Transplantation tolerance following conditioning with C1q-affinity anti-Thy-1 monoclonal antibodies.
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Animals↗
Biomedical subjects
S Thierfelder
Publications and source records attributed to S Thierfelder.
Lymphocyte surface markers profile in acute lymphoblastic leukaemia.
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Adolescent↗
Effective avidin coating of erythrocytes by disulfide bond formation. Production of a stable rosetting agent for multiple applications.
A method is described for covalently coupling avidin to the membrane of glutaraldehyde-treated erythrocytes. The method is based on the use of the heterobifunctional coupling reagent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and appears to be more effective than other protein coupling techniques. The resulting avidin-coated red cells are very stable and can be stored for more than 1 year without aggregation or loss of biotin-binding activity. They can be used in various rosetting assays and were tested for T cell depletion or negative cell selection with five biotinylated monoclonal antibodies.
Animals↗
Simultaneous demonstration of two antigens on single T cells using antibodies with contrasting labels.
We describe a double labeling method for the discrimination of 2 antigens on single cells. It consists of a combination of 3H-immunoautoradiography and immunocytochemistry applied to cells previously fixed on poly-L-lysine (PLL)-coated multispot slides. The method has been applied to various mouse cells contemporaneously labeled with 2 different monoclonal antibodies. In order to distinguish the attached antibodies unambiguously, they were labeled with contrasting markers. One of the antibodies was marked with tritium blackening the photographic film that covers the slide. The other was detected with the peroxidase-anti-peroxidase (PAP) method forming a reddish precipitate. The contrast between the reddish reaction product of the PAP-labeled antibody and the black silver grains allows cells, specifically labeled with both antibodies, to be distinguished from cells labeled with only one or neither of the antibodies. Tritium-labeled antibodies were introduced because of their advantage over antibodies labeled with iodine in the closer localization of the silver grains to the bound antibody and their much longer halflife (60 days versus 12 years). In this study we applied a tritium-labeled anti-Thy-1.1 together with anti-Lyt-1 monoclonal antibody for studying the distribution of the corresponding antigens on lymphocytes in the mouse thymus and lymph node cells.
Animals↗
Combined prophylactic suppression of graft-versus-host and host-versus-graft reactions following treatment of prospective bone marrow recipients with rat IgG 2b anti-mouse T cell antibodies.
A new approach to avoid typical complications from bone marrow transplantation into MHC different mice was studied. Rat monoclonal anti-Thy-1 antibodies of the IgG 2b isotype were identified, which inhibit T lymphocytes in vivo so that transplanted donor T cells as well as residual T cells of the conditioned marrow recipient were suppressed. A single injection of these antibodies after irradiation and before marrow transplantation did not only prevent graft-versus-host mortality but suppressed also host-versus-graft reactivity so that the radiation dose necessary for engraftment of donor cells differing in H-2, IA (both haplotypes) major histocompatibility antigens could be reduced to 6.0 Gy. In addition an anti-T leukemic cell effect from the injected monoclonal T cell antibodies was observed.
Animals↗
Improved detection of terminal transferase (TdT): the use of detergents on glutaraldehyde-fixed non-dehydrated cells prevents denaturation and diffusion artifacts.
TdT as an intranuclear enzyme mainly of immature lymphoid cells is commonly determined immunologically using air-dried cell smears fixed with methanol. Both cell dehydration and alcohol fixation were found here to denature TdT and surface antigens. This could be prevented by using non-dehydrated cells bound electrostatically to poly-L-lysine-coated slides, fixed minimally with glutaraldehyde and rendered permeable to antibodies by the non-ionic detergent Brij 56. Crosslinking glutaraldehyde in addition prevented diffusion of TdT to extranuclear sites. By avoiding artifacts of denaturation and diffusion, a higher sensitivity in the detection of TdT was achieved despite considerably lower quantities of antibody.
