Search

Objective Colony-stimulating factor 1 receptor (CSF-1R) essentially modulates monocyte proliferation, migration, and activation, which are considered important for the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine CSF-1R expression in human RA as well as the efficacy of a specific anti–CSF-1R monoclonal antibody (AFS98) in 2 different animal models of RA. Methods CSF-1R expression was examined in blood, synovium, and bone samples from RA patients, osteoarthritis (OA) patients, and healthy subjects. The efficacy of AFS98 was examined by clinical assessment, histology, and bone histomorphometry in collagen-induced arthritis (CIA) and serum-transfer arthritis. Results CSF-1R expression was increased in the synovium of RA patients compared to OA patients and healthy controls in fibroblast-like synoviocytes, follicular dendritic cells, macrophages, and osteoclasts. Circulating RA monocytes and neutrophils but not lymphocytes were CSF-1R+. In mice, blockade of CSF-1R abrogated cartilage damage, bone erosion, and systemic bone loss, and this was associated with the depletion of osteoclasts in both models. While blockade of CSF-1R did not affect inflammation in passive serum-transfer arthritis, it significantly reduced inflammation in CIA, and this was associated with the absence of synovial macrophages and reduced splenic CD11b+Gr-1− monocytes. Conclusion CSF-1R was broadly expressed in human RA. Blockade of CSF-1R protected against bone and cartilage destruction in both mouse models and also showed significant antiinflammatory effects in the CIA model. These data provide evidence for CSF-1R as a therapeutic target in RA.

The humanized monoclonal antibody H27K15 specifically targets human CD115, a type III tyrosine kinase receptor involved in multiple cancers and inflammatory diseases. Binding of H27K15 to hCD115 expressing cells inhibits the functional effect of colony-stimulating factor-1 (CSF-1), in a non-competitive manner. Both homology modeling and docking programs were used here to model the human CD115 extracellular domains, the H27K15 variable region and their interaction. The resulting predicted H27K15 epitope includes mainly the D1 domain in the N-terminal extracellular region of CD115 and some residues of the D2 domain. Sequence alignment with the non-binding murine CD115, enzyme-linked immunosorbent assay, nuclear magnetic resonance spectroscopy and affinity measurements by quartz crystal microbalance revealed critical residues of this epitope that are essential for H27K15 binding. A combination of computational simulations and biochemical experiments led to the design of a chimeric CD115 carrying the human epitope of H27K15 in a murine CD115 backbone that is able to bind both H27K15 as well as the murine ligands CSF-1 and IL-34. These results provide new possibilities to minutely study the functional effects of H27K15 in a transgenic mouse that would express this chimeric molecule.

Women showing normal cytology but diagnosed with a persistent high-risk human papillomavirus (HR-HPV) infection have a higher risk of developing high-grade cervical intraepithelial neoplasia and cervical cancer than noninfected women. As no therapeutic management other than surveillance is offered to these women, there is a major challenge to develop novel targeted therapies dedicated to the treatment of these patients. As such, E1 and E2 antigens, expressed early in the HPV life cycle, represent very interesting candidates. Both proteins are necessary for maintaining coordinated viral replication and gene synthesis during the differentiation process of the epithelium and are essential for the virus to complete its normal and propagative replication cycle. In the present study, we evaluated a new active targeted immunotherapeutic, a modified vaccinia virus Ankara (MVA) vector containing the E1 sequence of HPV16, aimed at inducing cellular immune responses with the potential to help and clear persistent HPV16-related infection. We carried out an extensive comparative time course analysis of the cellular immune responses induced by different schedules of immunization in C57BL/6 mice. We showed that multiple injections of MVA-E1 allowed sustained HPV16 E1-specific cellular immune responses in vaccinated mice and had no impact on the exhaustion phenotype of the generated HPV16 E1-specific CD8+T cells, but they led to the differentiation of multifunctional effector T cells with high cytotoxic capacity. This study provides proof of concept that an MVA expressing HPV16 E1 can induce robust and long-lasting E1-specific responses and warrants further development of this candidate.

Cancer progression has been associated with the presence of tumor-associated M2-macrophages (M2-TAMs) able to inhibit anti-tumor immune responses. It is also often associated with metastasis-induced bone destruction mediated by osteoclasts. Both cell types are controlled by the CD115 (CSF-1R)/colony-stimulating factor-1 (CSF-1, M-CSF) pathway, making CD115 a promising target for cancer therapy. Anti-human CD115 monoclonal antibodies (mAbs) that inhibit the receptor function have been generated in a number of laboratories. These mAbs compete with CSF-1 binding to CD115, dramatically affecting monocyte survival and preventing osteoclast and macrophage differentiation, but they also block CD115/CSF-1 internalization and degradation, which could lead to potent rebound CSF-1 effects in patients after mAb treatment has ended. We thus generated and selected a non-ligand competitive anti-CD115 mAb that exerts only partial inhibitory effects on CD115 signaling without blocking the internalization or the degradation of the CD115/CSF-1 complex. This mAb, H27K15, affects monocyte survival only minimally, but downregulates osteoclast differentiation and activity. Importantly, it inhibits monocyte differentiation to CD163(+)CD64(+) M2-polarized suppressor macrophages, skewing their differentiation toward CD14(-)CD1a(+) dendritic cells (DCs). In line with this observation, H27K15 also drastically inhibits monocyte chemotactic protein-1 secretion and reduces interleukin-6 production; these two molecules are known to be involved in M2-macrophage recruitment. Thus, the non-depleting mAb H27K15 is a promising anti-tumor candidate, able to inhibit osteoclast differentiation, likely decreasing metastasis-induced osteolysis, and able to prevent M2 polarization of TAMs while inducing DCs, hence contributing to the creation of more efficient anti-tumor immune responses.

After acute hepatitis C virus (HCV) infection, 20% of individuals clear the virus, which is dependent on sustained Th1 CD4+ T lymphocyte–mediated responses together with polyfunctional CD8+ T cells [1–4]. HCV is highly efficient in establishing persistent infections, and the specific T-cell responses are impaired during chronic infection [5–8] due to appearance of escape mutants [4, 9], inhibitory effects exerted by viral proteins [10], or expression of coinhibitory receptors resulting in T-cell exhaustion [11, 12]. One way to prime T cells and/or reactivate impaired T cells is to express HCV antigens under a more “immunogenic” setting than natural infection where massive antigen expression appears in the tolerogenic liver environment. Therapeutic vaccination has been tested with some success in HCV-infected patients showing evidence of T-cell activation [13, 14]. Vectored vaccines tailored to generate robust T-cell immunity have recently reached the clinic. MVATG16643, a modified virus Ankara (MVA)–based vaccine [15], has shown in a phase I trial good safety and the capacity to induce interferon γ (IFN-γ)–producing T cells with significant although transient viral load decrease in chronically infected patients [16]. Used in combination with pegylated (PEG)–interferon α (IFN-α)/ribavirin in a phase II clinical trial, MVATG16643 resulted in a significant early viral response [17]. The DNA vaccine ChronVac-C [18] has also shown the capacity to induce T cells, which had transient effects on viral load in a phase I/IIa trial [19], and it has been suggested that it improves cure rates when given before PEG–IFN-α/ribavirin treatment [19]. The combined use of DNA vaccines with viral vectors in a prime/boost regimen has been proven useful for enhancing response levels in clinical studies [20–22]. Similarly, in vivo electroporation (EP)–mediated delivery of DNA vaccines either as stand-alone or in a prime/boost setting with viral vectors has also served to enhance the development of polyfunctional CD8+ T-cell responses [23, 24]. Here we have performed a proof-of-concept study to define the extent of improvement that could result from a prime/boost approach based on ChronVac-C and MVATG16643—2 individual vaccines currently in the clinic. These 2 vaccine regimens have been optimized extensively individually previously, and we wanted to investigate whether the combination of 2 regimens could confer additional benefits over the individual regimens. This study thus combines for the first time in the HCV setting approaches previously shown individually to enhance immunogenicity (ie, DNA vaccine delivery with in vivo EP and prime/boost using viral vectors). We performed an exhaustive evaluation of this concept in wild-type C57BL/6J and human leucocyte antigen (HLA)–A2 transgenic mice. This strategy was found very potent for the improvement of polyfunctional CD4+ and CD8+ HCV-specific responses and resulted in a significant increase of epitope recognition.

