Plasma Epstein-Barr viral load predicting response after chemotherapy for post-transplant lymphoproliferative disease

Transplant patients are particularly at risk of developing B post-transplant lymphoproliferative disease (PTLD) related to intensive immunosuppressive treatment to prevent graft rejection. In EBV-positive PTLDs, EBV-DNA can be found in the patients' peripheral blood. Several methods have been described to assess peripheral blood EBV viral load. We report a case of a 13-year-old child who developed EBV-positive PTLD after renal transplantation. We assessed EBV plasma viral load by quantitative PCR and we found that the clearance of EBV-DNA correlated well with response to treatment.     

Post-immunosuppressive and immunomodulatory therapy lymphoproliferative disorders of the gastrointestinal tract

Post-immunosuppressive and immunomodulatory therapy lymphoproliferative disorders occur during immunosuppressive treatment following allogeneic solid organ or stem cell transplant (post-transplant lymphoproliferative disorder, PTLD) or for autoimmune disorders. These disorders commonly initially present in the gastrointestinal tract, and up to 30% of patients have been shown to have GI involvement. Post-immunosuppressive therapy lymphoproliferative disorders are frequently associated with Epstein–Barr virus (EBV) and encompass a wide spectrum of morphologic and clinical presentation. EBV-negative and T-cell lymphoproliferations occur to be increasing, and a high index of suspicion is required on the part of the pathologist for timely diagnosis and treatment.     

Differential cross-reactivity of monoclonal antibody OPD4 (anti-CD45RO) in macaques

Immunologic research in nonhuman primates is occasionally limited by the availability of reagents that cross-react in nonhuman primates. One major limitation has been the lack of a monoclonal antibody to CD45RO. Although the monoclonal antibody UCHL-1 is used to detect CD45RO isoforms in humans, it does not react with nonhuman primates, mandating the use of alternative strategies to define "memory" T cell responses in nonhuman primates. The current study examined the reactivity and specificity of another antibody against CD45RO, clone OPD4, in macaques. Here we demonstrate that OPD4 specifically labels memory CD4+ T cells in approximately 44% of rhesus macaques (Macaca mulatta) of Indian but not Chinese origin. In contrast, tissues from pigtail macaques (Macaca nemestrina) react with this clone, indicating that OPD4 may be useful for examining memory CD4+ T cells in certain macaques, but its utility may be limited in other species or even among individual macaques.     

移植后淋巴组织增生性疾病的临床病理分析

目的探讨移植后淋巴组织增生性疾病（PTLD）的临床及病理特征,提高其诊断和治疗水平。方法对4例移植后淋巴组织增生性疾病行HE和免疫组织化学EnVision法染色、原位杂交及聚合酶链反应,复习其临床资料并随访。结果4例中3例是肾移植后,其中2例为多形性PTLD,1例为单形性PTLD;另1例是异体骨髓移植后PTLD的“早期”病变。2例EB病毒阳性。4例移植后所用免疫抑制剂均以环孢A类药为主,辅以激素。例1～4从移植到诊断PTLD的时间为42、7、129、2个月。例3多形性PTLD的临床分期为Ⅱ期（诊断PTLD后2个月死亡）;其余均为Ⅰ期。均存活,诊断PTLD后生存期为40（例1）、26（例2）、5（例4）个月。结论PTLD是发生在器官移植后,具有独特的形态和临床特征的淋巴组织增生性疾病,部分病例与EB病毒感染有关。其预后与临床分期相关,免疫抑制剂减量可能有效。     

Determining virological, serological and immunological parameters of EBV infection in the development of PTLD

Post-transplant lymphoproliferative disease (PTLD) in Epstein-Barr virus (EBV) seronegative solid organ transplant recipients remains a significant problem, particularly in the first year post-transplant. Immune monitoring of a cohort of high-risk patients indicated that four EBV seronegative transplant recipients developed early-onset PTLD prior to evidence of an EBV humoral response. EBV status has been classically defined serologically, however these patients demonstrated multiple parameters of EBV infection, including the generation of EBV-specific CTL, outgrowth of spontaneous lymphoblastoid cell lines, and elevated EBV DNA levels, despite the absence of a classic EBV antibody response. As EBV serology is influenced by both immunosuppression and cytomegalovirus immunoglobulin treatment, both the EBV-specific CTL response and elevated EBV levels are more reliable indicators of EBV infection post-transplant.     

