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.     

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.     

Dynamics of bone marrow changes in patients with chronic idiopathic myelofibrosis following allogeneic stem cell transplantation

Scant knowledge exists about the dynamics of fibro-osteosclerotic bone marrow (BM) lesions and regeneration of hematopoiesis following allogeneic peripheral stem cell transplantation (SCT) in chronic idiopathic myelofibrosis. Therefore, an immunohistochemical and morphometric study was performed on BM biopsies in 20 patients before and at standardized intervals (days 30 through 384) following SCT. In responding patients, a total regression of the pretransplant increased fibrosis was completed in the posttransplant period after about six months, while the extent of osteosclerosis did not change significantly during observation time. The quantity of CD61+ megakaryocytes including precursors was strikingly variable after SCT and, by using planimetric methods, atypical microforms exhibiting a dysplastic aspect could be demonstrated. These anomalies may be responsible for posttransplant thrombocytopenia. CD34+ progenitor cells were increased before transplantation, however, their number declined rapidly to normal values in responding patients. Nucleated erythroid precursors revealed a decreased amount before and after SCT accounting for anemia. Large clusters of this cell lineage indicated an initial hematopoietic reconstitution comparable with the expansion of the neutrophil granulopoiesis. Proliferative activity and apoptosis showed an increase until one year after SCT that implied a still regenerating hematopoiesis in keeping with an enhanced cell turnover.     

The abrogation of allosensitization following the induction of mixed allogeneic chimerism

The association of preformed anti-donor Abs with the hyperacute rejection of bone marrow and solid organ allografts and the persistence of the anti-donor immune response secondary to immunologic memory make allosensitization an absolute contraindication to transplantation. Mixed allogeneic (A + B-->A) bone marrow chimerism has been demonstrated to confer donor-specific tolerance in nonsensitized recipients, but has not been evaluated in the setting of allosensitization. The current study documents that despite significant anti-donor sensitization, mixed allogeneic engraftment is possible and provides a marked advantage over fully allogeneic (B-->A) models. Moreover, the acceptance of donor skin grafts and loss of circulating anti-donor Abs suggest that allosensitization can be abrogated with the induction of stable mixed allogeneic chimerism.     

Development of a single probe for documentation of chimerism following bone marrow transplantation.

Although numerous genetic markers are available for studying chimerism after bone marrow transplantation (BMT), there remains a need for a practical and highly informative method that is applicable in the early posttransplantation period. Using DNA restriction-fragment-length polymorphisms (RFLPs), we have evaluated the feasibility of developing a single synthetic oligonucleotide probe to study post-BMT chimerism. We have thus tested three candidate probes, termed O-3315-32, O-3315-80, and O-AY-29, that are homologous to tandemly repetitive sequences. Our results demonstrated donor-specific and recipient-specific fragments in 11 of 11 HLA-matched sibling pairs tested using probes O-3315-32 and O-3315-80. When probe O-AY-29 was used, 14 of 17 sibling pairs showed both donor and recipient markers, one had only a recipient marker, and two were identical. We showed that each of the three synthetic probes was effective in documenting donor marrow engraftment, mixed hematopoietic chimerism, the patient's pre-BMT phenotype (by using cultured skin fibroblasts obtained after BMT), and the origin of the malignant hematopoietic cells (i.e., of donor or recipient origin) in patients who developed recurrent hematologic malignancy following BMT. Compared with the use of cloned genomic probes, there are several important advantages to the use of synthetic oligonucleotide probes in studying post-BMT chimerism. Synthetic probes have absolute hybridization specificity and can be designed to suit the purposes of an individual study, since they have adjustable specificity that can be altered by changes in the length of the probe and by changes in the hybridization temperature. A single synthetic probe analogous to several highly polymorphic loci can have a polymorphism information content sufficiently high so that all but a small percentage of BMT patients could be followed easily; for example, if a probe were complementary to three highly polymorphic unlinked loci, it would discriminate approximately 98% of sibling donor/recipient pairs. This would be accomplished using only one restriction-endonuclease digestion and only one gel electrophoresis. Since other genetic markers, e.g., red blood cell antigens, immunoglobulin allotypes, and chromosome analysis, are not uniformly informative and, in some cases, cannot be used in the early posttransplantation period, the use of synthetic oligonucleotide probes for analysis of DNA RFLP is emerging as the method of choice for studies of post-BMT chimerism. This method will allow for the development of new knowledge that has not been possible with previous methods.     

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.     

