Characterization of Tissue-Specific Immune Responses to Bronchoscopic Instillation of Mycobacterial Antigens Into the Human Lung
Phase: EARLY_PHASE1
Conditions: Healthy, Latent Tuberculosis Infection (LTBI)
Sponsor: National Heart, Lung, and Blood Institute (NHLBI)

 
 Protocol with Statistical Analysis Plan Cover Page:   
 
Official Title: 
Tissue-Specific Immune Responses to Tuberculin Purified Protein Derivative 
(PPD) in the Human Lung 
 
NCT number: 
 
NCT05027958 
Document Type:  
Protocol with Statistical Analysis Plan (SAP)  
Document Date: 
December 17, 2024 
 

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Abbreviated Title: Tissue-Specific Immune Responses to Tuberculin Purified Protein Derivative (PPD) in the 
Human Lung  Version Date: 12/17/2024
Template version date 07-29-2019
Abbreviated Title: Tissue-Specific Immune Responses to Tuberculin Purified Protein 
Derivative (PPD) in the Human Lung
Protocol #:  21H0027
Version Date: 12.17.2024
Title: Characterization of Tissue-Specific Immune Responses to Bronchoscopic Instillation of 
Mycobacterial Antigens into the Human Lung
NIH Principal Investigator:
Kevin P. Fennelly, M.D., MPH, ATSF
Pulmonary Branch
Division of Intramural Research, NHLBI
Building 10, Rm 5-1408A 
10 Center Drive
Bethesda, MD 20892
Phone: 301-385-0807
E-mail:  kevin.fennelly@nih.gov 
Investigational Agents (if applicable):
Drug Name:
Tuberculin Purified Protein 
Derivative
IND Number:
19720
Sponsor:
NHLBI OCD
Manufacturer:
Sanofi Pasteur

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Abbreviated Title: Tissue-Specific Immune Responses to Tuberculin Purified Protein Derivative (PPD) in the 
Human Lung  Version Date: 12/17/2024
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TABLE OF CONTENTS 
TABLE OF CONTENTS ................................................................................................................2
STATEMENT OF COMPLIANCE ................................................................................................6
1
PROTOCOL SUMMARY.......................................................................................................7
1.1
Synopsis ............................................................................................................................7
1.2
Schema..............................................................................................................................8
1.3
Schedule of Activities (SOA) .........................................................................................10
2
INTRODUCTION .................................................................................................................12
2.1
Study Rationale...............................................................................................................12
2.2
Background .....................................................................................................................12
2.3
Risk/Benefit Assessment.................................................................................................13
2.3.1
Known Potential Risks ............................................................................................13
2.3.2
Known Potential Benefits........................................................................................16
2.3.3
Assessment of Potential Risks and Benefits............................................................16
3
OBJECTIVES AND ENDPOINTS.......................................................................................17
4
STUDY DESIGN ..................................................................................................................18
4.1
Overall Design ................................................................................................................18
4.2
Scientific Rationale for Study Design.............................................................................19
4.3
Justification for Dose ......................................................................................................19
5
STUDY POPULATION........................................................................................................19
5.1
Inclusion Criteria.............................................................................................................19
5.2
Exclusion Criteria ...........................................................................................................20
5.3
Inclusion of Vulnerable Participants...............................................................................21
5.4
Lifestyle Considerations .................................................................................................21
5.5
Screen Failures................................................................................................................21
5.6
Strategies for Recruitment and Retention .......................................................................22
5.6.1
Costs ........................................................................................................................22
5.6.2
Compensation ..........................................................................................................22
6
STUDY INTERVENTION ...................................................................................................23
6.1
Study Interventions(s) Administration............................................................................23
6.1.1
Study Intervention Description................................................................................23
6.1.2
Dosing and Administration......................................................................................23

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6.1.3
Dose Escalation .......................................................................................................24
6.1.4
Dose Limiting Toxicity............................................................................................24
6.1.5
Dose Modifications..................................................................................................24
6.1.6
Drug Administration................................................................................................24
6.2
Preparation/Handling/Storage/Accountability................................................................24
6.2.1
Acquisition and Accountability ...............................................................................24
6.2.2
Formulation, Appearance, Packaging, and Labeling...............................................24
6.2.3
Product Storage and Stability ..................................................................................24
6.2.4
Preparation...............................................................................................................24
6.3
Measures to Minimize Bias: Randomization and Blinding ............................................25
6.4
Study Intervention Compliance ......................................................................................25
6.5
Concomitant Therapy......................................................................................................25
7
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL .....................................................................................25
7.1
Discontinuation of Study Intervention............................................................................25
7.2
Participant Discontinuation/Withdrawal from the Study................................................26
7.3
Lost to Follow-up............................................................................................................26
8
STUDY ASSESSMENTS AND PROCEDURES.................................................................26
8.1
Screening Procedures......................................................................................................26
8.2
Efficacy Assessments......................................................................................................27
8.2.1
Clinical Evaluations.................................................................................................27
8.2.2
Biospecimen Evaluations.........................................................................................28
8.2.3
Correlative Studies for Research/Pharmacokinetic Studies ....................................28
8.2.4
Samples for Genetic/Genomic Analysis..................................................................29
8.3
Safety and Other Assessments ........................................................................................29
8.4
Adverse Events and Serious Adverse Events .................................................................30
8.4.1
Definition of Adverse Event....................................................................................30
8.4.2
Definition of Serious Adverse Events (SAE) ..........................................................30
8.4.3
Classification of an Adverse Event..........................................................................30
8.4.4
Time Period and Frequency for Event Assessment and Follow-Up........................31
8.4.5
Adverse Event Reporting.........................................................................................32
8.4.6
Serious Adverse Event Reporting............................................................................32

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8.4.7
Events of Special Interest ........................................................................................32
8.4.8
Reporting of Pregnancy ...........................................................................................33
8.5
Unanticipated Problems ..................................................................................................33
8.5.1
Definition of Unanticipated Problems (UP) ............................................................33
8.5.2
Unanticipated Problem Reporting ...........................................................................33
8.5.3
NIH Intramural IRB Reporting of IND Safety Reports...........................................33
9
STATISTICAL CONSIDERATIONS ..................................................................................33
9.1
Statistical Hypothesis......................................................................................................33
9.2
Sample Size Determination.............................................................................................34
9.3
Populations for Analyses ................................................................................................34
9.3.1
Evaluable for toxicity ..............................................................................................34
9.3.2
Evaluable for objective response.............................................................................34
9.3.3
Evaluable Non-Target Disease Response................................................................34
9.4
Statistical Analyses .........................................................................................................34
9.4.1
General Approach....................................................................................................34
9.4.2
Analysis of the Primary and Secondary Endpoints .................................................35
9.4.3
Safety Analyses .......................................................................................................35
9.4.4
Baseline Descriptive Statistics.................................................................................35
9.4.5
Planned Interim Analyses........................................................................................35
9.4.6
Sub-Group Analyses................................................................................................35
9.4.7
Tabulation of individual Participant Data................................................................35
9.4.8
Exploratory Analyses...............................................................................................35
10
REGULATORY AND OPERATIONAL CONSIDERATIONS..........................................36
10.1
Informed Consent Process...........................................................................................36
10.1.1
Consent/Assent Procedures and Documentation.....................................................36
10.1.2
Consent for minors when they reach the age of majority........................................36
10.1.3
Telephone and or Telehealth platform consent .......................................................36
10.1.4
Telephone child assent.............................................................................................37
10.1.5
Participation of Subjects who are/become Decisionally Impaired..........................37
10.2
Study Discontinuation and Closure.............................................................................37
10.3
Confidentiality and Privacy.........................................................................................37
10.4
Future use of Stored Specimens and Data...................................................................38

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10.5
Safety Oversight..........................................................................................................38
10.6
Clinical Monitoring .....................................................................................................38
10.7
Quality Assurance and Quality Control ......................................................................39
10.8
Data Handling and Record Keeping............................................................................39
10.8.1
Data Collection and Management Responsibilities.................................................39
10.8.2
Study Records Retention .........................................................................................40
10.9
Protocol Deviations .....................................................................................................40
10.9.1
NIH Definition of Protocol Deviation .....................................................................41
10.10
Publication and Data Sharing Policy...........................................................................41
10.10.1 Human Data Sharing Plan .......................................................................................41
10.10.2 Genomic Data Sharing Plan.....................................................................................41
10.11
Collaborative Agreements...........................................................................................41
10.12
Conflict of Interest Policy ...........................................................................................41
11
ABBREVIATIONS ...............................................................................................................42
12
REFERENCES ......................................................................................................................44

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STATEMENT OF COMPLIANCE
The trial will be carried out in accordance with International Conference on Harmonization Good 
Clinical Practice (ICH GCP) and the following: 
• United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 
CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812) 
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded clinical trials have 
completed Human Subjects Protection and ICH GCP Training.
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (IRB) for review and approval.  Approval of both 
the protocol and the consent form must be obtained before any participant is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study.  In addition, all changes to the consent form will be IRB-approved; a 
determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent, using a previously approved consent form.

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Abbreviated Title: Tissue-Specific Immune Responses to Tuberculin Purified Protein Derivative (PPD) in the 
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1
PROTOCOL SUMMARY
1.1
SYNOPSIS
Title:
Characterization of Tissue-Specific Immune Responses to Bronchoscopic 
Instillation of Mycobacterial Antigens into the Human Lung
Study Description:
We propose a pilot study in which we employ directed bronchoscopic 
instillation of Mycobacterium tuberculosis (Mtb)- Tuberculin Purified Protein 
Derivative (PPD) to evaluate local airway immune response in adults with or 
without latent tuberculosis infection (LTBI).   
Objectives
Primary Objective: The primary objective of this study is to understand the 
pulmonary immune response to mycobacterial antigens by determining the 
persistence of antigen-specific immune cells in the airways versus circulation 
after local bronchoscopic instillation of Tuberculin Purified Protein 
Derivative (PPD) into the lungs of adults with or without LTBI.
Secondary Objective: The secondary objective is to characterize the location 
and persistence of immune cell activity in the pulmonary parenchyma and 
thoracic lymph nodes with positron emission tomography combined with 
chest computed tomography (PET-CT) after bronchoscopic Tuberculin 
Purified Protein Derivative (PPD) instillation in adults with or without LTBI. 
Exploratory Objectives: (a) An exploratory objective will be to perform 
phenotypic and functional analysis of immune cell populations in the airways 
vs circulation following bronchoscopic instillation of Tuberculin Purified 
Protein Derivative (PPD) into the lungs of individuals with or without LTBI. 
(b) An additional exploratory objective will be to characterize the production 
of soluble mediators (including but not limited to: cytokines, lipid mediators, 
and defensins) in BAL fluid vs blood within the same context.
Endpoints:
Primary Endpoint: The primary endpoint will be the enumeration of Mtb 
antigen-specific CD4+ and CD8+ T cell populations in the airway and 
peripheral blood at early and late time points after bronchoscopic Tuberculin 
Purified Protein Derivative (PPD)instillation.
Secondary Endpoint: The secondary endpoint will be the quantification of 
[18F]fluoro-D-glucose (FDG) uptake in adjacent pulmonary parenchyma 
and draining thoracic lymph nodes via PET-CT at early and late time points 
after PPD instillation. 
Exploratory Endpoints: (a) An exploratory endpoint will be the 
quantification of frequencies of cell surface molecules, intracellular cytokines 
and transcription factors among immune cell populations in the airways vs 
circulation following bronchoscopic instillation of Tuberculin Purified 
Protein Derivative  (PPD) into the lungs of individuals with or without LTBI.  
(b) An exploratory endpoint will involve the  identification and quantification 
of soluble mediators in the airway and peripheral blood after PPD instillation 
in the lungs within the same context.
Study Population:
Up to 100 male and female subjects will be screened in order to generate a 
Sample size: 13 confirmed latent tuberculosis infection (LTBI) cases and 7 
confirmed non-LTBI controls
Gender: male or female
Age: 18 – 64 years of age
Demographic group: no exclusions

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General health status: healthy volunteers (non-LTBI controls vs confirmed 
LTBI)
Phase:
Not applicable
Description of 
Sites/Facilities 
Enrolling 
Participants:
NIH Clinical Center: Procedures, Vascular Access and Conscious Sedation 
(PVCS) suite, Building 10; Radiology and Imaging Sciences Suite, Building 
10; Laboratories of Daniel Barber and Katrin Mayer-Barber, Building 33
Description of Study 
Intervention:
Bronchoscopic instillation of 0.5 tuberculin units (10 microliters) of 
Tuberculin Purified Protein Derivative  (PPD)(TUBERSOL®, Sanofi 
Pasteur Inc., Swiftwater, PA) diluted in 10 cc normal saline into the 
anterior segment of the right upper lobe. 
Study Duration:
5 years 
Participant 
Duration:
30 days
1.2
SCHEMA
General Schema Outline

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Screening Day 1
Screening Day 2
Visit 1
 
