Structural insights and biomedical potential of IgNAR scaffolds from sharks

In addition to antibodies with the classical composition of heavy and light chains, the adaptive immune repertoire of sharks also includes a heavy-chain only isotype, where antigen binding is mediated exclusively by a small and highly stable domain, referred to as vNAR. In recent years, due to their high affinity and specificity combined with their small size, high physicochemical stability and low-cost of production, vNAR fragments have evolved as promising target-binding scaffolds that can be tailor-made for applications in medicine and biotechnology. This review highlights the structural features of vNAR molecules, addresses aspects of their generation using immunization or in vitro high throughput screening methods and provides examples of therapeutic, diagnostic and other biotechnological applications.     

Isolation of a pH-Sensitive IgNAR Variable Domain from a Yeast-Displayed,Histidine-Doped Master Library

In recent years, engineering of pH-sensitivity into antibodies as well as antibody-derived fragments has become more and more attractive for biomedical and biotechnological applications. Herein, we report the isolation of the first pH-sensitive IgNAR variable domain (vNAR), which was isolated from a yeast-displayed, semi-synthetic master library. This strategy enables the direct identification of pH-dependent binders from a histidine-enriched CDR3 library. Displayed vNAR variants contained two histidine substitutions on average at random positions in their 12-residue CDR3 loop. Upon screening of seven rounds against the proof-of-concept target EpCAM (selection for binding at pH 7.4 and decreased binding at pH 6.0), a single clone was obtained that showed specific and pH-dependent binding as characterized by yeast surface display and biolayer interferometry. Potential applications for such pH-dependent vNAR domains include their employment in tailored affinity chromatography, enabling mild elution protocols. Moreover, utilizing a master library for the isolation of pH-sensitive vNAR variants may be a generic strategy to obtain binding entities with prescribed characteristics for applications in biotechnology, diagnostics, and therapy.     

Structural insights and biomedical potential of IgNAR scaffolds from sharks

In addition to antibodies with the classical composition of heavy and light chains, the adaptive immune repertoire of sharks also includes a heavy-chain only isotype, where antigen binding is mediated exclusively by a small and highly stable domain, referred to as vNAR. In recent years, due to their high affinity and specificity combined with their small size, high physicochemical stability and low-cost of production, vNAR fragments have evolved as promising target-binding scaffolds that can be tailor-made for applications in medicine and biotechnology. This review highlights the structural features of vNAR molecules, addresses aspects of their generation using immunization or in vitro high throughput screening methods and provides examples of therapeutic, diagnostic and other biotechnological applications.     

The EMAPII cytokine is released from the mammalian multisynthetase complex after cleavage of its p43/proEMAPII component

Endothelial-monocyte-activating polypeptide II (EMAPII) is an inflammatory cytokine released under apoptotic conditions. Its proEMAPII precursor proved to be identical to the auxiliary p43 component of the aminoacyl-tRNA synthetase complex. We show here that the EMAPII domain of p43 is released readily from the complex after in vitro digestion with caspase 7 and is able to induce migration of human mononuclear phagocytes. The N terminus of in vitro-processed EMAPII coincides exactly with that of the mature cytokine isolated from conditioned medium of fibrosarcoma cells. We also show that p43/proEMAPII has a strong tRNA binding capacity (K(D) = 0.2 microm) as compared with its isolated N or C domains (7.5 microm and 40 microm, respectively). The potent general RNA binding capacity ascribed to p43/proEMAPII is lost upon the release of the EMAPII domain. This suggests that after onset of apoptosis, the first consequence of the cleavage of p43 is to limit the availability of tRNA for aminoacyl-tRNA synthetases associated within the complex. Translation arrest is accompanied by the release of the EMAPII cytokine that plays a role in the engulfment of apoptotic cells by attracting phagocytes. As a consequence, p43 compares well with a molecular fuse that triggers the irreversible cell growth/cell death transition induced under apoptotic conditions.     

