Production, expansion, and clonal analysis of T cells with specific HLA-restricted male lysis.

A cytotoxic T cell (CT) lines grown as a population (CT line) was initiated from the peripheral blood lympocytes (PBL) of a female aplastic anemia patient who was known to express CT that were able to lyse HLA-A2-positive male cells. The anti-H-Y HLA-A2-restricted cytotoxic activity could be maintained over prolonged periods of time. The CT lines could be expanded and maintained in culture for >65 d by the use of mitogens and irradiated feeder cells. Out of 68 cultures obtained after cloning of the CT lines, 43 showed varying, but always specific, anti-H-Y HLA-A2-restricted lytic capacity on a per-cell basis. We could show that the cloned cultures were composed of >80% T cells that carry the HLA-A, -B, -C, and also the HLA-DR antigens identical to the original PBL.

Modifications of leukemic blast cells induced by in vivo high-dose recombinant interleukin-2.

High-dose recombinant human Interleukin-2 was given to 21 patients with acute myeloid (n = 11) or lymphoid (n = 10) leukemia in relapse. A rapid decrease in the peripheral leukemic blasts numbers was observed in six patients. We were unable to demonstrate at the bone marrow level a diminution in the percentage of leukemic blasts. However an increase in the expression of the adhesion molecule CD54/ICAM-1(LFA-1 ligand) affected the leukemic bone marrow blasts of these six patients. This increase in CD54 was found in eight of the 11 (73%) AML and four out of the ten (40%) ALL blasts and CD58/LFA-3 (CD2 ligand) to a lesser extent. This increased expression was not associated with modifications in the expression of MHC class II molecules. In vivo IL-2 also dramatically modified the bone marrow T-cell subsets via the increase of CD3+ cells expressing the CD45RO 'memory' marker (six out of the eight tested patients) or CD54 (seven out of the eight tested patients). Altogether these results demonstrate that leukemic blasts can be affected by in vivo IL-2 via mechanisms that could involve T cells.

Dissociation between early and late events in T cell activation mediated through CD28 surface molecule.

The regulation of early and late events of T cell activation via the CD28 molecule has been investigated, using as an indicator system the differentiated leukemic T cell line Jurkat. Both CD3 and CD28 mAbs induced an increase in (Ca2+)i in Jurkat cells, although with different kinetics, the latter being slower than the former. CD28-mediated (Ca2+)i mobilization was highly sensitive to cholera toxin (ID50 25 ng/ml, vs 300 ng/ml for CD3 stimulation). The inhibitory action of cholera toxin was neither merely due to the increase in intracellular cAMP concentrations, nor to decrease in cell surface expression of the CD28 molecule. To evaluate the effects of cholera toxin on late events of Jurkat cell activation induced by CD28 and CD3 mAbs, the action of cholera toxin and cAMP and CD3- and CD28-mediated IL-2 secretion was analyzed. CD3-induced IL-2 secretion was highly sensitive to cholera toxin (ID less than 5 ng/ml); on the other hand, CD28-induced IL-2 secretion was poorly sensitive to cholera toxin, in sharp contrast to (Ca2+)i mobilization. On the basis of these data, it is hypothesized that the CD28 pathway could be associated with at least two distinct transduction mechanisms, one responsible for the (Ca2+)i rise in Jurkat cells and highly sensitive to cholera toxin, and the other, whose second messenger is unknown, resistant to cholera toxin and responsible for IL-2 secretion.

Functional expression of human CD28 in murine T cell hybridomas.

