Bone Marrow Transplantation In Man Research Articles

Defective T‐cell stimulatory pathways in patients after allogeneic bone marrow transplantation (BMT) in man

Immunological reconstitution after allogeneic bone marrow transplantation in man is characterized by a decreased lymphocyte transformation response to various mitogens and antigens during a period of from months to years. One reason for the decreased proliferative capability could be an inverted CD4/CD8 ratio; however, the present investigation demonstrates that this is not the only explanation for the immunodeficiency, since the CD4 as well as the CD8 subset, when studied in isolation, have qualitative defects, as evidenced by a reduced response of both subsets to stimulation with PHA, anti-CD2 and anti-CD3 MABs. The reason for the qualitative defect is unknown but a distorted composition of the CD4+ as well as the CD8+ T-cell subsets is suggested by the present investigations. We also observed that the PHA response was almost completely reconstituted one year after BMT, while the PWM response was still severely affected. The present study suggests that T-cell subsets which differ in their capacity to respond to PHA and PWM have different kinetics of reconstitution after BMT.

Increased Frequencies of the CD29 and CD57 Markers and Decreased Frequency of CD45RA Within CD4+ and CD8+ Subsets after Allogeneic Bone Marrow Transplantation in Man

Two monoclonal antibodies, anti-CD45RA and anti-CD29, reciprocally divide the CD4+ and CD8+ lymphocytes into CD4+ CD45RA+, CD4+ CD29+, CD8+ CD45RA+ and CD8+ CD29+ subsets. The CD4+ CD45RA+, CD4+ CD29+ and CD8+ CD45RA+ possess suppressor-inducer, helper-inducer and suppressor-effector functions respectively. Since the role of these subsets has not been established after allogenic bone marrow transplantation we studied lymphocyte subpopulations in 12 patients 45-227 days after the procedure. The fraction of CD4+ lymphocytes was significantly (P = 0.0005) decreased to 20 +/- 9% versus 43 +/- 3% in controls. Within the CD4+ compartment, we found an increase in the fraction of CD4+ cells that co-expressed CD29 (CD29+/CD4+) to 92 +/- 10% versus 48 +/- 15% (P = 0.008) in controls and a concomitant decrease in CD45RA+/CD4+ to 16 +/- 12% versus 56 +/- 25% (P = 0.008). Patients were also noted to have an increase in the percentage of CD8+ lymphocytes to 41 +/- 5% compared to 23 +/- 4% in controls (P = 0.0004). Examination of the CD8+ subsets revealed a significant increase in the CD29+/CD8+ fraction to 97 +/- 3% versus 64 +/- 2% in controls (P = 0.008) and a decrease in the CD45RA+/CD8+ fraction to 36 +/- 11% versus 70 +/- 21% (P = 0.008). The number of cells co-expressing CD57 were also determined within the CD4+ and CD8+ subsets. In patients CD57+/CD4+ were increased to 29 +/- 7% versus 1 +/- 1% in controls (P = 0.04), and CD57+/CD8+ to 49 +/- 12% versus 23 +/- 9% (P = 0.02). Since CD29+ and CD57+ cells have a poor capability for IL-2 production and proliferation this shift in subset distribution may account for some of the defects in cellular immunity seen within the first year after allogeneic bone marrow transplantation.

Effect of thalidomide on the incidence of iodine-induced and spontaneous lymphocytic thyroiditis and spontaneous diabetes mellitus in the BB/Wor rat

Thalidomide, a derivative of glutamic acid, has immunosuppressive effects and suppresses graft-vs-host disease in the rat and following bone marrow transplantation in man. It is effectively used in the treatment of erythema nodosum leprosum and has a potential therapeutic effect in a variety of autoimmune diseases. In view of these observations, we evaluated the effect of thalidomide on the incidence of spontaneous and iodine-induced lymphocytic thyroiditis and spontaneous insulin dependent diabetes mellitus in the BB/Wor rat. Thalidomide did not suppress the incidence of lymphocytic thyroiditis and serum anti-thyroglobulin antibodies or affect the serum concentrations of T4, T3 and TSH in this rat model. Thalidomide also did not affect the incidence of insulin dependent diabetes mellitus. In contrast to preliminary studies in man and rat demonstrating efficacy in the therapy of autoimmune diseases, thalidomide did not prevent or suppress autoimmune lymphocytic thyroiditis or insulin-dependent diabetes mellitus in the BB/Wor rat.