Combined immunophenotyping and FISH with sex chromosome-specific DNA probes for the detection of chimerism in epidermal Langerhans cells after sex-mismatched bone marrow transplantation

 Langerhans cells (LC) of the skin represent bone marrow-derived dendritic antigen-presenting cells and are therefore important in pathophysiological processes such as rejection, graft-versus-host disease, and graft-versus-leukemia-reaction after bone marrow transplantation (BMT). For understanding of these diseases, the evaluation of the chimeric status of LC following BMT is of great interest. To analyze the sex chromosome constitution of LC in the skin, we established a modified and refined technique of combined immunophenotyping and fluorescence in situ hybridization (FISH) and investigated frozen sections of skin biopsies from nine patients after allogeneic sex-mismatched BMT and of two healthy donors for control. LC were specifically labeled using a fluorescent CD1a antibody and hybridized simultaneously with X and Y chromosome-specific DNA probes. The results of this practical application on nine leukemia patients show the appearance of donor-type LC and the persistence of host-type LC at various times (36 up to 1395 days) after sex-mismatched BMT. Complete chimerism of LC could not be detected in any case. The frequency of recipient-specific LC ranged from 7% to 92% and showed no correlation with time postgrafting. We conclude from our results of 1461 analyzed LC that combined immunophenotyping and interphase cytogenetic analysis by FISH is the method of choice for the assessment of chimerism in a particular cell type after sex-mismatched BMT. Its practical application on other tissues affected by BMT-related pathophysiological processes reveals further knowledge of the time-dependent course of chimeric patterns after BMT. Accepted: 18 July 1996     

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.     

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.     

Immune recovery following bone marrow transplantation

83 patients undergoing allogeneic or autologous BMT because of haematologic malignancies have been studied before and after transplantation at different intervals. The determinations consisted of lymphocyte counts, E-rosetting, lymphoblastic response, evaluation of serum immunoglobulin levels, skin testing, and in a smaller part of the patients surface marker studies using monoclonal antibodies of the BL-series. At first after BMT the lymphocyte and T cell counts went to normal between 4-18 weeks post transplant, about 4 weeks earlier in autologous than in allogeneic BMT. T suppressor cells showed an early increase compared to T helper cells which normalized much slower about 6 months after BMT. Lymphoblastic responses, however, tended to normal not before the second half of the first year both in autologous and allogeneic transplantation. Skin test reactivity became normal during the 2nd and 3rd year posttransplant, which was more complete in autologous than in allogeneic BMT. The IgG and IgM levels were depressed for half a year and IgA levels for 2 years. The most striking aspect was the multiphase course of lymphoblastic response in every individual patient. We suggest this to be the expression of sequential differentiation of donor lymphocytes.     

Acute graft-versus-host disease after allogeneic bone marrow transplantation.

In 25 patients receiving allogeneic bone marrow transplants methotrexate was used to prevent acute graft-versus-host disease (GVHD). Acute GVHD, grades 2 to 4, developed in only 5 (20%) of the patients. The incidence of acute GVHD in other series of recipients of bone marrow transplants has ranged from 5% to 76%. A review of the literature suggests that this variation cannot be completely accounted for by age, type of disease treated by transplantation or type of GVHD prophylaxis. However, transfusion of allogeneic lymphocytes that have not been completely inactivated by irradiation (e.g., in platelet and granulocyte preparations) and inadequate isolation-decontamination procedures may increase the probability of GVHD following bone marrow transplantation.     

The regulation of hematopoiesis following bone marrow transplantation

Allogeneic bone marrow transplantation requires that donor stem cells home to the recipient bone marrow, proliferate and differentiate under normal physiologic regulatory mechanisms. Recent observations that T cell depletion of donor bone marrow leads to a greatly increased incidence of graft failure mandate a detailed understanding of the engraftment process. Post-transplant hematopoietic deficiencies appear to be related to several sources: decreased number of stem cells, activation of donor hematopoietic suppressor cells, rejection of donor stem cells by residual recipient lymphocytes and abnormal function of accessory cells that produce hematopoietic growth factors. A better understanding of the relative roles of these factors should lead to a better understanding of engraftment as well as graft failure and its prevention.     

Status of allogeneic bone marrow transplantation in childhood in the GDR

Of 25 HLA-identical, MLC negative transplants 10 patients had acute lymphoblastic leukaemia (ALL), 8 acute nonlymphoblastic leukaemia (ANLL), 3 severe aplastic anaemia, 2 malignant histiocytosis, 1 patients neuroblastoma and 1 Fanconi anaemia. 3 HLA nonidentical, MLC positive transplants were performed, two children had malignant infantile osteopetrosis and 1 child had a severe combined immunodeficiency disease. Patients with ALL and ANLL received cyclophosphamide and single dose total body irradiation. 3 patients received fractionated TBI. The results for the allogeneic group overall indicate that the actuarial disease free survival rate is 0.62. 16 of 25 patients are in continuous complete remission (CCR) periods of 3-78 months posttransplant. All three transplanted children with severe aplastic anaemia alive disease-free for periods of 21-81 months. 10 patients with ALL were transplanted (2 in first remission for high risk ALL, 8 in second remission). 7 of 10 patients are alive and disease-free (CCR rate 0.67). 8 patients underwent BMT for ANNL while in first remission in 7 patients and in third partial remission in 1 patient. 4 of 8 patients are alive and disease-free for periods of 25-56 months (CCR rate 0.50). 1 patient with neuroblastoma stage IV survives 24 months, 1 child with Fanconi anemia died on day +25 of GVHD and septicaemia. 1 of the 2 patients transplanted for malignant histiocytosis relapsed 3 months posttransplant, 1 patient is alive and disease-free 5 months posttransplant. In none of the HLA-nonidentical and MLC positive transplantations T-cell depleted marrow engrafted.     

