Bone marrow transplantation. Therapeutic usefulness and complications

Eleven patients with a variety of fatal diseases received transplants of allogeneic bone marrow. Transplantation of human leukocyte group A (HL-A) identical marrow to three patients with lymphopenic immunologic deficiency was successful, although one died of a pre-transplant pneumonic process. After transplantation of HL-A nonidentical marrow obtained from his father, one patient with lymphopenic immunologic deficiency died from graft-versus-host disease (GVHD). A patient with chronic mucocutaneous candidiasis had a remission of her disease after bone marrow transplantation with HL-A nonidentical marrow but suffered from chronic diarrhea possibly caused by GVHD. Administration of cyclophosphamide was necessary for the marrow to “take” in patients with functional cellular immunity. One patient had transitory remission of her leukemia after bone marrow transplantation. A patient with grade IVB Hodgkin's disease died, with evidence of bone marrow take and of GVHD despite the fact that donor and recipient were identical at the HL-A locus. Two patients with terminal aplastic anemia died of sepsis shortly after bone marrow transplantation, probably due to a combination of their disease and the administration of cyclophosphamide. HL-A identical marrow failed to take in two patients who had received no preparatory immunosuppressive therapy.     

Experience with incompatible maternal donors for bone marrow transplantation

Summary Marrow transplantation in aplastic anemia and leukemia has generally been limited to siblings who have been histocompatible at both the serological (A and B) and lymphocyte determined (D or MLC) loci of the HLA system. We studied three male patients, two with aplastic anemia and one with acute myelogenous leukemia, who received transplants from their histoincompatible mothers. MLC studies between donors and recipients showed varying degrees of stimulation. Definite engraftment occurred in one patient and transient engraftment in another. Engraftment in the third patient could not be evaluated. In the patient with sustained engraftment, there was clinical evidence of severe graft versus host disease (GVHD) however, this was not substantiated by histologic findings. This preliminary study suggests that MLC incompatibility may be more of an indicator of the risk of GVHD than of bone marrow rejection. If more effective control of GVHD can be accomplished, marrow transplantation between MLC-reactive individuals may become feasible.This work was presented, in part, at the Fourth Congress of the International Society for Experimental Hematology, Trogir, Yugoslavia, September 24, 1975.     

New strategies in allogeneic stem cell transplantation: immunotherapy using irradiated allogeneic T cells

Recipients of T cell-depleted allogeneic bone marrow transplants have increased risks of relapse and graft rejection. The addition of donor T cells to the TCD allograft will decrease the risk of graft rejection but will increase the risk of graft-versus-host disease (GVHD). Relapse of leukemia or lymphoma following allogeneic bone marrow transplantation can be successfully treated with post-transplant infusions of donor lymphocytes. A relatively small number of donor T cells can have a profound anti-tumor effect and facilitate engraftment, but has an unpredictable potential for severe GVHD. An alternative to using viable immunocompetent donor immune cells to facilitate engraftment and to treat relapsed patients are donor lymphocytes that have been treated to limit their ability to proliferate and cause GVHD. T cells treated with irradiation retain cytotoxic activity against tumor cells and host immune cells. We have tested the hypothesis that allogeneic donor T cells treated with low-dose irradiation will facilitate engraftment and mediate an anti-leukemia effect in a mouse model of bone marrow transplantation. Multiple infusions of irradiated allogeneic donor lymphocytes in the peri-transplant period had graft-enhancing activity without resulting in GVHD. Murine recipients of irradiated allogeneic splenocytes and allogeneic bone marrow had stable donor-derived hematopoiesis without a significant contribution of irradiated donor cells to the T cell compartment. Removing T cells from the allogeneic splenocytes prior to irradiation largely eliminated their graft facilitating activity. Based upon the promising results of the pre-clinical murine studies, we initiated a phase I clinical trial of multiple infusions of irradiated allogeneic lymphocytes in patients who had relapsed after allogeneic BMT. Of 12 patients treated to date on this study, three have shown objective responses of their leukemia or lymphoma to multiple infusions of irradiated donor lymphocytes. We have initiated a new phase I clinical study to test the efficacy of multiple infusions of irradiated allogeneic cytotoxic T cells to facilitate engraftment in allogeneic transplantation. Successive cohorts of patients will be transplanted with allogeneic stem cells alone, or a combination of allogeneic stem cells and increasing numbers of irradiated allogeneic T cells. Irradiated allogeneic lymphocytes that retain short-term allo-specific cytotoxicity and lack the potential for clonal expansion in vivo can be considered as a novel form of immunotherapy with defined pharmacokinetics.     