Immunodeficiency after Allogeneic Bone Marrow Transplantation in Man.

This study was undertaken to clarify the mechanism behind the severely decreased lymphocyte proliferative response upon stimulation with mitogens and antigens seen after allogeneic bone marrow transplantation (BMT) in man. We investigated eight BMT patients and eight controls and found that the proliferative response of patient cells was reduced both when the cells were stimulated with phytohaemagglutinin (PHA) and when they were stimulated with a combination of phorbol myristate acetate (PMA), which is an activator of protein kinase C (PKC), and the calcium ionophore A23187, which irreversibly opens for calcium transport into the cell (median relative responses were 41 and 37%, respectively). However, the PHA-induced increase in the concentration of intracellular free calcium in post-BMT cells was not significantly different from the values found in control cells and the expression of interleukin 2 (IL-2) receptors (CD25) was only slightly decreased. However, the production of IL-2 was severely decreased in patient cells after stimulation with A23187/PMA (median 3541 units), although it was higher than in PHA-stimulated control cells (median 354 units). These results show that a direct activation of PKC by PMA combined with an increase in intracellular free calcium by A23187 cannot overcome the lymphocyte proliferation deficiency in cells from patients after allogeneic BMT. The data suggests that the defect is affecting the diacylglycerol pathway considerably more than the inositol triphosphate pathway.

Venocclusive disease of the liver after allogeneic bone marrow transplantation in man

One hundred and fifty-one consecutive patients underwent allogeneic bone marrow transplantation (B.M.T.) following high-dose chemotherapy and single dose total body irradiation (T.B.I.) for hematologic malignancies between September 1980 and December 1985. All patients included in this study were treated using a 60 Co beam to deliver a prescribed dose of 10 Gy to the mid-plane of the abdomen. Total body irradiation was performed the day before B.M.T. The mean instantaneous dose-rate was 3.5 cGy/min (range: 2.6 to 4.7). The real dose received was measured using thermoluminescent dosimeters (lithium borate). The difference between the doses delivered to the liver and to the mid-plane of the abdomen did not exceed 5%. The mean real dose delivered to the reference point was 10 Gy (range 8.3 to 11.7). Ninety five per cent of the patients received a dose ranging from 9.1 Gy to 10.9 Gy. High-dose cyclophosphamide was given to 126 patients with a “standard-risk” of relapse (60 mg/kg on day 5 and 4 before B.M.T.). Chemotherapy was intensified by the addition of other drugs in 25 patients with “higher-risk” of relapse. We analyzed the effect of the following pretransplant characteristics on the subsequent posttransplant development of V.O.D.: age, sex, ASAT and/or ALAT before conditioning regimen, diagnosis and status of malignant disease, history of liver disease, interval between diagnosis of hematologic malignancy and B.M.T., conditioning regimen (i.e., classical or intensified) and dose delivered to the liver during T.B.I. Seventeen patients were classified as having clinical V.O.D. giving a prevalence of 11.2%. In the first 2 months following B.M.T., death occurred respectively in 9 of 17 (53%) and 23 of 134 (17%) patients with and without clinical V.O.D. Univariate analysis showed that four characteristics were significantly related to an increased prevalence of V.O.D.: sex (11/62 females vs 6/89 males; p < 0.05); history of liver disease (7/28 vs 10/117 patients without antecedent; p < 0.01); ASAT and/or ALAT levels > 1.5 upper normal limit (11/49 vs 6/102 patients with levels < 1.5; p < 0.01) and intensified conditioning regimen (6/25 vs 11/126 patients with classical regimen; p < 0.05). The conditioning regimen and history of liver disease were highly correlated to transaminases levels. Only two factors, transaminases levels and female sex, remained significantly associated with V.O.D. after multivariate analysis.

