Bone-marrow purging with monoclonal antibodies and human complement in ALL and AML.
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Antibodies, Monoclonal↗
Biomedical subjects
H Stockinger
Publications and source records attributed to H Stockinger.
Mammalian T-lymphocyte antigen receptor genes: genetic and nongenetic potential to generate variability.
T lymphocytes of higher vertebrates are able to specifically recognize a seemingly unlimited number of foreign antigens via their receptors, the T cell antigen receptors (TCRs). T lymphocytes mature by passing through the thymus and acquire antigen specificity by expressing the TCR molecules on their cell surface. Genetic and somatic diversification mechanisms give rise to the enormous degree of TCR variability observed in mature T cells: germline and combinatorial diversity as well as junctional and the so-called N-region diversity. In contrast to the situation in immunoglobulin genes somatic hypermutation does not seem to play a significant role in TCR diversification. It is argued here that the enzyme terminal nucleotidyl-transferase is potentially a major factor in generating the immense diversity. We propose furthermore that this enzyme ensures the flexibility of T cell responses to novel antigens by random insertion of so-called N-region nucleotides. Apart from the physiological functions of TCR genes any involvement in the etiology of T cell neoplasia remains to be proven.
Animals↗
Induction of human complement activation without cytolysis by mouse monoclonal antibodies to human leukocyte antigens.
Ten monoclonal antibodies to human leukocyte subsets that had previously been shown to lyse their respective target cells in the presence of rabbit serum as complement source were evaluated for their cytolytic capacity with human complement. Four of the ten were lytic with human complement. All were of IgM type. Antibodies were also evaluated for their capacity to induce C3 binding to target cells. With this method we could demonstrate that, indeed, 3 of the 6 noncytolytic antibodies had the capacity to initiate the human complement activation process and to induce C3 binding. Two of these 3 antibodies were of IgM class (VIT3 and VIM13), one of IgG3 (562). From the practical point of view the most interesting of these 3 antibodies is the nonmitogenic anti-CD3 pan-T cell antibody VIT3. Therefore, this antibody was analyzed in more detail. VIT3 antibody concentrations needed to induce detectable C3 binding to human T cells are very low (down to 1 ng VIT3/ml). Human serum as complement source can also be considerably (100X) diluted before C3 binding becomes undetectable. Activation of C3 is a prerequesite for VIT3-induced C3 binding, and bound C3 seems to lack the C3a fragment. Bound C3, in contrast to the quickly occuring antigenic modulation of the CD3 complex and the simultaneous disappearance of the antibody coat, remains expressed also after prolonged incubation at 37 degrees C. C3 fragments bound to T cells after activation with VIT3 are also recognized by cells bearing C3 receptors of types CR1 and CR2.
Antibodies, Monoclonal↗
Human blood basophils display a unique phenotype including activation linked membrane structures.
To evaluate the membrane marker profile of human basophils a panel of well-established monoclonal antibodies (MoAbs, n = 60) was used for a combined toluidine/immunofluorescence staining procedure. Myeloid-associated MoAbs (particularly MoAbs against the LFA-1 family (CD11, CDw18), MoAbs directed against lactosylceramide (CDw17), anti-glycoprotein (gp) 150 MoAbs MCS 2 and MY 7 (CDw13), anti-gp 67 MoAb MY 9, anti Fc gamma-receptor (mol wt 40 kd) MoAb CIKM5, anti-CR 1 MoAb E 11, and the antiglycolipid MoAb VIM-2) were reactive with basophils, indicating a close relationship to other mature myeloid cells. Under normal conditions, basophils surprisingly express at least three activation-linked structures not detectable on mature neutrophils, ie, the p45 structure defined by MoAbs OKT-10 and VIP-2b, the p24 structure identified by the CD9 MoAb BA-2, and the receptor for interleukin 2 (IL 2) recognized by three different MoAbs (anti-TAC, IL2RI, anti-IL 2). Moreover, under short-term culture conditions basophils both in mononuclear cell (MNC) suspension and as purified fractions display the HLA-DR and T4 antigens. The neutrophilic/eosinophilic structure 3-fucosyl-N-acetyllactosamine is expressed on basophils only after neuraminidase treatment. Basophils were not stained at all by CD 16 MoAbs directed against the Fc gamma-receptor (mol wt 50 to 70 kd) of neutrophils, by the MoAb 63D3 (CDw12) recognizing the monocyte/granulocyte-associated p 200 antigen, and by the CDw 14 antibodies (VIM-13, Mo 2) defining the monocyte-specific structure p 55. Enriched basophils freshly obtained from chronic granulocytic leukemia (CGL) patients yielded identical results in FACS analyses. In summary, these data indicate that basophils generate a unique combination of surface determinants and possibly represent an activated cell population.
