Inhibition of NFAT promotes loss of tissue resident uterine natural killer cells and attendant pregnancy complications in humans.

Uterine natural killer cells (uNKs) are a tissue resident lymphocyte population that are critical for pregnancy success. Although mouse models have demonstrated that NK deficiency results in abnormal placentation and poor pregnancy outcomes, the generalizability of this knowledge to humans remains unclear. Here we identify uterus transplant (UTx) recipients as a human population with reduced endometrial NK cells and altered pregnancy phenotypes. We further show that the NK reduction in UTx is due to impaired transcriptional programming of NK tissue residency due to blockade of the transcription factor nuclear factor of activated T cells (NFAT). NFAT-dependent genes played a role in multiple molecular circuits governing tissue residency in uNKs, including early residency programs involving AP-1 transcription factors as well as TGFß-mediated upregulation of surface integrins. Collectively, our data identify a previously undescribed role for NFAT in uterine NK tissue residency and provide novel mechanistic insights into the biologic basis of pregnancy complications due to alteration of tissue resident NK subsets in humans. One Sentence Summary: Role of NFAT in uterine NK cell tissue residency.

Spatiotemporal immune atlas of a clinical-grade gene-edited pig-to-human kidney xenotransplant.

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. Here, we transplant a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and study the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells are uncommon in the porcine kidney cortex early after xenotransplantation and consist of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages express genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft is detectable. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression may be able to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.

Classification and treatment of vaginal strictures at the donor-recipient anastomosis after uterus transplant.

OBJECTIVE: To describe the incidence and management of vaginal stricture after uterus transplantation (UTx) in the US, to propose a grading system to classify stricture severity, and to identify risk factors for stricture formation. DESIGN: Prospective cohort study. SETTING: University Hospital. PATIENTS: Recipients undergoing UTx from 2016-2023 at Baylor University Medical Center in Dallas, Cleveland Clinic, the University of Pennsylvania, and the University of Alabama at Birmingham were monitored postoperatively with regular pelvic examinations. Stricture was defined as vaginal narrowing of <3 cm in patients with graft survival of at least 7 days. INTERVENTION: Demographic and surgery characteristics. MAIN OUTCOME MEASURES: Stricture development and severity (grade 1 for diameter 2-<3 cm, grade 2 for 1-<2 cm, or grade 3 for <1 cm). RESULTS: Of the 45 UTx from 2016-2023 (16 deceased donors and 29 living donors), 3 were excluded from the analysis because of graft loss within 7 days. Of the 42 remaining recipients, 39 (92.9%) had Mayer-Rokitansky-Küster-Hauser syndrome and 3 (7.1%) had a prior hysterectomy. Twenty-eight (66.7%) UTx recipients developed postoperative vaginal strictures with a median time to stricture of 33 days (interquartile range 19-53 days). Most strictures were of moderate severity, with 4 (14.3%) strictures categorized as grade 1, 19 (67.9%) as grade 2, and 5 (17.9%) as grade 3. History of Mayer-Rokitansky-Küster-Hauser syndrome and preoperative recipient vaginal length were significant risk factors for stricture, after adjustment for donor and recipient age and body mass index, anastomosis technique, total ischemia time, center, and year. Patients with longer preoperative vaginal length had a lower risk of stricture (hazard ratio 0.45, 0.29-0.70). The severity grading of the stricture was associated with the effectiveness of a nonoperative treatment approach (grade 1 vs. grade 3). No patients with grade 3 strictures improved with self-dilation alone; all required surgical repair and/or dilation under anesthesia. Conversely, for grade 1 or 2 strictures, self-dilation alone was successful in 47.8% (11/23), and no grade 1 strictures required surgical repair. CONCLUSIONS: Vaginal stricture is a common postoperative complication after UTx, affecting >65% of recipients. Short preoperative vaginal length and history of müllerian agenesis in the recipient are significant risk factors. Vaginal self-dilation was effective for some mild to moderate strictures, although dilation under anesthesia or surgical repair was required in most cases. CLINICAL TRIAL REGISTRATION NUMBERS: Dallas UtErus Transplant Study (DUETS) at Baylor University Medical Center (NCT02656550), Uterine transplantation for the treatment of uterine factor infertility at the Cleveland Clinic (NCT02573415), The University of Pennsylvania Uterus Transplant for Uterine Factor Infertility Trial (UNTIL) (NCT03307356).

Physiologic homeostasis after pig-to-human kidney xenotransplantation.