Effect of UVB radiation on the skin after allogeneic bone-marrow transplantation in man.

Effect of UVB radiation on the skin after allogeneic bone-marrow transplantation in man.

Fractionation and dose rate effects in mice: a model for bone marrow transplantation in man.

This study was designed to compare several fractionation and dose rate schedules to optimize the therapeutic ratio for total body irradiation (TBI). C3H/HeJ mice were given TBI and the bone marrow survival fraction was calculated using the CFUS assay. Irradiation was given at two dose rates: low dose rate (LDR) at 5 cGy/min or high dose rate (HDR) at 80 cGy/min in single fraction and fractionated regimens. The fractionated regimens were given as either 120 cGy three times daily, 200 cGy twice daily, or 200 cGy daily. The Do was 80 cGy for the single fraction, HDR group and 85 for the LDR group. For the fractionated regimens, the apparent Do's ranged from 55-65 indicating no sparing effect of fractionation for the normal bone marrow stem cells. Indeed, the Do's were smaller suggesting an increased sensitivity to irradiation with fractionation. Low dose rate (LDR) and fractionation were also studied for their influence on normal tissue toxicity following upper half body irradiation (UHBI). All the fractionated regimens had higher LD50/30 and LD50/30-180 values than those achieved by single fraction LDR alone. There was no significant dose rate effect for LD50/30 when 120 or 200 cGy fractions were used. However, dose rate was important for LD50/30-180 with 200 cGy but not with 120 cGy fractions. These results demonstrate protection of non-hematopoietic tissues with fractionation and low dose rate without protecting hematopoietic stem cells and may have implications for human bone marrow transplantation.

Systemic evaluation of Sjögren-like syndrome after bone marrow transplantation in man.

A systematic evaluation of Sjögren-like syndrome (SLS) was performed in 68 bone marrow transplant (BMT) recipients (60 allogeneic and 8 syngeneic recipients). At day 100, the patients underwent clinical evaluation, functional salivary scintigraphy, and lip biopsy. If any findings were abnormal, the examinations were repeated annually for 3 years. Twenty-two patients with SLS and extensive chronic graft-versus-host disease (CGVHD) had abnormal scintiscan and lip biopsy at day 100. Marked keratoconjunctivitis sicca and xerostomia developed between 12 and 24 months after BMT and, thereafter, progressively decreased. Twenty-seven irradiated recipients (7 syngeneic and 20 allogeneic recipients without CGVHD) had isolated xerostomia and disturbed scintiscan but normal biopsy. Seven other patients with limited CGVHD had a lymphocytic infiltrate on lip biopsy but no SLS and a normal scintiscan. Schirmer's test, functional salivary scintigraphy, and lip biopsy allowed us to distinguish SLS from radiotherapy sequelae. As early as day 100, these 3 tests have a predictive value for SLS, one of the criteria for extensive CGVHD.

Haemopoietic reconstitution after allogeneic bone marrow transplantation in man: recovery of haemopoietic progenitors (CFU-Mix, BFU-E and CFU-GM).

