Recipients Of Bone Marrow Allografts Research Articles

Expression and Targeted Inhibition of the Immunoregulatory Carbohydrate-Binding Lectin, Galectin 1, in EBV-Driven Post-Transplant Lymphoproliferative Disorders.

Abstract 96EBV-associated post-transplant lymphoproliferative disorders (PTLD) are potentially fatal EBV-driven B cell proliferations that develop in immunocompromised solid organ or bone marrow allograft recipients. Proliferation of EBV-infected B cells in PTLD is maintained by expression of EBV latent genes such as latent membrane protein 1 (LMP1) and LMP2A, viral immune evasion strategies, and impaired host immune surveillance. Management of early PTLD lesions is currently based on reduction or withdrawal of immunosuppression which increases the risk of graft rejection. For these reasons, alternative treatments that specifically boost host anti-EBV immune responses are needed.To identify novel T-cell inhibitory molecules induced by EBV and expressed in PTLDs, we compared the transcription profiles of a series of EBV-transformed lymphoblastoid B-cell lines (LCLs) and diffuse large B-cell lymphoma (DLBCL) cell lines. The LCLs overexpressed galectin-1 (Gal-1), a carbohydrate-binding lectin that induces tolerogenic dendritic cells and triggers the selective apoptosis of CD4+ Th1 and CD8+ cytotoxic T cells. Gal-1 protein expression was also uniformly high in LCLs and low or undetectable in DLBCL lines by western blotting. Immunohistochemical staining of primary tumors confirmed abundant Gal-1 expression in PTLD B-cells, whereas DLBCLs were negative.To elucidate the mechanisms responsible for Gal-1 overexpression in EBV-associated PTLD, we performed transcriptional reporter assays using cotransfected FLAG-tagged LMP1 and/or LMP2A and a luciferase reporter vector driven by Gal-1 promoter. LMP2A and LMP1 each increased Gal-1-driven luciferase expression 3- to 6-fold, respectively, and the combination of LMP2A and LMP1 was synergistic. In complementary experiments, siRNA-mediated depletion of LMP2A decreased Gal-1 protein abundance in EBV-transformed lymphoblastoid cells. The mechanism of LMP1 and LMP2A-induced Gal-1 expression in LCLs was both AP-1 and PI3K-dependent; the PI3K selective inhibitor, LY294002, markedly inhibited Gal-1 expression in these cells.Given the tolerogenic and immunosuppressive function of Gal-1 and the EBV-driven overexpression of the lectin in LCLs and primary PTLDs, we postulated that Gal-1 might be a targetable immune escape mechanism in EBV-associated malignancies. For these reasons, we assessed the neutralizing activity of a recently developed series of anti-Gal1 monoclonal antibodies. After confirming that these reagents selectively identified both recombinant and native Gal1, we asked whether certain Gal-1 monoclonal antibodies neutralized recombinant Gal-1-mediated apoptosis of EBV-specific polyclonal, largely CD8+, T-cells. Preincubation of human recombinant Gal-1 (hrGal-1) with Gal-1 monoclonal antibodies, but not with isotype-matched controls, selectively inhibited hrGal-1 induced apoptosis of EBV-specific CD8+ T-cells.Taken together, these studies indicate that EBV-transformed LCLs and primary EBV-associated PTLDs express Gal-1 in a LMP-1 and LMP2A-dependent manner. Given the acknowledged role of Gal-1 in mediating an immunopriviledged tumor microenvironment, the lectin likely inhibits the host anti-tumor response in these tumors. Antibody-mediated neutralization of secreted Gal-1 may represent a novel immunotherapeutic strategy in PTLD and other Gal1-expressing tumors. Disclosures:No relevant conflicts of interest to declare.

