Fibrotic progression from acute cellular rejection is dependent on secondary lymphoid organs in a mouse model of chronic lung allograft dysfunction.

Chronic lung allograft dysfunction (CLAD) remains one of the major limitations to long-term survival after lung transplantation. We modified a murine model of CLAD and transplanted left lungs from BALB/c donors into B6 recipients that were treated with intermittent cyclosporine and methylprednisolone postoperatively. In this model, the lung allograft developed acute cellular rejection on day 15 which, by day 30 after transplantation, progressed to severe pleural and peribronchovascular fibrosis, reminiscent of changes observed in restrictive allograft syndrome. Lung transplantation into splenectomized B6 alymphoplastic (aly/aly) or splenectomized B6 lymphotoxin-ß receptor-deficient mice demonstrated that recipient secondary lymphoid organs, such as spleen and lymph nodes, are necessary for progression from acute cellular rejection to allograft fibrosis in this model. Our work uncovered a critical role for recipient secondary lymphoid organs in the development of CLAD after pulmonary transplantation and may provide mechanistic insights into the pathogenesis of this complication.

Oral administration of the commensal Alistipes onderdonkii prolongs allograft survival.

Intestinal commensals can exert immunomodulatory effects on the host, with beneficial or detrimental consequences depending on underlying diseases. We have previously correlated longer survival of minor mismatched skin grafts in mice with the presence of an intestinal commensal bacterium, Alistipes onderdonkii. In this study, we investigated its sufficiency and mechanism of action. Oral administration of A onderdonkii strain DSM19147 but not DSM108265 was sufficient to prolong minor mismatched skin graft survival through inhibition of tumor necrosis factor production. Through metabolomic and metagenomic comparisons between DSM19147 and DSM108265, we identified candidate gene products associated with the anti-inflammatory effect of DSM19147. A onderdonkii DSM19147 can lower inflammation both at a steady state and after transplantation and may serve as an anti-inflammatory probiotic beneficial for transplant recipients.

TIGIT agonism alleviates costimulation blockade-resistant rejection in a regulatory T cell-dependent manner.

Belatacept-based immunosuppression in kidney transplantation confers fewer off-target toxicities than calcineurin inhibitors but comes at a cost of increased incidence and severity of acute rejection, potentially due to its deleterious effect on both the number and function of Foxp3+ regulatory T cells (Tregs). TIGIT is a CD28 family coinhibitory receptor expressed on several subsets of immune cells including Tregs. We hypothesized that coinhibition through TIGIT signaling could function to ameliorate costimulation blockade-resistant rejection. The results demonstrate that treatment with an agonistic anti-TIGIT antibody, when combined with costimulation blockade by CTLA-4Ig, can prolong allograft survival in a murine skin graft model compared with CTLA-4Ig alone. Further, this prolongation of graft survival is accompanied by an increase in the frequency and number of graft-infiltrating Tregs and a concomitant reduction in the number of CD8+ T cells in the graft. Through the use of Treg-specific TIGIT conditional knockout animals, we demonstrated that the TIGIT-mediated reduction in the graft-infiltrating CD8+ T cell response is dependent on signaling of TIGIT on Foxp3+ Tregs. Our results highlight both the key functional role of TIGIT on Foxp3+ Tregs under conditions in which CTLA-4 is blocked and the therapeutic potential of TIGIT agonism to optimize costimulation blockade-based immunosuppression.

Renin-angiotensin-aldosterone system function in the pig-to-baboon kidney xenotransplantation model.