Haemopoietic reconstitution was evaluated in 44 patients given HLA compatible sibling bone marrow transplants. The mean peripheral blood haemoglobin, neutrophil and platelet counts were markedly reduced early post-graft but returned to normal by 26 weeks post transplant. Bone marrow multipotent (CFU-Mix), erythroid (BFU-E) and myeloid (CFU-GM) progenitor cell reconstitution were also assessed at regular intervals up to 2 years post-graft. The mean value of CFU-GM increased gradually and attained a normal value by 52 weeks. The BFU-E value did not reach a normal value until after 52 weeks post-graft. However, CFU-Mix growth appeared to be impaired even up to 2 years post-transplant. The occurrence of graft versus host disease at 3 months post-transplant was associated with significantly lower mean numbers of platelets, marrow CFU-GM, BFU-E and CFU-Mix. Post-transplant patients who were on methotrexate therapy were also shown to have lower marrow CFU-GM and neutrophil values compared to those patients who received cyclosporin post-transplant. This study demonstrated that although peripheral blood counts were normal after 26 weeks post-graft, marrow stem cell reserve in these patients was reduced. This might in part explain the documented increase in risk of severe infections or thrombocytopenia in some of these patients, particularly during viral infection, graft-versus-host disease or immunosuppressive treatment.

Elimination of graft versus host disease in matched allogeneic leukemic transplant recipients using CAMPATH-1.

Bone marrow transplantation (BMT) offers one of the major advances in the treatment of certain malignant hematological disorders for which no specific effective alternative therapeutic modality exists, particularly leukemias. Although results of BMT in man have improved over the last few years, success rates have been limited mainly due to the reactivity of the graft against the host, a disease called graft versus host disease (GVHD). None of the treatment modalities studied thus far seems to be effective against established GVHD. Therefore, it appears that prevention of GVHD is the best approach to overcome the obstacle of GVHD. Since experimental data and recent pilot clinical studies suggest that acute GVHD results from donor type mature T cells present in the marrow aspirate (1–9), it would seem that the safest way to prevent GVHD is by elimination of T cells from the marrow allograft prior to transplantation. The present report summarizes our initial results in trying to prevent GVHD in patients undergoing BMT for leukemia using a monoclonal anti-lymphocyte antibody, CAMPATH-1 (10). Two major advantages of this novel antibody are: 1) its ability to deplete all lymphoid cells, including T cells, without damaging the stem cell compartment; and, 2) the unique characteristic of this antibody to bind human complement.

Allogeneic bone marrow transplantation in man: in vitro activity of FTS-Zn on T-cell markers and functions.

Thymulin (FTS-Zn) is a synthetic metallo-nonapeptide similar to the serum factor of thymic origin FTS, which induces the maturation of lymphoid cells. The activity of this compound on peripheral blood mononuclear cells from 12 bone marrow recipients was studied in vitro. It was demonstrated that thymulin was able to induce or modulate the expression of T-cell membrane markers, to enhance the proliferative responsiveness of lymphocytes to mitogens or allogeneic cells, and to increase mononuclear cells' natural killer activity. This in vitro responsiveness was contemporary to a transient decrease of FTS levels in the patients' serum, documented by sequential assays. These results suggest that thymulin could be of interest as a prophylactic therapy to speed up the immunological reconstitution of bone marrow recipients.

PROCEEDINGS OF THE AUSTRALIAN SOCIETY FOR MEDICAL RESEARCH

PROCEEDINGS OF THE AUSTRALIAN SOCIETY FOR MEDICAL RESEARCH

Progress in bone marrow transplantation in man.

Progress in bone marrow transplantation in man.

H-2-restriction of T cells mediating lethal graft-versus-host-disease to minor histocompatibility determinants.

Although the etiology and pathogenesis of graft-versus-host disease (GVHD) continues to excite controversy and speculation, it is now generally accepted that the disease reflects alloaggres-sion mediated by mature post-thymic T cells (1–3). The pioneering studies of Siraonsen (1) established that the most florid forms of GVHD occur when the donor and host differ at the major histocompatibility complex (MHC). In recent years, however, it has become clear that severe and often fatal forms of GVHD occur in MHC-com-patible combinations, e.g. following HLA-matched bone marrow transplantation in man (3). Although the etiology of this form of GVHD is still debated, our own studies in mice suggest that responses to minor histocompatibility antigens (minor HA)4 are the principal cause of the disease, at least in this species (4).

