This study is for people who have had a lung transplant and developed a condition called chronic lung allograft dysfunction (CLAD), which is a type of chronic rejection. Doctors often treat CLAD with a procedure called extracorporeal photopheresis (ECP), but it can take up to six months to know if the treatment is working. The goal of the study is to find early signs (biomarkers) that show whether ECP is helping, so patients can get the right care sooner.

For participants in the study, small blood samples will be collected at three points during ECP treatment and, for some participants, two MRI scans of the lungs will be performed-one before starting ECP and one after finishing treatment. The MRI uses a safe contrast dye to help us see changes in lung blood flow and tissue. Investigators will also look at certain immune cells in the blood.

This is not a study of a new drug or treatment-participants will receive the same ECP therapy their doctor already recommended. The study will help researchers understand how ECP works and identify markers that predict who benefits most. There is no direct benefit to participants, but participation may help improve care for future lung transplant patients.

I. ECP treatments The decision to prescribe ECP is independent of the study and will be determined by the subject's treating physician. ECP treatments will be performed at the subject's respective lung transplant center per standard participating center ECP treatment regimen and will not be altered for purposes of this study. Typically, procedures are performed using a treatment schedule to include a total of 24 procedures over 6 months. Two procedures are performed weekly for seven weeks (months 1 and 2), followed by paired procedures every-other-week for month 3, then once-a-month for months 4, 5, and 6.

II. Blood Sample Collection Study Visits and Parameters to be Measured When subjects present for their first ECP treatment at their respective lung transplant center, a completed informed consent form will be confirmed for participation in the study. Blood samples will be collected by the center performing the subject's ECP treatments at three time points over the 6-month course of an ECP treatment cycle: 1) immediately prior to the first ECP treatment, 2) immediately prior to the 15th ECP treatment at the start of month 3, and 3) immediately prior to the final ECP treatment of a full ECP treatment cycle.

a. Procedures i. Blood Sample Collection: Blood samples (up to 25 ml of venous blood, 15 ml for dd-cfDNA analysis and 10 ml for Treg studies) will be collected via venipuncture or at the time of intravenous catheter placement/access. Nonclinical samples will be labeled with a study number only to maintain patient confidentiality.

ii. dd-cfDNA Analysis: Whole blood samples will be collected in cfDNA BCT tubes (Streck Inc., Omaha, NE), and plasma will be isolated. cfDNA will be isolated using a customized protocol and quality-controlled with qPCR. Genomic DNA (gDNA) will be extracted, sheared and quality-controlled. Plasma cfDNA libraries will be prepared for shotgun sequencing and sequenced on Illumina HiSeq. Raw sequence reads will be trimmed, filtered and aligned to the human genome to survey for donor and recipient unique SNPs to compute % dd-cfDNA.

iii. cfDNA Methylation Analysis: cfDNA or gDNA will be treated with bisulfite, libraries constructed and sequenced with Illumina NovaSeq. Raw sequence read quality will be checked, reads will be trimmed and then aligned to a human reference genome. After deduplication, cytosine methylation signals will be extracted. The cellular composition for the origins of plasma cfDNA will be deconvoluted using the human cell-type methylation atlas algorithm.

iv. Treg analysis: Treg characteristics, function, and response before, during, and after ECP will be analyzed. Ten (10) mL of whole blood will be collected in heparinized tubes at specified time points at each center from subjects undergoing ECP. Peripheral blood mononuclear cells (PBMCs) will be isolated and Treg populations will be sorted and enriched for downstream functional and phenotypic analyses, including assessments of suppressive capacity, cytokine production, and surface marker expression.

III. DCE-MRI Study Visits and Parameters to be Measured Subjects will be screened to ensure that they meet the basic inclusion/exclusion criteria for the study and have no contraindications to gadolinium administration or MRI. Females of child-bearing potential will have a urine pregnancy test performed prior to each MRI scan to confirm eligibility. All patients will be asked to complete an MRI screening form prior to any research MRI. Weight will be obtained.

MRI: MRI of the thorax will be performed at two time points: 1) prior to the first ECP treatment and 2) following the final ECP treatment, ideally within 2 weeks of final ECP treatment but not more than 4 weeks later.

Spirometry: Spirometry will be performed on the day of and prior to MRI. If spirometry has been obtained within the prior 4 weeks for clinical care, spirometry may not be repeated on the date of MRI.