After pig-to-baboon kidney transplantation, episodes of hypovolemia and hypotension from an unexplained mechanism have been reported. This study evaluated the renin-angiotensin-aldosterone system post-kidney xenotransplantation. Kidneys from genetically-engineered pigs were transplanted into 5 immunosuppressed baboons after the excision of the native kidneys. Immunosuppressive therapy was based on the blockade of the CD40/CD154 costimulation pathway. Plasma renin, angiotensinogen (AGT), angiotensin II (Ang II), aldosterone levels, and urine osmolality and electrolytes were measured in healthy pigs, healthy nonimmunosuppressed baboons, and immunosuppressed baboons with life-supporting pig kidney grafts. After pig kidney transplantation, plasma renin and Ang II levels were not significantly different, although Ang II trended lower, even though plasma AGT and potassium were increased. Plasma aldosterone levels were unchanged. Urine osmolality and sodium concentration were decreased. Even in the presence of increasing AGT and potassium levels, lower plasma Ang II concentrations may be because of reduced, albeit not absent, the reactivity of pig renin to cleave baboon AGT, suggesting an impaired response of the renin-angiotensin-aldosterone system to hypovolemic and hypotensive episodes. The maintenance of aldosterone may be protective. The reduced urine osmolality and sodium concentration reflect the decreased ability of the pig kidney to concentrate urine. These considerations should not prohibit successful clinical pig kidney xenotransplantation.

Honoring the gift: The transformative potential of transplant-declined human organs.

For decades, transplantation has been a life-saving treatment for those fortunate enough to gain access. Nevertheless, many patients die waiting for an organ and countless more never make it onto the waitlist because of a shortage of donor organs. Concurrently, thousands of donated organs are declined for transplant each year because of concerns about poor outcomes post-transplant. The decline of any donated organ-even if medically justified-is tragic for both the donor family and potential recipients. In this Personal Viewpoint, we discuss the need for a new mindset in how we honor the gift of organ donation. We believe that the use of transplant-declined human organs in translational research has the potential to hasten breakthrough discoveries in a multitude of scientific and medical areas. More importantly, such breakthroughs will allow us to properly value every donated organ. We further discuss the many practical challenges that such research presents and offer some possible solutions based on experiences in our own research laboratories. Finally, we share our perspective on what we believe are the necessary next steps to ensure a future where every donated organ realizes its full potential to impact the lives of current and future patients.

Endothelial phosphoinositide 3-kinase-ß inactivation confers protection from immune-mediated vascular injury.

Heart transplant and recipient survival are limited by immune cell-mediated injury of the graft vasculature. We examined the role of the phosphoinositide 3-kinase-ß (PI3Kß) isoform in endothelial cells (EC) during coronary vascular immune injury and repair in mice. In minor histocompatibility-antigen mismatched allogeneic heart grafts, a robust immune response was mounted to each wild-type, PI3Kß inhibitor-treated, or endothelial-selective PI3Kß knockout (ECßKO) graft transplanted to wild-type recipients. However, microvascular EC loss and progressive occlusive vasculopathy only developed in control, but not PI3Kß-inactivated hearts. We observed a delay in inflammatory cell infiltration of the ECßKO grafts, particularly in the coronary arteries. Surprisingly, this was accompanied by an impaired display of proinflammatory chemokine and adhesion molecules by the ECßKO ECs. In vitro, tumor necrosis factor α-stimulated endothelial ICAM1 and VCAM1 expression was blocked by PI3Kß inhibition or RNA interference. Selective PI3Kß inhibition also blocked tumor necrosis factor α-stimulated degradation of inhibitor of nuclear factor kappa Bα and nuclear translocation of nuclear factor kappa B p65 in EC. These data identify PI3Kß as a therapeutic target to reduce vascular inflammation and injury.

Normalization of lipid oxidation defects arising from hypoxia early posthepatectomy prevents liver failure in mouse.