Post-transplant lymphoproliferative disorders

Post-transplant lymphoproliferative disorders (PTLD) are the second most frequent neoplasia in transplant patients after skin carcinomas. They occur following both solid organ transplants (SOT) and haematopoietic stem cell transplants (HSCT). Most PTLD in solid organ recipients are of host origin, whereas in HSCT recipients they are most often of donor origin. The EBV status of the recipient and the donor, the type of transplant, the type of immunosuppressive therapy used, and the time after transplant are all important parameters that have been associated with the incidence and the type of PTLD. Although most PTLD are B-cell lesions up to 15% may be T-cell or NK-cell type. Most PTLD are associated with EBV, but EBV-negative PTLD are also clearly recognized. In the 2008 WHO classification of lymphoid neoplasms (ref) PTLD are subclassified according to their histological and immunophenotypic characteristics into early lesions, polymorphic type, monomorphic type, and classical Hodgkin lymphoma-type. Overall PTLD mortality rates are around 50%, but new therapies that include early treatment with rituximab and novel anti-EBV therapies promise better outcomes.     

Monkey Models of Tuberculosis: Lessons Learned

The use of animal models has been invaluable for studying the pathogenesis of Mycobacterium tuberculosis infection, as well as for testing the efficacy of vaccines and drug regimens for tuberculosis. Among the applied animal models, nonhuman primates, particularly macaques, share the greatest anatomical and physiological similarities with humans. As such, macaque models have been used for investigating tuberculosis pathogenesis and preclinical testing of drugs and vaccines. This review focuses on published major studies which illustrate how the rhesus and cynomolgus macaques have enriched and may continue to advance the field of global tuberculosis research.     

Transplant-associated lymphoproliferation

Transplantation of solid organs and haematopoietic stem cells requires immunosuppressive drug therapy in order to prevent rejection or graft-versus-host disease. Depending on dosage and type of drug, the risk of developing an Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) is increased. The lesion spectrum ranges from hyperplastic lesions to manifest lymphomas, the latter being classified as monomorphic PTLD. Hyperplastic changes, which are not distinguishable from viral reactions, comprise early or mononucleosis-like lesions. Those with effaced lymph node architecture or extranodal manifestation without a lymphoma-like phenotype are designated polymorphic PTLD. Monomorphic PTLD are either high grade B cell lymphomas, plasma cell neoplasms or Hodgkin lymphomas and only very rarely T cell lymphomas. Low grade B cell lymphomas do not occur. In a subfraction of cases, including even monomorphic PTLD, reduction of immunosuppression alone is sufficient to induce remission of the pathological process.     

Complete resolution of posttransplant lymphoproliferative disorder (diffuse large B-cell lymphoma) with reduction of immunosuppressive therapy

Posttransplant lymphoproliferative disorder (PTLD) is a serious complication of organ transplantation. PTLD is the disorder arising from the combined effects of Epstein-Barr virus associated lymphoid proliferation with the disruption of the normal immune control by the cytotoxic T cells. The treatment for PTLD is one of the most controversial topics in solid organ transplantation. It is well known that the initial management of PTLD is a reduction of immunosuppression. Early diagnosis and the early reduction in immunosuppression are essential even for monomorphic lymphoma. We report here on a case of the complete resolution of PTLD (diffuse large B cell lymphoma) which occurred after a drastic reduction of immunosuppression in a renal transplant recipient.     

