Multiomic profiling of transplant glomerulopathy reveals a novel T-cell dominant subclass.

Kidney transplant (KTx) biopsies showing transplant glomerulopathy (TG) (glomerular basement membrane double contours (cg) > 0) and microvascular inflammation (MVI) in the absence of C4d staining and donor-specific antibodies (DSAs) do not fulfill the criteria for chronic active antibody-mediated rejection (CA-AMR) diagnosis and do not fit into any other Banff category. To investigate this, we initiated a multicenter intercontinental study encompassing 36 cases, comparing the immunomic and transcriptomic profiles of 14 KTx biopsies classified as cg+MVI DSA-/C4d- with 22 classified as CA-AMR DSA+/C4d+ through novel transcriptomic analysis using the NanoString Banff-Human Organ Transplant (B-HOT) panel and subsequent orthogonal subset analysis using two innovative 5-marker multiplex immunofluorescent panels. Nineteen genes were differentially expressed between the two study groups. Samples diagnosed with CA-AMR DSA+/C4d+ showed a higher glomerular abundance of natural killer cells and higher transcriptomic cell type scores for macrophages in an environment characterized by increased expression of complement-related genes (i.e., C5AR1) and higher activity of angiogenesis, interstitial fibrosis tubular atrophy, CA-AMR, and DSA-related pathways when compared to samples diagnosed with cg+MVI DSA-/C4d-. Samples diagnosed with cg+MVI DSA-/C4d- displayed a higher glomerular abundance and activity of T cells (CD3+, CD3+CD8+, and CD3+CD8-). Thus, we show that using novel multiomic techniques, KTx biopsies with cg+MVI DSA-/C4d- have a prominent T-cell presence and activity, putting forward the possibility that these represent a more T-cell dominant phenotype.

Tissue-resident memory T cells in human kidney transplants have alloreactive potential.

The extent to which tissue-resident memory T (TRM) cells in transplanted organs possess alloreactivity is uncertain. This study investigates the alloreactive potential of TRM cells in kidney explants from 4 patients who experienced severe acute rejection leading to graft loss. Alloreactive T cell receptor (TCR) clones were identified in pretransplant blood samples through mixed lymphocyte reactions, followed by single-cell RNA and TCR sequencing of the proliferated recipient T cells. Subsequently, these TCR clones were traced in the TRM cells of kidney explants, which were also subjected to single-cell RNA and TCR sequencing. The proportion of recipient-derived TRM cells expressing an alloreactive TCR in the 4 kidney explants varied from 0% to 9%. Notably, these alloreactive TCRs were predominantly found among CD4+ and CD8+ TRM cells with an effector phenotype. Intriguingly, these clones were present not only in recipient-derived TRM cells but also in donor-derived TRM cells, constituting up to 4% of the donor population, suggesting the presence of self-reactive TRM cells. Overall, our study demonstrates that T cells with alloreactive potential present in the peripheral blood prior to transplantation can infiltrate the kidney transplant and adopt a TRM phenotype.

Interactions of the Immune System with Human Kidney Organoids.

Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC), which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover, immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids, which will advance the use of kidney organoids for transplantation research.

Antibody and T-Cell Responses 6 Months After Coronavirus Disease 2019 Messenger RNA-1273 Vaccination in Patients With Chronic Kidney Disease, on Dialysis, or Living With a Kidney Transplant.

BACKGROUND: The immune response to COVID-19 vaccination is inferior in kidney transplant recipients (KTRs) and to a lesser extent in patients on dialysis or with chronic kidney disease (CKD). We assessed the immune response 6 months after mRNA-1273 vaccination in kidney patients and compared this to controls. METHODS: A total of 152 participants with CKD stages G4/5 (eGFR <30 mL/min/1.73 m2), 145 participants on dialysis, 267 KTRs, and 181 controls were included. SARS-CoV-2 Spike S1 specific IgG antibodies were measured using fluorescent bead-based multiplex-immunoassay, neutralizing antibodies to ancestral, Delta, and Omicron (BA.1) variants by plaque reduction, and T-cell responses by interferon-γ release assay. RESULTS: At 6 months after vaccination, S1-specific antibodies were detected in 100% of controls, 98.7% of CKD G4/5 patients, 95.1% of dialysis patients, and 56.6% of KTRs. These figures were comparable to the response rates at 28 days, but antibody levels waned significantly. Neutralization of the ancestral and Delta variants was detected in most participants, whereas neutralization of Omicron was mostly absent. S-specific T-cell responses were detected at 6 months in 75.0% of controls, 69.4% of CKD G4/5 patients, 52.6% of dialysis patients, and 12.9% of KTRs. T-cell responses at 6 months were significantly lower than responses at 28 days. CONCLUSIONS: Although seropositivity rates at 6 months were comparable to rates at 28 days after vaccination, significantly decreased antibody levels and T-cell responses were observed. The combination of low antibody levels, reduced T-cell responses, and absent neutralization of the newly emerging variants indicates the need for additional boosts or alternative vaccination strategies in KTRs. CLINICAL TRIALS REGISTRATION: NCT04741386.

