Cell-free DNA (cfDNA) is a marker of organ injury and immune response. DNA methylation is an epigenetic regulator of gene expression. Here, we elucidate total plasma cfDNA methylation from kidney transplant recipients in presence versus absence of rejection. In doing so, we exploit cfDNA as a real-time biomarker to define molecular pathways of rejection. Twenty plasma cfDNA samples from pediatric kidney transplant recipients were collected at allograft biopsy. Differentially methylated cytosine residues (>20 % methylation difference, q-value <0.05) were identified in presence (N = 7) versus absence (N = 9) of acute rejection. Separate analyses were performed comparing borderline rejection (N = 4) to rejection and non-rejection. In rejection versus non-rejection, there were 1269 differentially methylated genes corresponding to 533 pathways. These numbers were 4-13× greater than comparisons against borderline samples. Enriched pathways between rejection and non-rejection samples were related to immune cell/inflammatory response, lipid metabolism, and tryptophan-kynurenine metabolism, suggesting differential methylation of these pathways contributes to rejection.

Identifying additional immune checkpoints hindering antitumor T cell responses is key to the development of next-generation cancer immunotherapies. Here, we report the induction of serotonin transporter (SERT), a regulator of serotonin levels and physiological functions in the brain and peripheral tissues, in tumor-infiltrating CD8 T cells. Inhibition of SERT using selective serotonin reuptake inhibitors (SSRIs), the most widely prescribed antidepressants, significantly suppressed tumor growth and enhanced T cell antitumor immunity in various mouse syngeneic and human xenograft tumor models. Importantly, SSRI treatment exhibited significant therapeutic synergy with programmed cell death protein 1 (PD-1) blockade, and clinical data correlation studies negatively associated intratumoral SERT expression with patient survival in a range of cancers. Mechanistically, SERT functions as a negative-feedback regulator inhibiting CD8 T cell reactivities by depleting intratumoral T cell-autocrine serotonin. These findings highlight the significance of the intratumoral serotonin axis and identify SERT as an immune checkpoint, positioning SSRIs as promising candidates for cancer immunotherapy.

Recipients' age has emerged as a key factor that impacts on acute renal allograft rejection and graft survival. Age-related functional and structural changes in the immune system have been observed, yet the precise influence of aged immunity on kidney transplant remains unclear. In an initial retrospective analysis of clinical data gathered from two major centers in China and Germany, we found a correlation between aging and mitigated rejection outcomes in kidney recipients. To study the mechanism, we performed kidney transplantation on mice and observed attenuated allograft rejection in senescent recipients. Single-cell transcriptome analysis of allograft kidneys indicated a protective role of p21high macrophages in aged mice. Supernatant collected from p21high macrophage primary culture inhibited the cytotoxic function and proliferation of CD8+ T cells. Zfp36 is highly expressed in senescent p21high macrophages. To determine its role in renal allograft rejection, we studied mice with Zfp36 conditionally deleted in macrophages (Zfp36-cKO). These mice developed exacerbated allograft rejection with enhanced IL-27 production and CD8+ T cell hyperactivation. Inhibition of IL-27 with neutralizing antibody or deletion of IL-27 receptor on CD8+ T cells reversed acute renal allograft rejection in Zfp36-cKO mice. Moreover, in vitro silencing Zfp36 with siRNA led to impaired degradation of IL-27 p28 mRNA and a subsequent increase of IL-27 in p21high macrophages. In conclusion, senescent macrophages protect renal allograft rejection by suppressing CD8+ T cells via a Zfp36/IL-27-dependent mechanism. These findings may provide innovative therapeutic strategies for addressing kidney allograft rejection.

Heart transplantation, a crucial intervention for saving lives of those with end-stage cardiac failure, often faces complications from acute allograft rejection. This study focuses on the intricate dynamics of immune cell interactions and specific communication pathways between organs, which are not yet well understood. Our study investigates this interplay using a murine heterotopic transplant model, using single-cell RNA sequencing to examine CD45+ immune cells from both the heart grafts and spleens. We conduct a comprehensive analysis focused on functional enrichment, cell trajectory, and interorgan communication in heart transplants, highlighting dynamic interactions between monocyte/macrophage subtypes that is mediated by extracellular vesicles (EVs). We use unsupervised clustering and elucidate the complex cellular interactions that influence allograft outcomes. Notably, we discovered that microRNA-363 and microRNA-709, carried by EVs from CD63+ graft macrophages, can induce M1 polarization within the recipient's spleen via the Fcho2/Notch1 signaling pathway. These insights illuminate the nuanced immune responses during acute cardiac rejection and suggest that targeting EVs from graft-resident macrophages may offer a new strategy to mitigate transplant rejection.