CD28 is a 44 kDa Ig superfamily cell surface molecule expressed on most mature T cells. Through its interaction with the recently identified B7/BB1 counter-receptor, it is believed to play an important role as a co-stimulator of T cells along with the TCR-CD3 complex. Activation of T cells with CD28 mAbs synergizes with TCR-CD3 and CD2 stimulation, resulting in long term T cell proliferation, differentiation of cytotoxic T cells and production of large amounts of cytokines. In order to further delineate the role of CD28 in signal transduction and T cell activation, human CD28 was transfected into CD3+ murine T cell hybridomas. High levels of cell surface CD28 expression was achieved by protoplast fusion. The transfected molecule retained all the native CD28 mAb epitopes found on human T cells. In these transfectants, CD28 mAbs, similarly to CD3 mAbs, were able to induce Ca2+ mobilization, IL-2 promoter induction (measured as beta-galactosidase activity in T cells hybridomas pre-transfected with the IL-2-lac Z reporter gene), IL-2 secretion, TNF alpha production and apoptosis (observed as growth arrest and genome fragmentation). The parental host cells, or cells transfected with vector alone, responded only to mAbs to CD3. IL-2 secretion in the transfectants was obtained using either an IgM mAb to CD28 or IgG mAbs presented on the surface of IgG-FcR+ B lymphoma cells. Optimal activation via CD28 was inhibited by suboptimal concentrations of soluble CD3 mAb, suggesting an interaction between the two pathways. The immunosuppressive drugs Cyclosporin A and FK506 completely blocked CD28 and CD3 mediated IL-2 production in these transfectants whereas rapamycin had only a partial inhibitory effect. Finally, since the transfected human CD28 molecule confers full functional responsiveness to the murine T cell hybridomas without the need for costimulators such as PMA, this model is ideal for studying the structure-function relationships of the CD28 molecule as well as the transmembrane and cytoplasmic associations implied in CD28 signaling.

Pilot phase I study using zidovudine in association with a 10-day course of anti-CD4 monoclonal antibody in seven AIDS patients.

Experimental evidence has demonstrated that monoclonal antibody (MAb) 13B8.2, a workshop-qualified anti-CD4 MAb, (1) inhibits in vitro syncytium formation as well as in vitro HIV infection of CD4+ T cells; (2) delivers negative signals to T cells, thus preventing T-cell activation and viral replication; (3) contributes to CD4+ T-cell clearance by its Fc portion, and (4) induces an immune response by the patient, contributing potentially to an anti-idiotypic response of interest for the control of the immune parameters of the disease. On this basis a phase I study combining zidovudine treatment and a 10-day course of anti-CD4 MAb was performed in seven AIDS patients (Centers for Disease Control group IV). The treatment was well tolerated. MAb dosage and schedule were adjusted on the basis of circulating CD4+ cells and MAb pharmacokinetics; immunological and virological parameters were also monitored. One patient presented a transient increment in CD4+ T cells associated with augmented T-cell function, the suppression of p24 in the serum and a negative RT assay. A second patient had a steady increment of CD4+ T cells after completion of the treatment, with a transient decrease of serum p24 5 days after completion of the anti-CD4 protocol.

Cell surface expression of ICAM-1 (CD54) and LFA-3 (CD58), two adhesion molecules, is up-regulated on bone marrow leukemic blasts after in vivo administration of high-dose recombinant interleukin-2.

High-dose recombinant interleukin-2 (rIL-2) therapy can induce long-term remission in patients with melanoma and renal and colon cancer. More recently, in vivo IL-2 therapy was shown to induce complete or partial remission in some cases of relapsed chemotherapy-resistant acute myeloid leukemia. We have investigated the phenotypic modifications of bone marrow cells obtained from five patients with acute myeloid leukemia in relapse receiving high-dose i.v. rIL-2. We found that, in three of five patients, IL-2 could induce, in vivo, an increase in the expression of CD54/ICAM-1 and to a lesser extent of CD58/LFA-3 on bone marrow leukemic blasts. This demonstrates that rIL-2 modifies directly or indirectly the expression of the cell surface molecules of the tumor cells themselves. Upregulation of such adhesion molecules could account for the enhancement of cell interactions between the tumor and effector cells such as T, natural killer, and phagocytic cells as well as being indicators of differentiation signaling.

The effect of in vivo application of monoclonal antibodies specific for human cytotoxic T cells in rhesus monkeys.

