Antigen-binding Molecules Research Articles

Unleashing the power of shark variable single domains (VNARs): broadly neutralizing tools for combating SARS-CoV-2.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) generated a joint global effort to develop vaccines and other treatments that could mitigate the negative effects and the rapid spread of the virus. Single-domain antibodies derived from various sources, including cartilaginous fish, camelids, and humans, have gained attention as promising therapeutic tools against coronavirus disease 2019. Shark-derived variable new antigen receptors (VNARs) have emerged as the smallest naturally occurring antigen-binding molecules. Here, we compile and review recent published studies on VNARs with the capacity to recognize and/or neutralize SARS-CoV-2. We found a close balance between the use of natural immune libraries and synthetic VNAR libraries for the screening against SARS-CoV-2, with phage display being the preferred display technology for the selection of VNARs against this virus. In addition, we discuss potential modifications and engineering strategies employed to improve the neutralization potential of VNARs, such as exploring fusion with the Fc domain of human Immunoglobulin G (IgG) to increase avidity and therapeutic potential. This research highlights the potential of VNARs as powerful molecular tools in the fight against infectious diseases.

Crystal structures of N-myristoylated lipopeptide-bound HLA class I complexes indicate reorganization of B-pocket architecture upon ligand binding

Rhesus monkeys have evolved MHC-encoded class I allomorphs such as Mamu-B∗098 that are capable of binding N-myristoylated short lipopeptides rather than conventional long peptides; however, it remains unknown whether such antigen-binding molecules exist in other species, including humans. We herein demonstrate that human leukocyte antigen (HLA)-A∗24:02 and HLA-C∗14:02 proteins, which are known to bind conventional long peptides, also have the potential to bind N-myristoylated short lipopeptides. These HLA class I molecules shared a serine at position 9 (Ser9) with Mamu-B∗098, in contrast to most MHC class I molecules that harbor a larger amino acid residue, such as tyrosine, at this position. High resolution X-ray crystallographic analyses of lipopeptide-bound HLA-A∗24:02 and HLA-C∗14:02 complexes indicated that Ser9 was at the bottom of the B pocket with its small hydroxymethyl side chain directed away from the B-pocket cavity, thereby contributing to the formation of a deep hydrophobic cavity suitable for accommodating the long-chain fatty acid moiety of lipopeptide ligands. Upon peptide binding, however, we found the hydrogen-bond network involving Ser9 was reorganized, and the remodeled B pocket was able to capture the second amino acid residue (P2) of peptide ligands. Apart from the B pocket, virtually no marked alterations were observed for the A and F pockets upon peptide and lipopeptide binding. Thus, we concluded that the structural flexibility of the large B pocket of HLA-A∗2402 and HLA-C∗1402 primarily accounted for their previously unrecognized capacity to bind such chemically distinct ligands as conventional peptides and N-myristoylated lipopeptides.

Fluorescent Anti-CEA Nanobody for Rapid Tumor-Targeting and Imaging in Mouse Models of Pancreatic Cancer.

Tumor-specific targeting with fluorescent probes can enhance contrast for identification of cancer during surgical resection and visualize otherwise invisible tumor margins. Nanobodies are the smallest naturally-occurring antigen-binding molecules with rapid pharmacokinetics. The present work demonstrates the efficacy of a fluorescent anti-CEA nanobody conjugated to an IR800 dye to target and label patient derived pancreatic cancer xenografts. After intravenous administration, the probe rapidly localized to the pancreatic cancer tumors within an hour and had a tumor-to-background ratio of 2.0 by 3 h. The fluorescence signal was durable over a prolonged period of time. With the rapid kinetics afforded by fluorescent nanobodies, both targeting and imaging can be performed on the same day as surgery.

High MHC diversity confers no advantage for phenotypic quality and reproductive performance in a wild bird.