Cell Membrane Permeability↗
Antilymphocytic antibodies and marrow transplantation. VIII. Recipient conditioning with Clq-affine monoclonal anti-pan T antibodies prevents GVHD in homozygous fully mismatched mice.
An approach to suppressing secondary disease with antibodies was studied that differed from conventional antibody treatment of donor marrow in vitro. It consisted of the selection of anti-Thy-1 antibodies with high affinity for Clq, the first subunit of the complement cascade, and a single injection of such antibodies into prospective irradiated marrow recipients. Monoclonal mouse IgM and rat IgG 2c antibodies of high titers in complement-dependent test systems but with low affinity for Clq caused little immunosuppression. Monoclonal rat IgG2b or mouse IgG2a anti-Thy-1 antibodies with high affinity for Clq prevented acute and chronic mortality of graft-v-host disease (GVHD), however, when injected in irradiated CBA or AKR mice prior to C57BL/6 spleen and/or bone marrow cell transfusion. This treatment simultaneously suppressed residual host-v-graft reactivity of the irradiated mice, so that permanent hematopoietic engraftment ensued even at 5 or 6 Gy. Full chimerism and specific tolerance were obtained. Primary immune response to SRBC was clearly depressed in the chimeras; secondary immune response was not. Clearance of T cell antibody activity (greater than 6 days), timing, and dose of injected antibody, as well as other modalities of the conditioning treatment that may have contributed to the remarkable immunosuppression, are discussed.
Animals↗
In-vitro induction of some features of hairy cell leukemia in chronic lymphocytic leukemia and immunocytoma cells.
In an attempt to induce in vitro differentiation we exposed cells from patients with chronic lymphocytic leukemia (CLL) and with immunocytoma (IC) to 12-0-tetra-decanoylphorbol-13-acetate (TPA) at 160 nM. After 3 to 5 days of culture cells became enlarged and the CLL cells developed a basophilic cytoplasm with an excentric nucleus. In some instances cells with many fine projections were seen. We then employed the monoclonal antibody (MAB) HD 6, which was generated against hairy cell leukemia (HCL) cells and reacts most strongly with such cells but is unreactive with plasmocytoma cells and with most CLL cells. TPA treatment induced a greatly enhanced HD 6 binding in CLL and IC cells compared to controls as demonstrated by indirect immunofluorescence. Cytochemical studies revealed that at the same time an acid phosphatase was induced, which was found to be tartrate-resistant in every instance tested. Thus, several features of HCL can be induced in CLL and IC demonstrating the close relatedness of these entities.
Cell Differentiation↗
Quantitation of antibody uptake on A group erythrocytes using immunoautoradiography and monoclonal IgM anti-A.
The number of antibody molecules on individual erythrocytes was counted in A1, A2, A3 B and A group individuals using immunoautoradiography (IAR) and monoclonal IgM anti-A1. Quantitation was also done for A group pregnant women. The number of antibody molecules on different red cells of an individual varied widely. Gross variations were also noted in cells of different individuals from one and the same group. The mean values of the uptake of the number of antibody molecules showed the following range A1 greater than A2 greater than Ax greater than A3B. When compared to the average for total A1 adults, red cells of pregnant women and newborn infants showed a 10.7% and 19.7% reduction respectively, in antibody uptake. The mean number of antibody molecules per A1 adult red cells was 5.6 +/- 3 X 10(4), while A2 had 0.85 +/- 0.35 X 10(4) molecules, thus showing a significant quantitative variation.
ABO Blood-Group System↗
Antilymphocytic antibodies and marrow transplantation. VII. Two of nine monoclonal anti-Thy-1 antibodies used for pretreatment of donor marrow suppressed graft-versus-host reactions without added complement.
