Thymus-independent expansion of T lymphocytes in children after allogeneic bone marrow transplantation

The contribution of the thymus-dependent pathway and thymus-independent pathways for T cell regeneration after BMT in children is still unclear. We analyzed the kinetics of T cell regenerative pathways after allogeneic BMT. The number of CD4+CD45RA+ T cells, a thymus-dependent population, was very low until 3 months after BMT. The numbers of CD28- T cells and CD8+ T cells expressing CD8alpha/alpha homodimer (CD8alpha/alpha+ T cells), a thymus-independent population, increased shortly after BMT, beyond the levels of healthy children in some patients. The numbers of Vgamma9+Vdelta2+ and Valpha24+ T cells, which represent populations of extrathymic development, were less than 200/microl during the 6 months after BMT. There was a significant inverse correlation between the percentages of CD4+CD45RA+ and CD28-T cells at 1 month, and a positive correlation between the percentages of CD28- and CD8alpha/alpha+ T cells at 2 and 3 months after BMT. The mean age at BMT was higher in patients with a high level of CD8alpha/alpha+ T cells than in those without an increase in these cells, suggesting the influence of thymic function on the regenerative pathways. These results suggest that the thymus-independent pathway is the dominant source of T cells even in children shortly after allogeneic BMT.     

Autologous bone marrow transplantation for acute myeloid leukemia using monoclonal antibody-purged bone marrow

We report our experience from a clinical trial of autologous bone marrow transplantation (ABMT) in the treatment of 30 patients with acute myeloid leukemia (AML) using monoclonal antibody (MoAb) and complement-treated bone marrow. All patients were in complete remission (CR) at the time of transplant: 6 patients were in first CR, 18 in second CR, and 6 in third CR. The median age of all patients was 42 years (range 11 to 57 years). For marrow ablation, 28 patients were treated with cyclophosphamide and total body irradiation. One patient was treated with busulfan and cyclophosphamide and one was treated with busulfan and VP-16. Each patient was then transfused with autologous bone marrow that had been harvested previously and treated with two MoAbs, PM-81 and AML-2-23, and rabbit complement. Median time to recovery of neutrophils (500/microL) was 30 days, and platelets (20,000/microL) was 45 days. Median time for initial erythrocyte engraftment, assessed by a flow cytometric reticulocyte assay, was 13 days. Median overall and relapse-free survival of first CR patients was at least 17.4 months post-ABMT and the 2- and 3-year actuarial overall and relapse-free survival was 67% (+/- 19%). Median survival for the 24 patients in second or third CR was 6.8 months post-ABMT and 9.3 months since CR; however, six patients survived disease-free from 16 to 61 months post-ABMT. For the second and third CR group it was observed that six patients (5 of the 6 survivors) showed "inversions," when their post-ABMT remission lasted longer than any previous one. Actuarial 2- and 3-year disease-free and overall survival of patients in second and third CR was 25% (+/- 9%) and 18% (+/- 9%), and 29% (+/- 9%) and 23% (+/- 9%), respectively. ABMT avoids the problems of graft-versus-host disease and of finding suitable donors for allogeneic marrow transplantation.     

A large proportion of T lymphocytes lack CD5 expression after bone marrow transplantation

The lymphocyte cell surface molecule CD5 (T1, Leu 1, Tp67 in the human; Ly 1 in the mouse) is expressed on the majority of circulating T lymphocytes and a small population of B cells. We have analyzed CD5 expression on repopulating T cells in the peripheral blood of patients after allogeneic bone marrow transplantation (BMT). The frequency of CD3+ T cells that lack expression of CD5 is dramatically increased after BMT compared with the normal population. The percent of total CD3+ CD5- cells correlated with the presence of graft versus host disease and with time following transplant, but did not correlate with age, diagnosis, preparative regimen, T-cell depletion of the marrow, major histocompatibility complex compatibility, or the presence or absence of interstitial pneumonitis. Furthermore, the total number and percent of CD8+ CD5- cells was increased following BMT. CD3+ cells from BMT patients were sorted for the presence or absence of CD5 expression. CD3+ CD5- cells were capable of interleukin-2 production and of mediating cytolysis following lectin stimulation. We conclude that CD3+ CD5- T cells are functional and represent a significant proportion of circulating cells in patients after BMT.     