Visit 2 
Time: Day 0
Visit 3
Time: Day 2-6
Visit 4
Time: Day 3-7
Visit 5
Time: Day 15-25
Visit 6
Time: Day 16-26
Total N:  Obtain informed consent. Screen potential participants by inclusion and exclusion criteria; obtain history, 
physical, TST, IGRA, CMP, CBC, PT/ PTT, INR, ESR, CRP and serum for PBMC analysis, SARS CoV-2 PCR, 
EKG, CXR
Research blood collection; electrocardiogram (EKG)
Instillation bronchoscopy performed: BAL of LUL superior lingular segment (baseline), followed by saline instillation 
into anterior segment of LUL (control); BAL of RML medial segment, followed by PPD instillation into anterior 
EKG, Review of Meds, VS, Physical Exam, Pregnancy test
Collection bronchoscopy performed: BAL of anterior segment of LUL (saline control segment), BAL of anterior 
segment of RUL (PPD challenge segment)
Review of Meds, VS, Physical Exam, Pregnancy test, Chemistry/Hematology labs, Post-intervention PET-CT 
performed: SUV of FDG-avid areas calculated and compared to pre-intervention data
Final Assessments
Review of Meds, Physical Exam, VS, Repeat blood 
collection, IGRA and EKG performed: as above
Repeat BAL collection by bronchoscopy performed: as 
above
Arm 1: 
13 LTBI Patients 
TST+ IGRA+
Blood collection performed: CMP, CBC, PT/PTT, INR,  ESR, CRP and serum for PBMC analysis
Pre-intervention PET-CT performed: Baseline SUV of any FDG-avid areas calculated 
Group selection based on TST and IGRA status
Arm 2: 
7 non-LTBI Patients 
TST- IGRA-
Review of Meds, Physical Exam, Chemistry/Hematology, Pregnancy test Post-intervention PET-CT repeated: SUV of 
FDG-avid areas calculated and compared to Day 4 and pre-intervention data
Total N:  Have participants return for reading of TST and assessment of IGRA result. CMP, CBC, PT/PTT, INR,  
ESR, CRP and serum for PBMC analysis CXR, & EKG (can also be completed either screening day)  to rule out 
active disease, and or lung infection. 
     Telephone 
Follow-up Day 30

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1.3
 SCHEDULE OF ACTIVITIES (SOA)
Schedule of Activities (SOA)EVALUATION 
AND ASSESSMENTS 
Screen*
Baseline 
Study Visits 3 to 6
Telephone 
Follow- up
Day 30
PROTOCOL TIMEPOINT
 
V1
V2
V3
V4**
V5
V6**
Window +/- days 
-60 Days
-7 Days 
 
-2 
/+3 
D
 
-5  
/+6D
 
day /month
 
 
D0
D4
D5
D20 
D21
Assessments 
 
 
 
 
 
 
 
Review Inclusion/Exclusion Criteria 
X
 
 
 
 
 
 
Consent to study 
X
 
 
 
 
 
 
Review Medical History with Mtb Exposure
x1
 
 
 
 
 
 
Review of Medications
X
X
X
X
X
X
X
Physical Examination 
X
X
X
X
X
X
X
Weight, Height, and BMI
X
Vital Signs (BP, HR, Resp, Temp O2  Saturation)
X
X
X
X
X
Adverse Event Review 
 
X 
X
X
X
X
X
X
CHEMISTRY LABS
 
 
 
 
 
 
 
Female, Pregnancy Test (blood or urine) 2
X
X
X
X
X
X
X
Acute Care Panel
X
X
X
X
Mineral Panel 
X
X
 X
X
CRP
X
X
X
X
Hepatic Panel 
X
X
 X
X
LDH 
X
X
 X
X
International Normalized Ratio (INR), Prothrombin Time  (PT), and 
Partial Thromboplastin Time PTT
X3
X3
 X3
X3
COVID-19 (PCR)
X
HEMATOLOGY LABS
 
 
 
 
 
 
 
CBC with differential
X
X
 X
X 
ESR
X
X
X
X
Viral Serologies
 
 
 
 
 
 
 
Hepatitis B, C
X
 
 
 
 
 
 
HIV
X
 
 
 
 
 
 
Additional Tests
 
 
 
 
 
 
 
*QuantiFERON Gold Test (IGRA) 
X
 
 
 
 
 
X
*Tuberculin skin test (TST) 
X4
 
 
 
 
 
 
EVALUATIONS 
 
 
 
 
 
 
 
EKG
X
 
X
 
X
 
X
*Chest X-RAY   
X
 
 
 
 
 
 
Chest PET-CT
 
X
 
X
 
X
 
PROCEDURES 
 
 
 
 
 
 
 
Bronchoscopy 
 
 
X3
 
X3
 
X3

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* = Medical history screening activities may be done utilizing the Telehealth Visit Platform. Screening 
tests may be used as baseline if performed within 60 days. If re-screened after 60 days, the TST, IGRA, 
& chest x-ray will not be repeated unless there is a known tuberculosis exposure, including but not 
limited to travel to an endemic area or direct contact with an individual with known or 
suspected tuberculosis disease.
x1 = History of exposure to persons with active TB; History of residence in a TB-endemic country and 
Estimate of time when LTBI most likely occurred
 x2 = A plasma or urinary HCG in female subjects who are of child-bearing age will be collected within 7 
days of study participation. While on study women of child-bearing age will have pregnancy test at 
each visit unless test has been performed within 24 hours of study visit
x3 = Patient may not use platelet inhibitors (e.g. clopidogrel) within 7 days or systemic anticoagulants 
(e.g. warfarin, enoxaparin, or DOAC) within 14 days of bronchoscopy.
X4 = Tuberculin skin test (TST) to placed prior to screening labs. A second TST may be completed per PI 
or designee if needed to confirm negative results one to three weeks after the first TST.
** = Bronchoscopy for visit 4 and visit 6 will be performed within 2 days after the PET-CT
STUDY MEDICATION 
 
 
 
 
 
 
 
Installation of  Tuberculin Purified Protein Derivative (PPD) and saline 
 
 
X
 
 
 
 
Removal of Tuberculin Purified Protein Derivative (PPD) and saline cells 
 
 
 
 
X
 
X
Research Samples 
 
 
 
 
 
 
 
Immunology Assays = 40 mL via heparinized green top tubes 
 
 
X
X
X

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Abbreviated Title: Tissue-Specific Immune Responses to Tuberculin Purified Protein Derivative (PPD) in the 
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2
 INTRODUCTION
2.1
STUDY RATIONALE
Most of our understanding of human specific CD4 T cell responses to Mycobacterium 
tuberculosis (Mtb) is based on studies of circulating peripheral blood cells, and less is known 
about mycobacteria-specific CD4 T cells within the airways and alveoli. Recent studies in 
experimental mouse and macaque models by co-investigator Daniel Barber and colleagues have 
shown that this is a critical issue limiting our ability to understand protective cellular immune 
responses to Mtb (1-3). These functional differences between airway and blood T cells are 
poorly understood in humans within the context of tuberculosis infection (4). Furthermore, 
previous limited characterizations of the human airway immune cell phenotypes in the context of 
TB do not consistently correlate with mice and macaque models of Mtb pulmonary disease (5,6). 
For that reason, this proposal aims to address the gap in our knowledge of human Mtb airway 
immune responses not fully elucidated by animal models.
Additionally, the timing and location of early human immune responses to inhalation of M. 
tuberculosis are poorly understood. Data from animal studies have demonstrated that the 
mediastinal lymph nodes are the crucial primary site for the development of the adaptive antigen-
specific CD4 T cell activation post aerosol exposure to Mtb (7,8). Additional work has 
demonstrated that intralymphatic administration of Mycobacterium bovis-derived vaccine 
provides improved protection from Mtb infection in mice as compared to intradermal and 
subcutaneous routes, reinforcing the importance of the thoracic lymphatic network for the 
establishment of protective immunity to Mtb (9).
However, there are few data in humans that shed light on the CD4 T cell amnestic response 
in the lung tissue vs draining mediastinal lymph nodes in the context of Mtb exposure. It is 
unclear whether this process is solely driven by antigen-specific T cells which mobilize to the 
site of inflammation over time or by a population of resident antigen-nonspecific effector T cells 
which are recruited to inflamed lung tissue based on a chemokine gradient. In a small pilot study 
of household contacts of patients with active pulmonary TB who were recently diagnosed with 
latent TB infection, we observed increased cellular activity of the thoracic lymph nodes using 
positron emission tomography combined with chest tomography (PET-CT), in spite of otherwise 
normal appearing lung parenchyma and lymph nodes on CT (10). 18F-FDG PET-CT imaging is 
emerging as a very sensitive method for visualizing thoracic inflammation (11-13). For these 
reasons, longitudinal PET-CT images will provide critical non-invasive evaluations of cellular 
activity within the human lung parenchyma and draining mediastinal lymph nodes after exposure 
to Mtb antigen that provide insight into the location and timeline of anamnestic CD4 T cell 
immune response development.
2.2
BACKGROUND 
Tuberculosis is caused by the inhalation of airborne Mtb bacilli.  In animal models, 
alveolar macrophages are the first to engulf the bacilli, but eventually Mtb spreads to and grows 
within several other myeloid cell populations in the lungs (14,15). Approximately 1 to 2 weeks 
after initial infection, viable bacteria are transported via lymphatics to the draining hilar and 
mediastinal lymph nodes (16,17), and adaptive immune responses are then initiated (18). 
Recently, the use of PET-CT enabled us to demonstrate that there is early cellular activity in the 

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thoracic lymph nodes of recent household contacts of patients with active TB (10), but the early 
events of human Mtb infection remain poorly understood.
The treatment of active TB disease caused by drug-susceptible strains requires 4 
antimycobacterial drugs for the first 2 months, followed by 4 months of 2 drugs.  In the case of 
multidrug-resistant TB (MDR-TB), treatment requires more toxic drugs given for at least 18 
months and usually for 24 months or longer.  Extensively drug-resistant TB (XDR-TB) is often 
fatal in most of the world. In Cape Town, South Africa, where treatment is available, it is only 
successful in about 10-20% of cases (19). Thus, there is a pressing need for interventions to 
prevent infection or progression of disease. Vaccination with an attenuated strain of M. bovis 
BCG has been used for decades, but it is only useful in preventing disseminated disease, 
especially TB meningitis in infants.  It does not prevent pulmonary TB, which accounts for over 
85% of cases and is the form responsible for ongoing person-to-person airborne transmission.
Control of Mtb infection requires CD4 T cells, and if vaccination for Mtb is to be 
successful in the future, it will most likely be through the induction of CD4 T cell responses in 
the lungs. However, most of our understanding of human Mtb-specific CD4 T cell responses is 
based on studies of circulating peripheral blood cells, and little is known about mycobacteria-
specific CD4 T cells in the airways. In animal models of tuberculosis, the Barber lab has shown 
that there are major differences between the T cells that enter the lungs vs those in circulation (1-
3, 20). The primary purpose of this protocol is to study CD4 T cell responses in the human lung 
using bronchoscopic instillation of mycobacterial antigens in the form of tuberculin PPD, an 
established experimental approach that mimics exposure to inhaled Mtb.  This method was first 
pioneered and proven safe by Silver and colleagues (21-23).  Silver and colleagues have 
performed a limited immunological analysis on airway immune cells at day 2 post-instillation. 
Furthermore, this group routinely instilled PPD or control normal saline into the right middle 
lobe or lingula, the equivalent middle lung region on the left, where pulmonary TB rarely occurs. 
The major modifications of our approach is that we will perform a more comprehensive 
immunophenotyping of both peripheral and airway immune cells following PPD challenge at 
both early and late time points while evaluating local parenchymal and lymph node immune cell 
activity via PET-CT. Additionally, we will study PPD instillation and responses in the upper lobe 
of the lung, where most HIV-negative adults with pulmonary TB develop disease.
2.3
RISK/BENEFIT ASSESSMENT
2.3.1
Known Potential Risks
Risks Related to Tuberculin Purified Protein Derivative (PPD):  
PPD using the Mantoux method is the current standard for tuberculin skin tests (TSTs).  Rarely, 
patients may develop a necrotic skin reaction at the site of intradermal injection.  Screened 
subjects will be excluded from participating if they have a history of such skin reactions. 
The model of direct lung segment challenge with PPD was established at the Case Western 
Reserve University School of Medicine as discussed above. It has subsequently been used in 
multiple subsequent studies by this research group (21-24).  It results in a localized inflammatory 
response that is limited to the challenged sub-segment.  None of these studies have reported 
serious or unexpected side effects with this model.  

