Search

CMV infection is a significant complication after solid organ transplantation. We used single cell TCR αβ 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.

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.

Solid organ transplant remains a life-saving therapy for children with end-stage heart, lung, liver, or kidney disease; however, ∼25% of allograft recipients experience acute rejection within the first 12 months after transplant. Our ability to detect rejection early and to develop less toxic immunosuppressive agents is hampered by an incomplete understanding of the immune changes associated with rejection, particularly in the pediatric population. Here we used high-dimensional single-cell proteomic technologies (CyTOF) to generate the first detailed, multi-lineage analysis of the peripheral blood immune composition of pediatric solid organ transplant recipients. We report that the organ transplanted impacts the immune composition post-transplant. When taking these allograft-specific differences into account, we further observed that differences in the proportion of subsets of CD8 and CD4 T cells were significantly associated with allograft health. Together, these data form the basis for mechanistic studies into the pathobiology of rejection to develop less invasive tools to identify early rejection and new immunosuppressive agents with greater specificity and less toxicity.

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.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.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).Children with obesity have distinct immunophenotypes that may influence the tailoring of immunosuppression.

Post-transplant lymphoproliferative disorder (PTLD) is a serious complication of solid organ transplantation (SOT). Predisposing factors include primary Epstein-Barr virus (EBV) infection, reactivation of EBV in recipient B cells, and decreased T cell immunity due to immunosuppression. Previously, we demonstrated that EBV infection markedly reshapes the microRNA (miR) landscape in EBV+ B cell lines leading to increased IL-10 production. To establish the miRNAome of PTLD tumors we analyzed formalin-fixed, paraffin-embedded shavings of tumor tissues obtained from EBV+ PTLD SOT recipients by microarray analysis and quantitative PCR. The miRNAome of EBV+ PTLD tumors were distinctly different from EBV-PTLD tumors with reduced expression of miRs-17, 19 and 106a, and 194 among EBV+ PTLD tumors. miRs-17, 19, 106a, 155, and 194 were quantitated in the plasma and extracellular vesicles (EVs) from EBV+ PTLD+ SOT recipients and matched transplant controls. The plasma and EV levels of miRs-17, 19, 106a and 194 trended lower in the EBV+ PTLD+ group compared to matched controls, with miR-17 (plasma), miR-19 (EVs) and 106a (plasma and EVs) being significantly reduced. Importantly, the cell free miRs were contained primarily within the EVs. Further studies on the diagnostic, mechanistic, and therapeutic potential of these miRs in PTLD are warranted.

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.

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

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 TCRαβ 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.

Regulatory B cells (Bregs) can suppress immune responses through the secretion of IL-10 and other anti-inflammatory cytokines. 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. We therefore applied mass cytometry to simultaneously quantify cytokine production and immunophenotype in human peripheral B cells across a range of stimulatory conditions and timepoints. While multiple B cell subsets produced IL-10, no phenotype uniquely identified IL-10+ B cells and a significant portion of IL-10+ B cells co-expressed the proinflammatory cytokines IL-6 and TNFα. Despite this heterogeneity, we found operationally tolerant liver transplant recipients had a unique enrichment of IL-10+, but not TNFα+ or IL-6+, B cells as compared to 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.

Stem cell-based approaches have the potential to address the organ shortage in transplantation. Whereas both embryonic stem cells and induced pluripotent stem cells have been utilized as cellular sources for differentiation and lineage specification, their relative ability to be recognized by immune effector cells is unclear. We determined the expression of immune recognition molecules on hepatocyte-like cells (HLC) generated from murine embryonic stem cells and induced pluripotent stem cells, compared to adult hepatocytes, and we evaluated the impact on recognition by natural killer (NK) cells. We report that HLC lack MHC class I expression, and that embryonic stem cell-derived HLC have higher expression of the NK cell activating ligands Rae1, H60, and Mult1 than induced pluripotent stem cell-derived HLC and adult hepatocytes. Moreover, the lack of MHC class I renders embryonic stem cell-derived HLC, and induced pluripotent stem cell-derived HLC, susceptible to killing by syngeneic and allogeneic NK cells. Both embryonic stem cell-derived HLC, and induced pluripotent stem cell-derived HLC, are killed by NK cells at higher levels than adult hepatocytes. Finally, we demonstrate that the NK cell activation receptor, NKG2D, plays a key role in NK cell cytotoxicity of embryonic stem cell-derived HLC, but not induced pluripotent stem cell-derived HLC.

