Plasma and Radiologic Biomarkers of Response to ECP in Lung Transplant Recipients With CLAD

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.