The IPTA Nashville consensus conference on post-transplant lymphoproliferative disorders after solid organ transplantation in children: IV-consensus guidelines for the management of post-transplant lymphoproliferative disorders in children and adolescents.

The International Pediatric Transplant Association convened an expert consensus conference to assess current evidence and develop recommendations for various aspects of care relating to post-transplant lymphoproliferative disorders (PTLD) after pediatric solid organ transplantation. This report addresses the outcomes of deliberations by the PTLD Management Working Group. A strong recommendation was made for reduction in immunosuppression as the first step in management. Similarly, strong recommendations were made for the use of the anti-CD20 monoclonal antibody (rituximab) as was the case for chemotherapy in selected scenarios. In some scenarios, there is uncoupling of the strength of the recommendations from the available evidence in situations where such evidence is lacking but collective clinical experiences drive decision-making. Of note, there are no large, randomized phase III trials of any treatment for PTLD in the pediatric age group. Current gaps and future research priorities are highlighted.

Epstein-Barr virus-associated post-transplant lymphoproliferative disorders in pediatric transplantation: A prospective multicenter study in the United States.

BACKGROUND: Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disorders (PTLD) is the most common malignancy in children after transplant; however, difficulties for early detection may worsen the prognosis. METHODS: The prospective, multicenter, study enrolled 944 children (≤21 years of age). Of these, 872 received liver, heart, kidney, intestinal, or multivisceral transplants in seven US centers between 2014 and 2019 (NCT02182986). In total, 34 pediatric EBV+ PTLD (3.9%) were identified by biopsy. Variables included sex, age, race, ethnicity, transplanted organ, EBV viral load, pre-transplant EBV serology, immunosuppression, response to chemotherapy and rituximab, and histopathological diagnosis. RESULTS: The uni-/multivariable competing risk analyses revealed the combination of EBV-seropositive donor and EBV-naïve recipient (D+R-) was a significant risk factor for PTLD development (sub-hazard ratio: 2.79 [1.34-5.78], p = .006) and EBV DNAemia (2.65 [1.72-4.09], p < .001). Patients with D+R- were significantly more associated with monomorphic/polymorphic PTLD than those with the other combinations (p = .02). Patients with monomorphic/polymorphic PTLD (n = 21) had significantly more EBV DNAemia than non-PTLD patients (p < .001) and an earlier clinical presentation of PTLD than patients with hyperplasias (p < .001), within 6-month post-transplant. Among non-liver transplant recipients, monomorphic/polymorphic PTLD were significantly more frequent than hyperplasias in patients ≥5 years of age at transplant (p = .01). CONCLUSIONS: D+R- is a risk factor for PTLD and EBV DNAemia and associated with the incidence of monomorphic/polymorphic PTLD. Intensive follow-up of EBV viral load within 6-month post-transplant, especially for patients with D+R- and/or non-liver transplant recipients ≥5 years of age at transplant, may help detect monomorphic/polymorphic PTLD early in pediatric transplant.

Application of Mass Cytometry Platforms to Solid Organ Transplantation.

Transplantation serves as the cornerstone of treatment for patients with end-stage organ disease. The prevalence of complications, such as allograft rejection, infection, and malignancies, underscores the need to dissect the complex interactions of the immune system at the single-cell level. In this review, we discuss studies using mass cytometry or cytometry by time-of-flight, a cutting-edge technology enabling the characterization of immune populations and cell-to-cell interactions in granular detail. We review the application of mass cytometry in human and experimental animal studies in the context of transplantation, uncovering invaluable contributions of the tool to understanding rejection and other transplant-related complications. We discuss recent innovations that have the potential to streamline and standardize mass cytometry workflows for application to multisite clinical trials. Additionally, we introduce imaging mass cytometry, a technique that couples the power of mass cytometry with spatial context, thereby mapping cellular interactions within tissue microenvironments. The synergistic integration of mass cytometry and imaging mass cytometry data with other omics data sets and high-dimensional data platforms to further define immune dynamics is discussed. In conclusion, mass cytometry technologies, when integrated with other tools and data, shed light on the intricate landscape of the immune response in transplantation. This approach holds significant potential for enhancing patient outcomes by advancing our understanding and facilitating the development of new diagnostics and therapeutics.