Clinical Characteristics and Outcomes of Immunocompromised Patients With Coronavirus Disease 2019 Caused by the Omicron Variant: A Prospective, Observational Study.

BACKGROUND: Illness after infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant is less severe compared with previous variants. Data on the disease burden in immunocompromised patients are lacking. We investigated the clinical characteristics and outcomes of immunocompromised patients with coronavirus disease 2019 (COVID-19) caused by Omicron. METHODS: Organ transplant recipients, patients on anti-CD20 therapy, and allogenic hematopoietic stem cell transplantation recipients infected with the Omicron variant were included. Characteristics of consenting patients were collected and patients were contacted regularly until symptom resolution. To identify possible risk factors for hospitalization, a univariate logistic analysis was performed. RESULTS: 114 consecutive immunocompromised patients were enrolled. Eighty-nine percent had previously received 3 mRNA vaccinations. While only 1 patient died, 23 (20%) were hospitalized for a median of 11 days. A low SARS-CoV-2 immunoglobulin G (IgG) antibody response (<300 BAU [binding antibody units]/mL) at diagnosis, being older, being a lung transplant recipient, having more comorbidities, and having a higher frailty score were associated with hospital admission (all P < .01). At the end of follow-up, 25% had still not fully recovered. Of the 23 hospitalized patients, 70% had a negative and 92% had a low IgG (<300 BAU/mL) antibody response at admission. Sotrovimab was administered to 17 of these patients, and 1 died. CONCLUSIONS: While the mortality in immunocompromised patients infected with Omicron was low, hospital admission was frequent and the duration of symptoms often prolonged. In addition to vaccination, other interventions are needed to limit the morbidity from COVID-19 in immunocompromised patients.

The role of interleukin-21 in COVID-19 vaccine-induced B cell-mediated immune responses in patients with kidney disease and kidney transplant recipients.

T-cell-mediated help to B cells is required for the development of humoral responses, in which the cytokine interleukin (IL)-21 is key. Here, we studied the mRNA-1273 vaccine-induced SARS-CoV-2-specific memory T-cell IL-21 response, memory B cell response, and immunoglobulin (Ig)G antibody levels in peripheral blood at 28 days after the second vaccination by ELISpot and the fluorescent bead-based multiplex immunoassay, respectively. We included 40 patients with chronic kidney disease (CKD), 34 patients on dialysis, 63 kidney transplant recipients (KTR), and 47 controls. We found that KTR, but not patients with CKD and those receiving dialysis, showed a significantly lower number of SARS-CoV-2-specific IL-21 producing T cells than controls (P < .001). KTR and patients with CKD showed lower numbers of SARS-CoV-2-specific IgG-producing memory B cells when compared with controls (P < .001 and P = .01, respectively). The T-cell IL-21 response was positively associated with the SARS-CoV-2-specific B cell response and the SARS-CoV-2 spike S1-specific IgG antibody levels (both Pearson r = 0.5; P < .001). In addition, SARS-CoV-2-specific B cell responses were shown to be IL-21 dependent. Taken together, we show that IL-21 signaling is important in eliciting robust B cell-mediated immune responses in patients with kidney disease and KTR.

Kidney Organoids Are Capable of Forming Tumors, but Not Teratomas.

