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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.

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.

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.

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

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.

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.

A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.

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.

Purpose of review Natural killer (NK) cells are effector cells of the innate immune system that can lyse target cells without prior sensitization and are important in host defense to virally infected and transformed cells. Although the concept of ‘missing-self’ would suggest NK cells could target foreign allografts, the prevailing dogma has been that NK cells are not active participants in the rejection of solid organ allografts. This review summarizes recent studies that challenge this conclusion and instead suggest NK cells are important in outcomes posttransplant. Recent findings NK cells expressing specific cell surface receptors may promote graft damage and rejection. However, recent studies suggest some NK cell subsets have tolerogenic or immunoregulatory potential and promote graft stability, suggesting a dichotomous role for NK cells after transplant. Furthermore, NK cells respond to cells infected with cytomegalovirus and Epstein–Barr virus, and studies suggest some NK cells have immune memory. Summary Our understanding of the role of NK cells posttransplant has evolved from ‘no role’ to the current idea that NK cells may have ‘complex interactions’ that impact graft outcomes. Additional studies, using cutting edge techniques to comprehensively analyze the phenotypic and functional subsets of NK cells in transplant recipients, are clearly necessary.

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.

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.

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.

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.

Epstein Barr virus (EBV) is the etiological agent of acute infectious mononucleosis (IM). Since acute IM is a self-resolving disease with most patients regaining health in one to three weeks there have been few studies examining molecular signatures in early acute stages of the disease. microRNAs have been shown, however, to influence immune cell function and consequently the generation of antibody responses in IM. In this study, we performed a comprehensive analysis of differentially expressed microRNAs in early stage uncomplicated acute IM. microRNAs were profiled from patient peripheral blood obtained at the time of IM diagnosis and at subsequent time points, and pathway analysis performed to identify important immune and cell signaling pathways. We identified 215 differentially regulated microRNAs at the most acute stage of infection when the patients initially sought medical help. The number of differentially expressed microRNAs decreased to 148 and 68 at one and two months post-primary infection, with no significantly changed microRNAs identified at seven months post-infection. Interferon signaling, T and B cell signaling and antigen presentation were the top pathways influenced by the microRNAs associated with IM. Thus, a dynamic and regulated expression profile of miRNA accompanies the early acute immune response, and resolution of infection, in IM.

MicroRNAs (miRNAs) have emerged as highly evolutionarily conserved moieties that have very selective gene-regulatory functions. miRNAs are being researched for their use as potential biomarkers for diagnostics, routine prognostics as well as selective therapeutics in cancer, infectious diseases, autoimmune disorders and transplantation. This review summarizes how immune regulation by miRNAs affects the outcome of transplantation.Many miRNAs have been identified as selective markers for specific disease states and transplant conditions in the past two decades. In this review, we will discuss the recent advances and some seminal discoveries in miRNA research and their role as immune regulators in transplantation. Lastly, we will highlight the ongoing clinical trials for miRNA-based therapeutics for clinical applications and present our opinion on the future of miRNA-based diagnostics and therapeutics.miRNA-based diagnosis is a fast-moving field with new miRNA signatures being identified each day. Recent advances have also been successful at taking a few of these miRNAs to clinical trials for therapeutic interventions.

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.

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.

It is widely agreed that visuospatial orienting attention depends on a network of frontal and parietal areas in the right hemisphere. It is thought that the visuospatial orienting role of the right parietal lobe is related to its role in the production of overt eye movements. The experiments reported here test the possibility that other parietal regions may be important for directing attention in relation to response modalities other than eye movement. Specifically, we used positron emission tomography (PET) to test the hypothesis that a ‘left’ parietal area, the supramarginal gyrus, is important for attention in relation to limb movements (Rushworth et al., 1997; Rushworth, Ellison, & Walsh, in press). We have referred to this process as ‘motor attention’ to distinguish it from orienting attention. In one condition subjects spent most of the scanning period covertly attending to ‘left’ hand movements that they were about to make. Activity in this first condition was compared with a second condition with identical stimuli and movement responses but lacking motor attention periods. Comparison of the conditions revealed that motor attention related activity was almost exclusively restricted to the ‘left’ hemisphere despite the fact that subjects only ever made ipsilateral, left-hand responses. Left parietal activity was prominent in this comparison, within the parietal lobe the critical region for motor attention was the supramarginal gyrus and the adjacent anterior intraparietal sulcus (AIP), a region anterior to the posterior parietal cortex identified with orienting attention. In a second part of the experiment we compared a condition in which subjects covertly rehearsed verbal responses with a condition in which they made verbal responses immediately without rehearsal. A comparison of the two conditions revealed verbal rehearsal-related activity in several anterior left hemisphere areas including Broca's area. The lack of verbal rehearsal-related activity in the left supra-marginal gyrus confirms that this area plays a direct role in motor attention that cannot be attributed to any strategy of verbal mediation. The results also provide evidence concerning the importance of ventral premotor (PMv) and Broca's area in motor attention and language processes.

