Lawrence G. Lum

Methotrexate and Cyclosporine Compared with Cyclosporine Alone for Prophylaxis of Acute Graft versus Host Disease after Marrow Transplantation for Leukemia

We treated 93 patients who had acute nonlymphoblastic leukemia in the first remission or chronic myelocytic leukemia in the chronic phase (median age, 30 years) with high-dose cyclophosphamide and fractionated total-body irradiation, followed by infusion of marrow from an HLA-identical sibling. To evaluate postgrafting prophylaxis for graft versus host disease, we studied these patients in a sequential, prospective, randomized trial that compared the effect of a combination of methotrexate and cyclosporine (n = 43) with that of cyclosporine alone (n = 50). All patients had evidence of sustained engraftment. A significant reduction in the cumulative incidence of grades II to IV acute graft versus host disease was observed in the patients who received both methotrexate and cyclosporine (33 percent), as compared with those who were given cyclosporine alone (54 percent) (P = 0.014). Seven patients who received cyclosporine alone acquired grade IV acute graft versus host disease, as compared with none who received both methotrexate and cyclosporine. Thirty-five of the 43 patients given both methotrexate and cyclosporine and 31 of the 50 patients given cyclosporine are alive as of this writing, at 4 months to 2 years (median, 15 months); the actuarial survival rates in the two groups at 1.5 years were 80 percent and 55 percent, respectively (P = 0.042). We conclude that the combination of methotrexate and cyclosporine is superior to cyclosporine alone in the prevention of acute graft versus host disease after marrow transplantation for leukemia, and that this therapy may have a beneficial effect on long-term survival.

Leukemia of large granular lymphocytes: association with clonal chromosomal abnormalities and autoimmune neutropenia, thrombocytopenia, and hemolytic anemia

Three patients had leukocytosis of large granular lymphocytes and chronic neutropenia. Clonal chromosomal abnormalities (trisomy 8 and trisomy 14) and lymphocytic infiltration of splenic red pulp, hepatic sinusoids, and bone marrow indicated the neoplastic nature of the large granular lymphocytes. Demonstration of a T3+, T8+, HNK-1 + phenotype and low natural killer cell activity that was augmented by interferon treatment showed the leukemic cells to be immature natural killer cells. Multiple autoantibodies were present and included rheumatoid factor and antinuclear, antineutrophil, antiplatelet, and antierythrocyte antibodies, suggesting a defect of B-cell immunoregulation. In addition, in-vitro studies showed impaired suppression of immunoglobulin biosynthesis by abnormal cells from one patient. Antineutrophil antibodies and absence of direct cell-mediated inhibition of granulocyte-macrophage colony formation supported a humoral immune mechanism for the neutropenia. In these patients the syndrome of splenomegaly, multiple autoantibodies with neutropenia, and lymphocytosis of large granular lymphocytes is due to a neoplastic proliferation of immature natural killer cells.

Transfer of allergen-specific IgE-mediated hypersensitivity with allogeneic bone marrow transplantation

We investigated whether allergen-specific IgE-mediated hypersensitivity is transferred by bone marrow transplantation. Twelve patients, 14 to 47 years of age, undergoing allogeneic bone marrow transplantation for the treatment of hematologic cancer were selected, along with their donors, by a screening questionnaire for a history of atopy in the donor. We evaluated these donor-recipient pairs before transplantation and at several points afterward for immediate skin-test reactivity to 17 allergens. For allergens for which pretransplantation skin tests had been positive in the donors and negative in the recipients, 20 of 46 post-transplantation skin tests were positive in 8 of the 11 recipients who survived for more than one year after transplantation. For allergens for which both donors and recipients had had negative skin tests before transplantation, only 6 of 256 tests (2.3 percent) were positive in the recipients after transplantation. Long-term transfer of donor-derived mite-specific IgE was demonstrated by radioallergosorbent testing in two recipients. Seven recipients either acquired or had an exacerbation of allergic rhinitis, and two recipients without a history of asthma had asthma one year after transplantation. We conclude that allergen-specific IgE-mediated hypersensitivity is adoptively transferred by bone marrow transplantation from donor to recipient by B cells with allergen-specific memory.