Antibodies to lytic infection proteins in lymphocryptovirus-infected rhesus macaques: a model for humoral immune responses to epstein-barr virus infection

Humoral immune responses to rhesus lymphocryptovirus (rhLCV) lytic infection proteins were evaluated in the rhesus macaque animal model for Epstein-Barr virus (EBV) infection. We found a hierarchy of humoral responses to 14 rhLCV lytic infection proteins in naturally infected rhesus macaques, with (i) widespread and robust responses to four glycoproteins expressed as late proteins, (ii) frequent but less robust responses to a subset of early proteins, and (iii) low-level responses to immediate-early proteins. This hierarchy of humoral responses was similar to that reported for EBV-infected humans, with the notable exception of the response to rhBARF1. Serum antibodies to rhBARF1 were frequently detected in healthy rhLCV-infected macaques, but in humans, anti-BARF1 antibodies have been reported primarily in patients with EBV-positive nasopharyngeal carcinoma (NPC). The macaque data accurately predicted that serum antibodies against BARF1 are a normal response to EBV infection when human serum samples are analyzed. The rhesus macaque animal provides a unique perspective on humoral responses to EBV infection in humans and can be a valuable model for EBV vaccine development.     

Population genetics of the ABO locus within the rhesus (Macaca mulatta) and cynomolgus (M. fascicularis) macaque hybrid zone

Knowledge of the macaque ABO blood group system has been critical in the development of nonhuman primates (NHPs) as a translational model. Serving not only as a useful homologue of the disease‐linked ABO system in humans, macaque ABO blood groups must be typed in colonies prior to performing experimental procedures requiring blood transfusion or transplantation. While the rates of blood type incompatibility and the distributions of A, B and AB blood groups are known in large samples of rhesus (Macaca mulatta) and cynomolgus (M. fascicularis) macaques, there is a dearth of blood type data from macaque populations occupying the rhesus‐cynomolgus hybrid zone in Southeast Asia. Using molecular phenotyping, we profiled ABO blood group distributions of 232 macaques from 10 populations in the hybrid zone and compared them to pure blood populations of the two species. We found that while these distributions are significantly different in most populations, there was a lack of differentiation between the hybrid and cynomolgus macaques as well as between the Thai and neighbouring populations. This supports a more expansive model of hybridization between rhesus and cynomolgus macaques than often proposed and highlights the increased need for consideration of population genetic structure in biomedical studies that employ macaques as animal models. Additionally, we report an enrichment of indeterminate blood types in the hybrid populations.     

Inverse correlation of telomerase activity/proliferation of CD4+ T lymphocytes and disease progression in simian immunodeficiency virus-infected nonhuman primates

Both increased lymphocyte renewal with subsequent exhaustion of the immune system and impaired T-cell renewal have been put forth to account for CD4+ T-cell depletion and development of AIDS in HIV-1-infected humans and SIV-infected nonhuman primates. In the present study, telomeric terminal restriction fragment length and telomerase activity were used as measures of proliferative activity of T lymphocytes from three nonhuman primate species before and after being infected with SIV. In peripheral blood T cells, our data show both species and T-cell-subset-specific differences in proliferative activity accompanied by different patterns of disease progression. A significant postinfection increase in telomerase/proliferative activity in CD4+ T cells from seropositive sooty mangabeys and from normal progressor rhesus macaques was associated with asymptomatic infection or delayed disease progression, respectively, whereas a decrease in telomerase/proliferative activity detected in CD4+ T cells postinfection from SIVsmmPBj14-infected pigtailed macaques was associated with rapid CD4+ T-cell depletion and disease progression. The levels of telomerase activity observed in CD4+ T cells from peripheral blood closely parallelled those seen in CD4+ T cells in lymph node samples from selected animals. Our data suggest that an increase in proliferative activity of T lymphocytes in vivo may be associated with a favorable course of SIV infection in nonhuman primates.     

Vaccines for the prevention of HIV-1 disease

Clinical investigation in humans and experimental lentivirus infection in nonhuman primates have advanced our understanding of immune responses that control HIV-1 disease. Recently, immunization approaches in macaques have shown that the immune response can control viremia and improve clinical outcome. When such vaccine strategies are formulated to be similarly immunogenic in humans, they could form the basis for the development of candidate AIDS vaccines that would prevent infection, suppress progression to disease or reduce HIV-1 transmission in humans.     