Demand for kidney grafts outpaces supply, limiting kidney transplantation as a treatment for kidney failure. Xenotransplantation has the potential to make kidney transplantation available to many more patients with kidney failure, but the ability of xenografts to support human physiologic homeostasis has not been established. A brain-dead adult decedent underwent bilateral native nephrectomies followed by 10 gene-edited (four gene knockouts, six human transgenes) pig-to-human xenotransplantation. Physiologic parameters and laboratory values were measured for seven days in a critical care setting. Data collection aimed to assess homeostasis by measuring components of the renin-angiotensin-aldosterone system, parathyroid hormone signaling, glomerular filtration rate, and markers of salt and water balance. Mean arterial blood pressure was maintained above 60 mmHg throughout. Pig kidneys secreted renin (post-operative day three to seven mean and standard deviation: 47.3 ± 9 pg/mL). Aldosterone and angiotensin II levels were present (post-operative day three to seven, 57.0 ± 8 pg/mL and 5.4 ± 4.3 pg/mL, respectively) despite plasma renin activity under 0.6 ng/mL/hr. Parathyroid hormone levels followed ionized calcium. Urine output down trended from 37 L to 6 L per day with 4.5 L of electrolyte free water loss on post-operative day six. Aquaporin 2 channels were detected in the apical surface of principal cells, supporting pig kidney response to human vasopressin. Serum creatinine down trended to 0.9 mg/dL by day seven. Glomerular filtration rate ranged 90-240 mL/min by creatinine clearance and single-dose inulin clearance. Thus, in a human decedent model, xenotransplantation of 10 gene-edited pig kidneys provided physiologic balance for seven days. Hence, our in-human study paves the way for future clinical study of pig-to-human kidney xenotransplantation in living persons.

Regulatory T cell adoptive transfer alters uterine immune populations, increasing a novel MHC-IIlow macrophage associated with healthy pregnancy.

Intrauterine fetal demise (IUFD) - fetal loss after 20 weeks - affects 6 pregnancies per 1,000 live births in the United States, and the majority are of unknown etiology. Maternal systemic regulatory T cell (Treg) deficits have been implicated in fetal loss, but whether mucosal immune cells at the maternal-fetal interface contribute to fetal loss is under-explored. We hypothesized that the immune cell composition and function of the uterine mucosa would contribute to the pathogenesis of IUFD. To investigate local immune mechanisms of IUFD, we used the CBA mouse strain, which naturally has mid-late gestation fetal loss. We performed a Treg adoptive transfer and interrogated both pregnancy outcomes and the impact of systemic maternal Tregs on mucosal immune populations at the maternal-fetal interface. Treg transfer prevented fetal loss and increased an MHC-IIlow population of uterine macrophages. Single-cell RNA-sequencing was utilized to precisely evaluate the impact of systemic Tregs on uterine myeloid populations. A population of C1q+, Trem2+, MHC-IIlow uterine macrophages were increased in Treg-recipient mice. The transcriptional signature of this novel uterine macrophage subtype is enriched in multiple studies of human healthy decidual macrophages, suggesting a conserved role for these macrophages in preventing fetal loss.

How mothers tolerate their children.

Shifting pools of antigen can influence pregnancy-induced immune tolerance.

Normal Graft Function After Pig-to-Human Kidney Xenotransplant.

This case series examines the ability of a pig-to-human xenograft to provide life-sustaining kidney function.

B cell and antibody targeting of transplant alloantigen epitopes is supported by both self and allo-recognition.

Donor-specific antibody (DSA) responses against human leukocyte antigen (HLA) proteins mismatched between kidney transplant donors and recipients cause allograft loss. Using single-cell, molecular, structural, and proteomic techniques, we profiled the HLA-specific (alloreactive) B cell response in kidney and blood of a transplant recipient with antibody-mediated rejection (AMR). We identified 14 distinct alloreactive B cell lineages, which spanned the rejected organ and blood and expressed high-affinity anti-donor HLA-specific B cell receptors, many of which were clonally linked to circulating DSA. The alloreactive B cell response was focused on exposed, solvent-accessible mismatched HLA residues, while also demonstrating extensive contacts with self-HLA residues. Consistent with structural evidence of self-recognition, measurable self-reactivity by donor-specific B cells was common and positively correlated with anti-donor affinity maturation. Thus, allo- and self-reactive signatures appeared to converge, suggesting that during AMR, the recognition of non-self and breaches of tolerance conspire to produce a pathogenic donor-specific adaptive response.

Spatiotemporal immune atlas of the first clinical-grade, gene-edited pig-to-human kidney xenotransplant.

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. We transplanted a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and studied the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells were uncommon in the porcine kidney cortex early after xenotransplantation and consisted of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages expressed genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft was detected. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression is sufficient to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.