Post-transplant lymphoproliferative disorder (PTLD) is a serious complication of organ transplantation that often manifests as Epstein-Barr virus (EBV)-associated B cell lymphomas. Current treatments for PTLD have limited efficacy and can be associated with graft rejection or systemic toxicities. The mTOR inhibitor, rapamycin, suppresses tumor growth of EBV+ B cell lymphoma cells in vitro and in vivo; however, the efficacy is limited and clinical benefits of mTOR inhibitors for PTLD are variable. Here, we show constitutive activation of multiple nodes within the PI3K/Akt/mTOR pathway in EBV+ PTLD-derived cell lines. Inhibition of either PI3K or Akt, with specific inhibitors CAL-101 and MK-2206, respectively, diminished growth of EBV+ B cell lines from PTLD patients in a dose-dependent manner. Importantly, rapamycin combined with CAL-101 or MK-2206 had a synergistic effect in suppressing cell growth as determined by IC(50) isobolographic analysis and Loewe indices. Moreover, these combinations were significantly more effective than rapamycin alone in inhibiting tumor xenograft growth in NOD-SCID mice. Finally, both CAL-101 and MK-2206 also prolonged survival of heterotopic cardiac allografts in C57BL/6 mice. Thus, combination therapy with rapamycin and a PI3K inhibitor, or an Akt inhibitor, can be an efficacious treatment for EBV-associated PTLD, while simultaneously promoting allograft survival.

Epstein-Barr virus (EBV) is a ubiquitous, yet perplexing, gammaherpesvirus. Primary infection with EBV can have widely diverse outcomes ranging from asymptomatic infection to self-resolving infectious mononucleosis, to the development of EBV-associated malignancies including Burkitt lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma, and B cell lymphoma in transplant recipients and AIDS patients. In this chapter we will discuss the biology of EBV and its life cycle, viral genetics, and oncogenic mechanisms – and how these factors impact on the pathogenesis of PTLD.

In the spring of 2020, a series of reports from Europe and the USA described clusters of children and adolescents presenting with a life-threatening, hyperinflammatory syndrome — called ‘multi-system inflammatory syndrome in children’ (MIS-C) — that was seemingly linked to prior exposure to the coronavirus SARS-CoV-2. In June 2020, the US National Institutes of Health convened a workshop of immunologists and clinicians to discuss emerging knowledge and identify key questions surrounding MIS-C, with a focus on innate and adaptive immunity, genetics and epigenetics. This Meeting Report describes the main findings from the workshop.

Background The mouse is the most widely used animal for establishing in vivo models in transplant research. However, because of the advanced microsurgical skills required for these operations, the vascularized composite transplantation model in mouse has proven to be technically challenging. The purpose of this report is to describe novel modifications in surgical techniques to establish a consistent and reliable mouse model of hind limb transplantation. Methods Forty C57BL/6 male mice, half as donors and half as recipients, were used in this study. The donor hind limb was harvested and transplanted into the recipient’s ipsilateral cervical region by anastomosing the donor femoral artery to the recipient common carotid artery with a modified sleeve technique. The donor femoral vein was mounted with a modified cuff and inserted into the recipient external jugular vein. The graft was evaluated at 2 weeks postoperatively. Results The modified cuff and modified sleeve technique facilitated anastomoses. The time spent on either of the donor operation and recipient operation was about 45 minutes. The graft survival rate was 80% (16 of 20) at 2 weeks after transplant. There was minimal blood loss and no infections were noted. Conclusions Revised surgical techniques using a modified cuff proved to be a safe, reliable, and reproducible strategy in establishing a mouse model of hind limb heterotopic transplantation. The consistent graft survival in this syngeneic study demonstrates that this model can serve as a useful tool for further studies in vascularized composite transplantation.