In vitro regulation of immunoglobulin synthesis by T-cell subpopulations defined by a new human T-cell antigen (9.3)

Abstract The monoclonal antibody 9.3 was used to identify helper and suppressor phenotypes in the regulation of immunoglobulin production in the pokeweed mitogen-activated system for the induction of immunoglobulin secreting cells. Helper activity was confined to the 9.3 + subpopulation of T cells. Positive selection (“panning”) studies confirmed the findings of depletion of T-cell helper activity using antibody and complement. On the other hand, the 9.3 − subsets were found to be consistent suppressor cells. The functional activity of these new subsets was compared to the functional activity of the T4 and T8 subsets. The presence or absence of the 9.3 antigen on T lymphocytes defines two distinct T-cell subpopulations.

Chronic lymphocytosis with neutropenia: Evidence for a novel, abnormal T-cell population associated with antibody-mediated neutrophil destruction☆

A 67-year-old man with stable chronic neutropenia, lymphocytosis, multiple auto-antibodies, and recurring infections was studied in order to characterize the abnormal lymphocytes and the mechanism of neutropenia. One-half of the circulating mononuclear leukocytes were large granular lymphocytes that did not rosette with sheep erythrocytes and did not have surface immunoglobulin or receptors for C3 or IgG Fc. Virtually all of the mononuclear leukocytes, however, reacted with three monoclonal antibodies specific for mature T lymphocytes--OKT3, 3A1, and 12.1--the latter with a bimodal pattern on FACS analysis. The nonadherent, non-E-rosetting lymphocytes consisted of a homogeneous population of T cells with a novel phenotype: 3A1+, OKT3+, OKT8+, Ia+, 12.1+, OKT4-, 9.6-, 10.2-, Fc gamma receptor-. The abnormal phenotype of these cells suggested that they may have developed clonally. In spite of their OKT8+ phenotype, these lymphocytes were functionally abnormal, since they did not suppress B-cell immunoglobulin production in vitro. A humoral immune mechanism of neutropenia was indicated by increased levels of neutrophil-reactive IgG and in vivo kinetic studies with autologous neutrophils demonstrating shortened intravascular survival. The granulocyte turnover was normal, however, suggesting a blunted marrow response to the neutropenia. Corroborating the kinetic data, in vitro cultures of the patients blood and marrow cells showed reduced numbers of granulocytic progenitors (CFU-C). Neither blood lymphocytes nor serum from the patient suppressed growth of allogeneic CFU-C nor did removal of OKT8+ cells from the marrow increase CFU-C expression. Thus, the disorder in this patient is characterized by proliferation of abnormal OKT8+ lymphocytes with impaired suppressor function. Our findings suggest that the neutropenia was due to anti-neutrophil autoantibodies that may have resulted from abnormal T-cell immunoregulation.

Immunologic recovery in human marrow graft recipients given cyclosporine or methotrexate for the prevention of graft-versus-host disease.

Immunologic recovery was studied in ten patients with aplastic anemia and 23 patients with hematologic malignancy who received HLA-identical marrow grafts and cyclosporine postgrafting as prophylaxis against graft-versus-host disease. Cyclosporine, 12.5 mg/kg/ day, was administered beginning on the day before marrow infusion and continued for 50 days, when it was tapered and discontinued by 6 months postgrafting. Results were compared with data from concurrent and previously described patients receiving methotrexate as prophylaxis for graft-versus-hot disease. Patients treated with cyclosporine or methotrexate had lower-than-normal immunologic parameters and were not different from one another 3–5 months postgrafting. By 11 to 18 months after grafting lymphocyte counts had normalized in both groups. Serum IgA levels were low and IgG levels had normalized in methotrexate-treated patients, and IgM was normal in cyclosporine-treated patients. In vivo antibody production to T-dependent antigens and skin test responses to recall antigens continued to be impaired. The response to the neoantigen dinitrochlorobenzene was still impaired in patients treated with cyclosporine and normal in patients given methotrexate. These data suggest that immunologic recovery after marrow transplantation is similar in cyclo-sporine-treated and methotrexate-treated patients.

IgG anti-tetanus toxoid antibody production induced by Epstein-Barr virus from B cells of human marrow transplant recipients.

