Heart Transplantation Research: Investigation into Cardiac Allograft Rejection

Clinical Center

Investigators

Abstract

Animal Studies: A. ACR and infection (INF) are significant sources of morbidity & mortality after heart transplant (HT), accounting for nearly 50% of deaths. It can be difficult to clinically distinguish between ACR & INF as both are inflammatory processes with similar nonspecific symptoms. However, differentiation is essential for therapy. This ACUC approved protocol (n=124, closed 2012) studied whether gene microarray analysis of PBMCs would differentiate ACR from INF in a HT rat model. We initially studied the impact of animal strain on gene expression during ACR (BMC Genomics 10:280, 2009) & time course of post-surgical inflammatory changes to determine an opportune time to harvest transplanted hearts (i.e. when gene signatures from surgical inflammation are dissipating). The completed main protocol combined 2 established models, a heterotopic HT (HHT) model & an E. coli pneumonia model. On day 0 rats underwent HHT & received daily cyclosporine (CSA; 10 mg/kg SQ). On Day 6 rats were randomized to CSA withdrawn (initiate ACR) or continued (suppress ACR). On Day 13 (CSA discontinued) rats were randomized to intrabronchial E. coli or saline inoculation. Thus, 4 groups (2 by 2 design) were studied: no ACR with & without INF; ACR with & without INF. On day 14, rats were sacrificed & blood/heart removed for gene microarray analysis. Hearts/lungs/spleen/liver/thymus were procured & preserved for future analysis. We continue to analyze data related to metabolic effects of ACR on cellular energy metabolism (JHLT 36 (4S):S372-S373, 2017); temporal effects of surgical inflammation on gene expression; & effects of ACR and/or INF on gene expression. We continue to report due to ongoing collaborations & available sample library. As new genomic techniques become available we may do further data processing/analysis of the completed studies & their stored samples. B. Initial treatment in solid organ transplant is usually triple immunosuppression followed by maintenance with 1-3 immunosuppressive (IM) agents. One cannot withdraw drugs confidently even if the graft seems tolerant. Long term administration of IM agents results in an increase in morbidity & mortality. Establishment of tolerance without nonspecific IM drugs is a major goal. Induction of recipient (host) tolerance to histocompatibility antigens of the organ donor could eliminate need for long term usage of nonspecific IM drugs. This would have a major impact on quality/quantity of life for patients (pts) with long term surviving organ grafts by reducing the immunologic/non-immunologic complications associated with long term IM therapy. An established donor-based immunotolerance animal model involves injection of donor splenocytes plus a single injection of the T-cell suppressing agent anti-rat lymphocyte serum or RIB 5/2. Recipient based immunotolerance induction involves adoptive transfer (altering the hosts immune system using lymphoid cells from another subject) of ex vivo generated alloantigen-specific regulatory T cells. In HTs, recipient-based immunotolerance induction is clinically more applicable than donor-based as donor genotype is rarely known prior to transplant. The ACUC approved (2006-12; n=403) protocol examined donor & recipient-based immunotolerance induction protocols & evaluated for protein/gene changes that could serve as candidate biomarkers of tolerance. The protocol was 2 part: Part 1 (recipient-based tolerance); Part 2 (donor-based tolerance). In Part 1, Stage 1 we generated Th2.rapa cells from recipient BN rats. In Part 1, Stage 2 these adoptively transferred ex vivo generated BN Th2 cells were tested in culture using flow & cytokine phenotype tests, & an optimal rapamycin dose determined. In Part 1 Stage 3 we completed studies designed to determine if Th2-shifted hosts (recipient-based tolerance) have reduced rejection. In Part 2 (donor-base tolerance), Stage 1 (induction donor-based tolerance) we removed the spleen of DA rats donating hearts in stage 2 & successfully injected its processed splenocytes into the thymus of BN rats receiving the donor heart in stage 2. In Part 2, Stage 2 we completed studies designed to determine if donor-based tolerance induction reduces rejection. A manuscript was published (PloS One, 6(4): e18885, 2011) establishing the ability of host-type Th2. Rapa cell therapy given pre-transplant to shift post-transplant cytokines towards a Th2 phenotype & prolong allograft viability when used in combination with short course CSA. We continue to report on the project, since as new genomic techniques become available we may do further data processing/analysis of the completed studies & their stored samples. Human Study: We are applying functional genomics, detection of donor derived cell free DNA (ddcfDNA), and Peptidomics to study ACR & CAV. By correlating putative biomarkers with clinical, histological, and imaging evidence of allograft disease we hope to build a database of Genomic/Peptidomics data relevant to the immunologic relationship between donor organ & recipient. Blood/urine specimens were obtained serially from HT recipients during periods of allograft immunological tolerance (no ACR) and intolerance (ACR) and from HT recipients with & without CAV. Samples will be analyzed to determine whether unique gene and/or protein/peptide expression patterns are associated with each state. Detection of ddcfDNA in recipient blood can serve as a diagnostic tool of graft injury. Collaborating with the NHLBI Lab of Applied Precision Omics (APO), we embarked on biomarker discovery using our samples and their genomic approaches and also banked a subset of our plasma samples (639) to explore biomarker discovery schemes through nanotechnique-based peptidomic strategies. We shared a subset of plasma (1,205) & urine (74) samples with APO (2015-16). Urine samples were analyzed to determine the physical properties of cfDNA in urine to aid in protocol development (JHLT 35(4S):S16, 2016). From the plasma samples, 101 have been analyzed for cfDNA (JHLT 35(4S):S161, 2016; JHLT 36(9): 1004-12, 2017). We also reviewed histologic slides that were coincident to 350 biospecimen time-points, performing consensus histopathology reads (2016-17) by 2 expert transplant pathologists. Data from consensus reads and the original clinical histopathology dataset enabled concordance analyses. Through these analyses, we can now identify biosample time-points with concordance biopsy results for future collaborative studies. Aided by the consensus reads we identified 3 pts with allograft antibody-mediated rejection (AMR) and analyzed their plasma samples for %ddcfDNA (2017-18). APO is combining this cohort with theirs & a Stanford cohort (increased sample size for anticipated further analysis). APO developed 2 cfDNA approaches with increased specificity compared to SNP approach. APO methods rely on tissue specific methylation patterns (PMID: 33651717) or chromatin-immunoprecipitation signatures. APO will apply both to develop cfDNA tactics that detects rejection as reliably as the SNP method, and has increased specificity to 2 different rejection phenotypes:-AMR & ACR. GRAfT cohort (ClinicalTrials.gov: NCT02423070) will serve as derivation cohort and this cohort as part of a validation. The protocol (n=187; 145 pts/42 healthy volunteers (HV)) is closed to new HT candidates (2015), follow-up of enrolled pts (2015) and matched HV (completed 2019). The protocol created at NIH a biobank of samples from HT subjects with well-characterized clinical phenotypes. The protocol obtained a minimum of 1 yr of sample collections from all actively followed HT pts. We collected blood/urine samples during 621 EBxs. On average from each biopsy time point we stored 5 plasma/4 serum/1 RNA/1 urine sample. The study remains open for data analysis in collaboration with APO.