Acute graft-versus-host disease: clinical characteristics in the cyclosporine era

Graft-versus-host disease (GVHD) remains the major problem in allogeneic bone marrow transplantation. GVHD has limited the use of this technique to HLA-matched donor recipient pairs. Thus, only a quarter of patients who ultimately may have benefited from bone marrow transplantation are currently eligible. Even in matched patient recipient pairs, GVHD accounts for approximately 40% of the deaths following allogeneic bone marrow transplants. One of the major challenges for transplantation is to derive better strategies to prevent and treat GVHD while retaining the allogeneic benefit of graft-versus-leukemia. Current pharmacologic approaches have used cyclosporine, usually in combination with other drugs. More experimental approaches have removed lymphocytes from the marrow grafts. With either approach, maintaining the anti-leukemic benefit of an allogeneic transplant (i.e., immunologic attack of the leukemia resulting in a lower relapse rate), will need to be maintained if that approach will ultimately prove to be useful.     

Therapy for chronic myelogenous leukemia with unrelated donor bone marrow transplantation: results in 102 cases

From April, 1985, to February, 1989, 102 consecutive patients received unrelated donor bone marrow transplantation therapy for chronic myelogenous leukemia (CML) at four centers. Median age of the group was 31 years (range, 4.5 to 51 years). Fifty-four patients were in first chronic phase (CP) at time of transplantation, and 48 had evidence of more advanced disease (AD) (accelerated phase, 32; blast crisis, 9; second CP, 7). In 44 cases, the donor and recipient were identical at the HLA A, B, and DR loci and were nonreactive in bidirectional mixed leukocyte culture (MLC) ("matched"). In 58 cases, nonidentity between donor and recipient could be determined at at least one HLA locus or in bidirectional MLC ("mismatched"). Fifty-eight patients were prepared for transplantation with a combination of cyclophosphamide and fractionated total body irradiation (FTBI) and received acute graft-versus-host disease (GVHD) prophylaxis consisting of methotrexate alone or in combination with cyclosporine, prednisone, or antithymocyte globulin (ATG). In 44 cases, patients received preparative agents in addition to cyclophosphamide and FTBI, and marrow depleted of mature T lymphocytes by ex vivo incubation with either anti-CD3 antibody plus complement (n = 24) or Campath-1 (n = 20). Engraftment defined by a peripheral blood neutrophil count greater than 0.5 X 10(9)/L was demonstrated in 92 cases and occurred at a median of 22 days (range, 11 to 46 days). In 10 cases, peripheral blood evidence of engraftment did not occur, and in one case, engraftment was followed by aplasia. Hematologic relapse was seen in four cases. Recurrence or persistence of the Ph1 chromosome without evidence of hematologic relapse occurred in four additional cases. The incidence of grade II to IV acute GVHD is 65% (95% confidence interval [CI], +/- 10%). After adjustment for recipient age and donor matching status, recipients of T lymphocyte-depleted donor marrow had a significantly lower incidence of grade II to IV acute GVHD (P less than .01); however, T depletion was not significantly associated with improved survival (P = .34), disease-free survival (P = .51), or increased incidence of relapse (P = .39). Of 102 patients, 46 are alive, with a median survival of 12 months (range, 3 to 46 months), and the Kaplan-Meier estimate of disease-free survival is 29% (95% CI, +/- 9%) for the entire group at 2 1/2 years.(ABSTRACT TRUNCATED AT 400 WORDS)     

Generation of CD4+ cytotoxic T-lymphocyte clones from a patient with severe graft-versus-host disease after allogeneic bone marrow transplantation: implications for graft-versus-leukemia reactivity