Antibodies, Monoclonal↗
Use of a cocktail of monoclonal antibodies and human complement in selective killing of acute lymphocytic leukemia cells.
Autologous remission bone marrow is a potential source of repopulative stem cells after ablative chemoradiotherapy of tumor patients. Even with remission bone marrow, one major obstacle to use of autologous bone-marrow support is the danger of reinfusing viable tumor cells. This report describes a purging protocol with human complement, which seems suitable for eliminating acute lymphatic leukemia (ALL) cells of the common ALL type. Lysis of ALL blasts is induced with a cocktail of 3 monoclonal antibodies of IgM type (termed VIB-pool). These are directed against the CALLA (CD 10) antigen (VIL-AI antibody) and against 2 different epitopes of the CD24 surface structure (VIB-C5 and VIB-E3 antibodies). The purging efficiency was evaluated with leukemic cell lines of the common ALL type (Reh-6 and Nalm-6) and with blast cells from common ALL patients. Optimal lysis was obtained with antibody and human serum concentrations as low as 1 microgram/ml and 7% respectively. As a standard purging protocol we propose one 20-min incubation at room temperature with antibody followed by two 30-min incubations at 37 degrees C with 25% human complement. In dye exclusion test 99% purging efficiency and in clonogenic assays detecting elimination of up to 5 logs of clonogenic tumor cells 99.99% (= 4 logs) purging efficiency were achieved. Treatment with VIB-pool and human complement had no negative effect on the growth of the normal hemopoietic progenitor cells CFU-GM, CFU-E and BFU-E.
Animals↗
Recombinant interferon-gamma induces interleukin 2 receptors on human peripheral blood monocytes.
The present report describes the inducibility of IL 2 receptors on human peripheral blood monocytes. Although freshly isolated monocytes are IL 2 receptor negative, approximately one-third of the cells react with the anti-Tac antibody within 18 hr of culture. IFN-gamma is found to double both the number of positive cells and the number of binding sites, whereas IL 2 has no influence on the IL 2 receptor expression on monocytes. Anti-Tac precipitates from monocyte lysates several protein bands of similar m.w. to those previously found with activated T and B cells. Finally, IFN-gamma-induced, but not resting, monocytes are found to bind recombinant IL 2. We conclude that IFN-gamma induces peripheral blood monocytes to express IL 2 receptors similar in structure to those found on activated T and B lymphocytes.
Antibodies, Monoclonal↗
T cell stimulation via the erythrocyte receptor. Synergism between monoclonal antibodies and phorbol myristate acetate without changes of free cytoplasmic Ca++ levels.
We observed that certain E-receptor antibodies (CD2 antibodies) can induce proliferation of resting human T cells in the presence of PMA, while other CD2 antibodies fail to have such an effect. The same CD2 antibodies that were mitogenic in the presence of PMA (9.6, X11, VIT13), but not the nonreactive ones, were also able to induce T cell proliferation via the so-called alternative pathway of T cell activation, i.e., when added pairwise in certain combinations to T cells in the absence of PMA. While the simultaneous addition of two comitogenic CD2 antibodies (9.6 or X11 plus VIT13) or the addition of a single nonmitogenic CD3 antibody (VIT3) led to a clearcut elevation of intracellular Ca++ levels, no such effect could be observed after the addition of one CD2 antibody alone. Even in the presence of PMA, one comitogenic CD2 antibody alone was unable to trigger a significant Ca++ response, although this combination induced a proliferative response. These data indicate that, distinguishable by their influence on free cytoplasmic Ca++, there are two different mechanisms of T cell activation via CD2. While simultaneous triggering with two antibodies leads to cell proliferation preceded by an increase of Ca++ levels, stimulation with one antibody plus PMA results in proliferation without a measurable early Ca++ response. We conclude that T cells treated by certain CD2 antibodies alone already recognize an activation signal probably unrelated to Ca++ homeostasis, a signal that can further be developed by PMA to result in a completely developed proliferative response.
