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Four patients with lymphoproliferative disease of granular lymphocytes (LDGL) coexpressing CD3 and the natural killer (NK)-related “p58” receptor for HLA-C alleles were studied. These CD3+p58+ LDGLs have been detected among a series of 44 CD3+ LDGLs analyzed. Two patients with LDGL (GI and BA) expressed only the p58 molecule defined by the GL-183 and CH-L monoclonal antibodies (MoAbs), while the cases of patients PU and MA also coexpressed the molecular form identified by EB6 anti-p58 MoAb. Three LDGL cases (GI, MA, and PU) displayed the CD8+4-CD16+ T- cell receptor (TCR)alpha/beta+ phenotype, while one patient (BA) was CD8+4+CD16+ TCRalpha/beta+. Freshly isolated granular lymphocytes (GL) from these cases displayed cytolytic activity in an anti-CD3 MoAb- triggered redirected killing assay against the Fcgamma-receptor+ (Fcgamma-R+) P815 target cell line. Lysis of P815 target cells, triggered by an anti-CD3 or by anti-CD16 MoAb, could be inhibited by the addition of anti-p58 MoAb in three fresh or interleukin (IL)-2- cultured GL tested (GI, MA, and PU). Triggering of cytotoxicity against the HLA-DR+ Fcgamma-R+ Daudi cell line induced by appropriate superantigens could also be inhibited by anti-p58 MoAb in patients PU and GI with LDGL. These data indicate that activation through the CD16, CD3, and TCR molecules can be modulated by p58 receptors in these LDGLs. On the contrary, IL-2-expanded cells of patient BA were induced to lyse P815 target cells by anti-p58 MoAb. In addition, anti-p58 MoAB enhanced anti-CD16 MoAb triggered lysis and did not inhibit activation via CD3. These data indicate that, in this particular patient with LDGL, p58 displays a stimulatory effect on cell triggering, rather than the typical inhibitory effect previously observed in p58+ T-cell clones derived from healthy donors. The anti-p58 MoAb did not induce CA++ mobilization in p58+ LDGLs and in a p58+CD3+ normal T-cell clone equipped with inhibitory p58 molecules, while Ca++ mobilization could be observed in cultured GL from patient BA, which could be activated by anti-58 MoAb. These findings suggest that stimulatory and inhibitory p58 molecules are equipped with different signal transducing properties, thus contributing to a better knowledge of the normal counterpart.

We have analysed the expression of interleukin-2 receptor (IL-2R) on a panel of small-cell lung cancer (SCLC) cell lines. None of the 11 SCLC cell lines studied expressed detectable surface IL-2R alpha or beta chains by indirect immunofluorescence. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses indicated that only one out of 11 cell lines expressed detectable IL-2R beta mRNA while two expressed a weak positivity for IL-2R gamma. Five SCLC cell lines were transfected with the plasmid vector RSV.5 neo containing IL-2 cDNA coding sequence. Stable transfectants secreted biologically active IL-2 (ranging from 25 to 100 U ml-1 in the culture supernatant). IL-2 transfection did not produce significant modifications in the expression of surface molecules such as IL-2R alpha and beta chains, intercellular adhesion molecule-1 (ICAM-1), CD44, HLA class I and II or in IL-2R beta or gamma mRNA. More importantly, IL-2-transfected N592 and NCI H69 cell lines completely lost their tumorigenic potential in nude mice after subcutaneous injection, whereas experimental controls transfected with RSV.5 neo vector only, displayed an in vivo growth pattern identical to that of untransfected cells. In addition, in the N592 model, IL-2-producing N592 inhibited the growth of wild-type N592 injected at the same site, while injection of parental cells on the opposite side did not significantly affect the growth of wild-type tumour cells. Histopathological analysis of the rejection process of IL-2-transfected cells demonstrated the presence of MAC-1+, MAC-3+ macrophages and of RB68C5+ granulocytes, whereas T cells were undetectable and NK cells were scarcely represented. In addition, a reduction of the tumour blood vessels was observed. The possible relevance of these data for the development of vaccination strategies using cytokine-engineered tumour cells in SCLC is discussed.