HLA-identical bone marrow transplantation (BMT) is associated with both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) reactivity. Different T-cell subsets from the bone marrow (BM) graft may be responsible for GVHD and GVL reactivity after BMT. In the etiology of GVHD, not only CD8+ but also CD4+ donor T lymphocytes may play an important role. Here we report a patient with chronic myeloid leukemia (CML) who was transplanted with the BM from his HLA- genotypically identical sister. After BMT there was complete engraftment, but the patient died because of acute GVHD grade III-IV in complete remission. Cytotoxic T-lymphocyte (CTL) lines were generated after BMT using the irradiated leukemic cells from the patient as stimulator cells and the donor-originated peripheral blood mononuclear cells, procured from the patient after BMT, as responder cells. The generated CTL lines showed specific lysis of the recipient lymphocytes and leukemic cells in a 51Cr release assay. Two types of CTL clones could be established from these CTL lines, both phenotypically CD4+. Clone type I showed male-specific HLA-DQ5-restricted lysis of the recipient lymphocytes, but not of the circulating relatively mature leukemic cells from the patient. This may be explained by the low HLA- DQ5 expression of the more mature CML cells. Clone type II showed HLA- DR2-restricted minor histocompatibility antigen-specific lysis of the recipient lymphocytes and leukemic cells. Both types of CTL clones showed antigen-specific cell-mediated growth inhibition of the recipient clonogenic leukemic precursor cells. These CD4+ CTL clones produced several activating cytokines including tumor necrosis factor alpha, interferon gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF), and macrophage CSF. Our results illustrate that these CD4+ CTL clones may have induced GVHD directly by cytolysis and indirectly by activating cytokines. Because both types of CTL clones recognized the recipient leukemic progenitor cells, they may also contribute to GVL reactivity after BMT.     

Transplantation of HLA-haploidentical T-cell-depleted marrow for leukemia: autologous marrow recovery with specific immune sensitization to donor antigens

Autologous marrow recovery without engraftment of donor marrow was observed after bone marrow transplantation (BMT) for two patients with acute lymphoblastic leukemia. Each had received marrow from a haploidentical mixed lymphocyte culture (MLC) reactive donor after pretransplant conditioning with total body irradiation and high-dose cyclophosphamide. To minimize graft-vs-host disease, the marrow was depleted of T cells in vitro by treatment with a monoclonal anti-T-cell antibody and complement. Two weeks after each transplant, reactive lymphocytes were noted transiently in the blood of each patient. Analysis of karyotype, HLA type, and in vitro MLC responsiveness proved the lymphocytes to be of host, not donor, origin. MLC studies showed rapid proliferative responses specifically to stimulating cells from the BMT donor, indicating in vivo sensitization to donor antigens. Return of hematopoietic function was markedly delayed, but it eventually normalized after several months, without evidence of chimerism. These studies confirm that some immune and hematopoietic stem cells of host origin survive the high-dose chemoradiotherapy used as transplant conditioning. Because these immune cells are specifically reactive to donor alloantigens, more potent suppression of host immunity may be needed to prevent nonengraftment of T-cell-depleted, HLA-mismatched bone marrow.     

Mixed leukocyte culture reactivity and graft-versus-host disease in HLA-identical marrow transplantation for leukemia.

The results of pretransplant mixed leukocyte culture (MLC) assays were compared to subsequent risk of graft-versus-host disease (GVHD) in 783 patients receiving marrow transplants from HLA genotypically identical sibling donors. The mean MLC response observed between 1303 normal HLA identical sibling pairs was 0.0 +/- 4.2% RR. The donor anti-recipient MLC reaction, an in vitro response that presumably might be relevant to GVHD, was significantly increased (greater than mean + 2 sd) in 83 (10.6%) of the cases, most often in patients in relapse at the time of testing. No association was found, however, between this increased donor anti-recipient MLC reactivity pretransplant and the incidence or severity of subsequent acute or chronic GVHD. These data suggest that the increased MLC responses sometimes observed between leukemia patients and their HLA identical sibling donors prior to marrow transplantation do not represent genetic differences capable of causing GVHD.     

