Reactivity of acute lymphoblastic leukemia and normal bone marrow cells with the monoclonal anti-B-lymphocyte antibody, anti-Y 29/55.

Malignant lymphocyte populations in peripheral blood of patients with B-cell chronic lymphocytic leukemia, leukemic variant of B-cell non-Hodgkin's lymphoma, and hairy cell leukemia can be characterized by the use of a monoclonal murine antibody (anti-Y 29/55) which is directed against a cell membrane component normally confined to the sessile nonrecirculating cells of the B-lymphocyte population in lymphoid tissues. The present report describes the reactivity of the anti-Y 29/55 antibody with bone marrow cells obtained from children with acute lymphoblastic leukemia using an indirect immunofluorescence method in combination with morphological and cytokinetic studies. In 25 patients (acute lymphoblastic leukemia subtype: 14 common; 4 pre-B-cell; 4 null; and 3 T-cell), a maximum of 2% of cells (small lymphocytes) were stained. One patient presented with blasts exhibiting cytoplasmic and surface immunoglobulin M (IgM) (pre-B-B-cell acute lymphoblastic leukemia). About 11% of this patient's blast cells showed a positive reaction with anti-Y 29/55. They could not be differentiated by morphological criteria from the anti-Y 29/55-negative blast cell population. In another patient with pre-B-B-cell acute lymphoblastic leukemia, only 1% of anti-Y 29/55-positive cells was found. In bone marrow of children with relative lymphocytosis, 1.4 to 8.7% of mononuclear cells reacted with anti-Y 29/55. Morphologically, these cells were small lymphocytes and predominantly expressed surface IgM. In two of these children, a further subdivision of bone marrow cells could be achieved by combining anti-Y 29/55 and cytoplasmic IgM reactivity with [3H]thymidine pulse labeling. These studies revealed that the actively proliferating, normal pre-B-cell population was anti-Y 29/55-nonreactive, whereas a nonproliferating population of anti-Y 29/55-reactive, cytoplasmic IgM-positive cells probably represented B-cells with surface immunoglobulin M reacting when cytoplasmic IgM was assessed. We conclude that the reactivity of the monoclonal anti-B-cell antibody (anti-Y 29/55) is restricted to surface immunoglobulin-positive bone marrow cells and that neither leukemic or normal pre-B-cells nor common, null-cell, or T-cell acute lymphoblastic leukemia blasts react.

Monoclonal antibody against a membrane antigen characterizing leukemic human B-lymphocytes.

In the diagnosis of non-Hodgkin's lymphomas, the ready characterization of the neoplastic cell lineage by analysis of cell surface markers is of great importance. We present evidence for the existence of a human B-leukemia-associated antigen recognized by a complement-fixing monoclonal antibody (anti-Y 29/55). A hybridoma was produced by fusing mouse myeloma cells and splenocytes of a mouse immunized against lymphoid cells of a patient with B-cell chronic lymphocytic leukemia. Characterization of anti-Y 29/55-reactive normal and malignant leukocytes was demonstrated by cytolysis and indirect immunofluorescence. This revealed reactivity with an antigen on B-lymphoma cells (11 patients), on leukemic lymphocytes in B-cell chronic lymphocytic leukemia (13 patients), and on malignant cells in hairy-cell leukemia (two patients) but not on leukemic cells of T-cell acute lymphoblastic leukemia (one patient), on T-lymphoma cells (one patient), on cells of acute myeloblastic leukemia (four patients), or of chronic myeloid leukemia (four patients). No specific cytolysis occurred with B- and T-peripheral blood lymphocytes from (a) healthy donors (16 individuals), (b) patient with reactive lymphocytosis (one patient), (c) nonleukemic multiple myeloma (six patients), or (d) Hodgkin's disease (three patients). Surface immunoglobulin-positive, sheep RBC-negative lymphocytes isolated from human spleen (three individuals), tonsils (seven individuals), and lymph nodes (one individual), however, were recognized. It is concluded that leukemic B-cells carry a marker characteristic of nonrecirculating sessile B-lymphocytes.

Functional characterization of T lymphocytes grown in semisolid agar.