FcγRIIIa-dependent IFN-γ release in whole blood assay is predictive of therapeutic IgG1 antibodies safety

Immunomodulatory monoclonal IgG1 antibodies developed for cancer and autoimmune disease have an inherent risk of systemic release of pro-inflammatory cytokines. In vitro cytokine release assays are currently used to predict cytokine release syndrome (CRS) risk, but the validation of these preclinical tools suffers from the limited number of characterized CRS-inducing IgG1 antibodies and the poor understanding of the mechanisms regulating cytokine release. Here, we incubated human whole blood from naïve healthy volunteers with four monoclonal IgG1 antibodies with different proven or predicted capacity to elicit CRS in clinic and measured cytokine release using a multiplex assay. We found that, in contrast to anti-CD52 antibodies (Campath-1H homolog) that elicited high level of multiple inflammatory cytokines from human blood cells in vitro, other IgG1 antibodies with CRS-inducing potential consistently induced release of a single tested cytokine, interferon (IFN)-γ, with a smaller magnitude than Campath. IFN-γ expression was observed as early as 2–4 h after incubation, mediated by natural killer cells, and dependent upon tumor necrosis factor and FcγRIII. Importantly, the magnitude of the IFN-γ response elicited by IgG1 antibodies with CRS-inducing potential was determined by donor FcγRIIIa-V158F polymorphism. Overall, our results highlight the importance of FcγRIIIa-dependent IFN-γ release in preclinical cytokine release assay for the prediction of CRS risk associated with therapeutic IgG1 antibodies.     

Efficient tumor killing and minimal cytokine release with novel T-cell agonist bispecific antibodies

T-cell-recruiting bispecific antibodies (T-BsAbs) have shown potent tumor killing activity in humans, but cytokine release-related toxicities have affected their clinical utility. The use of novel anti-CD3 binding domains with more favorable properties could aid in the creation of T-BsAbs with improved therapeutic windows. Using a sequence-based discovery platform, we identified new anti-CD3 antibodies from humanized rats that bind to multiple epitopes and elicit varying levels of T-cell activation. In T-BsAb format, 12 different anti-CD3 arms induce equivalent levels of tumor cell lysis by primary T-cells, but potency varies by a thousand-fold. Our lead CD3-targeting arm stimulates very low levels of cytokine release, but drives robust tumor antigen-specific killing in vitro and in a mouse xenograft model. This new CD3-targeting antibody underpins a next-generation T-BsAb platform in which potent cytotoxicity is uncoupled from high levels of cytokine release, which may lead to a wider therapeutic window in the clinic.     

Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia coli cytoplasm

Single-chain variable fragment (scFv) antibodies have great potential for a range of applications including as diagnostic and therapeutic agents. However, production of scFvs is challenging because proper folding and activity depend on the formation of two intrachain disulfide bonds that do not readily form in the cytoplasm of living cells. Functional expression in bacteria therefore involves targeting to the more oxidizing periplasm, but yields in this compartment can be limiting due to secretion bottlenecks and the relatively small volume compared to the cytoplasm. In the present study, we evaluated an anti-HER2 scFv, which is specific for human epidermal growth receptor 2 (HER2) overexpressed in breast cancer, for functional expression in the cytoplasm of Escherichia coli strains BL21(DE3) and SHuffle T7 Express, the latter of which is genetically engineered for cytoplasmic disulfide bond formation. Specifically, we observed much greater solubility and binding activity with SHuffle T7 Express cells, which likely resulted from the more oxidative cytoplasm in this strain background. We also found that SHuffle T7 Express cells were capable of supporting high-level soluble production of anti-HER2 scFvs with intact disulfide bonds independent of variable domain orientation, providing further evidence that SHuffle T7 Express is a promising host for laboratory and preparative expression of functional scFv antibodies.     