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 the 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. Thus, blocking of the NKG2A-HLA-E axis with viral peptides may have therapeutic potential.

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 lack the capacity to resolve this high dimensionality. Single-cell mass cytometry was used to characterize the alloimmune response in early acute rejection, measuring 37 parameters simultaneously, across multiple time-points in two models; a well-established murine cardiac and a novel murine vascularized composite allotransplant (VCA). Unsupervised hierarchical clustering was 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 was mapped to visualize structural changes in antigen composition across cell types, revealing unique phenotypic signatures in alloimmune T cells, NK cells, and myeloid subsets that were conserved and that firmly distinguished rejecting from non-rejecting grafts.

BackgroundLiver failure of unknown etiology (LFUE) has a transplant-free survival rate MethodsWe 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.ResultsHHV-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).ConclusionsHHV-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.

Posttransplant lymphoproliferative disorder (PTLD) is a serious complication in organ transplant recipients and is most often associated with the Epstein Barr virus (EBV). EBV is a common gammaherpes virus with tropism for B lymphocytes and infection in immunocompetent individuals is typically asymptomatic and benign. However, infection in immunocompromised or immunosuppressed individuals can result in malignant B cell lymphoproliferations such as PTLD. EBV+ PTLD can arise following primary EBV infection, or because of reactivation of a prior infection, and represents a leading malignancy in the transplant population. The incidence of EBV+ PTLD is variable depending on the organ transplanted and whether the recipient has preexisting immunity to EBV but can be as high as 20%. It is generally accepted that impaired immune function due to immunosuppression is a primary cause of EBV+ PTLD. In this overview, we review the EBV life cycle and discuss our current understanding of the immune response to EBV in healthy, immunocompetent individuals, in transplant recipients, and in PTLD patients. We review the strategies that EBV utilizes to subvert and evade host immunity and discuss the implications for the development of EBV+ PTLD.

Depletional induction using anti-thymocyte 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 (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.

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.

Long-term IS in transplant patients has significant morbidity, poorer quality of life, and substantial economic costs. TOL, defined as graft acceptance without functional impairment in the absence of IS, has been achieved in some pediatric LT recipients. Using mass cytometry, peripheral blood immunotyping was performed to characterize differences between tolerant patients and patients who are stable on single-agent IS. Single-cell mass cytometry was performed using blood samples from a single-center pediatric LT population of operationally tolerant patients to comprehensively characterize the immune cell populations in the tolerant state compared with patients on chronic low-dose IS. Specific T-cell populations of interest were confirmed by flow cytometry. This high-dimensional phenotypic analysis revealed distinct immunoprofiles between transplant populations as well as a CD4+ TOT (CD4+ CD5+ CD25+ CD38-/lo CD45RA) that correlates with tolerance in pediatric LT recipients. In TOL patients, the TOT was significantly increased as compared to patients stable on low levels of IS. This TOT cell was confirmed by flow cytometry and is distinct from classic Treg cells. These results demonstrate the power of mass cytometry to discover significant immune cell signatures that have diagnostic potential.

Although the liver is less immunogenic than other solid organs, most liver transplant recipients receive lifelong immunosuppression. In both experimental models and clinical transplantation, total lymphoid irradiation (TLI) has been shown to induce allograft tolerance. Our goal was to identify the microRNAs (miRNAs) expressed in tolerant liver allograft recipients in an experimental model of TLI-induced tolerance.To identify the miRNAs associated with TLI-induced tolerance, we examined syngeneic recipients (Lewis→Lewis) and allogeneic recipients (Dark Agouti→Lewis) of orthotropic liver transplants that received posttransplant TLI, allogeneic recipients that were not treated posttransplantation and experienced acute rejection, and native Dark Agouti livers. Quantitative-polymerase chain reaction miRNA array cards were used to profile liver grafts.We identified 12 miRNAs that were specifically and significantly increased during acute rejection. In early tolerance, 33 miRNAs were altered compared with syngeneic livers, with 80% of the miRNAs increased. In established tolerance, 42 miRNAs were altered. In addition, miR-142-5p and miR-181a demonstrated increased expression in tolerant livers (both early and established tolerance) as compared with syngeneic livers. A principal component analysis of all miRNAs assayed demonstrated a profile in established tolerance that was closely related to that seen in syngeneic livers.The miRNA profile of established tolerant allografts is very similar to syngeneic grafts, suggesting tolerance may be a return to an immunological state of quiescence.