Bone marrow cell dose and kinetics of recovery following allogeneic marrow transplantation in man

In 50 patients transplanted for acute or chronic leukemia we studied the correlation between the number of infused bone marrow cells/kg recipient body weight and the time needed for engraftment. Engraftment was arbitrarily defined as the first day of 1 X 10(9) leukocytes/l and of 20 X 10(9) reticulocytes/l after the posttransplantation nadir. There is a negative non-linear correlation between the duration of leukopenia following marrow transplantation and the amount of transfused nucleated cells (p = 0.01). Since the incidence of infectious or hemorrhagic complications depends directly on the duration of aplasia it is justifiable to give a maximal cell dose.     

Linomide administration following bone marrow transplantation in mice

The effect of linomide, an immunomodulatory drug, on natural killer (NK) cells and T cell-dependent immune responses following syngeneic or allogeneic bone marrow transplantation (BMT) was investigated in BALB/c mice inoculated with B-cell leukemia (BCL1). Linomide given in the drinking water had no impact on graft survival or graft versus leukemia (GVL) effects. Although linomide regulates anti-self reactivity in mice with experimental and spontaneous autoimmune disorders, the anti-tumor effects induced by allogeneic donor lymphocytes were not affected. This indicates that different mechanisms regulate anti-self and anti-leukemia effects. Alternatively, linomide might affect the homing of self-reactive lymphocytes to specific target organs in autoimmune disorders, although the homing process may not be relevant to the control of leukemia by alloreactive lymphocytes.     

Somatic cell mutation in pediatric patients undergoing allogeneic bone marrow transplantation

In order to examine whether bone marrow transplantation (BMT) has genotoxic effects in vivo, mutant frequencies (Mfs) at the hypoxanthine-guanine phosphoribosyl transferase (Hprt) locus were evaluated. Thirty-seven pediatric patients who had received allogeneic BMT for various hematologic or immunologic disorders were enrolled. Nine out of the 37 patients (24.3%) were found to have Hprt-Mfs exceeding the 99% confidence limits calculated from observation of healthy controls. Among factors including gender, primary disease of the patient, donor-recipient histocompatibility relationship, age of donor, and total body irradiation as conditioning regimen, none was associated with an increased Hprt-Mf. In three patients who had chimerism in their peripheral blood after BMT, Hprt mutant clones turned out to be of donor- or recipient-origin. Mfs at the T-cell receptor (TCR) locus were examined in 28 patients. Four patients (14.3%) were found to have increased TCR-Mfs. However, there were not any patients who showed elevation of both Hprt-and TCR-Mfs. These data, taken together, suggest that BMT may cause genotoxicity in vivo in some patients.     

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.     

Sequential changes of thymocyte surface antigens with presence or absence of graft-vs-host reaction following allogeneic bone marrow transplantation

Lethally irradiated AKR mice were reconstituted with C57BL/6 bone marrow cells. Though the allogeneic marrow transplantation protected AKR recipients from acute irradiation deaths, the mice given unmanipulated marrow developed severe GVHR disease, and 80% died within 50 days. The thymus and spleen from the recipient mice, following recovery of body weight between the 10th and 20th days, gradually involuted and became miniscule after Day 30. Thymocytes from recipients were found to be entirely of donor cell type by Day 15. Thereafter, however, as the graft versus host reaction (GVHR) developed, changes in sensitivity of the thymocytes to four different alloantisera directed toward donor histocompatibility antigens (H-2^b, Thy 1.2, Lyt 1.2, and Lyt 2.2) were observed and these changes were associated with changes in antigen expression or quantity of Thy 1 antigens on the thymocytes. A different pattern of changes was observed in antigen expression on thymocytes in mice given B6 marrow cells that had been pretreated with anti-Thy 1 serum which prevented initiation of graft-vs-host disease and in the mice which received marrow not so treated and which regularly led to graft-vs-host disease. By contrast, the amount of H-2 antigen on the thymocytes from chimeras with or without GVHR was elevated equally. The mechanisms of these changes are discussed.