Antibodies, Monoclonal↗
A rapid and simple immunoperoxidase staining procedure for blood and bone marrow samples.
A fast and simple indirect immunoperoxidase staining technique is described, which can also be used for the characterization of cells with high endogenous peroxidase activity such as many haemopoietic cells. It is based on the combination of a newly developed glucose-oxidase plus glucose procedure for the inhibition of endogenous peroxidase activity with a standard 2 or 3 layer immunoperoxidase staining protocol. Glucose-oxidase plus glucose mixture completely inhibits endogenous peroxidase activity without having a detectable deleterious effect on any of the cellular antigens so far studied. In many instances this permits the use of only 1 layer of horseradish peroxidase (HRP)-labelled antibodies. The glucose-oxidase plus glucose mixture can also be added to cells together with the HRP-labelled antibody solution without losing its inhibitory effect for endogenous peroxidase activity and without leading to a visually detectable loss of the activity of the HRP conjugate. Consequently, the separate incubation step previously necessary for the inhibition of endogenous peroxidase activity becomes superfluous.
Antibodies, Monoclonal↗
Human DNA sequences isolated with an immunoglobulin switch region probe: sequence, chromosomal localization, and restriction fragment length polymorphisms.
We have screened a human genomic DNA library with an immunoglobulin (Ig) derived switch (S) region specific probe for homologous sequences. Five Ig independent phage clones were isolated and characterized. The S sequence homologous DNA fragments are short compared to the S region sequences. Ig independent S sequences are flanked by highly repetitive DNA elements and perfect inverted repeats can be demonstrated in their close vicinity. Using subclones of S homologous sequences restriction fragment length polymorphisms were shown within DNA of different T cell leukemias, Burkitt lymphomas, lymphoblastoid cell lines, and DNA of healthy individuals. One of the five clones isolated with the S region probe was evidently localized to chromosome 2 and/or 10 and showed a complex hybridisation pattern with several different human DNAs. S homologous sequences of another clone are most likely localized on chromosome 1. It is possible that these Ig independent S sequences have arisen by amplification and transposition and that they are involved in genetic recombination.
Base Sequence↗
Analysis of CD3-antibody-mediated inhibition of T-cell activation.
In this study the influence of a non-mitogenic anti-CD3 antibody on accessory cell-dependent antigen and mitogen-induced T-lymphocyte proliferation has been investigated. The antibody was found to completely inhibit PHA, Con A, PWM, and tetanus toxoid stimulation, with no effect on the proliferation induced by the calcium ionophore A23187. VIT3 completely abrogated the production of IL-2 by lectin-stimulated T cells. It had no effect, however, on the IL-2-dependent proliferation of preactivated T-cell blasts. In addition, the antibody was able to elevate free cytoplasmic Ca2+ levels within minutes after the addition to T cells. Detailed time kinetic analyses revealed that the time interval critical for inhibition was significantly dependent on the interaction between T cells and accessory cells. Under standard conditions, in the presence of 10% non-T cells as accessory cells 50% inhibition was still achieved when VIT3 was added to PHA-stimulated T cells as late as 8 hr after the onset of culture. Delayed addition or a decrease in the number of added accessory cells significantly prolonged this time period. Lectin-stimulated T cells can thus obviously be inhibited via CD3 as long as they have not received all signals including those delivered by accessory cells. Although the underlying mechanisms are not clear so far, the observation that VIT3 at the same time triggers an early cytoplasmic Ca2+ response might indicate that it thereby actively interferes with antigen and lectin-initiated activation processes.
Antibodies, Monoclonal↗
[Lineage infidelity of leukemia cells].