Identification of multiple isoforms of the low-affinity human IgG Fc receptor

Two varieties of similar, but structurally distinct, cDNA clones for the human low-affinity receptors for the Fc portion of immunoglobulin G (FcγRII) have been isolated. One type of clone was obtained from human B lymphocytes, and the other from PHA-activated peripheral T cells and monocytes. Transfection of both prototype clones into Cos-7 cells and subsequent specific staining with monoclonal antibodies of the CDw32 group confirmed the identification of the gene products. The nucleotide sequence of the cDNA clone from B lymphocytes contains an open reading frame that encodes a protein of relative mass (M _r) 27000 with an extracellular domain of 179 amino acids containing three potential N-glycosylation sites, a 26 amino acid transmembrane domain, and a 44 amino acid cytoplasmic domain. The clones from peripheral T cells and monocytes both encoded a protein ofM _r 31000 with a 179 amino acid extracellular domain containing two potential N-glycosylation sites and a 26 amino acid transmembrane domain. The two types of clones had similar sequences in their immunoglobulin-like extracellular and transmembrane domains, but differed in their leader sequences and 3′-untranslated regions. The most notable difference between the clones was the presence of a distinctive 76 amino acid cytoplasmic domain in those isolated from T cells and monocytes.     

Presence of IgM Antibodies Which Sensitize HIV-1-Infected Cells to Cytolysis by Homologous Complement in Long-Term Survivors of HIV Infection

Although human cells are resistant to homologous human complement due to the presence of species-specific membrane inhibitors, a naturally occurring IgM antibody which recognizes an asialo-oligosaccharide can sensitize HIV-1-infected cells for complement-mediated cytolysis. Therefore, we investigated whether long-term survivors of HIV-1 infection harbor such antibodies in their sera. Thirty of 31 sera from HIV-1 seropositive hemophilia patients who have survived HIV-1 infection 10 years or more showed appreciable cytolytic activity, while only 2 sera of 10 seropositive patients presumed to have been infected with HIV-1 (due to sexual contact) more recently showed cytolytic activity. On the other hand, only 7 out of 43 sera from seronegative hemophilia patients showed cytolytic activity. Immunofluorescence staining for IgM on HIV-L -infected cells essentially correlated with the cytolytic capacity of the sera. Therefore, naturally occurring IgM antibodies and/or generated IgM antibodies reactive with the HIV-L -infected cells in patients might have been responsible for long-term survival due to complement-mediated immune cytolysis which may, in conjunction with cytotoxic T lymphocytes, synergistically suppress the infected cells in vivo. Therefore, the transfusion of such IgM antibodies could be effective for the treatment of HIV-L -infected individuals.     

In vivo tumor delivery of a recombinant single chain Fv::tumor necrosis factor-alpha fusion [correction of factor: a fusion] protein.

Locoregional and intratumoral administration of tumor necrosis factor alpha (TNF alpha) has been successful in obtaining inhibition or regression of tumor growth in the clinic. This potent antitumor activity of TNF alpha has not yet been exploited as a systemic agent in cancer therapy, mainly due to high levels of toxicity to normal tissues before a therapeutic dose of TNF alpha in the tumor has been achieved. To address this, we have targeted TNF alpha using antitumor antibodies. We have used a genetic fusion of human recombinant TNF alpha with MFE-23, a single-chain Fv antibody fragment directed against carcinoembryonic antigen. MFE-23::TNF alpha fusion protein is isolated in high yields (28 mg/L) from bacterial inclusion bodies and purified to homogeneity by affinity chromatography. It is a 144 kDa trimer in native form and possesses the antigen-binding activity of the sFv and the cytotoxicity to both WEHI 164 and a human adenocarcinoma cell line (LoVo) of rhTNF alpha. Radiolabeled MFE-23::TNF alpha binds both human and mouse TNF receptor 1 in vitro and is able to localize effectively in nude (nu/nu) mice bearing human LS174T xenografts; tumor/tissue ratios of 21:1 and 60:1 are achieved 24 and 48 h after intravenous injection. These studies indicate that MFE-23::TNF alpha will provide an effective means for systemically administered cancer therapy with TNF alpha.     

