John A. Hansen

Probability of finding HLA-matched unrelated marrow donors.

Bone marrow transplantation has become the treatment of choice for certain hematologic diseases. However, only 30–40% of patients who might benefit from this procedure have a suitable family donor. Consequently, many centers have begun to explore the use of unrelated volunteer donors. Initial results have demonstrated the feasibility of this approach. As a result, a national effort has begun to recruit HLA-typed volunteers in order to establish a registry of individuals who would be willing to serve as bone marrow donors. This manuscript explores the potential impact of establishing such a registry. We find that a registry of attainable size could more than double the number of marrow transplants now being performed. However, even with a registry of enormous size, it will still not be possible to identify an HLA-matched donor for some patients.

A phase I-II study evaluating the murine anti-IL-2 receptor antibody 2A3 for treatment of acute graft-versus-host disease.

A murine IgG1 antibody specific for the IL-2-binding site on the human lymphocyte IL-2 receptor β chain (CD25) was evaluated in 11 patients who developed acute graft-versus-host disease following allogeneic marrow transplantation. All patients had received cyclosporine and methotrexate for prophylaxis of GVHD, either alone (4 cases), or in combination with antithymocyte globulin (4 cases) or with prednisone (3 cases). Patients had developed GVHD at 7–53 days (median 12) after transplantation and had failed treatment with corticosteroids for 3–44 days (median 19). Residual GVHD was of grade II severity in 4 patients, grade III in 5 patients, and grade IV in 2 patients. Sequential patients received monoclonal antibody in escalating doses from 0.1 mg/kg/day to 1.0 mg/kg/day for 7 days. Side effects were fever, respiratory distress, hypertension, hypotension, and chills occurring in 11 of 72 (14%) antibody infusions. Trough antibody levels greater than 6 μg/ml were achieved in patients treated with 0.5 or 1.0 mg/ kg/day. Four of eight evaluable patients had an IgM antibody response, and one had an IgG response to the murine immunoglobulin. Clinical response of GVHD was evaluated in 10 patients who received the entire course of the antibody treatment. Among 7 patients treated within 40 days from transplantation, one patient had a complete response in the skin as the only involved organ, and 3 patients had a partial response, 2 in the skin and one in the gastrointestinal tract. No responses were achieved with liver disease at anytime or in any organ in patients treated beyond 40 days after transplantation. Since administration of this antibody was well tolerated and some efficacy was observed in patients with acute GVHD treated early after transplantation, there is a rationale for testing this antibody as an agent for prophylaxis of GVHD.

Alloreactive T cell responses between HLA-identical siblings. Detection of anti-minor histocompatibility T cell clones induced in vivo.

Cytotoxic T cell (CTL) clones were isolated from the peripheral blood of a patient 13 days following marrow transplantation from her HLA-identical brother. Nineteen of the clones were specifically reactive with host--but not donor--cells, but one clone was reactive with donor but not host cells. Family studies using the 19 clones showed reactivity patterns consistent with three non-HLA, minor histocompatibility antigens (minor-HA). The frequency of reactivity on a large panel of unrelated cells indicated a relatively limited degree of polymorphism. Each of the clones was restricted in its activity by a class I epitope common to HLA-B7, B27, and B40. These data demonstrate that by clonal analysis it is possible to identify in vivo antidonor and antihost alloreactive CTL clones directed against HLA determinants following marrow transplantation.

Studies of the response in mixed leukocyte culture of cells from patients with aplastic anemia to cells from HLA-identical siblings.

We have studied the mixed leukocyte culture (MLC) reactions of 64 patients with severe aplastic anemia. Their peripheral blood mononuclear cells showed an increased relative response (RR) to cells from HLA-identical siblings as compared to cells from normal HLA-identical siblings, confirming the results reported in an earlier series of 34 patients. Elevated RRs were associated with patient antidonor lymphocyte antibodies as detected by the antibody-dependent cell-mediated cytotoxicity assay, but were not associated with antidonor complement-dependent cytotoxic antibodies or with antidonor cytotoxic T lymphocytes. Among 98 patients receiving marrow grafts from HLA-identical sibling donors, those with elevated RRs before transplantation were more apt to reject the transplant than those without (P less than 0.0001). There was no elevation of the RR in 10 untransfused patients, although positive RRs were noted in 2 patients within 12 to 24 hr of their first transfusions. Five patients with identical twins were also tested, and elevated RRs were noted in three. Although blood transfusion appears to be responsible for the increased RRs observed in some aplastic patients, genetic differences between donor and recipient were not always necessary for eliciting an increased MLC response, suggesting that mechanisms other than alloimmunization are involved.

