Phenotyping of canine lymphoma with monoclonal antibodies directed at cell surface antigens: classification, morphology, clinical presentation and response to chemotherapy.

Forty cases of naturally occurring canine lymphoma were studied using a panel of murine monoclonal antibodies which identify defined subsets of normal canine lymphocytes. The distribution of phenotypes was similar to that which is seen in man in that the majority (78 per cent) of canine lymphomas were of B-cell origin but a definite minority were phenotypically of T-cell (10 per cent) or non-B, non-T-cell (12 per cent) origin. The expression of Ia-like antigens was restricted to B-cell neoplasms. Within each histologic subgroup of canine lymphomas there was considerable heterogeneity of cell surface marker expression. Immunophenotype appeared to correlate with clinical presentation. Finally, the reactivity of lymphoma cells with murine monoclonal antibody DLy-6, an antibody which appears to react with a differentiation antigen on canine B and T cells, strongly predicted the outcome of initial induction chemotherapy in that all ten evaluable dogs with DLy-6-tumors achieved complete responses to initial chemotherapy while only four of 11 dogs with DLy-6+ tumors responded completely.

Monoclonal antibodies to canine cell surface antigens: a comparison of screening assays.

An ELISA for detection of monoclonal antibodies to surface antigens on canine lymphocytes was evaluated, and results were compared with three additional tests: a radioimmunoassay using 125I-labeled staphylococcal protein A (IPA), the fluorescence activated cell sorter (FACS II), and CdL. Three potentially interesting hybridomas were subsequently selected from two fusions.

Purified canine monocytes fail to confer concanavalin-A responsiveness to accessory-cell-depleted lymphocytes.

Monocytes were purified from canine peripheral blood by subjecting Ficoll-Hypaque-separated mononuclear cells to either complement-dependent cytolytic treatment with a panlymphocyte antibody (DLy-6) or rosette formation with human red blood cells (RBCs) followed by adherence to fetal-calf-serum-coated plastic dishes, or sedimentation over discontinuous Percoll gradients. These techniques resulted in monocyte enrichment of 83 +/- 3%, 89 +/-6%, and 42 +/- 3%, respectively. Monocytes purified by either the monoclonal antibody method or rosette formation with human RBCs failed to confer concanavalin-A responsiveness to lymphocytes depleted of accessory cells by discontinuous Percoll gradients. The most potent accessory activity was present among cells from low-density fractions of Percoll gradients and populations that did not form rosettes with human RBCs (monocyte-depleted), indicating that cells other than monocytes are involved in accessory function.

Canine bronchoalveolar cells: antigen-presenting macrophages are Ia-positive, lymphocytes are of non-B lineage.

Subsegmental bronchoalveolar lavages were performed in 18 healthy beagles. The average yield per lavage was 45 X 10(6) cells consisting on the average of 24% lymphocytes, 71% macrophages, and 4% granulocytes. Cells were further examined in cytofluorometric studies using monoclonal (anti-Ia, antilymphocyte, anti-T) and polyspecific (anti-Ig) antibodies. Sixty to 90% of lymphocytes were T cells as determined by the T cell antibody DT-2. No surface immunoglobulin-positive cells (B cells) could be detected. All macrophages expressed Ia antigens (p 29/34) whereas lymphocytes did not. In assays of concanavalin A-induced blastogenesis of thymocytes, alveolar macrophages functioned as accessory cells. The anti-Ia antibody 7.2 interfered with this function, indicating that Ia antigens on canine alveolar macrophages are involved in interaction with T cells resulting in T cell proliferation.

Analysis of alloreactive and cytotoxic canine lymphocytes and their precursors with murine monoclonal antibodies.

Four murine monoclonal antibodies were used in cytolytic assays to identify cell populations involved in canine T cell effector functions. Antibody DT-2, directed at T cells, and antibody DLy-6, a panlymphocyte antibody, inhibited mixed lymphocyte culture (MLC) responses and cytotoxic lymphocyte (CTL) activity only when lymphocytes were treated before culture (day 0), but they had no significant effect on these functions when cells were treated after 6 days in culture. Antibody DLy-1, reacting with canine lymphocytes and monocytes, abrogated MLC responses and CTL activity when responder cells were treated on day 0. When cells were treated after 6 days in culture, MLC responses were reduced to 47% of control, whereas CTL activity increased slightly. In contrast, the anti-Ia antibody 7.2 significantly reduced MLC responses and CTL activity of cells treated on either day 0 or day 6 of culture, suggesting that canine CTL express Ia antigens.

Transfusion requirements after HLA-identical marrow transplantation in 82 patients with aplastic anemia.

Red blood cell (RBC) and platelet (PLT) transfusion requirements during the first 13 weeks after HLA-identical marrow transplantation were studied in 82 patients with aplastic anemia. On the average, patients were given 9 units of RBCs (1-82) and 44 units of PLTS (6-468). The greatest need for support was during the first 4 weeks postgrafting. A multivariate statistical analysis of 22 variables showed that RBC and PLT requirements increased with age. In addition, RBC requirements increased if the patient had isohemagglutinins against the marrow donor's RBC, i.e., if the marrow transplant was across a major ABO barrier.

Stimulation of canine lymphocyte subpopulations separated nonlytically by monoclonal anti-T and polyclonal anti-B cell antibodies.