In vitro purging of bone marrow for autologous marrow transplantation in acute myelogenous leukemia using myeloid-specific monoclonal antibodies

Autologous bone marrow transplantation (ABMT) for acute myelogenous leukemia has gained new popularity recently due to the availability of techniques to purge the autograft of residual leukemia cells. One of the methods of purging involves the use of monoclonal antibodies (mAbs) directed to antigens expressed on the surface of leukemia cells. Several mAbs lend themselves to this use because of their high degree of reactivity with leukemia cells and their lack of reactivity with normal pluripotent hematopoietic progenitor cells. Candidate mAbs include those directed to the CD14, CD15, and CD33 molecules. This review focuses on the pre-clinical data concerning the use of mAbs to these antigens and on the results of on-going clinical trials of ABMT using mAb-mediated purging.     

T lymphocyte depletion of human peripheral blood and bone marrow using monoclonal antibodies and magnetic microspheres

It has previously been demonstrated that graft-versus-host disease can be overcome in patients receiving HLA-mismatched bone marrow transplants by prior in vitro depletion of T lymphocytes from the marrow. In this report we describe the use of monoclonal antibodies and magnetic microspheres for the depletion of T cells from peripheral blood and bone marrow. The target cells are sensitized with antibodies directed against the CD2, CD3, CD4 and/or CD8 cell surface antigens, captured by magnetic beads coated with sheep anti-mouse IgG antibody and collected by placing the cell suspension in a magnetic field. This simple, rapid procedure results in the efficient removal of T cells from peripheral blood and from bone marrow without affecting the colony-forming potential of normal hematopoietic stem cells. The procedure is capable of being scaled up for the treatment of larger volumes of marrow that are required for clinical transplantation.     

Loss of hepatitis A virus antibodies after bone marrow transplantation

Reimmunization guidelines have recommended the inactivated HAV vaccine for hematopoietic stem cell transplant (HSCT) recipients living in or traveling to areas where hepatitis A is endemic. As a shift from high to medium hepatitis A endemicity has been observed in several countries in Latin America, we conducted a retrospective study to evaluate the prevalence of hepatitis A pre-bone marrow transplant (BMT) and the loss of specific antibodies in consecutive stored serum samples from 77 BMT recipients followed up from 82 to 1530 days. The prevalence of HAV antibodies was 92.2% before BMT. As vaccine was not available in Brazil when the samples were taken, it was assumed that this prevalence reflects natural infection. Survival analysis showed that the probability of becoming seronegative was 4.5% (+/-2.6%), 7.9% (+/-3.4%), 10.1% (+/-4.0%), 23.4% (+/-9.6%) at 1, 2, 3 and 4 years after transplant, respectively. The loss of HAV antibodies was significantly associated with longer follow-up (P=0.0015), younger age (P=0.049) and acute graft-versus-host disease (P=0.035). As most reimmunization protocols start around day +365, in developing countries with similar HAV endemicity, BMT recipients should have serological screening before HAV vaccination and the inactivated vaccine should be advised to those seronegative.     

Immunohistological analysis of human bone marrow trephine biopsies using monoclonal antibodies

This paper describes the use of a recently developed immuno-alkaline phosphatase method (the 'APAAP' technique) for labelling frozen sections of undecalcified bone marrow biopsies with monoclonal antibodies, including reagents reactive with T cells and their subsets, B cells, glycophorin, HLA-DR antigen, common ALL antigen, epithelial cells and megakaryocytes. Use of an immuno-alkaline phosphatase technique avoids problems due to endogenous enzyme activity encountered when staining bone marrow by immunoperoxidase procedures. Immunohistological labelling of frozen trephine biopsies is of particular value when it is impossible to aspirate marrow particles and for identifying cells which do not readily enter suspension (e.g. dendritic reticulum cells or stromal cells). Details are given of cases in which immunohistological analysis was used for the phenotyping of acute leukaemias, for the differential diagnosis of intramedullary T and B cell proliferations, and for identifying bone marrow metastases.     

Recipient immune-competent T lymphocytes can survive intensive conditioning for bone marrow transplantation

Bone marrow transplantation is usually preceded by intensive chemotherapy and radiation therapy designed to completely eliminate recipient immune-competent cells that might reject the donor bone marrow. We show that seven of 14 bone marrow transplant recipients who received intensive conditioning retained circulating T lymphocytes that proliferate after incubation with interleukin 2 and phytohemagglutinin and function as effector cells in an in vitro model of graft rejection. These T cells may mediate graft rejection.     

Analysis of chimerism after bone marrow transplantation using specific oligonucleotide probes.