Cellular therapy: exploiting NK cell alloreactivity in transplantation

Allogeneic hematopoietic transplantation relies on T-cell alloreactions for engraftment and the graft-versus-leukemia (GVL) effect. In human leukocyte antigen (HLA) haplotype-mismatched transplants, extensive T-cell depletion of the graft is essential to prevent GVHD. This raises the question of whether mismatched transplants exert any GVL effect and whether it will ever be possible to reduce the intensity of preparative regimens. Because natural killer (NK) cells are negatively regulated by MHC class I-specific inhibitory receptors, mismatched transplants may trigger NK-cell alloreactivity. HLA class I disparities driving NK-cell alloreactions in the GVH direction mediate strong GVL effects, produce higher engraftment rates, and do not cause GVHD. In murine MHC-mismatched transplant models with no donor T-cell reactivity against the recipient, the pre-transplant infusion of donor-vs-recipient alloreactive NK cells conditioned the recipients to bone marrow transplantation without GVHD. NK-cell alloreactivity may be a unique therapeutic tool for tolerance induction and clearance of leukemia in hematopoietic transplantation.     

Allogeneic Marrow Transplantation for the Treatment of Leukaemia

22 HL-A antigen and mixed leukocyte culture-matched sibling bone marrow transplants were attempted in patients with acute leukaemia (at the National Cancer Institute) to define the toxicities of four different immunosuppressive regimens, the complications associated with warrow engraftment and antileukaemic effect. 73% (16/22) were engrafted as indicated by a change to donor red blood cells (RBC) type, leukocyte, immunoglobulin allotype or by the speed of marrow repopulation and the occurrence of the Graft Versus Host Disease (GVHD). 12 of 16 (75%) successful engrafted patients developed GVHD. The current published results of clinical bone marrow transplantation from major centers has been reviewed and will be discussed in relationship to current clinical complications associated with bone marrow transplantation.     

Bone marrow graft engineering by counterflow centrifugal elutriation: results of a phase I-II clinical trial

In an attempt to reduce the incidence and severity of acute graft-versus-host disease (GVHD), we have decreased the number of bone marrow (BM) lymphocytes in the donor marrow graft before bone marrow transplantation (BMT) using counterflow centrifugal elutriation (CCE). In a phase I-II clinical trial, 23 patients received lymphocyte-depleted BM allografts from their HLA-identical, mixed lymphocyte culture (MLC)-nonreactive sibling donors. The patients entered in the study were deemed to be at high risk for treatment failure on the basis of age (greater than 30 years; median, 39 years) and the result of our skin explant assay predictive of acute GVHD. Patients predicted not to develop acute GVHD by this assay were excluded from this study. All patients received a standard lymphocyte dose of 0.5 x 10(6) morphologic lymphocytes per kilogram ideal body weight (IBW) in the marrow graft and were maintained on cyclosporine A (CsA) immunosuppression for 170 days after BMT. Prompt hematopoietic recovery occurred in 22 of 23 patients with a median time to an absolute neutrophil count (ANC) greater than or equal to 500/microL of 21 days. Donor cell engraftment was subsequently verified by cytogenetic and/or DNA analysis in all of 21 evaluable patients. No patient developed systemic acute GVHD. Only five (22%) developed cutaneous GVHD (clinical stage 1) that required steroid treatment, including one patient who failed to engraft. The median follow-up of the patients enrolled in this study is 14 months (range, 5 to 20 months). Actuarial survival 1 year after BMT is 83%. Thus, in two consecutive clinical trials using CCE to deplete donor BM of alloreactive lymphocytes (1.0 x 10(6) versus 0.5 x 10(6) lymphocytes/kg), we have demonstrated that the procedure does not interfere with BM engraftment and is effective in decreasing the incidence and severity of acute GVHD. Furthermore, comparison of these studies has revealed a differential dose response relationship between the number of graft lymphocytes, protection of engraftment, and induction of acute GVHD. Although there appears to be a modest relationship between lymphocyte dose and time to hematopoietic recovery, the 50% reduction in lymphocyte dose from that used in our previous trial resulted in a marked decrease in acute GVHD without compromising engraftment.     