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Risks Related to Blood Collection:  Blood collection may cause some pain or bruising at the site 
on the arm from which the blood was drawn.  There is a small possibility of fainting and 
infection.  Similarly, there is a small risk of bleeding, blockage, or inflammation or infection of 
the vessel.  Discomfort does not usually last long and permanent damage is extremely rare.
Risks Related to Bronchoalveolar Lavage (BAL):  Subjects will sign a separate procedure 
consent for the BAL, which will further detail the potential risks of this procedure which include:
•
Discomfort due to coughing. This can be controlled by topical medication.
•
A decrease in the amount of oxygen in the blood.  Subjects will receive additional oxygen 
during the procedure and they will be closely monitored for oxygen levels, heart rate and 
blood pressure.  The risk of a serious problem occurring is very small (less than 1 out of 
10,000 procedures in published studies).
•
A vagal response may result in a transient slow heartbeat occurs (rare). These are self-
limited and do not require medication. A heart rhythm monitor (electrocardiograph) will 
be used throughout the procedure.
•
Mild bleeding from the nose can occur because the bronchoscope tube is sometimes 
placed through the nose to reach the large airways.  Placing medication and lubricant 
inside the nose will be done before the procedure to lower the risk of this occurring.  
Alternatively, the bronchoscope can be introduced through the mouth.
•
A sore throat may be present for several hours after the procedure (less than 10% of 
patients). This can be treated with topical anesthetics (e.g. throat lozenges)
•
A reaction to the topical anesthetics (i.e. lidocaine, tetracaine or cetacaine) occurs that 
may include confusion or, very rarely, seizures. This potential problem is minimized by 
using small, frequent doses of the medication.
•
Fever has been reported to develop in less than 5% of healthy volunteers six to eight 
hours after the bronchoscopy.  In participants who develop a fever after bronchoscopy, 
the symptoms can be ameliorated with acetaminophen.
•
Infection in the lungs resulting from aspirating gastric contents into the lung airways.  To 
minimize this risk, subjects do not eat or drink for at least six hours before the 
bronchoscopy. There is also a risk of aspiration in the post procedure period during which 
a patient is recovering from effects of sedative administration. For this reason, each 
subject will be monitored closely within the procedural unit until the level of conscious 
has returned to baseline. 
•
With the use of conscious sedation there is a minor risk of change in baseline respiratory 
status (hypoxia, respiratory depression) or cardiovascular complications (hypotension, 
dysrhythmias). Midazolam and Fentanyl are the medications that will be used for 
conscious sedation. With the use of midazolam there is minor risk of vomiting and 

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decreased respiratory rate. With the use of fentanyl there is a minor risk of abdominal 
pain, constipation, nausea and vomiting. Each study subject will be informed of this risk 
and presented with the alternative of undergoing this procedure with the use of local 
anesthesia alone. 
Risks Related to Chest PET-CT scans:  Subjects will sign a separate procedure consent 
for PET CT scans, which will further detail the potential risks of this procedure which 
include:
•
Radiation Risks: This study involves exposure to radiation from the Chest X-ray, and 
PET-CT scan and 18F radioisotope. At doses much higher than received in this study, 
radiation is known to increase the risk of developing cancer. Based on the doses of 
radiation planned for this study, the risk of inducing a neoplastic process is low. Each 
subject is planned to receive a total of  0.9 Roetgen equivalent man (REM) for each of the 
chest PET-CT Scan, thus receive a total of  2.7 REM during the study. Subjects will 
receive an additional 0.001 REM for the screening Chest X-ray, thus receive a total of 
2.71 REM during the study.  This is below the 5 REM annual dose limit recommended by 
the United States Nuclear Regulatory Commission (56 FR 23396). Each study participant 
will be informed of the magnitude of their radiation exposure and informed of their risk 
of exceeding the recommended annual dose limit if they receive additional significant 
radiation exposure outside of the study. A radiation exposure tracking tool, created by 
study personnel, will be completed to capture any radiation exposures in the prior 12 
months at the time of enrollment and any additional outside exposures between study 
visits. This tracking tool may be completed via a Excel document and or utilizing 
radiation calculator tool via https://www.xrayrisk.com/calculator/calculator-normal-
studies.php.
•
Allergy Risk: There is a risk of allergic or other adverse type reaction to PET-CT 
radiotracers. This is extremely rare. Fluorodeoxyglucose (FDG) is a natural sugar that is 
attached to the radiotracer. There have not been any allergic reactions reported to FDG 
alone in the past. However, if a patient were to develop an allergic reaction, treatment 
would be initiated immediately with diphenhydramine, ranitidine, and/or corticosteroids 
depending on the severity of the reaction.
•
Risk of Incidental Findings: Unanticipated clinically insignificant or potentially 
significant abnormalities may be detected from the results of the PET-CT scans 
performed. Such abnormalities due incur the risk of future potentially unnecessary 
additional diagnostic testing or therapeutic intervention, which can be associated with 
various complications.
•
Reproductive Risks: Since PET-CT scans involve exposure to radiation which can lead to 
teratogenic effects to a fetus. For this reason, women of child-bearing age will undergo 
pregnancy testing prior to PET-CT scan and be excluded from the study if testing is 
found to be positive. 

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2.3.2
Known Potential Benefits
There are currently no known direct immediate benefits of this study for individual subjects. 
There is the potential benefit of identification and subsequent treatment of latent tuberculosis 
infection in a subject without prior knowledge of this infection prior to this study. However, this 
study has the major benefit that this study will provide important data relevant to the 
development of TB vaccines, diagnostics, and host directed therapies, as well as basic 
understanding of human lung immunity.  For example, we expect to see different immune 
responses in those with LTBI versus those without.  If this is confirmed, it would suggest that 
strategies for vaccination should differ for those with and without LTBI.   Similarly, if we 
confirm that there is early cellular activity in the thoracic lymph nodes, it would be reasonable to 
develop drugs for early infection that target the thoracic lymph nodes, such as those in 
nanoparticles. 
2.3.3
Assessment of Potential Risks and Benefits
Rationale for exposing study participants to the risk of diagnostic bronchoscopy and 
bronchoalveolar lavage: As stated above the functional differences between airway and blood T 
cells are poorly understood in humans within the context of tuberculosis infection. Additionally, 
limited evaluations of the human airway immune cell phenotypes in the context of TB do not 
consistently correlate with mice and macaque models of Mtb pulmonary disease. For these 
reasons, in order to obtain direct characterization of the human airway immune response to this 
pathogen transmitted via aerosol exposure we propose the aforementioned bronchoscopic 
instillation model. The risks of bronchoscopic instillation and bronchoalveolar lavage to 
participants will be minimized by the following means:
•
Excluding participants with a past medical history or current medical issues which 
place them at risk of a decline in their baseline respiratory function during 
bronchoscopy
•
Use of M. tuberculosis protein derivative instead of active or inactivated bacilli in 
order to make infectious risk to participant negligible
•
Use of pulmonary arterial catheter for direct bronchial challenge, limiting lobar 
dissemination of PPD 
•
Absence of performance of bronchial or pulmonary parenchymal biopsy sampling 
to limit the risk of procedure-related pneumothorax
•
Excluding current use or inability to suspend use of anticoagulant including 
platelet inhibitors (e.g. clopidogrel)  within 7 days of bronchoscopy or systemic 
anticoagulants (e.g. warfarin, enoxaparin, DOAC) within 14 days of 
bronchoscopy 
•
Rationale for exposing study participants to the risk of PET-CT imaging: As 
stated above there are few data in humans that shed light on the CD4 T cell 
amnestic response in the lung tissue vs draining mediastinal lymph nodes in the 
context of Mtb exposure. PET-CT imaging is a very sensitive and non-invasive 
method for visualizing thoracic inflammation. For these reasons, longitudinal 
PET-CT images will provide critical non-invasive evaluations of cellular activity 
within the human lung parenchyma and draining mediastinal lymph nodes after 
exposure to Mtb antigen. In order to obtain evaluation of thoracic inflammation 

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over time, it will be optimal to perform PET-CT imaging prior to Mtb challenge, 
at an early time point and a late time point post challenge. The risks of evaluation 
of thoracic inflammation via repeat PET-CT imaging to the participant will be 
minimized by the following means: 
•
Absence of invasive lymph node sampling via endobronchial ultrasound and 
transbronchial biopsy
•
Limitation of the imaging field to include only the thorax and thus reduce overall 
radiation exposure
3
OBJECTIVES AND ENDPOINTS
OBJECTIVES
ENDPOINTS
JUSTIFICATION FOR 
ENDPOINTS
Primary
The primary objective of this study 
is to understand the pulmonary 
immune response to mycobacterial 
antigens by determining the 
persistence of antigen-specific 
immune cells in the airways versus 
circulation after local 
bronchoscopic instillation of PPD 
into the lungs of adults with or 
without LTBI.
The primary endpoint will be the 
enumeration of Mtb antigen-specific 
CD4+ and CD8+ T cell populations in 
the airway and peripheral blood at 
early and late time points after 
bronchoscopic PPD instillation.
This endpoint is chosen 
to determine the point 
after directed 
bronchoscopic 
exposure to Mtb 
peptide the antigen-
specific T cell response 
is strongest both locally 
in the lung and 
systemically in the 
blood.
Secondary
The secondary objective is to 
characterize the location and 
persistence of immune cell activity 
in the pulmonary parenchyma and 
thoracic lymph nodes with positron 
emission tomography combined 
with chest computed tomography 
(PET-CT) after bronchoscopic PPD 
instillation in adults with or without 
LTBI. 
The secondary study endpoint of this 
study is the quantification of FDG 
SUV throughout all lung fields and 
mediastinal lymph nodes of LTBI and 
non-LTBI patients at days -7, +4, and 
+20 after PPD bronchoscopic 
instillation. 
This endpoint is chosen 
in order to identify and 
track over time the 
geographically 
anatomic distribution of 
immune cell activity in 
the lungs after PPD 
instillation. 
Exploratory
The exploratory objectives of this 
study are (a) to perform phenotypic 
and functional analysis of immune 
cell populations in the airways vs 
circulation following bronchoscopic 
instillation of PPD into the lungs of 
individuals with or without LTBI, 
(b) to characterize the production of 
soluble mediators (i.e. cytokines, 
lipid mediators, defensins) in BAL 
(a) An exploratory endpoint will be 
the quantification of frequencies of 
cell surface molecules, intracellular 
cytokines and transcription factors 
among immune cell populations in the 
airways vs circulation following 
bronchoscopic instillation of PPD into 
the lungs of individuals with or 
without LTBI. (b) An exploratory 
endpoint will involve the 
These endpoints are 
chosen to characterize 
the cellular phenotype 
and function of the 
most influential 
immune cell 
populations within the 
context of pulmonary 
tuberculosis in the local 
lung micro-

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OBJECTIVES
ENDPOINTS
JUSTIFICATION FOR 
ENDPOINTS
fluid vs blood within the same 
context.
identification and quantification of 
soluble mediators in the airway and 
peripheral blood after PPD instillation 
in the lungs within the same context.
environment as 
compared to 
systemically at 
empirically selected 
early and late time 
points post Mtb antigen 
exposure.   
4
STUDY DESIGN
4.1
OVERALL DESIGN
Hypothesis 
The major hypothesis of this single-site pilot study is that recruitment of antigen-specific T cells 
and innate immune cell subsets following bronchoscopic instillation of purified protein 
derivative (PPD) from M. tuberculosis  into the lung will occur primarily in individuals with 
latent TB infection. These Mtb-specific T cell responses will occur only in the segments into 
which PPD was instilled and not in the control lung segments. Lastly, pulmonary immune cell 
activation and number will peak early after PPD instillation and subsequently wane both in the 
airways and peripheral blood. These responses will be much more pronounced in the local 
pulmonary micro-environment as opposed to the systemic vasculature. 
Using PET-CT imaging after PPD instillation, we hypothesize that it will be possible to identify 
increased cellular activity in draining thoracic lymph nodes, as well as in the pulmonary 
parenchyma. Furthermore, we expect that the above signals we are able to discern will 
progressively wane in the weeks following bronchoscopic PPD instillation.
Longitudinal Study Design
We plan to screen up to 100 non-LTBI controls vs confirmed LTBI to enroll up to 25 individuals 
in order to have a total of 20 individuals who are concordant on the PPD and IGRA test complete 
the study as follows:
Group A (LTBI Group): 13 PPD + and IGRA + subjects
Group B (non-LTBI Group): 7 PPD - and IGRA - subjects
We seek to enroll individuals at a 2:1 ratio of Group A:Group B in order to maximize the data 
derived from individuals with latent TB who will mount antigen-specific T cell responses post 
PPD instillation. This strategy will also decrease variance in Group A via relative oversampling 
compared to Group B.
Figure 1 outlines the experimental timeline. A baseline PET-CT study of the thoracic cavity will 
be obtained, and the first bronchoscopy will be performed in the following the week to collect 
the baseline BAL sample and to instill the PPD as outlined above.  Peripheral blood will be 
collected at the time of each bronchoscopy procedure before the BAL.  To examine early 
immune responses, on approximately day 4 the second PET-CT study will be obtained and the 
following day a second BAL will be performed, and peripheral blood samples will be collected.  
BAL will be done first in the control anterior segment of the left upper lobe and then again in the 

