(J-390) Predicting and Modeling Resistance to Organ Allograft Tolerance using a Minimally-Invasive Implant

While solid organ transplantation is the only life-saving procedure for many end-stage organ failures, patients require lifelong immunosuppression (IS) to prevent graft rejection, often leading to increased susceptibility to infections, malignancies, and renal impairment. These risks are particularly burdensome to pediatric and young adult heart transplant (HTx) recipients who must undergo aggressive IS for the duration of their lives. Current methods of monitoring allograft rejection are limited due to their high costs, invasiness, and reliance on lagging biomarkers of tissue damage. To address these limitations, the Shea lab has developed minimally invasive subcutaneous scaffold implants that can become vascularized and continuously sample immune cells from circulation. These scaffolds serve as an immunological niche and can be biopsied more frequently and less invasively, allowing for facile monitoring of acute cellular allograft rejection (ACAR). In this study, we sought to analyze changes in gene expression and cell phenotype from the scaffold to predict allograft rejection in murine heterotopic HTx recipients. Ultimately, a scaffold that predicts ACAR by capturing immune responses that precede graft injury could be used to personalize IS regiments to minimize rejections as well as their serious side effects. 

Scaffolds were manufactured by mixing poly(ε-caprolactone) (PCL) with NaCl porogen (250-425µm), pressed into molds (5mm diameter, 2mm thick), sintered at 135ºC, porogen-leached, and stored at -80°C until surgery. Heterotopic HTx surgeries were performed from BALB/c donors to B6 recipient mice in 3 cohorts: naive, highly sensitized, and lowly sensitized, which received either 0, 2 million, or 200,000 B6 splenic cells 28 days prior to HTx, respectively. Twenty-one days prior to HTx, 6 scaffolds were subcutaneously implanted into every mouse. Scaffolds were biopsied 1 day prior to HTx, as well as 10, 15, 20, 25, and 30 days after HTx, and stored in RNAse free tubes at -80°C. The mice received CTLA-4 Ig (500 μg) co-stimulatory blockade IS drug 2 days after HTx, as well as αCD40L (400 μg) on days 2, 4, 6, and 8 post-HTx. Transplanted grafts were monitored weekly for rejection progression using ECG and Doppler echocardiography and scored on the heart rate. The scaffolds were processed and sent to University of Michigan’s Advanced Genomic Core for bulk and single cell RNA sequencing. 

The naive mice had 100% graft survival at the end of the experiment time course 40 days post-HTx, while 30% of the highly sensitized mice began rejecting their grafts 18 days post-HTx. Twenty days post-HTx, 100% of the highly sensitized mice had rejected their grafts while 60% of lowly sensitized mice had rejected their allografts. Forty days post-HTx, the lowly sensitized mice continued to have a 40% graft survival rate. The highly sensitized mice rejected much quicker than the lowly sensitized mice due to prior exposure of the donor antigen, allowing their immune systems to recognize the foreign graft and reject it earlier. We are currently waiting for the Genomic Core to return our bulk and single cell RNA-seq results, but once we retrieve those data, we will perform an elastic net regression and through singular value decomposition and supervised machine learning, we will determine differential gene expression and the likelihood that a mouse will reject the transplanted graft. This information will show immune biomarkers associated with ACAR progression and gene expression of these scaffolds, which can indicate a therapeutic window in which the IS regiment can be altered to preserve graft functionality.