T lymphocyte colony forming cells (TL-CFC) grown in agar in the presence of PHA were assayed for their capacity to induce or suppress polyclonal PWM dependent B lymphocyte differentiation into plasma cells. This was measured by identifying cells containing intracytoplasmatic immunoglobulins by direct immunofluorescence. To validate the helper and suppressor system used in this paper, the inductive capacity of unfractionated T lymphocytes and their subpopulations bearing Fc-receptors for IgM (TM) and for IgG (TG) was measured. The unfractionated T cells and the TM fraction showed helper activity, whereas the TG cells expressed suppressor activity. The TL-CFC grown in agar in the presence of PHA manifested helper activity at low cell concentration. However, increasing the TL-CFC concentration finally caused suppression of B cell differentiation. The suppressor effect could be abolished by prior irradiation of the TL-CFC before seeding them in agar. These results indicate that T cells grown in agar have the functional capacity of T helper and T suppressor cells to induce and suppress polyclonal PWM dependent B lymphocyte differentiation into plasma cells.

[Determination of leukemic B-lymphoma cells with the monoclonal antibody anti-Y 29/55]. / Nachweis von leukämischen B-Lymphomzellen durch den monoklonalen Antikörper Anti-Y 29/55.

The monoclonal antibody anti-Y 29/55 recognizes a group specific antigen on sessile human B-lymphocytes which do not belong to the recirculating lymphocyte pool. The occurrence of this antigen in malignant NHL, with or without leukemic state, and in other leukemias has been studied. The antigen was expressed on cells of various histologic B-cell types but not on leukemic cells of ALL, T-lymphoma, AML or CML. It is concluded that in malignant B-lymphoma, B-CLL and HCL, cells appearing in blood carry a marker characteristic of virgin or activated sessile B-lymphocytes. Anti-Y 29/55 permits differentiation of such cells from normal recirculating B-cells and other leukemic cells including ALL, AML and CML. In follow-up studies this antibody may be helpful in detecting early leukemic output. B-lymphocytic leukemia may reflect a disproportion between binding sites on the lymphatic reticulum and the neoplastic cells bearing this antigen, which might be involved in binding of B-lymphocytes to the supporting lymphatic reticulum.

Reactivity of monoclonal antibodies LAU-A1 and anti-Y 29/55 in T and B cell malignancies of children: correlation with immunological markers and clinical data.

The monoclonal anti-pan-T cell antibody LAU-A1 stained neoplastic T cells arrested at different levels of maturation from all 21 children with T cell malignancies examined. Particularly in 7 patients with immature T cell neoplasia staining for LAU-A1 facilitated the recognition of a T cell origin of the malignant cells. Only 40% of these immature T cell malignancies were associated with an anterior mediastinal mass. A subdivision of T cell neoplasia into 4 differentiation-related subgroups did not permit to make predictions regarding the patients' survival. Despite the rather uniform clinical presentation the immunological phenotypes of tumor cells in 14 children with B cell non-Hodgkin's lymphoma (B NHL) were heterogeneous. Tumor cells lacked surface immunoglobulins (2 patients), expressed IgM only (7 patients), IgM and IgD (3 patients) or IgM, IgD and IgA (2 patients). Regardless of surface immunoglobulin expression anti-Y 29/55 stained practically all recognizable tumor cells of all B NHL examined. No correlation was found between the number of heavy-chain isotypes expressed on tumor cells and the survival of the patients. The only long-term survivors were 3 children transplanted with autologous bone marrow which had been purged in vitro with anti-Y 29/55 and complement.

Autologous bone-marrow transplantation for Burkitt's lymphoma: marrow purging with anti-Y 29/55 monoclonal antibody and complement.

Reinfusion of undetected tumour cells is a possible cause of relapse after autologous bone-marrow transplantation. In this paper, a system for in-vitro purging of bone marrow is presented which involves the B-cell neoplasia-associated monoclonal antibody anti-Y 29/55 and complement. Five patients with Burkitt's lymphoma were transplanted with purged marrow, demonstrating the clinical feasibility of the method. The pretransplant regimen included vincristine 2 mg/m2, adriamycin 60 mg/m2, four doses of cyclophosphamide 45 mg/kg and total body irradiation with 6 Gy. Tumour control appears to be better in patients with purged bone marrow as compared to an earlier patient group with unpurged marrow.