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anterior segment of the right upper lobe. To examine the persistence of this local pulmonary 
immune response, the next PET-CT image will be obtained on approximately day 20, and the 
following day 21, repeat blood and BAL samples will be collected as done previously. A repeat 
IGRA test will be collected on the final visit day of the study to assess for any change in the 
participant’s initial result. 
Figure 1. Experimental schematic.
4.2
SCIENTIFIC RATIONALE FOR STUDY DESIGN
Our study design is similar to that of Silver and colleagues in that we are proposing the 
evaluation of local airway responses in small groups of non-LTBI controls and confirmed LTBI 
cases. However, we anticipate that our immunological signals will be stronger due to our 
confirmation of LTBI status using concordant positive TST and positive IGRA tests, and 
similarly our confirmation of the status of controls without LTBI using concordant for negative 
TST and negative IGRA tests. In addition, we will be studying responses to antigen challenges in 
the upper lobes where most humans develop pulmonary TB, rather than the middle lobe as 
studied by Silver and colleagues. Furthermore, the novelty of our approach will involve the 
combination of immunological assays after bronchoscopic instillation of antigens directed to 
specific lung segments and PET-CT scanning to determine the location and kinetics of responses 
in the draining lymph nodes.  The PET-CT scanning component has the potential to provide the 
first localization of immunological challenge with non-infectious Mtb antigens in humans.  
4.3
JUSTIFICATION FOR DOSE
The PPD bronchoscopic instillation dose of 0.5 tuberculin units (10 uL Tubersol® in 10 mL 
normal saline) was determined by Silver and colleagues in 2003. They demonstrated that the 
minimally reactive dose of PPD administered through the airways to elicit the development of 
alveolar inflammation was a challenge dose of 0.5 TU or 1/10th of the standard TST dose (21).
5
STUDY POPULATION
Eligibility will be determined per two screening visits either in person or via a Telehealth visit. 
Alternatively, if a test result is available from any other NIH protocol within 60 days of 
enrollment, that test result can also be used for screening purposes. 
5.1
INCLUSION CRITERIA
Adults 18-64 years old will be recruited for the two groups (non-LTBI controls vs confirmed 
LTBI).  The lower limit of this age range is based on the need for invasive bronchoscopic 

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procedures and exposure to radiation, both of which carry more risk at younger ages. In addition, 
most adults present with post-primary or reactivation TB that most often occurs radiographically 
in the upper lobes of the lungs, often with cavitation.  Conversely, children and rare adults with 
primary TB have non-cavitary disease in the lower lobes.  The higher limit of this age range is 
based on the known property of immune senescence, i.e., the waning of the strength of immune 
responses with advancing age. 
In order to be eligible to participate in this study, an individual must meet all of the following 
criteria:
1. Ability of subject to understand and the willingness to sign a written informed consent 
document.
2. Stated willingness to comply with all study procedures and availability for the duration of 
the study 
3. Male or female, aged 18 – 64 years of age
4. No significant active medical problems. This would include but not limited to any cardiac 
disorder (e.g. arrhythmia, valvular disease), pulmonary disease (e.g. asthma requiring 
chronic medications, chronic bronchitis, emphysema, obstructive sleep apnea), kidney 
disease (e.g. nephritis, nephrosis), rheumatologic disorder (e.g. inflammatory arthritis), 
endocrine disorder (e.g. diabetes, thyroid disease), liver disease (e.g. hepatitis), 
gastrointestinal disorder (e.g. inflammatory bowel disease) or infectious disease (e.g. 
active tuberculosis). 
5. For females of reproductive potential: use of highly effective contraception for at least 1 
month prior to screening and agreement to use such a method during study participation 
6. For males of reproductive potential: use of condoms or other methods to ensure effective 
contraception with partner and agreement to use such a method during study participation
5.2
EXCLUSION CRITERIA
An individual who meets any of the following criteria will be excluded from participation in this 
study:
1. Pregnancy as assessed by urinary or plasma HCG or breastfeeding
2. History of clinically significant respiratory dysfunction 3 months prior to participating
a.
Evidence of new pulmonary infection 
b. History of any chronic lung infections or chronic lung disease 
c.
History of pulmonary hypertension
d. Need for supplemental oxygen administration at rest
3. Current use or inability to suspend use of anticoagulant therapy including platelet 
inhibitors (e.g. clopidogrel) within 7 days of study bronchoscopy or systemic 
anticoagulants (e.g. warfarin, enoxaparin, or DOAC) within 14 days of study 
bronchoscopy 
4. Any symptoms consistent with infection including fever, chills, night sweats, or 
unexplained weight loss
5. A history of a necrotic reaction to a tuberculin skin test, including during screening
6. A history of human immunodeficiency virus (HIV) infection
7. A history of coughing up blood in the last 3 months
8. Cigarette smoking, vaping or recreational drug use within the past 6 months (self- 
reported)

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9. If there is any discrepancy in tuberculin skin test and Interferon Gamma Release Assay 
test results (i.e. PPD+ but IGRA- or PPD- but IGRA+)
10. Refusal or inability to undergo bronchoscopy, or a history of poor tolerance of a 
bronchoscopy
11. If undergoing PET-CT imaging during this study places a participant over his/her annual 
radiation dose limit. Radiation exposure within the previous 12 months of > 2.3 rem
12. BMI > 40
13. Diabetes
14. Known life-threatening allergic reaction to Tuberculin, Lidocaine, Midazolam, Fentanyl 
or medications of similar classes
15. Presence of any immunosuppressive diseases, including cancers with the exception of 
non-melanomatous skin cancer. 
16. Use of any systemic immunosuppressive medications, including corticosteroids (e.g., 
prednisone) or biological agents in the last 6 months prior to enrollment
17. Any medical, psychiatric, social condition, occupational reason or other responsibility, in 
the judgement of the investigator, that is a contraindication to protocol participation or 
impairs a participant’s ability to give informed consent
18. Positive for COVID-19. (with-in 6 months prior to enrollment self-reported and or via 
PCR)
5.3
INCLUSION OF VULNERABLE PARTICIPANTS
NIH employees and members of their immediate families may participate in this protocol. We 
will follow the Guidelines, per Policy 404, for the Inclusion of Employees in NIH Research 
Studies and will give each employee a copy of the “NIH information sheet on Employee 
Research Participation”.
5.4
LIFESTYLE CONSIDERATIONS
Not applicable. 
5.5
SCREEN FAILURES
Screen failures will be defined as participants who consent to participate in the clinical trial but 
are not subsequently assigned to the study intervention or entered in the study. A minimal set of 
screen failure information is required to ensure transparent reporting of screen failure 
participants, to meet the Consolidated Standards of Reporting Trials (CONSORT) publishing 
requirements and to respond to queries from regulatory authorities. Minimal information 
includes demography, screen failure details, eligibility criteria, and any serious adverse event 
(SAE).
Individuals who do not meet the criteria for participation in this trial (screen failure) because of 
an evidence of active clinical illness, or radiation exposure within the last 12 months of  > 2.3 
rem, at the time of screening may be rescreened. Subject’s may be eligible to be rescreened to 
participate at a later date if at the time of enrollment they are found to have an active infection, 
vaccination, and or have been exposed to radiation within the last 12 months of  >2.3 rem, or met 
criteria, but subsequently not assigned to the study intervention within 60 days. 

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If re-screened after 60 days from screening visits, the TST, IGRA, & chest x-ray will not be 
repeated unless there is a known tuberculosis exposure, including but not limited to travel to an 
endemic area or direct contact with an individual with known or suspected tuberculosis disease.
The study team may contact the subject at a later date to determine if they would like to continue 
their participation and complete the screening again. 
5.6
STRATEGIES FOR RECRUITMENT AND RETENTION
The study will be listed on the ClinicalTrials.gov, Clinical Center (CC) Search the Studies, and 
the CC recruitment website. IRB-approved language will be used in all recruitment messaging 
and the following options for marketing the study may be used:
•
ResearchMatch; 
•
Official NIH social media accounts (including but not limited to Facebook, Twitter, 
Instagram);
•
Community newspapers, magazines, radio, TV ads and/or other appropriate print or 
electronic media;
•
NIH Listservs (GovDelivery, Clinical Fellows, Post Baccalaureate, OPR Study 
Volunteer), webpages, or other marketing platforms including Clinical Center TVs 
(CCTV) messaging;
•
Physician partnerships to include health specialists; 
•
Recruitment post cards and flyers printed will be designed for the study and distributed in 
the community, around the NIH campus, external partnering healthcare centers/offices, 
and/or any other locations where potential research volunteers may be found.
•
Metro train, bus, station ads (Outfront Media company partnership).  All messaging will 
be IRB approved in advance.
A letter to physicians may be used for electronic (and hard copy as appropriate) distribution to 
local physicians and specialists. All options mentioned in PSA (i.e. ResearchMatch, publications, 
etc.) may be used for recruitment. Social media posts may be used by the accounts of the Clinical 
Center and NHLBI, and/or other official NIH accounts. The Healthy Volunteer (HV) listserv will 
be used for outgoing communications and a regularly updated HV list will be obtained, all 
through a close partnership with the CC HV office within the Clinical Center Office of Patient 
Recruitment and Call Center. 
5.6.1
Costs
Not applicable
5.6.2
Compensation
Visits to NIH
Amount
Screening Visit #1 – 1hr
$80
Screening Visit #2 – 1hr
$80
On-Study Visit #1 (Baseline)– 3hrs
$120
On-Study Visit #2 (Day 0)– 5hrs
$120
On-Study Visit #3 (Day 4-5)- 4hrs 
$120
On-Study Visit #4 (Day 5-6)– 5hrs
$120
On-Study Visit #5 (Day 20-21)– 4hrs
$120
On-Study Visit #6 (Day 21-22)– 5hrs
$120
Total for Visits
$880

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Procedures
Amount
Tuberculin skin test (TST) - $50 x 2 2nd test may not be needed
$100
History and Physical
$20
EKG - $20 x 4
$80
Blood work – $30 x 4
$120
Research blood work - $30 x 3
$90
Screening chest X-ray
$20
PET-CT - $250 x 3
$750
Radioactive tracer - $80 x 3
$240
Bronchoscopy - $300 x 3
$900
PPD Study drug given with 1st Bronchoscopy
$50
Fentanyl and Midazolam IV admin - $20 x 3
$60
Escort fee for person staying with vol after bronch $20 x3
$60
Total for Procedures
$2,490
Total (Visits and Procedures) - $880 +2,490  $3,370
Recruitment, Selection and Compensation of Research Subjects per Policy 301.
Reimbursement of Travel
Reimbursement for travel will be in accordance with NHLBI travel policy reimbursement for 
food and lodging will be consistent with NIH and NHLBI guidelines. Car mileage will be 
reimbursed only if traveling >30 miles away at 0.40 cents per mile.  Reimbursement will be 
provided for long distance >50 miles for train and bus only.
6
STUDY INTERVENTION
6.1
STUDY INTERVENTIONS(S) ADMINISTRATION
6.1.1
Study Intervention Description
The normal saline control agent (serving as a comparison for PPD administration) for 
bronchoscopic instillation will be 0.9% sodium chloride solution.
Sterile commercially developed tuberculin PPD will be used for skin-testing and bronchoscopic 
instillation procedures. Per the Tubersol® Package insert: “Tuberculin Purified Protein Derivative 
(Mantoux) (PPD) for intradermal tuberculin testing is prepared from a large Master Batch 
Connaught Tuberculin (CT68) and is a cell-free purified protein fraction obtained from a human 
strain of Mycobacterium tuberculosis grown on a protein-free synthetic medium and inactivated. 
TUBERSOL is indicated to aid diagnosis of tuberculosis infection in persons at increased risk of 
developing active disease. It is currently only indicated for intradermal injection.
Endobronchial administration of tuberculin PPD may lead side effects including but not limited 
to cough, wheezing, bronchospasm, fever, pneumonitis, allergic reaction, or anaphylaxis. The 
possibility of the occurrence of such side effects will be monitored as detailed below.
This study will be conducted under an Investigational New Drug Application (IND). The IND 
for Tuberculin Purified Protein Derivative (PPD) TUBERSOL® is 19720. 
6.1.2
Dosing and Administration
The PPD bronchoscopic instillation dose of 0.5 tuberculin units (10 uL Tubersol® in 10 mL 
normal saline) was determined by Silver and colleagues in 2003. They demonstrated that the 
minimally reactive dose of PPD administered through the airways to elicit the development of 
alveolar inflammation was a challenge dose of 0.5 TU or 1/10th of the standard tuberculin skin 
test dose (15).