Engineering a human IgG2 antibody stable at low pH

IgG2 subclass antibodies have unique properties that include low effector function and a rigid hinge region. Although some IgG2 subclasses have been clinically tested and approved for therapeutic use, they have a higher propensity than IgG1 for aggregation, which can curtail or abolish their biological activity and enhance their immunogenicity. In this regard, acid‐induced aggregation of monoclonal antibodies during purification and virus inactivation must be prevented. In the present study, we replaced the constant domain of IgG2 with that of IgG1, using anti‐2,4‐dinitrophenol (DNP) IgG2 as a model antibody, and investigated whether that would confer greater stability. While the anti‐DNP IgG2 antibody showed significant aggregation at low pH, this was reduced for the IgG2 antibody containing the IgG1 CH2 domain. Substituting three amino acids within the CH2 domain—namely, F300Y, V309L, and T339A (IgG2_YLA)—reduced aggregation at low pH and increased CH2 transition temperature, as determined by differential scanning calorimetric analysis. IgG2_YLA exhibited similar antigen‐binding capacity to IgG2, low affinity for FcγRIIIa, and low binding ability to C1q. The same YLA substitution also reduced the aggregation of panitumumab, another IgG2 antibody, at low pH. Our engineered human IgG2 antibody showed reduced aggregation during bioprocessing and provides a basis for designing improved IgG2 antibodies for therapeutic applications.     

Evidence for the expression of two distinct MHC class II DRβ like molecules in the sheep

This study used monoclonal antibodies to sheep MHC class II molecules as well as an L cell transfectant (T8.1) which expresses DRA and DRB genes to show that two distinct DRβ chains are expressed in the sheep. Two anti-β chain specific monoclonal antibodies VPM37 and VPM43 react with DR antigen but not DQ antigen by ELISA. These two antibodies do not react with the DRβ chain expressed in the T8.1 cell line. Two-dimensional immunoblotting shows that these antibodies recognize a subgroup of the spots recognized by the DR-specific monoclonal antibody VPM57 which does react with the T8.1 β chain. Amino-terminal sequence analysis of the α chain associated with VPM37β chain shows that this α chain is homologous to the human DRα chain strongly indicating that the β chain is DR-like. VPM37 and VPM43 are shown to be directed against different epitopes on sheep MHC class II molecules so it is highly unlikely that the data can be explained by the presence of posttranslational modifications or the existence of a very common allele. These data provide clear evidence for the expression of two distinct DRP chains in the sheep.     

Vκ polymorphisms in NOD mice are spread throughout the entire immunoglobulin kappa locus and are shared by other autoimmune strains

The diversity of immunoglobulin (Ig) and T cell receptor (TCR) genes available to form the lymphocyte repertoire has the capacity to produce a broad array of both protective and harmful specificities. In type 1 diabetes (T1D), the presence of antibodies to insulin and other islet antigens predicts disease development in both mice and humans, and demonstrate that immune tolerance is lost early in the disease process. Anti-insulin T cells isolated from T1D-prone non-obese diabetic (NOD) mice use polymorphic TCRα chains, suggesting that the available T cell repertoire is altered in these autoimmune mice. To probe whether insulin-binding B cells also possess polymorphic V genes, Ig light chains were isolated and sequenced from NOD mice that harbor an Ig heavy chain transgene. Three insulin-binding Vκ genes were identified, all of which were polymorphic to the closest germline sequence matches present in the GenBank database. Additional analysis of over 300 light chain sequences from multiple sources, including germline DNA, shows that polymorphisms are spread throughout the entire NOD Igκ locus, as these polymorphic sequences represent 43 distinct Vκ genes which belong to 14 Vκ families. Database searches reveal that a majority of polymorphic Vκ genes identified in NOD are identical to Vκ genes isolated from SLE-prone NZBxNZW F1 or MRL strains of mice, suggesting that a shared Igκ haplotype may be present. Predicted amino acid changes preferentially occur in CDR, and thus could alter antigen recognition by the germline B cell repertoire of autoimmune versus non-autoimmune mouse strains.     