The IPTA Nashville Consensus Conference on Post-Transplant lymphoproliferative disorders after solid organ transplantation in children: III - Consensus guidelines for Epstein-Barr virus load and other biomarker monitoring.

The International Pediatric Transplant Association convened an expert consensus conference to assess current evidence and develop recommendations for various aspects of care relating to post-transplant lymphoproliferative disorders after solid organ transplantation in children. In this report from the Viral Load and Biomarker Monitoring Working Group, we reviewed the existing literature regarding the role of Epstein-Barr viral load and other biomarkers in peripheral blood for predicting the development of PTLD, for PTLD diagnosis, and for monitoring of response to treatment. Key recommendations from the group highlighted the strong recommendation for use of the term EBV DNAemia instead of "viremia" to describe EBV DNA levels in peripheral blood as well as concerns with comparison of EBV DNAemia measurement results performed at different institutions even when tests are calibrated using the WHO international standard. The working group concluded that either whole blood or plasma could be used as matrices for EBV DNA measurement; optimal specimen type may be clinical context dependent. Whole blood testing has some advantages for surveillance to inform pre-emptive interventions while plasma testing may be preferred in the setting of clinical symptoms and treatment monitoring. However, EBV DNAemia testing alone was not recommended for PTLD diagnosis. Quantitative EBV DNAemia surveillance to identify patients at risk for PTLD and to inform pre-emptive interventions in patients who are EBV seronegative pre-transplant was recommended. In contrast, with the exception of intestinal transplant recipients or those with recent primary EBV infection prior to SOT, surveillance was not recommended in pediatric SOT recipients EBV seropositive pre-transplant. Implications of viral load kinetic parameters including peak load and viral set point on pre-emptive PTLD prevention monitoring algorithms were discussed. Use of additional markers, including measurements of EBV specific cell mediated immunity was discussed but not recommended though the importance of obtaining additional data from prospective multicenter studies was highlighted as a key research priority.

High-dimensional profiling of pediatric immune responses to solid organ transplantation.

Solid organ transplant remains a life-saving therapy for children with end-stage heart, lung, liver, or kidney disease; however, ∼33% of allograft recipients experience acute rejection within the first year after transplant. Our ability to detect early rejection is hampered by an incomplete understanding of the immune changes associated with allograft health, particularly in the pediatric population. We performed detailed, multilineage, single-cell analysis of the peripheral blood immune composition in pediatric solid organ transplant recipients, with high-dimensional mass cytometry. Supervised and unsupervised analysis methods to study cell-type proportions indicate that the allograft type strongly influences the post-transplant immune profile. Further, when organ-specific differences are considered, graft health is associated with changes in the proportion of distinct T cell subpopulations. Together, these data form the basis for mechanistic studies into the pathobiology of rejection and allow for the development of new immunosuppressive agents with greater specificity.

Mutations in latent membrane protein 1 of Epstein-Barr virus are associated with increased risk of posttransplant lymphoproliferative disorder in children.

Epstein-Barr virus (EBV)-positive posttransplant lymphoproliferative disorder (PTLD) results in significant morbidity and mortality in pediatric transplant recipients. Identifying individuals at an increased risk of EBV-positive PTLD could influence clinical management of immunosuppression and other therapies, improving posttransplant outcomes. A 7-center prospective, observational clinical trial of 872 pediatric transplant recipients evaluated the presence of mutations at positions 212 and 366 of EBV latent membrane protein 1 (LMP1) as an indicator of risk of EBV-positive PTLD (clinical trials: NCT02182986). DNA was isolated from peripheral blood of EBV-positive PTLD case patients and matched controls (1:2 nested case:control), and the cytoplasmic tail of LMP1 was sequenced. Thirty-four participants reached the primary endpoint of biopsy-proven EBV-positive PTLD. DNA was sequenced from 32 PTLD case patients and 62 matched controls. Both LMP1 mutations were present in 31 of 32 PTLD cases (96.9%) and in 45 of 62 matched controls (72.6%) (P = .005; OR = 11.7; 95% confidence interval, 1.5, 92.6). The presence of both G212S and S366T carries a nearly 12-fold increased risk of development of EBV-positive PTLD. Conversely, transplant recipients without both LMP1 mutations carry a very low risk of PTLD. Analysis of mutations at positions 212 and 366 of LMP1 can be informative in stratifying patients for risk of EBV-positive PTLD.

