Nontuberculous mycobacterial pulmonary disease (NTM-PD) is a chronic lung infection caused by environmental mycobacteria. The clinical course of NTM-PD varies widely among patients. Some individuals remain stable for long periods without treatment, while others experience worsening lung disease that requires antibiotic therapy or may lead to death. Currently available clinical tools are limited in their ability to predict which patients will experience disease progression.

This observational study aims to better understand how the body's immune response and nutritional status are related to disease progression in adults with confirmed or suspected NTM-PD. In particular, the study focuses on T cells, a type of immune cell that plays an important role in controlling mycobacterial infections. Prior research suggests that impaired T-cell function may contribute to disease progression in NTM-PD, but most studies have relied on blood samples rather than immune cells from the lung, where the infection occurs.

In this study, immune cells obtained from bronchoalveolar lavage fluid during clinically indicated bronchoscopy will be analyzed to assess inhibitory and exhausted T-cell profiles in the lung. In addition, systemic T-cell function will be evaluated using the mitogen response from the QuantiFERON-TB Gold Plus blood test. Nutritional status and body composition will also be assessed, as poor nutrition is known to affect immune function and disease outcomes.

Participants will be followed over time as part of routine clinical care. The primary outcome of the study is the time from enrollment to the initiation of antibiotic treatment due to clinical disease progression. Secondary outcomes include identifying immune predictors of treatment initiation, examining the relationship between nutritional status and immune activity, evaluating changes in body composition and immune markers with disease progression, and determining whether immune and nutritional measures improve prediction of mortality beyond established clinical risk scores.

By integrating lung immune profiling, blood-based immune testing, nutritional assessment, and clinical data, this study seeks to improve risk stratification in NTM-PD. The results may help identify patients at higher risk for disease progression and poor outcomes, support more personalized monitoring strategies, and inform future studies targeting immune and nutritional pathways in NTM-PD.

Nontuberculous mycobacterial pulmonary disease (NTM-PD) is a chronic and heterogeneous infectious lung disease caused by a variety of environmental mycobacteria, including Mycobacterium avium complex (MAC), Mycobacterium abscessus subspecies, Mycobacterium kansasii, and other rapidly or slowly growing species. Despite increasing global prevalence, the clinical course of NTM-PD remains highly variable. Some patients experience long-term stability without treatment, whereas others develop progressive lung destruction, recurrent exacerbations, treatment failure, or death. This heterogeneity highlights limitations in current prognostic tools and underscores the need for biologically informed risk stratification.

The BACES score (Body mass index <18.5 kg/m², Age ≥65 years, Cavitary disease, Elevated erythrocyte sedimentation rate, and male Sex) is a validated clinical scoring system for mortality prediction in NTM-PD. While clinically practical, BACES relies exclusively on demographic, nutritional, inflammatory, and radiographic variables and does not incorporate direct measures of host immune function. Increasing evidence suggests that host immune dysregulation-particularly impairment of adaptive T-cell-mediated immunity-plays a central role in disease progression and may explain outcome differences among patients with similar clinical features.

Recent translational studies using lung tissue, spatial transcriptomics, and peripheral blood immunophenotyping have demonstrated a characteristic immune imbalance in NTM-PD. These studies consistently show hyperactivation of innate immune pathways (e.g., macrophage and monocyte inflammatory signaling) alongside functional suppression of T-cell-mediated adaptive immunity. This T-cell dysfunction is characterized by reduced antigen-specific interferon-gamma (IFN-γ) production, decreased co-stimulatory signaling (such as CD28 downregulation), and increased expression of inhibitory immune checkpoint receptors, including programmed death-1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3). Additionally, CD39-expressing T cells-associated with adenosine-mediated immunosuppression and terminal exhaustion-have been implicated in chronic infectious and inflammatory states.

Importantly, increased frequencies of PD-1-positive or TIM-3-positive CD4+ T cells have been associated with higher mycobacterial burden, cavitary disease, low body mass index, and adverse clinical outcomes in NTM-PD. These inhibitory and exhausted T-cell phenotypes appear to reflect chronic antigenic stimulation and impaired immune control of infection. However, most prior studies have relied on peripheral blood samples, which may not accurately represent the immune microenvironment within the lung, the primary site of disease activity.