We phenotyped 548 blast populations from patients with acute de novo leukemia with a panel of 27 monoclonal antibodies and an antiserum against the enzyme terminal deoxynucleotidyl-transferase (TdT). In 56 cases (10,2%) we found a marker profile consisting of both myeloid and lymphoid characteristics (biphenotypic) leukemia = interlineage infidelity). In 49 out of the 56 cases the blast populations were clearly related to the myeloid (n = 41) or the lymphatic (n = 8) system. In those blast populations only one characteristic of the other differentiation could be detected. However, in 7 cases more than one myeloid as well as lymphatic characteristics were found on the same blast cells. In 4 cases (0.7%) the immunological phenotyping indicated the presence of two separate blast populations. The occurrence of a stem cell leukemia, an abnormal derepression of a genome, a defect of the membrane synthesis and the existence of a normal counterpart are taken into consideration as a cause of an "atypical" marker profile.
Acute Disease↗
[Considerations on the therapeutic use of monoclonal antibodies].
The availability of monoclonal antibodies (MoAb) decisively improved and extended the analytical and diagnostic possibilities both in science and in clinical medicine. Monoclonal antibodies are also important tools for the isolation and purification of biologically active components. However, the therapeutic applications of monoclonal antibodies require manifold clarification. In this manuscript an attempt is made to present a short survey of the current stage of development in the therapeutic applications of MoAb and to discuss the foreseeable future in this research area. Possible fields of application, such as selective cell manipulation, drug targeting, detoxification by neutralization and/or immune clearance, specific antibody substitution are summarized and the negative and positive results so far achieved are discussed. Ex vivo therapy with monoclonal antibodies, which has already been used in a number of hospitals, is dealt with in detail. Problems already encountered, as well as anticipated in the course of the therapeutic implementation of monoclonal antibodies are discussed.
Antibodies, Monoclonal↗
Kinetics of activation antigen expression by in vitro-stimulated human T lymphocytes.
In this study a panel of monoclonal antibodies was used to investigate the kinetics of the appearance of activation-linked surface determinants as well as cytoplasmic and nuclear determinants in human T cells following lectin stimulation. Well known activation markers, such as Ia/DR, transferrin receptor, IL-2 receptor, T10, and gp24, were compared and investigated together with the T13 structure, recently found in this laboratory. T13, not demonstrable on resting T cells, could be seen within 24 hr after lectin stimulation. Kinetics of the appearance were similar to IL-2 receptor and transferrin receptor expression. Ia/DR synthesis was investigated separately for each polypeptide and the cytoplasmic invariant gamma-chain expression could be demonstrated for the first time with a gamma-chain-specific monoclonal antibody VIC-Y1. Moreover, gamma-chain synthesis seems to precede alpha- and beta-chain occurrence in human T cells. In addition, data from quantitative studies on antigenic densities are presented.
Antibodies, Monoclonal↗
Unexpected absence of a myeloid surface antigen (3-fucosyl-N-acetyllactosamine) in promyelocytic leukemia.
According to the criteria of the FAB classification we diagnosed acute promyelocytic leukemia in 19 out of 191 AML blast cell populations. The reaction pattern with the anti-myeloid monoclonal antibodies VIM2, MCS2 and MY9 and the MHC-class II antibody VID1 in all cases showed the characteristics which were expected from the normal granulopoiesis. The monoclonal antibody VIM-D5, which recognizes the myeloid cell surface structure 3-fucosyl-N-acetyllactosamine (3-FAL), was not reactive with the majority of promyelocytic blast cells, however. In 13 cases 3-FAL was not detectable and in 6 cases only low or moderate reactivity of VIM-D5 with blast cells was observed. The phenotype of leukemic promyelocytes thus differs from the majority of normal promyelocytes in this respect.
Antibodies, Monoclonal↗
Analysis of T cell activation with a non-mitogenic anti CD3 antibody and the phorbol ester TPA.
