The explosion of genetic information from recent advances in sequencing technologies, bioinformatics and genomics highlights the importance of understanding mechanisms involved in gene expression and ...regulation. Over the last decade, it has become clear that small ribonucleic acids (RNAs) are a central component of the cellular gene regulatory network. MicroRNAs (miRNAs) are a family of endogenous, small, noncoding single‐stranded RNA of ∼22 nucleotides in length that act as posttranscriptional gene regulatory elements. MicroRNAs can inhibit de novo protein synthesis by blocking translation through base‐pairing with complementary messenger RNA (mRNA) and also suppress translation by promoting degradation of target mRNA. MicroRNAs are intimately involved in a variety of biologic processes including development, hematopoietic cell differentiation, apoptosis and proliferation. To date, over 800 human miRNAs have been identified, though the biologic function of only a fraction of miRNAs has been elucidated. Here, we discuss how miRNAs are produced, identified and quantitated, and focus on several key miRNAs that govern expression of genes relevant to allograft rejection, tolerance induction and posttransplant infection. Finally, we discuss potential ways in which the miRNA network can be modulated that ultimately may offer new strategies to promote long‐term graft survival.
The biogenesis, function, and analysis of microRNA will become increasingly relevant to transplantation.
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.
MicroRNAs are differentially expressed between allogeneic and syngeneic heart grafts, suggesting potentially important regulatory functions in graft rejection.
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 ...after transplant. Mass cytometry, or cytometry by time of flight, 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 >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 compared with multiparameter flow cytometry, introduce novel methods of high‐dimensional data analysis and visualization, and review some recent studies using mass cytometry to profile the immune systems of healthy people and transplant recipients.
This review discusses the current state of mass cytometry, the methods of data analysis, and potential applications of this technology to both basic science and clinical studies in transplantation.
Posttransplant lymphoproliferative disorder (PTLD) continues to be a devastating and potentially life‐threatening complication in organ transplant recipients. PTLD is associated with EBV infection ...and can result in malignant B cell lymphomas. Here we demonstrate that the PI3K/Akt/mTOR pathway is highly activated in EBV+ B cell lymphoma lines derived from patients with PTLD. Treatment with the mTORC1 inhibitor Rapamycin (RAPA) partially inhibited the proliferation of EBV+ B cell lines. Resistance to RAPA treatment correlated with high levels of Akt phosphorylation. An mTORC1/2 inhibitor and a PI3K/mTOR dual inhibitor suppressed Akt phosphorylation and showed a greater anti‐proliferative effect on EBV+ B lymphoma lines compared to RAPA. EBV+ B cell lymphoma lines expressed high levels of PI3Kδ. We demonstrate that PI3Kδ is responsible for Akt activation in EBV+ B cell lymphomas, and that selective inhibition of PI3Kδ by either siRNA, or a small molecule inhibitor, augmented the anti‐proliferative effect of RAPA on EBV+ B cell lymphomas. These results suggest that PI3Kδ is a novel, potential therapeutic target for the treatment of EBV‐associated PTLD and that combined blockade of PI3Kδ and mTOR provides increased efficacy in inhibiting proliferation of EBV+ B cell lymphomas.
The authors demonstrate that targeting PI3Kδ with a small molecule inhibitor can enhance the antiproliferative effect of mTOR inhibitors in the Epstein‐Barr virus positive B cell lymphomas associated with posttransplant lymphoproliferative disorder.
The 12th Banff Conference on Allograft Pathology was held in Comandatuba, Brazil, from August 19–23, 2013, and was preceded by a 2‐day Latin American Symposium on Transplant Immunobiology and ...Immunopathology. The meeting was highlighted by the presentation of the findings of several working groups formed at the 2009 and 2011 Banff meetings to: (1) establish consensus criteria for diagnosing antibody‐mediated rejection (ABMR) in the presence and absence of detectable C4d deposition; (2) develop consensus definitions and thresholds for glomerulitis (g score) and chronic glomerulopathy (cg score), associated with improved inter‐observer agreement and correlation with clinical, molecular and serological data; (3) determine whether isolated lesions of intimal arteritis (“isolated v”) represent acute rejection similar to intimal arteritis in the presence of tubulointerstitial inflammation; (4) compare different methodologies for evaluating interstitial fibrosis and for performing/evaluating implantation biopsies of renal allografts with regard to reproducibility and prediction of subsequent graft function; and (5) define clinically and prognostically significant morphologic criteria for subclassifying polyoma virus nephropathy. The key outcome of the 2013 conference is defining criteria for diagnosis of C4d‐negative ABMR and respective modification of the Banff classification. In addition, three new Banff Working Groups were initiated.
The 2013 Banff Transplant Conference defines revised, consensus criteria for acute/active and chronic, active antibody‐mediated rejection in renal allograft biopsies, criteria that include C4d‐negative antibody‐mediated rejection and antibody‐associated arterial lesions. Also see comprehensive review by Djamali et al on page 255.
EBV-infected B-cell lymphomas are a potentially life-threatening complication in bone marrow and solid organ transplant recipients. Immunosuppressive drugs required to prevent allograft rejection ...also impair anti-EBV T-cell immunity, thereby increasing the risk of EBV-associated disease. Here we demonstrate that the immunosuppressant rapamycin (RAPA) has a strong antiproliferative effect in vitro on B-cell lines derived from organ transplant recipients with EBV-associated posttransplant lymphoproliferative disorder (PTLD). Furthermore, RAPA significantly inhibits or delays the growth of solid tumors established from EBV-infected B-cell lines in a xenogeneic mouse model of PTLD. RAPA acts via cell cycle arrest, induction of apoptosis, and, most importantly, inhibition of interleukin 10 secretion, a necessary autocrine growth factor. The reduced interleukin 10 production is accompanied by corresponding decreases in the constitutive activation of the growth-promoting transcription factors signal transducer and activator of transcription 1 and 3. Thus, RAPA can limit B-cell lymphoma growth while simultaneously providing immunosuppression to prevent graft rejection in patients who are otherwise at risk for EBV-associated PTLD. Moreover, these findings may have application to other EBV-associated malignancies.
