Pre-Transplant T-Cell Clonal Analysis Identifies CD8+ Donor Reactive Clones That Contribute To Kidney Transplant Rejection Following Two Different Induction Modalities

Introduction: Responses to allogeneic human leukocyte antigen (HLA) molecules limit the survival of transplanted organs. The changes in T-cell alloreactivity that contribute to this process, however, are not fully understood. We defined a set of donor reactive T-cell clones (DRTC) with the goal to elucidate signatures of kidney allograft rejection. Methods: DRTC were identified pretransplant using an anti-donor mixed lymphocyte reaction assay: CFSE-diluting CD4+ and CD8+ DRTC were flow-sorted, and the TCR sequences were identified using Adaptive Immunosequencing. DRTC were then tracked in post-transplant biopsies, blood, and urine samples in a cohort of kidney transplant recipients. Results: In patients with an abnormal biopsy, the majority of CD8+ DRTC found within the allograft were detected in the circulating pre-transplant repertoire. Circulating CD8+ DRTC were more abundant pre- and post-transplant in patients that received non-lymphodepletional induction and developed an abnormal biopsy when compared to stable patients. Additionally, DRTC were detected as early as two weeks post-transplant in the urine of some patients, with some of these clones subsequently identified in follow-up kidney biopsy samples. Discussion: The findings of our study add to our understanding of T-cell alloreactivity following kidney transplantation and provide evidence for the role of pre-defined alloreactive T-cells in the development of allograft rejection. Study Subjects: This was a single-center, non-randomized prospective observational study performed at the Comprehensive Transplant Center at Northwestern University Feinberg School of Medicine. Kidney transplant recipients were enrolled between January 2018 and December 2019. Enrolled subjects were followed for one year with sample collections obtained at designated protocol visits. Clinical laboratory values, post-transplant complications, and outcomes were assessed at 3 and 12 months post-transplant. Informed consent was obtained from all subjects. The study protocol was approved by the Northwestern Institutional Review Board (IRB number: STU00206157), and no organs/tissues were procured from prisoners. Informed consent was obtained from all subjects after the nature and possible consequences of the study were explained. Samples: Blood samples were obtained from donors and recipients prior to transplantation, and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque gradient centrifugation. During the post-transplant period, recipient blood samples were serially collected at 3-months, 6-months, 12-months and any potential rejection episode(s). 50 to 100 milliliters of urine were collected from recipients at 2 weeks post-transplant (as a baseline post-transplant), 3-months, 6-months and 12-months post-transplant, and any possible rejection episode(s). Cell components were isolated by centrifugation at 300g and were frozen as cell pellets at -80oC for DNA isolation and Adaptive immunosequencing. Protocol allograft biopsies were acquired at the back-table before implantation, 3-months and 12-months post-transplant, as well as any potential rejection episode (i.e., “for-cause” biopsy). The decision to perform a “for-cause” biopsy was left up to the treating physician based upon clinical presentation and laboratory values. A second pass biopsy was also obtained, which was frozen dry at -80oC for DNA isolation and Adaptive immunosequencing. Mixed Lymphocyte Reaction: PBMCs were isolated from recipient and donor blood samples obtained before transplantation. Recipient PBMCs were labeled with CFSE, and donor cells were labeled with PKH26 and irradiated (3000 rad). The labeled recipient and irradiated donor cells were then cultured in bulk in RPMI-1640 supplemented with 15% AB serum, at 37oC, 5% CO2, and a concentration of 1x106 cells/mL. After 7 days of culture, cells were harvested and labeled with anti-CD3, anti-CD4, and anti-CD8 monoclonal antibodies (Beckman-Coulter, Miami, FL, or Becton-Dickinson [BD], San Jose, CA). CD8+ and CD4+ donor reactive T-cells were then sorted on FACSAria (BD, San Jose, CA) by gating on the CFSE dim population (i.e., proliferating responder cells), after gating out both CFSE high cells (i.e., recipient non-proliferating cells) and residual PKH26+ donor stimulator cells. Flow-sorted T-cells were frozen as cell pellets at -80oC for future DNA isolation and Adaptive immunosequencing. DRTC Identification and Monitoring: Donor reactive T cells clones (DRTC) were identified utilizing differential abundance analyses generated for each subject. In brief, each rearrangement with a combined total count of at least 5 is treated as a fixed number of “trials” (Bernoulli experiments) in a two-sided binomial test. The probability, p, of the observed template counts in each sample is calculated under the null hypothesis that these templates are evenly distributed between the two samples, relative to their respective repertoire sizes (i.e., the total productive template count of each sample). Rearrangements that are more unequally distributed relative to this expected proportion will result lower probabilities. A rearrangement is considered differentially abundant if it satisfies two criteria: 1) p < 0.01 after applying the Benjamini-Hochberg adjustment procedure to control false discovery rate (FDR); and 2) the rearrangement has a frequency at least 2-fold higher in one sample than in the other. In the context of DRTC, the proliferated CD8+ or CD4+ sorted cell populations were compared against the unstimulated, pre-transplant PBMC sample. DRTC were then defined as clones that were significantly, and at least 2-fold, more abundant in the MLR sort relative to the unstimulated pre-transplant sample in order to identify those that may be most biologically relevant (i.e., expand more readily post-transplant). Once DRTC were identified, they were then monitored over time in the blood, urine, and allograft biopsy samples obtained from recipients (described above). DRTC metrics used for analysis included the absolute number of DRTC, the frequency of these clones within the entire T cell repertoire (i.e., depth), and proportion of all unique TCR clones detected that were DRTC (i.e., breadth). The Morisita Index (MI) and Jaccard Index (JI) were used to compare the similarities of the bulk repertoire between two, independent samples. Both indices are scored 0-1, with 0 being completely divergent repertoires and 1 being identical. *************************************************************** Adaptive Biotechnologies data ***************************************************************