1. Drugs to be Used Each subject will receive a single dose of gadoterate meglumine (Dotarem®), an FDA-approved, gadolinium-based MR contrast agent. Gadoterate meglumine will be given at a dose of 0.05 mmol/kg as a single intravenous administration.
2. Devices to be Used MR images will be acquired using commercial, FDA-approved 3 Tesla scanners using conventional, FDA-approved, and/or work-in progress sequences for image acquisition.
3. Procedures

i. MR Contrast Administration: An intravenous catheter will be placed, and subjects will be administered gadoterate meglumine (Dotarem®) as a single intravenous injection of 0.05 mmol/kg, which is half of the recommended dose, at a rate of 4 cc/second followed by 20 cc of saline. Gadoterate meglumine is an FDA-approved, gadolinium-based MR contrast agent. A study staff physician will be present during contrast administration to monitor for signs and symptoms of hypersensitivity and hypotension.

ii. Lung MRI Protocol: MR images focused on the thorax will be acquired using commercial, FDA-approved 3 Tesla scanners using conventional, FDA-approved sequences and/or work-in-progress sequences for image acquisition which may include but are not limited to dynamic contrast-enhanced, ventilation/perfusion, and diffusion sequences as well as sequences primarily used to assess anatomic information. Prior to scanning the subjects will be asked to remove metal (including jewelry, etc.) from their clothing and person. Subjects will wear earplugs as hearing protection during scanning. Subjects will lie still in a supine position in the scanner with their head positioned comfortably on a pillow for the duration of the study. Subjects will be in communication with the investigator(s) via a two-way intercom at all times during the imaging study. Subjects will be encouraged to communicate any feelings of discomfort to the investigator immediately. Periodically during the duration of the MRI exam, the investigators will confirm that the subject is comfortable and wishes to continue. The entire imaging session may last up to 60 minutes. A study staff physician will be present during MR image acquisition.

iii. MR Image Analysis: The investigators will perform analyses of contrast kinetics to measure changes in the microvascular and the extravascular extracellular space. Additional analyses may be added based on the sequences obtained. Any of the above findings may be compared to chest imaging that had been obtained clinically on the post-transplant patients.

The images will not be routinely reviewed by a radiologist. If upon image analysis, a potential abnormality happens to be detected, a formal consultation by a radiologist will be requested. If the presence of an abnormality is confirmed, the subject will be contacted by one of the physician investigators. The physician investigator will inform the subject as to the abnormality, answer all questions, and recommend follow-up or additional testing if needed. The physician investigator will also ask for permission from the subject to contact his or her primary care physician or pulmonologist, if applicable. Documentation of the MRI abnormality may be placed in the subject's medical record.

III. Data Collection In addition to information obtained from cfDNA and MR image analyses as detailed above, investigators will review and record clinical data for the subjects enrolled in the study. This includes review of pathology reports from lung biopsies. The collected information will allow us to determine the relationship, if any, between cfDNA and lung MRI findings with outcomes. This data will be stored in a password-protected REDCap (Research Electronic Data Capture) database. For this study, investigators will record basic demographic data (age, sex, gender, race/ethnicity), clinical data (including pathology reports, other diagnoses, medications, smoking history), as well as measurements of pulmonary function (diffusion capacity of carbon monoxide, FEV1, FVC, total lung capacity, performance on six-minute walk test) and various blood or serological markers routinely measured in the clinical management of lung transplant recipients as available. Investigators may also record the results of clinically obtained pulmonary imaging scans to monitor the progression of lung disease and/or additional testing (such as echocardiogram or cardiopulmonary exercise testing) that may have been performed. In addition to recording the above data at study entry (baseline), investigators will also access the patient's electronic medical records periodically to record subsequent pulmonary function test (PFT) results and other relevant follow-up data, such as hospitalizations and vital status approximately every three months up to an indefinite amount of time from the time of study completion.

Inclusion Criteria:

• Adults (≥18 years) or pediatric patients (7-17 years) who have had a single or double lung transplant
• Diagnosed with CLAD (any stage) and prescribed ECP therapy
• Ability to provide informed consent (or assent with parental consent for minors)

Exclusion Criteria:

• Previously received ECP treatment
• Receipt of anti-thymocyte globulin within 3 months or alemtuzumab within 12 months of ECP initiation
• Contraindications to MRI (for MRI subgroup), including metal implants, claustrophobia, seizure disorder
• Known allergy to gadolinium or impaired kidney function (eGFR <30 ml/min)
• Pregnant or breastfeeding

Patients who have undergone lung transplantation, developed CLAD and been prescribed ECP therapy in addition to standard of care immunosuppression by their transplant clinician.

We will make a decision in the future.