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Background: Monoclonal CD4 antibodies have been proposed as a new immunosuppressant drug in the treatment of inflammatory bowel disease. We report our experience of treatment with a monoclonal anti-CD4 (B-F5) antibody in severe refractory Crohn's disease., Methods: Twelve patients with severe refractory Crohn's disease were treated in an open clinical trial. B-F5 was given intravenously at a dose of 0.5 mg. day/kg for 7 consecutive days (patients 1-8). For patients 9-12, B-F5 was given at a dose of 0.5 mg. day/kg on the first day (day 0) and of 1 mg.day/kg on days 1-6. Follow-up examinations were carried out at days 8, 15, 22 and 30. Endoscopic evaluation was performed on days 0 and 30 in eight of 12 patients., Results: Immediately after the first infusion, one patient had dyspnoea and tachycardia requiring cessation of the treatment. Among the 11 patients who received the complete course of treatment, two had prolonged clinical improvement and two had partial clinical improvement. Significant endoscopic improvement was observed in only one patient. No sustained depletion of CD4+ cells could be observed., Conclusion: In this uncontrolled open trial, monoclonal anti-CD4 B-F5 antibody was not successful in severe Crohn's disease.

Cytokines may be helpful in promoting hematopoietic reconstitution but have also an impact on the cellular interactions that contribute to GvHD and immunologic graft rejection. Because IL-12 is emerging as a central cytokine in immune response, we have investigated its levels in serum samples of patients undergoing bone marrow transplantation and transplant-related events. A double-antibody radioimmunoassay method for monitoring levels of endogenous IL-12, before and after allogeneic (27 patients) or autologous (19 patients) bone marrow transplantation, was used. The serum levels of IL-12 after allogeneic BMT were found to be relatively low (140-300 pg/ml) and similar to the IL-12 levels in the healthy donors (183 pg/ml). Seric IL-12 levels following autologous BMT (350 pg/ml) were higher than those observed in patients receiving an allogeneic BMT and in healthy donors. Our data indicate that the occurrence of GvHD and the development of infection after allogeneic BMT are not associated with IL-12 induction which suggests a possible down-regulation due to immunosuppressive treatment.

CD4 monoclonal antibodies (mAbs) are potent immunosuppressive agents which have been shown to induce in vivo tolerance to antigens and alloantigens and are presently evaluated in humans in the treatment of autoimmune diseases. In previous studies, we observed that clinical improvement of CD4 mAb-treated psoriasis patients was achieved without depletion of CD4+ lymphocytes and at nonsaturating CD4 mAb concentrations, suggesting a functional blockade of CD4+ lymphocyte responses. In this study, we demonstrate that priming of normal human CD4+ T cells by immobilized OKT3 (iOKT3) in the presence of CD4 mAbs in soluble phase induces a hyporesponsiveness following subsequent restimulation by iOKT3 in the absence of CD4 mAbs. This hyporesponsiveness was not associated with increased cell death during priming or restimulation cultures and could be reversed by the combination of phorbol ester + ionomycin, demonstrating a functional blockade of viable cells by CD4 mAbs. Following iOKT3 restimulation, hyporesponsive cells showed a lack of blast transformation and CD25 expression and were not able to respond to IL-2 since addition of high doses of exogenous IL-2 +/- CD28 mAbs did not reverse the hyporesponsiveness. However, costimulation with CD45RA mAb completely reversed the hyporesponsiveness, suggesting that CD45 controlled the CD4-mediated hyporesponsiveness.

When administered to patients, various biotechnology products may induce early toxic effects mediated by a cytokine cascade in which Tumor Necrosis Factor-alpha (TNF-alpha) plays a central role. The mechanisms of these toxic reactions have been extensively documented in the clinical model of the CD3 monoclonal antibody (mAb), OKT3. In order to develop a preclinical test for assessing the potential of mAbs or recombinant molecules to induce acute reactions when administered to patients, we investigated different in vitro culture systems for TNF-alpha secretion, using human blood cells. OKT3 and bacterial lipopolysaccharide (LPS) were used as positive controls. By comparing different culture conditions and kinetics, we concluded that 6-h supernatants of plasma-depleted whole blood contained high amounts of TNF-alpha in cultures stimulated by OKT3 or LPS, but not in those performed in the absence of exogenous stimulant or in the presence of mAbs which do not induce toxic reactions in vivo. This in vitro assay may be applied to the preclinical evaluation of the risk of cytokine-mediated toxicity in vivo. Owing to its simplicity, it could be used for preclinical investigation of inter-individual differences in the susceptibility to 'activation syndrome'.

Natural killer (NK) cells can be differentiated into lymphokine-activated killer (LAK) effectors following stimulation with interleukin (IL)-2. This induction can be negatively regulated by IL-4. In this study, we demonstrate that the stimulation of NK cells through the CD2 pathway with (9-1 + 9.6) monoclonal antibodies can also induce these cells to secrete tumor necrosis factor-alpha (TNF-alpha) and to differentiate into LAK effectors. More importantly, our data indicate that, in contrast to the IL-2-induced LAK generation, the anti-CD2-triggered LAK activity was not regulated by IL-4. IL-4 was found to enhance the LAK activity as well as NK cell proliferation following activation with anti-CD2 by a mechanism involving, at least in part, an increased TNF-alpha production. Using immobilized monoclonal antibodies against the Fc receptor (Fc gamma RIII or CD16) for NK stimulation, we also observed that the anti-CD16-induced LAK activity was not inhibited by IL-4. These data further point to a pivotal role of TNF-alpha as a regulatory cytokine in anti-CD2-induced LAK generation, and suggest that IL-4 could serve as a discriminatory factor between two distinct pathways involved in the activation of non-MHC-restricted cytotoxicity.