Cancer immunotherapy is hampered by the immunosuppression maintained by regulatory T cells (Tregs) in tumor-bearing hosts. Stimulation of the Toll-like receptor 2 (TLR2) by Pam3Cys is known to affect Treg-mediated suppression. We found that Pam3Cys increases the proliferation of both CD4(+) effector T cells (Teffs) and Tregs co-cultured in vitro, but did not induce the proliferation of Tregs alone upon CD3 and CD28 stimulation. In a mouse model of RMA-MUC1 tumors, Pam3Cys was administered either alone or in combination with a modified vaccinia ankara (MVA)-based mucin 1 (MUC1) therapeutic vaccine. The combination of Pam3Cys with MVA-MUC1 (1) diminished splenic Treg/CD4(+) T-cell ratios to those found in tumor-free mice, (2) stimulated a specific anti-MUC1 interferon γ (IFNγ) response and (3) had a significant therapeutic effect on tumor growth and mouse survival. When CD4(+) Teffs and Tregs were isolated from Pam3Cys-treated mice, Teffs had become resistant to Treg-mediated suppression while upregulating the expression of BclL-x(L). Tregs from Pam3Cys-treated mice were fully suppressive for Teffs from naïve mice. Bcl-x(L) was induced by Pam3Cys with different kinetics in Tregs and Teffs. Teff from Pam3Cys-treated mice produced increased levels of Th1 and Th2-type cytokines and an interleukin (IL)-6-dependent secretion of IL-17 was observed in Teff:Treg co-cultures, suggesting that TLR2 stimulation had skewed the immune response toward a Th17 profile. Our results show for the first time that in a tumor-bearing host, TLR2 stimulation with Pam3Cys affects both Tregs and Teffs, protects Teff from Treg-mediated suppression and has strong therapeutic effects when combined with an MVA-based antitumor vaccine.

International audience; BACKGROUND: Chemotherapy is the standard of care for advanced stages of non-small-cell lung cancer (NSCLC). TG4010 is a targeted immunotherapy based on a poxvirus (modified vaccinia virus Ankara) that codes for MUC1 tumour-associated antigen and interleukin 2. This study assessed TG4010 in combination with first-line chemotherapy in advanced NSCLC. METHODS: 148 patients with advanced (stage IIIB [wet] or IV) NSCLC expressing MUC1 by immunohistochemistry, and with performance status 0 or 1, were enrolled in parallel groups in this open-label, phase 2B study. 74 patients were allocated to the combination therapy group, and received TG4010 (10(8) plaque forming units) plus cisplatin (75 mg/m(2) on day 1) and gemcitabine (1250 mg/m(2) on days 1 and 8) repeated every 3 weeks for up to six cycles. 74 patients allocated to the control group received the same chemotherapy alone. Patients were allocated using a dynamic minimisation procedure stratified by centre, performance status, and disease stage. The primary endpoint was 6-month progression-free survival (PFS), with a target rate of 40% or higher in the experimental group. Analyses were done on an intention-to-treat basis. This study is completed and is registered with ClinicalTrials.gov, number NCT00415818. FINDINGS: 6-month PFS was 43*2% (32 of 74; 95% CI 33*4-53*5) in the TG4010 plus chemotherapy group, and 35*1% (26 of 74; 25*9-45*3) in the chemotherapy alone group. Fever, abdominal pain, and injection-site pain of any grade according to National Cancer Institute Common Toxicity Criteria were more common in the TG4010 group than in the chemotherapy alone group: 17 of 73 patients (23*3%) versus six of 72 (8*3%), 12 (16*4%) versus two (2*8%), and four (5*5%) versus zero (0%), respectively. The most common grade 3-4 adverse events were neutropenia (33 [45*2%] of patients in the TG4010 plus chemotherapy group vs 31 [43*1%] in the chemotherapy alone group) and fatigue (18 [24*7%] vs 13 [18*1%]); the only grade 3-4 events that differed significantly between groups were anorexia (three [4*1%] vs 10 [13*9%]) and pleural effusion (none vs four [5*6%]). 38 of 73 patients (52*1%) in the TG4010 plus chemotherapy group and 34 of 72 (47*2%) in the chemotherapy alone group had at least one serious adverse event. INTERPRETATION: This phase 2B study suggests that TG4010 enhances the effect of chemotherapy in advanced NSCLC. A confirmatory phase 2B-3 trial has been initiated. FUNDING: Transgene SA, Advanced Diagnostics for New Therapeutic Approaches (ADNA)/OSEO.

L’immunotherapie des cancers a longtemps ete consideree comme une approche therapeutique seduisante mais peu efficace. Deux nouvelles approches d’immunotherapie, l’une vaccinale chez des patients atteints de cancers de la prostate ou de melanomes, et l’autre utilisant un anticorps anti-CTLA4 qui cible les lymphocytes T de patients atteints de melanomes, ont demontre leur efficacite dans des essais cliniques de phase III. La prise en compte de l’immunosuppression associee au cancer et la combinaison de l’immunotherapie avec les traitements conventionnels (chimiotherapie, traitement antiangiogenique) devraient encore ameliorer cette approche, aussi bien dans son efficacite que dans ses indications cliniques.

Fighting viral infections typically includes two types of action: prevention, notably through wide use of vaccination, or treatment by means of small molecule-based therapeutic interventions. Unfor...

Any drug-based treatment, be it prophylactic or therapeutic, is bound to suffer from two limitations. The fi rst one is that the drug will never be active in 100% of individuals. In fact, even in classes of drugs such as prophylactic vaccines, which exhibit very high levels of effi cacy, a full effi cacy rate is very rarely obtained. In the case of small chemical drugs, the range of effi cacy is approximately 50‐70% and overall, 30% of patients do not benefi t from current medicines. The second limitation of any drug is its safety profi le. An ‘active’ drug is likely to induce side effects that will vary in severity from one individual to another. It is noteworthy in this context that the number of reported serious adverse

Tumor-associated antigens (TAAs) have been formulated into vaccines that, combined with adjuvants, cytokines or other strategies to boost the immune response, are now in clinical development. Both humoral and cellular immune responses to TAAs have been generated using these vaccines. This approach relies on the patient's own immune system generating an effective anti-tumor immune response. The advantage of this over therapy with monoclonal antibodies for the treatment of cancer is that multiple antigenic epitopes can be involved and the immune system is able to adapt to the most effective antigenic specificity for tumor growth control and rejection. In this article, we describe the clinical use of vaccinia virus, in particular modified vaccinia virus Ankara (MVA), to express TAAs in vivo and to stimulate an effective immune response to the cancer antigens.

