Guido Moll ^1* Andreas Beilhack ²

• ¹ Charité University Medicine Berlin, Berlin, Baden-Wurttemberg, Germany
• ² University Hospital Würzburg, Würzburg, Bavaria, Germany

This Research Topic is part of the "Methods in Immunology" series, which highlights cuttingedge techniques and methods used to investigate fundamental questions in immunology research, with a focus on Alloimmunity and Transplantation (Figure 1) [1,2]. Alloimmunity is the immune response to alloantigens -immunogenic molecules from members of the same species, including blood group antigens and the highly polymorphic antigens of the major histocompatibility complex / human leukocyte antigens (MHC / HLA) (Figure 1A) [3][4][5].Alloantigens can be classified as major and minor mismatch antigens, which is distinguished from xenoantigens / xenoreactivity against different species, and autoantigens / autoimmunity against self-antigens [1, [3][4][5][6][7][8][9]. Alloantigens can trigger the formation of alloantibodies through alloantigen-primed B-cells and mature plasma cells (e.g. panel-reactive vs. donor-specific antibodies, PRA vs. DSA, respectively), as part of the humoral immune response, and the activation of effector T-lymphocytes, as part of the cellular immune response, with further amplification through secondary immune cell activation and infiltration [1,[3][4][5][6][7][8][9]. Both, humoral and cellular alloimmune responses can lead to acute and chronic graft rejection in solid organ transplantation (SOT), and graft failure or graft-versus-host disease (GVHD) in hematopoietic (stem) cell transplantation (HCT/HSCT) [10][11][12][13][14]. The most common SOT modalities include transplantation of kidneys (KTx), liver (LiTx), lungs (LuTx), heart (HTx), and vascularized composite allografts (VCA, e.g. hand, and face Tx) [1,2,15,16]. Successful allo-Tx requires precise "tissue matching" and optimal "IS protocols", to reduce the risk of immune rejection and to minimize IS toxicity [17]. Current advancements in alloimmunity and transplantation (Figure 1B) include next generation sequencing (NGS) for transcriptome analysis at both bulk or single cell levels (RNAseq and scRNAseq) [18,19], T-and B-cell receptor repertoire sequencing (TCRseq and BCRseq) [20][21][22], NGS analysis of donor-derived cell-free DNA (dd-cfDNA) [23][24][25][26], sophisticated in vitro and vivo models to study transplant rejection [14,27,28], but also novel concepts of transplantation (Tx), immunosuppression (IS), and patient care [17,29], including advanced therapy medicinal products and cell and gene therapies (ATMPs and CGTs) [1,12,17,[30][31][32][33][34][35]. Adjunct technologies include machine perfusion of donor organs, novel renal replacement therapies (RRTs), but also the exponentially increasing use of advanced bioinformatics, systems biology, and artificial intelligence, for optimal analysis and interpretation of increasingly complex / large data sets [1,[35][36][37][38][39][40][41][42]. recipients to assess the relevance of dd-cfDNA for detecting acute and chronic rejection, or infection one month post LuTx [23][24][25]. Total cfDNA was quantified with fluorimetry and digital PCR, cfDNA fragment size with BIABooster (Adelis), and dd-cfDNA with NGS (AlloSeq). While total cfDNA levels did not correlate with patient outcomes, higher dd-cfDNA were linked to graft injuries at d30 after LuTx (P=0.0004). A threshold of 1,72% dd-cfDNA effectively identified patients with healthy grafts, while higher levels of small dd-cfDNA indicated chronic injection or infection with 100% specificity.