Background Chronic immunosuppressive therapy in transplant patients can be associated with opportunistic infections and risks of cancer development. There is an important need to better measure the immune status of patients in a transplant setting to improve patient monitoring and guide development of appropriate immunosuppressive agents and regimen. Results In this study, CyTOF was performed to comprehensively characterize the immune differences between 10 renal transplant recipients and 11 age-matched healthy donors, longitudinally over 6 months. Overall, the immune cells of transplant patients showed signs of increased CD8+ T cell activation/differentiation. Higher levels of CD57 on CD8+ T cells were observed. CD8+ T cells and their memory subsets also showed increased production of MIP-1β and IFNγ. Further, CD107a and Granzyme B expression were also increased in CD8+ T cells and CD56hi NK cells, while Tregs had decreased IL-10 production. These changes are consistent with a more stimulated immune system, despite the administration of immune suppressants to the patients. However, there were also some signs of immune suppression in the transplant patients consistent with the treatment regimen. γδT cells showed decreased TNFα and IFNγ expression, while MIP-1β expression was reduced in NKT, CD56hi NK and γδT cells. CD8+ T memory cell subsets also showed decreased expression of IL-2, IL-17 and GM-CSF. Conclusions This mosaic pattern of functional changes highlights the importance of comprehensive immune monitoring of these patients. Such information will aid in optimizing individual treatment strategies and development of improved immunosuppressants that specifically target overly activated populations.

T cell receptor (TCR) sequences are very diverse, with many more possible sequence combinations than T cells in any one individual1–4. Here we define the minimal requirements for TCR antigen specificity, through an analysis of TCR sequences using a panel of peptide and major histocompatibility complex (pMHC)-tetramer-sorted cells and structural data. From this analysis we developed an algorithm that we term GLIPH (grouping of lymphocyte interactions by paratope hotspots) to cluster TCRs with a high probability of sharing specificity owing to both conserved motifs and global similarity of complementarity-determining region 3 (CDR3) sequences. We show that GLIPH can reliably group TCRs of common specificity from different donors, and that conserved CDR3 motifs help to define the TCR clusters that are often contact points with the antigenic peptides. As an independent validation, we analysed 5,711 TCRβ chain sequences from reactive CD4 T cells from 22 individuals with latent Mycobacterium tuberculosis infection. We found 141 TCR specificity groups, including 16 distinct groups containing TCRs from multiple individuals. These TCR groups typically shared HLA alleles, allowing prediction of the likely HLA restriction, and a large number of M. tuberculosis T cell epitopes enabled us to identify pMHC ligands for all five of the groups tested. Mutagenesis and de novo TCR design confirmed that the GLIPH-identified motifs were critical and sufficient for shared-antigen recognition. Thus the GLIPH algorithm can analyse large numbers of TCR sequences and define TCR specificity groups shared by TCRs and individuals, which should greatly accelerate the analysis of T cell responses and expedite the identification of specific ligands.

Natural killer (NK) cells are effector cells of the innate immune system that can lyse target cells without prior sensitization and have an important role in host defense to pathogens and transformed cells. A balance between negative and positive signals transmitted via germ line-encoded inhibitory and activating receptors controls the function of NK cells. Although the concept of "missing-self" would suggest that NK cells could target foreign allografts, the prevailing dogma has been that NK cells are not active participants in the mechanisms that culminate in the rejection of solid organ allografts. Recent studies, however, challenge this conclusion and instead implicate NK cells in contributing to both graft rejection and tolerance to an allograft. In this review, we highlight recent studies with the goal of understanding the complex NK cell interactions that impact alloimmunity.