Antibody-drug conjugates with HER2-targeting antibodies from synthetic antibody libraries are highly potent against HER2-positive human gastric tumor in xenograft models

HER2-ECD (human epidermal growth factor receptor 2 – extracellular domain) is a prominent therapeutic target validated for treating HER2-positive breast and gastric cancer, but HER2-specific therapeutic options for treating advanced gastric cancer remain limited. We have developed antibody-drug conjugates (ADCs), comprising IgG1 linked via valine-citrulline to monomethyl auristatin E, with potential to treat HER2-positive gastric cancer in humans. The antibodies optimally selected from the ADC discovery platform, which was developed to discover antibody candidates suitable for immunoconjugates from synthetic antibody libraries designed using antibody-antigen interaction principles, were demonstrated to be superior immunoconjugate targeting modules in terms of efficacy and off-target toxicity. In comparison with the two control humanized antibodies (trastuzumab and H32) derived from murine antibody repertoires, the antibodies derived from the synthetic antibody libraries had enhanced receptor-mediated internalization rate, which could result in ADCs with optimal efficacies. Along with the ADCs, two other forms of immunoconjugates (scFv-PE38KDEL and IgG1-AL1-PE38KDEL) were used to test the antibodies for delivering cytotoxic payloads to xenograft tumor models in vivo and to cultured cells in vitro. The in vivo experiments with the three forms of immunoconjugates revealed minimal off-target toxicities of the selected antibodies from the synthetic antibody libraries; the off-target toxicities of the control antibodies could have resulted from the antibodies’ propensity to target the liver in the animal models. Our ADC discovery platform and the knowledge gained from our in vivo tests on xenograft models with the three forms of immunoconjugates could be useful to anyone developing optimal ADC cancer therapeutics.     

Phage display of intact domains at high copy number: a system based on SOC, the small outer capsid protein of bacteriophage T4.

Peptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning. However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self-assemble. We describe here a system in which the molecules to be displayed are bound to pre-assembled polymers. The polymers are T4 capsids and polyheads (tubular capsid variants) and the display molecules are derivatives of the dispensable capsid protein SOC. In one implementation, SOC and its fusion derivatives are expressed at high levels in Escherichia coli, purified in high yield, and then bound in vitro to separately isolated polyheads. In the other, a positive selection vector forces integration of the modified soc gene into a soc-deleted T4 genome, leading to in vivo binding of the display protein to progeny virions. The system is demonstrated as applied to C-terminal fusions to SOC of (1) a tetrapeptide; (2) the 43-residue V3 loop domain of gp120, the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein; and (3) poliovirus VP1 capsid protein (312 residues). SOC-V3 displaying phage were highly antigenic in mice and produced antibodies reactive with native gp120. That the fusion protein binds correctly to the surface lattice was attested in averaged electron micrographs of polyheads. The SOC display system is capable of presenting up to approximately 10(3) copies per capsid and > 10(4) copies per polyhead of V3-sized domains. Phage displaying SOC-VP1 were isolated from a 1:10(6) mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated.     

Immunolocalization of an extracellular domain of connexin43 in rat heart gap junctions.

Antipeptide antibodies directed to residues 55 to 66 (NTQQPGCENVCY) of connexin43 (cx43) specifically recognize this protein on Western blots of intact and urea-split gap junctions isolated from rat heart. These antibodies detect a single protein of 43 kDa, corresponding to cx43, on Western blots of whole fractions of various vertebrate hearts. Immunogold labeling by electron microscopy shows that the epitopes recognized by these antibodies are not localized on the cytoplasmic surfaces of intact gap junctions but only at the edges of these junctions. In urea-split gap junctions the gold particles are seen in the junctional space, associated with the extracellular faces of junctional membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses of rat heart gap junctions treated with trypsin show that they are constituted with either two polypeptides of Mr 12,000 and 14,000 or a single polypeptide of Mr 22,000 according to whether the analyses are performed under reducing or non-reducing conditions, respectively. The antibodies directed to residues 55 to 66 of cx43 cross-react with both the 12 and 22 kDa polypeptides. These results suggest that the two protected domains of 12 and 14 kDa which contain the first extracellular loop and a putative second extracellular loop, respectively, are linked by disulfide bonds. In adult rat heart sections analyzed by indirect immunofluorescence the intercalated discs are labeled with antibodies directed to a cytoplasmic carboxy-terminal domain of cx43 (El Aoumari et al., J. Membr. Biol. 115, 229-240 (1990)). The same intercalated discs are also labeled in adjacent sections incubated with the antibodies directed to residues 55 to 66. Two hypotheses might explain these results: either the antibodies have access to the extracellular domain of cx43 molecules localized at the edges of the gap junctions, or cx43 molecules are present in the non-junctional membranes of the intercalated discs.     