Rhesus monkeys were treated in vivo with monoclonal antibodies specific for human cytotoxic T cells. These antibodies reacted with rhesus lymphocytes as they do with human lymphocytes. Injection of a pool of monoclonal antibodies resulted in rapid elimination of the relevant T cell subpopulation from the circulation. Injection of a single monoclonal antibody did not result in elimination of the subpopulation, but the cells were coated with the injected monoclonal antibody. Injection of the single monoclonal antibody did not prolong the allogeneic skin graft. These results indicate that the rhesus monkey is a useful model for testing antihuman monoclonal antibodies.

Elimination of leukemia cells from human bone marrow using floating beads.

A new in vitro immunophysical method of removing leukemia or lymphoma cells from autologous bone marrow is described. This new technique makes use of low-density polypropylene beads (density: 0.91) coated with a monoclonal antibody anti-CALLA (antibody ALB2). To ascertain its ability to selectively remove human B/pre-B hematopoietic cells, this technique was applied to normal human bone marrow cell suspensions contaminated with 1-5% of tumor cells. Samples were incubated with the floating beads at 4 degrees C on a rotating wheel for 60 min, followed by a 10-min decantation period, after which the beads bearing the tumor cells floated on the surface, whereas unbound normal marrow cells remained in suspension and were easily recovered free of beads. To demonstrate the feasibility of our method, 2 types of assays were carried out, one using target cell radiolabeled with 111indium, and the other a clonogenic assay. The first assays were to calibrate the different parameters (cellular density, quantity of beads, incubation time) with tumor cell lines: Namalwa (CALLA+) and Molt 4 (CALLA-). These 2 cells lines being able to clone, it is hard to envisage clonogenic assays. In this case, it is very hazardous to evaluate correctly the remaining clonogenic units of Namalwa cells. It is why radiolabelling assays were used for these first experiments. The second assays were to study a model close to the clinical setting and to control the safety of the beads on normal bone marrow cells. In this case, the mixture experiments in which only Namalwa cells were able to clone were evaluated with clonogenic assays, which are more sensitive than radiolabeling assays. A 3- to 4-log reduction of tumor load was achieved with 1-step treatment, and an average of 5-log depletion was obtained by repeating the process twice, as ascertained by the clonogenic assay. Viability, average recovery of nucleated cells, and stem cells potential following the purge were excellent.

Therapeutic applications of anti-CD4 antibodies.

The CD4 molecule represents a major functional T-cell surface molecule, defining an important T-cell subset, which also is expressed on monocyte, dendritic, and Langerhans cells. Various in vivo studies have demonstrated its implication in various steps of physiological T-cell activation: 1. CD4 interacts with its physiological ligand, the class II molecules, thus increasing the affinity of the conjugation between CD4+CD(3+)-TCR+ and class II+ antigen-presenting cells. 2. Through CD4, the signal transduction machinery is stimulated via its association with p56lck. In addition, CD4 has proved to be the receptor for gp120, the surface glycoprotein of HIV, that allows the virus to penetrate the CD4+ T cells and monocytes. Based on in vitro studies in various animal models, CD4 mAbs have proved to be efficient in the prevention and/or therapy of a variety of immunologically based diseases: 1. When injected early in the prodromic phase of autoimmune diseases (AID) such as diabetes, either delay or prevention is achieved with or without maintenance after therapy. 2. These mAbs have proved to be self-tolerogenic, thus allowing prolonged in vivo therapy and suppression of immunogenicity of mAb of a distinct specificity. In humans, CD4 mAbs are, or could be, used and evaluated in AID (lupus, diabetes, rheumatoid arthritis, etc.), transplantation, leukemias and lymphomas expressing CD4, and, finally, in AIDS patients, in whom CD4 mAbs can block HIV-CD4 binding and deliver a negative signal to T cell, thus blocking T-cell activation and HIV transcription. CD4 mAbs at least provide evidence that the CD4 molecules are suitable for immunomodulation and could be the target for a new pharmacological antagonist.