Murine models have shown that IL-18 has antiangiogenic and antitumor effects, but little is known about IL-18 production in human tumors. We investigated IL-18 expression in clinically localized prostate cancers by immunohistochemistry and showed that 75% of the prostate cancers studied (27/36 cases) presented with tumor cells producing IL-18. Prostate tumor cell lines PC-3, DU 145 and LNCaP synthesized the immature form of IL-18 (p24). IFN-gamma produced in prostate cancers induced caspase-1 mRNA and IL-18 secretion of tumor cell lines, which was inhibited by the cell-permeable Tyr-Val-Ala-Asp-aldehyde caspase-1 inhibitor (YVAD-CHO). Interestingly, IFN-alpha also induced IL-18 secretion of the poorly differentiated cell line PC-3. PC-3 and DU 145, but not the well-differentiated cell line LNCaP, expressed IL-18R alpha (IL-1Rrp) protein and transcripts for IL-18R beta (AcPL). Exogenous IL-18 increased mitochondrial activity of both cell lines evaluated by the tetrazolium (MTT) assay but did not influence their proliferation. This indicated that prostate tumor cells could secrete IL-18 in response to IFN-gamma in the tumor microenvironment and that IL-18 could act as a autocrine/paracrine factor for the tumor. In the cohort of patients studied, IL-18 expression in prostate cancers (with up to 10% of tumor cells stained) was associated with a favorable outcome and equally predictive as pathologic stage on multivariate analysis (log rank test, p = 0.02). Tumor IL-18 production is a novel physiopathologic feature of prostate cancer and appears to be a favorable event in the course of the disease. Modulation of IL-18 production by interferons could have a beneficial clinical effect, which deserves further investigation.

Prevention of respiratory syncytial virus (RSV) disease will implicate neonatal priming. However, neonatal antigen exposure frequently results into Th2-like responses, some of which are critical for formalin-inactivated RSV (FI-RSV)-associated lung immunopathology. Neonatal immunization of mice may thus represent a more stringent model of RSV-enhanced pathology than adults. Indeed, after RSV challenge, lung cell infiltration, lymphocyte activation, and eosinophilia were higher following neonatal compared with adult FI-RSV priming of BALB/c mice. Unexpectedly, similar findings were obtained with Al(OH)(3)-adsorbed live RSV. In contrast, neonatal priming with BBG2Na, a recombinant RSV subunit vaccine candidate, formulated in either Al(OH)(3) or TiterMax (a Th1-driving adjuvant) resulted in predominant Th2- or Th1-like responses, respectively, but never elicited lung immunopathology post-challenge. Importantly, our data emphasize that the induction of Th2-like responses by RSV subunit vaccines do not necessarily imply lung immunopathology.

The protective efficacy of the influenza matrix protein epitope 58–66 (called M1), recognized in the context of human HLA-A2 molecules, was evaluated in a HLA-A2/Kb transgenic mouse model of lethal influenza infection. Repeated subcutaneous immunizations with M1 increased the percentage of survival. This effect was mediated by T cells since protection was abolished following in vivo depletion of all T lymphocytes, CD8+, or CD4+ T cells. The survival correlated with the detection of memory CD8+ splenocytes able to proliferate in vitro upon stimulation with M1 and to bind M1-loaded HLA-A2 dimers, as well as with M1-specific T cells in the lungs, which were directly cytotoxic to influenza-infected cells following influenza challenge. These results demonstrated for the first time that HLA-A2-restricted cytotoxic T cells specific for the major immunodominant influenza matrix epitope are protective against the infection. They encourage further in vivo evaluation of T cell epitopes recognized in the context of human MHC molecules.

Summary Outer membrane protein (Omp)A is highly represented and conserved in the Enterobacteriaceae family. Using a recombinant OmpA from Klebsiella pneumoniae (kpOmpA), we have analysed the interaction between this bacterial cell wall protein and human Langerhans cells (LC), the antigen-presenting cells of the epidermis and mucosa. We showed that biotinylated kpOmpA binds to human LC freshly isolated from epidermis. kpOmpA up-regulated MHC class II, CD86 and CCR7 expression, enhanced migration in response to macrophage inflammatory protein-3β (MIP-3β) through a reconstituted basement membrane mimicking the prerequisite passage through the dermal-epidermal basement membrane on the way to lymph nodes. The allostimulatory function of kpOmpA-treated LC was more potent than that of untreated cells. Even though the proportion of LC which binds kpOmpA was shown to vary between individuals, our data indicate that kpOmpA binds to and activates LC, and suggest that recognition of OmpA by LC may be an initiating event in the antibacterial host response.

Outer membrane protein A (OmpA) is a class of proteins highly conserved among the Enterobacteriaceae family and throughout evolution. We have observed that antigen presenting cells (APCs) recognize and are activated by the recombinant OmpA from Klebsiella pneumoniae (KpOmpA). KpOmpA triggers cytokine production by macrophages and dendritic cells (DC), induces DC maturation and signals via Toll-like receptor 2. KpOmpA also interacts with endocytic receptor(s) expressed on DC and macrophages. Tumor antigens coupled to KpOmpA are taken up by APCs and gain access to the MHC class I pathway, triggering the initiation of protective anti-tumor cytotoxic responses in the absence of CD4 T cell help and adjuvant. Thus, OmpA appears as a new type of pathogen-associated molecular pattern (PAMP) usable as a vector in anti-infectious and therapeutic anti-tumor vaccines to elicit CTLs.

International audience; Some exogenous antigens, such as heat shock proteins or apoptotic bodies, gain access to the MHC class I processing pathway and initiate CTL responses, a process called cross-priming. To be efficient in vivo, this process requires endocytosis of the antigen by dendritic cells via receptors which remain unidentified. Here, we report that scavenger receptors are the main HSP binding structures on human dendritic cells and identify LOX-1 as one of these molecules. A neutralizing anti-LOX-1 mAb inhibits Hsp70 binding to dendritic cells and Hsp70-induced antigen cross-presentation. In vivo, to target LOX-1 with a tumor antigen using an anti-LOX-1 mAb induces antitumor immunity. Thus, the scavenger receptor LOX-1 is certainly a promising target for cancer immunotherapy.

Human respiratory syncytial virus (hRSV) is one of the most common causes of respiratory infection in infants and the elderly. Previous attempts to vaccinate children against RSV failed and the induction of an aberrant Th2-type immune response was shown to induce severe to fatal pulmonary disease characterised in part by eosinophilia. BBG2Na is a promising human RSV subunit vaccine candidate which successfully passed phase II clinical trials in adults in association with Adju-Phos ® . However, this formulation is not the most suitable for use in children since aluminium salts are known to induce a Th2-based immune response. In this study, we describe a potent and safe adjuvant formulation for BBG2Na in dimethyldioctadecylammonium bromide (DDA) that induces a mixed Th1/Th2 immune response in BALB/c mice. Furthermore, BBG2Na showed the same protective efficacy against RSV challenge when formulated either in DDA or in alum in mice and cotton rats.

Background Nuclear factor kappa B (NF-κB) is a ubiquitously expressed transcription factor that regulates cytokine and immunoglobulin (Ig) gene expression. In most cell types, the inactive p50/p65 NF-κB heterodimer is located in the cytoplasm, complexed to its IκB inhibitory unit. Stimulation of cells by various reagents such as bacterial endotoxin or cytokines leads to a dissociation of NF-κB from IκB and a rapid translocation of free NF-κB to the nucleus. The aim of this article is to define optimal conditions for the measurement of NF-κB translocation by both confocal microscopy and flow cytometry. Methods Four commercial anti–NF-κB antibodies were evaluated by confocal microscopy, after using two methods of fixation and permeabilization of the cells. These antibodies were examined further by flow cytometry on purified nuclei. Results Paraformaldehyde-methanol treatment of dendritic cells is a good combination to visualize NF-κB translocation by confocal microscopy. Three of the four antibodies tested gave good results on nonactivated and on lipopolysaccharide (LPS)-activated dendritic cells. The measurement of NF-κB translocation by flow cytometry on purified nuclei is a quick and sensitive method. Only one of the four evaluated antibodies showed a significant difference between nonactivated and activated cells. Conclusions Microscopy and flow cytometry are quick and reproducible methods to measure NF-κB translocation and can be adapted to identify new molecules that activate dendritic cells. Cytometry 48:71–79, 2002. © 2002 Wiley-Liss, Inc.

