Graves-Basedow disease after allogeneic bone marrow transplantation

One severe aplastic anaemia case who presented autoimmune thyroid disease after allogeneic bone marrow transplantation (alloBMT) is described. A 19 year old Polish girl developed Graves' hyperthyroidisms 19 months after allogeneic BMT for severe aplastic anaemia (SAA) donated from her brother. Her serum was positive for thyroid stimulating antibody (TSAb) and anti-thyroid peroxidase autoantibodies (aTPO) while her brother remained euthyroid, seronegative for TSAb, and showed no clinical signs of thyroid pathology. The genetic studies of lymphocytes FISH (fluorescence in situ hybridization) and analysis of STR (short tandem repeated) fragments suggested, that lymphocytes responsible for hyperthyroidisms were of donor origin.     

Graft-versus-host disease in experimental allogeneic bone marrow transplantation

Considerable progress has been made in defining the relative contributions of CD4+ and CD8+ cells to GVHD. Studies in mice have shown that, in isolation, each T cell subset is able to induce lethal GVHD in irradiated hosts. For hosts differing at the MHC (H-2), CD4+ cells cause GVHD directed to H-2 class II antigens whereas CD8+ cells produce GVHD to H-2 class I antigens. With H-2-matched hosts expressing multiple minor H antigens, induction of lethal GVHD is largely under the control of CD8+ cells. Which particular minor H antigens provide the targets for GVHD in mice is still unclear. Clarifying this question is complicated by the finding that the target antigens for GVHD do not necessarily correlate with the targets for cytotoxicity measured in vitro; moreover, immunodominance occurs when T cells are exposed to multiple minor H antigens in vivo. In terms of clinical application, there is a need to devise animal models for improving the success of HLA-matched bone marrow transplantation. Selectively depleting the marrow inoculum of CD8+ cells and preimmunizing the donor against viral pathogens are two procedures which are currently under study.     

Treatment of Niemann-Pick disease type B by allogeneic bone marrow transplantation.

Allogenic bone marrow transplantation was carried out on a 3 year old girl with Niemann-Pick disease type B. Successful engraftment was achieved, and nine months after the procedure there was definite clearing of the sphingomyelin from the liver and pronounced clearing from the bone marrow. Any patient with Niemann-Pick disease type B complicated by early or severe hepatic impairment should be considered for bone marrow transplantation.     

Elimination of leukemia in the absence of lethal graft-versus-host disease after allogenic bone marrow transplantation

Donor T cells are able to effect a graft-vs-leukemia (GVL) response but also induce graft-vs-host disease (GVHD) after allogeneic bone marrow transplantation. We used an AKR leukemia murine transplant model, analogous to human acute lymphoblastic leukemia, in which donor T cells expressed a thymidine kinase suicide gene, to test whether separation of GVL and graft-vs-host (GVH) responses was feasible by selectively eliminating alloactivated donor T cells at defined time points posttransplant. Under experimental conditions where untreated mice could not be cured of disease without dying from GVHD, mice transplanted with thymidine kinase-positive T cells and subsequently administered ganciclovir (GCV) could eliminate leukemia without lethal GVHD. Timing of GCV administration, donor T cell dose, and preexisting leukemia burden were observed to be critical variables. Eradication of leukemia without lethal GVHD in GCV-treated mice implied that the kinetics of GVL and GVH responses were asynchronous and could therefore be temporally dissociated by timely GCV administration. That this strategy was feasible in a murine leukemia model in which GVHD and GVL reactivity are tightly linked suggests that this approach may be relevant to the treatment of selected human leukemias where similar constraints exist. This strategy represents an alternative approach to separating GVL and GVH reactivity and challenges the current paradigm that separation of these responses is dependent upon the administration of donor T cells with restricted specificity for leukemia as opposed to host Ags.     