Human allospecific T cell proliferative clones: different T cell clones expressing an apparently identical HLA class II specificity are heterogeneous in regard to their proliferative inhibition by a panel of monoclonal antibodies against human Ia-like antigens.

Four independent PLT clones displaying an apparently identical class II specificity (i.e., Dw/DR3) were found to give a heterogeneous pattern of inhibition in relation to 15 anti-class II mAb and an anti-beta 2m mAb used as a control. Some clones were inhibited by all anti-class II mAbs, irrespective of the cluster of molecular subsets with which they reacted. Such clones were also inhibited by the control anti-beta 2m mAb. Other clones were inhibited by only a few of the mAb tested. Within this group of T clones following the addition of a limiting amount of conditioned medium the inhibitory data of all independent clones with an identical specificity were inhibited by the same mAb; under these conditions, it was possible to relate the mAb inhibition patterns with the specificity of the T cell clones and these T cell specificities with the epitopic cluster/molecular subsets defined by these mAbs. A new level of T cell subset heterogeneity within T cell clones with apparent identical proliferative specificities is demonstrated, in relation to the T cell clone "susceptibility" or "resistance" to the effects of anti-class II mAbs directed towards their own Ia-like antigens.

Expansion of human lymphocyte populations expressing specific immune reactivities. III. Specific colonies, either cytotoxic or proliferative, obtained from a population of responder cells primed in vitro. Preliminary immunogenetic analysis.

Human alloreactive cell lines were maintained in culture over prolonged periods of time using conditioned medium. Primed lymphocyte typing reactivity was observed in these T cell lines for only 1 mo, but these T cell lines have remained for more than 7 mo highly and specifically cytotoxic. Using as growth promoter an irradiated autologous feeder consisting of irradiated peripheral blood lymphocytes and the lectin leucoagglutinin, we have derived by limiting dilution cloning of in vitro primed allogeneic combinations, primary colonies (or primary clones) with monofunctional immune reactivities: either cytotoxic (the rarest observed) or PLT reactive (the majority of the colonies). Furthermore, each monofunctional primary colony when tested for PLT or CML reactivity on a panel of unrelated PBL, always showed a restricted specificity when compared to the original primed population. The PLT reactivity of each of the primary clones was short lasting in contrast to their growth potential. The CML reactivity of the primary clones, as for the T cell lines, was long lasting as was their growth potential.

A 55,000 Mr surface antigen on activated human T lymphocytes defined by a monoclonal antibody.

A T cell growth factor-dependent alloreactive human T cell line has been used to generate a monoclonal antibody B1.49.9 that reacts with an antigen present on most if not all mitogen or alloantigen activated T cells but not on resting T cells. The T lymphoblastoid cell line HUT-102 is also strongly reactive with B1.49.9 but all other T and non-T leukemia-lymphoma cell lines tested were negative. The B1.49.9 antigen is a glycoprotein of 55,000 Mr on mitogen or alloantigen activated T cells and 50,000 Mr on the cell line HUT-102. Pulse labeling experiments showed that a 40,000 Mr precursor (at approximately 0.7 h) which does not bind to ricin lectin precedes the appearance of the ricin-binding 55,000 Mr form. Comparisons of the monoclonal antibody anti-Tac, which recognizes the IL-2 receptor, to B1.49.9 suggest that B1.49.9 also recognizes a structure similar or identical to the IL-2 receptor.

Anti-T11.1 and -T11.2 monoclonal antibodies play a different role in CD2-mediated signal transduction.