There is a discrepancy between the results of imaging studies in which subjects learn motor sequences. Some experiments have shown decreases in the activation of some areas as learning increased, whereas others have reported learning-related increases as learning progressed. We have exploited fMRI to measure changes in blood oxygen leve-dependent (BOLD) signal throughout the course of learning. T2*-weighted echo-planar images were acquired over the whole brain for 40 min while the subjects learned a sequence eight moves long by trial and error. The movements were visually paced every 3.2 s and visual feedback was provided to the subjects. A baseline period followed each activation period. The effect due to the experimental conditions was modeled using a square-wave function, time locked to their occurrence. Changes over time in the difference between activation and baseline signal were modeled using a set of polynomial basis functions. This allowed us to take into account linear as well as nonlinear changes over time. Low-frequency changes over time common to both activation and baseline conditions (and thus not learning related) were modeled and removed. Linear and nonlinear changes of BOLD signal over time were found in prefrontal, premotor, and parietal cortex and in neostriatal and cerebellar areas. Single-unit recordings in nonhuman primates during the learning of motor tasks have clearly shown increased activity early in learning, followed by a decrease as learning progressed. Both phenomena can be observed at the population level in the present study.

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.

Background and Objective: Human major histocompatibility complex class I-related gene A (MICA) is reportedly associated with poor transplant outcomes and a high risk of acute and chronic rejection in solid organ transplantation. However, studies on these risks have found conflicting results. In order to identify areas in which additional research is needed, we have undertaken the first systematic review of evidence concerning the risk of anti-MICA antibodies in recipients’ sera. Methods: We searched MEDLINE, EMBASE, and the Cochrane Library for original reports of clinical studies involving detection of MICA abs in transplant recipients’ sera which used survival rate, acute rejection, and/or chronic rejection as outcome measures. RevMan 5.0.15 was used to calculate relative risk (RR), odds ratios (ORs), and 95% confidence intervals (95%CIs). Results: We found 18 relevant articles, with a total of 6,607 recipients. Follow-up duration ranged from 1 to 15 years. In studies with more than 2 years of follow-up, anti-MICA abs positive in kidney recipients’ post-transplant sera was associated with a lower graft survival rate (4 years: RR = 2.04, 95%CI 1.30 to 3.22; 3 years: OR = 3.56, 95%CI 1.47 to 8.62; 2 years: RR = 2.17, 95%CI 1.09 to 4.31) and a higher acute rejection rate (RR = 1.92, 95%CI 1.27 to 2.91), but there was no clear association with chronic rejection. Similar conclusions could not be drawn for heart or liver transplantation due to possible confounding by anti-HLA abs and the small sample sizes of the available studies. Conclusion: Anti-MICA antibodies in recipients’ sera may associated with poor graft survival rates and high risk of acute and chronic rejection in solid organ transplantation, but more rigorous studies are needed to confirm or refute this relationship. Current immunosuppressive therapy may fail to suppress the harmful effect of MICA antigens.

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

Epstein Barr virus (EBV) is associated with B-cell lymphomas in posttransplant lymphoproliferative disease (PTLD). Latent membrane protein 1 (LMP1), the major oncogenic protein of EBV, promotes tumorigenesis through activation of NF-kappaB, Erk, p38, JNK and Akt. The Jak/STAT signal transduction pathway is also constitutively active in PTLD-associated EBV(+) B-cell lymphomas. Here we determine the mechanism of Jak/STAT activation in EBV(+) B-cell lymphomas and the role of LMP1 in this process. Immunoprecipitation studies revealed no direct interaction of LMP1 and JAK3, but known associations between JAK3 and common gamma chain, and between LMP1 and TRAF3, were readily detected in EBV(+) B cell lines from patients with PTLD. An inducible LMP1 molecule expressed in EBV(-) BL41 Burkitt's cells demonstrated STAT activation only after prolonged LMP1 signaling. While LMP1 induced IFN-gamma production in BL41 cells, IFN-gamma receptor blockade and IFN-gamma neutralization prior to LMP1 activation markedly decreased STAT1 activation and expression of LMP1-driven IFN-gamma inducible genes. Understanding the mechanisms by which EBV induces cellular signal transduction pathways may facilitate development of new treatments for PTLD.