The IPTA Nashville consensus conference on Post-Transplant lymphoproliferative disorders after solid organ transplantation in children: II-consensus guidelines for prevention.

The International Pediatric Transplant Association (IPTA) convened an expert consensus conference to assess current evidence and develop recommendations for various aspects of care relating to post-transplant lymphoproliferative disorder after solid organ transplantation in children. In this report from the Prevention Working Group, we reviewed the existing literature regarding immunoprophylaxis and chemoprophylaxis, and pre-emptive strategies. While the group made a strong recommendation for pre-emptive reduction of immunosuppression at the time of EBV DNAemia (low to moderate evidence), no recommendations for use could be made for any prophylactic strategy or alternate pre-emptive strategy, largely due to insufficient or conflicting evidence. Current gaps and future research priorities are highlighted.

Host microRNAs are decreased in pediatric solid-organ transplant recipients during EBV+ Post-transplant Lymphoproliferative Disorder.

Post-transplant lymphoproliferative disorder (PTLD) is a serious complication of solid organ transplantation. Predisposing factors include primary Epstein-Barr virus (EBV) infection, reactivation of EBV in recipient B cells, and decreased T cell immunity due to immunosuppression. In our previous studies EBV infection was demonstrated to markedly alter the expression of host B cell microRNA (miR). Specifically, miR-194 expression was uniquely suppressed in EBV+ B cell lines from PTLD patients and the 3'untranslated region of IL-10 was determined to be targeted by miR-194. Although EBV has been shown to regulate host miR expression in B cell lymphoma cell lines, the expression of miRs in the circulation of patients with EBV-associated PTLD has not been studied. The objective of this study was to determine if changes in miR expression are associated with EBV+ PTLD. In this study, we have shown that miR-194 is significantly decreased in EBV+PTLD tumors and that additional miRs, including miRs-17, 19 and 106a are also reduced in EBV+PTLD as compared to EBV-PTLD. We quantitated the levels of miRs-17, 19, 106a, 155, and 194 in the plasma and extracellular vesicles (EV; 50-70 nm as determined by nanoparticle tracking analysis) from pediatric recipients of solid organ transplants with EBV+ PTLD+ that were matched 1:2 with EBV+ PTLD- pediatric transplant recipients as part of the NIH-sponsored Clinical Trials in Organ Transplantation in Children, (CTOTC-06) study. Levels of miRs-17, 19, 106a, and 194 were reduced in the plasma and extracellular vesicles (EV) of EBV+ PTLD+ group compared to matched controls, with miRs-17 (p = 0.034; plasma), miRs-19 (p = 0.029; EV) and miR-106a (p = 0.007; plasma and EV) being significantly reduced. Similar levels of miR-155 were detected in the plasma and EV of all pediatric SOT recipients. Importantly, ~90% of the cell-free miR were contained within the EV supporting that EBV+ PTLD tumor miR are detected in the circulation and suggesting that EVs, containing miRs, may have the potential to target and regulate cells of the immune system. Further development of diagnostic, mechanistic and potential therapeutic uses of the miRs in PTLD is warranted.

Malnutrition and immune cell subsets in children undergoing kidney transplantation.