The heterodimeric cytokine composed of the soluble ciliary neurotrophic factor receptor (sCNTFR) and the IL-6 family member cardiotrophin-like cytokine (CLC) was recently identified as a new ligand for gp130–leukemia inhibitory factor receptor (LIFR) complex [Plun-Favreau, H., Elson, G., Chabbert, M., Froger, J., deLapeyriere, O., Lelievre, E., Guillet, C., Hermann, J., Gauchat, J. F., Gascan, H. & Chevalier, S. (2001) EMBO J.20, 1692–1703]. This heterodimer shows overlapping biological properties with LIF. Although CLC contains a putative signal peptide and therefore should enter into the classical secretory pathway, the protein has been shown to be retained within transfected mammalian cells, unless coexpressed with either sCNTFR or cytokine like factor (CLF) [Elson, G. C., Lelievre, E., Guillet, C., Chevalier, S., Plun-Favreau, H., Froger, J., Suard, I., de Coignac, A. B., Delneste, Y., Bonnefoy, J. Y., Gauchat, J. F. & Gascan, H. (2000) Nat. Neurosci.3, 867–872]. In the present study, we demonstrate that a fusion protein comprising CLC covalently coupled through a glycine/serine linker to sCNTFR (CC–FP) is efficiently secreted from transfected mammalian cells. CC–FP shows enhanced activities in respect to the CLC/sCNTFR native complex, on a number of cells expressing gp130 and LIFR on their surface. In addition, CC–FP is able to compete with CNTF for cell binding, indicating that both cytokines share binding epitope(s) expressed by their receptor complex. Analysis of the downstream signaling events revealed the recruitment by CC–FP of the signal transducer and activator of transcription (STAT)-3, Akt and mitogen-activated protein (MAP) kinase pathways. The monomeric bioactive CLC/sCNTFR fusion protein is therefore a powerful tool to study the biological role of the recently described cytokine CLC.

To identify novel stimulators of the innate immune system, we constructed a panel of eight HEK293 cell lines double positive for human Toll-like receptors (TLRs) and an NF-κB-inducible reporter gene. Screening of a large variety of compounds and cellular extracts detected a TLR3-activating compound in a microsomal yeast extract. Fractionation of this extract identified an RNA molecule of 4.6 kb, named nucleic acid band 2 (NAB2), that was sufficient to confer the activation of TLR3. Digests with single- and double-strand-specific RNases showed the double-strand nature of this RNA, and its sequence was found to be identical to that of the genome of the double-stranded RNA (dsRNA) L-BC virus of Saccharomyces cerevisiae . A large-scale process of production and purification of this RNA was established on the basis of chemical cell lysis and dsRNA-specific chromatography. NAB2 complexed with the cationic lipid Lipofectin but neither NAB2 nor Lipofectin alone induced the secretion of interleukin-12(p70) [IL-12(p70)], alpha interferon, gamma interferon-induced protein 10, macrophage inflammatory protein 1β, or IL-6 in human monocyte-derived dendritic cells. While NAB2 activated TLR3, Lipofectin-stabilized NAB2 also signaled via the cytoplasmic sensor for RNA recognition MDA-5. A significant increase of RMA-MUC1 tumor rejection and survival was observed in C57BL/6 mice after prophylactic vaccination with MUC1-encoding modified vaccinia virus Ankara (MVA) and NAB2-Lipofectin. This combination of immunotherapies strongly increased at the injection sites the percentage of infiltrating natural killer (NK) cells and plasmacytoid dendritic cells (pDCs), cell types which can modulate innate and adaptive immune responses. IMPORTANCE Virus-based cancer vaccines offer a good alternative to the treatment of cancer but could be improved. Starting from a screening approach, we have identified and characterized an unexplored biological molecule with immunomodulatory characteristics which augments the efficacy of an MVA-based immunotherapeutic agent. The immune modulator consists of the purified dsRNA genome isolated from a commercially used yeast strain, NAB2, mixed with a cationic lipid, Lipofectin. NAB2-Lipofectin stimulates the immune system via TLR3 and MDA-5. When it was injected at the MVA vaccination site, the immune modulator increased survival in a preclinical tumor model. We could demonstrate that NAB2-Lipofectin augments the MVA-induced infiltration of natural killer and plasmacytoid dendritic cells. We suggest indirect mechanisms of activation of these cell types by the influence of NAB2-Lipofectin on innate and adaptive immunity. Detailed analysis of cell migration at the vaccine injection site and the appropriate choice of an immune modulator should be considered to achieve the rational improvement of virus vector-based vaccination by immune modulators.

Human respiratory syncytial virus (hRSV) is a major pathogen responsible for bronchiolitis and severe pulmonary disease in very young children, immunodeficient patients and the elderly. BBG2Na, a recombinant chimeric protein produced in Escherichia coli, is a promising subunit vaccine candidate against this respiratory pathogen, composed of G2Na, the central domain of RSV G glycoprotein, and BB, an albumin binding domain of streptococcal protein G. BBG2Na has a basic isoelectric point (pI 9.3) and as expected, is strongly adsorbed by aluminium phosphate (AP). Surprisingly, BBG2Na is also strongly adsorbed by aluminium hydroxide (AH), which normally binds molecules with acidic isoelectric points. This behaviour was unexpected according to the well established adsorption model of Hem and co-workers. Our observations may be explained by the bipolar two-domain structure of the BBG2Na chimera which is not reflected by the global basic isoelectric point of the whole protein: the BB domain has an acidic isoelectric point (pI 5.5) and the G2Na domain a highly basic one (pI 10.0). Importantly, formulation in either aluminium salt resulted in equally high immunogenicity and protective efficacy against RSV in mice. From a physicochemical point of view, this unique property of BBG2Na makes it eminently suitable for combination to either paediatric or elderly multivalent AH- or AP-containing vaccines already in the market or in development.

Respiratory syncytial virus (RSV) is an important respiratory pathogen in man, against which no vaccine is available. However, recent evidence suggests that antibodies to the RSV F and G proteins may play an important role in disease prevention. We previously demonstrated that BBG2Na, a subunit vaccine candidate including residues 130-230 of the Long strain G protein, protects rodents against RSV challenge. Using a panel of monoclonal antibodies (MAb) and synthetic peptides, five linear B cell epitopes were identified that mapped to residues 152-163, 165-172, 171-187 (two over-lapping epitopes) and 196-204. Antibody passive transfer and peptide immunisation studies revealed that all were protective. Pepscan analyses of anti-RSV-A and BBG2Na murine polyclonal sera suggested stronger immunogenicity of some protective epitopes (protectopes) in the context of BBG2Na compared with live virus. However, all the identified murine B cell protectopes were conserved in RSV seropositive humans. Should these protectopes correspond with protection in humans, BBG2Na may constitute a very interesting vaccine candidate against RSV.

CD86 is a costimulatory molecule constitutively expressed by human antigen presenting cells which interacts with CD28 and CTLA-4 expressed by T cells. We have recently reported the identification of an alternatively spliced CD86 mRNA variant (CD86deltaTM) characterized by the deletion of exon 6 which encodes for the transmembrane domain. We report here the identification of an alternatively spliced variant (called CD86deltaEC) expressed by nonstimulated human monocytes and characterized by the deletion of exons 4 and 5 which encode for the extracellular V-like and C-like domains, respectively. The activation of monocytes by IFNgamma (i) induces the preferential expression of the transcript encoding for the membrane form and (ii) increases the expression of CD86 and of the accessory molecules CD40, CD49d and CD54. These results suggest that resting human monocytes may constitutively express different forms of CD86 which can then influence the activation of T cells.