MicroRNAs (miRNAs) are important regulators in many biologic processes and have been implicated in the control of genes relevant to acute rejection and liver functions. Here we review the miRNAs specifically expressed in allografts during acute rejection and discuss potential roles for these miRNAs in liver dysfunction. We focus on miRNAs dysregulated both in the liver and in peripheral blood mononuclear cells and include a discussion of the potential for these miRNAs as non-invasive biomarkers to reflect liver status posttransplant.

Cell replacement therapy holds promise for a number of untreatable neurological or psychiatric diseases but the immunogenicity of cellular grafts remains controversial. Emerging stem cell and reprogramming technologies can be used to generate autologous grafts that minimize immunological concerns but autologous grafts may carry an underlying genetic vulnerability that reduces graft efficacy or survival. Healthy allogeneic grafts are an attractive and commercially scalable alternative if immunological variables can be controlled. Stem cells and immature neural progenitor cells (NPC) do not express major histocompatibility complex (MHC) antigens and can evade adaptive immune surveillance. Nevertheless, in an experimental murine model, allogeneic NPCs do not survive and differentiate as well as syngeneic grafts, even when traditional immunosuppressive treatments are used. In this study, we show that natural killer (NK) cells recognize the lack of self-MHC antigens on NPCs and pose a barrier to NPC transplantation. NK cells readily target both syngeneic and allogeneic NPC, and killing is modulated primarily by NK-inhibiting “self” class I MHC and NK-activating NKG2D-ligand expression. The absence of NKG2D signaling in NK cells significantly improves NPC-derived neuron survival and differentiation. These data illustrate the importance of innate immune mechanisms in graft outcome and the potential value of identifying and targeting NK cell-activating ligands that may be expressed by stem cell derived grafts.

Single-cell flow cytometric techniques have been indispensable to improving our understanding of the phenotype and function of immune cell subsets that are important in both rejection and tolerance post-transplant. Mass cytometry, or Cytometry by Time-of-Flight (CyTOF), is a single cell–based platform that utilizes antibodies conjugated to rare heavy metal ions for analysis of cellular proteins by a time-of-flight mass spectrometer. This new technology allows for the evaluation of over 40 simultaneous cellular parameters in a single sample because the limitation of spectral overlap, seen in conventional flow cytometry, is eliminated. In this review we discuss the current state of mass cytometry, describe the advantages and disadvantages over multi-parameter flow cytometry, introduce novel methods of high-dimensional data analysis and visualization, and review some recent studies using mass cytometry to profile the immune system of healthy people, and transplant recipients.

Epstein-Barr virus (EBV)-encoded BamHI-A rightward frame 1 (BARF1) is a putative viral oncogene in EBV-infected stomach cancer. The aim of the present study was to investigate BARF1-induced cellular protein and microRNA alterations. In this study, BARF1-expressing stomach cancer cells showed a high rate of proliferation, high levels of NFκB, and miR-146a upregulation, which was reversed by NFκB knockdown. During BARF1-induced NFκB upregulation, hCSF1 receptor level was unchanged. Knockdown of BARF1 in the naturally EBV-infected YCCEL1 stomach cancer cells suppressed cell proliferation, and downregulated NFκB and miR-146a. SMAD4 was identified as a miR-146a target and was downregulated in BARF1-expressing cells, whereas SMAD4 expression was restored by anti-miR-146a. Knockdown of BARF1 in YCCEL1 cells upregulated SMAD4, and this effect was reversed by miR-146a overexpression. Transfection of BARF1-expressing cells with pCEP4-SMAD4 abolished the cell proliferating effect of BARF1. In stomach cancer tissues, miR-146a was expressed at higher levels, and more frequent NFκB nuclear positivity immunohistochemically, but not of SMAD4 nuclear loss was found in the EBV-positive group compared with the EBV-negative group. In conclusion, EBV-encoded BARF1 promotes cell proliferation in stomach cancer by upregulating NFκB and miR-146a and downregulating SMAD4, thereby contributing to EBV-induced stomach cancer progression.

MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate the posttranscriptional expression of target genes and are important regulators in immune responses. Previous studies demonstrated that the miRNA, miR-182 was significantly increased during allograft rejection. Further, the transcription factor Forkhead box (FOX) protein 1, (FOXO1) was shown to be a target of miR-182. The aim of this study is to further examine the role of miR-182 in alloimmune responses.Transplantation of BALB/c cardiac allografts was performed in C57BL/6, miR-182, B6.129S-H2 (MHC II and CD4 T cell-deficient) and B6.129S2-Tap1 (MHC I and CD8 T cell-deficient) mice, with or without CTLA-4Ig administration. T cell phenotype, FOXO1 protein levels and graft infiltrating lymphocytes were determined in C57BL/6 or miR-182 mice by flow cytometric analysis, Western blot, and immunohistochemistry, respectively.We now show that T cells, mainly CD4 are the main cellular source of miR-182 during allograft rejection. In the absence of miR-182, CTLA-4Ig treatment significantly increased allograft survival (31.5 days C57BL/6 vs 60 days miR-182; P0.01). Further, CTLA4-Ig treatment inhibits miR-182 expression, increases FOXO1 levels, and reduces the percentage of CD4CD44 T cells after transplantation. Fewer T cells infiltrate the cardiac allografts, and memory T cells are significantly decreased in allograft recipients deficient in miR-182 with CTLA4-Ig treatment (P0.01).Our findings suggest that miR-182 contributes to the T-cell responses to alloantigen especially under costimulation blockade. Therapeutics that target specific miRNAs may prove beneficial in transplantation.

Epstein-Barr virus (EBV) is a human -herpesvirus that establishes latency and lifelong infection in host B cells while achieving a balance with the host immune response. When the immune system is perturbed through immunosuppression or immunodeficiency, however, these latently-infected B cells can give rise to aggressive B cell lymphomas. Natural killer (NK) cells are regarded as critical in the early immune response to viral infection, but their role in controlling expansion of infected B cells is not understood. Here we report that NK cells from healthy human donors display increased killing of autologous B lymphoblastoid cell lines (LCL) harboring latent EBV compared to primary B cells. Co-culture of NK cells with autologous EBV+ LCL identifies an NK cell population that produces IFN-gamma and mobilizes the cytotoxic granule protein CD107a. Multi-parameter flow cytometry and Boolean analysis reveals that these functional cells are enriched for expression of the NK cell receptor NKG2A. Further, NKG2A+ NK cells more efficiently lyse autologous LCL than do NKG2A- NK cells. More specifically, NKG2A+2B4+CD16-CD57-NKG2C-NKG2D+ cells constitute the predominant NK cell population that responds to latently-infected autologous EBV+ B cells. Thus, a subset of NK cells is enhanced for the ability to recognize and eliminate autologous, EBV-infected transformed cells, laying the groundwork for harnessing this subset for therapeutic use in EBV+ malignancies.

Post-transplant lymphoproliferative disorders (PTLDs) are a group of conditions that involve uncontrolled proliferation of lymphoid cells as a consequence of extrinsic immunosuppression after organ or haematopoietic stem cell transplant. PTLDs show some similarities to classic lymphomas in the non-immunosuppressed general population. The oncogenic Epstein-Barr virus (EBV) is a key pathogenic driver in many early-onset cases, through multiple mechanisms. The incidence of PTLD varies with the type of transplant; a clear distinction should therefore be made between the conditions after solid organ transplant and after haematopoietic stem cell transplant. Recipient EBV seronegativity and the intensity of immunosuppression are among key risk factors. Symptoms and signs depend on the localization of the lymphoid masses. Diagnosis requires histopathology, although imaging techniques can provide additional supportive evidence. Pre-emptive intervention based on monitoring EBV levels in blood has emerged as the preferred strategy for PTLD prevention. Treatment of established disease includes reduction of immunosuppression and/or administration of rituximab (a B cell-specific antibody against CD20), chemotherapy and EBV-specific cytotoxic T cells. Despite these strategies, the mortality and morbidity remains considerable. Patient outcome is influenced by the severity of presentation, treatment-related complications and risk of allograft loss. New innovative treatment options hold promise for changing the outlook in the future.