Comparison of DNA Amplification, mRNA Amplification, and DNA Hybridization Techniques for Detection of Cytomegalovirus in Bone Marrow Transplant Recipients

A total of 676 specimens from 63 recipients of bone marrow allografts were tested for cytomegalovirus (CMV) by the following assays: CMV pp67 NucliSens (NS), AMPLICOR CMV MONITOR (RA), and the Digene CMV DNA test (DG). In a consensus analysis, the sensitivities and specificities were 60 and 99% (NS), 96 and 98% (RA), and 90 and 76% (DG), respectively; for detection of symptomatic CMV infection, they were 60 and 97% (NS), 65 and 97% (RA), and 95 and 77% (DG), respectively. In multivariate analysis, the major risk factor for symptomatic CMV infection was an increase in the viral load in the DG assay; in univariate analyses, maximum viral loads in both DG and RA assays and a rising viral load in the RA assay were also significant. The earliest detection of CMV replication was provided by the RA assay (mean, 39 days posttransplantation), followed by the DG assay (mean, 48 days) and the NS assay (mean, 58 days).

Prevalence and clinical features of hepatitis G virus infection in bone marrow allograft recipients.

To study the prevalence and clinical features of hepatitis G virus (HGV)/GB virus C (GBV-C) infection in bone marrow transplantation (BMT), we examined frozen serum samples from 95 bone marrow allograft patients for HGV/GBV-C RNA by RT-PCR. Twenty-eight out of 95 (29.5%) were positive and 14 of the HGV+ patients were already positive before transplantation. The mean numbers of blood donors to whom the HGV and HGV+ populations were exposed before BMT were not significantly different (Kruskal-Wallis test, P = 0.08, NS) but did reveal that the HGV+ population had been transfused more often. Moreover, all but one of the patients who were HGV+ before graft, had had hematological diseases which needed heavy transfusion protocols suggesting, a role of blood products in HGV transmission. Fifty out of the 95 patients received Gammagard intravenous immunoglobulin (i.v.IG) batches suspected of having transmitted HCV. However, no significant difference appeared between these recipients and those receiving other i.v.IG. Despite their immunodeficiency, no clinical or biological evidence of liver disease potentially linked to HGV infection has as yet been observed. The clinical outcome, in terms of acute GVHD, chronic GVHD or veno-occlusive disease was similar in HGV+ and HGV- recipients suggesting the absence of adverse effects of HGV infection on the early outcome of allogenic BMT. Long-term evolution remains to be prospectively studied.

Posttransplantation lymphoproliferative disorders in solid organ recipients are predominantly aggressive tumors of host origin.

Patients immunosuppressed after organ transplantation have an increased frequency of lymphoproliferative disorders, known as posttransplantation lymphoproliferative disorders (PTLDs). In recipients of bone marrow allografts. PTLDs are often of donor origin. In only a few cases of lymphoma arising in solid-organ transplant recipients has the origin from host or donor lymphocytes been established. The authors have analyzed 11 cases of PTLD from Massachusetts General Hospital, arising in seven male and four female patients, aged 8 to 63, five with renal, four with cardiac, and two with hepatic allografts. Using the polymerase chain reaction (PCR) to investigate genetic polymorphism at the D4S174 locus on chromosome 4, the Rb1.20 locus on chromosome 13, and the D19S178 locus on chromosome 19, only one tumor (previously reported) was of donor origin, whereas 10 were of host origin. Follow-up revealed that six patients died of PTLD, one was alive with recurrent PTLD, and four were alive and well or had died of other causes, including the patient with donor-origin PTLD. Based on these cases and on a review of previously reported cases, the authors conclude that the majority of PTLDs in solid organ recipients are of host origin. There appears to be a trend toward a greater likelihood of persistent or recurrent PTLD among solid organ recipients with host-origin tumors than among those with donor-origin tumor.