A novel recombinant respiratory syncytial virus (RSV) subunit vaccine, designated BBG2Na, was administered to 108 healthy adults randomly assigned to receive 10, 100, or 300 microg of BBG2Na in aluminum phosphate or saline placebo. Each subject received 1, 2, or 3 intramuscular injections of the assigned dose at monthly intervals. Local and systemic reactions were mild, and no evidence of harmful properties of BBG2Na was reported. The highest ELISA and virus-neutralizing (VN) antibody responses were evident in the 100- and 300-microg groups; second or third injections provided no significant boosts against RSV-derived antigens. BBG2Na inducedor 2-fold andor =4-fold increases in G2Na-specific ELISA units in up to 100% and 57% of subjects, respectively; corresponding RSV-A-specific responses were 89% and 67%. Furthermore, up to 71% of subjects hador =2-fold VN titer increases. Antibody responses to 2 murine lung protective epitopes were also highly boosted after vaccination. Therefore, BBG2Na is safe, well tolerated, and highly immunogenic in RSV-seropositive adults.

Colony-stimulating factor 1 receptor (CSF-1R) essentially modulates monocyte proliferation, migration, and activation, which are considered important for the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine CSF-1R expression in human RA as well as the efficacy of a specific anti-CSF-1R monoclonal antibody (AFS98) in 2 different animal models of RA.CSF-1R expression was examined in blood, synovium, and bone samples from RA patients, osteoarthritis (OA) patients, and healthy subjects. The efficacy of AFS98 was examined by clinical assessment, histology, and bone histomorphometry in collagen-induced arthritis (CIA) and serum-transfer arthritis.CSF-1R expression was increased in the synovium of RA patients compared to OA patients and healthy controls in fibroblast-like synoviocytes, follicular dendritic cells, macrophages, and osteoclasts. Circulating RA monocytes and neutrophils but not lymphocytes were CSF-1R+. In mice, blockade of CSF-1R abrogated cartilage damage, bone erosion, and systemic bone loss, and this was associated with the depletion of osteoclasts in both models. While blockade of CSF-1R did not affect inflammation in passive serum-transfer arthritis, it significantly reduced inflammation in CIA, and this was associated with the absence of synovial macrophages and reduced splenic CD11b+Gr-1- monocytes.CSF-1R was broadly expressed in human RA. Blockade of CSF-1R protected against bone and cartilage destruction in both mouse models and also showed significant antiinflammatory effects in the CIA model. These data provide evidence for CSF-1R as a therapeutic target in RA.

Outer membrane protein (Omp)A is highly represented and conserved in the Enterobacteriaceae family. Using a recombinant OmpA from Klebsiella pneumoniae (P40), we have analyzed the interaction between OmpA and macrophages. We report that Alexa488-labeled P40 binds (at 4°C) to murine and human macrophages in a dose-dependent manner and is rapidly internalized (at 37°C). No binding or internalization of the Alexa488-labeled glycophorin A control protein is observed under the same conditions. Furthermore, P40 up-regulates the production of IL-1β, IL-8, IL-10, IL-12, and TNF-α by human macrophages and of NO by the RAW 264.7 murine macrophage cell line. P40 also synergizes with IFN-γ and suboptimal concentrations of LPS to up-regulate the production of these mediators. In conclusion, P40 binds to and activates macrophages. These data suggest that recognition of OmpA by macrophages may be an initiating event in the antibacterial host response.

2B4 is a surface molecule involved in activation of the natural killer (NK) cell–mediated cytotoxicity. It binds a protein termed Src homology 2 domain–containing protein (SH2D1A) or signaling lymphocyte activation molecule (SLAM)-associated protein (SAP), which in turn has been proposed to function as a regulator of the 2B4-associated signal transduction pathway. In this study, we analyzed patients with X-linked lymphoproliferative disease (XLP), a severe inherited immunodeficiency characterized by critical mutations in the SH2D1A gene and by the inability to control Epstein-Barr virus (EBV) infections. We show that, in these patients, 2B4 not only fails to transduce triggering signals, but also mediates a sharp inhibition of the NK-mediated cytolysis. Other receptors involved in NK cell triggering, including CD16, NKp46, NKp44, and NKp30, displayed a normal functional capability. However, their activating function was inhibited upon engagement of 2B4 molecules. CD48, the natural ligand of 2B4, is highly expressed on the surface of EBV+ B cell lines. Remarkably, NK cells from XLP patients could not kill EBV+ B cell lines. This failure was found to be the consequence of inhibitory signals generated by the interaction between 2B4 and CD48, as the antibody-mediated disruption of the 2B4–CD48 interaction restored lysis of EBV+ target cells lacking human histocompatibility leukocyte antigen (HLA) class I molecules. In the case of autologous or allogeneic (HLA class I+) EBV+ lymphoblastoid cell lines, restoration of lysis was achieved only by the simultaneous disruption of 2B4–CD48 and NK receptor–HLA class I interactions. Molecular analysis revealed that 2B4 molecules isolated from either XLP or normal NK cells were identical. As expected, in XLP-NK cells, 2B4 did not associate with SH2D1A, whereas similar to 2B4 molecules isolated from normal NK cells, it did associate with Src homology 2 domain–containing phosphatase 1.

A live bacterial vaccine-delivery system based on the food-grade bacterium Staphylococcus carnosus was used for delivery of peptides from the G glycoprotein of human respiratory syncytial virus, subtype A (RSV-A). Three peptides, corresponding to the G protein amino acids, 144-159 (denoted G5), 190-203 (G9) and 171-188 (G4 S), the latter with four cysteine residues substituted for serines, were expressed by recombinant means as surface-exposed on three different bacteria, and their surface accessibility on the bacteria was verified by fluorescence-activated cell sorting (FACS). Intranasal immunization of mice with the live recombinant staphylococci elicited significant anti-peptide as well as anti-virus serum IgG responses of balanced IgG1/IgG2a isotype profiles, and upon viral challenge with 10(5) tissue culture infectious doses(50) (TCID(50)), lung protection was demonstrated for approximately half of the mice in the G9 and G4 S immunization groups. To our knowledge, this is the first study in which protective immunity to a viral pathogen has been evoked using food-grade bacteria as vaccine-delivery vehicles.

We analyzed the protective mechanisms induced against respiratory syncytial virus subgroup A (RSV-A) infection in the lower and upper respiratory tracts (LRT and URT) of BALB/c mice after intraperitoneal immunization with a recombinant fusion protein incorporating residues 130 to 230 of RSV-A G protein (BBG2Na). Mother-to-offspring antibody (Ab) transfer and adoptive transfer of BBG2Na-primed B cells into SCID mice demonstrated that Abs are important for LRT protection but have no effect on URT infection. In contrast, RSV-A clearance in the URT was achieved in a dose-dependent fashion after adoptive transfer of BBG2Na-primed T cells, while it was abolished in BBG2Na-immunized mice upon in vivo depletion of CD4+, but not CD8+, T cells. Furthermore, the conserved RSV-A G protein cysteines and residues 193 and 194, overlapping the recently identified T helper cell epitope on the G protein (P. W. Tebbey et al., J. Exp. Med. 188:1967–1972, 1998), were found to be essential for URT but not LRT protection. Taken together, these results demonstrate for the first time that CD4+T cells induced upon parenteral immunization with an RSV G protein fragment play a critical role in URT protection of normal mice against RSV infection.