Genes of the major histocompatibility complex (MHC) encode antigen‐binding molecules and are an integral part of the acquired immune response of vertebrates. In general, high individual MHC diversity is expected to increase fitness by broadening the spectrum of pathogens recognized by the immune system, in accordance with the heterozygote advantage mechanism. On the other hand, the optimality hypothesis assumes that individuals with optimal (intermediate), rather than maximum, diversity of the MHC will achieve the highest fitness because of inherent costs associated with expressing diverse MHC alleles.Here, we tested for associations between individual diversity of the MHC class I and class II genes (binding antigens of intra‐ and extracellular pathogens respectively) and a range of fitness‐related traits (condition, ornament expression and reproduction) in an urban population of the Eurasian coot Fulica atra.Contrary to our expectation, we found that high within‐individual allelic diversity of MHC genes (both class I and II) was associated with poorer condition (lower blood haemoglobin concentrations), weaker expression of the putative ornament (smaller frontal shield), later onset of breeding and smaller clutches. An analysis of functional MHC allele clusters (supertypes) provided further support for negative associations of MHC diversity with phenotypic quality and reproductive performance, but most of these relationships could not be explained by the presence of specific maladaptive supertypes. Finally, we found little empirical support for the optimality hypothesis in the Eurasian coot.Our results suggest that the costs of high MHC diversity outweighed any benefits associated with broad MHC repertoire, which could be driven by depauperate pathogen diversity in an urban landscape. To the best of our knowledge, this is one of the first studies providing consistent evidence for negative associations of MHC diversity with a range of fitness‐related traits in a natural avian population.

The interplay between the immune system and viruses.

The human immune response can be divided into two arms: innate and adaptive immunity. The innate immune system consists of "hard-wired" responses encoded by host germline genes. In contrast, the adaptive response consists of gene elements that are somatically rearranged to assemble antigen-binding molecules with specificity for individual foreign structures. In contrast to the adaptive immune system, which depends upon T and B lymphocytes, innate immune protection is a task performed by cells of both hematopoietic and non-hematopoietic origin. Hematopoietic cells involved in innate immune responses include macrophages, dendritic cells, mast cell, neutrophils, eosinophils, natural killer (NK) cells and natural killer T cells. The induction of an adaptive immune response begins when a pathogen is ingested by an Antigen Presenting Cell (APC), such as the Dendritic cell (DC), in the infected tissue. DCs bridge the gap between first line innate responses and powerful adaptive immune responses, by internalizing, processing and presenting antigens on Major Histocompatibility Complex (MHC) and MHC-like molecules to the adaptive immune cells In addition to DCs, macrophages and B cells are deemed antigen presenting cells (Llewelyn & Cohen, 2002).

Selection and Characterization of CSFV-Specific Single-Domain Antibodies and Their Application along with Immunomagnetic Nanobeads and Quantum Dots.

Outbreak of classical swine fever (CSF) results in high mortality and thus causes severe economic losses in the swine industry. Single-domain antibody (sdAb) is the smallest antigen-binding molecule derived from camelid heavy-chain antibodies and has the potential to be used as a molecular probe for detection of CSF virus (CSFV). In this study, two sdAb fragments against the E2 antigen of CSFV were obtained, expressed in vitro. The functional characteristics analysis indicated that the recombinant sdAbE2-1 and sdAbE2-2 have excellent binding activity, specificity, and high affinity with equilibrium constant value of 3.34 × 10−7 and 1.35 × 10−8 M to E2 protein. Then, sdAbE2s were conjugated with quantum dots (QD)/AF488 to synthesize two molecular probes for imaging CSFV distribution in cells. The sdAbE2-1 was also labeled with carboxyl-magnetic beads to construct immunomagnetic nanobeads (IMNBs) able to capture CSFV virions and recombinant E2 protein. QD/AF455-sdAbE2s probes colocalised with CSFV virions in swine testis cells, and IMNBs were used as a detection template and proved to bind specifically with CSFV virions and E2 protein. The selected sdAb fragments and sdAb-based molecular probes may be used for the rapid identification of CSFV during field outbreaks and for research on CSFV and host interactions.

The Camel Adaptive Immune Receptors Repertoire as a Singular Example of Structural and Functional Genomics.