Surgical liver failure (SLF) develops when a marginal amount of hepatic mass is left after surgery, such as following excessive resection. SLF is the commonest cause of death due to liver surgery; however, its etiology remains obscure. Using mouse models of standard hepatectomy (sHx) (68%, resulting in full regeneration) or extended hepatectomy (eHx) (86%/91%, causing SLF), we explored the causes of early SLF related to portal hyperafflux. Assessing the levels of HIF2A with or without oxygenating agent inositol trispyrophosphate (ITPP) indicated hypoxia early after eHx. Subsequently, lipid oxidation (PPARA/PGC1α) was downregulated and associated with persisting steatosis. Mild oxidation with low-dose ITPP reduced the levels of HIF2A, restored downstream PPARA/PGC1α expression along with lipid oxidation activities (LOAs), and normalized steatosis and other metabolic or regenerative SLF deficiencies. Promotion of LOA with L-carnitine likewise normalized the SLF phenotype, and both ITPP and L-carnitine markedly raised survival in lethal SLF. In patients who underwent hepatectomy, pronounced increases in serum carnitine levels (reflecting LOA) were associated with better recovery. Lipid oxidation thus provides a link between the hyperafflux of O2-poor portal blood, the metabolic/regenerative deficits, and the increased mortality typifying SLF. Stimulation of lipid oxidation-the prime regenerative energy source-particularly through L-carnitine may offer a safe and feasible way to reduce SLF risks in the clinic.

miR-449a ameliorates acute rejection after liver transplantation via targeting procollagen-lysine1,2-oxoglutarate5-dioxygenase 1 in macrophages.

Acute rejection (AR) is an important factor that leads to poor prognosis after liver transplantation (LT). Macrophage M1-polarization is an important mechanism in AR development. MicroRNAs play vital roles in disease regulation; however, their effects on macrophages and AR remain unclear. In this study, rat models of AR were established following LT, and macrophages and peripheral blood mononuclear cells were isolated from rats and humans, respectively. We found miR-449a expression to be significantly reduced in macrophages and peripheral blood mononuclear cells. Overexpression of miR-449a not only inhibited the M1-polarization of macrophages in vitro but also improved the AR of transplant in vivo. The mechanism involved inhibiting the noncanonical nuclear factor-kappaB (NF-κB) pathway. We identified procollagen-lysine1,2-oxoglutarate5-dioxygenase 1 (PLOD1) as a target gene of miR-449a, which could reverse miR-449a's inhibition of macrophage M1-polarization, amelioration of AR, and inhibition of the NF-κB pathway. Overall, miR-449a inhibited the NF-κB pathway in macrophages through PLOD1 and also inhibited the M1-polarization of macrophages, thus attenuating AR after LT. In conclusion, miR-449a and PLOD1 may be new targets for the prevention and mitigation of AR.

Oral alloantigen exposure promotes donor-specific tolerance in a mouse model of minor-mismatched skin transplantation.

Oral antigen exposure is a powerful, non-invasive route to induce immune tolerance to dietary antigens, and has been modestly successful at prolonging graft survival in rodent models of transplantation. To harness the mechanisms of oral tolerance for promoting long-term graft acceptance, we developed a mouse model where the antigen ovalbumin (OVA) was introduced orally prior to transplantation with skin grafts expressing OVA. Oral OVA treatment pre-transplantation promoted permanent graft acceptance and linked tolerance to skin grafts expressing OVA fused to the additional antigen 2W. Tolerance was donor-specific, as secondary donor-matched, but not third-party allografts were spontaneously accepted. Oral OVA treatment promoted an anergic phenotype in OVA-reactive CD4+ and CD8+ conventional T cells (Tconvs) and expanded OVA-reactive Tregs pre-transplantation. However, skin graft acceptance following oral OVA resisted partial depletion of Tregs and blockade of PD-L1. Mechanistically, we revealed a role for the proximal gut draining lymph nodes (gdLNs) in mediating this effect, as an intestinal infection that drains to the proximal gdLNs prevented tolerance induction. Our study extends previous work applying oral antigen exposure to transplantation and serves as proof of concept that the systemic immune mechanisms supporting oral tolerance are sufficient to promote long-term graft acceptance.

Mesenchymal stem cells augment regulatory T cell function via CD80-mediated interactions and promote allograft survival.