Imbalance of T-cell subpopulations and defective pokeweed mitogen-induced B-cell differentiation after bone marrow transplantation in man

Peripheral blood T-cell subpopulations were studied in eight patients undergoing bone marrow transplantation (BMT) for severe aplastic anemia (SAA) or acute leukemia (AL). Seven received an allogeneic graft and one a syngeneic graft: seven showed clinical and pathological signs of graft versus host disease (GvHD) and a marked imbalance in T-cell subpopulations. In spite of normal numbers of circulating unfractionated T cells, T lymphocytes with receptors for IgG (T G cells) were increased 2- to 5-fold, whereas T cells with receptors for IgM (T M cells) showed a 2- to 20-fold decrease. The T M-T G imbalance paralleled the development of GvHD. The patient who received the syngeneic graft showed no T M-T G imbalance and no signs of GvHD. In addition pokeweed mitogen (PWM)-induced B-cell differentiation in vitro was severely impaired in these patients in the presence of either autologous or normal allogeneic T cells. Patients' T cells did not provide adequate help for autologous or normal B cells either in the presence or absence of T G cells. The patients' purified T G cells were capable of suppressing normal B-cell responses to PWM but to a lesser degree than normal T G cells. The imbalance of T-cell subpopulations with defective helper activity and the impaired B-cell function may play an important role in the development of immunodeficiency after bone marrow transplantation and particularly during GvHD.

Immunity to human haematopoietic stem cells.

Graft rejection is an important problem in human bone marrow transplantation, with a 25–60% incidence in grafts between HLA–identical siblings1–6. In mice, two models of rejection of bone marrow grafts have been described: T-lymphocyte-mediated immunity and hybrid resistance (for reviews see refs 7, 8). The first model involves the recognition of foreign histo-compatibility (H–2) antigens by selected subsets of T lymphocytes. These antigen-reactive cells undergo clonal expansion and differentiate into cytotoxic effector cells which mediate graft rejection. In the hybrid resistance model, mononuclear cells, probably identical to natural killer (NK) cells, react against haematopoietic histocompatibility antigens on parental marrow cells9. Cells involved in hybrid resistance exhibit ‘spontaneous’ cytotoxicity to disparate haematopoietic cells and, in contrast to T-lymphocyte-mediated immunity, do not require a sensitisation phase before differentiating into cytotoxic effector cells. The mechanism of graft rejection following bone marrow transplantation in man is unknown. In transplants between HLA-identical siblings the target antigens of graft rejection are not HLA or antigens closely linked to the major histocompatibility complex (MHC). To study the problem of graft rejection in man we developed and describe here an in vitro model to detect immunity to haematopoietic stem cells committed to granulocyte–macrophage differentiation (CFU-C; colony-forming unit culture). We found that human peripheral blood and bone marrow but not thymus contain a population of cells which are cytotoxic to haematopoietic stem cells following in vitro sensitisation. The effector cell population is not a conventional T or B lymphocyte, monocyte–macrophage or NK cell.

Lethal graft-versus-host disease after bone marrow transplantation across minor histocompatibility barriers in mice. Prevention by removing mature T cells from marrow.

In two situations, transfer of normal unsensitized bone marrow cells into heavily irradiated H-2-identical allogeneic mice caused a high incidence of lethal chronic graft-versus-host disease (GVHD), i.e. mortality occuring between days of 20 and 80 postirradiation. Minor histocompatibility determinants appeared to be the main target for eliciting GVHD. Removing mature T cells from the marrow with anti-Thy 1.2 serum and complement before injection prevented GVHD. On the basis of adding purified T cells to T-cell-depleted marrow cells, it was concluded that contamination of the marrow with as few as 0.3% T cells was sufficient to cause a high incidence of lethal GVHD in certain situations. No GVHD was found with the injection of non-T cells (Thy 1.2-negative cells) or with tolerant T cells. Irradiated recipients of T-cell-depleted marrow cells remained in good health for prolonged periods. These mice showed extensive chimerism with respect to the donor marrow, normal numbers of T and B cells and were immunocompetent. The data provide no support for the view that chronic GVHD developing after bone marrow transplantation in man is the result of an attack by the progeny of the donor stem cells. The results imply that mature T cells contaminating marrow inocula are probably the main cause of GVHD seen in the clinical situation.