Induced pluripotent stem cell (iPSC)-derived kidney organoids are a potential tool for the regeneration of kidney tissue. They represent an early stage of nephrogenesis and have been shown to successfsully vascularize and mature further in vivo. However, there are concerns regarding the long-term safety and stability of iPSC derivatives. Specifically, the potential for tumorigenesis may impede the road to clinical application. To study safety and stability of kidney organoids, we analyzed their potential for malignant transformation in a teratoma assay and following long-term subcutaneous implantation in an immune-deficient mouse model. We did not detect fully functional residual iPSCs in the kidney organoids as analyzed by gene expression analysis, single-cell sequencing and immunohistochemistry. Accordingly, kidney organoids failed to form teratoma. Upon long-term subcutaneous implantation of whole organoids in immunodeficient IL2Ry-/-RAG2-/- mice, we observed tumor formation in 5 out of 103 implanted kidney organoids. These tumors were composed of WT1+CD56+ immature blastemal cells and showed histological resemblance with Wilms tumor. No genetic changes were identified that contributed to the occurrence of tumorigenic cells within the kidney organoids. However, assessment of epigenetic changes revealed a unique cluster of differentially methylated genes that were also present in undifferentiated iPSCs. We discovered that kidney organoids have the capacity to form tumors upon long-term implantation. The presence of epigenetic modifications combined with the lack of environmental cues may have caused an arrest in terminal differentiation. Our results indicate that the safe implementation of kidney organoids should exclude the presence of pro-tumorigenic methylation in kidney organoids.

Isolation-free measurement of single urinary extracellular vesicles by imaging flow cytometry.

Urinary extracellular vesicles (uEVs) are promising biomarkers for various diseases. However, many tools measuring uEVs rely on time-consuming uEV isolation methods, which could induce sample bias. This study demonstrates the detection of single uEVs without isolation using imaging flow cytometry (IFCM). Unstained urine samples contained auto-fluorescent (A-F) particles when characterized with IFCM. Centrifugation successfully removed A-F particles from the unprocessed urine. Based on the disappearance of A-F particles, a gate was defined to distinguish uEVs from A-F particles. The final readouts of IFCM were verified as single EVs based on detergent treatment and serial dilutions. When developing this protocol to measure urine samples with abnormally high protein levels, 25 mg/mL dithiothreitol (DTT) showed improved uEV recovery over 200 mg/mL DTT. This study provides an isolation-free protocol using IFCM to quantify and phenotype single uEVs, eliminating the hindrance and influence of A-F particles, protein aggregates, and coincidence events.

High Tacrolimus Intrapatient Variability and Subtherapeutic Immunosuppression are Associated With Adverse Kidney Transplant Outcomes.

BACKGROUND: Kidney transplant recipients with high intrapatient variability (IPV) in tacrolimus (Tac) exposure experience more rejection and reduced graft survival. To understand the underlying pathophysiology of this association, the authors investigated whether patients with high tacrolimus IPV have a more activated immune system than patients with low IPV. In addition, exposure to tacrolimus and mycophenolic acid (MPA) was studied in relation to rejection and graft survival. METHODS: At the time of patient inclusion (5-7 years post-transplantation), the frequency of donor-reactive cells was determined by enzyme-linked immunosorbent assay, and the development of donor-specific anti-Human Leukocyte Antigen antibodies (DSA) was measured by Luminex Single Antigen assay. Tacrolimus IPV was retrospectively calculated between 6 and 12 months and the exposure to tacrolimus and MPA was determined between 1 and 5 years post-transplantation. RESULTS: A total of 371 kidney transplant recipients were included in this study, of whom 56 developed a rejection episode after 12 months and 60 experienced graft failure after 5-7 years. No correlations were found between tacrolimus IPV or immunosuppression exposure and the number of donor-reactive cells after 5 years of transplantation. DSA were detected more often in patients with low exposure to both tacrolimus and MMF [4/21 (19%) versus 17/350 (4.9%), P = 0.04]. In this cohort, neither tacrolimus IPV nor low overall immunosuppression exposure was associated with a higher incidence of rejection. However, regression analysis showed that a higher tacrolimus IPV was associated with an increased incidence of graft failure (odds ratio = 1.03, P = 0.02). CONCLUSIONS: This study verifies the relationship between high tacrolimus IPV and impaired kidney allograft survival in long-term follow-up. DSA was also found to be more prevalent in patients with subtherapeutic concentrations of tacrolimus and MPA. An increased prevalence of donor-specific alloreactivity is yet to be demonstrated in patients with high IPV.

Association Between the Intracellular Tacrolimus Concentration in CD3 + T Lymphocytes and CD14 + Monocytes and Acute Kidney Transplant Rejection.