Immunohistological detection of human herpes virus 6 in formalin‐fixed, paraffin‐embedded lung tissues

Human herpes virus type 6 (HHV-6) infection is widespread in healthy individuals. The only definite disease association is with exanthem subitum in infants though the virus has been linked with a variety of other diseases including interstitial pneumonitis in bone marrow allograft recipients. In order to investigate the role of HHV-6 in the latter disease we have developed an optimised staining method for the demonstration of specific antigen in routinely processed post-mortem tissues. Formalin-fixed, paraffin-embedded lung tissue from 8 immunocompromised patients who died from interstitial pneumonitis was subjected to immunoperoxidase staining with monoclonal antibodies against HHV-6, cytomegalovirus (CMV), and adenovirus, using a modified avidin-biotin complex (ABC) method. Staining for HHV-6 was obtained in 6 of the 8 patients studied and was present in pneumocytes and macrophages. CMV and adenovirus antigens were identified in 4 and 6 patients, respectively. Whilst the lung tissue of 6 patients contained more than one virus, there was no evidence of cross-reactivity between the monoclonal antibodies. We demonstrated that accurate localisation of HHV-6 using monoclonal antibodies is possible in post-mortem lung tissue and conclude that either HHV-6 alone or in combination with other viruses may play a role in the development of interstitial pneumonitis following bone marrow transplantation or chemotherapy.

Induction of donor-type chimerism in murine recipients of bone marrow allografts by different radiation regimens currently used in treatment of leukemia patients

Three radiation protocols currently used in treatment of leukemia patients before bone marrow transplantation (BMT) were investigated in a murine model (C57BL/6----C3H/HeJ) for BM allograft rejection. These include (a) a single dose of total body irradiation (8.5 Gy TBI delivered at a dose rate of 0.2 Gy/min), (b) fractionated TBI (12 Gy administered in six fractions, 2 Gy twice a day in 3 days, delivered at a dose rate of 0.1 Gy/min, and (c) hyperfractionated TBI (14.4 Gy administered in 12 fractions, 1.2 Gy three times a day in 3 days, delivered at a dose rate of 0.1 Gy/min). Donor-type chimerism 6 to 8 weeks after BMT and hematologic reconstitution on day 12 after BMT found in these groups were compared with results obtained in mice conditioned with 8 Gy TBI delivered at a dose rate of 0.67 Gy/min, routinely used in this murine model. The results in both parameters showed a marked advantage for the single dose 8.5 Gy TBI over all the other treatments. This advantage was found to be equivalent to three- to fourfold increment in the BM inoculum when compared with hyperfractionated radiation, which afforded the least favorable conditions for development of donor-type chimerism. The fractionated radiation protocol was equivalent in its efficacy to results obtained in mice irradiated by single-dose 8 Gy TBI, both of which afforded a smaller but not significant advantage over the hyperfractionated protocol. This model was also used to test the effect of radiation dose rate on the development of donor-type chimerism. A significant enhancement was found after an increase in dose rate from 0.1 to 0.7 Gy/min. Further enhancement could be achieved when the dose rate was increased to 1.3 Gy/min, but survival at this high dose rate was reduced. These results demonstrated indirectly that dose rate affects the expression of host-type pluripotent stem cells, the progeny of which appear 3 to 6 weeks after treatment with 8 Gy TBI delivered at a dose rate of 0.1 Gy/min, but which are eradicated if radiation is delivered at a dose rate of 1.3 Gy/min.

Induction of Donor-Type Chimerism in Murine Recipients of Bone Marrow Allografts by Different Radiation Regimens Currently Used in Treatment of Leukemia Patients

