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    Biomaterials

    (B-64) Tissue Engineering Scaffolds Remotely Surveil Presymptomatic Allograft Rejection and Heterologous Immunity

    Friday, October 13, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row B - Poster # 64
    Introduction:: As there is no assay to predict the risk of acute cellular allograft rejection (ACAR), clinicians rely on graft biopsy and aggressive, one-size-fits-all immunosuppression. Endomyocardial biopsy (EMB) is a flawed standard for heart transplant surveillance and diagnosing ACAR, as histological evidence of rejection inherently lags behind molecular biomarkers and suffers from variability. Non-invasive alternatives to EMB in ACAR surveillance, including gene profiling and donor-derived cell-free DNA, also measure lagging indicators of ACAR. A minimally invasive surveillance method is urgently needed to quantify early risk of rejection for minimizing biopsy and personalizing immunosuppression. We have developed porous biomaterial “scaffolds” for minimally-invasive sampling (Figure 1). These scaffolds amass immune cells which produce biomarkers predictive of ACAR. In this work, we expand on prediction of acute rejection to identify sensitivity to tolerance-inducing costimulatory blockade treatment.

    Materials and Methods:: Microporous Scaffold Generation. Microporous polycaprolactone scaffolds were generated as described previously28,33,34. Briefly, microporous polycaprolactone (PCL) scaffolds were generated by first mixing 99 gm of sieved NaCl (250-425μm) with 3 gm of PCL pellets in a Ross Mixer at 85°C for one hour. 77.5 mg of the PCL-NaCl melt dispersion was measured and pressed into 5 or 6-mm diameter scaffolds. Scaffolds were heated at 65°C for 5 min on each side to anneal the PCL. Upon cooling, the scaffolds were immersed in water three times for one hour to dissolve the NaCl porogen.

    Scaffold Implants in Transplant Recipients. Mice were anesthetized with isoflurane for subcutaneous implantation of scaffolds and serial scaffold explants. Mice received carprofen immediately before surgery and 24 hours after surgery. Four to six scaffolds were implanted subcutaneously 14 days prior to inducing graft rejection in Rag2KO mice or prior to transplant in immunocompetent mice. After vascularization and cell in-growth, scaffolds were then explanted before and during graft rejection. Blood (serial points) and primary grafts (terminal points) were also collected.


    RNA Sequencing. For high-throughput gene expression analysis, bulk RNA sequencing analysis and quality control were performed on a fee-for-service basis by the UM Advanced Genomics Core. For RNAseq a total of n = 6 samples per condition were analyzed from each time point (day 0, 5, 9, or 14).
    Mixed Lymphocyte Assay.     T cells derived by scaffolds from C57Bl/6 (B6) mice previously infected by influenza A virus (IAV) or sensitized by an i.p. injection of BALB/c splenic cells.



    Results, Conclusions, and Discussions:: We identified predictive biomarkers of ACAR conserved in both murine heterotopic heart transplant and skin transplant through the remote scaffold. We identified 17 highly differentially-expressed genes as graft-agnostic biomarkers of T cell-mediated rejection. These novel biomarkers differentiate between pre-symptomatic ACAR and recipients with healthy allografts, without invasive biopsy. Scaffold cell capture technology provides a leading indicator of ACAR by monitoring tissue-specific immune responses associated with allograft rejection (Figure 2). This implantable scaffold enables minimally invasive histological evaluation and molecular calculation of the early risk of rejection to reduce the frequency of routine EMB and personalize immunosuppression that could prolong transplant life while minimizing risks.

    In reality, humans would have been exposed to various pathogens prior to organ transplant which may lead to subsequent graft cross-reactivity after transplant. Therefore, we used scaffolds to study the influence of previous viral infections on allograft rejection. We performed mixed lymphocyte reactions using T cells derived from C57Bl/6 (B6) mice previously infected by influenza A virus (IAV) or sensitized by an i.p. injection of BALB/c splenic cells. These B6 T cells were co-cultured in vitro with irradiated dendritic cells from BALB/c mice, representative of our graft donor strain. The results indicated that compared to naive mice, IAV-infected mice have a higher population of T cells that are reactive to allograft antigens (Figure 3). Thus, we have confirmed that virus-induced T cells have cross reactivity to allografts. We are now underway studying how the implantable scaffold captures this history of viral infection and its influence on allograft tolerance induction in an anti-CD40L, CTLA-4 Ig costimulatory blockade model of murine heterotopic heart transplants.

     



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