BACKGROUND: Intracellular tacrolimus concentration in peripheral blood mononuclear cells (PBMCs) (TAC [PBMC] ) has been proposed to better represent its active concentration than its whole blood concentration. As tacrolimus acts on T lymphocytes and other white blood cells, including monocytes, we investigated the association of tacrolimus concentration in CD3 + T lymphocytes (TAC [CD3] ) and CD14 + monocytes (TAC [CD14] ) with acute rejection after kidney transplantation. METHODS: From a total of 61 samples in this case-control study, 28 samples were obtained during biopsy-proven acute rejection (rejection group), and 33 samples were obtained in the absence of rejection (control group). PBMCs were collected from both cryopreserved (retrospectively) and freshly obtained (prospectively) samples. CD3 + T lymphocytes and CD14 + monocytes were isolated from PBMCs, and their intracellular tacrolimus concentrations were measured. RESULTS: The correlation between tacrolimus whole-blood and intracellular concentrations was poor. TAC [CD3] was significantly lower than TAC [CD14] (median 12.8 versus 81.6 pg/million cells; P < 0.001). No difference in TAC [PBMC] (48.5 versus 44.4 pg/million cells; P = 0.82), TAC [CD3] (13.4 versus 12.5 pg/million cells; P = 0.28), and TAC [CD14] (90.0 versus 72.8 pg/million cells; P = 0.27) was found between the rejection and control groups. However, freshly isolated PBMCs showed significantly higher TAC [PBMC] than PBMCs from cryopreserved samples. Subgroup analysis of intracellular tacrolimus concentrations from freshly isolated cells did not show a difference between rejectors and nonrejectors. CONCLUSIONS: Differences in TAC [CD3] and TAC [CD14] between patients with and without rejection could not be demonstrated. However, further optimization of the cell isolation process is required because a difference in TAC [PBMC] between fresh and cryopreserved cells was observed. These results need to be confirmed in a study with a larger number of patients.

Donor-Derived Cell-Free DNA for the Detection of Heart Allograft Injury: The Impact of the Timing of the Liquid Biopsy.

Background: In heart transplant recipients, donor-derived cell-free DNA (ddcfDNA) is a potential biomarker for acute rejection (AR), in that increased values may indicate rejection. For the assessment of ddcfDNA as new biomarker for rejection, blood plasma sampling around the endomyocardial biopsy (EMB) seems a practical approach. To evaluate the effect of the EMB procedure on ddcfDNA values, ddcfDNA values before the EMB were pairwise compared to ddcfDNA values after the EMB. We aimed at evaluating whether it matters whether the ddcfDNA sampling is done before or after the EMB-procedure. Methods: Plasma samples from heart transplant recipients were obtained pre-EMB and post-EMB. A droplet digital PCR method was used for measuring ddcfDNA, making use of single-nucleotide polymorphisms that allowed both relative quantification, as well as absolute quantification of ddcfDNA. Results: Pairwise comparison of ddcfDNA values pre-EMB with post-EMB samples (n = 113) showed significantly increased ddcfDNA concentrations and ddcfDNA% in post-EMB samples: an average 1.28-fold increase in ddcfDNA concentrations and a 1.31-fold increase in ddcfDNA% was observed (p = 0.007 and p = 0.03, respectively). Conclusion: The EMB procedure causes iatrogenic injury to the allograft that results in an increase in ddcfDNA% and ddcfDNA concentrations. For the assessment of ddcfDNA as marker for AR, collection of plasma samples before the EMB procedure is therefore essential.

Human kidney organoids produce functional renin.

Renin production by the kidney is of vital importance for salt, volume, and blood pressure homeostasis. The lack of human models hampers investigation into the regulation of renin and its relevance for kidney physiology. To develop such a model, we used human induced pluripotent stem cell-derived kidney organoids to study the role of renin and the renin-angiotensin system in the kidney. Extensive characterization of the kidney organoids revealed kidney-specific cell populations consisting of podocytes, proximal and distal tubular cells, stromal cells and endothelial cells. We examined the presence of various components of the renin-angiotensin system such as angiotensin II receptors, angiotensinogen, and angiotensin-converting enzymes 1 and 2. We identified by single-cell sequencing, immunohistochemistry, and functional assays that cyclic AMP stimulation induces a subset of pericytes to increase the synthesis and secretion of enzymatically active renin. Renin production by the organoids was responsive to regulation by parathyroid hormone. Subcutaneously implanted kidney organoids in immunodeficient IL2Ry-/-Rag2-/- mice were successfully vascularized, maintained tubular and glomerular structures, and retained capacity to produce renin two months after implantation. Thus, our results demonstrate that kidney organoids express renin and provide insights into the endocrine potential of human kidney organoids, which is important for regenerative medicine in the context of the endocrine system.