Human B cell development. I. Phenotypic differences of B lymphocytes in the bone marrow and peripheral lymphoid tissue.

The phenotype of B lineage cells (TdT+, pre-B, IgM+, IgD-, and IgM+,IgD+) in infant and adult human bone marrow was compared with that of B cells seen in peripheral tissues such as tonsil and blood. The range of B cell-associated antibodies used included four reagents with greater than 90% reactivity on peripheral B cells: RFB4 and To15 (both p135, corresponding to CD22), RFB6 (p140 corresponding to CD21), and Y29/55, a unique B cell-specific antibody. In addition, AL-1, an antibody with virtually no reactivity against peripheral B cells was also used. The BM cell subpopulations were heterogeneous in respect of antibody reactivity. The TdT+, pre-B and IgM+, IgD- cells were AL-1+ but did not express membrane antigens recognized by the antibodies To15, RFB4 (CD22), and RFB6 (CD21). TdT+, pre-B cells, and 50% of IgM+, IgD- BM B cells were also unreactive with antibody Y29/55, the other 50% being Y29/55+. In contrast, the IgM+,IgD+ BM B cells, like peripheral B cells, were positive with antibodies To15, RFB4, RFB6, and Y29/55, but reacted only in small numbers with AL-1. The orderly differentiation-linked display of these antigens was also suggested by the findings that normal TdT+, pre-B, and IgM+,IgD- cells expressed the To15 and RFB4 (CD22) antigens in their cytoplasm (in the Golgi region). This observation was confirmed in malignant common acute lymphoblastic and pre-B blast cells, as well as in the corresponding permanent cell lines KM3 and NALM-6. In these lines the membrane expression of To15 and RFB4 could be induced by phorbol ester during a 48 to 72 hr culture period.

Tissue distribution and ultrastructure of B lymphocytes reacting with the monoclonal antibody anti-Y29/55.

The reactivity of normal tonsilar cells with the monoclonal antibody anti-Y29/55 is characterized at the tissue and ultrastructural cytological level. Using an indirect immuno-alkaline phosphatase method on frozen sections the antibody labels mantle zone and germinal center lymphocytes. This staining reaction is more generalized in B-lymphocyte areas than that obtained with antibodies to IgM and IgD. By indirect immunoperoxidase staining, as well as by an indirect rosetting procedure in cell suspensions, the reactive cell population were either small resting lymphocytes or activated lymphocytes corresponding to centrocytes, centroblasts, immunoblasts and plasmoblasts; some plasma cells were also labeled. These results characterize the monoclonal antibody anti-Y29/55 as a pan-B-marker antibody, useful for labeling resting and activated peripheral B-lymphocytes in frozen tissue sections and cell suspensions.

Humoral immune function in pediatric patients treated with autologous bone marrow transplantation for B cell non-Hodgkin's lymphoma. The influence of ex vivo marrow decontamination with anti-Y 29/55 monoclonal antibody and complement.

Elimination of neoplastic B cell populations from autologous bone marrow grafts also removes normal B lymphocytes. This is potentially hazardous for the reconstitution of the immune system in patients undergoing high-dose chemotherapy and total body irradiation followed by autologous marrow rescue. Five pediatric patients with B cell non-Hodgkin's lymphoma in first remission undergoing such a regimen were studied. They received bone marrow pretreated with anti-Y 29/55 monoclonal antibody and complement. B and T lymphocyte subpopulations reached normal levels within 6 months after autologous bone marrow transplantation (ABMT), and serum immunoglobulin levels became normal within 4 to 9 months. Vaccination with diphtheria and tetanus toxoid, trivalent poliomyelitis vaccine of the Salk type, and pneumococcal capsular antigens (38 to 54 months after transplantation) gave rise to specific antibody production. ABO isoagglutinins could be demonstrated in all patients. The response pattern was similar to that of patients who received unmanipulated autologous bone marrow. It is concluded that ex vivo anti-Y 29/55 depletion of the marrow graft does not induce relevant disturbances of humoral immune functions.