Mesenchymal stem cells (MSCs) and regulatory T cells (Tregs) both have been shown to modulate the alloimmune response and promote transplant survival. Mounting evidence suggests that MSCs augment Treg function, but the mechanisms underlying this phenomenon have not been fully deciphered. Here, we identified that MSCs express substantial levels of CD80 and evaluated its immunoregulatory function using in vivo and in vitro experiments. Our in vitro culture assays demonstrated that MSCs induce expression of FoxP3 in Tregs in a contact-dependent manner, and the blockade of CD80 abrogates this FoxP3 induction and Treg-mediated suppression of T cell proliferation. Moreover, supplementation of soluble CD80 significantly upregulated FoxP3 expression. Using a well-characterized murine model of corneal transplantation, we show that silencing CD80 in MSCs diminishes the capacity of MSCs to promote selective graft infiltration of Tregs, promote FoxP3 expression and upregulate suppressive function of Tregs. Consequently, MSCs, following CD80 knockdown, failed to promote corneal allograft survival.

Inhibition of mevalonate metabolism by statins augments the immunoregulatory phenotype of vascular endothelial cells and inhibits the costimulation of CD4+ T cells.

The statin family of therapeutics is widely used clinically as cholesterol lowering agents, and their effects to target intracellular mevalonate production is a key mechanism of action. In this study, we performed full transcriptomic RNA sequencing and qPCR to evaluate the effects of mevalonate on the immunoregulatory phenotype of endothelial cells (EC). We find that mevalonate-dependent gene regulation includes a reduction in the expression of multiple pro-inflammatory genes including TNFSF4 (OX40-L) and TNFSF18 (GITR-L) and a co-incident induction of immunoregulatory genes including LGALS3 (Galectin-3) and LGALS9 (Galectin-9). In functional assays, pretreatment of EC with simvastatin to inhibit mevalonate metabolism resulted in a dose-dependent reduction in the costimulation of CD45RO+ CD4+ T cell proliferation as well as IL-2, IFNγ and IL-6 production versus vehicle-treated EC. In contrast, pre-treatment of EC with L-mevalonate in combination with simvastatin reversed phenotypic and functional responses. Collectively, these results indicate that relative mevalonate metabolism by EC is critical to sustain EC-dependent mechanisms of immunity. Our findings have broad relevance for the repurposing of statins as therapeutics to augment immunoregulation and/or to inhibit local tissue pro-inflammatory cytokine production following transplantation.

The molecular phenotypes of injury, steatohepatitis, and fibrosis in liver transplant biopsies in the INTERLIVER study.

To extend previous molecular analyses of rejection in liver transplant biopsies in the INTERLIVER study (ClinicalTrials.gov #NCT03193151), the present study aimed to define the gene expression selective for parenchymal injury, fibrosis, and steatohepatitis. We analyzed genome-wide microarray measurements from 337 liver transplant biopsies from 13 centers. We examined expression of genes previously annotated as increased in injury and fibrosis using principal component analysis (PCA). PC1 reflected parenchymal injury and related inflammation in the early posttransplant period, slowly regressing over many months. PC2 separated early injury from late fibrosis. Positive PC3 identified a distinct mildly inflamed state correlating with histologic steatohepatitis. Injury PCs correlated with liver function and histologic abnormalities. A classifier trained on histologic steatohepatitis predicted histologic steatohepatitis with cross-validated AUC = 0.83, and was associated with pathways reflecting metabolic abnormalities distinct from fibrosis. PC2 predicted histologic fibrosis (AUC = 0.80), as did a molecular fibrosis classifier (AUC = 0.74). The fibrosis classifier correlated with matrix remodeling pathways with minimal overlap with those selective for steatohepatitis, although some biopsies had both. Genome-wide assessment of liver transplant biopsies can not only detect molecular changes induced by rejection but also those correlating with parenchymal injury, steatohepatitis, and fibrosis, offering potential insights into disease mechanisms for primary diseases.