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This instillation will be performed via introduction of a balloon-tipped 5-French pulmonary 
artery (PA) catheter within the working channel of the bronchoscope, subsequent occlusion of 
the subsegment of interest, instillation of normal saline control, followed by a  saline flush (20). 
Next, the bronchoscope will be moved to the right lung, where 0.5 tuberculin units (10 
microliters of Tubersol®) diluted in 10 cc normal saline will be instilled into the anterior segment 
of the right upper lobe via a separate 5-French balloon-tipped PA catheter similarly followed by 
saline flush.
6.1.3
Dose Escalation
Not applicable.
6.1.4
Dose Limiting Toxicity
Not applicable.
6.1.5
Dose Modifications
Not applicable. 
6.1.6
Drug Administration
See section 6.1.2
6.2
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY 
6.2.1
Acquisition and Accountability
Sterile commercially developed TUBERSOL® will be used for skin-testing and bronchoscopic 
instillation procedures. Please refer to the Tubersol® Package insert.
PPD for clinical and in vitro use will be purchased as Tubersol® from Sanofi Pasteur, 1 
Discovery Drive, Swiftwater, PA 18370. 
6.2.2
Formulation, Appearance, Packaging, and Labeling
As above in section 6.2.1
6.2.3
Product Storage and Stability
Intact vials will be stored in the refrigerator (2˚C - 8˚C) (36˚F - 46˚F). The vials will be protected 
from freezing and light exposure in order to maintain product integrity.
Stability studies have demonstrated that PPD containing the stabilizer of Tween 80 and 
preservative of phenol are stable for at least 3 years at 4 degrees C, 2 years at room temperature 
(24 degrees C) and even at 37 degrees C are stable for at least 1 year (27). Per Canadian and U.S. 
regulations PPD will be stored at 2 to 8 degrees C in their original containers. The reconstituted 
solution should be stored in the refrigerator (2˚C - 8˚C). . The pharmacy will be notified of when 
the subject will be ready for the procedure and prepare the syringe at this time.  The Syringe will 
be transferred to the procedural unit per the Pharmacy Drug Preparation Manual Standard of 
Practice (SOP). 
6.2.4
Preparation
Tubersol® contains 0.0005% Tween 80 as a stabilizer and 0.28% phenol as a preservative. In 
order to administer the experimentally established bronchoscopic instillation dose of 0.5 

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tuberculin units (TU), 10 ul of the commercial Tubersol preparation will be diluted in 10 ml of 
saline, creating final concentrations of 0.0000025% Tween 80 of and 0.0014% phenol. To ensure 
sterility dilutions of PPD will be prepared in a laminar flow hood immediately prior to 
bronchoscopic instillation using previously unopened vials of Tubersol® and sterile saline. For 
intradermal injection each standard dose of 5 TU will be administered in the standard volume of 
0.1 ml using 26G needles. 
6.3
MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING
In order to minimize observer and confirmation bias, research specimens collected will be coded 
so that the principal investigator will be blinded to which group (LTBI vs. non-LTBI) subjects 
are enrolled, after subject’s completion of the schedule of activities but prior to primary data 
analysis has been completed. Designated AIs and study research nurses will coordinate the 
process of coding and labeling the samples collected into respective LTBI and non-LTBI groups 
in a manner that will blind the principal investigator to study group. These specimen code group 
will only be released to the principal investigator after flow cytometry, and PET-CT data have 
been collected and undergone primary analysis. 
6.4
STUDY INTERVENTION COMPLIANCE
Adherence to the protocol will be assessed by consistent patient presentation to all pre-described 
appointments including blood collection, PET-CT scans and bronchoscopies.  
6.5
CONCOMITANT THERAPY
For this protocol, a prescription medication is defined as a medication that can be prescribed only 
by a properly authorized/licensed clinician. Medications to be reported in the medical record that 
include concomitant prescription medications, over-the-counter medications and supplements.
After the completion of the study protocol, all study participants within the LTBI (PPD +, IGRA 
+) group will be recommended for referral to their local county health department or an 
infectious disease outpatient clinic for initiation of LTBI therapy as outlined by the WHO with 
the regimen selected per the recommendation of their clinician (19). 
7
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL 
7.1
DISCONTINUATION OF STUDY INTERVENTION
Participants are free to withdraw from participation in the study at any time upon request.
An investigator may discontinue or withdraw a participant from the study for the following 
reasons:
•
In the event of a single serious adverse event (SAE) or Grade 3 or higher adverse event or 
laboratory abnormality that is assessed as related or possibly related to the study 
procedures.  Events will be followed until resolution or stabilization. 
•
If a study participant misses one or more study appointments
•
Investigator discretion
•
Positive pregnancy test
•
Positive COVID-19  PCR

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•
Current use of anticoagulant therapy including platelet inhibitors (e.g. clopidogrel), or 
systemic anticoagulants (e.g. warfarin, enoxaparin, or DOAC).
7.2
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY
Participants are free to withdraw from participation in the study at any time upon request. 
An investigator may discontinue or withdraw a participant from the study for the following 
reasons:
•
Significant study intervention non-compliance 
•
If any clinical adverse event (AE), laboratory abnormality, or other medical condition or 
situation occurs such that continued participation in the study would not be in the best 
interest of the participant
•
Subject has completed the study follow-up period
•
Death
•
Screen Failure
The reason for participant discontinuation or withdrawal from the study will be recorded on the 
Case Report Form (CRF). Subjects who sign the informed consent form and are assigned to a 
cohort but do not receive the study intervention may be replaced.  Subjects who sign the 
informed consent form and subsequently withdraw, or are withdrawn or discontinued from the 
study, will be replaced.
7.3
LOST TO FOLLOW-UP
A participant will be considered lost to follow-up if he or she fails to return for one scheduled 
visit and is unable to be contacted by the study site staff. 
The following actions must be taken if a participant fails to return to the clinic for a required 
study visit:
•
The site will attempt to contact the participant and reschedule the missed visit within 
three days and counsel the participant on the importance of maintaining the assigned visit 
schedule and ascertain if the participant wishes to and/or should continue in the study.
•
Before a participant is deemed lost to follow-up, the investigator or designee will make 
every effort to regain contact with the participant (where possible, 3 telephone calls and, 
if necessary, a certified letter to the participant’s last known mailing address or local 
equivalent methods). These contact attempts should be documented in the participant’s 
medical record or study file. 
•
Should the participant continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up.
8
STUDY ASSESSMENTS AND PROCEDURES
8.1
SCREENING PROCEDURES
Screening activities performed prior to obtaining informed consent
Minimal risk activities that may be performed before the subject has signed a consent include the
following:
• Email, written, in person or telephone communications with prospective subjects
• Review of existing medical records to include H&P, laboratory studies, etc.

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Once the research team has identified a potential subject for the study, the subject will be asked 
to come to the NIH.  The study team will consent the subject via in person, telephone and or 
Telehealth visit platform, allowing time for the subjects to ask questions and make a voluntary 
decision. 
The following tests will be performed to determine eligibility. 
•
Review of medical history including history of Mtb Exposure 
o
History of exposure to persons with active TB
o
History of residence in a TB-endemic country
o
Estimate of time when LTBI most likely occurred
o
Exposure to Bacillus Calmette–Guérin (BCG) vaccine
•
Review of concurrent medications and vaccinations performed within the last year
o
Of note, eligibility review and procedures will be completed at least 2 weeks 
following most recent vaccination
The following tests will be performed after informed consent is obtained to determine eligibility. 
•
Vital signs 
•
Complete blood count with diff, acute care panel, hepatic care panel, LDH, International 
Normalized Ratio (INR), Prothrombin Time (PT), and Partial Thromboplastin Time 
(PTT) , interferon gamma release assay (IGRA), HIV (I/II), Hepatitis B and C 
•
Plasma or urinary HCG in female subjects who are of child-bearing age will be collected 
(after informed consent and within 7 days of any research study procedure )
•
Tuberculin skin test (TST) will be performed and read within 48 to 72 hours. A second 
TST (two-step) may be performed on individuals who have a positive IGRA result, but 
negative TST result on initial screening. Two-step TST will be performed 1 to 3 weeks 
after the 1st  test.
•
Chest X-Ray 
•
EKG
•
COVID-19 PCR 
8.2
EFFICACY ASSESSMENTS
8.2.1
Clinical Evaluations
Physical examination 
History and physical examination with vital signs (weight, height, BMI, blood pressure, 
respirations, heart rate, temperature and oxygen saturation) will performed at screening with 
focus on respiratory and cardiovascular systems. Repeat physical examinations with focus on 
respiratory and cardiovascular systems with vital sign measurements (blood pressure, 
respirations, heart rate, temperature and oxygen saturation) will be performed prior to each 
bronchoscopy.
Radiographic or other imaging assessments. 
Chest PET-CT Scan

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Subjects will undergo a total of 3 PET/CT scans. A baseline PET/CT of the chest will be 
performed no more than one week prior to the initial bronchoscopy (10). In order to examine 
early immune responses, on day 4 post instillation bronchoscopy, a second PET-CT scan will be 
obtained. Furthermore, in order to examine the persistence of immune cell metabolic activity in 
the pulmonary parenchyma and thoracic lymph nodes, a PET-CT image will be obtained on 
approximately day 20 post initial bronchoscopic challenge. 
Each participant will be asked to provide an estimate of their radiation exposure within the 
calendar in order to assess if participation in the study will cause a participant to exceed his/her 
annual dose limit. 
8.2.2
Biospecimen Evaluations
Biological specimen collection and laboratory evaluations. 
A peripheral intravenous catheter will be placed to obtain blood for analysis (up to 120 mL). The 
whole blood volume will be divided into aliquots for myeloid cell flow cytometry analysis, 
peripheral blood mononuclear cell (PBMC) isolation and laboratory testing. PBMC isolation and 
subsequent in-vitro stimulation with PPD antigen is detailed below. Blood for clinical serologic 
testing will be sent to the NIH chemistry lab for analysis via complete metabolic panel (CMP) 
and c-reactive protein (CRP) and the hematology lab for analysis via complete blood count with 
differential count (CBC w/ diff) and erythrocyte sediment count (ESR). The amount of blood 
that may be drawn from adult patients and volunteers (i.e., those persons 18 years of age or 
older) for research purposes shall not exceed 10.5 mL/kg or 550 mL, whichever is smaller, over 
any eight-week period.
In order to assess for any concurrent lower respiratory tract infection in study subjects, a 2 mL 
aliquot of bronchoalveolar lavage (BAL) fluid from day 0, day 5 and day 21 will be set aside 
from the research analysis sample. This BAL aliquot will be sent to the NIH Clinical Center 
Microbiology department for bacterial gram stain and culture in addition to BioFire respiratory 
pathogen panel for detection of the following viral and bacterial organisms: Adenovirus, 
Coronavirus HKU1/NL63/229E/OC43, Human Metapneumovirus, Human 
Rhinovirus/Enterovirus, Influenza A, Influenza B, Parainfluenza, Respiratory Syncytial Virus, 
Bordetella pertussis, Chlamydia pneumoniae, and Mycoplasma pneumoniae.
8.2.3
Correlative Studies for Research/Pharmacokinetic Studies
Special assays or procedures required 
Blood will be collected into heparinized Green Top tubes on the day of each bronchoscopy (days 
0, 5 and 21).  Peripheral Blood Mononuclear Cell (PBMC) and BAL cells will be isolated, 
stimulated and cell surface molecules, intracellular cytokines and transcription factors will be 
evaluated via flow cytometry. Peripheral blood and BAL soluble mediators will be analyzed via 
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8.2.4
Samples for Genetic/Genomic Analysis
Not applicable. 
8.3
SAFETY AND OTHER ASSESSMENTS
First participants will undergo history and physical assessment, blood or urine pregnancy test and 
a confirmatory TB skin test, and have blood drawn for an interferon gamma release assay 
(IGRA; Quantiferon Gold In-Tube, Qiagen) screening. Subjects will undergo a chest x-ray 
(CXR) to rule out for active disease. This is to ensure their eligibility prior to undergoing 
PET/CT scans, and bronchoscopies.
Eligible study subjects will be given a username and password to access a web-based patient 
self-reporting diary to record any evidence of self- assessed adverse events. At each study visit 
this will be reviewed with the subject by a designated research study personnel.  If a subject 
reports a possible serious adverse event, then the subject will be contacted immediately for 
review of event(s) by designated research study personnel. Subjects will also be directed to 
contact research study nurse if they have any concerns or questions.
Biological specimen collection and laboratory evaluations.
Bronchoscopy, BAL and Bronchoscopic Instillation
Study subjects will undergo three bronchoscopies and bronchoalveolar lavages for the evaluation 
of airway and alveolar immune cells.
The study subject will be required to be without solid food or drink after midnight or six hours 
prior to each bronchoscopy. 
First, topical anesthesia will be achieved via administration of inhaled medications containing 
lidocaine. Following completion of local anesthesia, sedative medications will be administered to 
achieve a level of moderate sedation. 
A bronchoscope will be introduced via the trans-oral or trans-nasal route. Lidocaine will be 
instilled through the bronchoscope at the levels of the epiglottis, vocal cords, carina and 
mainstem bronchi to provide additional anesthesia during the procedure. A baseline (prior to 
control or PPD instillation) bronchoalveolar lavage (BAL) will be performed in the left superior 
lingular segment of the left upper lobe. The control instillation of normal saline will then be 
placed into the anterior segment of the left upper lobe. This instillation will be performed via 
introduction of a balloon-tipped 5-French pulmonary artery (PA) catheter (26). Next, the 
bronchoscope will be moved to the right lung, similarly, a baseline BAL will be performed in the 
right middle lobe medial segment. Immediately afterwards, tuberculin purified protein derivative 
will be instilled into the anterior segment of the right upper lobe via a separate 5-French balloon-
tipped PA catheter. The bronchoscope will then be removed and the subject will recover and be 
monitored. If a subject requires additional monitoring they will be transferred to the Day Unit of 
the Clinical Center. Signs and symptoms for expected and unexpected responses from 
bronchoscopy and PPD instillation will be assessed during this time. At time of discharge their 
vital signs must return to baseline, the subject must be able to eat and drink without difficulty 
and have a responsible adult available for transport from the Clinical Center. Each participant 
will be called the following day to assess their respiratory status and evaluate for the 
development of any new pulmonary symptoms. If necessary, they will be re-evaluated at the 
Clinical Center by Dr. Fennelly.
Provide Study Monitoring Plan:

