This Research Topic is part of the “Methods in Immunology” series, which highlights cutting-edge 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 ) (3–5). 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, 3–8). 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, 3–8). 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) (9–12). 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, 13, 14). Successful allo-Tx requires precise “tissue matching” and optimal “IS protocols”, to reduce the risk of immune rejection and to minimize IS toxicity (15). 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) (16, 17), T- and B-cell receptor repertoire sequencing (TCRseq and BCRseq) (18–20), NGS analysis of donor-derived cell-free DNA (dd-cfDNA) (21–24), sophisticated in vitro and vivo models to study transplant rejection (12, 25), but also novel concepts of transplantation (Tx), immunosuppression (IS), and patient care (15), including advanced therapy medicinal products and cell and gene therapies (ATMPs and CGTs) (1, 11, 15, 26–31). 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, 31–37).

Wong et al. from the University of British Columbia and Mc Gill University in Canada, reviewed the clinical use of TCRseq and BCRseq to monitor dynamic changes in donor-reactive clonal cell populations following Tx (20), which may enable therapy adjustments to prevent rejection, reduce excessive IS, and indicate the development of tolerance. The authors reviewed 37 articles - 16 on KTx (43%) and 21 on other types (57%) and they concluded that immune repertoire sequencing is a valuable emerging tool for pre- and post-Tx monitoring.

Pedini et al. from Marseille in France conducted a prospective single-center study on 62 LuTx recipients to assess the relevance of dd-cfDNA for detecting acute and chronic rejection, or infection one month post LuTx (21–23). 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.

Burke et al. from the Miami Transplant Institute in Florida reviewed the role of podocytes in in focal segmental glomerulosclerosis (FSGS), a common glomerular disorder that manifests as nephrotic syndrome after KTx. They focused on podocytes as targets of circulating factors which promote recurrence of proteinuria following KTx. They discussed the potential of pre-/post-reperfusion biopsies and podocyte in vitro assays to develop new treatments for FSGS.

Mahler et al. from several Eurotransplant centers in Germany analyzed the outcomes from 328 cadaveric KTx recipients using 164 paired donor kidneys. They aimed to distinguish donor related risks from recipient and procedural variables, e.g. (a)symmetry of partner graft function, defined as early graft loss or impaired graft function (eGFR <30 ml/min) 3 months post KTx. They found that while donor factors impact early graft outcomes, they may play a limited role in long-term graft survival once the kidney graft has been accepted.

Bae et al. from the Catholic University of Korea investigated whether pre-KTx polyomavirus (BKV) serostatus and BK-specific cell mediated immunity (IFNg-ELISPOT against five BK viral antigens, LT, St, VP1, VP2, and VP3) could predict post-KTx BKV infection by evaluating 93 donor-recipient pairs who underwent KTx vs. 44 healthy controls. A combination of elevated donor BKV-IgG, low recipient BKV-IgG, and low BKV ELISPOT accurately predicted BKV infections in KTx recipients, helping clinicians to intervene earlier.

Moll et al. from Charité Berlin discovered that non-HLA-directed, protease activated receptor 1 (PAR1)-/G-protein coupled receptor (GPCR)-targeting regulatory autoantibodies (RABs) from KTx patients with transplant vasculopathy, but not IgG from KTx patients without vasculopathy or healthy controls, can exert immune stimulatory effects, triggering intracellular, and extracellular signaling in human microvascular endothelial cells and monocytic cells that may contribute to vasculopathy and graft failure, irrespective of alloantigen-directed responses.

Zhao et al. from Charité Berlin found that expanded hemodialysis (HDx) with medium-cutoff (MCO) membranes can reduce endothelial dysfunction caused by uremia in HD patients. In turn, HDx therapy preserved the vasculoprotective Krüppel-like factor 2 (KLF2), which counteracts inflammation and promotes vascular health.

Ringdén et al. from Karolinska Institutet in Stockholm, Sweden, reviewed their >20-year “Karolinska Experience” providing home care to HSCT patients starting in 1998. Analyzing 252 allo-HSCT patient outcomes they found that home care is safe, reduces the risk of developing acute GVHD, lowers transplant-related mortality, improves survival, and decreases proinflammatory cytokine levels compared to hospital-treated controls.

Zhang et al. from Tongji Medical College in Wuhan, China, reported a case of neuronal surface antibody syndrome (NSAS)-related autoimmune neurological disorder, with presentation of autoimmune encephalitis (AE), in a 7-year-old girl following HSCT, diagnosed with anti-metabotropic glutamate receptor-5 (mGluR5) autoimmunity, a less common form of NSAS-related autoimmunity. Treatment with IVIG and methylprednisolone, followed by oral prednisone tablets, and levetiracetam as antiepileptic therapy led to significant improvement.

Matthe et al. from the University Hospital Erlangen and University of Erlangen-Nuremberg in Germany developed a novel T-cell-organoid (co)culture system to study lympho-epithelial interactions in intestinal GvHD, which provides a valuable ex vivo platform for screening new therapeutic strategies on cellular and molecular level.

Verlaat et al. from Charité Berlin report the development of two murine cGvHD models, which display high long-term morbidity, but low mortality, and depict heterogeneous clinical manifestations seen of cGVDH pathophysiology seen in patients.