Kinetics of in vivo elimination of suicide gene-expressing T cells affects engraftment, graft-versus-host disease, and graft-versus-leukemia after allogeneic bone marrow transplantation

Suicide gene therapy is one approach being evaluated for the control of graft-vs-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). We recently constructed a novel chimeric suicide gene in which the entire coding region of HSV thymidine kinase (HSV-tk) was fused in-frame to the extracellular and transmembrane domains of human CD34 (DeltaCD34-tk). DeltaCD34-tk is an attractive candidate as a suicide gene in man because of the ensured expression of HSV-tk in all selected cells and the ability to rapidly and efficiently purify gene-modified cells using clinically approved CD34 immunoselection techniques. In this study we assessed the efficacy of the DeltaCD34-tk suicide gene in the absence of extended ex vivo manipulation by generating transgenic animals that express DeltaCD34-tk in the peripheral and thymic T cell compartments using the CD2 locus control region. We found that DeltaCD34-tk-expressing T cells could be purified to near homogeneity by CD34 immunoselection and selectively eliminated ex vivo and in vivo when exposed to low concentrations of GCV. The optimal time to administer GCV after allogeneic BMT with DeltaCD34-tk-expressing transgenic T cells was dependent on the intensity of the conditioning regimen, the leukemic status of the recipient, and the dose and timing of T cell infusion. Importantly, we used a controlled graft-vs-host reaction to promote alloengraftment in sublethally irradiated mice and provide a graft-vs-leukemia effect in recipients administered a delayed infusion of DeltaCD34-tk-expressing T cells. This murine model demonstrates the potential usefulness of DeltaCD34-tk-expressing T cells to control GVHD, promote alloengraftment, and provide a graft-vs-leukemia effect in man.     

Dissociation of hemopoietic chimerism and allograft tolerance after allogeneic bone marrow transplantation.

Creation of stable hemopoietic chimerism has been considered to be a prerequisite for allograft tolerance after bone marrow transplantation (BMT). In this study, we demonstrated that allogeneic BMT with bone marrow cells (BMC) prepared from either knockout mice deficient in both CD4 and CD8 T cells or CD3E-transgenic mice lacking both T cells and NK cells maintained a high degree of chimerism, but failed to induce tolerance to donor-specific wild-type skin grafts. Lymphocytes from mice reconstituted with T cell-deficient BMC proliferated when they were injected into irradiated donor strain mice, whereas lymphocytes from mice reconstituted with wild-type BMC were unresponsive to donor alloantigens. Donor-specific allograft tolerance was restored when donor-type T cells were adoptively transferred to recipient mice given T cell-deficient BMC. These results show that donor T cell engraftment is required for induction of allograft tolerance, but not for creation of continuous hemopoietic chimerism after allogeneic BMT, and that a high degree of chimerism is not necessarily associated with specific allograft tolerance.     

Host NKT cells can prevent graft-versus-host disease and permit graft antitumor activity after bone marrow transplantation

Allogeneic bone marrow transplantation is a curative treatment for leukemia and lymphoma, but graft-vs-host disease (GVHD) remains a major complication. Using a GVHD protective nonmyeloablative conditioning regimen of total lymphoid irradiation and antithymocyte serum (TLI/ATS) in mice that has been recently adapted to clinical studies, we show that regulatory host NKT cells prevent the expansion and tissue inflammation induced by donor T cells, but allow retention of the killing activity of donor T cells against the BCL1 B cell lymphoma. Whereas wild-type hosts given transplants from wild-type donors were protected against progressive tumor growth and lethal GVHD, NKT cell-deficient CD1d-/- and Jalpha-18-/- host mice given wild-type transplants cleared the tumor cells but died of GVHD. In contrast, wild-type hosts given transplants from CD8-/- or perforin-/- donors had progressive tumor growth without GVHD. Injection of host-type NKT cells into Jalpha-18-/- host mice conditioned with TLI/ATS markedly reduced the early expansion and colon injury induced by donor T cells. In conclusion, after TLI/ATS host conditioning and allogeneic bone marrow transplantation, host NKT cells can separate the proinflammatory and tumor cytolytic functions of donor T cells.     