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We will use the Common Terminology Criteria for Adverse Events for Adverse Events 
(CTCAE) Version 5.0 to assess for any adverse events related to endobronchial PPD instillation. 
Adverse events (AE) will be graded by their level of severity (Grade 1 mild, Grade 2 moderate, 
Grade 3 severe, Grade 4 life-threatening consequences, Grade 5 Death related to AE).  We will 
specifically assess for evidence of respiratory-related adverse events commonly associated with 
bronchoscopy and/or endobronchial administration of a non-endogenous agent which includes 
but is not limited do: dyspnea, cough, sore throat, stridor, wheezing, bronchospasm, hypoxia, 
laryngeal inflammation, laryngospasm, pneumonitis, or pneumothorax. We will similarly assess 
for evidence of systemic immune- or infectious-related adverse events including but not limited 
to: allergic reaction, anaphylaxis, bronchial infection, lung infection, pharyngitis and/or pleural 
infection. 
8.4
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS
8.4.1
Definition of Adverse Event
Any untoward medical occurrence in a human subject, including any abnormal sign (e.g., 
abnormal physical exam or laboratory finding), symptom, or disease, temporally associated with 
the subject’s participation in the research, whether or not considered related to the subject’s 
participation in the research (21 CFR 312.32 (a)).
8.4.2
Definition of Serious Adverse Events (SAE)
An adverse event (AE) or suspected adverse reaction is considered "serious" if, in the view of 
either the investigator or sponsor, it results in any of the following outcomes: death, a life-
threatening adverse event, inpatient hospitalization or prolongation of existing hospitalization, a 
persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions, or a congenital anomaly/birth defect. Important medical events that may not result in 
death, be life-threatening, or require hospitalization may be considered serious when, based upon 
appropriate medical judgment, they may jeopardize the participant and may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition. Examples of such 
medical events include allergic bronchospasm requiring intensive treatment in an emergency 
room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, 
or the development of drug dependency or drug abuse.
8.4.3
Classification of an Adverse Event
8.4.3.1
Severity of Event
This study will utilize the Common Terminology Criteria for Adverse Events version 5.0 (CTCAE 
v5.0) for toxicity and adverse event reporting.  A copy of the CTCAE v5.0 can be downloaded 
from the https://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm.
AEs will be recorded, verified, and followed until satisfactory resolution or stabilization. Severity 
definitions found in the CTCAE v5.0 will be used for grading the severity (intensity) of AEs:
•
Mild – Events require minimal or no treatment and do not interfere with the participant’s 
daily activities. 
•
Moderate – Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning.

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•
Severe – Events interrupt a participant’s usual daily activity and may require systemic 
drug therapy or other treatment. Severe events are usually potentially life-threatening or 
incapacitating.  Of note, the term “severe” does not necessarily equate to “serious”.
•
Life-threatening – Events with life-threatening consequences in which urgent 
intervention is indicated
•
Death – Death related to an adverse event
8.4.3.2
Relationship to Study Intervention
All adverse events (AEs) must have their relationship to study intervention assessed by the 
investigator who examines and evaluates the participant based on temporal relationship and 
his/her clinical judgment. The degree of certainty about causality will be graded using the 
categories below. In a clinical trial, the study product must always be suspect. 
•
Related – The AE is known to occur with the study intervention, there is a reasonable 
possibility that the study intervention caused the AE, or there is a temporal relationship 
between the study intervention and event. Reasonable possibility means that there is 
evidence to suggest a causal relationship between the study intervention and the AE.
•
Not Related – There is not a reasonable possibility that the administration of the study 
intervention caused the event, there is no temporal relationship between the study 
intervention and event onset, or an alternate etiology has been established.
8.4.3.3
Expectedness 
The principal investigator will be responsible for determining whether an adverse event (AE) is 
expected or unexpected.  An AE will be considered unexpected if the nature, severity, or 
frequency of the event is not consistent with the risk information previously described for the 
study intervention. The expected risks will be primarily related to bronchoscopy and 
endobronchial manipulations. These risks include: dyspnea, cough, sore throat, stridor, 
wheezing, bronchospasm, hypoxia, laryngeal inflammation, laryngospasm, pneumonitis, 
pneumothorax, allergic reaction, and anaphylaxis. We expect some vasovagal symptoms during 
blood draws (expected frequency 50%) and transient bruising at the site of blood draws 
(expected frequency 50%).
8.4.4
Time Period and Frequency for Event Assessment and Follow-Up
The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to the 
attention of study personnel during study visits and interviews of a study participant presenting 
for medical care, or upon review by a study monitor.
All AEs including clinically significant laboratory tests, local and systemic reactions not meeting 
the criteria for SAEs will be captured on the appropriate case report form (CRF). Information to 
be collected includes event description, time of onset, clinician’s assessment of severity, 
relationship to study product/research procedure (assessed only by those with the training and 
authority to make a diagnosis), and time of resolution/stabilization of the event. All AEs 
occurring while on study must be documented appropriately regardless of relationship. All AEs 
will be followed to adequate resolution.

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Any medical condition that is present at the time that the participant is screened will be 
considered as baseline and not reported as an AE. However, if the study participant’s condition 
deteriorates at any time during the study, it will be recorded as an AE. 
Changes in the severity of an AE will be documented to allow an assessment of the duration of 
the event at each level of severity to be performed. AEs characterized as intermittent require 
documentation of onset and duration of each episode.
The principal investigator will record all reportable events with start dates occurring any time 
after informed consent is obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after the 
last day of study research procedure or until resolution or stabilization of an SAE whichever 
comes later .  At each study visit, the investigator will inquire about the occurrence of AE/SAEs 
since the last visit.  Events will be followed for outcome information until resolution or 
stabilization via in person, telephone and or e-mail.
8.4.5
Adverse Event Reporting 
The study investigator will be responsible for reporting all AEs, regardless of attribution, to the 
sponsor at the time of NIH IRB continuing review and FDA annual report.
If at any time the study investigator believes that a frequency of an AE is greater than 
anticipated, the investigator will report these findings to the sponsor within 7 days.
8.4.6
Serious Adverse Event Reporting 
The study investigator will report to the sponsor any serious adverse event, whether or not 
considered study intervention related, including those listed in the protocol and must include an 
assessment of whether there is a reasonable possibility that the study intervention caused the 
event. 
All serious adverse events (SAEs) will be followed until satisfactory resolution or until the 
investigator deems the event to be chronic or the participant is stable. 
The study sponsor will be responsible for notifying the Food and Drug Administration (FDA) of 
any unexpected fatal or life-threatening suspected adverse reaction as soon as possible, but in no 
case later than 7 calendar days after the sponsor's initial receipt of the information.  In addition, 
the sponsor must notify FDA and all participating investigators in an Investigational New Drug 
(IND) safety report of potential serious risks, from clinical trials or any other source, as soon as 
possible, but in no case later than 15 calendar days after the sponsor determines that the 
information qualifies for reporting.
All SAEs will be reported to the DSMB at the time of review.  However, if an SAE is 
considered, unexpected and related to the intervention, the study investigator will notify the 
DSMB of the event within 48 hours.
8.4.7
Events of Special Interest 
Any incidental radiographic findings discovered on PET-CT imaging will be reported to a study 
subject no later than 7 days after the PI first learns of the result. 

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8.4.8
Reporting of Pregnancy 
If the serum or urinary beta-HCG result of a female participant is positive at the time of 
screening, the individual will be informed of this result and excluded from the study.
All female subjects will receive pregnancy testing prior to radiation exposure.  Should a subject 
find out that she became pregnant while participating in this study, she should notify the team 
immediately.  The team will provide counseling on the risks of radiation to the fetus.  The 
investigators will report the pregnancy to the IRB and NHLBI Clinical Director at the 
Continuning Review as a reason subject was withdrawn early from the study 
8.5
UNANTICIPATED PROBLEMS
8.5.1
Definition of Unanticipated Problems (UP)
Any incident, experience, or outcome that meets all of the following criteria:
•
Unexpected in terms of nature, severity, or frequency given (a) the research procedures 
that are described in the protocol-related documents, such as the Institutional Review 
Board (IRB)-approved research protocol and informed consent document; and (b) the 
characteristics of the participant population being studied; and
•
Related or possibly related to participation in the research (“possibly related” means there 
is a reasonable possibility that the incident, experience, or outcome may have been 
caused by the procedures involved in the research); and
•
Suggests that the research places participants or others (which many include research 
staff, family members or other individuals not directly participating in the research) at a 
greater risk of harm (including physical, psychological, economic, or social harm) than 
was previously known or expected.
8.5.2
 Unanticipated Problem Reporting 
The investigator will report unanticipated problems (UPs) to the NIH Institutional Review Board 
(IRB) as per Policy 801.
8.5.3
NIH Intramural IRB Reporting of IND Safety Reports
Only IND Safety Reports that meet the definition of an unanticipated problem will need to be 
reported to the NIH Intramural IRB.
9
STATISTICAL CONSIDERATIONS
9.1
STATISTICAL HYPOTHESIS
•
Primary Endpoint: 
We hypothesize that antigen-specific CD4+ T cells response will be highest in the BAL 
five days post challenge in LTBI subjects alone and may subsequently wane 21 days post 
challenge.
•
Secondary Endpoint: 
We hypothesize that there will be significant FDG-avidity within the lung parenchyma 
and adjacent thoracic lymph node within the area of the lung (right upper lobe) in which 
PPD is selectively instilled. We expect that the SUV quantities will be most pronounced 
in LTBI subjects at day 4 post challenge and subsequently wane at day 20.  
•
Exploratory Endpoints:

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We hypothesize that the phenotype and function of Mtb-specific T cells in the BAL will 
differ from that of circulating T cells.  We hypothesize that bronchoscopic instillation of 
PPD will induce production of TH1 predominant pro-inflammatory cytokines, eicosanoid 
lipid mediators and defensins in the local airway environment more so than the systemic 
vasculature. We hypothesize that these soluble mediators will be primarily present in 
LTBI subjects on day 5 post challenge and wane at day 20. 
9.2
SAMPLE SIZE DETERMINATION
This is a pilot study which will be hypothesis-generating in nature. For this reason we will not be 
pursuing the formal sample size calculation that corresponds to a specified statistical power. 
However, the sample size of 13 confirmed LTBI cases and 7 confirmed non-LTBI controls (a 
total of 20 subjects) is slightly larger than those of the original studies of the current 
bronchoscopic instillation model pioneered by Silver and colleagues (21-23). Silver’s 2003, 2005 
and 2011 PPD bronchoscopic instillation studies included a total of 9, 15, and 14 total subjects 
respectively. Each study was able to demonstrate a statistically significant increase in the 
frequency of Mtb-specific CD4+ T cells in the BAL of LTBI subjects as compared to the BAL of 
non-LTBI controls within each study. Furthermore, we anticipate that our immunological signals 
will likely stand out due to confirmation of LTBI status using concordant TST and IGRA testing. 
We will enroll up to an additional 5 subjects in order to achieve 20 completed studies. 
9.3
POPULATIONS FOR ANALYSES
Not applicable. 
9.3.1
Evaluable for toxicity 
Not applicable.
9.3.2
Evaluable for objective response 
Not applicable. 
9.3.3
Evaluable Non-Target Disease Response
Not applicable
9.4
STATISTICAL ANALYSES
9.4.1
General Approach
Statistical analysis will be performed in R version 3.6.1 or later (CRAN.R-Project.org). For the 
analysis of primary and secondary endpoints, mixed effects models with repeated measure will 
be used to compare the LTBI and non-LTBI responses after bronchoscopic PPD instillation. 
For the primary hypothesis, the cell count for Mtb antigen-specific CD4+ and CD8+ T cells will 
be used as the response variable. Two separate statistical models will be entertained to explain 
the local pulmonary response and systemic vasculature, respectively. To guard against non-
normal responses, a suitable data transformation on the response variable, e.g., log transform, 
will be performed if the original scale is highly skewed. 