Infection of rhesus monkeys and chimpanzees with Epstein-Barr virus

Summary Seventy-two nonhuman primates were entered into a long-term study to evaluate the pathogenicity of Epstein-Barr virus (EBV). Infectious virus was inoculated into 42 rhesus monkeys, 4 chimpanzees and 1 cynomolgus monkey. Immunostimulation or immunosuppression was attempted in 34 of these animals to enhance the oncogenic potential of the virus. Eleven inoculated animals were followed for more than 3 years and two were observed for 8 years. No tumors were observed in any of the animals; however, serological evaluation of the 47 inoculated primates and 25 matched controls indicated that at least 14 rhesus monkeys and the cynomolgus monkey were successfully infected with EBV. The potential use of rhesus monkeys as a model for EBV-induced disease in humans is discussed.With 2 FiguresSupported in part by Contracts NO 1-CP-4333 and NO 1-CP-61029 with the Carcinogenesis Extramural Program, Division of Cancer Cause and Prevention.     

Strategies for preclinical evaluation of dendritic cell subsets for promotion of transplant tolerance in the nonhuman primate

A role for dendritic cells (DC) as critical regulators of immune reactivity has become increasingly recognized. There is evidence in rodent models that donor-derived DC, particularly in the immature state, can prolong organ allograft survival and even induce donor-specific tolerance. To allow the potential tolerogenic properties of these cells to be evaluated more fully with a view to clinical testing, it is necessary to identify DC subsets in nonhuman primates. We have identified the putative rhesus monkey equivalents of circulating human DC subset precursors as lineage(-), HLA-DR(+), CD123(lo),CD11c(hi)(pDC1) and lineage(-), HLA-DR(+), CD123(hi),CD11c(lo)(pDC2). Testing of these DC populations both in vitro and in vivo, as well as in transplant models in combination with conventional or experimental immunosuppressive reagents, will aid the development of novel strategies for the promotion of allo-antigen specific tolerance in transplantation.     

Epstein‐Barr virus related post‐transplant lymphoproliferative disorder prevention strategies in allogeneic hematopoietic stem cell transplantation

Epstein‐Barr virus associated post‐transplant lymphoproliferative disorders (EBV PTLD) are recognized as a significant cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). The number of patients at risk of developing EBV PTLD is increasing, partly as a result of highly immunosuppressive regimens, including the use of anti‐thymocyte globulin (ATG). Importantly, there is heterogeneity in PTLD management strategies between alloHSCT centers worldwide. This review summarizes the different EBV PTLD prevention strategies being utilized including the alloHSCT and T‐cell depletion regimes and the risk they confer; monitoring programs, including the timing and analytes used for EBV virus detection, as well as pre‐emptive thresholds and therapy with rituximab. In the absence of an institution‐specific policy, it is suggested that the optimal pre‐emptive strategy in HSCT recipients with T‐cell depleting treatments, acute graft vs host disease (GVHD) and a mismatched donor for PTLD prevention is (a) monitoring of EBV DNA post‐transplant weekly using plasma or WB as analyte and (b) pre‐emptively reducing immune suppression (if possible) at an EBV DNA threshold of >1000 copies/mL (plasma or WB), and treating with rituximab at a threshold of >1000 copies/mL (plasma) or >5000 copies/mL (WB). There is emerging evidence for prophylactic rituximab as a feasible and safe strategy for PTLD, particularly if pre‐emptive monitoring is problematic. Future management strategies such as prophylactic EBV specific CTLs have shown promising results and as this procedure becomes less expensive and more accessible, it may become the strategy of choice for EBV PTLD prevention.     

Preclinical evaluation of cellular immune responses elicited by a polyvalent DNA prime/protein boost HIV-1 vaccine

While DNA vaccines have been shown to prime cellular immune responses, levels are often low in nonhuman primates or humans. Hence, efforts have been directed toward boosting responses by combining DNA with different vaccination modalities. To this end, a polyvalent DNA prime/protein boost vaccine, consisting of codon optimized HIV-1 env (A, B, C, E) and gag (C) and homologous gp120 proteins in QS-21, was evaluated in rhesus macaques and BALB/c mice. Humoral and cellular responses, detected following DNA immunization, were increased following protein boost in macaques and mice. In dissecting cellular immune responses in mice, protein-enhanced responses were found to be mediated by CD4+ and CD8+ T cells with a Th1 cytokine bias. Our study reveals that, in addition to augmenting humoral responses, protein boosting of DNA-primed animals augments cellular immune responses mediated by CD8+ CTL, CD4+ T-helper cells and Th1 cytokines; thus, offering much promise in controlling HIV-1 in vaccinees.     