The adaptive immune receptors repertoire is highly plastic, with its ability to produce antigen-binding molecules and select those with high affinity for their antigen. Species have developed diverse genetic and structural strategies to create their respective repertoires required for their survival in the different environments. Camelids, until now, considered as a case of evolutionary innovation because of their only heavy-chain antibodies, represent a new mammalian model particularly useful for understanding the role of diversity in the immune system function. Here, we review the structural and functional characteristics and the current status of the genomic organization of camel immunoglobulins (IG) or antibodies, α/ß and γ/δ T cell receptors (TR), and major histocompatibility complex (MHC). In camelid humoral response, in addition to the conventional antibodies, there are IG with “only-heavy-chain” (no light chain, and two identical heavy gamma chains lacking CH1 and with a VH domain designated as VHH). The unique features of these VHH offer advantages in biotechnology and for clinical applications. The TRG and TRD rearranged variable domains of Camelus dromedarius (Arabian camel) display somatic hypermutation (SHM), increasing the intrinsic structural stability in the γ/δ heterodimer and influencing the affinity maturation to a given antigen similar to immunoglobulin genes. The SHM increases the dromedary γ/δ repertoire diversity. In Camelus genus, the general structural organization of the TRB locus is similar to that of the other artiodactyl species, with a pool of TRBV genes positioned at the 5’ end of three in tandem D-J-C clusters, followed by a single TRBV gene with an inverted transcriptional orientation located at the 3’ end. At the difference of TRG and TRD, the diversity of the TRB variable domains is not shaped by SHM and depends from the classical combinatorial and junctional diversity. The MHC locus is located on chromosome 20 in Camelus dromedarius. Cytogenetic and comparative whole genome analyses revealed the order of the three major regions “Centromere-ClassII-ClassIII-ClassI”. Unexpectedly low extent of polymorphisms and haplotypes was observed in all Old World camels despite different geographic origins.

Ultrafast image-based ELISA for sensitive detection of cytokines in allergen-induced asthmatic samples

Rapid measurement of cytokines, key mediators of immunity and inflammation is important for diagnosis of several inflammatory diseases including asthma, autoimmune and infectious diseases. Here, we report ultrafast detection of cytokines involving ELISA method by microwave irradiation in a domestic microwave oven in just 3min50s. We have detected interleukin-5 (IL-5), interleukin-13 (IL-13) and interferon-ϒ (IFN-ϒ) by covalently immobilizing capture antibodies of IL-5, IL-13 and IFN-ϒ respectively, on activated polypropylene microtest plates (APPμTP) by microwave irradiation in 20s followed by blocking, analyte (antigen) binding and detection molecule binding in 10s, 100s and 100s respectively by microwave irradiation. The limit of detection (LOD) for IL-13 is 0.39ng/ml, IL-5 is 0.078ng/ml and IFN-ϒ is 0.125ng/ml. The present method is further used for detection of IL-13 and IFN-ϒ in diseased asthmatic and control healthy mice samples. In case of IL-13, diseased samples show increased cytokine level compared to normal healthy samples; for IFN-ϒ, the result is reverse. As the present method for detection of cytokines is low cost, ultrafast, specific and sensitive, it can be used as an excellent substitute for conventional - ELISA based cytokine detection.

Silane-modified surfaces in specific antibody-mediated cell recognition.

The immobilization of antibodies on various surfaces has been the subject of advanced research in various immunoassay-based diagnostic devices. The physical and chemical stabilities of the immobilized antibodies on a solid surface still cause many problems because upon immobilizing antibody molecules, the antigen-binding ability usually decreases. The silanization of surfaces with organosilanes carrying chemically active groups such as (3-aminopropyl) triethoxysilane (APTES) can accommodate these antigen-binding molecules in an appropriate orientation so that their functionality and binding activity are essentially retained. In this study, n-butyltrimethoxysilane (BMS) and 3-(octafluoropentyloxy)-propyltriethoxysilane (OFPOS) were used as "blocking silanes". The aims of this study were to compare the effectiveness of specific antibody binding of APTES, APTES + BMS and APTES + OFPOS and to characterize the modified surfaces by contact angle measurements and immunofluorescence measurements prior to and after immobilizing proteins. Additionally, we have evaluated the functionality of the immobilized antibodies by their abilities to bind EpCAM-positive human colon adenocarcinoma cell line (LoVo) and EpCAM-negative mouse embryonic fibroblast cell line (3T3). Cell enumeration was conducted on the basis of DAPI-positive signals and recorded using a confocal laser scanning biological microscope. The results of our study showed that the immobilization capability and reactivity of APTES, APTES + BMS and APTES + OFPOS differ. The modification of APTES with unreactive silanes (BMS,OFPOS) is recommended to improve the antibody binding efficiency. However, using OFPOS resulted in more effective antibody and cell binding, and it appears to be the most useful compound in specific antibody-mediated cell recognition.

