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Graft Source-dependent Biomarkers for Risk of Chronic Graft-Versus-Host Disease and Death

ABSTRACT: We identify risk markers and graft specific markers at day 90 post-HCT (hematopoietic cell transplant). For this, we developed a 4-plex TMT plasma proteomic approach comparing day-90 samples from PB (peripheral-blood stem cells) graft recipients who developed cGVHD by day 180, PB graft recipients who did not develop cGVHD, BM (bone marrow) graft recipients who developed cGVHD by day 180, BM graft recipients who did not developed cGVHD. Five markers were candidate risk markers and selected to move to the validation phase. Using ELISA assays these 5 markers were measured along with nine previously identified cGVHD proteomic biomarkers (ST2, CXCL9, MMP3, OPN, CXCL10, soluble CD163, IL17, B-cell activating factor (BAFF), and C-C-motif-chemokine-15 (CCL15)16,23-29 in 335 recipients (from CTN0201 to test if they were markers of risk and graft-specific markers). Finally, we measured the six significant candidate risk markers in CTN0201 in 653 patients from the CTN1202 study, an independent contemporary cohort.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Sophie Paczesny

PROVIDER: MSV000088585 | MassIVE | Fri Dec 17 12:28:00 GMT 2021

REPOSITORIES: MassIVE

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Project description:Measurement of specific gene expression in clinical samples is a promising approach for monitoring the recipient immune status to the graft in organ transplantation. Identification of biomarker genes closely associated with tolerance or rejection is critical for this monitoring protocol. Unlike previous studies, our microarray analysis focused on donor antigen-reactive T cells, which were prepared by collecting CD69+ T cells from cocultures of recipient peripheral T cells and donor antigen-presenting cells. A comparison of different recipient groups enabled us to identify several tolerance- and rejection-correlated biomarker genes, including previously unknown genes. By measuring biomarker gene expression in the CD69+ T cell fraction using quantitative reverse-transcription polymerase chain reaction, we were able to precisely detect the immune status of recipients relative to their graft. Full-thickness donor (BDF1; C57BL/6 x DBA/2) tail skin was grafted onto recipient (C57BL/6) mice. Tolerance to the graft was induced by donor specific transfusion combined with administration of anti-CD40L Ab (MR-1). Total T lymphocytes were prepared from spleen and lymph nodes of tolerant recipient mice on day 21 and 100 after skin transplantation, untreated recipient mice on day 7, and ungrafted, healthy C57BL/6 mice and used as early tolerant, tolerant, rejecting and naïve T lymphocyte samples, respectively. In order to isolate alloantigen-reactive cells, T lymphocytes were cocultured with donor BM-DCs for 20-21 hours and then separated into two subpopulations based on CD69 expression using fluorescence cell sorter. Total RNA was extracted from these CD69+ and CD69- T cells and subjected to microarray using Illunima MouseRef-8 Expression BeadChip. All data analysis and visualization of differentially expressed genes was conducted using Illumina BeadStudio v3.1.3 (Gene Expression Module v3.3.8), Array Assist 5.5.1 (Stratagene) and R 2.4.1(www.r-project.org). Candidate tolerance genes were defined as the common genes satisfying the following criteria; 1) 1.5 fold or higher expression in CD69+ T cells isolated from tolerant recipients than those from naïve mice, 2) 1.5 fold or higher expression in CD69+ T cells than CD69- T cells from tolerant mice, 3) 500 or higher average signal measured in CD69+ T cells from tolerant mice. The criteria for candidate rejection genes were 1) 1.5 fold or higher expression in CD69+ T cells isolated from rejecting recipients than those from tolerant mice, 2) 1.5 fold or higher expression in CD69+ T cells than CD69- T cells from rejecting mice, 3) 500 or higher average signal measured in CD69+ T cells from rejecting mice.

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Graft Source-dependent Biomarkers for Risk of Chronic Graft-Versus-Host Disease and Death

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