The in vitro and in vivo stability and anti-tumour efficacy of the anti-EGFR/anti-CD3 bispecific monoclonal antibody (biMAb), M26.1, were analysed. The interaction of the intact biMAb with Fc receptor I (Fc gamma RI) present on human leucocytes was not observed when the antibody was used as an F(ab')2 fragment. A CD8+ T-cell clone coated with M26.1 F(ab')2 was as effective as the intact biMAb in inducing IGROV1 target cell lysis when tested in a 51Cr-release assay. Variable levels of reduction of F(ab')2 to monovalent F(ab') were observed upon incubation with human ovarian cancer ascitic fluid (OCAF) or with human glioblastoma cavity fluid (GCF), but not with mouse or human sera. Activated lymphocytes coated with F(ab')2 and incubated in vitro with GCF or OCAF for 24 and 48 h respectively maintained their targeting. Thus, the F(ab')2, when present as a soluble molecule, but not when bound to T cells, might lose some functional activity as a consequence of partial reduction to F(ab'). In normal mice, M26.1 F(ab')2 retained full cytotoxic activity in the circulation, and clearance values were similar to those obtained with parental and other MAb F(ab')2. Treatment of IGROV1 tumour-bearing mice with activated human lymphocytes coated with the M26.1 F(ab')2 significantly prolonged survival of the animals compared with tumour-bearing untreated and control mice treated with lymphocytes or F(ab')2 alone. Together, these results suggest the clinical usefulness of bispecific M26.1 F(ab')2 as a targeting agent for local treatment of tumours such as glioma and ovarian cancers that express variable levels of epidermal growth factor receptor (EGFR).

This study has been designed to verify the specific toxicity of saporin, a type 1 ribosome-inactivating protein (RIP), with the same activity as ricin A chain, targeted by a bispecific monoclonal antibody (bimAb) recognising both the CD25 antigen and the RIP. The CD25 antigen is expressed by lymphoid populations upon activation and by leukaemias and lymphomas with an activated membrane phenotype (Hodgkin's lymphoma, anaplastic large cell lymphoma, adult T cell leukaemia). The bimAb-saporin mixture was tested on CD25+ targets at different bimAb and saporin concentrations. Saporin, in the presence of a bimAb concentration of 10(-9) M, inhibited protein synthesis by CD25+ neoplastic lymphocytes (L540 and MT2 cell lines) with IC50S (concentrations giving 50% of inhibition) ranging from 8 x 10(-12) M to 3 x 10(-11) M. The saporin-bimAb mixture was also effective in blocking the phytohaemagglutinin-driven proliferation of normal lymphocytes, whereas it displayed the same level of toxicity exerted by saporin alone on an irrelevant CD25-negative cell line (EBV-infected B lymphoblastoid cell line). From these results it is possible to envisage a clinical use of this bimAb as a cytotoxic agent for CD25+ leukaemias and lymphomas, as well as an immunosuppressive agent for severe immune disorders such as graft-vs-host disease (GVHD) and transplanted organ rejection.

127 randomly selected patients on haemodialysis showed a high prevalence of ventricular arrhythmias, the frequency of which rose significantly during and after dialysis. These patients have now been followed up for 4 years. Only age and ischaemic heart disease correlated independently with mortality. Although ventricular arrhythmias are often associated with cardiac disease in patients on chronic haemodialysis, they do not seem to predict overall mortality.

Aim of this study was the characterization of SCLC lines genetically engineered to express the immunostimulatory cytokine IL-2. Preliminarily, we examined by immunofluorescence and RT-PCR the expression of IL-2 receptor on 10 different SCLC lines to evaluate possible effects of IL-2 on the tumour cells. Only one SCLC cell line (GLC1) out of 10 tested expressed IL-2R α, βand γchain while the others were negative. Human IL-2 gene was cloned by RT-PCR and inserted into the RSV.5 expression vector containing the neo resistance gene. By electroporation or lyposome-mediated transfection we have selected stable transfectants of 4 different SCLC lines (GLCI, N592, NCI-H69 and NCI-H164) secreting 25–200U/ml of biologically active IL-2 in the supernatant. All IL-2 transfected tumour cell lines, including the IL-2R+ GLC1, displayed an “in vitro” proliferative potential (by MTT proliferation assay) similar to that of untransfected cell lines. Further analyses of the tumourigenic potential in nude mice are in progress. IL-2 transfected cells were able to stimulate the proliferation and to induce anti-tumour cytotoxic activity in peripheral blood lymphocytes.