We have evaluated the expression and the involvement of membrane-associated TNF-alpha (mTNF-alpha) in human NK cell-mediated cytotoxicity. Results from FCM analysis show that peripheral blood NK cells constitutively express mTNF-alpha. In contrast, mTNF-alpha expression is undetectable on resting T cells, B cells and monocytes. Western blotting analysis confirmed that freshly purified NK cells express the 17-kDa soluble form (sTNF-alpha) and the 26-kDa transmembrane form of TNF-alpha. Stimulation with IL-2, IL-15 and IL-18 up-regulates TNF-alpha mRNA, sTNF-alpha and mTNF-alpha expression in NK cells. The role of mTNF-alpha in the cytotoxic activity of resting NK cells has been evaluated in in vitro cytotoxic assays using freshly purified NK cells fixed with paraformaldehyde as effector cells (in order to avoid the participation of cytotoxic soluble mediators such as perforin, granzymes or sTNF-alpha) and the TNF-alpha-sensitive Fas ligand- and TRAIL-resistant cell line KYM-1-D4 as target cell. Results show that fixed NK cells kill the KYM-1-D4 cells and that neutralizing anti-TNF-alpha antibodies partly prevent this effect. In contrast to the other types of peripheral blood mononuclear cells NK cells from adult blood constitutively express functional mTNF-alpha in the absence of prior contact with target cells or activation. These data demonstrate a novel mechanism of cell-mediated cytotoxicity by non-acitvated human peripheral blood NK cells.

We have recently cloned a new protein, recombinant P40 (rP40). When tested in vivo after conjugation to a B-cell epitope, rP40 induces an important antibody response without the need for adjuvant. To characterize its potency, this carrier protein was coupled to a peptide derived from respiratory syncytial virus attachment G protein (G1′). After immunization of mice with the rP40-G1′ conjugate, strong antipeptide antibodies were detected, whereas peptide alone was not immunogenic. To emphasize the carrier properties of rP40, a polysaccharide derived from Haemophilus influenzae type b (Hib) was coupled to it. Immunoglobulin G responses against the Hib polysaccharide were observed after coupling to rP40. Interestingly, an antipeptide antibody response was observed despite preexisting anti-rP40 antibodies generated by preimmunization with rP40. In addition, rP40 compares well with the reference carrier protein, tetanus toxoid (TT), since antibody responses of equal intensity were observed when a peptide or a polysaccharide was coupled to TT and rP40. Moreover, rP40 had advantages compared to TT; e.g., it induced a mixed Th1/Th2 response, whereas TT induced only a Th2 profile. Together, the results indicate that rP40 is a novel carrier protein with potential for use as an alternative carrier for human vaccination.

Background: Current cytotoxic assays, including Cr release and fluorescent assays, do not directly measure the proportion of target cells which are killed by apoptosis. Cell-mediated cytotoxicity induced by CTLs and NK cells is mainly regulated by the perforin-granzyme, the Fas ligand (Fas L), and the Tumor Necrosis Factor (TNF)-a pathways. Perforin generates pores in the membrane of target cells, allowing granzyme B to enter and initiate apoptosis. The other effectors, Fas L and TNF-a act by an apoptosis mechanism, leading to DNA fragmentation. A three color flow cytometric method to measure cell-mediated cytotoxicity induced by CTLs or NK cells is described. Methods: The fluorochromes used are: PKH-26, a stable membrane dye for the labeling of the effector cells, annexin V-FITC which allows the direct evaluation of early apoptotic cells and propidium iodide which distinguishes membrane permeabilized and late apoptotic cells. Results: By eliminating through gating PKH-26 positive effector cells, we obtain a direct estimation of the percentage of target cells in the early stages of apoptosis as well as the percentage of target cells dying after late apoptosis and membrane permeabilization. The cytotoxic activity of IL-2 stimulated PBL against K562, Jurkat and KYM-1 was evaluated. Conclusions: This rapid and novel assay permits the discrimination of the cell death mechanisms occurring during a cytotoxic response and to precisely evaluate the contribution of apoptosis in the early phases of cell-mediated cytotoxicity. Cytometry 37: 197‐204, 1999. r 1999 Wiley-Liss, Inc. Key terms: cytotoxicity; apoptosis; annexin V; PKH-26; flow cytometry; TUNEL

CTLA-4, expressed by activated T cells, transduces an inhibitory signal. We show here that PCR amplification of the coding sequence of CTLA-4 in nonstimulated human T lymphocytes results in the amplification of two transcripts of 650 and 550 bp. Sequencing shows that the larger form codes for membrane CTLA-4 and the 550-bp transcript is a spliced variant in which exon 2 coding for the transmembrane region is deleted. This spliced cDNA has been named CTLA-4delTM. The splicing induces a frame shift which results in the addition of 22 extra amino acids before a translational termination. Activation of T cells with phorbol 12-myristate 13-acetate plus ionomycin or anti-CD3 plus anti-CD28 monoclonal antibodies induces a suppression of CTLA-4delTM mRNA expression associated with a preferential expression of the membrane CTLA-4 mRNA, showing that CTLA-4delTM mRNA expression is restricted to nonactivated T cells. A soluble immunoreactive form of CTLA-4 was detected in the serum of 14 / 64 healthy subjects. These results suggest that nonstimulated T cells may constitutively produce a soluble form of CTLA-4 which may have an important role in the regulation of immune homeostasis.

Using the sequence of cosmids derived from chromosome 19p12, we have identified a gene encoding a novel protein, BSMAP (brain-specific membrane-anchored protein) and cloned cDNA encoding the full-length open reading frame. Northern blot analysis revealed that BSMAP mRNA is preferentially expressed at a high level in the brain. BSMAP has a putative transmembrane domain and is predicted to be a type-I membrane glycoprotein. Genomic sequence analysis revealed that the gene encoding BSMAP consists of eight exons spanning approximately 8 kb and lies 6 kb away from the gene encoding CLF-1 in a reverse orientation. Although no candidate genetic disorders were found to map either to this precise region of chromosome 19 or to the syntenic region of the mouse genome, the highly specific expression of BSMAP mRNA suggests a role for the protein in CNS function.

The possibility of improving the antibody responses to a model streptococcal antigen, administered by intranasal immunization as surface-displayed on the food-grade bacterium Staphylococcus carnosus, by co-exposure of a peptide (CTBp) comprising amino acids 50-75 of the cholera toxin B subunit, was investigated. It was found that the introduction of the CTBp into the chimeric surface proteins, containing a serum albumin binding protein (ABP) from streptococcal protein G as model antigen, significantly increased serum IgG responses upon intranasal immunization. Similarly, elicited local IgA responses were also found to be improved. Furthermore, it was demonstrated that live delivery of the staphylococci was required to obtain this effect, since UV-irradiated or heat-killed bacteria exposing the same chimeric surface proteins did not show increased anti-ABP IgG responses.

Alum-adsorbed BBG2Na, a recombinant vaccine derived in part from the respiratory syncytial virus (RSV) subgroup A G protein, induced moderate antibody titers after 1 immunization in 1-week-old mice but conferred complete lung protection upon RSV challenge. The anti-BBG2Na IgG1-IgG2a neonatal isotype profile was suggestive of dominant Th2 responses compared with those in adults. Formulation of BBG2Na with a Th1-driving adjuvant efficiently shifted neonatal responses toward a more balanced and adultlike IgG1-IgG2a profile without compromising its protective efficacy. BBG2Na-induced protective immunity was maintained even after early life immunization in the presence of high titers of maternal antibodies. Under these conditions, the protective efficacy (86%-100%) reflected the high capacity of the nonglycosylated G2Na immunogen to escape inhibition by RSV-A-induced maternal antibodies. Thus, immunization with BBG2Na protected against viral challenge despite neonatal immunologic immaturity and the presence of maternal antibodies, two major obstacles to neonatal RSV vaccine development.

BBG2Na is a protein comprising residues 130-230 of the respiratory syncytial virus subgroup A (RSV-A) G protein (G2Na) fused to the albumin-binding domain of streptococcal G protein (BB). BBG2Na was cloned, expressed in Escherichia coli and renaturated. In rodent models, this subunit RSV vaccine adjuvanted in Alhydrogel induced specific antibodies and conferred protection to RSV infection. Comparison of the antibody production in a BALB/c mouse model revealed that BBG2Na induced a stronger and earlier G2Na antibody response than G2Na alone, without altering the IgG subclass distribution. To address the role of the BB part, we explored its carrier properties and showed that it is a Th dependent antigen, generating a more potent G2Na-specific B cell memory response and able to generate Th cells that provide help for G2Na antibody production.

