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.

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.

Donor-derived regulatory dendritic cell infusion modulates effector CD8+ T cell and NK cell responses after liver transplantation.

Immune cell-based therapies are promising strategies to facilitate immunosuppression withdrawal after organ transplantation. Regulatory dendritic cells (DCreg) are innate immune cells that down-regulate alloimmune responses in preclinical models. Here, we performed clinical monitoring and comprehensive assessment of peripheral and allograft tissue immune cell populations in DCreg-infused live-donor liver transplant (LDLT) recipients up to 12 months (M) after transplant. Thirteen patients were given a single infusion of donor-derived DCreg 1 week before transplant (STUDY) and were compared with 40 propensity-matched standard-of-care (SOC) patients. Donor-derived DCreg infusion was well tolerated in all STUDY patients. There were no differences in postoperative complications or biopsy-confirmed acute rejection compared with SOC patients up to 12M. DCreg administration was associated with lower frequencies of effector T-bet+Eomes+CD8+ T cells and CD16bright natural killer (NK) cells and an increase in putative tolerogenic CD141+CD163+ DCs compared with SOC at 12M. Antidonor proliferative capacity of interferon-γ+ (IFN-γ+) CD4+ and CD8+ T cells was lower compared with antithird party responses in STUDY participants, but not in SOC patients, at 12M. In addition, lower circulating concentrations of interleukin-12p40 (IL-12p40), IFN-γ, and CXCL10 were detected in STUDY participants compared with SOC patients at 12M. Analysis of 12M allograft biopsies revealed lower frequencies of graft-infiltrating CD8+ T cells, as well as attenuation of cytolytic TH1 effector genes and pathways among intragraft CD8+ T cells and NK cells, in DCreg-infused patients. These reductions may be conducive to reduced dependence on immunosuppressive drug therapy or immunosuppression withdrawal.

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.

Activated-memory T cells influence naïve T cell fate: a noncytotoxic function of human CD8 T cells.

T cells are endowed with the capacity to sense their environment including other T cells around them. They do so to set their numbers and activation thresholds. This form of regulation has been well-studied within a given T cell population - i.e., within the naïve or memory pool; however, less is known about the cross-talk between T cell subsets. Here, we tested whether memory T cells interact with and influence surrounding naïve T cells. We report that human naïve CD8 T cells (TN) undergo phenotypic and transcriptional changes in the presence of autologous activated-memory CD8 T cells (TMem). Following in vitro co-culture with activated central memory cells (TCM), ~3% of the TN acquired activation/memory canonical markers (CD45RO and CD95) in an MHC-I dependent-fashion. Using scRNA-seq, we also observed that ~3% of the TN acquired an activated/memory signature, while ~84% developed a unique activated transcriptional profile hybrid between naïve and activated memory. Pseudotime trajectory analysis provided further evidence that TN with an activated/memory or hybrid phenotype were derived from TN. Our data reveal a non-cytotoxic function of TMem with potential to activate autologous TN into the activated/memory pool. These findings may have implications for host-protection and autoimmunity that arises after vaccination, infection or transplantation.

Dendritic Cell-Mediated Regulation of Liver Ischemia-Reperfusion Injury and Liver Transplant Rejection.

Liver allograft recipients are more likely to develop transplantation tolerance than those that receive other types of organ graft. Experimental studies suggest that immune cells and other non-parenchymal cells in the unique liver microenvironment play critical roles in promoting liver tolerogenicity. Of these, liver interstitial dendritic cells (DCs) are heterogeneous, innate immune cells that appear to play pivotal roles in the instigation, integration and regulation of inflammatory responses after liver transplantation. Interstitial liver DCs (recruited in situ or derived from circulating precursors) have been implicated in regulation of both ischemia/reperfusion injury (IRI) and anti-donor immunity. Thus, livers transplanted from mice constitutively lacking DCs into syngeneic, wild-type recipients, display increased tissue injury, indicating a protective role of liver-resident donor DCs against transplant IRI. Also, donor DC depletion before transplant prevents mouse spontaneous liver allograft tolerance across major histocompatibility complex (MHC) barriers. On the other hand, mouse liver graft-infiltrating host DCs that acquire donor MHC antigen via "cross-dressing", regulate anti-donor T cell reactivity in association with exhaustion of graft-infiltrating T cells and promote allograft tolerance. In an early phase clinical trial, infusion of donor-derived regulatory DCs (DCreg) before living donor liver transplantation can induce alterations in host T cell populations that may be conducive to attenuation of anti-donor immune reactivity. We discuss the role of DCs in regulation of warm and liver transplant IRI and the induction of liver allograft tolerance. We also address design of cell therapies using DCreg to reduce the immunosuppressive drug burden and promote clinical liver allograft tolerance.