Allogeneic marrow transplantation for the treatment of leukaemia. A review.

22 HL-A antigen and mixed leukocyte culture-matched sibling bone marrow transplants were attempted in patients with acute leukaemia (at the National Cancer Institute) to define the toxicities of four different immunosuppressive regimens, the complications associated with warrow engraftment and antileukaemic effect. 73% (16/22) were engrafted as indicated by a change to donor red blood cells (RBC) type, leukocyte, immunoglobulin allotype or by the speed of morrow repopulation and the occurrence of the Graft Versus Host Disease (GVHD). 12 of 16 (75%) successful engrafted patients developed GVHD. The current published results of clinical bone marrow transplantation from major centers has been reviewed and will be discussed in relationship to current clinical complications associated with bone marrow transplantation.     

Immunotherapy of hematologic malignancies and metastatic solid tumors in experimental animals and man

Following engraftment of donor hematopoietic cells and induction of host-versus-graft tolerance, immunocompetent lymphocytes of donor origin can induce graft-versus-leukemia (GVL) and graft-versus-tumor (GVT) effects. Engraftment of allogeneic bone marrow cells can be accomplished following non-myeloablative conditioning while possibly controlling graft-versus-host disease (GVHD). GVL and GVT effects may thus be successfully accomplished following non-myeloablative stem cell transplantation (NST) as shown by data derived from experimental animals and man.     

Result of allogeneic bone marrow transplantation from unrelated donor

Two patients treated by unrelated bone marrow transplantation were reported. Case 1 was an eight-year-old boy with Morquio's disease received bone marrow graft from an HLA one-locus mismatched, MLC non reactive unrelated donor. The patient was prepared with conventional dose of cyclophosphamide, and thoracoabdominal irradiation. For the prophylaxis of GVHD, three drug regimen consisted of methotrexate (MTX), cyclosporine A (CsA), and prednisolone (PSL) was administered. Engraftment was prompt, and grade I of acute GVHD developed, which resolved with increased dose of PSL. Case 2 was a ten-month-old boy with juvenile chronic myelogenous leukemia received bone marrow graft from an HLA fully matched, MLC non reactive unrelated donor. Preconditioning regimen consisted of total body irradiation, VP-16, and cytosine arabinoside. MTX, CsA, and methylprednisolone were administered to prevent GVHD, but grade II of acute GVHD developed, which resolved with prolonged course of PSL. Both cases are alive and well, without chronic GVHD. In conclusion, unrelated donor bone marrow transplantation may be a useful to treat hematologic malignancies, aplastic anemia, and some inherited diseases.     

Lethal graft-versus-host disease: modification with allogeneic cultured donor cells

The use of the bone marrow culture technique was studied as a means to prepare donor marrow for bone marrow transplantation to avoid lethal graft-versus-host disease (GVHD). Preliminary experiments demonstrated the rapid loss of theta-positive cells in such cultures, so that theta- positive cells were not detected after 6 days. Initial experiments in C3H/HeJ (H-2k, Hbbd) recipients prepared with 900 rad demonstrated improved survival when 3-day cultured C57BL/6 (H-2b, Hbbs) donor cells were used in place of hind limb marrow for transplantation. However, hemoglobin typing of recipient animals revealed only short-term donor engraftment, with competitive repopulation of recipient marrow occurring. Subsequent experiments were done in 1,200-rad prepared recipients, with long-term donor engraftment demonstrated. The majority of 1,200-rad prepared animals receiving cultured allogeneic cells died of GVHD, but animals receiving 28-day cultured cells had an improved 90- day survival and a delay in GVHD development over animals receiving hind limb marrow or marrow from shorter times in culture. In addition, animals receiving anti-theta-treated, 3-day nonadherent cells had an improved survival (44%) over animals receiving anti-theta-treated hind limb marrow (20%). These experiments demonstrate modest benefit for the use of cultured cells in bone marrow transplantation across major H-2 histocompatibility complex differences.     