DNA hybridization with synthetic oligonucleotide probes was used to follow 18 leukemia patients who received bone marrow transplantation from HLA-identical siblings. Five oligomers complementary to the tandem repetitive sequences of different hypervariable regions of human DNA were designed to produce simple restriction fragment length polymorphism patterns. Each probe hybridized to one or two bands in Hinf I-digested genomic DNA. Combined use of these probes enabled us to distinguish all sibling pairs. DNA analysis early post-transplant (15 days) detected donor-specific fragments in 14 of 18 subjects; two patients had a combination of recipient and donor fragments. Later post-transplant, (102-15 days), one of these two showed only recipient-specific fragments, and the other donor-specific fragments. These data are in accord with other markers of engraftment including cytogenetics and red blood cell phenotyping.     

T cell regeneration after allogeneic and autologous bone marrow transplantation

Venous blood T cell phenotypes were analysed with monoclonal antibodies after 11 allogeneic and 17 autologous bone marrow transplants. In seven cases studied in the early regenerative period, cells with a thymocyte phenotype were present in the blood. In the large majority of patients treated with both allografts and autografts there was an imbalance of phenotypic 'helper' and 'suppressor' T cell subsets with initially a relative and later an absolute increase of 'suppressor' T cells. This imbalance was still present at over 250 d in eight out nine cases. Suppressor T cells bearing HLA-Dr antigens were abundant in one case of fatal GVHD but not in another, and were also increased following two autografts. It is concluded that T cell phenotyping is not of diagnostic value in sick patients following bone marrow transplantation when graft-versus-host disease is suspected.     

Selective depletion of CD8+ T lymphocytes for prevention of graft-versus-host disease after allogeneic bone marrow transplantation

The effects of selectively depleting CD8+ cells from donor bone marrow were assessed in 36 patients receiving transplantation from an HLA-identical sibling as treatment for leukemia. Donor bone marrow underwent ex vivo treatment using anti-Leu-2 monoclonal antibody and complement. Patients received cyclosporine post-transplant for 6 months. Thirty-three patients had initial engraftment. Three failed to have hematologic recovery, and one patient with initial engraftment had late graft failure. The actuarial incidence of grade greater than or equal to 2 acute graft-versus-host disease was 28% +/- 18% and was usually confined to the skin. Of 33 patients with engraftment, 32 were complete chimeras and one had mixed chimerism. The tempo of hematologic and immunologic recovery was comparable with that reported with transplantation of unmodified bone marrow, although CD4+ and CD8+ T cells recovered at comparable rates. The actuarial rate of leukemia relapse was 11% +/- 10%, occurring in three patients with acute leukemia but in none of 13 patients transplanted for chronic myelogenous leukemia. Actuarial survival was 57% +/- 17% at 2 years. These data indicate that after transplantation of marrow depleted of CD8+ cells, engraftment with prompt hematologic and immunologic recovery generally occurs, with a relatively low rate of acute graft-versus-host disease. Graft failure remains a problem despite retention of CD4+ cells within the donor marrow. The lack of leukemia relapse in patients with chronic myelogenous leukemia suggests retention of a graft-versus-leukemia effect, at least for this malignancy.     

Autologous bone marrow transplantation in non-Hodgkin's lymphoma: monoclonal antibodies plus complement for ex vivo marrow treatment

PURPOSE: Patients with non-Hodgkin's lymphoma who fail to achieve a complete remission or who relapse are rarely cured with conventional therapies. For this group of patients, intensive therapy and bone marrow rescue may be curative. Our goal was to assess the effects of autologous transplantation in patients with B-cell lymphomas and a poor prognosis. To avoid occult or overt contamination with lymphoma, monoclonal antibodies BA-1, BA-2, and BA-3 were used for ex vivo marrow treatment. PATIENTS AND METHODS: Seventeen patients underwent intensive therapy and autologous bone marrow transplant using the aforementioned marrow. Ten of the 17 patients (Group I) had disease that was in complete or partial remission. The other seven patients either had disease that was not responsive to treatment or had bone marrow transplant as their initial therapy at relapse. RESULTS: The ex vivo treatment did not adversely affect engraftment. For those patients who could be evaluated, the median time to white cell engraftment was 24 days; the median durations of red cell and platelet support were 24 and 29 days, respectively. Eleven of the 17 patients had complete remissions at the evaluation 28 days after transplant. Three patients subsequently experienced a relapse, three died while their disease was in complete remission, and five are alive and disease-free 405 to 1,674 days after transplant. Group I patients had an estimated 40 percent disease-free survival rate at three years compared with 0 percent for Group II patients (p less than 0.01). CONCLUSION: Our data support autologous bone marrow transplantation as an important treatment modality for the non-Hodgkin's lymphomas. With the current preparative regimens available, however, its use should be limited to patients with disease that is still responding to conventional therapies.     