Mesenchymal stromal cell treatment of donor kidneys during ex vivo normothermic machine perfusion: A porcine renal autotransplantation study.

Normothermic machine perfusion (NMP) of injured kidneys offers the opportunity for interventions to metabolically active organs prior to transplantation. Mesenchymal stromal cells (MSCs) can exert regenerative and anti-inflammatory effects in ischemia-reperfusion injury. The aims of this study were to evaluate the safety and feasibility of MSC treatment of kidneys during NMP using a porcine autotransplantation model, and examine potential MSC treatment-associated kidney improvements up to 14 days posttransplant. After 75 min of kidney warm ischemia, four experimental groups of n = 7 underwent 14 h of oxygenated hypothermic machine perfusion. In three groups this was followed by 240 min of NMP with infusion of vehicle, 10 million porcine, or 10 million human adipose-derived MSCs. All kidneys were autotransplanted after contralateral nephrectomy. MSC treatment did not affect perfusion hemodynamics during NMP or cause adverse effects at reperfusion, with 100% animal survival. MSCs did not affect plasma creatinine, glomerular filtration rate, neutrophil gelatinase-associated lipocalin concentrations or kidney damage assessed by histology during the 14 days, and MSCs retention was demonstrated in renal cortex. Infusing MSCs during ex vivo NMP of porcine kidneys was safe and feasible. Within the short posttransplant follow-up period, no beneficial effects of ex vivo MSC therapy could be demonstrated.

Monitoring the tacrolimus concentration in peripheral blood mononuclear cells of kidney transplant recipients.

AIMS: Tacrolimus is a critical dose drug and to avoid under- and overexposure, therapeutic drug monitoring is standard practice. However, rejection and drug-related toxicity occur despite whole-blood tacrolimus pre-dose concentrations ([Tac]blood ) being on target. Monitoring tacrolimus concentrations at the target site (within peripheral blood mononuclear cells; [Tac]cells ) may better correlate with drug-efficacy. The aim of this study was to (1) investigate the relationship between [Tac]blood and [Tac]cells , (2) identify factors affecting the tacrolimus distribution in cells and whole-blood, and (3) study the relationship between [Tac]cells and clinical outcomes after kidney transplantation. METHODS: A total of 175 renal transplant recipients were prospectively followed. [Tac]blood and [Tac]cells were determined at Months 3, 6 and 12 post-transplantation. Patients were genotyped for ABCB1 1199G>A and 3435C>T, CYP3A4 15389C>T, and CYP3A5 6986G>A. Data on rejection and tacrolimus-related nephrotoxicity and post-transplant diabetes mellitus were collected. RESULTS: Correlations between [Tac]blood and [Tac]cells were moderate to poor (Spearman's r = 0.31; r = 0.41; r = 0.61 at Months 3, 6 and 12, respectively). The [Tac]cells /[Tac]blood ratio was stable over time in most patients (median intra-patient variability 39.0%; range 3.5%-173.2%). Age, albumin and haematocrit correlated with the [Tac]cells /[Tac]blood ratio. CYP3A5 and CYP3A4 genotype combined affected both dose-corrected [Tac]blood and [Tac]cells . ABCB1 was not significantly related to tacrolimus distribution. Neither [Tac]blood nor [Tac]cells correlated with clinical outcomes. CONCLUSIONS: The correlation between [Tac]blood and [Tac]cells is poor. Age, albumin and haematocrit correlate with the [Tac]cells /[Tac]blood ratio, whereas genetic variation in ABCB1, CYP3A4 and CYP3A5 do not. Neither [Tac]blood nor [Tac]cells correlated with clinical outcomes.

Urinary Extracellular Vesicles Are a Novel Tool to Monitor Allograft Function in Kidney Transplantation: A Systematic Review.