Donor-derived regulatory dendritic cell infusion results in host cell cross-dressing and T cell subset changes in prospective living donor liver transplant recipients.

Regulatory dendritic cells (DCreg) promote transplant tolerance following their adoptive transfer in experimental animals. We investigated the feasibility, safety, fate, and impact on host T cells of donor monocyte-derived DCreg infused into prospective, living donor liver transplant patients, 7 days before transplantation. The DCreg expressed a tolerogenic gene transcriptional profile, high cell surface programed death ligand-1 (PD-L1):CD86 ratios, high IL-10/no IL-12 productivity and poor ability to stimulate allogeneic T cell proliferation. Target DCreg doses (range 2.5-10 × 106 cells/kg) were achieved in all but 1 of 15 recipients, with no infusion reactions. Following DCreg infusion, transiently elevated levels of donor HLA and immunoregulatory PD-L1, CD39, and CD73 were detected in circulating small extracellular vesicles. At the same time, flow and advanced image stream analysis revealed intact DCreg and "cross-dressing" of host DCs in blood and lymph nodes. PD-L1 co-localization with donor HLA was observed at higher levels than with recipient HLA. Between DCreg infusion and transplantation, T-bethi Eomeshi memory CD8+ T cells decreased, whereas regulatory (CD25hi CD127- Foxp3+ ): T-bethi Eomeshi CD8+ T cell ratios increased. Thus, donor-derived DCreg infusion may induce systemic changes in host antigen-presenting cells and T cells potentially conducive to modulated anti-donor immune reactivity at the time of transplant.

Regulatory dendritic cells for human organ transplantation.

Current immunosuppressive (IS) regimens used to prevent organ allograft rejection have well-recognized side effects, that include enhanced risk of infection and certain types of cancer, metabolic disorders, cardiovascular disease, renal complications and failure to control chronic allograft rejection. The life-long dependency of patients on these IS agents reflects their inability to induce donor-specific tolerance. Extensive studies in rodent and non-human primate models have demonstrated the ability of adoptively-transferred regulatory immune cells (either regulatory myeloid cells or regulatory T cells) to promote transplant tolerance. Consequently, there is considerable interest in the potential of regulatory immune cell therapy to allow safe minimization/complete withdrawal of immunosuppression and the promotion of organ transplant tolerance in the clinic. Here, we review the properties of regulatory dendritic cells (DCreg) with a focus on the approaches being taken to generate human DCreg for clinical testing. We also document the early phase clinical trials that are underway to assess DCreg therapy in clinical organ transplantation as well as in autoimmune disorders.

High PD-L1/CD86 MFI ratio and IL-10 secretion characterize human regulatory dendritic cells generated for clinical testing in organ transplantation.

Human regulatory dendritic cells (DCreg) were generated from CD14 immunobead-purified or elutriated monocytes in the presence of vitamin D3 and IL-10. They exhibited similar, low levels of costimulatory CD80 and CD86, but comparatively high levels of co-inhibitory programed death ligand-1 (PD-L1) and IL-10 production compared to control immature DC (iDC). Following Toll-like receptor 4 ligation, unlike control iDC, DCreg resisted phenotypic and functional maturation and further upregulated PD-L1:CD86 expression. Whereas LPS-stimulated control iDC (mature DC; matDC) secreted pro-inflammatory tumor necrosis factor but no IL-10, the converse was observed for LPS-stimulated DCreg. DCreg weakly stimulated naïve and memory allogeneic CD4+ and CD8+ T cell proliferation and IFNγ, IL-17A and perforin/granzyme B production in MLR. Their stimulatory function was enhanced however, by blocking PD-1 ligation. High-throughput T cell receptor (TCR) sequencing revealed that, among circulating T cell subsets, memory CD8+ T cells contained the most alloreactive TCR clonotypes and that, while matDC expanded these alloreactive memory CD8 TCR clonotypes, DCreg induced more attenuated responses. These findings demonstrate the feasibility of generating highly-purified GMP-grade DCreg for systemic infusion, their influence on the alloreactive T cell response, and a key mechanistic role of the PD1 pathway.