We comparatively evaluated (Ca2+)i mobilization after triggering with a stimulatory pair of CD2 (CD2.9, anti-T11.1 + CD2.1, anti-T11.2) or CD3 mAbs in the differentiated T-cell line Jurkat, using INDO-1 labeling and cytofluorimetry. The results obtained showed different (Ca2+)i mobilization kinetics following CD2 or CD3 stimulation (the former being slower than the latter), not due to different association kinetics of mAbs. In a nonreciprocal manner, however, preliminary interaction with CD2.1 (anti-T11.2) followed by CD2.9 (anti-T11.1) induces a rapid (Ca2+)i rise, similar to CD3 stimulation, as shown by preincubation experiments. There is no interference between CD2.9 and CD2.1 mAb binding. CD2.1 mAb by itself is unable to induce (Ca2+)i mobilization; in addition, preincubation with CD2.1 mAb did not modify the CD2, CD3, CD45, or CD28 immunoprecipitation patterns. Triggering of the epitope recognized by CD2.1 mAb may favor, possibly via conformational changes of CD2 molecule or (Ca2+)i-unrelated metabolic effect(s), optimal signal transduction.

Parameters of interaction of a novel monoclonal antibody (33B3.1) with the human IL2-receptors: interrelationship between 33B3.1, anti-Tac, and IL2 binding sites.

This report describes the molecular parameters of interaction of a new antibody (33B3.1) with the human membrane RIL2 expressed by ConA-activated T lymphocytes or allogeneic T-cell clones established from a rejected kidney allograft: the 33B3.1 immunoprecipitates a membrane protein of 55000 MW. It inhibits IL2-driven proliferation of activated T cells. This inhibition occurred in the nanomolar range when low concentrations of recombinant IL2 (rec-IL2) were used. The (125I)-33B3.1 binds in a specific way to a single class of receptor sites on activated T cells. The rate constants of association and dissociation at 37 degrees C of the labeled 33B3.1 were k*1 = 12 X 10(5) M-1 s-1 and k*-1 = 7 X 10(-4) s-1, respectively, and its equilibrium dissociation constant was KD = 0.65 nM. Maximal binding capacities were fairly variable among T-cell clones, as high as 300,000 sites/cell for some of them. Competition experiments demonstrate that the 33B3.1 and anti-Tac interact with the RIL2 in a competitive manner, suggesting that they recognize closely associated epitopes on the RIL2. However, the 33B3.1 inhibits the binding of (35S)-recombinant IL2 to its high affinity RIL2 in a noncompetitive way. The 33B3.1 seems therefore to interact with an epitope close but distinct from the IL2 binding site. Our data could suggest either that the 33B3.1 is able to convert high affinity RIL2 towards low affinity conformations or that there is more than one IL2 binding site per molecule of high affinity RIL2.

Generation of CD8 cytolytic T cells early after autologous or allogeneic bone marrow transplantation.

Longitudinal in vitro assays related to cell-mediated immunity were performed in patients following allogeneic (32) or autologous (15) bone marrow transplantation (BMT). In both groups of reconstituted patients, low CD4+/CD8+ T cell ratio and weak allogeneic mixed lymphocyte reactions were found in the first 6 months after BMT, progressively reaching values similar to controls (bone marrow donors or unrelated individuals). In contrast, a strong generation of allogeneic cytotoxic cells, assessed by the number of lytic units per 10(6) cells, was frequently found (18/38 patients tested in both groups) in the first 4 months, despite the quantitative deficit of the CD4+ subset. This in vitro differentiation was found to be independent of in vivo acute graft-versus-host disease (GVHD) and chronic GVHD in allo-transplanted patients. As also documented in autologous recipients, this observation suggests that this phenomenon could be, at least partially, related to the transplantation per se. Preliminary characterization of the effector cells indicates that they belong to the CD8+ subset and that their differentiation is interleukin-2-dependent. Experimental depletion of the CD4+ subset in normal subjects did not increase the number of lytic units in allogeneic cultures. This implies qualitative differences between BMT recipients and normal subjects, namely in CD8+ subset: i.e. that following BMT early CD8+ T cells appear to produce their own growth factor (IL-2), while in normal adult individuals, such autocrine CD8+ T cells, if present, are very rare.

Anti LFA1 monoclonal antibody for the prevention of graft rejection after T cell-depleted HLA-matched bone marrow transplantation for leukemia in adults.