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In regard to PET CT analysis, calculations for standardized uptake value will be made and 
normalized for scan differences using OsiriX MD (11). For the secondary hypothesis, the FDG 
SUV values from the PET CT image, measured at days -7, +4, and +20 days will be used as 
response in similar mixed effects models to study the group difference in average immunologic 
responses.
For the exploratory hypotheses, both numerical and graphical summary tools will be used to 
study the phenotypic and functional analysis of immune cell populations in the airways and 
circulation of individuals with or without LTBI. Also, descriptive statistics will be used to 
characterize the production of soluble mediators in BAL fluid and blood.
9.4.2
Analysis of the Primary and Secondary Endpoints
For the analysis of primary and secondary endpoints, mixed effects models with repeated 
measure will be used to compare the LTBI and non-LTBI responses after bronchoscopic PPD 
instillation. 
For the primary hypothesis, the cell count for Mtb antigen-specific CD4+ and CD8+ T cells will 
be used as the response variable. Two separate statistical models will be entertained to explain 
the local pulmonary response and systemic vasculature, respectively. To guard against non-
normal responses, a suitable data transformation on the response variable, e.g., log transform, 
will be performed if the original scale is highly skewed. 
9.4.3
Safety Analyses
N/A
9.4.4
Baseline Descriptive Statistics
In this pilot study, various descriptive statistics will be used to describe and compare baseline 
patient demographics per group. 
9.4.5
Planned Interim Analyses
None planned at this time
9.4.6
Sub-Group Analyses
The primary, secondary and exploratory endpoints will be analyzed within group stratified for 
age in order to determine if there is evidence of a differential response in the setting of 
immunosenescence.  
9.4.7
Tabulation of individual Participant Data
Each individual participant data will be listed by measurement evaluated, location of 
measurement acquisition and day of measurement acquisition relative to bronchoscopic 
challenge date. 
9.4.8
Exploratory Analyses
For the exploratory hypotheses, both numerical and graphical summary tools will be used to 
study the phenotypic and functional analysis of immune cell populations in the airways and 

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circulation of individuals with or without LTBI. Also, descriptive statistics will be used to 
characterize the production of soluble mediators in BAL fluid and blood.
10 REGULATORY AND OPERATIONAL CONSIDERATIONS
10.1 INFORMED CONSENT PROCESS
10.1.1 Consent/Assent Procedures and Documentation
Informed Consent Processes and Procedures
Informed consent will be conducted following OHSRP Policy 301. An IRB-approved consent 
form, will be provided to the subject electronically or by hard copy for review prior to 
consenting. The initial consent process as well as re-consent, when required, may take place in 
person or remotely (e.g., via telephone, telehealth or other NIH approved platforms). The 
investigational nature and objectives of this trial, the procedures, and their attendant risks and 
discomforts and potential benefits will be carefully explained to the subject in a private setting.  
The subject will be given as much time as they need to review the document and to consult with 
their family, friends, and personal health care providers. In addition, a study team member will 
be available to answer any questions. 
A signed and dated informed consent document will be obtained by any investigator authorized 
to consent (See Key Study Personnel) prior to entry onto the study.  Consent may be obtained 
with required signatures on the hard copy of the consent or on the electronic document.  
When a document that is in electronic format is used for obtaining  consent, this study may use 
the iMed platform which is 21 CFR, Part 11 compliant, to obtain the required signatures.
During the consent process, participants and investigators may view the same approved consent 
document simultaneously when participant is being consented in person at the Clinical Center or 
both may view individual copies of the approved consent document on screens in their respective 
locations remotely. Signatures may be obtained either by both directly signing on the device that 
the consenting investigator is using (when in person) or through iMed Mobile Signature Capture  
(remotely) which allows texting or emailing a link to the participant. That link allows the 
participant to review the consent, then proceed to sign on the device they are using.
Whether hard copy or electronic, both the investigator and the participant will sign the document 
with a hand signature using a pen (if using hard copy), finger, stylus, or mouse (if electronic).
When done remotely, if the participant prefers to sign a hard copy, they may be instructed to sign 
and date the consent document during the discussion and mail, secure email or fax the signed 
document to the consenting investigator.
 Whether in person or remote, the privacy of the subject will be maintained. 
The fully signed informed consent document will be stored in the electronic medical record, and 
the subject will receive a copy of the signed informed consent document.
10.1.2 Consent for minors when they reach the age of majority 
Not applicable.
10.1.3 Telephone and or Telehealth platform consent 
Participants may be contacted by telephone and or Telehealth NIH- approved platform per Policy 
303 to obtain informed consent.  For initial consent and for subjects actively participating in the 
study who may need to be re-consented due to changes to the informed consent or procedures.

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10.1.4 Telephone child assent
Not applicable
10.1.5 Participation of Subjects who are/become Decisionally Impaired
Not applicable. Adults who are unable to provide initial informed consent will be excluded. 
10.2 STUDY DISCONTINUATION AND CLOSURE
This study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause.  Written notification, documenting the reason for study suspension or 
termination, will be provided by the suspending or terminating party to study participants. If the 
study is prematurely terminated or suspended, the Principal Investigator (PI) will promptly 
inform study participants, the Institutional Review Board (IRB), and sponsor and will provide 
the reason(s) for the termination or suspension.  Study participants will be contacted, as 
applicable, and be informed of changes to study visit schedule.
 Circumstances that may warrant termination or suspension include, but are not limited to:
•
Determination of unexpected, significant, or unacceptable risk to participants
a.
A single SAE in a subject that is assessed as related or possibly related to the study 
drug
b. Grade 3 or higher laboratory abnormality in 3 or more subjects with a reasonable 
possibility of a causal relationship to the administration of study drug
c.
Grade 3 or higher with the same or similar Adverse Event in 2 or more subjects 
assessed as possibly related to study drug.
d. Will restart study if investigation determines study drug not to be the cause
•
Insufficient compliance to protocol requirements
•
Data that are not sufficiently complete and/or evaluable
•
Determination that the primary endpoint has been met
Study may resume once concerns about safety, protocol compliance, and data quality are 
addressed, and satisfy the sponsor, IRB and/or Food and Drug Administration (FDA).
10.3 CONFIDENTIALITY AND PRIVACY
Participant confidentiality and privacy is strictly held in trust by the participating investigators 
and their staff. This confidentiality is extended to cover testing of biological samples and genetic 
tests in addition to the clinical information relating to participants. Therefore, the study protocol, 
documentation, data, and all other information generated will be held in strict confidence. No 
information concerning the study or the data will be released to any unauthorized third party 
without prior written approval of the sponsor. 
All research activities will be conducted in as private a setting as possible.
The study monitor, representatives of the Institutional Review Board (IRB), and/or regulatory 
agencies may inspect all documents and records required to be maintained by the investigator, 
including but not limited to, medical records (office, clinic, or hospital) and pharmacy records 
for the participants in this study. The clinical study site will permit access to such records.
The study participant’s contact information will be securely stored at each clinical site for 
internal use during the study. At the end of the study, all records will continue to be kept in a 

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secure location for as long a period as dictated by the reviewing IRB, Institutional policies, or 
sponsor requirements.
Study participant research data, which is for purposes of statistical analysis and scientific 
reporting, will be transmitted to and stored at the Barber Lab data server. This will not include 
the participant’s contact or identifying information. Rather, individual participants and their 
research data will be identified by a unique study identification number. The study data entry and 
study management systems used will be secured and password protected. At the end of the study, 
all study databases will be de-identified and archived at the at the Barber Lab data server.
To further protect the privacy of study participants, a Certificate of Confidentiality has been 
issued by the National Institutes of Health (NIH).  This certificate protects identifiable research 
information from forced disclosure. It allows the investigator and others who have access to 
research records to refuse to disclose identifying information on research participation in any 
civil, criminal, administrative, legislative, or other proceeding, whether at the federal, state, or 
local level. By protecting researchers and institutions from being compelled to disclose 
information that would identify research participants, Certificates of Confidentiality help achieve 
the research objectives and promote participation in studies by helping assure confidentiality and 
privacy to participants.
10.4 FUTURE USE OF STORED SPECIMENS AND DATA
After analyzing the biospecimens for primary research purposes as described in the protocol, 
remaining samples suitable for future research will be stored in the Principal Investigator’s 
laboratory freezers in a coded manner to ensure data protection. Biospecimens may be destroyed 
only when permitted by the clinical director and approved by the IRB. Any future research use of 
biospecimens not defined in the research protocol in which NHLBI investigators are engaged in 
research (e.g., they are undertaking research activities and hold the key that identifies research 
subjects) requires IRB review and approval.  Coded biospecimens (NHLBI investigators hold the 
key that identifies research subjects) to be shared outside of NIH for future research use where 
results will not be returned to the Principal Investigator does not require IRB review. Refusal of 
a research subject participant to allow for future use of biospecimens will be honored.
10.5 SAFETY OVERSIGHT
Safety oversight will be under the direction of a Data and Safety Monitoring Board (DSMB) 
composed of individuals with the appropriate expertise. Members of the DSMB should be 
independent from the study conduct and free of conflict of interest, or measures should be in 
place to minimize perceived conflict of interest.  The DSMB will meet at least annually to assess 
safety and efficacy data on each arm of the study. The DMSB will operate under the rules of an 
approved charter. 
10.6 CLINICAL MONITORING
Principal Investigator:  
Accrual will be monitored by the principal investigator who will provide oversight and will 
monitor accrual and safety data. The investigator (and/or designee) will make study documents 
(e.g., consent forms and pertinent hospital or clinical records) readily available for inspection by 

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the local IRB and the NHLBI Office of the Clinical Director staff for confirmation of the study 
data. 
The monitoring of this study will be conducted by clinical  research associates (CRAs)/monitors 
employed by an independent contract organization working under an agreement with NHLBI to 
monitor aspects of the study in accordance with the appropriate regulations and the approved 
protocol. The objectives of a monitoring visit will be: 1) to verify the existence of signed informed 
consent form (ICF) and documentation of the ICF process for each monitored subject; 2) to verify 
the prompt and accurate recording of all monitored data points, and prompt reporting of all SAEs; 
3) to compare abstracted information with individual subjects’ records and source documents 
(subject’s charts, laboratory analyses and test results, physicians’ progress notes, nurses’ notes, 
and any other relevant original subject information); and 4) to help ensure investigators are in 
compliance with the protocol. The monitors also will inspect the clinical site regulatory files to 
ensure that regulatory requirements (Office for Human Research Protections-OHRP) and 
applicable guidelines (ICH-GCP) are being followed.  Monitoring will be conducted according to 
the OCD schedule.  During the monitoring visits, the investigator (and/or designee) and other study 
personnel will be available to discuss the study progress and monitoring visit.
The investigator (and/or designee) will make study documents (e.g., consent forms and pertinent 
hospital or clinical records readily available for inspection by the local IRB, the site monitors, 
and the NHLBI staff for confirmation of the study data.
10.7 QUALITY ASSURANCE AND QUALITY CONTROL
Each clinical site will perform internal quality management of study conduct, data and biological 
specimen collection, documentation and completion.  An individualized quality management 
plan will be developed to describe a site’s quality management.
Quality control (QC) procedures will be implemented beginning with the data entry system and 
data QC checks that will be run on the database will be generated. Any missing data or data 
anomalies will be communicated to the site(s) for clarification/resolution.
Following written Standard Operating Procedures (SOPs), the monitors will verify that the 
clinical trial is conducted and data are generated and biological specimens are collected, 
documented (recorded), and reported in compliance with the protocol, International Conference 
on Harmonization Good Clinical Practice (ICH GCP), and applicable regulatory requirements 
(e.g., Good Laboratory Practices (GLP), Good Manufacturing Practices (GMP)). 
The investigational site will provide direct access to all trial related sites, source data/documents, 
and reports for the purpose of monitoring and auditing by the sponsor, and inspection by local 
and regulatory authorities.
10.8 DATA HANDLING AND RECORD KEEPING
10.8.1 Data Collection and Management Responsibilities
Data collection is the responsibility of the clinical trial staff at the site under the supervision of 
the site investigator. The investigator is responsible for ensuring the accuracy, completeness, 
legibility, and timeliness of the data reported.

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All source documents should be completed in a neat, legible manner to ensure accurate 
interpretation of data.  
Hardcopies of the study visit worksheets will be provided for use as source document worksheets 
for recording data for each participant enrolled in the study.  Data recorded in the electronic case 
report form (eCRF) derived from source documents should be consistent with the data recorded 
on the source documents. 
Data Management
The PI will be responsible for overseeing entry of data into an in-house password protected 
electronic system and ensuring data accuracy, consistency and timeliness. The PI, associate 
investigators/research nurses and/or a contracted data manager will assist with the data 
management efforts to ensure that data is verifiable and evaluable. Data will be abstracted from 
Clinical Center progress notes and investigations as well as from outside medical records. 
Laboratory data from NIH will be imported electronically from CRIS into an in-house clinical 
trial database. Laboratory values from outside physicians will be entered into the system. 
Research data will be prospectively collected by authorized personnel and entered into an 
NHLBI, 21 CFR 11 compliant, database which will consist of the study specific set of electronic 
CRFs (e-CRFs) used for capturing, managing and reporting clinical research data.
We will maintain the confidentiality of identifiable private information collected in this Clinical 
Trial and protect the privacy of the individual human subjects. Primary data containing 
individually identifiable information obtained during the conduct of the protocol will be kept in 
secure network drives or in approved alternative sites that comply with NIH information security 
standards.
10.8.2 Study Records Retention
Study documents should be retained for a minimum of 2 years after the last approval of a 
marketing application in an International Conference on Harmonization (ICH) region and until 
there are no pending or contemplated marketing applications in an ICH region or until at least 2 
years have elapsed since the formal discontinuation of clinical development of the study 
intervention, or as per the NIH Intramural Records Retention Schedule. No records will be 
destroyed without the written consent of the sponsor, if applicable. It is the responsibility of the 
sponsor to inform the investigator when these documents no longer need to be retained.
10.9 PROTOCOL DEVIATIONS
It is the responsibility of the investigator to use continuous vigilance to identify and 
report deviations to the NIH Institutional Review Board as per Policy 801.  All deviations 
must be addressed in study source documents, reported to a NHLBI Program Official. 
The investigator is responsible for knowing and adhering to the reviewing IRB 
requirements. 
Reports to the CD:
The PI or designee will refer to NHLBI DIR guidelines to determine CD reporting 
requirements.