Impact of pretransplant conditioning and donor T cells on chimerism, graft-versus-host disease, graft-versus-leukemia reactivity, and tolerance after bone marrow transplantation

Graft rejection, mixed chimerism, graft-versus-host disease (GVHD), leukemia relapse, and tolerance are interrelated manifestations of immunologic reactivity between donor and host cells that significantly affect survival after allogeneic bone marrow transplantation (BMT). In this report, a mouse model of BMT, in which the donor and host were compatible at the major histocompatibility complex (MHC), was used (1) to examine the interrelationship of pretransplant conditioning and T-cell content of donor BM with regard to lymphoid chimerism and GVHD and (2) to determine how these factors affected graft-versus-leukemia (GVL) reactivity and donor-host-tolerance. AKR (H-2k) host mice were administered optimal or suboptimal total body irradiation (TBI) as pretransplant conditioning followed by administration of BM cells from B10.BR (H-2k) donor mice with or without added spleen cells as a source of T lymphocytes. Transplanted mice were injected with a supralethal dose of AKR leukemia cells 20 and 45 days post-BMT to assess GVL reactivity in vivo. The pretransplant conditioning of the host and T-cell content of the donor marrow affected the extent of donor T-cell chimerism and the severity of GVH disease. GVL reactivity was dependent on transplantation of mature donor T cells and occurred only in complete chimeras. Transplantation of T-cell-deficient BM resulted in the persistence of host T cells, ie, incomplete donor T-cell chimerism, even when lethal TBI was used. Mixed chimerism was associated with a lack of GVL reactivity, despite the fact that similar numbers of donor T cells were present in the spleens of mixed and complete chimeras. In this model, moderate numbers of donor T cells facilitated complete donor T-cell engraftment, caused only mild GVHD, and provided a significant GVL effect without preventing the subsequent development of tolerance after conditioning with suboptimal TBI. In contrast, severe, often lethal, GVHD developed when the dose of TBI was increased, whereas tolerance and no GVH/GVL reactivity developed when the T-cell content of the marrow was decreased.     

Augmentation of donor bone marrow engraftment in histoincompatible murine recipients by granulocyte/macrophage colony-stimulating factor

T cell depletion of donor bone marrow can prevent graft v host disease (GVHD) in human and murine marrow graft recipients. However, engraftment in the recipient may be compromised as a consequence of donor marrow T cell depletion. The effect of recombinant murine granulocyte/macrophage colony-stimulating factor (rmu GM-CSF) on engraftment and hematologic reconstitution was evaluated in a murine allogeneic bone marrow transplantation (BMT) model involving T cell depletion of marrow. Before transplantation into irradiated mice differing at major and minor histocompatibility loci, rmu GM-CSF was preincubated with T cell-depleted donor marrow. When low degrees of engraftment were noted in control recipients, treatment of donor marrow with high concentrations of rmu GM-CSF led to enhanced engraftment. Ex vivo donor graft incubation with rmu GM-CSF or a single in vivo injection of rmu GM-CSF were both effective means of promoting engraftment. When the engraftment rate in control recipients was high, rmu GM-CSF did not have an identifiable effect. Only slight increases in hematologic recovery were detected regardless of the rate of engraftment. Neither post-BMT survival nor marrow stem cell capacity was affected by rmu GM-CSF incubation. Furthermore, growth factor administration did not have a significant effect on the incidence of GVHD in recipients of non-T cell-depleted bone marrow splenocyte preparations. In vitro natural killer-mediated target cell lysis was not altered by incubation of effector cells with rmu GM-CSF. These results demonstrate the potential of ex vivo rmu GM-CSF treatment of donor marrow to facilitate engraftment across extensive histo- compatibility barriers.     

Chronic graft versus host disease: a syndrome of disordered immunity.