While the Banff classification dichotomizes kidney allograft rejection based on the localization of the cells in the different compartments of the cortical kidney tissue [schematically interstitium for T cell mediated rejection (TCMR) and glomerular and peritubular capillaries for antibody-mediated rejection (AMR)], there is a growing evidences that subtyping the immune cells can help refine prognosis prediction and treatment tailoring, based on a better understanding of the pathophysiology of kidney allograft rejection. In the last few years, multiplex IF techniques and automatic counting systems as well as transcriptomics studies (bulk, single-cell and spatial techniques) have provided invaluable clues to further decipher the complex puzzle of rejection. In this review, we aim to better describe the inflammatory infiltrates that occur during the course of kidney transplant rejection (active AMR, chronic active AMR and acute and chronic active TCMR). We also discuss minor components of the inflammatory response (mastocytes, eosinophils, neutrophils, follicular dendritic cells). We conclude by discussing whether the over simplistic dichotomy between AMR and TCMR, currently used in clinical routine, remains relevant given the great diversity of immune actors involved in rejections.

The late embryonic development of the liver, a major metabolic organ, remains poorly characterized at single cell resolution. Here, we used single-nucleus RNA-sequencing (snRNA-seq) to characterize the chicken liver cells at 2 embryonic development time points (E14 and D1). We uncovered 8 cell types including hepatocytes, endothelial cells, hepatic stellate cells, erythrocytes, cholangiocytes, kupffer cells, mesothelial cells, and lymphocytes. And we discovered significant differences in the abundance of different cell types between E14 and D1. Moreover, we characterized the heterogeneity of hepatocytes, endothelial cells, and mesenchymal cells based on the gene regulatory networks of each clusters. Trajectory analyses revealed 128 genes associated with hepatocyte development and function, including apolipoprotein genes involved hepatic lipid metabolism and NADH dehydrogenase subunits involved hepatic oxidative phosphorylation. Furthermore, we identified the differentially expressed genes (DEGs) between E14 and D1 at the cellular levels, which contribute to changes in liver development and function. These DEGs were significantly enriched in PPAR signaling pathways and lipid metabolism related pathways. Our results presented the single-cell mapping of chick embryonic liver at late stages of development and demonstrated the metabolic changes across the 2 age stages at the cellular level, which can help to further study the molecular development mechanism of embryonic liver.

Acute kidney injury (AKI) transformed to chronic kidney disease (CKD) is a critical clinical issue characterized by tubulointerstitial inflammation (TII) and fibrosis. However, the exact mechanism remains largely unclear. In this study, we used single-cell RNA sequencing (scRNA-seq) to obtain a high-resolution profile of T cells in AKI to CKD transition with a mice model of unilateral ischemia-reperfusion injury (uIRI). We found that T cells accumulated increasingly with the progression of AKI to CKD, which was categorized into 9 clusters. A notably increased proportion of CD8 T cells via self-proliferation occurred in the early stage of AKI was identified. Further study revealed that the CD8 T cells were recruited through CXCL16-CXCR6 pathway mediated by macrophages. Notably, CD8 T cells induced endothelial cell apoptosis via Fas ligand-Fas signaling. Consistently, increased CD8 T cell infiltration accompanied with peritubular capillaries (PTCs) rarefaction was observed in uIRI mice. More impressively, the loss of PTCs and renal fibrosis was remarkably ameliorated after the elimination of CD8 T cells. In summary, our study provides a novel insight into the role of CD8 T cells in the transition from AKI to CKD via induction of PTCs rarefaction, which could suggest a promising therapeutic target for AKI.

Single-cell RNA sequencing is a high-throughput novel method that provides transcriptional profiling of individual cells within biological samples. This method typically uses microfluidics systems to uncover the complex intercellular communication networks and biological pathways buried within highly heterogeneous cell populations in tissues. One important application of this technology sits in the fields of organ and stem cell transplantation, where complications such as graft rejection and other post-transplantation life-threatening issues may occur. In this review, we first focus on research in which single-cell RNA sequencing is used to study the transcriptional profile of transplanted tissues. This technology enables the analysis of the donor and recipient cells and identifies cell types and states associated with transplant complications and pathologies. We also review the use of single-cell RNA sequencing in stem cell implantation. This method enables studying the heterogeneity of normal and pathological stem cells and the heterogeneity in cell populations. With their remarkably rapid pace, the single-cell RNA sequencing methodologies will potentially result in breakthroughs in clinical transplantation in the coming years.