Localized scleroderma-like lesions after bone marrow transplantation in man.

Localized scleroderma-like skin lesions which developed in two children, from 8 to 10 months after successful bone marrow transplantation for aplastic anaemia, showed histopathological features resembling those of scleroderma. This finding, like the animal models described in the literature, provides additional support for the auto-immune nature of scleroderma.

Successful Allogenic Bone Marrow Transplantation in Man: Chimerism, Induced Specific Tolerance and Possible Anti-Leukemic Effects

Abstract A patient suffering from acute lymphoblastic leukemia in the third progressive phase, refractory to the majority of available treatments, was given total-body irradiation of 800 r. dosage, after administration of methyl-nitro-imidazolyl-mercaptopurine (300 mg. daily for 4 days). One week after irradiation, he received an infusion of a mixture of equal parts of bone marrow from six donors (mother, sister, father and three brothers), a total of 5.8 x 1010 nucleated cells. Myeloid restoration began 2 weeks after the infusion and continued to progress rapidly. The reality of the graft was demonstrated by study of the erythrocytic antigens. Nearly all the erythrocytes found in the blood after 6 months belonged to the phenotype of one of the male donors. Studies of chromosomes and leukocyte sex chromatin suggested that a small proportion of leukocytes were being produced by the cells of one of the female donors. The patient was found to produce beta 2A, beta 2M and gamma globulins of the Inv type of the donors, suggesting that lymphoid cells may also have been successfully grafted. A skin graft from the male donor whose marrow graft had apparently been accepted was still perfectly intact 7 months after grafting, whereas the grafts from other donors were rejected, indicating a specific tolerance to tissues from this donor. Previous studies of compatibility between the recipient and the various donors by a histocompatibility test described by us, or by means of similarities in leukocyte antigens, indicated that the accepted donor had been antigenically more nearly compatible than the others. What appeared to be a secondary syndrome became manifest 1 week after marrow transfusion; it consisted of weight loss, digestive disorders (anorexia, nausea, vomiting, diarrhea), hepatic disturbances (hepatomegaly, increased serum concentrations of various enzymes), transient polyadenopathy (histologically composed of histiocytic proliferation and lymphocytic aplasia), desquamative erythrodermia, eosinophilia exceeding 50 per cent, and superinfection (including a probable miliary tuberculosis). The syndrome was controlled by careful symptomatic treatment; its intensity gradually abated. Reactivation of the secondary syndrome was accomplished by reinjection of leukocytes from the donor whose graft had been tolerated. This was controlled by Δ-1-cortisone and symptomatic treatment. The patient was still alive and in apparently complete hematologic remission from the leukemia 12 months after the treatment described above. Particular reasons for this success possibly include the absence of previous blood transfusions and the use of multiple marrow donors.

The application of stored autologous bone marrow in the treatment of advanced malignant disease. (B) Haematological aspets.

Most rapid advances in a particular branch of medicine are seen to follow the publication of a particular paper containing an idea or suggestion which is sufficiently brilliant in its conception to stimulate even the most conservative. In 1956, Barnes, Corp, Loutit and Neal published such a paper and the great interest at present shown in America, France and in this country in Bone Marrow Transplantation in Man, has undoubtedly sprung from this. We have been particularly interested in the application of bone marrow transplantation to overcome the severe haemopoietic and lymphopoietic depression which follows large volume high dosage radiotherapy and also high dosage chemotherapy for malignant disease.