3+CD4+ and TIM3+CD8+ lymphocytes regardless of age and gender. TIM3+ expression on T lymphocytes positively correlated with high Annexin V+ apoptosis. In PMA plus ionomycin stimulation, high TIM3+ expression was associated with low production of IL-2, TNF-α, and a lower percentage of triple-positive T cells on CD4+ cells and lower percentages of IFN-γ and TNFα-positive CD8+ lymphocytes. In terms of clinical characteristics, TIM3+ expression on CD4+ and CD8+ lymphocytes was higher in patients with higher bacilli burden, determined by a positive result for AFS; more disease extent, determined by radiographic score; and lower BMI than those of their counterpart groups, respectively.

TIM3 is located on the immune cell membrane and transduces inhibitory signals primarily on T cells, like other suppressive receptors such as PD-1 (12, 13). In viral infections, tuberculosis, or tumors (14–16), increasing expression of TIM3 on T cells is reportedly associated with cytokine reduction and poor prognosis (14, 17). However, the role of TIM3 has been rarely studied in NTM-LD. One Korean study showed that TIM-3-expressing T cells increased in MAC-LD patients in response to MAC stimulation and speculated that the increase might be related to attenuated cellular immunity (22). However, the mechanism leading from TIM3+ expression to immunity attenuation is unclear.

The present study also showed that TIM3+ over CD4+ and CD8+ both increased in patients with NTM-LD. After anti-NTM treatment, TIM3 expression significantly decreased, indicating that eradication of NTM might reduce TIM3 expression. At the initial stage of NTM-LD, TIM3 may have an anti-inflammatory effect. Patients with persistent NTM-LD might have subsequent TIM3 overexpression and T cell exhaustion, like the report of PD-1 in NTM-LD (10). The vicious cycle of NTM–TIM3 might be a possible pathogenesis of persistent infection of NTM-LD. However, such TIM3+ over-responsiveness is only hypothetical at present and will require future study to determine the responsible host pathogenesis.

For the possible role of TIM3 increment in NTM-LD, increasing apoptosis, inhibited proliferation, and suppression of cytokine production are the main ways that immune checkpoint receptors perform negative regulation of T cells (23, 24). Although the present study did not check lymphocyte proliferation status according to TIM3 expression, we confirmed that in NTM-LD, TIM3 expression was associated with increasing cellular apoptosis. In addition, blocking TIM3 could reverse cell apoptosis status significantly.

In regard to cytokine production from T cells, TIM3+ expression also demonstrated its role in cytokine attenuation. For CD4+ T cells, IL-2 and TNF-α were decreased significantly, as were polyfunctional T cells. IL-2 induces proliferation and differentiation (25). By contrast, TNF-α helps lymphoid tissue development and cellular differentiation to fight against intracellular pathogens (26). For CD8+ T lymphocytes, TNF-α and IFN-γ were attenuated in high expression of TIM3+ on T cells. IFN-γ upregulates pathogen recognition, processing, and activation of microbicidal effector functions (27). Overall, TIM3+-related decreased cytokine might lead to attenuation of CD4+-related proliferation and coordination, as well as CD8+-related cytotoxicity and pathogen defense.

In terms of clinical features, we found that TIM3 expression was higher in patients with high-grade AFS, indicating greater bacilli burden and severity of disease status. In cases of cellular impact by TIM3+ expression, TIM3+ might lead to decreased immunity, also known as exhaustion, which is more prevalent in patients with high AFS grade and severe NTM-LD. On the other hand, low BMI represents nutrition status and is correlated with low leptin (28), which is an upregulator for toll-like receptors (29) and a promotor of lymphocyte survival (30). Leptin deficiency might induce worse immunity upon NTM-LD and possible subsequent TIM3+ overexpression, but the details of the mechanism await further study.

Several limitations existed in this study. First, participants were enrolled at medical centers, so selection bias might exist, although patients with major underlying diseases were excluded. Second, the sample size was not large. Third, only TIM3, and no other immune checkpoints, was studied. Cross-linking between them should be studied in the future. Fourth, some analysis identified cross-sectional associations, but no causal relationships can be inferred. Determination of the direct mechanism may require future bench and animal studies. Last, the study was conducted in Taiwan, so generalization of the findings to other ethnicities and areas can be performed only after validation.

In conclusion, NTM-LD patients had higher expression of TIM3+ over CD4+ and CD8+ as compared with controls. The expression of TIM3 decreased after anti-NTM treatment. High TIM3+ might lead to cell exhaustion in NTM-LD through increased apoptosis and attenuated cytokine production. In addition, high TIM3 expression might result from a high bacilli burden, greater disease extent of NTM-LD, and lower BMI. Although many details are still understudied, we suggest that TIM3+ might be overexpressed in NTM-LD with T-cell exhaustion. Future study for TIM3 overexpression in NTM-LD is warranted.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Research Ethics Committees of National Taiwan University Hospital (NTUH IRB No. 201407079RIND, 201505069RINC, 201701014RINC and 201705087RINA), Taipei Veterans General Hospital (IRB No. 2014-09-008BC) and Far Eastern Memorial Hospital (IRB: 107162-E,109004-E). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

C-CS and M-FW were involved in performing the experiment and collecting data. S-WP, C-CS, P-HW, S-LC, M-FW, and C-YH contributed to the data analysis and manuscript writing. C-CS was responsible for the study conceptualization and coordination. All authors contributed to the article and approved the submitted version.

This study was supported in part by grants from National Taiwan University Hospital (NTUH. 109-S4552, 110-S5053, 110-T07), the Ministry of Science and Technology Taiwan (MOST 109-2628-B-075-026 and 109-2326-B-002-009-MY3), and the Far Eastern Memorial Hospital National Taiwan University Hospital Joint Research Program (108-FTN01 and 109-FTN04). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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