The Trend of TIM3 Expression on T Cells in Patients With Nontuberculous Mycobacterial Lung Disease: From Immune Cell Dysfunction to Clinical Severity

Background The incidence of nontuberculous mycobacterial lung disease (NTM-LD) is increasing worldwide. Immune exhaustion has been reported in NTM-LD, but T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), a co-inhibitory receptor on T cells, has been scarcely studied. Methods Patients with NTM-LD and healthy controls were prospectively recruited from July 2014 to August 2019 at three tertiary referral centers in Taiwan. We examined TIM3 expression on the T cells from the participants using flow cytometry. TIM3 expression was analyzed for different disease statuses and after treatment. The apoptosis and cytokine profiles were analyzed according to the TIM3 expression. Results Among enrolled subjects (47 patients and 46 controls), TIM3 on CD4+ cells (6.44% vs. 4.12%, p = 0.028) and CD8+ cells (18.47% vs. 9.13%, p = 0.003) were higher in NTM-LD patients than in the controls. The TIM3 level on CD4+ and CD8+ T cells was positively associated with T-cell apoptosis in the NTM-LD patients. In stimulating peripheral blood mononuclear cells using PMA plus ionomycin, a high TIM3 level on T cells correlated with low interleukin-2 and tumor necrosis factor-alpha (TNF-α) on CD4+ cells and interferon-gamma and TNF-α on CD8+ T cells. For clinical manifestation, low body mass index (BMI), positive sputum acid-fast smear, and high radiographic score correlated with high TIM3 expression on T cells. After NTM treatment, TIM3+ decreased significantly on CD4+ and CD8+ T cells. Conclusions In patients with NTM-LD, TIM3+ expression increased over CD4+ and CD8+ T cells and correlated with cell apoptosis and specific cytokine attenuation. Clinically, TIM3+ T cells increased in patients with low BMI, high disease extent, and high bacilli burden but decreased after treatment.


INTRODUCTION
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 coreceptor 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-a and IFN-g in viral infections, mycobacterial infection, or tumors (14)(15)(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.

Patient Enrollment
This prospective study was conducted at the National Taiwan University Hospital (NTUH) and Taipei  Adult patients (≥20 years old) with at least two sputum cultures positive for the same NTM were recruited for the NTM-LD group based on the guidelines from the American Thoracic Society (ATS) (1). We excluded patients with active tuberculosis and human immunodeficiency virus infection. We included healthy controls with sputum-negative results for NTM and negative findings of NTM-LD by chest radiographic images. All final enrolled participants provided written informed consent.

Collection and Staining of Peripheral Blood Mononuclear Cells
We sampled patients' peripheral blood into heparin-containing tubes and isolated the mononuclear cells immediately using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Sweden). We then stained PBMCs for CD4, CD8, TIM3, Annexin V, and Sytox Orange. The expression was measured using flow cytometry (FACSVerse, BD Biosciences, USA). The lymphocyte population could be discriminated by forward scatter (FSC) and side scatter (SSC), and the subgroups of CD4 + and CD8 + T lymphocytes could be gated. We measured the expressions of TIM3, Annexin V, and Sytox Orange.

TIM3 Blocking Assay
Because previous studies showed that healthy controls had response to the NTM antigen (10,18,19) and we aimed to investigate the general effect of TIM3 functional blocking on apoptosis occurrence, we enrolled MAC-LD patients and the healthy controls for the assay. After we collected the blood sample and isolated PBMCs, we cultivated PBMCs in 24-well plates (5 × 10 5 cells per well) and added heat-killed M. avium bacilli (10) with MOI of 10 for 24 h with or without pretreatment of antagonistic TIM3 (10 mg/ml for 1 h) (clone F38-2E2, eBiosciences, USA). Apoptosis in the cocultured PBMCs was defined by Annexin V and Sytox Orange and was measured by flow cytometry.

Data Collection and Statistical Analysis
We recorded clinical data, radiographic findings and laboratory data at enrollment. The clinical data included age, sex, body mass index (BMI), and comorbidities. An expert interpreted chest imaging by radiographic score (20). We recorded grade of acidfast bacilli staining (AFS), which ranged from 0 to 4+, and results of Mycobacterium cultures of the sputum samples. Positive AFS indicated a high burden of NTM bacilli (21). We then classified those with low BMI, positive AFS, and high radiographic score for analysis of the role of TIM3 in disease severity.
We used the Mann-Whitney U test and Fisher's exact test for continuous and categorical variables, respectively. A p value < 0.05 indicated statistical significance in the univariate analysis. All analyses were conducted in SPSS version 19.0 (Chicago, IL).

DISCUSSION
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-a, and a lower percentage of triple-positive T cells on CD4+ cells and lower percentages of IFN-g and TNFa-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)(15)(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  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.  The scatter plots are shown with median levels. We analyzed the data using the unpaired t test. One star indicates p < 0.05; two stars mean p < 0.01.
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-a were decreased significantly, as were polyfunctional T cells. IL-2 induces proliferation and differentiation (25). By contrast, TNF-a helps lymphoid tissue development and cellular differentiation to fight against intracellular pathogens (26). For CD8+ T lymphocytes, TNF-a and IFN-g were attenuated in high expression of TIM3+ on T cells. IFN-g 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.

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

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Research Ethics Committees of National Taiwan  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.

AUTHOR CONTRIBUTIONS
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 FIGURE 6 | The expression of TIM3+ on CD4+ and CD8+ lymphocytes before and after anti-mycobacterial treatment. The intra-subject changes (before and after treatment) were compared using the Wilcoxon test. Three stars indicate p < 0.001. 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.

FUNDING
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.