Creation of recombinant antigen-binding molecules derived from hybridomas secreting specific antibodies

This protocol describes the design and development of recombinant monovalent antigen-binding molecules derived from monoclonal antibodies through rapid identification and cloning of the functional variable heavy (VH) and variable light (VL) genes and the design and cloning of a synthetic DNA sequence optimized for expression in recombinant bacteria. Typically, monoclonal antibodies are obtained from mouse hybridomas, which most often result from the fusion of B lymphocytes from immunized mice with murine myeloma cells. The protocol described here has previously been exploited for the successful development of multiple antibody-based molecules targeting a wide range of biomolecular targets. The protocol is accessible for research groups who may not be specialized in this area, and should permit the straightforward reverse engineering of functional, recombinant antigen-binding molecules from hybridoma cells secreting functional IgGs within 50 working days. Furthermore, convenient strategies for purification of antibody fragments are described.

Rugged Single Domain Antibody Detection Elements for Bacillus anthracis Spores and Vegetative Cells

Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs) were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.

Novel approaches to antiviral and anticancer immunotherapy

In this review we discuss existing as well as new approaches to immunotherapy directed against infected or cancerous cells. These approaches traditionally exploit either natural components of immune system (such as cytokines, chemokines, co-stimulatory molecules and adjuvants), or monoclonal antibodies designed to target foreign agents and/or diseased cells through their molecular markers. Additional strategies in development include therapeutic vaccines, oncolytic viruses and T-cell therapies. In addition, we briefly describe a novel strategy called ReDIT (Re-Directed ImmunoTherapy), based on re-orienting the existing long-lasting immune responses (e.g. induced by measles vaccination or natural infection) towards new target molecules on the surface of infected or malignant cells. This can be principally achieved by using bi-functional protein constructs that contain an antigen carrier component and a re-directing component. The antigen carrier component can consist of the ectodomain of the measles hemagglutinin that can be recognized by antibodies and memory cells generated during previous infection or vaccination. The re-directing component consists of the specific virus- or tumor antigen-binding molecule. The fusion constructs are expected to boost existing anti-measles immunity and re-direct it against a new target, engaging the existing anti-measles immunity as an effector mechanism. Thus, ReDIT is a promising novel approach that may represent a valuable addition to immunotherapy of difficult to treat infections and tumors, as it exploits a mechanism distinct from other available therapies.

Isolation of a Highly Thermal Stable Lama Single Domain Antibody Specific for Staphylococcus aureusEnterotoxin B

BackgroundCamelids and sharks possess a unique subclass of antibodies comprised of only heavy chains. The antigen binding fragments of these unique antibodies can be cloned and expressed as single domain antibodies (sdAbs). The ability of these small antigen-binding molecules to refold after heating to achieve their original structure, as well as their diminutive size, makes them attractive candidates for diagnostic assays.ResultsHere we describe the isolation of an sdAb against Staphyloccocus aureus enterotoxin B (SEB). The clone, A3, was found to have high affinity (Kd = 75 pM) and good specificity for SEB, showing no cross reactivity to related molecules such as Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin D (SED), and Shiga toxin. Most remarkably, this anti-SEB sdAb had an extremely high Tm of 85°C and an ability to refold after heating to 95°C. The sharp Tm determined by circular dichroism, was found to contrast with the gradual decrease observed in intrinsic fluorescence. We demonstrated the utility of this sdAb as a capture and detector molecule in Luminex based assays providing limits of detection (LODs) of at least 64 pg/mL.ConclusionThe anti-SEB sdAb A3 was found to have a high affinity and an extraordinarily high Tm and could still refold to recover activity after heat denaturation. This combination of heat resilience and strong, specific binding make this sdAb a good candidate for use in antibody-based toxin detection technologies.

Adapter-Directed Display: A Modular Design for Shuttling Display on Phage Surfaces

A novel adapter-directed phage display system was developed with modular features. In this system, the target protein is expressed as a fusion protein consisting of adapter GR1 from the phagemid vector, while the recombinant phage coat protein is expressed as a fusion protein consisting of adapter GR2 in the helper phage vector. Surface display of the target protein is accomplished through specific heterodimerization of GR1 and GR2 adapters, followed by incorporation of the heterodimers into phage particles. A series of engineered helper phages were constructed to facilitate both display valency and formats, based on various phage coat proteins. As the target protein is independent of a specific phage coat protein, this modular system allows the target protein to be displayed on any given phage coat protein and allows various display formats from the same vector without the need for reengineering. Here, we demonstrate the shuttling display of a single-chain Fv antibody on phage surfaces between multivalent and monovalent formats, as well as the shuttling display of an antigen-binding fragment molecule on phage coat proteins pIII, pVII, and pVIII using the same phagemid vectors combined with different helper phage vectors. This adapter-directed display concept has been applied to eukaryotic yeast surface display and to a novel cross-species display that can shuttle between prokaryotic phage and eukaryotic yeast systems.