We have investigated tumor necrosis factor-alpha levels in serum samples of patients before and after allogenic (16 patients) or autologous (8 patients) bone marrow transplantation. A sensitive immunoradiometric assay for monitoring levels of endogenous tumor necrosis factor-alpha was used. The serum levels of tumor necrosis factor-alpha were found to be relatively low (ranging from less than 15 to 77 pg/ml). Among 13 patients having graft-versus-host disease following allogeneic bone marrow transplantation 8 patients did not have detectable tumor necrosis factor-alpha (less than 15 pg/ml) while 4 out of 8 patients undergoing autologous bone marrow transplantation had detectable tumor necrosis factor-alpha levels (15 pg/ml), indicating a lack of correlation between tumor necrosis factor-alpha serum levels and the occurrence of graft-versus-host disease. Because the tumor necrosis factor-alpha levels detected in patient sera could be regulated by TNF-receptor expression, the presence of TNF-receptor on patients' peripheral blood mononuclear cells was also studied using fluorescent liposome-conjugated tumor necrosis factor-alpha and immunofluorescence analysis. Our data indicate that peripheral blood mononuclear cells of some patients receiving either autologous or allogeneic bone marrow transplantation expressed significant levels of TNF-receptors, suggesting a lack of correlation between TNF-receptor expression and graft-versus-host disease development.

Originally described for its capacity to induce hemorrhagic necrosis of transplantable tumors in mice, TNF-alpha also exerts cytotoxic effects against some tumor cell lines in vitro. It is now known that TNF is an essential mediator of cellular immunity and a wide variety of biological activities of TNF in vitro and in vivo has been reported. TNF is an important mediator of inflammation and is involved during the pathogenesis of several auto-immune, infectious or cancer diseases. While some immunomodulatory properties of TNF have at least been partially elucidated, the biochemical basis of TNF cytotoxic action remains largely unknown. Furthermore, the molecular mechanisms of TNF susceptibility have yet to be clarified. Clinical studies with recombinant TNF as an anticancer agent are encouraging. Multiple phase I and phase II trials have been carried out without major therapeutic effect. In fact, TNF resistance and TNF-induced systemic toxicity are two major limitations for the use of TNF as an antineoplastic agent. The clinical application of human TNF remains an area of active research and innovative approaches such as gene therapy need to be elaborated. The elucidation of the process of lysis and the modulation of TNF resistance are crucial to the future development of TNF and its use in antitumor therapy.

We have investigated the functional interaction between IL-2 and TNF on the generation of alloreactive CTL. The study was performed by using primary mixed cultures of lymphocytes from a MHC-recombinant sibling identical for MHC class II Ag (DR, DP, DQ) and displaying MHC class I disparity. Our data show that MHC class I disparity can trigger the induction of TNF receptor without promoting significant TNF production. Addition of exogenous TNF at the sensitizing phase of the primary mixed lymphocyte reaction did not result in CTL activation. However, when simultaneously added with IL-2, TNF could promote an optimal induction of cytotoxic T cell generation. The enhanced lytic ability of MHC class-I primed CTL by TNF was associated with a selective up-regulation of Tac Ag and subsequent amplification of cell proliferation. Furthermore, TNF was also found to induce a considerable increase in IL-2-induced intracellular benzyloxycarbonyl-L-lysine thiobenzylester-esterase activity by MHC class I-primed cells. TNF did not affect the expression of LFA1, CD2, CD4, and CD8, molecules that are associated with CTL-target interactions, on responder cells. These results extend our earlier observations on the role of class I MHC molecules that may function to transduce activation signals and suggest that TNF may be a potent mediator involved in the IL-2-induced acquisition of optimal lytic competence by precursor cytotoxic T cells.

In previous studies, IL-4 has been reported to interfere with IL-2-driven generation of lymphokine-activated killer (LAK) activity. In this investigation, we have demonstrated that IL-4 inhibited the IL-2-induced differentiation of large granular lymphocytes (LGL) into LAK effectors by a mechanism involving, at least in part, an increase in LGL intracellular cAMP levels. In contrast, with its capacity to induce cAMP accumulation in resting LGL, IL-4 had a very negligible effect on LAK activity induction, and cAMP levels increase in LGL that had been preincubated with IL-2. Furthermore, the inhibitory effect of IL-4 on LAK activity generation also correlated with a marked decrease in N-CBZ-L-lysine thiobenzylester esterase activity, with an inhibition of tumor necrosis factor (TNF) mRNA expression and TNF production by IL-2-stimulated LGL. These results strongly suggest that complex signaling processes could be ascribed to the dual activities of cytokines and their interplay in LAK promotion.

The purpose of the present study was to explore the interaction between interleukin-2 (IL-2), interleukin-4 (IL-4), and tumor necrosis factor-alpha (TNF) on the differentiation of human large granular lymphocytes (LGL) into lymphokine-activated killer cells (LAK). The data show that recombinant human IL-4 (100-1,000/ml) was able to induce the differentiation of human LGL into LAK effectors. The levels of the IL-4-induced cytotoxicity are significantly lower than those observed after stimulation of LGL by optimal doses of IL-2. This LAK activity generation by IL-4 was not associated with LGL proliferation. When TNF was added in LGL culture in the presence of suboptimal concentrations of IL-4, the lytic capacity of the activated killer cells was significantly enhanced, suggesting an apparent synergy between these two factors. Most interestingly, our data indicate that exogenous TNF can partially overcome the known inhibitory effect of IL-4 on IL-2-induced LGL differentiation into LAK effectors. These findings suggest a role for TNF in the process of LAK induction.