Hemopoietic progenitor cell function after HLA-identical sibling bone marrow transplantation: influence of chronic graft-versus-host disease

We examined hemopoietic reconstitution during the first 12 months post-transplant in 31 patients given high-dose cyclophosphamide, total body irradiation and an HLA-identical sibling marrow transplant for hematological malignancy. Unexpectedly, we found marrow CFU-gm and marrow CFU-e cells to be denser than normal throughout the first year post-transplant. While functionally adequate neutrophil and platelet counts were achieved in the first six weeks post-transplant, there were defects in hemopoietic progenitor cell function during the first year post-transplant. Although we could detect no influence from acute graft-versus-host disease (GVHD), chronic GVHD adversely affected the growth of both myeloid and erythroid blood progenitor cells.     

Serum megakaryocytopoietic activity after allogeneic bone marrow transplantation (BMT).

Platelet recovery after allogeneic bone marrow transplantation for treatment of different haematologic disorders was monitored by ability of sera from BMT recipients to support the clonal growth of normal megakaryocyte progenitors. The resultant megakaryocyte colonies were expressed as a Serum Factor Index-Megakaryocyte (SFI-Mk). In two patients with whole and in one with partial haematological reconstitution SFI-Mk was normal, and in others SFI-Mk value was increased. Our results indicate that sera from BMT recipients contain megakaryocytopoietic activity which inversely correlate with recovery of megakarycytic lineage following bone marrow transplantation.     

NK cells in allogeneic bone marrow transplantation

NK cells, until recently an ignored subset of lymphocytes, have begun to emerge as important cytotoxic effectors. It is now accepted that NK cells together with T cells constitute major actors in graft-versus-leukemia reaction after allogeneic bone marrow transplantation (BMT). Over the last several years the mechanisms regulating the activation of NK cells have been the subject of intense investigations encouraged by the clinical implications that these studies will have. This article provides a general overview of NK-cells biology and regulation pertinent to their function in allogeneic BMT, followed by a review of the in vivo preclinical and clinical evidence for the beneficial effect of NK cells in the adoptive immunotherapy of leukemia.     

Immune reconstitution in patients with Fanconi anemia after allogeneic bone marrow transplantation

Background aimsFanconi anemia is an autosomal recessive or X-linked genetic disorder characterized by bone marrow (BM) failure/aplasia. Failure of hematopoiesis results in depletion of the BM stem cell reservoir, which leads to severe anemia, neutropenia and thrombocytopenia, frequently requiring therapeutic interventions, including hematopoietic stem cell transplantation (HSCT). Successful BM transplantation (BMT) requires reconstitution of normal immunity.MethodsIn the present study, we performed a detailed analysis of the distribution of peripheral blood subsets of T, B and natural killer (NK) lymphocytes in 23 patients with Fanconi anemia before and after BMT on days +30, +60, +100, +180, +270 and +360. In parallel, we evaluated the effect of related versus unrelated donor marrow as well as the presence of graft-versus-host disease (GVHD).ResultsAfter transplantation, we found different kinetics of recovery for the distinct major subsets of lymphocytes. NK cells were the first to recover, followed by cytotoxic CD8⁺ T cells and B cells, and finally CD4⁺ helper T cells. Early lymphocyte recovery was at the expense of memory cells, potentially derived from the graft, whereas recent thymic emigrant (CD31⁺ CD45RA⁺) and naive CD4⁺ or CD8⁺ T cells rose only at 6 months after HSCT, in the presence of immunosuppressive GVHD prophylactic agents. Only slight differences were observed in the early recovery of cytotoxic CD8⁺ T cells among those cases receiving a graft from a related donor versus an unrelated donor. Patients with GVHD displayed a markedly delayed recovery of NK cells and B cells as well as of regulatory T cells and both early thymic emigrant and total CD4⁺ T cells.ConclusionsOur results support the utility of post-transplant monitoring of a peripheral blood lymphocyte subset for improved follow-up of patients with Fanconi anemia undergoing BMT.     