Genetically targeting the BATF family transcription factors BATF and BATF3 in the mouse abrogates effector T cell activities and enables long-term heart allograft survival.

T cells must be activated and become effectors first before executing allograft rejection, a process that is regulated by diverse signals and transcription factors. In this study, we studied the basic leucine zipper ATF-like transcription factor (BATF) family members in regulating T cell activities in a heart transplant model and found that mice deficient for both BATF and BATF3 (Batf-/- Batf3-/- mice) spontaneously accept the heart allografts long-term without tolerizing therapies. Similarly, adoptive transfer of wild type T cells into Rag1-/- hosts induced prompt rejection of heart and skin allografts, whereas the Batf-/- Batf3-/- T cells failed to do so. Analyses of graft-infiltrating cells showed that Batf-/- Batf3-/- T cells infiltrate the graft but fail to acquire an effector phenotype (CD44high KLRG1+ ). Co-transfer experiments in a T cell receptor transgenic TEa model revealed that the Batf-/- Batf3-/- T cells fail to expand in vivo, retain a quiescent phenotype (CD62L+ CD127+ ), and unable to produce effector cytokines to alloantigen stimulation, which contrasted sharply to that of wild type T cells. Together, our data demonstrate that the BATF and BATF3 are critical regulators of T effector functions, thus making them attractive targets for therapeutic interventions in transplant settings.

Molecular diagnosis of ABMR with or without donor-specific antibody in kidney transplant biopsies: Differences in timing and intensity but similar mechanisms and outcomes.

We studied the clinical, histologic, and molecular features distinguishing DSA-negative from DSA-positive molecularly defined antibody-mediated rejection (mABMR). We analyzed mABMR biopsies with available DSA assessments from the INTERCOMEX study: 148 DSA-negative versus 248 DSA-positive, compared with 864 no rejection (excluding TCMR and Mixed). DSA-positivity varied with mABMR stage: early-stage (EABMR) 56%; fully developed (FABMR) 70%; and late-stage (LABMR) 58%. DSA-negative patients with mABMR were usually sensitized, 60% being HLA antibody-positive. Compared with DSA-positive mABMR, DSA-negative mABMR was more often C4d-negative; earlier by 1.5 years (average 2.4 vs. 3.9 years); and had lower ABMR activity and earlier stage in molecular and histology features. However, the top ABMR-associated transcripts were identical in DSA-negative versus DSA-positive mABMR, for example, NK-associated (e.g., KLRD1 and GZMB) and IFNG-inducible (e.g., PLA1A). Genome-wide class comparison between DSA-negative and DSA-positive mABMR showed no significant differences in transcript expression except those related to lower intensity and earlier time of DSA-negative ABMR. Three-year graft loss in DSA-negative mABMR was the same as DSA-positive mABMR, even after adjusting for ABMR stage. Thus, compared with DSA-positive mABMR, DSA-negative mABMR is on average earlier, less active, and more often C4d-negative but has similar graft loss, and genome-wide analysis suggests that it involves the same mechanisms. SUMMARY SENTENCE: In 398 kidney transplant biopsies with molecular antibody-mediated rejection, the 150 DSA-negative cases are earlier, less intense, and mostly C4d-negative, but use identical molecular mechanisms and have the same risk of graft loss as the 248 DSA-positive cases.

Interleukin 24 promotes cell death in renal epithelial cells and is associated with acute renal injury.