TCR gamma/delta positive lymphocytes after allogeneic bone marrow transplantation.

Peripheral gamma/delta+ T cells were studied in patients following allogeneic bone marrow transplantation (BMT) by indirect immunofluorescence utilizing two monoclonal antibodies (G1 and A13) able to recognize the two major subpopulations (V delta 2+ and V delta 1+, respectively) of these cells. We found that the relative percentage of 'total' (gamma/delta+ T lymphocytes) (V delta 2 + V delta 1 positive cells), and particularly of G1+ (V delta 2+) cells, in CD3+ lymphocytes was higher in transplanted patients, and especially in those presenting with acute graft-versus-host disease (aGVHD), than in normal controls. This finding was confirmed by the analysis of the V delta 2+/V delta 1+ cell ratio which was again significantly higher in patients with aGVHD as compared to controls. Similarly, the absolute number of 'total' gamma/delta+ and V delta 2+ cells was also significantly increased in patients with aGVHD. TCR gamma/delta+ T cells increased as a function of time after BMT reaching a plateau value at about day 60 post-BMT. When patients were stratified for the presence or absence of aGVHD this correlation was maintained only for patients with aGVHD. Finally, most V delta 2+ cells expressed surface T cell activation markers such as CD25 (IL-2 receptor) and DR (MHC class II) antigens. Our results suggest a possible involvement of gamma/delta+ T cells and particularly of V delta 2+ cells in the clinical and immunological events (aGVHD) occurring after allogeneic BMT.     

Cytolytic function of clonable T cells after human bone marrow transplantation

We evaluated T-cell mediated lymphokine activated killer (LAK) function during the late (greater than 5 months) reconstitution phase after T cell-depleted allogeneic bone marrow transplantation (BMT) for hematologic malignancy. Since LAK cells are sustained by interleukin-2 (IL-2), we also investigated the ability of post-BMT T cells to produce IL-2. These functions were investigated at the clonal level. More than 200 T-cell clones from six long-term BMT recipients were generated and compared with 60 T-cell clones derived from two normal controls. Almost all the CD8+ clonal cultures from BMT recipients expressed cytolytic activity in a lectin-dependent cellular cytoxicity assay. Interestingly, a higher proportion of BMT recipient-derived cytolytic clones were able to mediate LAK activity in comparison with control clones (28% versus 4%, P less than .05). However, T-cell clones from BMT recipients, as opposed to control clones, were largely incapable of producing IL-2. Given the high proportions of post-BMT circulating CD8+ T cells, it appears that, in long-term BMT recipients, the precursors of nonspecific LAK effectors are present at above normal levels. However, their function may be defective in vivo due to poor IL-2 production.     

Improved outcome for high-risk acute myeloid leukemia patients using autologous bone marrow transplantation and monoclonal antibody-purged bone marrow

We have conducted a 9-year multicenter trial of autologous bone marrow transplantation (ABMT) for acute myeloid leukemia (AML). Remission BM was purged in vitro using monoclonal antibodies (MoAbs; PM-81, AML-2- 23) and complement targeting myeloid differentiation antigens (CD15, CD14). In 1988, the preparative regimen changed from 60 mg/kg/d cyclophosphamide x 2 and fractionated total body irradiation (TBI) total dose, 1,200 cGy (Cy/fTBI), to 4 mg/kg/d busulfan x 4 and 60 mg/kg/d Cy x 2 (Bu/Cy2). Recent analysis (October 1, 1993) shows that the Bu/Cy2 regimen along with the same MoAb purging method yields an improved outcome. Seven first complete-remission (CR) (CR1), 45 second- or third-CR (CR2/3), and 11 first-relapse (R1) patients were treated with chemotherapy and TBI or chemotherapy alone followed by ABMT with MoAb-purged BM. Median age at ABMT for those patients in CR 2/3 and R1 patients was 36 years. Twenty-nine CR 2/3 and R1 patients were conditioned with Cy/fTBI, and 27 CR2/3 and R1 patients were conditioned with Bu/CY. Using the Kaplan-Meier method, the CY/fTBI, CR2/3, and R1 patients have a 3-year disease-free survival (DFS) of 21%. On the other hand, the Bu/Cy2, CR2/3, and R1 patients have a 3-year DFS of 48%. Nineteen CR2/3 and R1 patients relapsed post-ABMT. On analysis by conditioning regimen, those treated with Cy/fTBI have a 3-year relapse rate (RR) of 58%, whereas the patients conditioned with Bu/Cy2 have a 39% 3-year RR. Long-term DFS can be achieved in about 50% of patients with advanced remissions and relapsed AML using Bu/Cy2 with MoAb-purged BM.     

