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A Lanzavecchia

Publications and source records attributed to A Lanzavecchia.

At least 145 records · Page 8Linked to original sources
PubMed1988

Characterization of granzymes A and B isolated from granules of cloned human cytotoxic T lymphocytes.

A human CD8+ CTL clone with cytolytic potential was shown to express two serine proteases, a 50-kDa homodimer and a 27-kDa monomer, which were purified from cytoplasmic granules. N-terminal sequencing of the purified proteins revealed that the 50-kDa homodimer is the gene product of the human Hanukah factor cDNA clone and that it represents the human homologue to granzyme A. Similarly, the 27-kDa protein was shown to be the serine esterase encoded by the human lymphocyte protease cDNA clone and corresponds to granzyme B. There was no evidence for the presence of other granzymes, in particular for the human homologues to murine granzymes C, D, E, and F. The substrate best cleaved by granzyme A was Gly-Pro-Arg-amido-4-methyl-coumarin after the Arg residue and the pH optimum was 8 to 8.5. Upon triggering of the TCR-CD3 complex with an anti-CD3 mAb, granzyme A was released into the culture medium. Furthermore, a granule-associated hemolytic activity was detected after salt extraction and partial purification of granule proteins. This suggests that hemolytically active human perforin can be obtained from inactive granules.

Amino Acid Sequence↗
PubMed1988

Activation of mononuclear cells to be used for hybrid monoclonal antibody-induced lysis of human ovarian carcinoma cells.

Recently we reported that cytotoxic T-cell clones can be retargeted to unrelated tumor cells by bispecific monoclonal antibodies (MAbs), anti-CD3 and anti-ovarian carcinoma (alpha OC/TR) (Mezzanzanica et al., 1988). In the perspective of in vivo tumor immunotherapy, as an alternative to cytotoxic T lymphocytes (CTL) from T-cell clones, since human peripheral blood mononuclear cells (PBMCs) without stimulation were quite ineffective, a suitable in vitro activation method was developed to render PBMCs lytic for relevant targets in the presence of the bispecific hybrid MAb alpha OC/TR. This activation protocol was applied to PBMCs from 9 healthy donors (HD) and 6 ovarian carcinoma patients (P) and to tumor-associated lymphocytes (TAL) from 4 ovarian carcinoma P. The method consisted of in vitro stimulation with phytohemagglutinin (PHA) for 2 days, followed by culture with a low dose of recombinant human interleukin-2 (rIL-2) for 6 to 10 days. The antibody-mediated lysis of CTL from HD PBMCs was found to be specifically directed against cells expressing the relevant ovarian tumor antigen when different tumor cell lines and short-term cultures of tumor and normal cells were tested. The antibody-mediated lysis of CTL from P PBMCs or TAL was efficient both on autologous and allogeneic ovarian tumor cells, whereas no reactivity with autologous normal cells was observed and LAK activity was only evident in 1 out of 4 cases. The hybrid antibody induced cytotoxic activity of CTL from P was, however, lower than that of CTL from HD.

Antibodies, Monoclonal↗
PubMed1988

T cells can present antigens such as HIV gp120 targeted to their own surface molecules.

To trigger class II-restricted T cells, antigen presenting cells have to capture antigens, process them and display their fragments in association with class II molecules. In most species, activated T cells express class II molecules; however, no evidence has been found that these cells can present soluble antigens. This failure may be due to the inefficient capture, processing or display of antigens in a stimulatory form by T-cells. The capture of a soluble antigen, which is achieved by nonspecific mechanisms in macrophages and dendritic cells, can be up to 10(3) times more efficient in the presence of surface receptors, such as surface immunoglobulin on B cells that specifically bind antigen with high affinity. We asked whether T cells would be able to present soluble antigens that bind to their own surface molecules. Here we show that such antigens can be effectively processed and presented by both CD4+- and CD8+-bearing human T cells. This indicates that T cells are fully capable of processing and displaying antigens and are mainly limited in antigen presentation by their inefficiency at antigen capture.

Antibodies, Monoclonal↗
PubMed1988

Kinetics of MHC-antigen complex formation on antigen-presenting cells.