Regulatory dendritic cells for promotion of liver transplant operational tolerance: Rationale for a clinical trial and accompanying mechanistic studies.

Dendritic cells (DC) are rare, bone marrow (BM)-derived innate immune cells that critically maintain self-tolerance in the healthy steady-state. Regulatory DC (DCreg) with capacity to suppress allograft rejection and promote transplant tolerance in pre-clinical models can readily be generated from BM precursors or circulating blood monocytes. These DCreg enhance allograft survival via various mechanisms, including promotion of regulatory T cells. In non-human primates receiving minimal immunosuppressive drug therapy (IS), infusion of DCreg of donor origin, one week before transplant, safely prolongs renal allograft survival and selectively attenuates anti-donor CD8+ memory T cell responses in the early post-transplant period. Based on these observations, and in view of the critical need to reduce patient dependence on non-specific IS agents that predispose to cardiometabolic side effects and renal insufficiency, we will conduct a first-in-human safety and preliminary efficacy study of donor-derived DCreg infusion to achieve early (18 months post-transplant) complete IS withdrawal in low-risk, living donor liver transplant recipients receiving standard-of-care IS (mycophenolate mofetil, tacrolimus and steroids). We will test the hypothesis that, although donor-derived DCreg are short-lived, they will induce robust donor-specific T cell hyporesponsiveness. We will examine immunological mechanisms by sequential analysis of blood and tissue samples, incorporating cutting-edge technologies.

Phenotype, function, and differentiation potential of human monocyte subsets.

Human monocytes have been grouped into classical (CD14++CD16-), non-classical (CD14dimCD16++), and intermediate (CD14++CD16+) subsets. Documentation of normal function and variation in this complement of subtypes, particularly their differentiation potential to dendritic cells (DC) or macrophages, remains incomplete. We therefore phenotyped monocytes from peripheral blood of healthy subjects and performed functional studies on high-speed sorted subsets. Subset frequencies were found to be tightly controlled over time and across individuals. Subsets were distinct in their secretion of TNFα, IL-6, and IL-1ß in response to TLR agonists, with classical monocytes being the most producers and non-classical monocytes the least. Monocytes, particularly those of the non-classical subtype, secreted interferon-α (IFN-α) in response to intracellular TLR3 stimulation. After incubation with IL-4 and GM-CSF, classical monocytes acquired monocyte-derived DC (mo-DC) markers and morphology and stimulated allogeneic T cell proliferation in MLR; intermediate and non-classical monocytes did not. After incubation with IL-3 and Flt3 ligand, no subset differentiated to plasmacytoid DC. After incubation with GM-CSF (M1 induction) or macrophage colony-stimulating factor (M-CSF) (M2 induction), all subsets acquired macrophage morphology, secreted macrophage-associated cytokines, and displayed enhanced phagocytosis. From these studies we conclude that classical monocytes are the principal source of mo-DCs, but all subsets can differentiate to macrophages. We also found that monocytes, in particular the non-classical subset, represent an alternate source of type I IFN secretion in response to virus-associated TLR agonists.

Prospective Clinical Testing of Regulatory Dendritic Cells in Organ Transplantation.