Eleven monoclonal antibodies of rat or mouse origin against the mouse pan T antigen Thy-1 were compared for their ability to reduce mortality from graft-versus-host disease (GVHD) when incubated with donor marrow. Spleen and bone marrow cells were transferred to F1 hybrids or to fully allogeneic (H-2 I-A incompatible) mice. Particular attention was paid to whether complement (rabbit) enhanced the anti-GVHD effect of the antibodies in homozygous histoincompatible chimeras: without complement, 5 IgM anti-Thy-1 and 2 IgG2a anti-Thy-1 did not reduce GVHD. With complement, acute GVHD was completely suppressed. Two of two rat IgG2b anti-Thy-1, however, suppressed acute GVHD without the need for added complement. One of the two also prevented chronic mortality following two haplotype-unmatched transplantation. This antibody, in contrast to other complement-fixing anti-Thy-1 antibodies, had previously been shown to delay rejection of skin allografts. Its specificity did not differ from other complement-dependent Thy-1 antibodies when tested in a cross-blocking radioimmunoassay, and it also had the lower affinity for Thy-1. It seems therefore that only a minority of the antibodies were able to fully exploit the marrow recipients' opsonizing capacity for suppression of GVHD. The important clinical implications of the remarkable difference in immunosuppression of various monoclonal antibodies with comparable specificity and capacity to fix complement in vitro are discussed.
Animals↗
Autologous bone marrow transplantation in leukemia.
In patients without HLA-identical donors autologous bone marrow transplantation should be considered as an alternative to conservative treatment of recurrent malignancies. Any patient in complete remission is suitable for an autologous bone marrow transplantation. Bone marrow is aspirated from the patient during complete remission, frozen, and stored at -196 degrees C, and it can be used for hematopoietic reconstitution after high-dose chemotherapy and radiotherapy of the patient. In patients with solid malignant tumors or leukemia the elimination of clonogenic tumor cells from the graft by means of physical separation techniques has not been successful. However, studies in mice and more recently in patients indicate that antibodies destroy residual leukemic cells of the bone marrow transplant without jeopardizing the capacity for regeneration. During our first study in children with acute lymphoblastic leukemia, bone marrow was taken from 50 patients in remission and cryopreserved. Preservation time ranged from 1 to 60 months and the mean number of aspirated nucleated bone marrow cells was 4 X 10(8)/kg body weight (1.9 - 7.4 X 10(8)/kg). After cryopreservation the stem cell viability of the standard samples was 80% - 100%. Autologous bone marrow was transplanted in five patients with common ALL (cALL). The transplant was prepared by incubation with specific antisera of high cytotoxic, selective activity against cALL cells. Our preliminary results show that bone marrow cells aspirated in remission and prepared with antileukemic antisera stimulate repopulation of the recipient's bone marrow and effect hematopoietic regeneration.
Animals↗
Serological identification and separation of canine lymphocyte subpopulations.
In vitro treatment of bone marrow grafts with absorbed rabbit-antidog thymocyte globulin (ATG) prevented graft-versus-host disease in a substantial number of the dogs differing by one DLA haplotype. Absorbed ATG has now been used for serological identification of canine lymphocyte populations. Specific labeling of canine T-lymphocytes by absorbed ATG could be demonstrated by (a) a distribution of ATG-positive cells in suspensions of canine lymphoid organs similar to that of T cells observed in other species and by specific staining of paracortical thymus-dependent lymph node areas in immunohistochemistry, (b) complementary labeling of nylon-wool-separated cells by ATG and antiimmunoglobulin sera, and (c) correlation of ATG surface labeling with responder activities in mixed lymphocyte cultures.
Animals↗
[Lymphocyte markers in sarcoidosis and tuberculosis].
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Adult↗
Serological inhibition of graft versus host disease: recent results in 28 patients with leukemia.
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Acute Disease↗
Crossreaction of monoclonal anti-T-cell antibodies: implications for classifying B-cell leukemias.