Extracellular vesicles (EVs) are nanoparticles that transmit molecules from releasing cells to target cells. Recent studies link urinary EVs (uEV) to diverse processes such as infection and rejection after kidney transplantation. This, and the unmet need for biomarkers diagnosing kidney transplant dysfunction, has led to the current high level of interest in uEV. uEV provide non-intrusive access to local protein, DNA, and RNA analytics without invasive biopsy. To determine the added value of uEV measurements for detecting allograft dysfunction after kidney transplantation, we systematically included all related literature containing directly relevant information, with the addition of indirect evidence regarding urine or kidney injury without transplantation. According to their varying characteristics, uEV markers after transplantation could be categorized into kidney-specific, donor-specific, and immune response-related (IR-) markers. A few convincing studies have shown that kidney-specific markers (PODXL, ion cotransporters, SYT17, NGAL, and CD133) and IR-markers (CD3, multi-mRNA signatures, and viral miRNA) could diagnose rejection, BK virus-associated nephropathy, and calcineurin inhibitor nephrotoxicity after kidney transplantation. In addition, some indirect proof regarding donor-specific markers (donor-derived cell-free DNA) in urine has been demonstrated. Together, this literature review provides directions for exploring novel uEV markers' profiling complications after kidney transplantation.

Proteomic Analysis of Mesenchymal Stromal Cell-Derived Extracellular Vesicles and Reconstructed Membrane Particles.

Extracellular vesicles (EV) derived from mesenchymal stromal cells (MSC) are a potential therapy for immunological and degenerative diseases. However, large-scale production of EV free from contamination by soluble proteins is a major challenge. The generation of particles from isolated membranes of MSC, membrane particles (MP), may be an alternative to EV. In the present study we generated MP from the membranes of lysed MSC after removal of the nuclei. The yield of MP per MSC was 1 × 105 times higher than EV derived from the same number of MSC. To compare the proteome of MP and EV, proteomic analysis of MP and EV was performed. MP contained over 20 times more proteins than EV. The proteins present in MP evidenced a multi-organelle origin of MP. The projected function of the proteins in EV and MP was very different. Whilst proteins in EV mainly play a role in extracellular matrix organization, proteins in MP were interconnected in diverse molecular pathways, including protein synthesis and degradation pathways and demonstrated enzymatic activity. Treatment of MSC with IFNγ led to a profound effect on the protein make up of EV and MP, demonstrating the possibility to modify the phenotype of EV and MP through modification of parent MSC. These results demonstrate that MP are an attractive alternative to EV for the development of potential therapies. Functional studies will have to demonstrate therapeutic efficacy of MP in preclinical disease models.

Variations in DNA methylation and allograft rejection.

PURPOSE OF REVIEW: DNA methylation is involved in gene transcription and as such important for cellular function. Here, the literature on DNA methylation in relation to acute rejection is summarized with a focus on the potential clinical utility of DNA methylation for monitoring transplant rejection. RECENT FINDINGS: The tight transcriptional control of DNA methylation in immune cell function, e.g. demethylation in regulatory T-cell-specific genes for stable immunosuppressive capacities, suggests an important role for DNA methylation variations in the antidonor-directed immune response. Until today, differentially methylated DNA in immune cells, however, has not been described at the moment of allograft rejection. The ability to locus-specific modify DNA methylation could facilitate the generation of stable cells for cellular therapy purposes. The unique cell-specific characteristics of DNA methylation provide the opportunity to identify its cellular origin. Examining methylation of cell-free DNA in blood or urine may serve as a 'liquid biopsy' enabling minimally invasive detection of allograft rejection. SUMMARY: Actual research publications on DNA methylation in relation to allograft rejection are scarce, which makes it challenging to determine its potential clinical value. Extensive research is needed to investigate the value of DNA methylation in early recognition, diagnosis, and/or successful treatment of allograft rejection.

Immunomodulation By Therapeutic Mesenchymal Stromal Cells (MSC) Is Triggered Through Phagocytosis of MSC By Monocytic Cells.

Mesenchymal stem or stromal cells (MSC) are under investigation as a potential immunotherapy. MSC are usually administered via intravenous infusion, after which they are trapped in the lungs and die and disappear within a day. The fate of MSC after their disappearance from the lungs is unknown and it is unclear how MSC realize their immunomodulatory effects in their short lifespan. We examined immunological mechanisms determining the fate of infused MSC and the immunomodulatory response associated with it. Tracking viable and dead human umbilical cord MSC (ucMSC) in mice using Qtracker beads (contained in viable cells) and Hoechst33342 (staining all cells) revealed that viable ucMSC were present in the lungs immediately after infusion. Twenty-four hours later, the majority of ucMSC were dead and found in the lungs and liver where they were contained in monocytic cells of predominantly non-classical Ly6Clow phenotype. Monocytes containing ucMSC were also detected systemically. In vitro experiments confirmed that human CD14++ /CD16- classical monocytes polarized toward a non-classical CD14++ CD16+ CD206+ phenotype after phagocytosis of ucMSC and expressed programmed death ligand-1 and IL-10, while TNF-α was reduced. ucMSC-primed monocytes induced Foxp3+ regulatory T cell formation in mixed lymphocyte reactions. These results demonstrate that infused MSC are rapidly phagocytosed by monocytes, which subsequently migrate from the lungs to other body sites. Phagocytosis of ucMSC induces phenotypical and functional changes in monocytes, which subsequently modulate cells of the adaptive immune system. It can be concluded that monocytes play a crucial role in mediating, distributing, and transferring the immunomodulatory effect of MSC. Stem Cells 2018;36:602-615.