Targeting of Epidermal Growth Factor Receptor (EGFR)-Expressing Tumor Cells with Sterically Stabilized Affibody Liposomes (SAL)

Affibody molecules are small and stable antigen-binding molecules derived from the B domain of protein A. We applied a bivalent, high-affinity epidermal growth factor receptor (EGFR)-specific affibody molecule for the generation of targeted PEGylated liposomes. These sterically stabilized affibody liposomes (SAL) were produced by chemical coupling of the cysteine-modified affibody molecule to maleimide-PEG(2000)-DSPE and subsequent insertion into PEGylated liposomes. These SAL showed strong and selective binding to EGFR-expressing tumor cell lines. Binding was dependent on the amount of inserted affibody molecule-lipid conjugates and could be blocked by soluble EGF. Approximately 30% of binding activity was still retained after 6 days of incubation in human plasma at 37 degrees C. Binding of SAL to cells led to efficient internalization of the liposomes. Using mitoxantrone-loaded liposomes, we observed for SAL, compared to untargeted liposomes, an enhanced cytotoxicity toward EGFR-expressing cells. In summary, we show that SAL can be easily prepared from affibody molecules and thus may be suitable for the development of carrier systems for targeted delivery of drugs.

Single-Chain Antibodies as New Antithrombotic Drugs

Antibodies are the most rapidly growing class of human therapeutics and the second largest class of drugs after vaccines. At present, several antibodies are approved for therapeutic use in diverse clinical settings, including oncology, chronic inflammatory diseases, transplantation, infectious diseases, and cardiovascular medicine. These approved antibody therapeutics include unmodified immunoglobulin G molecules, radioimmunoconjugates, antibody-drug conjugates, and fragment antigen-binding molecules. At least 150 additional antibodies are in clinical development. A major strength of therapeutic antibodies is their established properties as a drug class with high success rates from clinical trials to regulatory approvals. Much of the experience gained from the generation and optimization of one antibody is applicable to other antibodies. Antibody fragments are a subclass with growing clinical importance. This review focuses on single-chain antibodies as the smallest possible format for recombinant antibodies, and their use as antithrombotic drugs. We describe different antibody formats, the current applications of antibody fragments, and their generation by cloning from hybridoma cell lines as well as their selection from antibody libraries. We review the use of antibody fragments for thrombus targeting using fibrin and platelet-specific single-chain antibodies in combination with anticoagulants and thrombolytic agents as antithrombotic drugs.

Increased Levels ofCandida albicansMannan-Specific T-Cell-Derived Antigen Binding Molecules in Patients with Invasive Candidiasis

In addition to cytokines, CD4+ T cells have been found to secrete soluble, T-cell-derived antigen binding molecules (TABMs). These antigen-specific immunoproteins are thought to have immunoregulatory properties in the suppression of cell-mediated immunity (CMI) because they often associate with interleukin-10 (IL-10) and transforming growth factor beta. Decreased CMI causes susceptibility to infections caused by organisms which are normally nonpathogenic. In this situation, e.g., Candida albicans saprophytism may develop into invasive candidiasis. The difficult diagnosis of invasive candidiasis is based on the findings obtained from blood cultures and with tissue biopsy specimens, with some additional diagnostic value gained by the detection of Candida albicans mannan antigenemia and antimannan antibodies. In the present study, Candida albicans mannan-specific TABM (CAM-TABM) levels in the sera of patients with invasive candidiasis (n = 11), Candida colonization (n = 11) and noncolonization (n = 10), recurrent vulvovaginal candidiasis (n = 30), and atopic eczema dermatitis syndrome (n = 59) and healthy controls (n = 30) were analyzed. For 14 participants, the effect of mannan stimulation on TABM production and gamma interferon (IFN-gamma) and IL-4 mRNA expression by peripheral blood lymphocytes was also studied. It was demonstrated that CAM-TABM production was the highest in patients with invasive candidiasis and that CAM-TABM levels could distinguish Candida-colonized patients from noncolonized patients. In addition, the CAM-TABM level was directly related to mRNA expression for IL-4 but not IFN-gamma. These results reinforce the view that TABMs are associated with decreased CMI, immunoregulation, and the T-helper cell 2-type immune response.