Canine blood lymphocytes were nonlytically separated on antibody-coated petri dishes into surface immunoglobulin-positive (SIg+) and -negative (SIg-) populations. SIg- cells were further separated into cells reactive or non-reactive with monoclonal antibody DT-2 recognizing canine T lymphocytes. The purity of the three enriched lymphocyte populations exceeded 90% as assessed by immunofluorescence. Mitogen stimulation showed a vigorous response of SIg+ cells to pokeweed mitogen and concanavalin A but only a weak response to phytohemagglutinin. In mixed DT-2- and DT-2+ cells responded to phytohemagglutinin, concanavalin A and pokeweed mitogen, and both populations were good responders in mixed leukocyte culture. Only DT-2- cells were potent stimulators; DT-2+ cells were not. Hence, canine blood T cells can be divided into two subsets, DT-2+ and DT-2-, both of which are responsive to mitogens and alloantigens.

Two monoclonal antibodies (DLy-1 and DLy-6) directed against canine lymphocytes.

Two murine monoclonal antibodies directed against canine lymphocytes are described. DLy-1, raised against puppy thymocytes, and DLy-6, raised against bronchoalveolar lymphocytes, both react with most lymphocytes in peripheral blood, thoracic duct lymph, and bronchoalveolar lavage fluids. DLy-1 also recognizes monocytes and granulocytes. However, it is not reactive with erythrocytes, megakaryocytes, or platelets. Expression of DLy-1 antigen on thymocytes ranged from 5--30%. The distribution of DLy-6 antigens seems to be confined to lymphoid cells. Ten to 60% puppy thymocytes were positive. Interestingly, lymphoblasts formed in response to stimulation with mitogens or alloantigens lacked DLy-6 in contrast to DLy-1 cell surface antigen expression.

A monoclonal antibody (DT-2) recognizing canine T lymphocytes.

A murine monoclonal antibody (DT-2) is described which has been raised against canine thymocytes. DT-2 activates complement and is reactive with most canine thymocytes, peripheral blood T cells, thoracic duct lymphocytes, bronchoalveolar lymphocytes, and T chronic lymphatic leukemia cells. The antibody is nonreactive with surface immunoglobulin-positive blood lymphocytes, monocytes, bronchoalveolar macrophages, null acute lymphatic leukemia cells, granulocytes, erythrocytes, and platelets. In mixed lymphocyte cultures DT-2 and complement eliminate the responding but not the stimulating cell population. Mitogen stimulation (phytohemagglutinin, concanavalin A) experiments revealed that the responding cell affected by DT-2 is of lymphoid and not of monocyte/macrophage origin. All our data suggest that DT-2 is an antibody reacting specifically with canine T cells.

Unusual distribution of Ia-like antigens on canine lymphocytes.

The murine monoclonal antibody 7.2, specific for a framework determinant of human Ia antigens, cross-reacts with canine cell membranes recognizing a bimolecular complex (29,000 and 34,000 daltons) similar to that described in man. We investigated the distribution of these Ia-like antigens on mononuclear cells in peripheral blood, thoracic-duct lymph, marrow, alveolar lavage fluids, lymph nodes, and thymuses from normal dogs. By complement-mediated cytotoxicity and indirect immunofluorescence, virtually all lymphocytes expressing surface immunoglobulin (B lymphocytes), monocytes/macrophages, dendritic cells, and many thymus-epithelial cells were Ia-positive. Furthermore, most non-B-lymphocytes in peripheral blood, thoracic-duct lymph, and lymph nodes expressed Ia antigens. Alveolar (T) lymphocytes and most thymocytes were Ia-negative. Generally, fluorescence intensity was higher on monocytes/macrophages and B lymphocytes than on non-B-lymphocytes. In mixed leukocyte cultures and concanavalin A-induced blastogenesis assays, treatment of responder cells with antibody 7.2 and complement abolished proliferation. Proliferative responses could not be restored by adding untreated accessory cells, indicating that cytolytic treatment had eliminated responder T-lymphocytes. However, addition of antibody alone to cultures had no significant effect. These studies indicate that most mature canine T-lymphocytes express Ia-like antigens. Whether this is an intrinsic property of canine cells or possibly related to continuous in vivo stimulation remains to be determined.

Nonspecific acid esterase activity as a marker for canine T lymphocytes.

Canine T lymphocytes are as yet poorly defined. Since focal paranuclear positivity for nonspecific esterase (alpha-naphthyl acetate esterase--ANAE) has been shown to be a reliable marker for T cells in humans and mice, canine lymphoid tissue sections, and cell suspensions were stained for this enzyme. Sections of five lymph nodes and 11 puppy thymuses displayed the same distribution patterns of focally ANAE-positive cells as were found in humans and mice in both organs: in thymuses only the medulla contained positive cells, and in lymph nodes only the thymus-dependent, paracortical zones, but not central areas of germinal centers, displayed focal ANAE reactivity. Furthermore, 56-78% of peripheral blood mononuclear cells, 60-71% of thoracic duct lymphocytes, and 10-15% of suspended thymus cells were positive for ANAE. It is concluded that focal ANAE reactivity is a marker for a canine T cell subpopulation.

Purification and characterization of human bone marrow lymphocyte subpopulations: evidence for the existence of an alloreactive immature T cell.

This study was conducted in order to clarify further the role of bone marrow lymphocyte (BML) subsets in graft-versus-host disease (GVHD) after clinical BM transplantation. Human BML separated by velocity sedimentation through a continuous sucrose gradient were found to consist of 38% T, 21% B and 41% null cells. T cells were purified from BML or peripheral blood lymphocytes (PBL) by rosette formation with sheep red blood cells (SRBC) and subsequent gradient centrifugation. Mitogenic stimulation of BML, PBL and the respective T cell subsets revealed the well-known reactivity of mature T lymphocytes, whereas non-T lymphocytes comprising T cell precursors showed only a weak response. In allogeneic stimulation kinetics, however, non-T-BML disclosed a delayed but marked responsiveness as compared to BML and T-BML suggesting that a T cell precursor matures under culture conditions. This cell type could be involved in GVHD in addition to mature T cells after clinical bone marrow transplantation.