This investigation shows that Epstein-Barr virus (EBV)-activated human B cells from marrow transplant recipients can produce in vitro IgG anti-tetanus toxoid antibody (anti-TT) without booster immunizations with tetanus toxoid (TT). Purified B cells (E-rosette negative) from 8 normal subjects, 6 healthy long-term marrow graft recipients, and 15 long-term marrow graft recipients with chronic graft-vs-host disease (GVHD), were stimulated for 12 days with EBV to induce anti-TT production in culture supernatants. The amount of anti-TT in culture supernatants was quantitated using a enzyme-linked immunosorbent assay. B cells from all 8 normal controls produced in vitro IgG anti-TT after EBV stimulation. Five of 6 healthy recipients had B cells that produced anti-TT after EBV stimulation. Four of 15 recipients with chronic GVHD had B cells capable of producing anti-TT after EBV stimulation. The number of cultures making anti-TT responses was less in those with chronic GVHD than in those without chronic GVHD or normal individuals (P less than 0.001). B cells from patients with chronic GVHD had fewer responses exceeding the overall median of 0.7 ng/ml when compared with the other two groups (P less than 0.03). These data show that B cells of donor origin can produce in vitro IgG anti-TT antibody to tetanus toxoid antigen in a T-independent fashion.

The regulatory roles of T4 and T8 subsets in tetanus toxoid-induced in vitro immunoglobulin production

A new mitogenic system for in vitro immunoglobulin production induced by tetanus toxoid is presented and the role of T4 and T8 subsets in tetanus toxoid-induced in vitro immunoglobulin production is investigated. Purified T, T4, T8, and B cells from normal individuals previously immunized but not boosted with tetanus toxoid were cultured in helper and suppressor assays and the number of immunoglobulin-secreting cells were enumerated after culture using a hemolytic plaque assay. The regulatory roles of T4 and T8 cells in this tetanus toxoid system were compared with the role of these subsets after pokeweed mitogen stimulation. Although most of the immunoglobulin produced in the tetanus toxoid system was polyclonal, there were differences in the time course, the magnitude of the responses, the radiosensitivity of the subsets, and optimal T- to B-cell ratios for immunoglobulin production which distinguish the tetanus toxoid and pokeweed mitogen systems.

Immunologic and Hematologic Reconstitution After Allogeneic Bone Marrow Transplantation

Chronic mucocutaneous candidiasis (CMC) is typically associated with the inability of T lymphocytes to proliferate and produce lymphokines in response to Candida antigen. A 7-year-old girl with CMC developed severe aplastic anemia and, after conditioning with cyclophosphamide, 200 mg/kg, underwent bone marrow transplantation from her HLA-identical sister. Engraftment was prompt and complete. The patient is surviving more than 3 years after transplantation with normal donor-derived hemopoiesis and immune function. Manifestations of CMC have resolved completely and she has not received antifungal therapy for more than 2 years.

Regulatory roles of human OKT4/OKT8 subsetsin polyclonal immunoglobulin production induced by herpes simplex type 1 virus

T cells, OKT4 cells and OKT8 cells from the peripheral blood of normal individuals seropositive for herpes simplex type 1 virus (HSV) were studied for their capacity to regulate in vitro polyclonal immunoglobulin (Ig) production induced by inactivated HSV. Polyclonal Ig production induced by HSV has been demonstrated to be T-cell dependent. T cells, OKT4 cells and OKT8 cells were co-cultured with autologous non-T cells in the presence of HSV or pokeweed mitogen (PWM) and the number of plaque-forming cells (PFC) was measured with an hemolytic plaque assay after 6 days of culture. The results in the HSV system show that the OKT4 cells provided significantly more helper activity than OKT8 cells (p = 0.002); and the OKT8 cells exhibited more suppressor activity than OKT4 cells for Ig production (p = 0.02). The helper activity of OKT4 cells after HSV stimulation was significantly less than that obtained after pokeweed mitogen stimulation (p = 0.01). The in vitro polyclonal immunoglobulin response to HSV antigen is regulated by the balance of helper/suppressor activity exerted by OKT4 and OKT8 cell subsets.