Dendritic cells (DC) are rare, professional antigen-presenting cells with ability to induce or regulate alloimmune responses. Regulatory DC (DCreg) with potential to down-modulate acute and chronic inflammatory conditions that occur in organ transplantation can be generated in vitro under a variety of conditions. Here, we provide a rationale for evaluation of DCreg therapy in clinical organ transplantation with the goal of promoting sustained, donor-specific hyporesponsiveness, while lowering the incidence and severity of rejection and reducing patients' dependence on anti-rejection drugs. Generation of donor- or recipient-derived DCreg that suppress T cell responses and prolong transplant survival in rodents or non-human primates has been well-described. Recently, good manufacturing practice (GMP)-grade DCreg have been produced at our Institution for prospective use in human organ transplantation. We briefly review experience of regulatory immune therapy in organ transplantation and describe our experience generating and characterizing human monocyte-derived DCreg. We propose a phase I/II safety study in which the influence of donor-derived DCreg combined with conventional immunosuppression on subclinical and clinical rejection and host alloimmune responses will be examined in detail.

Mathematical Modeling of Early Cellular Innate and Adaptive Immune Responses to Ischemia/Reperfusion Injury and Solid Organ Allotransplantation.

A mathematical model of the early inflammatory response in transplantation is formulated with ordinary differential equations. We first consider the inflammatory events associated only with the initial surgical procedure and the subsequent ischemia/reperfusion (I/R) events that cause tissue damage to the host as well as the donor graft. These events release damage-associated molecular pattern molecules (DAMPs), thereby initiating an acute inflammatory response. In simulations of this model, resolution of inflammation depends on the severity of the tissue damage caused by these events and the patient's (co)-morbidities. We augment a portion of a previously published mathematical model of acute inflammation with the inflammatory effects of T cells in the absence of antigenic allograft mismatch (but with DAMP release proportional to the degree of graft damage prior to transplant). Finally, we include the antigenic mismatch of the graft, which leads to the stimulation of potent memory T cell responses, leading to further DAMP release from the graft and concomitant increase in allograft damage. Regulatory mechanisms are also included at the final stage. Our simulations suggest that surgical injury and I/R-induced graft damage can be well-tolerated by the recipient when each is present alone, but that their combination (along with antigenic mismatch) may lead to acute rejection, as seen clinically in a subset of patients. An emergent phenomenon from our simulations is that low-level DAMP release can tolerize the recipient to a mismatched allograft, whereas different restimulation regimens resulted in an exaggerated rejection response, in agreement with published studies. We suggest that mechanistic mathematical models might serve as an adjunct for patient- or sub-group-specific predictions, simulated clinical studies, and rational design of immunosuppression.

Upper-extremity transplantation using a cell-based protocol to minimize immunosuppression.

OBJECTIVE: To minimize maintenance immunosuppression in upper-extremity transplantation to favor the risk-benefit balance of this procedure. BACKGROUND: Despite favorable outcomes, broad clinical application of reconstructive transplantation is limited by the risks and side effects of multidrug immunosuppression. We present our experience with upper-extremity transplantation under a novel, donor bone marrow (BM) cell-based treatment protocol ("Pittsburgh protocol"). METHODS: Between March 2009 and September 2010, 5 patients received a bilateral hand (n = 2), a bilateral hand/forearm (n = 1), or a unilateral (n = 2) hand transplant. Patients were treated with alemtuzumab and methylprednisolone for induction, followed by tacrolimus monotherapy. On day 14, patients received an infusion of donor BM cells isolated from 9 vertebral bodies. Comprehensive follow-up included functional evaluation, imaging, and immunomonitoring. RESULTS: All patients are maintained on tacrolimus monotherapy with trough levels ranging between 4 and 12 ng/mL. Skin rejections were infrequent and reversible. Patients demonstrated sustained improvements in motor function and sensory return correlating with time after transplantation and level of amputation. Side effects included transient increase in serum creatinine, hyperglycemia managed with oral hypoglycemics, minor wound infection, and hyperuricemia but no infections. Immunomonitoring revealed transient moderate levels of donor-specific antibodies, adequate immunocompetence, and no peripheral blood chimerism. Imaging demonstrated patent vessels with only mild luminal narrowing/occlusion in 1 case. Protocol skin biopsies showed absent or minimal perivascular cellular infiltrates. CONCLUSIONS: Our data suggest that this BM cell-based treatment protocol is safe, is well tolerated, and allows upper-extremity transplantation using low-dose tacrolimus monotherapy.