The liver is the most tolerogenic of transplanted organs. However, the mechanisms underlying liver transplant tolerance are not well understood. The comparison between liver transplantation tolerance and heart/kidney transplantation rejection will deepen our understanding of tolerance and rejection in solid organs. Here, we built a mouse model of liver, heart and kidney allograft and performed single-cell RNA sequencing of 66,393 cells to describe the cell composition and immune cell interactions at the early stage of tolerance or rejection. We also performed bulk RNA-seq of mouse liver allografts from Day 7 to Day 60 post-transplantation to map the dynamic transcriptional variation in spontaneous tolerance. The transcriptome of lymphocytes and myeloid cells were characterized and compared in three types of organ allografts. Cell-cell interaction networks reveal the coordinated function of Kupffer cells, macrophages and their associated metabolic processes, including insulin receptor signalling and oxidative phosphorylation in tolerance induction. Cd11b+ dendritic cells (DCs) in liver allografts were found to inhibit cytotoxic T cells by secreting anti-inflammatory cytokines such as Il10. In summary, we profiled single-cell transcriptome analysis of mouse solid organ allografts. We characterized the immune microenvironment of mouse organ allografts in the acute rejection state (heart, kidney) and tolerance state (liver).

In recent years, there has been growing concern over the rising incidence of liver diseases, with increasing exposure to environmental toxins as a significant contributing factor. However, the mechanisms of liver injury induced by environmental pollutants are largely unclear. Here, using tetrabromobisphenol A (TBBPA), a widely used brominated flame retardant, as an example, environmental toxin-induced liver toxicity in mice is characterized via single-cell sequencing technology. Heterogeneous gene expression profiles after exposure to TBBPA in major cell types of the liver are demonstrated. In hepatocytes, pathway analysis of differentially expressed genes reveals the enhanced interferon response and diminished metabolic processes. The disrupted endothelial functions in TBBPA-treated cells are then shown. Moreover, the activation of M2-polarization in Kupffer cells, as well as activated effector T and B cells are unveiled in TBBPA-treated cells. Finally, ligand-receptor pair analysis shows that TBBPA disrupts cell-cell communication and induces an inflammatory microenvironment. Overall, the results reveal that TBBPA-induced dysfunction of hepatocytes and endothelial cells may then activate and recruit other immune cells such as Kuffer cells, and T/NK cells into the liver, further increasing inflammatory response and liver injury. Thus, the results provide novel insight into undesiring environmental pollutant-induced liver injury.

Preventing kidney graft dysfunction and rejection is a critical step in addressing the nationwide organ shortage and improving patient outcomes. While kidney transplants (KT) are performed more frequently, the overall number of patients on the waitlist consistently exceeds organ availability. Despite improved short-term outcomes in KT, comparable progress in long-term allograft survival has not been achieved. Major cause of graft loss at 5 years post-KT is chronic allograft dysfunction (CAD) characterized by interstitial fibrosis and tubular atrophy (IFTA). Accordingly, proactive prevention of CAD requires a comprehensive understanding of the immune mechanisms associated with either further dysfunction or impaired repair. Allograft rejection is primed by innate immune cells and carried out by adaptive immune cells. The rejection process is primarily facilitated by antibody-mediated rejection (ABMR) and T cell-mediated rejection (TCMR). It is essential to better elucidate the actions of individual immune cell subclasses (e.g. B memory, Tregs, Macrophage type 1 and 2) throughout the rejection process, rather than limiting our understanding to broad classes of immune cells. Embracing multi-omic approaches may be the solution in acknowledging these intricacies and decoding these enigmatic pathways. A transition alongside advancing technology will better allow organ biology to find its place in this era of precision and personalized medicine.

Multi-omics approach has advanced our knowledge on transplantation-associated clinical outcomes, such as acute rejection and infection, and emerging omics data are becoming available in kidney transplant and COVID-19. Herein, we discuss updated findings of multi-omics data on kidney transplant outcomes, as well as COVID-19 and kidney transplant.

Transcriptomics, proteomics, and metabolomics revealed various inflammation pathways associated with kidney transplantation-related outcomes and COVID-19. Although multi-omics data on kidney transplant and COVID-19 is limited, activation of innate immune pathways and suppression of adaptive immune pathways were observed in the active phase of COVID-19 in kidney transplant recipients.