We used a non mitogenic anti CD3 antibody, termed VIT3, to study the signals required for the activation of normal resting T lymphocytes. Besides being not mitogenic, this antibody completely inhibits mitogen induced proliferative responses. In the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), however, VIT3 induces DNA replication and cell proliferation comparable to PHA responses. In addition, T cells cultured with VIT3 plus TPA but not with VIT3 or TPA alone express high levels of interleukin 2 (IL-2) receptors and transferrin receptors. This co-stimulation appears to be accessory cell independent. Purified T cells respond equally well to VIT3 plus TPA as do unseparated mononuclear cells and addition of non-T cells has no enhancing effect. We conclude that the IgM antibody VIT3, although non-mitogenic by itself, still delivers a first and for the activation essential signal. In resting T cells this signal does not induce demonstrable anti-Tac antibody binding nor does it lead to a fully developed proliferative response in the presence of recombinant IL-2. Together with a second signal provided by TPA it serves, however, as a potent inducer of T cell growth.
Animals↗
Lymphocytes of haemophilia patients treated with clotting factor concentrates display activation-linked cell-surface antigens.
Peripheral blood lymphocytes from 30 patients with haemophilia A were investigated for the expression of six activation-linked cell surface antigens as well as with regard to the relative proportions and total numbers of Leu-3a and Leu-2a positive cells. Twenty-nine of the haemophilia patients showed no clinical symptoms of immunodeficiency or infection whereas one patient presented the typical symptomatology of the acquired immunodeficiency syndrome (AIDS). The proportions and total numbers of circulating lymphocytes displaying Ia antigens, the p45 protein and/or the two recently defined surface antigens VIP-4 and VIP-5 were significantly increased in haemophilia patients when compared to healthy individuals of the same age group. No such increases could be observed for transferrin receptor and IL-2 receptor expression. After the observation of depressed helper/suppressor T-cell ratios in many haemophiliacs, the expression of activation linked surface antigens represents a further lymphocyte abnormality which resembles the findings in AIDS and its prodromal stages and can also be found in certain viral and parasitic diseases.
Acquired Immunodeficiency Syndrome↗
Studies on differentiation of committed hemopoietic progenitor cells with monoclonal antibodies directed against myeloid differentiation antigens.
In the present study we evaluated the reactivity of monoclonal cytotoxic antibodies directed against myeloid differentiation antigens with hemopoietic precursor cells. VIM-D5 and VIM-2 inhibit the proliferation of clusters and colony formation after seven days of incubation. Day-14 CFU-GM are not affected by these antibodies. After complement-mediated cytolysis with VIM-2, the number of BFU-e was significantly reduced; however, this effect was largely abrogated by addition of leukocyte-conditioned medium to the cultures as an exogenous source of burst-promoting activity. Furthermore, the maturation of myeloid progenitor cells has been examined by delayed treatment with VIM-D5 and complement during the in vitro culture period. In these experiments a different maturation behavior of day-7 and day-14 CFU-GM was demonstrated. To study whether a cryptic carbohydrate structure is present on more immature CFU-GM, the effect of neuraminidase treatment of myeloid progenitor cells on reactivity with VIM-D5 was tested.
Antibodies, Monoclonal↗
M2, a novel myelomonocytic cell surface antigen and its distribution on leukemic cells.
The selectivity of a novel myelomonocytic cell surface antigen, designated M2, has been assessed in a series of 208 leukemias. The M2 antigen is defined by a monoclonal antibody (VIM-2) of the IgM class. Its expression within the normal hemopoietic system is restricted to myelomonocytic cells. Lymphocytes, erythrocytes, thrombocytes and their morphologically recognizable precursors are negative. Sixty of the 66 acute myeloblastic leukemias (= 91%) and 28 of the 30 myeloid blast crises of CML patients (= 93%) were M2-positive. As expected from our findings with normal myeloid cells, the myeloid cells found in stable phase of CML were also in all instances, M2-positive. Quite in contrast, lymphoid cells from patients with B-CLL, T-CLL, prolymphocytic leukemia, hairy-cell leukemia, lymphoblastic lymphoma, Sézary syndrome, from CML patients in lymphoid blast crisis and from the majority of patients with ALL, were completely M2-negative. Also negative were the blast cells of patients with acute megakaryoblastic leukemia and acute erythroleukemia. A direct comparison of M2 expression with the display of the 3-fucosyl-N-acetyllactosamine determinant, the structure recognized by most of the anti-myeloid monoclonal antibodies reported so far, shows that more AMLs are M2-positive and the proportion of M2-positive blast cells in individual AML samples is higher.
Antibodies, Monoclonal↗