BACKGROUND: Malnutrition, including obesity and undernutrition, among children is increasing in prevalence and is common among children on renal replacement therapy. The effect of malnutrition on the pre-transplant immune system and how the pediatric immune system responds to the insult of both immunosuppression and allotransplantation is unknown. We examined the relationship of nutritional status with post-transplant outcomes and characterized the peripheral immune cell phenotypes of children from the Immune Development of Pediatric Transplant (IMPACT) study. METHODS: Ninety-eight patients from the IMPACT study were classified as having obesity, undernutrition, or normal nutrition-based pre-transplant measurements. Incidence of infectious and alloimmune outcomes at 1-year post-transplantation was compared between nutritional groups using Gray's test and Fine-Gray subdistribution hazards model. Event-free survival was estimated by Kaplan-Meier method and compared between groups. Differences in immune cell subsets between nutritional groups over time were determined using generalized estimating equations accounting for the correlation between repeated measurements. RESULTS: We did not observe that nutritional status was associated with infectious or alloimmune events or event-free survival post-transplant. We demonstrated that children with obesity had distinct T-and B-cell signatures relative to those with undernutrition and normal nutrition, even when controlling for immunosuppression. Children with obesity had a lower frequency of CD8 Tnaive cells 9-month post-transplant (p < .001), a higher frequency of CD4 CD57 + PD1- T cells, and lower frequencies of CD57-PD1+ CD8 and CD57-PD1- CD8 T cells at 12-month transplant (p < .05 for all). CONCLUSIONS: Children with obesity have distinct immunophenotypes that may influence the tailoring of immunosuppression.

Human IL-10-producing B cells have diverse states that are induced from multiple B cell subsets.

Regulatory B cells (Bregs) suppress immune responses through the secretion of interleukin-10 (IL-10). This immunomodulatory capacity holds therapeutic potential, yet a definitional immunophenotype for enumeration and prospective isolation of B cells capable of IL-10 production remains elusive. Here, we simultaneously quantify cytokine production and immunophenotype in human peripheral B cells across a range of stimulatory conditions and time points using mass cytometry. Our analysis shows that multiple functional B cell subsets produce IL-10 and that no phenotype uniquely identifies IL-10+ B cells. Further, a significant portion of IL-10+ B cells co-express the pro-inflammatory cytokines IL-6 and tumor necrosis factor alpha (TNFα). Despite this heterogeneity, operationally tolerant liver transplant recipients have a unique enrichment of IL-10+, but not TNFα+ or IL-6+, B cells compared with transplant recipients receiving immunosuppression. Thus, human IL-10-producing B cells constitute an induced, transient state arising from a diversity of B cell subsets that may contribute to maintenance of immune homeostasis.

Functional Consequences of Memory Inflation after Solid Organ Transplantation.

CMV is a major infectious complication following solid organ transplantation. Reactivation of CMV leads to memory inflation, a process in which CD8 T cells expand over time. Memory inflation is associated with specific changes in T cell function, including increased oligoclonality, decreased cytokine production, and terminal differentiation. To address whether memory inflation during the first year after transplantation in human subjects alters T cell differentiation and function, we employed single-cell-matched TCRαß and targeted gene expression sequencing. Expanded T cell clones exhibited a terminally differentiated, immunosenescent, and polyfunctional phenotype whereas rare clones were less differentiated. Clonal expansion occurring between pre- and 3 mo posttransplant was accompanied by enhancement of polyfunctionality. In contrast, polyfunctionality and differentiation state were largely maintained between 3 and 12 mo posttransplant. Highly expanded clones had a higher degree of polyfunctionality than rare clones. Thus, CMV-responsive CD8 T cells differentiated during the pre- to posttransplant period then maintained their differentiation state and functional capacity despite posttransplant clonal expansion.

Evolution of Cytomegalovirus-Responsive T Cell Clonality following Solid Organ Transplantation.

CMV infection is a significant complication after solid organ transplantation. We used single cell TCR αß sequencing to determine how memory inflation impacts clonality and diversity of the CMV-responsive CD8 and CD4 T cell repertoire in the first year after transplantation in human subjects. We observed CD8 T cell inflation but no changes in clonal diversity, indicating homeostatic stability in clones. In contrast, the CD4 repertoire was diverse and stable over time, with no evidence of CMV-responsive CD4 T cell expansion. We identified shared CDR3 TCR motifs among patients but no public CMV-specific TCRs. Temporal changes in clonality in response to transplantation and in the absence of detectable viral reactivation suggest changes in the repertoire immediately after transplantation followed by an expansion with stable clonal competition that may mediate protection.

High-resolution phenotyping of early acute rejection reveals a conserved alloimmune signature.