Bronchoalveolar lavage (BAL) fluid provides direct access to immune cells within affected lung segments and offers a unique opportunity to characterize local pulmonary immune responses. BAL-derived T-cell immunophenotyping may therefore yield more clinically relevant insights into disease pathogenesis and progression than blood-based analyses alone. Nonetheless, systematic evaluation of inhibitory and exhausted T-cell subsets in BAL fluid and their association with clinically meaningful outcomes in NTM-PD remains limited.

In parallel, systemic T-cell functional capacity may influence disease trajectory. The QuantiFERON-TB Gold Plus assay includes a mitogen control that measures nonspecific T-cell interferon-gamma production. The Mitogen minus Nil value reflects overall T-cell immune reserve independent of antigen specificity. Low mitogen responses have been associated with immune suppression, severe infection, prolonged hospitalization, and increased mortality in other clinical contexts. Despite its widespread clinical use, the prognostic relevance of the QuantiFERON mitogen response has not been systematically evaluated in NTM-PD.

Nutritional status represents another key modulator of immune function. Low body mass index, reduced muscle mass, and diminished fat reserves are well-established risk factors for NTM-PD development and progression. Nutritional deficiency is also known to impair T-cell metabolism, proliferation, cytokine production, and memory formation. Thus, nutritional status may interact with immune dysfunction to influence disease outcomes. Integrating nutritional, immunologic, and clinical data may therefore enable a more comprehensive understanding of NTM-PD progression.

Study Objectives

This prospective observational study is designed to investigate whether bronchoalveolar lavage-derived inhibitory and exhausted T-cell profiles and systemic T-cell functional capacity, assessed by the QuantiFERON mitogen response, are associated with time to initiation of antimicrobial treatment due to clinical disease progression in adults with confirmed or suspected NTM-PD. Furthermore, the study aims to determine whether incorporating these immune and nutritional parameters into established clinical risk models, including the BACES score, improves prognostic discrimination for clinically meaningful outcomes, including treatment initiation and all-cause mortality.

Study Design and Population

This is a single-center, prospective observational cohort study enrolling adults aged 18 years or older with confirmed or suspected NTM-PD in whom active pulmonary tuberculosis has been excluded. Eligible participants are those undergoing bronchoscopy as part of routine clinical evaluation or disease monitoring according to established NTM management guidelines. No additional invasive procedures are performed solely for research purposes.

Patients with active tuberculosis, current malignancy requiring treatment, solid organ or stem cell transplantation, systemic immunosuppressive therapy, autoimmune disease, recent acute lower respiratory tract infection, or pregnancy are excluded to minimize confounding effects on immune function.

Sample Collection and Immunologic Assessment

Bronchoalveolar lavage is performed according to standard clinical protocols. After completion of all clinically indicated diagnostic testing, residual BAL fluid is processed for flow cytometric analysis. BAL cells are filtered, washed, and stained with a multicolor antibody panel designed to characterize T-cell lineage, activation status, regulatory features, and inhibitory and exhausted phenotypes.

Key immunologic parameters include:

• Frequencies of CD4+ and CD8+ T cells expressing PD-1, TIM-3, and CD39
• Co-expression patterns of inhibitory immune checkpoint receptors
• Regulatory T-cell populations defined by CD25^high and CD127^low expression
• Co-stimulatory marker expression (CD28)
• T-cell subset differentiation states relevant to chronic antigen exposure and immune exhaustion

Peripheral blood laboratory data are obtained from routine clinical testing. QuantiFERON-TB Gold Plus results are collected, with particular emphasis on the Mitogen minus Nil value as a measure of global T-cell functional capacity.

Nutritional and Radiographic Assessment

Nutritional status is assessed using anthropometric measurements (body mass index, waist circumference), the Mini Nutritional Assessment questionnaire, and body composition metrics derived from clinically indicated chest computed tomography. Quantitative measurements include skeletal muscle area and subcutaneous and visceral fat compartments at standardized anatomical levels using validated image analysis software.