Peripheral blood lymphocyte depletion after hepatic arterial 90Yttrium microsphere therapy for hepatocellular carcinoma.

PURPOSE: The short- and long-term effects of (90)Yttrium microspheres therapy for hepatocellular carcinoma (HCC) on peripheral blood lymphocytes are unknown and were therefore examined. METHODS AND MATERIALS: Ninety-two HCC patients were enrolled in a (90)Yttrium therapy study and routine blood counts were examined as part of standard clinical monitoring. RESULTS: We found an early, profound, and prolonged lymphopenia. In a subsequent cohort of 25 additional HCC patients, prospective flow cytometric immune-monitoring analysis was performed to identify specific changes on distinct lymphocyte subsets (i.e., CD3, CD4, CD8 T, and CD19 B lymphocytes) and NK cells absolute numbers, in addition to the granulocytes and platelets subsets. We found that the pretreatment lymphocyte subset absolute numbers (with the exception of NK cells) had a tendency to be lower compared with healthy control values, but no significant differences were detected between groups. Posttherapy follow-up revealed that overall, all lymphocyte subsets, except for NK cells, were significantly (>50% from pretherapy values), promptly (as early as 24 h) and persistently (up to 30 months) depleted post-(90)Yttrium microspheres therapy. In contrast, granulocytes increased rapidly (24 h) to compensate for lymphocyte depletion, and remained increased at 1-year after therapy. We further stratified patients into two groups, according to survival at 1 year. We found that lack of recovery of CD19, CD3, CD8, and especially CD4 T cells was linked to poor patient survival. No fungal or bacterial infections were noted during the 30-month follow-up period. CONCLUSIONS: The results show that lymphocytes (and not granulocytes, platelets, or NK cells) are sensitive to hepatic arterial (90)Yttrium without associated clinical toxicity, and lack of lymphocyte recovery (possibly leading to dysregulation of adaptive cellular immunity) posttherapy indicates poor survival.

Porcine cell microchimerism but lack of productive porcine endogenous retrovirus (PERV) infection in naive and humanized SCID-beige mice treated with porcine peripheral blood mononuclear cells.

Pigs are considered a suitable source of cells and organs for xenotransplantation. All known strains of pigs contain porcine endogenous retrovirus (PERV) and PERV released by porcine cells may infect human cells in vitro and severe-combined immunodeficient (SCID) mice in vivo. Humanized SCID (hu-SCID) mice develop immune response to porcine antigens. Here we investigated PERV transmission in humanized SCID-beige mice using porcine peripheral blood mononuclear cells (PBMC) as the donor tissue (and the source of PERV). Mice were infused in the peritoneal cavity with 1.5-3.0 x 10(7) unfractionated human PBMC. Unfractionated porcine PBMC (1.5-3.0 x 10(7) cell/mouse) were infused to the mice simultaneously with human PBMC or 3 weeks after human PBMC infusion. The treated mice were monitored for weight and skin changes, donor cell chimerism, anti-pig antibodies and PERV transmission. All humanized mice tested 5-12 weeks after human PBMC transplantation were macrochimeric (up to 40% of cells in blood) for human cells, where 99% of the human cells were T-lymphocytes. Although human B lymphocytes were very rare in the blood of humanized mice at that point, the mice were positive for human anti-pig natural antibodies. The control SCID-beige mice or mice treated with porcine PBMC alone were negative for anti-porcine antibodies. Approximately 70% of the humanized mice treated with porcine PBMC were also microchimeric for porcine cells. Although some tissue samples of these mice were positive for PERV DNA in the absence of porcine DNA indicating PERV infection, the infection was non-productive as PERV transcripts were not detectable in those tissues. PERV infection of human and mouse cells in vitro by co-culturing with porcine PBMC was also non-productive. Humanized SCID-beige mice suffered weight loss and occasional minor skin changes due to graft vs. host disease caused by human PBMC but none of the mice showed observable effect attributable to the apparent PERV infection alone.