Alloimmune responses in acute rejection are complex, involving multiple interacting cell types and pathways. Deep profiling of these cell types has been limited by technology that lacks the capacity to resolve this high dimensionality. Single-cell mass cytometry is used to characterize the alloimmune response in early acute rejection, measuring 37 parameters simultaneously, across multiple time points in two models: a murine cardiac and vascularized composite allotransplant (VCA). Semi-supervised hierarchical clustering is used to group related cell types defined by combinatorial expression of surface and intracellular proteins, along with markers of effector function and activation. This expression profile is mapped to visualize changes in antigen composition across cell types, revealing phenotypic signatures in alloimmune T cells, natural killer (NK) cells, and myeloid subsets that are conserved and that firmly distinguish rejecting from non-rejecting grafts. These data provide a comprehensive, high-dimensional profile of cellular rejection after allograft transplantation.

Relationship between antithymocyte globulin, T cell phenotypes, and clinical outcomes in pediatric kidney transplantation.

Depletional induction using antithymocyte globulin (ATG) reduces rates of acute rejection in adult kidney transplant recipients, yet little is known about its effects in children. Using a longitudinal cohort of 103 patients in the Immune Development in Pediatric Transplant (IMPACT) study, we compared T cell phenotypes after ATG or non-ATG induction. We examined the effects of ATG on the early clinical outcomes of alloimmune events (development of de novo donor specific antibody and/or biopsy proven rejection) and infection events (viremia/viral infections). Long-term patient and graft outcomes were examined using the Scientific Registry of Transplant Recipients. After ATG induction, although absolute counts of CD4 and CD8 T cells were lower, patients had higher percentages of CD4 and CD8 memory T cells with a concomitant decrease in frequency of naïve T cells compared to non-ATG induction. In adjusted and unadjusted models, ATG induction was associated with increased early event-free survival, with no difference in long-term patient or allograft survival. Decreased CD4+ naïve and increased CD4+ effector memory T cell frequencies were associated with improved clinical outcomes. Though immunologic parameters are drastically altered with ATG induction, long-term clinical benefits remain unclear in pediatric patients.

Epstein-Barr virus peptides derived from latent cycle proteins alter NKG2A + NK cell effector function.

Natural killer (NK) cells control viral infection through the interaction between inhibitory receptors and human leukocyte antigen (HLA) ligands and bound peptide. NK cells expressing the inhibitory receptor NKG2A/CD94 recognize and respond to autologous B cells latently infected with Epstein-Barr virus (EBV). The mechanism is not yet understood, thus we investigated peptides derived from seven latent proteins of EBV in the interaction of NKG2A and its ligand HLA-E. Functional analysis demonstrated that EBV peptides can bind to HLA-E and block inhibition of NK cell effector function. Moreover, analysis of DNA from 79 subjects showed sequence variations in the latent protein, LMP1, which alters NK responses to EBV. We provide evidence that peptides derived from EBV latent cycle proteins can impair the recognition of NKG2A despite being presented by HLA-E, resulting in NK cell activation.

Genomic variations in EBNA3C of EBV associate with posttransplant lymphoproliferative disorder.

Epstein-Barr Virus (EBV) is a ubiquitous virus linked to a variety of lymphoid and epithelial malignancies. In solid organ and hematopoietic stem cell transplant recipients, EBV is causally associated with posttransplant lymphoproliferative disorder (PTLD), a group of heterogeneous lymphoid diseases. EBV+ B cell lymphomas that develop in the context of PTLD are generally attributed to the immunosuppression required to promote graft survival, but little is known regarding the role of EBV genome diversity in the development of malignancy. We deep-sequenced the EBV genome from the peripheral blood of 18 solid organ transplant recipients, including 6 PTLD patients. Sequences from 6 EBV+ spontaneous lymphoblastoid B cell lines (SLCL) were similarly analyzed. The EBV genome from PTLD patients had a significantly greater number of variations than EBV from transplant recipients without PTLD. Importantly, there were 15 nonsynonymous variations, including 8 in the latent cycle gene EBNA3C that were associated with the development of PTLD. One of the nonsynonymous variations in EBNA3C is located within a previously defined T cell epitope. These findings suggest that variations in the EBV genome can contribute to the pathogenesis of PTLD.

Biomarkers for PTLD diagnosis and therapies.