Epstein–Barr virus (EBV) is a γ‐herpesvirus that is linked to the development of posttransplant lymphoproliferative disorder (PTLD) in solid organ recipients. We previously demonstrated that EBV+ B ...cell lymphoma cell lines isolated from patients with PTLD produce human IL‐10 as an autocrine growth factor. However, little is known regarding IL‐10 regulation in B cells. Here we show that EBV infection markedly alters the expression of host B cell microRNA, a class of small noncoding RNA that is an important regulator of transcriptional and posttranscriptional gene expression. Gene arrays reveal unique microRNA profiles in EBV+ B cell lymphoma lines from patients with PTLD, compared to normal B cells or in vitro generated EBV+ lymphoblastoid cell lines. We show that microRNA‐194 expression is uniquely suppressed in EBV+ B cell lines from PTLD patients and that the 3'untranslated region of IL‐10 is targeted by microRNA‐194. Overexpression of microRNA‐194 attenuates IL‐10 production and increases apoptosis of EBV+ B cell lymphoma lines. Together, these data indicate that EBV co‐opts the host B cell microRNA network and specifically suppresses microRNA‐194 to override control of IL‐10 expression. Thus, modulation of microRNA‐194 may constitute a novel approach to inhibiting proliferation of EBV+ B cell lymphomas in PTLD.
Epstein‐Barr virus modulates B cell microRNA‐194 expression during latent infection to promote the production of IL‐10, an important autocrine growth factor for EBV+ B cell lymphomas in posttransplant lymphoproliferative disorder.
Posttransplant lymphoproliferative disorder (PTLD)‐associated Epstein–Barr virus (EBV)+ B cell lymphomas are serious complications of solid organ and bone marrow transplantation. The EBV protein ...LMP2a, a B cell receptor (BCR) mimic, provides survival signals to virally infected cells through Syk tyrosine kinase. Therefore, we explored whether Syk inhibition is a viable therapeutic strategy for EBV‐associated PTLD. We have shown that R406, the active metabolite of the Syk inhibitor fostamatinib, induces apoptosis and cell cycle arrest while decreasing downstream phosphatidylinositol‐3′‐kinase (PI3K)/Akt signaling in EBV+ B cell lymphoma PTLD lines in vitro. However, Syk inhibition did not inhibit or delay the in vivo growth of solid tumors established from EBV‐infected B cell lines. Instead, we observed tumor growth in adjacent inguinal lymph nodes exclusively in fostamatinib‐treated animals. In contrast, direct inhibition of PI3K/Akt significantly reduced tumor burden in a xenogeneic mouse model of PTLD without evidence of tumor growth in adjacent inguinal lymph nodes. Taken together, our data indicate that Syk activates PI3K/Akt signaling which is required for survival of EBV+ B cell lymphomas. PI3K/Akt signaling may be a promising therapeutic target for PTLD, and other EBV‐associated malignancies.
The authors demonstrate that small molecule inhibition of the PI3K/Akt pathway reduces tumor formation and tumor burden in a xenotransplantation model of Epstein—Barr virus—positive posttransplant lymphoproliferative disorder.
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‐κB, 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‐γ production in BL41 cells, IFN‐γ receptor blockade and IFN‐γ neutralization prior to LMP1 activation markedly decreased STAT1 activation and expression of LMP1‐driven IFN‐γ inducible genes. Understanding the mechanisms by which EBV induces cellular signal transduction pathways may facilitate development of new treatments for PTLD.
The Epstein Barr virus encoded protein, latent membrane protein 1, induces production of interferon‐gamma that results in activation of STAT1 and expression of STAT‐1 inducible genes in B cell lymphomas.
Keratin 8 and 18 (K8/K18) mutations are found in patients with cryptogenic cirrhosis, but the role of keratin mutations in noncryptogenic cirrhosis and the incidence of keratin mutations in the ...general population are not known. We screened for K8/K18 mutations in genomic DNA isolated from 314 liver explants of patients who primarily had noncryptogenic cirrhosis, and from 349 blood bank volunteers. Seven unique K8/K18 mutations were found in 11 independent patients with biliary atresia, hepatitis B/C, alcohol, primary biliary cirrhosis, and fulminant hepatitis. Seven of the 11 patients had mutations previously described in patients with cryptogenic cirrhosis: K8 Tyr-53 → His, K8 Gly-61 → Cys, and K18 His-127 → Leu. The four remaining patients had mutations at one K8 and three other K18 new sites. Of the 349 blood bank control samples, only one contained the Tyr-53 → His and one the Gly-61 → Cys K8 mutations (P < 0.004 when comparing cirrhosis versus control groups). Two additional mutations were found in both the liver disease and blood bank groups and, hence, likely represent polymorphisms. Livers with keratin mutations had cytoplasmic filamentous deposits that were less frequent in livers without the mutations (P = 0.03). Therefore, K8/K18 are likely susceptibility genes for developing cryptogenic and noncryptogenic forms of liver disease.
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