Targeted Proteomic Analysis Detects Acute T Cell-Mediated Kidney Allograft Rejection in Belatacept-Treated Patients.

BACKGROUND: There is an unmet need for reliable minimally invasive diagnostic biomarkers for immunological allograft monitoring and for the detection of acute kidney transplant rejection. Here, targeted proteomic analysis was applied to compare 92 proteins in sera of belatacept-treated patients who had biopsy-proven, acute T-cell-mediated rejection (aTCMR) with patients without aTCMR. METHODS: Proximity extension immunoassay was used to measure 92 inflammation-related protein concentrations in the prerejection and rejection sera of 11 patients with aTCMR and 9 patients without aTCMR. This assay uses 2 matched oligonucleotide-labeled antibody probes for each protein and polymerase chain reaction to measure normalized protein expression values. RESULTS: Five proteins (CD5, CD8A, NCR1, TNFRSF4, and TNFRSF9) were expressed significantly higher in samples with aTCMR compared with samples without aTCMR (adjusted P-value < 0.014) and had a good predictive capacity for aTCMR [area under the curve in a receiver-operator curve ranged from 0.83 to 0.91 (P < 0.014)]. These proteins are associated with CD8 cytotoxic T-cell and NK cell functions. Nonhierarchical clustering analysis showed distinct clustering of samples with aTCMR and samples without aTCMR. This clustering was not found in prerejection samples (1 month after transplantation). In prerejection samples, IFN-γ was expressed at a significantly lower level (normalized protein expression value median -0.15, interquartile range: -0.27 to 0.04) than in samples of patients without rejection (median 0.13, interquartile range: -0.07 to 0.15, adjusted P-value = 0.00367). CONCLUSIONS: Targeted proteomic analysis with proximity extension immunoassay is a promising minimally invasive technique to diagnose aTCMR in kidney transplant recipients.

Epigenetic changes in umbilical cord mesenchymal stromal cells upon stimulation and culture expansion.

BACKGROUND: Mesenchymal stromal cells (MSCs) are studied for their immunotherapeutic potential. Prior to therapeutic use, MSCs are culture expanded to obtain the required cell numbers and, to improve their efficacy, MSCs may be primed in vitro. Culture expansion and priming induce phenotypical and functional changes in MSCs and thus standardisation and quality control measurements come in need. We investigated the impact of priming and culturing on MSC DNA methylation and examined the use of epigenetic profiling as a quality control tool. METHODS: Human umbilical cord-derived MSCs (ucMSCs) were cultured for 3 days with interferon (IFN)γ, transforming growth factor (TGF)ß or a multi-factor combination (MC; IFNγ, TGFß and retinoic acid). In addition, ucMSCs were culture expanded for 14 days. Phenotypical changes and T-cell proliferation inhibition capacity were examined. Genome-wide DNA methylation was measured with Infinium MethylationEPIC Beadchip. RESULTS: Upon priming, ucMSCs exhibited a different immunophenotype and ucMSC(IFNγ) and ucMSC(MC) had an increased capacity to inhibit T-cell proliferation. DNA methylation patterns were minimally affected by priming, with only one significantly differentially methylated site (DMS) in IFNγ- and MC-primed ucMSCs associated with autophagy activity. In contrast, 14 days after culture expansion, ucMSCs displayed minor phenotypical and functional changes but showed >4000 significantly DMSs, mostly concerning genes involved in membrane composition, cell adhesion and transmembrane signalling. DISCUSSION: These data show that DNA methylation of MSCs is only marginally affected by priming, whereas culture expansion and subsequent increased cellular interactions have a large impact on methylation. On account of this study, we suggest that DNA methylation analysis is a useful quality control tool for culture expanded therapeutic MSCs.