Neovascularization frequently accompanies chronic immune responses characterized by T cell infiltration and activation. Angiogenesis requires endothelial cells (ECs) to penetrate extracellular matrix, a process that involves matrix metalloproteinases (MMPs). We report here that activated human T cells mediate contact-dependent expression of MMPs in ECs through CD40/CD40 ligand signaling. Ligation of CD40 on ECs induced de novo expression of gelatinase B (MMP-9), increased interstitial collagenase (MMP-1) and stromelysin (MMP-3), and activated gelatinase A (MMP-2). Recombinant human CD40L induced expression of MMPs by human vascular ECs to a greater extent than did maximally effective concentrations of interleukin-1β or tumor necrosis factor-α. Moreover, activation of human vascular ECs through CD40 induced tube formation in a three-dimensional fibrin matrix gel assay, an effect antagonized by a MMP inhibitor. These results demonstrated that activation of ECs by interaction with T cells induced synthesis and release of MMPs and promoted an angiogenic function of ECs via CD40L-CD40 signaling. As vascular cells at the sites of chronic inflammation, such as atherosclerotic plaques, express CD40 and its ligand, our findings suggest that ligation of CD40 on ECs can mediate aspects of vascular remodeling and neovessel formation during atherogenesis and other chronic immune reactions.

To study the expression of IL-13 receptor alpha1 (IL-13Ralpha1), specific monoclonal antibodies (mAb) were generated. Surface expression of the IL-13Ralpha1 on B cells, monocytes and T cells was assessed by flow cytometry using these specific mAb. Among tonsillar B cells, the expression was the highest on the IgD+ CD38- B cell subpopulation which is believed to represent naive B cells. Expression was also detectable on a large fraction of the IgD-CD38- B cells but not on CD38+ B cells. Activation under conditions which promote B cell Ig class switching up-regulated the expression of the receptor. However, the same stimuli had an opposite effect for IL-13Ralpha1 expression levels on monocytes. While IL-13Ralpha1 mRNA was clearly detectable in T cell preparations, no surface expression was detected. However, permeabilization of the T cells showed a clear intracellular expression of the receptor. A soluble form of the receptor was immunoprecipitated from the supernatant of activated peripheral T cells, suggesting that T cell IL-13Ralpha1 might have functions unrelated to the capacity to form a type II IL-4/IL-13R with IL-4Ralpha.

In the present study we have compared the binding of two monoclonal antibodies to CD23, EBVCS1 and mAb25, which recognize the stalk and the lectin domain, respectively, on the CD23 molecule. At 4 degreesC, EBVCS1 binds to about 10% of the receptors recognized by mAb25 on the B cell surface. At 37 degreesC, whereas mAb25 reaches its maximal binding within a few seconds, EBVCS1 requires 60 min to bind to the same extent. Stabilization of the oligomeric structure of CD23 with IgE strongly affects in a dose-dependent fashion the number of binding sites seen by EBVCS1 but not the t1/2 to reach them, suggesting that EBVCS1 binds to the coiled coil region through an allosteric mechanism. EBVCS1 rapidly down-modulates the membrane CD23 expression with a coincident increase of CD23-soluble fragments in the culture medium, an effect that is inhibited by IgE. In contrast, mAb25, as well as IgE, protects CD23 from proteolytic cleavage and stimulates its endocytosis. These results suggest that EBVCS1 unravels the coiled coil structure of CD23, rendering it more susceptible to proteolytic attack. This supports the oligomeric model proposed previously (Gould, H., Sutton, B., Edmeades, R., and Beavil, A. (1991) Monogr. Allergy 29, 28-49). The biological significance of these observations is discussed.

Interleukin-7 (IL-7) is a B-cell growth factor produced by both bone marrow stroma cells and follicular dendritic cells (FDCs) located in primary lymphoid follicles and germinal centers. In this study, we have evaluated the role of IL-7 on human Ig class switching. IL-7 was added to peripheral blood mononuclear cells (PBMCs) or tonsillar B cells in the absence or presence of IL-4 and/or anti-CD40 monoclonal antibody (MoAb). Alone, IL-7 did not affect Ig production by PBMCs or by anti-CD40 MoAb-stimulated B cells. Rather, IL-7 potentiated IL-4–induced IgE and IgG4 production by PBMCs. In parallel, IgG3 production was also enhanced but to a lesser extent, whereas the production of the other isotypes was unaltered. The activity of IL-2, IL-9, or IL-15, which share usage of the common γ chain for signaling, was also assessed. IL-9, like IL-7, potentiated mainly IgE and IgG4 production by IL-4–stimulated PBMCs. IL-15, in contrast, was ineffective, whereas IL-2 enhanced the production of all isotypes. More precisely, IL-7 potentiation of IgE and IgG4 production required the presence of T cells and was accompanied by an increase of the expression of two soluble molecules favoring preferentially IgE and IgG4 synthesis: CD23 (sCD23) and IL-9. Moreover, neutralizing anti-CD23 and anti–IL-9 antibodies partly inhibited the increase of IgE synthesis induced by IL-7. Thus, IL-7 produced locally in the germinal centers by FDCs may interact with T cells and potentiate human IgE and IgG4 switching by favoring IL-9 and sCD23 production.

CD23 is expressed on a variety of hemopoietic cells. Recently, we have reported that blocking CD23 interactions in a murine model of arthritis resulted in a marked improvement of disease severity. Here, we demonstrate that CD11b, the alpha chain of the beta 2 integrin adhesion molecule complex CD11b/CD18 expressed on monocytes interacts with CD23. Using a recombinant fusion protein (ZZ-CD23), murine CD23 was shown to bind to peritoneal macrophages and peripheral blood cells isolated from mice as well as the murine macrophage cell line, RAW. The interactions between mouse ZZ-CD23 and CD11b/CD18-expressing cells were significantly inhibited by anti-CD11b monoclonal antibodies. A functional consequence was then demonstrated by inducing an up-regulation of interleukin-6 (IL-6) production following ZZ-CD23 incubation with monocytes. The addition of Fab fragments generated from the monoclonal antibody CD11b impaired this cytokine production by 50%. Interestingly, a positive autocrine loop was identified as IL-6 was shown to increase CD23 binding to macrophages. These results demonstrate that similar to findings using human cells, murine CD23 binds to the surface adhesion molecule, CD11b, and these interactions regulate biological activities of murine myeloid cells.

Abstract Physical disruption of an atheromatous lesion often underlies acute coronary syndromes. Matrix-degrading enzymes, eg, matrix metalloproteinases (MMPs), may cause loss in mechanical integrity of plaque tissue that favors rupture. T lymphocytes accumulate at sites where atheromata rupture, but the mechanisms by which these immune cells may contribute to plaque destabilization are unknown. This study tested the hypothesis that the T-lymphocyte surface molecule CD40 ligand (CD40L), recently localized in atherosclerotic plaques, regulates the expression of MMPs in human vascular smooth muscle cells (SMCs), the most numerous cell type in arteries. We report here that stimulated human T lymphocytes induced the expression of the matrix-degrading enzymes, ie, interstitial collagenase (MMP-1), stromelysin (MMP-3), gelatinase B (MMP-9), and activated gelatinase A (MMP-2), in human vascular SMCs by cell contact via CD40 ligation, as demonstrated by Western blot analysis, zymography, and antibody neutralization. Recombinant human CD40L (rCD40L) induced de novo synthesis of MMP-1, MMP-3, and MMP-9 on vascular SMCs and stimulated the expression of these enzymes to a greater extent than did maximally effective concentrations of tumor necrosis factor-α or interleukin-1β, established agonists of MMP expression. Interferon gamma, another T-lymphocyte–derived cytokine, inhibited the induction of MMPs by rCD40L. Immunohistochemical analysis of human coronary atheromata colocalized MMP-1 and MMP-3 with CD40-positive SMCs. These results demonstrated that CD40 ligand, expressed on T lymphocytes, promoted the expression of matrix-degrading enzymes in vascular SMCs and thus established a new pathway of immune-modulated destabilization in human atheromata.