Serum immunoglobulin levels following allogeneic bone marrow transplantation

In order to study the posttransplant evolution of serum immunoglobulin levels, we measured serum IgG, IgA and IgM levels in 50 recipients of allogeneic bone marrow before transplantation and at different intervals thereafter (days 39, 120, 365 and 730). IgG and IgM levels were depressed for 1 year and IgA levels for 2 years posttransplant. Immunoglobulin deficiency was more severe and prolonged in patients with graft versus-host-disease. Hypogammaglobulinemia may contribute to the frequent infections observed in these patients, especially those with chronic graft-versus-host disease.     

T-cell depletion of allogeneic bone marrow prevents acceleration of graft-versus-host disease induced by exogenous interleukin 2

Highly purified human recombinant interleukin 2 (IL-2) markedly accelerated lethal GVHD in the H-2-identical B10.BR----CBA combination, but had no effect when the donor cells were depleted of mature (Thy-1.2-positive) T lymphocytes, indicating a strong immunopotentiating effect of IL-2 on mature T cells causing GVHD. In the same donor-host combination, IL-2 did not influence the recovery from the post-transplantation bone marrow aplasia. The results suggest that IL-2 could be considered for adjuvant hormonal therapy to enhance immune recovery in recipients of T-cell-depleted allogeneic marrow.     

Inflammatory cytokines and dendritic cells in acute graft-versus-host disease after allogeneic stem cell transplantation

The wider use of allogeneic stem cell transplantation (allo-SCT) is still limited by the immunologic recognition and destruction of host tissues, termed graft-versus-host disease (GVHD). The role of inflammatory cytokines such as TNF-alpha and IL-1, and their impact on immune effectors (mainly CD4+ and CD8+ T) cells has been extensively studied in the context of GVHD occurring after standard myeloablative allo-SCT. However, recent data suggested that GVHD pathophysiology is likely to involve more complex interactions where antigen-presenting cells, especially dendritic cells (DCs), may play a major role at time of initiation of acute GVHD. In addition, the wider use of reduced intensity and less toxic conditioning (RIC) regimens prior to allo-SCT would allow better visualization of the fine functions of immune effectors, thereby offering a window of opportunities to better decipher the intimate pathophysiological mechanisms underlying GVHD. The aim of this work is to review the available research evidence on the role of DCs as in vivo regulators of alloimmune reactivity, and their interactions with other immune effectors.     

Ceacam1 separates graft-versus-host-disease from graft-versus-tumor activity after experimental allogeneic bone marrow transplantation

Background

Allogeneic bone marrow transplantation (allo-BMT) is a potentially curative therapy for a variety of hematologic diseases, but benefits, including graft-versus-tumor (GVT) activity are limited by graft-versus-host-disease (GVHD). Carcinoembryonic antigen related cell adhesion molecule 1 (Ceacam1) is a transmembrane glycoprotein found on epithelium, T cells, and many tumors. It regulates a variety of physiologic and pathological processes such as tumor biology, leukocyte activation, and energy homeostasis. Previous studies suggest that Ceacam1 negatively regulates inflammation in inflammatory bowel disease models.

Methods

We studied Ceacam1 as a regulator of GVHD and GVT after allogeneic bone marrow transplantation (allo-BMT) in mouse models. In vivo, Ceacam1^−/− T cells caused increased GVHD mortality and GVHD of the colon, and greater numbers of donor T cells were positive for activation markers (CD25^hi, CD62L^lo). Additionally, Ceacam1^−/− CD8 T cells had greater expression of the gut-trafficking integrin α₄β₇, though both CD4 and CD8 T cells were found increased numbers in the gut post-transplant. Ceacam1^−/− recipients also experienced increased GVHD mortality and GVHD of the colon, and alloreactive T cells displayed increased activation. Additionally, Ceacam1^−/− mice had increased mortality and decreased numbers of regenerating small intestinal crypts upon radiation exposure. Conversely, Ceacam1-overexpressing T cells caused attenuated target-organ and systemic GVHD, which correlated with decreased donor T cell numbers in target tissues, and mortality. Finally, graft-versus-tumor survival in a Ceacam1⁺ lymphoma model was improved in animals receiving Ceacam1^−/− vs. control T cells.