Microsatellite analysis in post-transplantation lymphoproliferative disorder to determine donor/recipient origin.

Post-transplantation lymphoproliferative disorders (PTLD) are a group of heterogeneous diseases that occur after organ transplantation. Determination of the origin of the tumor cells not only provides clues to its possible pathogenetic mechanism, but also gives prognostic guidance in the clinical management of patients. We reviewed the clinicopathological features of four cases of PTLD that developed after solid organ transplantation. Using microsatellite analysis performed on paraffin-embedded tissue and using multiple, highly polymorphic markers, we have successfully determined the recipient/donor origin of the tumor cells in all of them. The time of onset of the PTLD ranged from 5 to 11 mo. All cases were diffuse large cell lymphomas of B-cell lineage, and the two cases that have been tested for EBV by in situ hybridization were positive. Three of the 4 PTLD were of donor origin and these three patients died of diseases unrelated to PTLD. The single patient with PTLD of recipient origin died of disseminated PTLD. The mean survival length of the three patients with donor origin was 26.3 mo, whereas that of the patient with recipient origin was 12 mo. Our results indicate a relatively high incidence of PTLD of donor origin among our patients with solid organ transplantation, as compared to other reported series. Moreover, the finding of the relatively indolent nature of PTLD of donor origin supports that determination of the donor/recipient origin of PTLD is of prognostic significance.     

Novel cynomolgus macaque MHC-DPB1 polymorphisms in three South-East Asian populations

Cynomolgus macaques (Macaca fascicularis, Mafa), alias the crab-eating monkeys or long-tailed macaques, live across a vast range of South-East Asia. These non-human primates have emerged as important animal models in infectious and chronic diseases and transplantation studies, necessitating a more extensive characterization of their major histocompatibility complex polymorphic regions. The current information on the polymorphic variation or diversity of the Mafa-DPB1 locus is largely limited in comparison with the more commonly studied rhesus macaque DPB1 locus. In this article, to better elucidate the degree and types of polymorphisms and genetic differences of Mafa-DPB1 locus among three South-East Asian populations and to investigate how the allele differences between macaques and humans might affect their respective immune responses, we identified 40 alleles within exon 2 of the Mafa-DPB1 locus by DNA sequencing using 217 individuals. We also performed evolutionary and population analyses using these sequences to reveal some population-specific alleles and trans-species allelic conservation between the cynomolgus macaques and the rhesus macaques. Of the 40 new alleles, eight belong to a newly identified lineage group not previously found in the rhesus macaque species. This allele information will be useful for medical researchers using the cynomolgus macaques in disease and immunological studies.     

Treatment options for post-transplant lymphoproliferative disorder and other Epstein-Barr virus-associated malignancies

Epstein-Barr virus (EBV) is associated with a range of malignancies that largely arise from a defect in EBV-specific cytotoxic T lymphocyte (CTL) immunity and function. Much work has focused on the reconstitution of CTL immunity to EBV in transplant patients, in whom immunosuppression modalities render them susceptible to post-transplant lymphoproliferative disease (PTLD). Adoptive transfer of autologous CTLs is effective at both preventing and curing PTLD in solid organ transplant recipients and can produce a long-term memory response and protection against recurring disease. In this review, the benefits and restrictions of administering EBV-specific CTLs for the treatment of PTLD are discussed and compared with emerging therapies including the generation of allogeneic human leukocyte antigen-matched CTL banks and the anti-CD20 monoclonal antibody therapy, MabThera. Furthermore, studies involving other EBV-associated disorders have described the potential benefit of adoptive transfer of EBV-specific CTLs for Hodgkin's disease, nasopharyngeal carcinoma, chronic active EBV infection, and Burkitt's lymphoma. The challenges of tailor-making therapies for individual diseases and EBV antigen expression latencies are highlighted, in addition to considering vaccination strategies for optimal treatment.