A chronic, debilitating syndrome related to graft-versus-host disease (GVHD) has been recognized in long-term survivors following allogeneic bone marrow transplantation. In six of 20 marrow graft recipients who survived for more than one year after receiving a transplant, this complication developed; they were studied to better define the syndrome. There was no association between the sex of either donor or recipient, HLA type, blood group, conditioning regimen or marrow cell dose and subsequent development of chronic GVHD. All six patients had mild to moderate manifestations of acute GVHD following prompt engraftment. Chronic GVHD was characterized in each patient by progression to scleroderma-like skin involvement with hyperkeratosis, reticular hyperpigmentation, atrophy with ulceration and fibrosis with limitation of joint movement. A sicca syndrome was prominent in five patients. Four patients had idiopathic interstitial pneumonitis. Infectious complications were frequent, and DNA viral infections were prominent. Autoimmune hemolytic anemia was present in three patients, and one patient had antinuclear antibody (ANA). A spectrum of immune abnormalities was observed including hypergammaglobulinemia, immunoglobulin M (IgM) paraprotein, elevated circulating immune complexes, plasma cell hyperplasia, lymphocytotoxic antibodies and autoantibodies to autologous or donor lymphocytes. Despite clinical similarity to collagen vascular diseases, none of these patients had anti-DNA antibodies or antibodies to smooth muscle, thyroid or extractable nuclear antigens. In one patient, a skin graft from the marrow donor remained healthy despite progressive involvement in recipient skin, whereas unrelated skin grafts were rejected. Immunosuppressive therapy and plasmapheresis have not been effective. Four patients have died (median survival 458 days from transplantation). Chronic GVHD appears to be a syndrome of disordered immune regulation features of immunodeficiency and autoimmunity.     

Separation of rat bone marrow cells by counterflow centrifugal elutriation: a model for studying the effects of lymphocyte depletion

We have developed a simple three flow-rate, fixed rotor speed, counterflow centrifugal elutriation (CCE) procedure that permits the isolation of an engraftable lymphocyte-depleted (greater than 98%) fraction from ACI rat bone marrow. The different cell fractions were characterized by morphology, alloreactivity in mixed lymphocyte culture and limiting dilution analysis, colony-forming capacity, and their capacity to reconstitute hematopoiesis and effect a graft-versus-host reaction in lethally irradiated allogeneic hosts. After CCE fractionation of ACI rat marrow, transplantation of the lymphocyte-depleted marrow fraction resulted in sustained engraftment without evidence of clinical or histologic acute graft-versus-host disease (GVHD). CCE fractionation of rat bone marrow may be a useful preclinical model for studying lympho-hematopoietic and immune reconstitution after transplantation with lymphocyte-depleted donor marrow, as well as for studying the role of lymphocyte subpopulations on engraftment, acute GVHD, and leukemia relapse in syngeneic and allogeneic bone marrow transplantation.     

Reduction of lethal graft-versus-host disease: transplantation of cultured murine bone marrow across minor histocompatibility differences

The murine bone marrow culture technique was used to prepare donor marrow for bone marrow transplantation across minor histocompatibility complex differences. Previous studies have shown that theta-positive cells are rapidly lost from such cultures and that transplantation of cultured marrow across major histocompatibility complex differences results in a delay in the development of lethal graft-v-host disease (GVHD). In this study, a total of 1 to 2 X 10(7) nonadherent cells (740 to 1560 CFUs [colony-forming units]) from three-day-old cultures were used as a source of donor marrow. Three strain combinations were evaluated; LP/J into C57BL/6; BIO.BR into CBA/J; and C57BL/6 into LP/J. Donor mice were immunized with recipient spleen cells prior to culture in order to increase the graft-v-host response. For LP/J marrow into C57BL/6 mice, 5 X 10(7) donor spleen cells transplanted along with the marrow were needed to induce lethal GVHD. However, lethal GVHD was seen without the addition of spleen cells for BIO.BR into CBA/J and C57BL/6 into LP/J strain combinations. Most animals receiving fresh marrow were dead of GVHD five weeks after transplantation. With the use of cultured marrow the three-month survival was 80%, 51%, and 93%, respectively, for LP/J into C57BL/6, BIO.BR into CBA/J, and C57BL/6 into LP/J strain combinations. Long-term donor engraftment in all recipient animals receiving cultured marrow was confirmed by analyzing hemoglobin polymorphisms between the strain combinations. These results demonstrate that in contrast to transplantation across major histocompatibility complex differences, the use of cultured cells for bone marrow transplantation across minor histocompatibility complex differences allows for engraftment while reducing the risk of lethal GVHD.