Conclusions

We conclude that Ceacam1 regulates T cell activation, GVHD target organ damage, and numbers of donor T cells in lymphoid organs and GVHD target tissues. In recipients of allo-BMT, Ceacam1 may also regulate tissue radiosensitivity. Because of its expression on both the donor graft and host tissues, this suggests that targeting Ceacam1 may represent a potent strategy for the regulation of GVHD and GVT after allogeneic transplantation.     

Acute graft-versus-host disease-challenge for a broader application of allogeneic hematopoietic cell transplantation

Allogeneic hematopoietic cell transplanation (aHCT) has been a successful treatment option for malignant disease based on the graft-versus-tumor effect. However, the overall clinical success of aHCT is impaired by the high morbidity and mortality caused by acute graft-versus-host disease (aGVHD). aGVHD can also be seen as the major limitation of aHCT for a broader clinical applicability of this treatment, particularly for non-malignant disease conditions. Recent murine studies have shed more light on the kinetics of aGVHD development by tracking donor T cells in vivo. These data define two functionally distinct stages in aGVHD pathogenesis taking place in different anatomical compartments. The aGVHD initiation phase is confined to T cell areas in secondary lymphoid organs in contrast to the later aGVHD effector phase at target sites. This temporal pattern may explain the clinical observation that when acute aGVHD is clinically overt, treatment with intensified immunosuppression often remains ineffective. This review will focus on the immune-pathogenesis of aGVHD, conventional and novel treatment strategies including the removal of na?ve T cells, tolerance induction by mesenchymal stem cells, regulatory T cells, genetic manipulation of donor cells and the potential of memory T cells for improving immune reconstitution without aGVHD. A better understanding of the mechanisms involved in aGVHD pathogenesis might allow for a broader application of novel stem cell therapies.     

Myeloperoxidase deficiency enhances inflammation after allogeneic marrow transplantation

Myeloperoxidase (MPO)-derived oxidants participate in the respiratory antimicrobial defense system but are also implicated in oxidant-mediated acute lung injury. We hypothesized that MPO contributes to lung injury commonly observed after bone marrow transplantation (BMT). MPO-sufficient (MPO+/+) and -deficient (MPO-/-) mice were given cyclophosphamide and lethally irradiated followed by infusion of inflammation-inducing donor spleen T cells at time of BMT. Despite suppressed generation of nitrative stress, MPO-/- recipient mice unexpectedly exhibited accelerated weight loss and increased markers of lung dysfunction compared with MPO+/+ mice. The increased lung injury during MPO deficiency was a result of donor T cell-dependent inflammatory responses because bronchoalveolar lavage fluids (BALF) from MPO-/- mice contained increased numbers of inflammatory cells and higher levels of the proinflammatory cytokine TNF-alpha and the monocyte chemoattractant protein-1 compared with wild-type mice. Enhanced inflammation in MPO-/- mice was associated with suppressed apoptosis of BALF inflammatory cells. The inflammatory process in MPO-/- recipients was also associated with enhanced necrosis of freshly isolated alveolar type II cells, critical for preventing capillary leak. We conclude that suppressed MPO-derived oxidative/nitrative stress is associated with enhanced lung inflammation and persistent alveolar epithelial injury.     

Estimating the size of the dividing stem cell pool after allogeneic bone marrow transplantation

Currently the problem of estimation of the number of pluripotent stem cells reconstituting marrow grafts following bone marrow transplantation, or studies looking at questions of clonal dominance in hematopoietic cell populations, rely on indirect measurement and a simple application of the formula for the sampling variation of a binomial proportion. This approach, from a statistical viewpoint, can be seen to be flawed. It is very easily remedied though and only requires appropriate use of variance stabilizing transformations. These lead to a very simple estimator for the number of hematopoietic stem cells involved in repopulating the marrow and require little in the way of additional calculation. We give the distribution theory for this estimator as well as simple approximations for practical application. As an illustration we rework data recently gathered to address the question as to whether or not reconstitution of marrow grafts in the clinical setting is oligoclonal.