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10.9.1 NIH Definition of Protocol Deviation
A protocol deviation is any changed, divergence, or departure from the IRB-approved research 
protocol. 
•
Major deviations: Deviations from the IRB approved protocol that have, or may have the 
potential to, negatively impact the rights, welfare or safety of the subject, or to 
substantially negatively impact the scientific integrity or validity of the study.
•
Minor deviations: Deviations that do not have the potential to negatively impact the 
rights, safety or welfare of subjects or others, or the scientific integrity or validity of the 
study.
10.10 PUBLICATION AND DATA SHARING POLICY
10.10.1Human Data Sharing Plan
This study will be conducted in accordance with the following publication and data sharing 
policies and regulations:
National Institutes of Health (NIH) Public Access Policy, which ensures that the public has 
access to the published results of NIH funded research. It requires scientists to submit final peer-
reviewed journal manuscripts that arise from NIH funds to the digital archive PubMed Central 
upon acceptance for publication.
This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination of 
NIH-Funded Clinical Trial Information and the Clinical Trials Registration and Results 
Information Submission rule. As such, this trial will be registered at ClinicalTrials.gov, and 
results information from this trial will be submitted to ClinicalTrials.gov. In addition, every 
attempt will be made to publish results in peer-reviewed journals.  Data from this study may be 
requested from other researchers 10 years after the completion of the primary endpoint by 
contacting Kevin Fennelly.
10.10.2Genomic Data Sharing Plan
N/A
10.11 COLLABORATIVE AGREEMENTS 
Not applicable.
10.12 CONFLICT OF INTEREST POLICY
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical.  Therefore, any actual conflict of interest of persons who 
have a role in the design, conduct, analysis, publication, or any aspect of this trial will be 
disclosed and managed. Furthermore, persons who have a perceived conflict of interest will be 
required to have such conflicts managed in a way that is appropriate to their participation in the 
design and conduct of this trial.  The study leadership in conjunction with the NHLBI has 
established policies and procedures for all study group members to disclose all conflicts of 
interest and will establish a mechanism for the management of all reported dualities of interest.

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11 ABBREVIATIONS
The list below includes abbreviations utilized in this template.  However, this list should be 
customized for each protocol (i.e., abbreviations not used should be removed and new 
abbreviations used should be added to this list).
AE
Adverse Event
BAL
Bronchoalveolar Lavage
BALF
Bronchoalveolar Lavage Fluid
CBC
Complete Blood Count
CFR
Code of Federal Regulations
CMP
Complete Metabolic Panel 
CRF
Case Report Form
CRP
C-Reactive Protein
CXR
Chest X-Ray
DSMB
Data Safety Monitoring Board
EKG
Electrocardiogram
eCRF
Electronic Case Report Forms
ESR
Erythrocyte Sedimentation Rate
FDA
Food and Drug Administration
FDG
Fluorodeoxyglucose
GCP
Good Clinical Practice
GLP
Good Laboratory Practices
GMP
Good Manufacturing Practices
HIPAA
Health Insurance Portability and Accountability Act 
ICH
International Conference on Harmonisation 
IGRA
Interferon Gamma Release Assay
IND
Investigational New Drug Application
IRB
Institutional Review Board
LTBI
Latent Tuberculosis Infection
LUL
Left Upper Lobe
MOP
Manual of Procedures
MSDS
Material Safety Data Sheet
Mtb
Mycobacterium tuberculosis
NCT
National Clinical Trial
NHLBI
National Heart, Lung and Blood Institute
NIH 
National Institutes of Health
NIH IC
NIH Institute or Center
OHRP
Office for Human Research Protections
PBMC
Peripheral Blood Mononuclear Cell
PET-CT
Positron Emission Tomography-Computed Tomography
PI
Principal Investigator
PPD
Purified Protein Derivative
PT
Prothrombin Time
PTT
Partial Thromboplastin Time

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QA
Quality Assurance
QC
Quality Control
RML
Right Middle Lobe
RUL
Right Upper Lobe
SAE
Serious Adverse Event
SOA
Schedule of Activities
SOP
Standard Operating Procedure
SUV
Standardized Uptake Value
TST
Tuberculin Skin Test
UP
Unanticipated Problem
US
United States

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12 REFERENCES 
1. Sakai S, Mayer-Barber KD, Barber DL. Defining features of protective CD4 T cell 
responses to Mycobacterium tuberculosis. Curr Opin Immunol. 2014 Aug;29:137-42.
2. Kauffman KD, Sallin MA, Hoft SG, Sakai S, Moore R, Wilder-Kofie T, Moore IN, Sette 
A, Arlehamn CSL, Barber DL. 2018. Limited pulmonary mucosal-associated invariant T 
cell accumulation and activation during Mycobacterium tuberculosis infection in rhesus 
macaques. Infect Immun 86:e00431-18.
3. Hoft SG, Sallin MA, Kauffman KD, Sakai S, Ganusov VV, Barber DL. The Rate of CD4 
T Cell Entry into the Lungs during Mycobacterium tuberculosis Infection Is Determined 
by Partial and Opposing Effects of Multiple Chemokine Receptors. Infect Immun. 2019 
May 21;87(6).
4. Ogongo P, Porterfield JZ, Leslie A. Lung Tissue Resident Memory T-Cells in the 
Immune Response to Mycobacterium tuberculosis. Front Immunol. 2019 May 3;10:992.
5. Silver RF, Myers AJ, Jarvela J, Flynn J, Rutledge T, Bonfield T, Lin PL. Diversity of 
Human and Macaque Airway Immune Cells at Baseline and during Tuberculosis 
Infection. Am J Respir Cell Mol Biol. 2016 Dec;55(6):899-908.
6. Dharmadhikari AS1, Nardell EA. What animal models teach humans about tuberculosis. 
Am J Respir Cell Mol Biol. 2008 Nov;39(5):503-8.
7. Wolf AJ, Desvignes L, Linas B, Banaiee N, Tamura T, Takatsu K, Ernst JD. Initiation of 
the adaptive immune response to Mycobacterium tuberculosis depends on antigen 
production in the local lymph node, not the lungs. J Exp Med. 2008 Jan 21;205(1):105-
15.
8. Reiley WW, Calayag MD, Wittmer ST, Huntington JL, Pearl JE, Fountain JJ, Martino 
CA, Roberts AD, Cooper AM, Winslow GM, Woodland DL. ESAT-6-specific CD4 T 
cell responses to aerosol Mycobacterium tuberculosis infection are initiated in the 
mediastinal lymph nodes. Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10961-6.
9. Waeckerle-Men Y1, Bruffaerts N, Liang Y, Jurion F, Sander P, Kündig TM, Huygen K, 
Johansen P. Lymph node targeting of BCG vaccines amplifies CD4 and CD8 T-cell 
responses and protection against Mycobacterium tuberculosis. Vaccine. 2013 Feb 
4;31(7):1057-64.
10. Ghesani N, Patrawalla A, Lardizabal A, Salgame P, Fennelly KP. Increased cellular 
activity in thoracic lymph nodes in early human latent tuberculosis infection. Am J Respir 
Crit Care Med. 2014 Mar 15;189(6):748-50.
11. Coleman MT, Chen RY, Lee M, Lin PL, Dodd LE, Maiello P, Via LE, Kim Y, Marriner 
G, Dartois V, Scanga C, Janssen C, Wang J, Klein E, Cho SN, Barry CE 3rd, Flynn JL. 

45
Abbreviated Title: Tissue-Specific Immune Responses to Tuberculin Purified Protein Derivative (PPD) in the 
Human Lung  Version Date: 12/17/2024
Template version date 07-29-2019
PET/CT imaging reveals a therapeutic response to oxazolidinones in macaques and 
humans with tuberculosis. Sci Transl Med. 2014 Dec 3;6(265).
12. Ankrah AO, van der Werf TS, de Vries EF, Dierckx RA, Sathekge MM, Glaudemans 
AW. PET/CT imaging of Mycobacterium tuberculosis infection. Clin Transl Imaging. 
2016;4:131-144.
13. Geadas C, Acuna-Villaorduna C, Mercier G, Kleinman MB, Horsburgh CR Jr, Ellner JJ, 
Jacobson KR. FDG-PET/CT activity leads to the diagnosis of unsuspected TB: a 
retrospective study. BMC Res Notes. 2018 Jul 13;11(1):464.
14. Behar SM1, Divangahi M, Remold HG. Evasion of innate immunity by Mycobacterium 
tuberculosis: is death an exit strategy? Nat Rev Microbiol. 2010 Sep;8(9):668-74.
15. O'Garra A, Redford PS, McNab FW, Bloom CI, Wilkinson RJ, Berry MP. The immune 
response in tuberculosis. Annu Rev Immunol. 2013;31:475-527.
16. Srivastava S, Ernst JD. Cell-to-Cell Transfer of M. tuberculosis Antigens Optimizes CD4 
T Cell Priming. Cell Host Microbe. 2014 Jun 11; 15(6): 741–752.
17. Samstein M1, Schreiber HA, Leiner IM, Susac B, Glickman MS, Pamer EG. Essential 
yet limited role for CCR2⁺ inflammatory monocytes during Mycobacterium tuberculosis-
specific T cell priming. Elife. 2013 Nov 12;2:e01086.
18. Gallegos AM, Pamer EG, Glickman MS. Delayed protection by ESAT-6-specific effector 
CD4+ T cells after airborne M. tuberculosis infection. J Exp Med. 2008 Sep 
29;205(10):2359-68.
19. Sakai, S., K. D. Kauffman, J. M. Schenkel, C. C. McBerry, K. D. Mayer-Barber, D. 
Masopust, and D. L. Barber. 2014. Cutting Edge: Control of Mycobacterium tuberculosis 
Infection by a Subset of Lung Parenchyma-Homing CD4 T Cells. J Immunol 192: 2965–
2969.
20. World Health Organization. Global Tuberculosis Report 2017. Available at: 
http://www.who.int/tb/publications/global_report/en/ (Accessed on November 03, 2017).
21. Silver, R. F., L. Zukowski, S. Kotake, Q. Li, F. Pozuelo, A. Krywiak, and R. Larkin. 
2003. Recruitment of antigen-specific Th1-like responses to the human lung following 
bronchoscopic segmental challenge with purified protein derivative of Mycobacterium 
tuberculosis. Am. J. Respir. Cell Mol. Biol. 29: 117–123.
22. Walrath, J., L. Zukowski, A. Krywiak, and R. F. Silver. 2005. Resident Th1-like effector 
memory cells in pulmonary recall responses to Mycobacterium tuberculosis. Am. J. 
Respir. Cell Mol. Biol. 33: 48–55.

46
Abbreviated Title: Tissue-Specific Immune Responses to Tuberculin Purified Protein Derivative (PPD) in the 
Human Lung  Version Date: 12/17/2024
Template version date 07-29-2019
23. Walrath, J. R., and R. F. Silver. 2011. The α4β1 integrin in localization of 
Mycobacterium tuberculosis-specific T helper type 1 cells to the human lung. Am. J. 
Respir. Cell Mol. Biol. 45: 24–30.
24. Jarvela JR, Tuscano L, Lee H, Silver RF. Pulmonary responses to pathogen-specific 
antigens in latent Mycobacterium tuberculosis infection. Tuberculosis (Edinb). 2016 
Jan;96:158-64.
25. Latent tuberculosis infection: updated and consolidated guidelines for programmatic 
management. Geneva: World Health Organization; 2018. License: CC BY-NC-SA 3.0 
IGO.
26. O'Grady NP, Preas HL, Pugin J, Fiuza C, Tropea M, Reda D, Banks SM, Suffredini AF. 
Local inflammatory responses following bronchial endotoxin instillation in humans. Am 
J Respir Crit Care Med. 2001 Jun;163(7):1591-8.
27. Landi S, Held HR. Stability of dilute solutions of tuberculin purified protein derivative. 
Tubercle. 1978 Jun;59(2):121-33.
28. Jones-López EC, White LF, Kirenga B, Mumbowa F, Ssebidandi M, Moine S, 
Mbabazi,O, Mboowa G, Ayakaka I, Kim S, Thornton CS, Okwera A, Joloba M, Fennelly 
KP.  Cough Aerosol Cultures of Mycobacterium tuberculosis: Insights on TST / IGRA 
Discordance and Transmission Dynamics. PLoS One. 2015 Sep 22;10(9):e0138358.
29. Kauffman KD, Sallin MA, Sakai S, Kamenyeva O, Kabat J, Weiner D, Sutphin M, 
Schimel D, Via L, Barry CE 3rd, Wilder-Kofie T, Moore I, Moore R, Barber DL. 
Defective positioning in granulomas but not lung-homing limits CD4 T-cell interactions 
with Mycobacterium tuberculosis-infected macrophages in rhesus macaques. Mucosal 
Immunol. 2018 Mar;11(2):462-473.