Multi-omics analysis has led us to a deeper exploration and a more comprehensive understanding of key biological pathways in complex clinical settings, such as kidney transplantation and COVID-19. Future multi-omics analysis leveraging multi-center biobank collaborative will further advance our knowledge on the precise immunological responses to allograft and emerging pathogens.

This study investigated whether exosomes from LPS pretreated bone marrow mesenchymal stem cells (LPS pre-MSCs) could prolong skin graft survival.

The exosomes were isolated from the supernatant of MSCs pretreated with LPS. LPS pre-Exo and rapamycin were injected via the tail vein into C57BL/6 mice allografted with BALB/c skin; graft survival was observed and evaluated. The accumulation and polarization of macrophages were examined by immunohistochemistry. The differentiation of macrophages in the spleen was analyzed by flow cytometry. For in vitro, an inflammatory model was established. Specifically, bone marrow-derived macrophages (BMDMs) were isolated and cultured with LPS (100 ng/ml) for 3 h, and were further treated with LPS pre-Exo for 24 h or 48 h. The molecular signaling pathway responsible for modulating inflammation was examined by Western blotting. The expressions of downstream inflammatory cytokines were determined by Elisa, and the polarization of macrophages was analyzed by flow cytometry.

LPS pre-Exo could better ablate inflammation compared to untreated MSC-derived exosomes (BM-Exo). These loaded factors inhibited the expressions of inflammatory factors via a negative feedback mechanism. In vivo, LPS pre-Exo significantly attenuated inflammatory infiltration, thus improving the survival of allogeneic skin graft. Flow cytometric analysis of BMDMs showed that LPS pre-Exo were involved in the regulation of macrophage polarization and immune homeostasis during inflammation. Further investigation revealed that the NF-κB/NLRP3/procaspase-1/IL-1β signaling pathway played a key role in LPS pre-Exo-mediated regulation of macrophage polarization. Inhibiting NF-κB in BMDMs could abolish the LPS-induced activation of inflammatory pathways and the polarization of M1 macrophages while increasing the proportion of M2 cells.

LPS pre-Exo are able to switch the polarization of macrophages and enhance the resolution of inflammation. This type of exosomes provides an improved immunotherapeutic potential in prolonging graft survival.

Mouse kidney allografts are spontaneously accepted in select, fully mismatched donor-recipient strain combinations, like DBA/2J to C57BL/6 (B6), by natural tolerance. We previously showed accepted renal grafts form aggregates containing various immune cells within 2 weeks posttransplant, referred to as regulatory T cell-rich organized lymphoid structures, which are a novel regulatory tertiary lymphoid organ. To characterize the cells within T cell-rich organized lymphoid structures, we performed single-cell RNA sequencing on CD45+ sorted cells from accepted and rejected renal grafts from 1-week to 6-months posttransplant. Analysis of single-cell RNA sequencing data revealed a shifting from a T cell-dominant to a B cell-rich population by 6 months with an increased regulatory B cell signature. Furthermore, B cells were a greater proportion of the early infiltrating cells in accepted vs rejecting grafts. Flow cytometry of B cells at 20 weeks posttransplant revealed T cell, immunoglobulin domain and mucin domain-1+ B cells, potentially implicating a regulatory role in the maintenance of allograft tolerance. Lastly, B cell trajectory analysis revealed intragraft differentiation from precursor B cells to memory B cells in accepted allografts. In summary, we show a shifting T cell- to B cell-rich environment and a differential cellular pattern among accepted vs rejecting kidney allografts, possibly implicating B cells in the maintenance of kidney allograft acceptance.

Extracellular regulated protein kinases (ERK) signaling is a master regulator of cell behavior, life, and fate. Although ERK pathway is shown to be involved in T-cell activation, little is known about its role in the development of allograft rejection. Here, it is reported that ERK signaling pathway is activated in allograft-infiltrating T cells. On the basis of surface plasmon resonance technology, lycorine is identified as an ERK-specific inhibitor. ERK inhibition by lycorine significantly prolongs allograft survival in a stringent mouse cardiac allotransplant model. As compared to untreated mice, lycorine-treated mice show a decrease in the number and activation of allograft-infiltrated T cells. It is further confirmed that lycorine-treated mouse and human T cells are less responsive to stimulation in vitro, as indicated by their low proliferative rates and decreased cytokine production. Mechanistic studies reveal that T cells treated with lycorine exhibit mitochondrial dysfunction, resulting in metabolic reprogramming upon stimulation. Transcriptome analysis of lycorine-treated T cells reveals an enrichment in a series of downregulated terms related to immune response, the mitogen-activated protein kinase cascade, and metabolic processes. These findings offer new insights into the development of immunosuppressive agents by targeting the ERK pathway involved in T-cell activation and allograft rejection.