Radiographic disease severity is evaluated using chest CT-based scoring systems that account for the extent of nodules, bronchiectasis, consolidation, and cavitary lesions. Disease phenotype (e.g., nodular bronchiectatic or fibrocavitary form) is recorded.

Follow-up and Outcomes

Participants are followed longitudinally according to routine clinical practice, typically at 3- to 6-month intervals. Follow-up evaluations include sputum mycobacterial cultures, radiographic imaging, and clinical assessments.

The primary outcome is time to initiation of antimicrobial treatment due to clinical disease progression, defined as the interval from study enrollment to the start of guideline-based therapy, as determined by the treating physician based on worsening respiratory symptoms, microbiologic findings, and/or radiographic deterioration requiring treatment.

Secondary outcomes include:

• Immune predictors of treatment initiation, including bronchoalveolar lavage-derived inhibitory and exhausted T-cell profiles and systemic T-cell functional capacity assessed by the QuantiFERON mitogen response
• Associations between nutritional status and T-cell immune activity
• Within-participant changes in body composition and T-cell immune activity between baseline and the time of disease progression requiring treatment
• Incremental prognostic value of immune and nutritional parameters beyond the BACES score for predicting all-cause mortality

Statistical Analysis

Associations between immune parameters, nutritional variables, and clinical outcomes are evaluated using appropriate univariate and multivariable statistical methods. Time-to-event analyses are performed using Kaplan-Meier methods and Cox proportional hazards models, with time to initiation of treatment due to disease progression as the primary time-to-event outcome. Logistic regression or Cox regression models are used, as appropriate, to identify independent immune and nutritional predictors of treatment initiation.

To assess prognostic performance for mortality, models incorporating BAL-derived immune variables, QuantiFERON mitogen responses, and nutritional parameters are compared with clinical models based on the BACES score alone. Discrimination and reclassification performance are evaluated using measures such as the area under the receiver operating characteristic curve (AUROC), concordance index, and reclassification metrics where appropriate.

Significance

By integrating local pulmonary immune profiling, systemic T-cell functional assessment, nutritional evaluation, and established clinical risk scores, this study seeks to advance biologically informed risk stratification in NTM-PD. The findings may help identify patients at higher risk for treatment-requiring disease progression and mortality, support personalized monitoring strategies, and provide a foundation for future interventional studies targeting immune and nutritional pathways in NTM-PD.

Inclusion Criteria:

• Adults aged 18 years or older
• Confirmed or suspected nontuberculous mycobacterial pulmonary disease
• Active pulmonary tuberculosis excluded by standard microbiologic testing
• Undergoing bronchoscopy as part of routine clinical evaluation or disease monitoring
• Able and willing to provide written informed consent

Exclusion Criteria:

• Active pulmonary tuberculosis confirmed by microbiologic testing
• Positive latent tuberculosis infection test
• Current active malignancy requiring treatment
• History of solid organ transplantation or hematopoietic stem cell transplantation
• Use of systemic immunosuppressive medications
• Diagnosis of autoimmune disease
• Use of biologic agents or planned initiation of biologic therapy
• Acute lower respiratory tract infection requiring treatment within 4 weeks prior to bronchoscopy
• Pregnancy

Participants will be recruited from adults with confirmed or suspected nontuberculous mycobacterial pulmonary disease who are scheduled to undergo bronchoscopy as part of routine clinical evaluation or disease monitoring according to established NTM management guidelines. The study population consists of patients receiving standard-of-care clinical assessment and follow-up, without any additional procedures performed solely for research purposes.

Individual participant data will not be shared publicly. The study includes sensitive clinical, immunologic, and imaging data derived from bronchoalveolar lavage samples and other patient-specific assessments, and no formal data-sharing infrastructure or governance plan has been established at the time of trial registration. However, de-identified individual participant data that underlie the results reported in peer-reviewed publications may be made available after publication upon reasonable request to the corresponding author, subject to appropriate data use agreements, institutional approval, and compliance with applicable ethical and privacy regulations.