Three radiation protocols currently used in treatment of leukemia patients before bone marrow transplantation (BMT) were investigated in a murine model (C57BL/6----C3H/HeJ) for BM allograft rejection. These include (a) a single dose of total body irradiation (8.5 Gy TBI delivered at a dose rate of 0.2 Gy/min), (b) fractionated TBI (12 Gy administered in six fractions, 2 Gy twice a day in 3 days, delivered at a dose rate of 0.1 Gy/min, and (c) hyperfractionated TBI (14.4 Gy administered in 12 fractions, 1.2 Gy three times a day in 3 days, delivered at a dose rate of 0.1 Gy/min). Donor-type chimerism 6 to 8 weeks after BMT and hematologic reconstitution on day 12 after BMT found in these groups were compared with results obtained in mice conditioned with 8 Gy TBI delivered at a dose rate of 0.67 Gy/min, routinely used in this murine model. The results in both parameters showed a marked advantage for the single dose 8.5 Gy TBI over all the other treatments. This advantage was found to be equivalent to three- to fourfold increment in the BM inoculum when compared with hyperfractionated radiation, which afforded the least favorable conditions for development of donor-type chimerism. The fractionated radiation protocol was equivalent in its efficacy to results obtained in mice irradiated by single-dose 8 Gy TBI, both of which afforded a smaller but not significant advantage over the hyperfractionated protocol. This model was also used to test the effect of radiation dose rate on the development of donor-type chimerism. A significant enhancement was found after an increase in dose rate from 0.1 to 0.7 Gy/min. Further enhancement could be achieved when the dose rate was increased to 1.3 Gy/min, but survival at this high dose rate was reduced. These results demonstrated indirectly that dose rate affects the expression of host-type pluripotent stem cells, the progeny of which appear 3 to 6 weeks after treatment with 8 Gy TBI delivered at a dose rate of 0.1 Gy/min, but which are eradicated if radiation is delivered at a dose rate of 1.3 Gy/min.

Cytogenetic studies of leukemic recurrence in recipients of bone marrow allografts

Cytogenetic studies were made in 20 leukemic patients who relapsed after treatment by allogeneic bone marrow transplantation (BMT). Seven of the eight patients in whom no chromosomal abnormalities were detected in leukemic cells before BMT developed clonal abnormalities after BMT, and in two of these patients two independent clones were observed. Most patients in whom clones were detected before BMT showed evidence of clonal evolution after BMT. Nonclonal abnormalities were also observed in clonal cells. These additional abnormalities, both clonal and nonclonal, were attributed to the effects of total body irradiation received by the patient before BMT. There was no evidence of recurrence of leukemia in donor cells among these patients. We concluded that the cells found in leukemic recurrence were derived from the original leukemic clone.

Ganciclovir treatment of cytomegalovirus disease in immunocompromised children

Twelve immunocompromised children were treated with 13 courses of intravenously administered ganciclovir for severe cytomegalovirus disease. All children were allograft recipients; 6 received organ transplants (5 liver, 1 kidney) and 6 received bone marrow. They presented with one or more of the following forms of cytomegalovirus disease: pneumonitis, 9; hepatitis, 3; colitis, 2; peritonitis, 1; and retinitis, 1. Clinical improvement was observed in 7 (58%) of 12 patients during ganciclovir therapy. Cessation of active viral replication during therapy accompanied 69% of the treatment courses. Mild and transient increases in creatinine and liver function tests and/or decreases in neutrophil count accompanied 77% of treatment courses but neutropenia (less than 1000 cells/mm3) did not occur. Transient decreases in platelet counts accompanied therapy in 3 bone marrow allograft recipients, but greater than 50% decrease in lymphocyte count was not seen. We conclude that ganciclovir is safe and appears to have a beneficial effect on cytomegalovirus disease in some pediatric transplant recipients.