Long-Term Comparison of Immunosuppressive Therapy with Antithymocyte Globulin to Bone Marrow Transplantation in Aplastic Anemia

Treatment recommendations for aplastic anemia are based on long-term survival data for recipients of syngeneic or allogeneic bone marrow transplants (BMT) and the more recent results of “immunosuppressive therapy” (1ST), which usually includes antihuman thymocyte globulin (ATG) or antihuman lymphoblast globulin (ALG). Patient age and availability of a suitable marrow donor limit the number of patients who are potential candidates for marrow grafting. Many centers will not recommend an allogeneic BMT for patients with aplasia who are over 40 years of age, although some extend the upper age limit to 50 years. Suitable marrow donors include identical twins, genotypically HLA-identical siblings, or phenotypically HLA-identical family members. Transplants using HLA-mismatched family members or phenotypically identical, unrelated donors are usually reserved for “salvage” therapy after failure of a nontransplant treatment regimen.

T-Cell Recognition of HLA Class I Molecules

The purpose of the Class I T-Cell Clone component of the Tenth Workshop (10WS) was to determine the extent to which HLA class I molecules express polymorphisms recognizable by alloreactive or antigen-specific class I-restricted cloned T cells. A standard protocol for testing available proliferative and cytolytic T-cell clones for reactivity against the 10WS core cell line panel was developed in parallel to the Class II T-Cell Clone component, with the exception that monoclonal antibody blocking studies were not performed with the anti-class I T-cell clones. The same method for data reduction (whereby a specific proliferative or cytolytic activity was converted into simple numerical scores (“1”=negative; “6” or “8”=strong positive) was used.

Pretransplant lymphocytotoxins do not predict bone marrow graft rejection.

SUMMARY Ninety multitransfused patients with severe aplastic anemia were conditioned with cyclophosphamide and given marrow transplants from HLA-identical siblings. Thirty patients had complement-dependent lymphocytotoxins against a panel of cells from unrelated individuals immediately before transplantation while 60 did not. Lymphocytotoxins were more frequent among patients who had received more blood product transfusions, and these patients were more likely to be refractory to random donor platelet support. Twenty-seven patients rejected the marrow transplant while 63 had sustained engraftment. The presence of pretransplant lymphocytotoxins did not correlate with graft rejection and hence does not appear to be useful as a test for identification of patients at high risk of rejection.

Studies of T Cell Proliferation Induced by Monoclonal Antibodies of the Second International Workshop

Monoclonal antibodies have proved useful as probes for identifying and studying molecules that mediate or regulate cell function. For example, certain CD3 antibodies are known to activate T cells and cause T cell proliferation (1). These antibodies bind to a noncovalently associated complex of polypeptides with molecular weights between 19,000–28,000 daltons (2–4). The CD3 complex in turn is noncovalently associated with other cell surface molecules that function as antigen receptors for T lymphocytes (5–6). The cross-linking of CD3/T cell antigen receptor complexes by CD3 antibodies is now understood to trigger T cell activation and proliferation in a way that mimics triggering by specific antigen (7). Binding of CD3 antibodies and cross-linking of CD3 on the cell surface can lead to expression of interleukin-2 (IL-2) receptors and secretion of IL-2 into the medium (8).

Retransplantation of marrow and hematopoietic stem cells from normal donors

Transplantation of marrow or hematopoietic stem cells (HSC) from a normal donor requires high-dose marrow ablative conditioning in order to achieve full and sustained engraftment [1]. Graft failure is an uncommon problem following transplantation of marrow from an HLA identical sibling, but occurs more frequently in recipients of marrow from an HLA incompatible donor [2]. Graft failure can have catastrophic consequences, and prolonged pancytopenia may place the patient at high risk of infection or hemorrhage. Although severe graft failure is often fatal, autologous recovery of hematopoiesis can occur in some patients. The absence of donor cells and presence of host lymphoid cells suggests rejection as the cause of graft failure. Severe graft failure refractory to treatment with hematopoietic growth factors is a primary indication for retransplantation. Retransplantation may also be necessitated by recurrence of hematological malignancy. Additional immune suppression is usually necessary before retransplantation, and patients are usually very sensitive to the toxicity of additional conditioning therapy, especially if the time interval after initial transplantation is short. The feasibility of retransplantation and the optimal method depend on the primary disease and the condition of the patient.

Allogeneic and Syngeneic Marrow Transplantation for Aplastic Anemia: Overview of Seattle

Marrow transplantation for the treatment of severe aplastic anemia has to be viewed in the context of alternate therapies for this disease. An earlier study of the International Aplastic Anemia Study Group showed that newly diagnosed patients with severe aplastic anemia treated by supportive therapy with or without androgens had a survival of only 20% at 5 years, with most patients dying within the first 6 months of diagnosis [1]. Most surviving patients had partial or complete spontaneous hematologic recoveries sufficient to live without the need for transfusions. These results were subsequently confirmed by the study group in another cohort of patients [2].