At least one YMHCI molecule in the chicken is alloimmunogenic and dynamically expressed on spleen cells during development

Transcriptionally active, MHC class I (MHCI) loci are located in two separate polymorphic genomic regions in the chicken called B and Y. The YMHCI gene sequences encode molecules with uncommon substitutions in the antigen-binding region indicating that YMHCI molecules are likely unique and may bind a specialized form of antigen distinct from that of other antigen-binding MHCI molecules. To learn whether YMHCI gene expression results in the production of alloantigens at the cell surface, we immunized 15I(5) x 7(2) : chickens using syngeneic RP9 cells expressing transduced YF1w*7.1, a potentially alloimmunogenic YMHCI allele from the Y7 haplotype present in line C. The resulting antisera show that YF1w*7.1 MHCI molecules are immunogenic and expressed on the surfaces of cells in blood and spleen of line C chickens. Virtually all CD3+, CD4+, and CD8+ cells circulating in line C blood are positive, as are BU1+ cells. The YF1w*7.1 MHCI allele is dynamically expressed at levels comparable to but transcriptionally independent of classical BMHCI on erythrocytes, lymphocytes, granulocytes, monocytes, and thrombocytes within the spleen pre- and post-hatching. The antisera react with cells from two among four haplotypes segregating in closed populations of lines N and P. YMHCI shares features associated with both classical and non-classical MHCI. It is becoming increasingly likely that YMHCI has a fundamental role in avian immunity and thereby needs to be included in the growing spectrum of functionally active, diverse MHCI molecules no longer adequately described by the classical/non-classical dichotomy.

Cytotoxic T Cells

The immune system is a complex arrangement of cells and molecules that preserve the integrity of the organism by elimination of all elements judged dangerous. Within the immune system, a humoral and a cellular as well as an innate and an adaptive arm can be differentiated. The key players of adaptive cellular immune responses are T lymphocytes in general and, for the effector function, cytotoxic T lymphocytes (CTLs) in particular. T lymphocytes arise in the bone marrow and migrate to the thymus for maturation. During this process, T cells somatically rearrange gene segments, eventually leading to the expression of a unique antigen-binding molecule, the T-cell receptor (TCR). This receptor allows them to monitor all cells of the body, ready to destroy any cell posing a threat to the organism. Cytotoxicity is exerted directly through the Fas or perforin pathway and/or indirectly by the release of cytokines. Obviously, the activity of such a potent cell is tightly regulated. Indeed, a predominance of stimulatory over inhibitory signals is required for effective immune responses to pathogens, and a predominance of inhibitory over stimulatory signals is required for maintenance of self-tolerance. Still, several situations occur in which an inappropriate CTL response leads to either autoimmune disease or persistence of pathogens.

The pre-B-cell receptor induces silencing of VpreB and lambda5 transcription.

The pre-B-cell receptor (pre-BCR), composed of Ig heavy and surrogate light chain (SLC), signals pre-BII-cell proliferative expansion. We have investigated whether the pre-BCR also signals downregulation of the SLC genes (VpreB and lambda5), thereby limiting this expansion. We demonstrate that, as BM cells progress from the pre-BI to large pre-BII-cell stage, there is a shift from bi- to mono-allelic lambda5 transcription, while the second allele is silenced in small pre-BII cells. A VpreB1-promoter-driven transgene shows the same pattern, therefore suggesting that VpreB1 is similarly regulated and thereby defines the promoter as a target for transcriptional silencing. Analyses of pre-BCR-deficient mice show a temporal delay in lambda5 downregulation, thereby demonstrating that the pre-BCR is essential for monoallelic silencing at the large pre-BII-cell stage. Our data also suggest that SLP-65 is one of the signaling components important for this process. Furthermore, the VpreB1/lambda5 alleles undergo dynamic changes with respect to nuclear positioning and heterochromatin association, thereby providing a possible mechanism for their transcriptional silencing.