Enhancement by dimethyl myleran of donor type chimerism in murine recipients of bone marrow allografts

A major problem in using murine models for studies of bone marrow allograft rejection in leukemia patients is the narrow margin in which graft rejection can be analyzed. In mice irradiated with greater than 9 Gy total body irradiation (TBI) rejection is minimal, whereas after administration of 8 Gy TBI, which spares a significant number of clonable T cells, a substantial frequency of host stem cells can also be detected. In current murine models, unlike in humans, bone marrow allograft rejection is generally associated with full autologous hematopoietic reconstitution. In the present study, we investigated the effect of the myeloablative drug dimethyl myleran (DMM) on chimerism status following transplantation of T cell-depleted allogenic bone marrow (using C57BL/6 donors and C3H/HeJ recipients, conditioned with 8 Gy TBI). Donor type chimerism 1 to 2 months post-transplant of 1 to 3 x 10(6) bone marrow cells was markedly enhanced by using DMM one day after TBI and prior to transplantation. Conditioning with cyclophosphamide instead of DMM, in combination with 8 Gy TBI, did not enhance engraftment of donor type cells. Artificial reconstitution of T cells, after conditioning with TBI plus DMM, by adding mature thymocytes, or presensitization with irradiated donor type spleen cells 1 week before TBI and DMM, led to strong graft rejection and consequently to severe anemia. The anti-donor responses in these models were proportional to the number of added T cells and to the number of cells used for presensitization, and they could be neutralized by increasing the bone marrow inoculum. These results demonstrate the potential of DMM to facilitate engraftment in unsensitized mice in which the host stem cells may compete with donor type cells; the use of DMM to create models in which mechanisms of immune rejection can be studied without interference due to stem cell competition; and that bone marrow allograft rejection may be overcome by increasing the bone marrow inoculum in these stringent models.

Enhancement by Dimethyl Myleran of Donor Type Chimerism in Murine Recipients of Bone Marrow Allografts

A major problem in using murine models for studies of bone marrow allograft rejection in leukemia patients is the narrow margin in which graft rejection can be analyzed. In mice irradiated with greater than 9 Gy total body irradiation (TBI) rejection is minimal, whereas after administration of 8 Gy TBI, which spares a significant number of clonable T cells, a substantial frequency of host stem cells can also be detected. In current murine models, unlike in humans, bone marrow allograft rejection is generally associated with full autologous hematopoietic reconstitution. In the present study, we investigated the effect of the myeloablative drug dimethyl myleran (DMM) on chimerism status following transplantation of T cell-depleted allogenic bone marrow (using C57BL/6 donors and C3H/HeJ recipients, conditioned with 8 Gy TBI). Donor type chimerism 1 to 2 months post-transplant of 1 to 3 x 10(6) bone marrow cells was markedly enhanced by using DMM one day after TBI and prior to transplantation. Conditioning with cyclophosphamide instead of DMM, in combination with 8 Gy TBI, did not enhance engraftment of donor type cells. Artificial reconstitution of T cells, after conditioning with TBI plus DMM, by adding mature thymocytes, or presensitization with irradiated donor type spleen cells 1 week before TBI and DMM, led to strong graft rejection and consequently to severe anemia. The anti-donor responses in these models were proportional to the number of added T cells and to the number of cells used for presensitization, and they could be neutralized by increasing the bone marrow inoculum. These results demonstrate the potential of DMM to facilitate engraftment in unsensitized mice in which the host stem cells may compete with donor type cells; the use of DMM to create models in which mechanisms of immune rejection can be studied without interference due to stem cell competition; and that bone marrow allograft rejection may be overcome by increasing the bone marrow inoculum in these stringent models.

Ganciclovir and Immune Globulin in Cytomegalovirus Pneumonia

Excerpt To the editor:In two recent trials, the mortality from cytomegalovirus pneumonia was lower in recipients of bone marrow allografts given combined ganciclovir and cytomegalovirus immune glob...

Cytomegalovirus hyperimmunoglobulin and substitution with blood products from antibody-negative donors. A pilot study in bone marrow transplant recipients.

A major cause of death after allogeneic mar­row transplantation is interstitial pneumoni­tis (IP), frequently associated with cytomegalovirus (CMV) infection [1]. It is presumed that most CMV infections are either acquired by transfusion of blood products or represent reactivation of a latent endogenous virus. Because the prospects for treatment of CMV are relatively remote, attention has centered on prevention. Recently, controlled trials of passive immunization for the prevention of CMV disease in bone marrow allograft recipients have been done [2–4].