Cell samples from 62 patients with B-cell leukemias (33 CLL, 8 IC, 6 PLL, 4 HCL, 2 ALL and 9 other NHL) were tested with a series of monoclonal antibodies (A50, T101, Lyt2, Leu 1, M203) directed against T cells and shown to crossreact with B-CLL. The results demonstrate a heterogeneity of B-cell leukemias as all typical cases of CLL were reactive whereas most other cases were negative. Using the fluorescence-activated cell sorter, a somewhat stronger reaction was seen in early or benign cases of CLL, compared to advanced cases. All B-cell leukemias tested expressed the Ia antigen.
Adult↗
Immunofluorescence with monoclonal antibodies on poly-L-lysine coated slides: an alternative to conventional methods.
It is sometimes difficult to perform immunofluorescence tests because of low cell numbers. Therefore a simple method using poly-L-lysine coated slides was applied to immunofluorescence and compared with the routine suspension method. Peripheral blood lymphocytes from normal persons, samples of normal bone marrow, thymus and tonsil as well as samples from patients with leukemia or lymphoma were tested with these two methods using 20 different monoclonal antibodies. The PLL-slide method was shown to be comparable with the suspension method and is an alternative in case of low cell numbers, since as few as 3 X 10(4) cells can be tested for one antigen.
Antibodies, Monoclonal↗
Immunosuppression with monoclonal antibodies. A model to determine the rules for effective serotherapy.
Despite the range of available T cell specific monoclonal antibodies, there are no established rules to predict which might be immunosuppressive. We here describe a series of five rat monoclonal antibodies to a defined T cell antigen (mouse Thy-1) and evaluate their ability to immunosuppress mice. When compared with rabbit anti-lymphocyte globulin, only one of these monoclonal antibodies was able to delay skin allograft rejection and eliminate antibody responses to sheep red blood cells. This antibody was immunosuppressive following intra-peritoneal administration, even though it did not eliminate all of the T cells in vivo. Two factors may be relevant in determining the immunosuppressive properties of this reagent. First, the monoclonal antibody is of the rat IgG2b sub-class, and second, the specificity of the antibody is different to the other monoclonal antibodies in that it reacts with sub-populations of peripheral T cells, thymocytes and non-T cells. In practice, this suggests that to derive suitable monoclonal antibodies for human serotherapy, one should give attention to both the subclass and the fine specificity of the antibody for the target molecule.
Animals↗
Antilymphocytic antibodies and marrow transplantation. VI. Absence of immunosuppression in vivo after injection of monoclonal antibodies blocking graft-versus-host reactions and humoral antibody formation in vitro.
The in vivo and in vitro effectiveness of several monoclonal antimouse T and B cell antibodies, of anti-Th-1 and of Iak serum, as well as of ATG were compared. The parameters were prolongation of skin graft survival, prevention of graft-versus-host disease (GVHD), antibody and primary and secondary plaque formation against sheep redblood cells (RBCs), and T cell depletion of lymphoid tissues. In general, in vitro effectiveness of the monoclonal antibodies exceeded their in vivo effectiveness. Skin graft survival was prolonged by ATG, but not by monoclonal anti-T, or anti-T plus anti-B antibody. GVHD was prevented by in vitro incubation of donor bone marrow with monoclonal anti-Th-1, but in vivo treatment of marrow donors was ineffective. Treatment with ATG was successful. Anti Iak antibody blocked plaque formation by spleen cells incubated with sheep RBCs, but had no effect on secondary plaque formation when given in vivo. Neither was there any in vivo effect of anti-Iak or anti-Th-1 on antisheep RBC agglutinin formation. ATG was effective in both of these assays, although its cytotoxic and complement-fixing titer did not exceed that of anti-Th-1 or anti-Iak. Although anti-Th-1 was cleared more rapidly from the serum of mice expressing the corresponding Th-1 alloantigen, than from mice with the noncorresponding alloantigen and although anti-Th-1 was shown to bind to the T cell areas of the lymphoid tissue, it did not--unlike ATG--deplete these areas of T cells. Possible reasons for the difference in effectiveness of in vitro and in vivo application of these monoclonal antibodies are discussed.
Animals↗