Because of its anticarcinogenic and antimutagenic properties, N-acetyl-L-cysteine (NAC) has been proposed for cancer treatment. Here we present a mechanism of action for NAC in cancer. Our data show that NAC (1) induces an early and sustained increase of membrane tumor necrosis factor α (TNFα) expression on human stimulated-peripheral blood (PB) T cells and (2) increases membrane TNF-RI and TNF-RII on tumoral cell lines and on T cells after stimulation. These effects result from an early inhibition of both TNFα and TNF-R shedding, as well as a later increase of the respective mRNA expression. Consequently, NAC confers cytotoxic properties to human PB T cells through a membrane TNFα-dependent pathway. In vivo, NAC given orally inhibits tumor appearance in more than a third (18 out of 50) B6D2F1 mice injected with L1210 lymphoma cells. Spleen cells from protected mice killed L1210 lymphoma cells in vitro in a membrane TNFα-dependent manner. Furthemore these mice were resistant to a second inoculation of L1210 cells without further treatment with NAC. Thus, NAC exhibits a potent antitumoral activity by modulating TNFα and TNF-R processing without showing any in vitro and in vivo toxicity.

Inflammation contributes to a variety of arterial diseases including atherosclerosis. Interleukin 1beta (IL-1beta) in its activated mature 17-kDa form may mediate aspects of vascular inflammation. As shown previously, human vascular wall cells, such as smooth muscle cells (SMC), express the IL-1beta precursor upon stimulation and the IL-1beta-converting enzyme (ICE) constitutively but do not produce mature IL-1beta or express ICE activity. How SMC, the most numerous cell type in arteries, may release active IL-1beta has therefore remained a perplexing problem. We report here that stimulation of human vascular SMC and endothelial cells (EC) through CD40 ligand, a mediator recently localized in human atheroma, induced elaboration of the IL-1beta precursor as well as activation of cell-associated ICE. In addition to the constitutively expressed 45- and 30-kDa immunoreactive ICE proteins, vascular cells incubated with recombinant human CD40 ligand (rCD40L) (but not IL-1 or TNF) showed an increase of a 20-kDa immunoreactive ICE protein by Western blot analysis. Furthermore, SMC and EC stimulated through rCD40L processed recombinant human IL-1beta precursor (pIL-1beta), generating a cleavage product of approximately 17 kDa. Appearance of both the 20-kDa immunoreactive ICE protein and pIL-1beta processing activity required at least 6 h of stimulation with 0.3 or 1.0 microg/ml rCD40L, respectively, and was inhibited by pre-incubation of the ligand with an anti-CD40L antibody. Stimulation of vascular SMC and EC through rCD40L resulted in the release of biologically active IL-1beta, indicating processing of the native IL-1beta precursor induced by the ligand. These findings establish a novel mechanism of IL-1beta activation in human vascular cells and, moreover, indicate a new pathway of ICE-activation, which could participate in inflammatory aspects of atherogenesis and other disease states.

Background Plaque disruption with thrombosis commonly causes the acute coronary syndromes. Macrophages, abundant at sites of plaque rupture, release proteinases that weaken plaques and express tissue factor (TF), which initiates thrombosis. The signals that induce expression of these macrophage functions, particularly TF, remain obscure. Recent studies have localized the receptor CD40 and its ligand in human atheroma. This study tested the hypothesis that ligation of CD40 can activate key mononuclear phagocyte functions related to clinical manifestations of atheroma. Methods and Results Stimulation of human monocytes/macrophages through CD40 by either membranes from activated T cells or recombinant CD40L (rCD40L) induced expression of interstitial collagenase, stromelysin, and TF protein and activity. In contrast, the soluble cytokines interleukin-1 or tumor necrosis factor-α did not induce or weakly induced TF expression. Neutralization with anti-CD40L antibody markedly inhibited these actions of both T-cell membranes and rCD40L. Conclusions By inducing the expression of matrix-degrading proteinases and of TF procoagulant, CD40 signaling may contribute to the triggering of acute coronary events.

Human CD23 is a 45-kD type II membrane glycoprotein, which functions as a low-affinity receptor for IgE and as a ligand for the CD21 and CD11b/CD11c differentiation antigens. CD23 is released from the surface of cells as soluble fragments, and a 25-kD species of soluble CD23 (sCD23) appears to act as a multifunctional cytokine. In this report, sCD23 is shown to sustain the growth of low cell density cultures of a human pre-B–acute lymphocytic leukemia cell line, SMS-SB: no other cytokine tested was able to induce this effect. Flow cytometric analysis indicates that sCD23 acts to prevent apoptosis of SMS-SB cells. SMS-SB cells cultured at low cell density possess low levels of bcl-2 protein. Addition of sCD23 to cells at low cell density maintained bcl-2 expression at levels equivalent to those observed in SMS-SB cells cultured at higher cell densities. No CD23 mRNA was found in SMS-SB cells, ruling out an autocrine function for CD23 in this cell line model. Although SMS-SB cells do not express the known receptors for CD23, namely CD21, CD11b-CD18, or CD11c-CD18, the cells specifically bind CD23-containing liposomes, but not glycophorin-containing liposomes. Binding of CD23-containing liposomes is inhibited by anti-CD23 but not by anti-CD21 or anti-CD11b/c monoclonal antibodies. The data show that sCD23 prevents apoptosis of the SMS-SB cell line by acting through a novel receptor.

Immunoglobulin (Ig) E production by B cells requires two primary signals provided by T cells, interleukin (IL)-4 or IL-13 and CD40 ligand (CD40L). In addition, costimulatory signals, such as CD23-CD21 interaction, contribute further ensuring a selective control over this production. Recently, CD28, expressed on T cells, has been reported to be involved in this process. The CD28 ligands, CD80 (B7-1) and CD86 (B7-2), are expressed on human tonsillar B cells, and their expression is up-regulated by IL-4, IL-13, and/or an anti-CD40 monoclonal antibody (mAb). We have investigated whether signaling via the B7 molecules affects IgE synthesis. Human B cells were stimulated by IL-4 plus anti-CD40 mAb in the presence of different anti-B7 mAbs. Cross-linking of CD86 with IT2.2 potentiated IgE and IgG4 production and e transcripts expression. The production of the other isotypes was not modulated. Conversely, the anti-CD80 and the other anti-CD86 mAbs tested had no effect. The increase of IgE and IgG4 production induced by IT2.2 was accompanied by an increase in proliferation, in cell surface density of CD23, and in CD23 binding to CD21-expressing B cells. In contrast, the expression of other B cell surface molecules such as CD11a, CD30, and CD58 remained unaffected. Since IT2.2 favors CD23-CD21 pairing, we tested whether blocking this interaction affected IT2.2-increased IgE production. The neutralizing anti-CD23 mAb, Mab 25, caused a dose-dependent inhibition of the effect of IT2.2 on IgE synthesis. Finally, IT2.2 potentiation on B cell proliferation and IgE production required the two primary signals, IL-4 and anti-CD40 mAb, since IT2.2 alone or in combination with only one of these stimuli did not show any effect on B cells. This study is the first demonstration of a signaling role for CD86. Together with IL-4 or IL-13 and CD40L, CD86 favors CD23-CD21 pairing and consequently functions as a selective and potent costimulus for human IgE and IgG4 synthesis.