COVID-19 Vaccination and Remdesivir are Associated With Protection From New or Increased Levels of Donor-Specific Antibodies Among Kidney Transplant Recipients Hospitalized With COVID-19.

Alloimmune responses in kidney transplant (KT) patients previously hospitalized with COVID-19 are understudied. We analyzed a cohort of 112 kidney transplant recipients who were hospitalized following a positive SARS-CoV-2 test result during the first 20 months of the COVID-19 pandemic. We found a cumulative incidence of 17% for the development of new donor-specific antibodies (DSA) or increased levels of pre-existing DSA in hospitalized SARS-CoV-2-infected KT patients. This risk extended 8 months post-infection. These changes in DSA status were associated with late allograft dysfunction. Risk factors for new or increased DSA responses in this KT patient cohort included the presence of circulating DSA pre-COVID-19 diagnosis and time post-transplantation. COVID-19 vaccination prior to infection and remdesivir administration during infection were each associated with decreased likelihood of developing a new or increased DSA response. These data show that new or enhanced DSA responses frequently occur among KT patients requiring admission with COVID-19 and suggest that surveillance, vaccination, and antiviral therapies may be important tools to prevent alloimmunity in these individuals.

A multi-institutional study of renal outcomes and renal-related pregnancy outcomes in uterus transplant recipients.

Uterus transplantation (UTx) is an effective treatment option for uterine factor infertility. However, the need for immunosuppression and congenital renal anomalies that coexist with uterine agenesis in about 30% of women with Mayer-Rokitansky-Kuster-Hauser syndrome create a risk for renal dysfunction. We therefore examined renal function trajectory and related pregnancy complications in an international cohort of 18 UTx recipients from September 2016-February 2020 who had at least one live birth. All UTx recipients had a diminution in their renal function that was apparent starting at 30 days posttransplant and in half the reduction in eGFR was at least 20%; the decrease in eGFR persisted into the early post-partum period. Half met criteria for Stage 1 acute kidney injury (AKI) as defined by the AKI Network criteria during their pregnancy. Overall, 28% of UTx recipients developed pre-eclampsia. eGFR was lower at embryo transfer and throughout pregnancy among those who developed pre-eclampsia, reaching statistical significance at week 16 of pregnancy. This effect was independent of tacrolimus levels. Mean eGFR remained significantly lower in the first 1-3 months after delivery. In the subgroup who reached 12 months of postpartum follow up and had a graft hysterectomy (n = 4), there was no longer a statistical difference in eGFR (pretransplant 106.7 ml/m ± 17.7 vs. 12 mos postpartum 92.6 ml/m ± 21.7, p = .13) but the number was small. Further study is required to delineate long term renal risks for UTx recipients, improve patient selection, and make decisions regarding a second pregnancy.

The science of xenotransplantation for nephrologists.

PURPOSE OF REVIEW: The field of xenotransplantation has seen remarkable progress since its inception with recent preclinical trials in human recipients pushing kidney xenotransplantation one-step closer to clinical reality. In this review, we update practicing clinicians on recent advances in kidney xenotransplantation given the proximity of clinical trials in humans. RECENT FINDINGS: Early studies in the field established the physiologic basis of xenotransplantation and suggested that the pig kidney will support human physiology. Genetic engineering of source pigs has greatly reduced the immunogenicity of kidney grafts, and studies in nonhuman primates have demonstrated the viability of kidney xenotransplants for months after transplantation. Finally, a recent study in a novel preclinical human model demonstrated that key findings in NHP experiments are generalizable to humans, namely, the absence of hyperacute rejection. SUMMARY: Overall, it appears that critical physiologic, immunologic and technical barriers to implementation of clinical trials in humans have been overcome.

Immunologic and Infectious Concerns in Uterus Transplantation.

Pharmacologic immunosuppression is required for the success of uterus transplantation but can provoke several complications for the transplant recipient. In this review, we discuss the immunologic complications that can occur in the uterus transplant recipient. First, we provide the latest update on immunosuppression regimens used by programs throughout the world. Next, we discuss the prevalence, mechanisms, treatment, and outcome of rejection in uterus transplant recipients. Finally, we discuss infectious complications of varying severity alongside their treatment and impact.

First clinical-grade porcine kidney xenotransplant using a human decedent model.