Type 1 regulatory T cells specific for collagen type II as an efficient cell-based therapy in arthritis

Introduction

Regulatory T (Treg) cells play a crucial role in preventing autoimmune diseases and are an ideal target for the development of therapies designed to suppress inflammation in an antigen-specific manner. Type 1 regulatory T (Tr1) cells are defined by their capacity to produce high levels of interleukin 10 (IL-10), which contributes to their ability to suppress pathological immune responses in several settings. The aim of this study was to evaluate the therapeutic potential of collagen type II–specific Tr1 (Col-Treg) cells in two models of rheumatoid arthritis (RA) in mice.

Methods

Col-Treg clones were isolated and expanded from collagen-specific TCR transgenic mice. Their cytokine secretion profile and phenotype characterization were studied. The therapeutic potential of Col-Treg cells was evaluated after adoptive transfer in collagen-antibody– and collagen-induced arthritis models. The in vivo suppressive mechanism of Col-Treg clones on effector T-cell proliferation was also investigated.

Results

Col-Treg clones are characterized by their specific cytokine profile (IL-10^highIL-4^negIFN-γ^int) and mediate contact-independent immune suppression. They also share with natural Tregs high expression of GITR, CD39 and granzyme B. A single infusion of Col-Treg cells reduced the incidence and clinical symptoms of arthritis in both preventive and curative settings, with a significant impact on collagen type II antibodies. Importantly, injection of antigen-specific Tr1 cells decreased the proliferation of antigen-specific effector T cells in vivo significantly.

Conclusions

Our results demonstrate the therapeutic potential of Col-Treg cells in two models of RA, providing evidence that Col-Treg could be an efficient cell-based therapy for RA patients whose disease is refractory to current treatments.     

IL-15 is highly expressed in inflammatory bowel disease and regulates local T cell-dependent cytokine production

IL-15 shares biological activities but no significant sequence homology with IL-2. It induces T cell recruitment to sites of inflammation, T cell proliferation, and cytokine production and rescue from apoptosis. The aim of this study was to investigate expression of IL-15 and its effects on proinflammatory cytokine production in inflammatory bowel disease (IBD). Immunohistochemistry demonstrated local IL-15 production by macrophages in inflamed mucosa from IBD patients. Isolated lamina propria mononuclear cells from these patients but not from controls produced IL-15 when stimulated with LPS or IFN-gamma. Moreover, lamina propria T cells (LP-T) from IBD patients were more responsive to IL-15 as compared with controls, and IL-15 alone without a primary T cell stimulus induced IFN-gamma and TNF production by isolated IBD LP-T cells, especially by LP-T cells from patients with Crohn's disease. LP-T cells from IBD patients could induce CD40-CD40 ligand (CD40L) interaction-dependent TNF and IL-12 production by monocytes in a coculture system. This capacity of LP-T cells was strongly enhanced by preincubation in IL-15 and was the result of higher CD40L expression after culture in IL-15. These data indicate that IL-15 is overexpressed in the inflamed mucosa in IBD and that IL-15 enhances local T cell activation, proliferation, and proinflammatory cytokine production by both T cells and macrophages, the latter via a CD40-CD40L interaction-dependent mechanism. Treatment directed against IL-15 may have therapeutic potential in IBD.     