MicrorRNA are small noncoding RNA molecules that regulate the posttranscriptional expression of target genes. In addition to being involved in many biologic processes, microRNAs are important regulators in innate and adaptive immune responses. Distinct sets of expressed microRNAs are found in different cell types and tissues and aberrant expression of microRNAs is associated with many disease states. MicroRNA expression was examined in a model of heterotopic heart transplantation by microarray analyses and a unique profile was detected in rejecting allogeneic transplants (BALB/c → C57BL/6) as compared to syngeneic transplants (C57BL/6 → C57BL/6). The microRNA miR-182 was significantly increased in rejecting cardiac allografts and in mononuclear cells that infiltrate the grafts. Forkhead box (FOX) proteins are a family of important transcription factors and FOXO1 is a target of miR-182. As miR-182 increases after transplant, there is a concomitant posttranscriptional decrease in FOXO1 expression in heart allografts that is localized to both the cardiomyocytes and CD3(+) T cells. The microRNA miR-182 is significantly increased in both peripheral blood mononuclear cells and plasma during graft rejection suggesting potential as a biomarker of graft status. Our results identify microRNAs that may regulate alloimmune responses and graft outcomes.

NK cells are important in the immune response against tumors and virally infected cells. A balance between inhibitory and activating receptors controls the effector functions of NK cells. We examined the fate of circulating NK cells and the expression of the NK cell-activating receptors in pediatric liver transplant recipients. Blood specimens were collected from 38 pediatric liver transplant recipients before transplant, and at one wk, one, three, six, and nine months, and one yr post-transplant. PBMCs were isolated and analyzed for the levels of NK cell activation receptors NKp30, NKp46, and NKG2D in the CD56(dim) CD16(+) and CD56(bright) CD16(+/-) subsets of NK cells. We demonstrated that there is a significant decrease in the percentage of circulating NK cells post-transplant (pretransplant 7.69 ± 1.54 vs. one wk post-transplant 1.73 ± 0.44) in pediatric liver transplant recipients. Interestingly, NKp30 expression is significantly increased, while NKp46 and NKG2D levels remain stable on the NK cells that persist at one wk post-transplant. These data indicate that the numbers and subsets of circulating NK cells are altered in children after liver transplantation.

EBV is a gammaherpes virus that has infected >95% of the world’s population (1). In healthy individuals, EBV infection is usually asymptomatic and the virus establishes a latent infection, residing in memory B cells for the lifetime of the host. However, when the immune system is debilitated as in immunosuppressed transplant recipients, opportunistic EBV-associated B-cell lymphomas can arise (2). These lymphomas are the most serious manifestation of PTLD (3). The incidence of EBV-associated PTLD is known to vary widely with the organ transplanted and the serologic status of the recipient (4). Nevertheless, EBV-associated B-cell lymphomas are the most common cancer in children that receive solid organ grafts, representing well over half of the de novo, post-transplant malignancies. Children are at particular risk for PTLD, in part, because they are often immunologically naive at the time of transplant and can acquire a primary infection when receiving a graft from a seropositive donor in the setting of immunosuppression (5). Although uncommon, PTLD may also arise from T cells; however, this review will be focused on EBV-induced PTLD, which accounts for 93% of lymphoproliferative disorders in pediatric organ transplant recipients. The current standard therapeutic approaches for the treatment of EBV-associated PTLD include reduction or elimination of immunosuppression, chemotherapy, radiotherapy, the use of anti-B-cell antibodies such as rituximab, and surgical resection when feasible. Determining the optimal treatment for individual patients, however, remains difficult and requires extensive interaction between transplant specialists and oncologists. The recent emergence of cellular-based immunotherapy holds promise for alternate approaches and underscores the need for new strategies to manage children with EBV-associated B-cell lymphomas.

PurposeIn transplant recipients, Epstein-Barr Virus (EBV) is associated with the development of B cell lymphomas in post-transplant lymphoproliferative disorder (PTLD). We utilized next-generation sequencing to characterize the diversity of the EBV genome in transplant recipients with low EBV viral