We examined CD4(+)CD25(hi)FOXP3(+) cells Treg in children following liver transplantation and determined the relationship between Treg cell levels in the blood and in the graft. Peripheral blood was obtained from pediatric liver transplant patients at sequential time points: pre-transplant, one month, 3-4 months, 6-7 months, and 11-12 months post-transplant. PBMC were isolated, labeled for CD4, CD25 and FOXP3 expression and analyzed by flow cytometry for CD4(+)CD25(hi)FOXP3(+) cells. Sorted CD4(+)CD25(hi) cells were assessed for functional activity. Pretransplant blood levels of CD4(+)CD25(hi)FOXP3(+) Treg cells were not significantly different from post-transplant blood levels of CD4(+)CD25(hi)FOXP3(+) Treg cells. However, the blood levels of CD4(+)CD25(hi)FOXP3(+) Treg cells were significantly decreased during acute rejection compared with levels when graft function was stable. Immunohistochemistry revealed that FOXP3(+) cells were increased in the portal region of livers with histopathologic evidence of acute graft rejection compared with livers without evidence of rejection and were localized primarily within the inflammatory infiltrate. These data indicate that Treg cells are found at the site of allograft rejection and may play a role in regulation of alloreactivity. Moreover, monitoring peripheral CD4(+)CD25(hi)FOXP3(+) Treg cell levels may be useful in improving the post-transplant management of pediatric liver allograft recipients.

To summarize recent advances that contribute to our understanding of the pathobiology of Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD), the host immune response to virally infected B cells, and the molecular basis for the effects of mammalian target of rapamycin inhibitors on EBV+ B-cell lymphomas.Cytogenetic and genomic analyses support the concept that the underlying biology of EBV-associated PTLD is complex. Transplant recipients can generate and maintain significant populations of EBV-specific CD8+ memory T cells but the function of these cells may be impaired. EBV invokes multiple strategies to subvert and evade the host immune response. The phosphoinositide-3 kinase/Akt/mammalian target of rapamycin signal transduction pathway is a nexus for growth and survival signals in PTLD-associated EBV+ B-cell lymphomas.Multiple factors influence the development of EBV-associated PTLD including the host immune response to EBV, virally induced effects on the infected cell and the host immune system, and the type and intensity of immunosuppression.

Nicotine has wellknown, unpleasant side effects, e.g., transient dizziness, nausea, and nicotine-induced nystagmus (NIN). To investigate factors influencing these effects, we addressed three questions: (1) Is the intensity of dizziness, nausea, NIN, and unsteadiness dependent on nicotine dosage? (2) Does the intensity of perceptual, ocular motor, vegetative effects, and postural imbalance correlate? (3) Do visual or vestibular motion stimuli produce and/or aggravate distressing dizziness and nausea? Sixty healthy non-smokers or occasional smokers participated; 40 were tested once before and six times after application of a nicotine nasal spray in doses of 1 mg or 2 mg with or without motion stimulation; 20 received a placebo nasal spray. Plasma nicotine concentrations were significantly related to nicotine dosage. Dizziness, nausea, NIN, and unsteadiness also depended on the nicotine dosage (p < 0.01).Nicotine blood concentration was a better predictor for the temporal dependence of nystagmus than nicotine dosage. Dizziness correlated highly with nausea (R = 0.63, p < 0.001). The degree of nicotine-induced nausea significantly correlated with postural imbalance. The time course of postural sway differed according to nicotine dosage and gender: for women, there was no clear relationship between sway magnitude and nicotine dosage, while men showed increased sway with higher dosage. Motion stimulation increased nicotine-induced dizziness and nausea, but did not significantly influence NIN or postural imbalance. Our data support the view that all measured adverse effects reflect dose-dependent nicotine-induced vestibular dysfunction. Additional motion stimulation aggravates dizziness and nausea, i.e., nicotine increases sensitivity to motion sickness.