A mouse IgG monoclonal antibody (MoAb) directed against the human LFA1 molecule (25.3 MoAb) was used in nine adult leukemic patients to prevent graft rejection after T cell-depleted HLA matched bone marrow transplantation. Based on the results of a previous study in children 0.1 mg/kg of 25.3 was given on days -3, -1, +1, +3, +5 in addition to a standard conditioning regimen with cyclophosphamide (120 mg/kg) and fractionated total body irradiation. The marrow transplant was T cell-depleted using T101 Fab immunotoxin ricin A chain. Seven patients received post-graft immunosuppression with methotrexate and cyclosporine A; two patients received no immunosuppression post-graft. A mean T cell depletion of 98.3% (80-100%) was achieved. Tolerance to the infusions of 25.3 MoAb was excellent. No patient developed any form of graft-versus-host disease. However two patients failed to engraft and three patients had delayed graft failures. These results show that this regimen of anti LFA1 MoAb, which was extremely good at permitting engraftment of HLA mismatched T cell-depleted transplant in children with constitutional diseases, is not able to prevent graft failure and rejection of T cell-depleted HLA matched transplants in adults with leukemia. Further efforts are needed to overcome graft failures in this clinical situation.

Autologous bone marrow transplantation for B cell malignancies after in vitro purging with floating immunobeads.

We report here 16 autologous bone marrow transplantations (ABMT) for poor prognosis B or pre-B malignancies (16 acute lymphoblastic leukemias (ALL), three Burkitt lymphomas, one multiple myeloma) in 11 adults and five children where in vitro purging was accomplished by means of floating immunobeads. This method was developed to avoid non-specific killing by complement or toxin or batch-to-batch variability and provides a 3 log reduction of tumor in a model of B lymphoid malignancies. Low density bone marrow mononuclear cells were incubated for 30 min at 4 degrees C with anti CD10 (ALB2 Immunotech) and/or anti CD19 (Bg4) monoclonal antibodies (MoAb) and then mixed with low density polypropylene beads precoated with a rat antimouse MoAb. After 1 h at 4 degrees C the beads with target cells were decanted; the depleted marrow was collected through a microfilter and cryoperserved. After immunodepletion the recovery of nucleated cells was 75% with a median of 0.75 x 10(8) cells/kg (range 0.3-3.6) and the recovery of hematopoietic progenitors was 83% with a median of 2.9 x 10(4) CFU-GM/kg. The conditioning regimen consisted of busulfan 16 mg/kg and melphalan 140 mg/m2 for three patients, fractionated total body irradiation (TBI) following melphalan 140 mg/m2 for nine patients, TBI and cyclophosphamide 120 mg/m2 for two patients and TBI associated with melphalan and cyclophosphamide for two patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential immuno-suppressive effects of metabolic inhibitors on T-lymphocyte activation.

An important challenge in the field of auto-immune diseases, bone marrow and organ transplantation is the control of T-lymphocyte activation. To gain more insight into the in vitro correlation of immunosuppression, we investigated the effects of cyclosporin A (CSA) and two other metabolic inhibitors on cytokine secretion and T-cell proliferation. Secretion of TNF-alpha and GM-CSF was much more resistant to metabolic inhibitors than proliferation or synthesis of IL-1 alpha or IL-2. Moreover, our data suggested that the regulation of IL-1 alpha production in T-cells was CSA and protein kinase C (PKC)-dependent, as opposed to monocytes regulation. The receptivity to the epithelial cell-derived cytokine IL-7, associated either with antigen-dependent or independent triggering, was almost similarly inhibited by cyclosporin A, forskolin or PKC inhibitor, in sharp contrast to IL-2 receptivity. In this latter case, CD28+ IL-2 stimulation was more sensitive to both forskolin and PKC inhibition than that of CD2 or CD3+ IL-2. With regard to CSA effects, limiting dilution analysis provided evidence for some heterogeneity at the clonal level. This strongly suggested that T-cell functional monitoring at the population level does not truly reflect the actual immunosuppression. Additional experiments are required to evaluate the sensitivity to metabolic inhibitors of T-lymphocyte activation via the natural ligands of CD2 and CD28.