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Research Highlights

Flossmann, Viktoria1; Issa, Fadi DPhil, FRCS2

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doi: 10.1097/TP.0000000000003794
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Cell-free DNA to Detect Heart Allograft Acute Rejection

Agbor-Enoh S, Shah P, Tunc I, et al. Circulation. 2021;143(12):1184–1197.

For surveillance, heart allograft recipients are required to undergo endomyocardial biopsies as the gold standard for detection and grading of acute rejections.1 However, the procedure is invasive, prone to sampling errors, and results show high variability. These limitations warrant the search for new diagnostic tools. In this regard, cell-free DNA is gaining interest as a method for noninvasive monitoring.2

In their multicenter prospective cohort study, Agbor-Enoh et al, including the GRAfT investigators, validated an elegant technique to detect both acute cellular rejection (ACR) and antibody-mediated rejection (AMR) by measuring the amount of donor-derived cell-free DNA (ddcfDNA) in recipients’ plasma.3 One hundred seventy-one heart transplant recipients were included in the study and followed up for a median of 18 months. ddcfDNA was measured by shotgun sequencing at several timepoints with concurrent ECG to screen for graft dysfunction, and endomyocardial biopsies to diagnose and grade acute rejection episodes. The ratio of ddcfDNA/total cell-free DNA in recipients was correlated with histologically confirmed ACR, AMR, and controls.

The authors were able to demonstrate a strong positive correlation between the level of ddcfDNA and the severity of acute rejection graded by histology (AUROC 0.92). Levels of ddcfDNA were 13-fold higher in acute rejection compared with mild-to-no rejection. This correlation was also observed separately for ACR and AMR, as well as for the severity of allograft dysfunction with an 81% sensitivity and 85% specificity for overall acute rejection, leading to a high-negative predictive value of 99.2%. Seventy-six percent of “biopsy-negative” cases with increased levels of ddcfDNA were found to either occur with simultaneous graft dysfunction, preceding acute rejections or graft dysfunction. The authors convincingly show that this diagnostic tool could possibly avoid 81% of routine endomyocardial biopsies. One crucial finding was the ability to distinguish episodes of ACR and AMR and to predict their occurrence based on higher ddcfDNA levels and an earlier increase in AMR (30-fold higher ddcfDNA levels; 3.2 mo before biopsy-proven rejection) versus ACR (9-fold higher levels; 0.5 mo before biopsy-proven rejection). Fragment lengths and genomic composition also differed allowing early discrimination between rejection types and appropriate earlier treatment mitigating damage to the graft.

The presented results support the use of ddcfDNA as a noninvasive biomarker to detect acute rejection with excellent evidence supporting its integration into clinical practice in the future.

Graft-derived Extracellular Vesicles Transported Across Subcapsular Sinus Macrophages Elicit B-cell Alloimmunity After Transplantation

Zeng F, Chen Z, Chen R, et al. Sci Transl Med. 2021;13(585):eabb0122.

Allosensitization reflects priming of the recipient immune system upon contact with alloantigens, resulting in lymphocyte activation, and, most commonly, the production of detrimental donor-specific antibodies (DSAs). Treatment of antibody-mediated rejection is challenging and the risk for graft loss is high.4 In recent years, extracellular vesicles (EVs) have gained attention as potentially contributing to this sensitization.

Using mouse models for heart and skin transplantation, Zeng and coworkers show that EVs loaded with intact donor major histocompatibility complex are released by the graft and travel into recipient graft-draining lymph nodes where they are received by specific macrophages, then further proceed into B-cell follicles where they initiate allosensitization and DSA production.5

The authors demonstrated that donor antigen-presenting cells (APCs) circulating through the recipient lymphatic tissue as passenger leukocytes, as generally assumed, were not responsible for inducing rejection since no fluorochrome-marked donor APCs were retrievable in recipient’s graft-draining lymph nodes. In knockout allografts in which leukocytes were not able to leave the graft, rejection was still observed. The authors next confirmed crossdressing of recipient APCs with donor EVs by ImageStream, a combination of high-resolution microscopy, flow cytometry, and immunoelectron microscopy. The involvement of graft-infiltrating leukocytes in crossdressing was ruled out in the study. Instead, donor alloantigen was detected bound to EVs within subcapsular sinus macrophages (SSMs) and in association with B and dendritic cells in B-cell follicles.

The insignificance of passenger leukocytes in this process, and the presence of crossdressed EVs and donor-derived cell-free content in SSMs, was also demonstrated for human skin grafts on humanized mice. In the nonhumanized mouse studies, EVs were immediately taken up by SSMs, trafficked unidirectionally through these cells by forming tunnel-like structures, and recognized by underlying B cells via their B-cell receptors. Only physically intact EVs were bound by B cells. The relevance of SSMs to allosensitization of B cells was demonstrated by depletion of SSMs with low-dose clodronate liposomes resulting in less uptake of EVs, reduced B-cell activation, and lower DSA titers in recipient’s serum. Importantly, pretreatment with a pharmacological inhibitor of small EV biogenesis led to reduced crossdressing, decreased serum DSA levels, and prolonged graft survival.

This study challenges widely held theories on allorecognition while providing crucial information about the role of EVs in this process. Moreover, targeting EVs may pave the way for new therapeutic strategies controlling rejection and preventing allosensitization.


1. Nikolova AP, Kobashigawa JA. Cardiac allograft vasculopathy: the enduring enemy of cardiac transplantation. Transplantation. 2019;103:1338–1348.
2. Knuttgen F, Beck J, Dittrich M, et al. Graft-derived cell-free DNA as a noninvasive biomarker of cardiac allograft rejection: a cohort study on clinical validity and confounding factors. Transplantation. [Epub ahead of print. February 25, 2021]. doi:10.1097/TP.0000000000003725
3. Agbor-Enoh S, Shah P, Tunc I, et al.; GRAfT Investigators. Cell-free DNA to detect heart allograft acute rejection. Circulation. 2021;143:1184–1197.
4. Jordan SC, Ammerman N, Choi J, et al. Novel therapeutic approaches to allosensitization and antibody-mediated rejection. Transplantation. 2019;103:262–272.
5. Zeng F, Chen Z, Chen R, et al. Graft-derived extracellular vesicles transported across subcapsular sinus macrophages elicit B cell alloimmunity after transplantation. Sci Transl Med. 2021;13:eabb0122.
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