With the use of flow cytometry, we recorded changes in intracellular ionized calcium [Ca2+]i of Indo-1 loaded T cells that were triggered by contact with APC. This rapid readout of TCR perturbation enabled us to monitor the formation of stimulatory Ag-MHC complexes on EBV-transformed B cells that were either pulsed with native tetanus toxoid (TT) or with a 12-amino-acid fragment of this protein. Neither unpulsed APC nor Ag-specific APC that were pulsed with native Ag and kept at +4 degrees C were able to induce changes in basal T cell [Ca2+]i in TT-specific T cell clones. After 1 h at 37 degrees C, however, the Ag-pulsed APC were able to induce a three-to-fourfold increase in [Ca2+]i. This length of time appeared to be almost independent of the concentration of Ag with which the APC were pulsed, suggesting that the lag time was due more to intracellular transit than to association of the processed Ag with the MHC molecule. Furthermore, the same lag time and independence of Ag concentration were found when the EBV-transformed B cells were pulsed with a mouse-anti-transferrin receptor mAb and tested for their capacity to trigger a T cell clone specific for processed mouse Ig. This indicates that, in addition to surface Ig, other receptors that are internalized can function in the same fashion in the uptake and processing of a soluble Ag. In contrast to what was found with intact native Ag, no lag time was observed when the APC were pulsed with high concentrations of a 12-amino-acid peptide, containing the amino acid sequence recognized by a TT-specific T cell clone, suggesting that the formation of MHC-peptide complexes occurs instantly. Pulsing with a lower peptide concentration, however, caused the appearance of a time-dependent increase in efficacy of Ag presentation, suggesting a slow accumulation of MHC-peptide complexes on the B cell membrane.

Animals↗
PubMed1988

Human ovarian carcinoma lysis by cytotoxic T cells targeted by bispecific monoclonal antibodies: analysis of the antibody components.

In the perspective of in vivo therapeutic applications, the monoclonal antibody (MAb) MOv18 was selected for its restricted reactivity with human ovarian carcinoma. Using the pH 2.8 desorption assay, we found that the antigen recognized by MOv18 had a high stability on the cell membrane and poor internalization. Therefore, a therapeutic approach which does not require internalization, i.e., the re-targeting of cytotoxic T lymphocytes (CTL) by bispecific MAbs, was investigated. MOv18 and anti-CD3 MAbs were used to produce bispecific reagents, obtained either by chemical cross-linkage (hetero-conjugates) or by somatic hybridization techniques (hybrid MAbs). The maintenance of the binding reactivity and specificity of the bispecific MAbs was analyzed by solid-phase radioimmunoassay, immunofluorescence and cross-competition tests on the relevant target cells (ovarian carcinoma cell line OVCA 432 for MOv18 and PHA-stimulated peripheral blood mononuclear cells for anti-CD3 MAbs), and on 2 irrelevant tumor cell lines. Bv a 51Cr-release assay the bispecific MAbs were found to efficiently promote, at picomolar concentration, cell lysis by CTL clones, but the specificity pattern was wider than that predicted by the binding studies. The F(ab')2 fragment of one hybrid MAb mediated a lysis which was just as efficient as the entire MAb on the relevant target cells and allowed specific lysis to be distinguished from Fc-receptor-mediated lysis. Human immunoglobulins were unable to compete with the Fc receptor binding of the hybrid MAbs and therefore, in the perspective of in vivo applications, Fc fragment removal seems to be an essential step. Analysis of the bispecific reagents indicated that hybrid MAbs are superior to the heteroconjugate as far as storage stability is concerned.

Animals↗
PubMed1988

Anti-ovarian carcinoma anti-T3 heteroconjugates or hybrid antibodies induce tumor cell lysis by cytotoxic T-cells.

