Heart Transplantation Research: Investigation into Cardiac Allograft Rejection

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Animal Studies: A. ACR and infection are significant sources of morbidity and mortality after heart transplant, accounting for nearly 50% of deaths. It can be difficult to clinically distinguish between ACR and infection as both are inflammatory processes with similar nonspecific symptoms. However, differential is essential for therapy. This ACUC approved protocol (n=124, closed 2012) studied whether gene microarray analysis of PBMCs would differentiate ACR from infection in the transplanted rat. We initially studied the impact of animal strain on gene expression during ACR (BMC Genomics 10:280, 2009) and the time course of post-surgical inflammatory changes to determine an opportune time to harvest transplanted hearts (i.e. when gene signatures due to surgical inflammation are dissipating). The completed main protocol combined 2 established models, a heterotopic heart transplant model and an E. coli pneumonia model. On day 0 rats underwent heart transplant and received daily cyclosporine (CSA; 10 mg/kg SQ). On Day 6 rats were randomized to have CSA withdrawn (initiate ACR) or continued (suppress ACR). Day 13 after transplant (CSA discontinued) rats were randomized to receive intrabronchial E. coli or saline inoculation. Thus, 4 groups (2 by 2 design) were studied: no ACR with and without infection; ACR with and without infection. On day 14, rats were sacrificed and the blood and heart removed for gene microarray analysis. The hearts, lungs, spleen, liver, and thymus were procured and 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); the temporal effects of surgical inflammation on gene expression; and the effects of ACR and/or infection on gene expression. We continue to report due to ongoing collaborations and available sample library. As new genomic techniques become available we may do further data processing and analysis of the completed studies and 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 and mortality. Establishment of tolerance without nonspecific IM drugs is a major goal. The induction of recipient (host) tolerance to histocompatibility antigens of the organ donor could eliminate the need for long term administration of nonspecific IM drugs. This would have a major impact on the quality/quantity of life of patients with long term surviving organ grafts by reducing the immunologic and 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 heart transplants, 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 and recipient-based immunotolerance induction protocols and evaluated for protein/gene changes that could serve as candidate biomarkers of tolerance. The protocol was 2 part: Part 1 (recipient-based tolerance); and 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 and cytokine phenotype tests, and 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 and 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 and 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 and analysis of the completed studies and their stored samples. Human Study: We are applying functional genomics, detection of donor derived cell free DNA (ddcfDNA), and peptidomics to study ACR and CAV. By correlating putative biomarkers with clinical, histological, and imaging evidence of allograft disease we hope to build a database of genomic/peptidomic data relevant to the immunologic relationship between donor organ and recipient. Blood/urine specimens were obtained serially from heart transplant recipients during periods of allograft immunological tolerance (no ACR) and intolerance (ACR) and from transplant recipients with and 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 Laboratory of Transplantation Genomics (LTG), we embarked on biomarker discovery using our collected samples and their genomic approaches. Peptidomics is also a promising diagnostic tool. We are planning a collaboration to explore biomarker discovery schemes using a subset of our plasma samples (639) and nanotechnique-based peptidomic strategies. We shared a subset of plasma (1,205) and urine (74) samples with LTG (2015-16). The 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 and performed consensus histopathology reads (2016-17) by 2 expert transplant pathologists. The data from the 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 subjects with allograft antibody-mediated rejection and analyzed their plasma samples for %ddcfDNA (2017-18). LTG is combining this cohort with theirs and a Stanford cohort in anticipation of increasing sample size for further analysis. The protocol (n=187; 145 patients/42 healthy volunteers (HV)) is closed to new transplant candidates (2015), follow-up of enrolled patients (2015) and matched HV (completed 2019). The protocol created at NIH a biobank of samples from transplant subjects with well-characterized clinical phenotypes. The protocol obtained a minimum of 1 yr of sample collections from all actively followed transplanted patients. We collected blood/urine samples during 621 EBxs. On average from each biopsy time point we stored 5 plasma, 4 serum, 1 RNA, and 1 urine sample. The study remains open for data analysis.