Post-transplant lymphoproliferative disorder (PTLD) represents a spectrum of lymphoproliferative disorders and is a serious complication of pediatric transplantation. The majority of PTLD are associated with Epstein Barr virus (EBV) and the characteristic EBV+ B cell lymphomas are the leading post-transplant malignancy in children. EBV+ PTLD remains a formidable issue in pediatric transplantation and is thought to result from impaired immunity to EBV as a result of immunosuppression. However, the key viral and immune factors that determine whether EBV+ PTLD develops remain unknown. Recently, there has been much interest in developing biomarkers in order to improve and achieve more personalized approaches, in the clinical diagnosis, management, and treatment of EBV+ PTLD. Here, we review the status of immune-, viral-, and B cell lymphoma-derived candidates for biomarkers of EBV+ PTLD.

Epstein-Barr Virus Latent Membrane Protein 1 Regulates Host B Cell MicroRNA-155 and Its Target FOXO3a via PI3K p110α Activation.

Epstein-Barr Virus (EBV) is associated with potentially fatal lymphoproliferations such as post-transplant lymphoproliferative disorder (PTLD), a serious complication of transplantation. The viral mechanisms underlying the development and maintenance of EBV+ B cell lymphomas remain elusive but represent attractive therapeutic targets. EBV modulates the expression of host microRNAs (miRs), non-coding RNAs that regulate gene expression, to promote survival of EBV+ B cell lymphomas. Here, we examined how the primary oncogene of EBV, latent membrane protein 1 (LMP1), regulates host miRs using an established model of inducible LMP1 signaling. LMP1 derived from the B95.8 lab strain or PTLD induced expression of the oncogene miR-155. However, PTLD variant LMP1 lost the ability to upregulate the tumor suppressor miR-193. Small molecule inhibitors (SMI) of p38 MAPK, NF-κB, and PI3K p110α inhibited upregulation of miR-155 by B95.8 LMP1; no individual SMI significantly reduced upregulation of miR-155 by PTLD variant LMP1. miR-155 was significantly elevated in EBV+ B cell lymphoma cell lines and associated exosomes and inversely correlated with expression of the miR-155 target FOXO3a in cell lines. Finally, LMP1 reduced expression of FOXO3a, which was rescued by a PI3K p110α SMI. Our data indicate that tumor variant LMP1 differentially regulates host B cell miR expression, suggesting viral genotype as an important consideration for the treatment of EBV+ B cell lymphomas. Notably, we demonstrate a novel mechanism in which LMP1 supports the regulation of miR-155 and its target FOXO3a in B cells through activation of PI3K p110α. This mechanism expands on the previously established mechanisms by which LMP1 regulates miR-155 and FOXO3a and may represent both rational therapeutic targets and biomarkers for EBV+ B cell lymphomas.

Evaluating for Human Herpesvirus 6 in the Liver Explants of Children With Liver Failure of Unknown Etiology.

BACKGROUND: Liver failure of unknown etiology (LFUE) has a transplant-free survival rate <25%. Human herpesvirus 6 (HHV-6) may be associated with LFUE, but studies are limited by small sample size. METHODS: We identified all children who underwent liver transplant for LFUE at a single quaternary children's hospital; 51/65 cases could be age matched with controls (children who underwent liver transplant for metabolic liver disease). Quantitative polymerase chain reaction for HHV-6 was performed on DNA from formalin-fixed paraffin-embedded liver explant tissue. RESULTS: HHV-6 was detected in 34/51 cases (66.7%) and 19/51 controls (37.3%) (P = .005). Average HHV-6 viral load was 213207 copies/106 cells in positive cases (range: 7293-1102030) and 38115 copies/106 cells in positive controls (range: 1382-122375) (P = .0008). HHV-6 was present significantly more often in cases compared to controls in patients younger than 6 years. In particular, in patients younger than 3 years, HHV-6 was present in 13/27 cases (48.1%) and 2/27 controls (7.4%) (P = .0009). CONCLUSIONS: HHV-6 was detected in liver explants significantly more often and in higher quantities in children transplanted for LFUE compared to controls, suggesting HHV-6 should be evaluated in young children who present with LFUE.