In the perspective of therapeutic in vivo targeting for T-cell attack, the monoclonal antibody (MAb) MOv18, selected for its restricted reactivity with human ovarian carcinoma, and an anti-T3 MAb were used to produce heteroconjugate or hybrid antibodies derived by fusion of relevant hybridomas. Specificity and activity of bispecific MAbs were analyzed by solid-phase RIA, immunofluorescence and a 51Cr-release assay on the ovarian carcinoma cell line OVCA 432, which expresses the relevant tumor-associated antigen, and on several irrelevant tumor cell lines. Both reagents efficiently promoted, at picomolar concentration, target cell lysis by cytotoxic T-cell (CTL) clones. Although the pattern of tumor cell lines which were lysed was wider than that predicted by binding studies, further studies using a double-determinant immunoradiometric assay confirmed the specificity of MAb targeting. Analysis of reagents indicated that the hybrid MAb was superior to the heteroaggregate as far as purification recovery and storage stability were concerned. Besides CTL clones, peripheral blood lymphocytes could also be used as cytolytic effectors, provided that a suitable in vitro activation scheme was used.

Antibodies, Heterophile↗
PubMed1987

The use of hybrid hybridomas to target human cytotoxic T lymphocytes.

This study describes a general strategy to produce hybrid monoclonal antibodies that are capable of targeting human cytotoxic T lymphocytes (CTL) against any cell carrying the appropriate target antigen. This is done by fusing a HAT-sensitive, G418-resistant anti-T3 hybridoma with immune spleen cells (or with other hybridomas) that produce antibodies against the desired target antigen. In the hybrid hybridomas the reassortment of Ig heavy and light chains results in the production of bifunctional antibody molecules. Because of their double specificity, these antibodies are able to bridge human CTL to target cells and trigger cytotoxic function. We have isolated several stable hybrid hybridomas in which one specificity is against T3 and one either against HLA antigens (class II, DC-1, A3), human Ig (IgM, IgE, kappa), Toxoplasma gondii or an ovary carcinoma-associated antigen. In all of these cases we show that culture supernatants can efficiently and specifically target any CTL clone against any target cell that possesses the relevant surface antigen recognized by the antibody. Furthermore, the killing requires as little as 0.1 ng/ml of antibody, occurs at effector to target ratios comparable to those used in conventional cytotoxic assays and does not affect bystander cells. Nonspecific killing of Fc receptor-positive cells can be avoided using F(ab')2 fragments. As an example, we show that it is possible to change the major histocompatibility complex class and allospecificity of a CTL clone and target CTL against non-major histocompatibility complex antigens such as Ig, parasites and tumor-associated antigens.

Antibodies, Monoclonal↗
PubMed1987

Lysis of nonnucleated red blood cells by cytotoxic T lymphocytes.

To establish the role of direct membrane damage in cytotoxic T lymphocytes (CTL)-mediated lysis we investigated whether CTL would be capable of lysing nonnucleated target cells. By antibody-mediated targeting we show that CTL are indeed capable of lysing sheep and human red blood cells.

Animals↗
PubMed1987

Antigen uptake and accumulation in antigen-specific B cells.

Using EBV-transformed antigen-specific B-cell clones as APC, we have started to analyze the factors that govern the uptake of antigen by B cells and lead to its efficient presentation to T cells. The binding of antigen to SIg can be blocked by soluble antibodies that react with the same epitope that is seen by the B cell's SIg, but not by antibodies that bind to different epitopes. By studying the antigen presenting capacity of B cells that had been pulsed with antigen in different conditions, we came to the conclusion that specific B cells work as vacuum cleaners, i.e. over time they accumulate the antigen that binds to SIg. This effect results from the difference between the rate of influx (approximately 5-10 times the amount of antigen bound is internalized every hour by receptor-mediated endocytosis) and the rate of loss (the half-life of processed antigen relevant for T-cell activation is about 1 d). T cells specific for mouse Ig are triggered much more efficiently by mouse anti-human Ig than by mouse antibodies directed against other B-cell surface antigens, suggesting that SIg are extremely efficient for antigen internalization and processing.

Antibody Specificity↗
PubMed1985

In vitro synthesis of human IgE: reappraisal of a 5-year study.

In the last 5 years some models of human IgE production in vitro have been investigated in our laboratory. Spontaneous IgE synthesis was found in cultures of B cells from most patients with atopic dermatitis or atopic patients with multiple sensitivities and from some patients with pollenosis, but only during the pollination period. A small and variable increase of the spontaneous IgE synthesis was induced by soluble factor(s) produced by T cells from patients with severe atopy. Selected helper T cell clones were also able to induce IgE synthesis in vitro by both atopic and normal B cells.

Antibody Formation↗