Antibody-mediated rejection (ABMR) is the major cause of chronic allograft dysfunction and loss in kidney transplantation. The immunological mechanisms of ABMR that have been featured in the latest studies indicate a highly complex interplay between various immune and nonimmune cell types. Clinical diagnostic standards have long been criticized for being arbitrary and the lack of accuracy. Transcriptomic approaches, including microarray and RNA sequencing of allograft biopsies, enable the identification of differential gene expression and the continuous improvement of diagnostics. Given that conventional bulk transcriptomic approaches only reflect the average gene expression but not the status at the single-cell level, thereby ignoring the heterogeneity of the transcriptome across individual cells, single-cell RNA sequencing is rising as a powerful tool to provide a high-resolution transcriptome map of immune cells, which allows the elucidation of the pathogenesis and may facilitate the development of novel strategies for clinical treatment of ABMR.

Despite recent advances made in short-term outcomes; minimal improvements have been observed in long-term kidney transplantation outcomes. Due to an imbalance between organ transplant availability and patient waiting list, expanding kidney allograft longevity is a critical need in the field. Prior studies have either focused on early ischemic and immunological conditions affecting kidney allografts (e.g., delayed graft function, acute rejection) or late stage chronic injury when interventions are no longer feasible. However, studies characterizing kidney allografts with normal function by its cellular distribution, cell-cell interactions, and associated molecular pathways are lacking. Herein, we used single nuclei RNA-sequencing to uncover the cellular landscape and transcriptome of the normal kidney allograft. We profiled 40,950 nuclei from seven human kidney biopsies (normal native, N = 3; normal allograft, N = 4); normal allograft protocol biopsies were collected ≥15-months post-transplant. A total of 17 distinct cell clusters were identified with proximal tubules (25.70 and 21.01%), distal tubules (15.22 and 18.20%), and endothelial cells (EC) (4.26 and 9.94%) constituting the major cell populations of normal native and normal allograft kidneys, respectively. A large proportion of cycling cells from normal native kidneys were in G1-phase (43.96%) whereas cells from normal allograft were predominantly in S-phase (32.69%). This result suggests that transcriptional differences between normal native and normal allograft biopsies are dependent on the new host environment, immunosuppression, and injury-affliction. In the normal allograft, EC-specific genes upregulated metabolism, the immune response, and cellular growth, emphasizing their role in maintaining homeostasis during the ongoing alloreactive stress response. Immune cells, including B (2.81%), macrophages (24.96%), monocytes (15.29%), natural killer (NK) (12.83%), neutrophils (8.44%), and T cells (14.41%, were increased in normal allografts despite lack of histological or clinical evidence of acute rejection. Phenotypic characterization of immune cell markers supported lymphocyte activation and proinflammatory cytokines signaling pathways (i.e., IL-15, IL-32). The activation of B, NK, and T cells reveals potential immune cells underlying subclinical inflammation and repair. These single nuclei analyses provide novel insights into kidney and immune cell associated signaling pathways that portray kidney grafts with normal allograft function beyond 2-years post-transplant, revealing a novel perspective in understanding long-term allograft graft survival.

Ischemia reperfusion injury(IRI) is an important factor affecting the early function and long-term survival of transplanted kidney. Single cell RNA sequencing (scRNA-seq) is a powerful method for investigating cell-specific transcriptome changes in the kidney. This study aimed to identify the significant cell type and potential biomarkers in IRI. First, we downloaded the IRI related scRNA dataset GSE139506 from the GEO database. Then, classification of cell type was characterized and proximal tubule cell (PTC) was identified as a significant cell type. The functional enrichment analysis indicated that PTC were related to kidney function and is significant in the ferroptosis of IRI. Analyses of three-dimensional structure and iron binding substructure of protein was carried out basing on SWISS-MODEL database. Finally, we constructed the murine model with IRI and verify the higher expression of PHYH in IRI by PCR, Western blot (WB) and Immunohistochemistry (IHC) experiments. In conclusion, this study provided novel insights on the cell-type-specific expression gene biomarker in IRI pathogenesis.