Ischemia-reperfusion injury is a major cause of acute kidney injury. Many cytokines are involved in the pathogenesis of renal ischemia-reperfusion injury. IL24 is a member of the IL10 family and has gained importance because of its apoptosis-inducing effects in tumor disease besides its immunoregulative function. Littles is known about the role of IL24 in kidney disease. Using a mouse model, we found that IL24 is upregulated in the kidney after renal ischemia-reperfusion injury and that tubular epithelial cells and infiltrating inflammatory cells are the source of IL24. Mice lacking IL24 are protected from renal injury and inflammation. Cell culture studies showed that IL24 induces apoptosis in renal tubular epithelial cells, which is accompanied by an increased endoplasmatic reticulum stress response. Moreover, IL24 induces robust expression of endogenous IL24 in tubular cells, fostering ER-stress and apoptosis. In kidney transplant recipients with delayed graft function and patients at high risk to develop acute kidney injury after cardiac surgery IL24 is upregulated in the kidney and serum. Taken together, IL24 can serve as a biomarker, plays an important mechanistic role involving both extracellular and intracellular targets, and is a promising therapeutic target in patients at risk of or with ischemia-induced acute kidney injury.

Normothermic ex situ pancreas perfusion for the preservation of porcine pancreas grafts.

Pancreas transplantation improves and extends the life of patients with insulin-dependent diabetes. Pancreata from extended criteria donors have been increasingly used due to the scarcity of available grafts. Normothermic ex situ pancreas perfusion (NESPP) can keep grafts metabolically active, potentially allowing for assessment and organ repair, and could improve outcomes of marginal grafts. A novel NESPP technique was developed and tested. Porcine pancreata were removed after a short period of warm ischemia and subjected to 6 h of NESPP. Perfusion parameters, potential graft assessment markers and graft injury were measured. Next, pancreata subjected to 3 h of NESPP were transplanted and animals were followed for up to 3 days. Graft function and injury post-transplantation were evaluated. Using this novel system of perfusion, pancreata were perfused for an extended period of time with minimal edema. Histology at the end of perfusion showed intact islet cells with only mild signs of tissue injury. NESPP transplanted grafts showed immediate function after transplantation, with glucose levels in normal range. NESPP maintains a physiologic environment and excellent graft function without causing significant graft injury. Porcine pancreas transplantation is feasible and allows for in vivo graft assessment of pancreas function and injury after NESPP.

Novel role for tumor suppressor gene, liver kinase B1, in epithelial-mesenchymal transition leading to chronic lung allograft dysfunction.

Epithelial-mesenchymal transition (EMT) has been implicated to play a role in chronic lung allograft dysfunction (CLAD). Liver kinase B1 (LKB1), a tumor suppressor gene, can regulate EMT. However, its role in CLAD development following lung transplantation remains unknown. Using qRT-PCR, biopsies from lung transplant recipients with bronchiolitis obliterans syndrome (BOS) demonstrated significant downregulation of LKB1 (p = .0001), compared to stable biopsies. To determine the role of LKB1 in EMT development, we analyzed EMT in human bronchial epithelial cell line BEAS-2B. Knockdown of LKB1 by siRNA significantly dysregulated mesenchymal markers expression in BEAS-2B cells. Following incubation of human primary bronchial epithelial cell or BEAS-2B cells with exosomes isolated from BOS or stable lung transplant recipients, LKB1 expression was inhibited when incubated with BOS-exosome. Incubation with BOS-exosomes also decreased LKB1 expression and induced EMT markers in air-liquid interface culture method. Our results provide novel evidence that exosomes released from transplanted lungs undergoing chronic rejection are associated with inactivated tumor suppressor gene LKB1 and this loss induces EMT leading to the pathogenesis of CLAD following human lung transplantation.

Blockade or deficiency of PD-L1 expression in intestinal allograft accelerates graft tissue injury in mice.