Treatment of severe Epstein-Barr virus-induced polyclonal B-lymphocyte proliferation by anti-B-cell monoclonal antibodies. Two cases after HLA-mismatched bone marrow transplantation

We treated two children who developed Epstein-Barr virus-induced polyclonal B-cell proliferation after HLA-mismatched bone marrow transplantation for congenital immunodeficiency with two monoclonal anti-B-cell antibodies. Lymphoproliferative syndrome occurred between 50 and 60 days after bone marrow infusion, and was diagnosed by the presence of spontaneously growing B cells containing Epstein-Barr-nuclear antigen in the blood and bone marrow. The mouse monoclonal anti-B-cell antibodies used were a CD21-specific antibody recognizing the CR2 receptor on B cells (BL13, IgG1) and a CD24-specific antibody binding B cells at all steps of differentiation (ALB9 IgG1). Both antibodies were given intravenously (0.2 mg/kg/body weight.d for 10 days). All clinical and biological manifestations resolved within 3 weeks of treatment. Recurrence was not seen at 18- and 15-month follow-ups. T-cell function developed normally; B-cell function remained partially deficient in one patient 21 months after bone marrow transplantation. These results suggest that monoclonal anti-B-cell antibodies could be useful in controlling severe polyclonal lymphoproliferative syndrome in profoundly immunodeficient patients after bone marrow transplantation.     

Elimination of myeloma cells from bone marrow by using monoclonal antibodies and magnetic immunobeads

The efficacy of immunomagnetic beads to purge human myeloma cells from bone marrow ex vivo was evaluated. The optimal conditions for purging were studied first by using three myeloma cell lines: RPMI-8226, SKO- 007, and SKMM-2. Myeloma cells labeled with the vital fluorescent dye Hoechst 33342 were admixed with normal bone marrow cells, and two monoclonal antibodies reactive with the myeloma cells (PCA-1 and BL-3) were added alone or in combination with the cells. Magnetic beads coated with goat antimouse immunoglobulin G were then added, and the tumor cells to which beads were attached were separated from the mixture with a magnet. The efficacy of tumor cell removal was dependent on the bead-to-tumor ratio; a ratio of more than 500 was optimal in the presence of excess normal marrow cells. The combination of monoclonal antibodies PCA-1 and BL-3 increased the tumor cell removal as compared with either antibody alone. Two cycles of treatment were more effective than one cycle was. Under optimal conditions, 2.3 to 4 logs of tumor cells could be removed from the mixture containing 10% myeloma cells without a significant loss of normal hematopoietic progenitors as measured by CFU-GM, CFU-GEM, and BFU-E. When the efficacy of this procedure was tested on fresh bone marrow from patients with multiple myeloma (MM) by using the combination of PCA-1, BL-3, and J-5, 1.6 to 2.5 logs of tumor cells could be removed by one cycle of treatment, even from marrows containing less than 10% myeloma cells. These observations support the use of monoclonal antibody combinations and immunobeads as a reliable and nontoxic method to eliminate contaminating myeloma cells ex vivo in preparation for autologous bone marrow transplantation in patients with MM.     

Chimerism studies using in situ hybridization for the Y chromosome after T cell-depleted bone marrow transplantation

In situ hybridization for the Y chromosome (Y-ISH) was used to monitor engraftment in 10 patients with hematological malignancies who had received T cell-depleted marrow transplants from sex-mismatched donors, seven of whom were only partially HLA-matched. In the three patients who engrafted, as the peripheral counts rose, the percentage of host peripheral blood and marrow mononuclear cells decreased steadily, although host cells (less than 1%) could still be detected as late as day 252. The percentage of host granulocytes fell rapidly to less than 0.2%. Seven patients did not achieve full engraftment by day 28. Those with a low percentage of host cells (less than 1%) improved with observation or treatment with steroids, while those with a high or increasing percentage of host cells did not improve even after treatment with GM-CSF or with repeat marrow infusion without reconditioning. In one patient with graft failure, the residual host cells were predominantly CD8+ CD57+ and CD3+ CD56+, phenotypes consistent with non-MHC-restricted cytotoxic T cells. Lack of full engraftment in recipients of T cell-depleted marrow is not always associated with autologous reconstitution and does not always require retransplantation. Y-ISH may be useful for monitoring patients at high risk for graft failure in order to detect adverse trends in mixed chimerism that will alter therapy early after transplantation.