A radical solution is needed for the organ supply crisis, and the domestic pig is a promising organ source. In preparation for a clinical trial of xenotransplantation, we developed an in vivo pre-clinical human model to test safety and feasibility tenets established in animal models. After performance of a novel, prospective compatible crossmatch, we performed bilateral native nephrectomies in a human brain-dead decedent and subsequently transplanted two kidneys from a pig genetically engineered for human xenotransplantation. The decedent was hemodynamically stable through reperfusion, and vascular integrity was maintained despite the exposure of the xenografts to human blood pressure. No hyperacute rejection was observed, and the kidneys remained viable until termination 74 h later. No chimerism or transmission of porcine retroviruses was detected. Longitudinal biopsies revealed thrombotic microangiopathy that did not progress in severity, without evidence of cellular rejection or deposition of antibody or complement proteins. Although the xenografts produced variable amounts of urine, creatinine clearance did not recover. Whether renal recovery was impacted by the milieu of brain death and/or microvascular injury remains unknown. In summary, our study suggests that major barriers to human xenotransplantation have been surmounted and identifies where new knowledge is needed to optimize xenotransplantation outcomes in humans.

NFAT-dependent and -independent exhaustion circuits program maternal CD8 T cell hypofunction in pregnancy.

Pregnancy is a common immunization event, but the molecular mechanisms and immunological consequences provoked by pregnancy remain largely unknown. We used mouse models and human transplant registry data to reveal that pregnancy induced exhausted CD8 T cells (Preg-TEX), which associated with prolonged allograft survival. Maternal CD8 T cells shared features of exhaustion with CD8 T cells from cancer and chronic infection, including transcriptional down-regulation of ribosomal proteins and up-regulation of TOX and inhibitory receptors. Similar to other models of T cell exhaustion, NFAT-dependent elements of the exhaustion program were induced by fetal antigen in pregnancy, whereas NFAT-independent elements did not require fetal antigen. Despite using conserved molecular circuitry, Preg-TEX cells differed from TEX cells in chronic viral infection with respect to magnitude and dependency of T cell hypofunction on NFAT-independent signals. Altogether, these data reveal the molecular mechanisms and clinical consequences of maternal CD8 T cell hypofunction and identify pregnancy as a previously unappreciated context in which T cell exhaustion may occur.

Uterus transplantation: the importance of uterine natural killer cells.

PURPOSE OF REVIEW: Murine studies have established that uterine natural killer (uNK) cells are critical regulators of normal placentation and fetal development in mammals. However, the biology of uNK cells in humans remains poorly understood. This ignorance represents a costly knowledge gap, as disordered placentation is thought to underpin a variety of pregnancy complications that impact maternal and neonatal health. In the context of uterus transplantation (UTx), uNK cells are anticipated to play a critical role within the allograft. Here, we review the current understanding of uNK cells in pregnancy biology and explore how this critically important cell population may contribute to pregnancy and graft outcomes in uterus transplant recipients. RECENT FINDINGS: Recent studies have characterized differences in NK cell populations between anatomic compartments in humans. In the endometrium, at least five phenotypically and functionally distinct subpopulations of uNK cells have been identified, with research into mechanisms regulating their differentiation and function currently underway. SUMMARY: Further elucidating uNK cell biology has the potential to influence the outcomes of pregnancy and UTx and benefit human health. UTx is a unique opportunity to study uNK cell biology and may shed light on mechanisms by which immunological tolerance is established at the maternal-fetal interface.

IFNγ-Producing γ/δ T Cells Accumulate in the Fetal Brain Following Intrauterine Inflammation.

Intrauterine inflammation impacts prenatal neurodevelopment and is linked to adverse neurobehavioral outcomes ranging from cerebral palsy to autism spectrum disorder. However, the mechanism by which a prenatal exposure to intrauterine inflammation contributes to life-long neurobehavioral consequences is unknown. To address this gap in knowledge, this study investigates how inflammation transverses across multiple anatomic compartments from the maternal reproductive tract to the fetal brain and what specific cell types in the fetal brain may cause long-term neuronal injury. Utilizing a well-established mouse model, we found that mid-gestation intrauterine inflammation resulted in a lasting neutrophil influx to the decidua in the absence of maternal systemic inflammation. Fetal immunologic changes were observed at 72-hours post-intrauterine inflammation, including elevated neutrophils and macrophages in the fetal liver, and increased granulocytes and activated microglia in the fetal brain. Through unbiased clustering, a population of Gr-1+ γ/δ T cells was identified as the earliest immune cell shift in the fetal brain of fetuses exposed to intrauterine inflammation and determined to be producing high levels of IFNγ when compared to γ/δ T cells in other compartments. In a case-control study of term infants, IFNγ was found to be elevated in the cord blood of term infants exposed to intrauterine inflammation compared to those without this exposure. Collectively, these data identify a novel cellular immune mechanism for fetal brain injury in the setting of intrauterine inflammation.