Correlation between donor cytomegalovirus immunity and chronic graft-versus-host disease after allogeneic bone marrow transplantation.

Chronic graft-versus-host disease (GvHD) in bone marrow allograft recipients is frequently preceded by a cytomegalovirus (CMV) infection. To elucidate whether an immune reaction of transplanted donor cells against CMV was involved in the pathogenesis of chronic GvHD, the effect of donor pretransplant CMV immune status on chronic GvHD incidence was analyzed. In 43 bone marrow recipients at risk, the 2-yr cumulative chronic GvHD probability was 55% when the donor was immune, in contrast to 16.5% when the donor was non-immune (p less than 0.002). No correlation between recipient pretransplant CMV immunity and chronic GvHD was observed. Donor CMV immunity did not correlate with acute GvHD or posttranplant CMV infection and seemed to predispose for chronic GvHD regardless of donor and recipient age. However, the proportion of CMV immune donors increased with increasing donor and recipient age. This may account for the higher incidence of chronic GvHD in older patients. The present study suggests that chronic GvHD may be mediated by a reaction of immune donor cells against CMV infected recipient cells.

Virological and serological diagnosis of cytomegalovirus infection in bone marrow allograft recipients

To detect cytomegalovirus (CMV) infections, a total of 1,074 cultures of urine, saliva, or blood were collected weekly from 43 consecutive patients undergoing allogeneic bone marrow transplantation. Twenty-three patients were seronegative before transplant and primary infection occurred in 2 (9%). Twenty patients were initially seropositive and recurrent infections occurred in 5 (25%). Three patients in the recurrent group had proven CMV pneumonitis; viraemia was detected in two recipients, while the third had CMV isolated only from bronchial lavage fluid. The serological response of the 43 patients was defined by testing 559 serial sera for specific IgG and IgM antibodies by radioimmunoassay. Passive acquisition of IgG antibodies from blood products was found in 78% of initially seronegative recipients. One patient with primary infection responded in a pattern typical of immunocompetent individuals with long-term production of specific IgG and transient production of specific IgM antibodies. The second patient also had a typical response, but this was delayed until several weeks after the start of virus excretion. In patients with recurrent infections, specific IgM production did not correlate with episodes of virus excretion. Three of five such patients failed to mount a specific IgM response, and these were the only patients in the study to develop CMV pneumonitis. We conclude that CMV infection in bone marrow recipients can only be diagnosed by detection of virus; therefore, the ability of these patients to mount humoral immune responses should not be relied upon for diagnostic purposes.

Human bone marrow allograft recipients: production of, and responsiveness to, interleukin 2.

The production and utilization of interleukin 2 (IL 2) by peripheral blood mononuclear cells (PBMC) from 14 bone marrow allograft recipients was examined. PBMC from all patients were impaired in their ability to produce IL 2 when stimulated by phytohemagglutinin (PHA). On the average, the IL 2 activity produced by patients' PBMC was 8% of that from normal donors' PBMC. To examine the basis for this impaired T cell function in marrow recipients, the ability of a resting T lymphocyte population isolated from PBMC to respond to PHA and exogenously supplied IL 2 was analyzed. The proliferative response of resting T cells to PHA and IL 2, although low at early times post-transplant, reached near normal levels by 8 mo. Only two of 11 patients had normal numbers of precursor T cells that could respond. For all other patients the average number of precursor T cells was 10-fold lower than the average determined for normal donors. The impaired production of IL 2 by patients' PBMC may be due to this low precursor frequency. For some patients the rate and/or extent of clonal expansion of activated T cells appears to be greater than that of normal donors. The data suggest that the therapeutic application of IL 2 to such patients is unlikely to be successful in overcoming defects of T cell function before 8 mo post-marrow transplant.