The importance of PD-1/PD-L1 interaction to alloimmune response is unknown in intestinal transplantation. We tested whether PD-L1 regulates allograft tissue injury in murine intestinal transplantation. PD-L1 expression was observed on the endothelium and immune cells in the intestinal allograft. Monoclonal antibody treatment against PD-L1 led to accelerated allograft tissue damage, characterized by severe cellular infiltrations, massive destruction of villi, and increased crypt apoptosis in the graft. Interestingly, PD-L1-/- allografts were more severely rejected than wild-type allografts, but the presence or absence of PD-L1 in recipients did not affect the degree of allograft injury. PD-L1-/- allografts showed increased infiltrating Ly6G+ and CD11b+ cells in lamina propria on day 4, whereas the degree of CD4+ or CD8+ T cell infiltration was comparable to wild-type allografts. Gene expression analysis revealed that PD-L1-/- allografts had increased mRNA expressions of Cxcr2, S100a8/9, Nox1, IL1rL1, IL1r2, and Nos2 in the lamina propria cells on day 4. Taken together, study results suggest that PD-L1 expression in the intestinal allograft, but not in the recipient, plays a critical role in mitigating allograft tissue damage in the early phase after transplantation. The PD-1/PD-L1 interaction may contribute to immune regulation of the intestinal allograft via the innate immune system.

Multiplexed droplet single-cell sequencing (Mux-Seq) of normal and transplant kidney.

Maintenance of systemic homeostasis by kidney requires the coordinated response of diverse cell types. The use of single-cell RNA sequencing (scRNAseq) for patient tissue samples remains fraught with difficulties with cell isolation, purity, and experimental bias. The ability to characterize immune and parenchymal cells during transplant rejection will be invaluable in defining transplant pathology where tissue availability is restricted to needle biopsy fragments. Herein, we present feasibility data for multiplexing approach for droplet scRNAseq (Mux-Seq). Mux-Seq has the potential to minimize experimental batch bias and variation even with very small sample input. In this first proof-of-concept study for this approach, explant tissues from six normal and two transplant recipients after multiple early post-transplant rejection episodes leading to nephrectomy due to aggressive antibody mediated rejection, were pooled for Mux-Seq. A computational tool, Demuxlet was applied for demultiplexing the individual cells from the pooled experiment. Each sample was also applied individually in a single microfluidic run (singleplex) to correlate results with the pooled data from the same sample. Our applied protocol demonstrated that data from Mux-Seq correlated highly with singleplex (Pearson coefficient 0.982) sequencing results, with the ability to identify many known and novel kidney cell types including different infiltrating immune cells. Trajectory analysis of proximal tubule and endothelial cells demonstrated separation between healthy and injured kidney from transplant explant suggesting evolving stages of cell- specific differentiation in alloimmune injury. This study provides the technical groundwork for understanding the pathogenesis of alloimmune injury and host tissue response in transplant rejection and normal human kidney and provides a protocol for optimized processing precious and low input human kidney biopsy tissue for larger scale studies.

Clonal hematopoiesis confers an increased mortality risk in orthotopic heart transplant recipients.

Novel risk stratification and non-invasive surveillance methods are needed in orthotopic heart transplant (OHT) to reduce morbidity and mortality post-transplant. Clonal hematopoiesis (CH) refers to the acquisition of specific gene mutations in hematopoietic stem cells linked to enhanced inflammation and worse cardiovascular outcomes. The purpose of this study was to investigate the association between CH and OHT. Blood samples were collected from 127 OHT recipients. Error-corrected sequencing was used to detect CH-associated mutations. We evaluated the association between CH and acute cellular rejection, CMV infection, cardiac allograft vasculopathy (CAV), malignancies, and survival. CH mutations were detected in 26 (20.5%) patients, mostly in DNMT3A, ASXL1, and TET2. Patients with CH showed a higher frequency of CAV grade 2 or 3 (0% vs. 18%, p < .001). Moreover, a higher mortality rate was observed in patients with CH (11 [42%] vs. 15 [15%], p = .008) with an adjusted hazard ratio of 2.9 (95% CI, 1.4-6.3; p = .003). CH was not associated with acute cellular rejection, CMV infection or malignancies. The prevalence of CH in OHT recipients is higher than previously reported for the general population of the same age group, with an associated higher prevalence of CAV and mortality.