Background. Posttransplant lymphoproliferative disease (PTLD) is a serious complication of solid organ and bone marrow transplantation and is closely associated with Epstein-Barr virus (EBV) infection. We have previously shown that rapamycin (RAPA) directly inhibits the in vitro and in vivo proliferation of EBV-infected B lymphoblastoid cell lines (SLCL), derived from patients with PTLD, by arresting cells in the G1 phase of the cell cycle. The aim of this study is to elucidate the mechanism by which RAPA causes cell cycle arrest in EBV + B cells. Methods. SLCL were cultured without or with RAPA (10 ng/ml) and G1-associated cell cycle proteins were analyzed by immunoblot and densitometric analysis. CDK complexes were immunoprecipitated and incubated with retinoblastoma protein (Rb) substrate. Kinase activity of the complex was determined by Western blot with anti-phospho-Rb antibodies. Results. We show that RAPA decreased both Cyclin D2 and Cyclin D3 protein levels. Furthermore, RAPA decreased the protein levels of cyclin dependent kinase 4 (CDK4) and increased the expression of the CDK inhibitor p27. In contrast, expression of the CDK inhibitor p21 was markedly inhibited by RAPA in the SLCL. Finally, in vitro kinase assays revealed that downstream hyperphosphorylation of Rb by CDK complexes was also decreased by RAPA. Conclusion. The results presented here elucidate key targets of RAPA-induced cell cycle arrest, provide insight into the growth pathways or EBV + B-cell lymphomas, and demonstrate the potential for RAPA as a therapeutic option in the treatment of PTLD and other EBV + lymphomas.

The relationship between EBV infection and sensitivity to death receptor (DR)-induced apoptosis is poorly understood. Using EBV− and EBV+ BJAB cells, we provide the first evidence that EBV can protect latently infected B cell lymphomas from apoptosis triggered through Fas or TRAIL receptors. Caspase 8 activation was impaired and cellular FLIP recruitment was enriched in death-inducing signaling complexes formed in EBV-infected BJAB cells relative to parent BJAB cells. Furthermore, latent membrane protein 1 expression alone could reduce caspase activation and confer partial resistance to DR apoptosis in BJAB cells. This protective effect was dependent on C-terminal activating region 2-driven NF-κB activation, which in turn up-regulated cellular FLIP expression in latent membrane protein 1+ BJAB cells. Thus, the ability of latent EBV to block DR apoptosis may help to ensure the survival of host cells during B cell differentiation, and contribute to the development of B cell lymphomas, especially in immunocompromised individuals.

NKp30 is a stimulatory receptor on human NK cells implicated in tumor immunity, and is capable of promoting or terminating dendritic cell maturation. To gain a better understanding of NKp30 biology, we have investigated the expression and function of rat NKp30 (rNKp30). We generated stable transfectants of rNKp30 in RNK16 cells, a rat NK lymphoma line, and used a novel panel of mAb against rNKp30 to study this receptor. Using agonistic rNKp30 mAb, we demonstrated that rNKp30 mediates robust IFN-gamma production and cytolytic responses from rNKp30-transfected RNK16 cells. We determined by flow cytometry that rNKp30 is expressed by a subset of primary NK cells isolated from the blood and spleen, and to a lesser extent also on liver NK cells. Stimulation of rNKp30 on primary NK cells led to IFN-gamma production. Liver NK cells expressed low levels of NKp30 and had reduced rNKp30-mediated IFN-gamma responses. During an alloimmune response in vivo, the proportion of the rNKp30(+) NK cell subset in the peripheral blood significantly increased, suggesting that rNKp30 may play an important role during alloactivation. Thus, our data demonstrate that NKp30 is indeed expressed in rodents and is a functional stimulatory receptor in a subset of rat NK cells.

Many transplant physicians are faced with questions from their patients about the safety and long-term consequences of pregnancy following transplantation. To better understand how pregnancies are managed and to clarify the outcome of pregnancy after transplantation, a survey questionnaire was developed and mailed to all medical and surgical directors of transplant centers throughout the United States; responses were obtained from 59.1% of the transplant centers. Although many opinions were collected, most respondents conceded that their opinions were based on personal experience rather than evidence-based. The underutilization of existing information was revealing and highlighted a need for an evidence-based approach to care of the pregnant transplant recipient and her offspring. The survey results, reported in this article, led to formation of a consensus conference to determine the optimal approach to pregnant transplant recipients and to define what is currently known and unknown about reproduction and transplantation.