Evaluation of murine interleukin 4 (IL-4) receptor expression using anti-receptor monoclonal antibodies and S1 nuclease protection analyses

Anti-receptor antibodies have previously been used in two cytokine systems (IL-1 and TNF alpha) to identify the existence of different cytokine receptors on different cell types. In this study, we have similarly used two approaches to evaluate whether IL-4 receptors on different cell types are identical, or whether more than one species of IL-4 receptor exists. The first approach involved production of monoclonal antibodies specific for the IL-4 receptor expressed by the murine mast cell line, MC/9. Six anti-IL-4 receptor monoclonal antibodies were produced against the purified soluble extracellular domain of the recombinant IL-4 receptor derived from MC/9 cells. These antibodies were capable of binding to and specifically immunoprecipitating the soluble extracellular domain of the recombinant mast cell IL-4 receptor. Following biotinylation of the antibodies and addition of phycoerythrin-streptavidin, their binding to cell associated IL-4 receptors on MC/9 mast cells could be readily visualized by immunofluorescence. Using this approach, the anti-mast cell IL-4R antibodies were found to specifically bind IL-4 receptors expressed on a variety of other murine cell types, including T cells, B cells, macrophages, fibroblasts, and L cells. The antibodies did not bind to two human cell lines known to bind human but not murine IL-4. The intensity of staining was directly related to the number of IL-4 binding sites identified previously by receptor-ligand equilibrium binding analyses. As a second approach to evaluating potential receptor heterogeneity, we constructed S1 nuclease protection assay probes for two separate regions of the mast cell IL-4 receptor, one located in the extracellular domain and one in the intracellular domain. Subsequent S1 analyses showed that both regions are expressed by the following types of cells: T cells, B cells, macrophages, myeloid cells, L cells, and stromal cells. The two approaches used in this study therefore indicate that the same or highly similar IL-4 receptor species is expressed by a wide variety of hemopoietic and nonhemopoietic cells. Since the anti-IL-4 receptor antibodies produced in this study did not block binding of IL-4 to its receptor, we cannot exclude the possible existence of a second type of IL-4R coexpressed on the cells tested in this study, or expressed uniquely by other cell types that were not investigated.     

Prior stimulation of antigen-presenting cells with Lactobacillus regulates excessive antigen-specific cytokine responses in vitro when compared with Bacteroides

The development of allergy is related to differences in the intestinal microbiota. Therefore, it is suggested that the immune responses induced by different genera of bacteria might be regulated through adaptive as well as innate immunity. In this study, we examined whether antigen-specific immune responses were affected by stimulation with the different genera of intestinal bacteria in vitro. Mesenteric lymph node (MLN) cells isolated from germ-free ovalbumin (OVA)-specific T cell receptor transgenic (OVA-Tg) mice were stimulated with OVA and intestinal bacteria. Cecal contents from conventional mice but not germ-free mice could induce OVA-specific cytokine production. Among the murine intestinal bacteria, Bacteroides acidofaciens (BA) enhanced OVA-specific IFN-γ and IL-10 production while Lactobacillus johnsonii (LA) increased OVA-specific IL-10 production only. The expression of cell surface molecules and cytokine production by antigen-presenting cells (APCs) from germ-free Balb/c mice were analyzed. BA increased the expression of MHC II and co-stimulatory molecules on APCs compared with LA. BA increased IL-6 and IL-10 production but induced less IL-12p40 than LA. To examine the effects of prior stimulation of APCs by intestinal bacteria on the induction of antigen-specific immune responses, cytokine production was determined following co-culture with OVA, CD4⁺ T cells from OVA-Tg mice, and APCs which were pre-stimulated with the bacteria or not. APCs pre-stimulated with LA did not enhance OVA-specific cytokine production while BA stimulated OVA-specific IL-10 production. These results suggest that the prior stimulation of intestinal immunocytes by Lactobacillus might regulate excessive antigen-specific cytokine responses via APCs when compared with prior stimulation by Bacteroides.