Central B-cell tolerance: where selection begins

The development of an adaptive immune system based on the random generation of antigen receptors requires a stringent selection process that sifts through receptor specificities to remove those reacting with self-antigens. In the B-cell lineage, this selection process is first applied to IgM⁺ immature B cells. By using increasingly sophisticated mouse models, investigators have identified the central tolerance mechanisms that negatively select autoreactive immature B cells and prevent inclusion of their antigen receptors into the peripheral B-cell pool. Additional studies have uncovered mechanisms that promote the differentiation of nonautoreactive immature B cells and their positive selection into the peripheral B-cell population. These mechanisms of central selection are fundamental to the generation of a naïve B-cell repertoire that is largely devoid of self-reactivity while capable of reacting with any foreign insult.B-cell generation in the bone marrow of adult mammals occurs through a tightly controlled developmental process (Fig. 1). Productive rearrangement of immunoglobulin heavy (IgH) and light (IgL) chain gene segments in B lymphocyte precursor cells, in addition to the expression of Ig-α (CD79a) and Ig-β (CD79b), result in the generation and expression on the cell surface of a mature B-cell antigen receptor (BCR). Whereas the combination of Ig H and L chains determines the antigenic specificity of the newly formed BCR, their association with Ig-α and Ig-β allows transduction of a signal inside the cell that directs cell fate. Developing B cells first express a mature BCR on the cell surface in the form of IgM and as such are classified as immature B cells (Fig. 1) (Hardy et al. 1991; Pelanda et al. 1996). It is at the immature B-cell stage that the BCR is tested for the first time for reactivity against autoantigens. This test determines whether the immature B cell and the antibody it expresses on the surface will be selected into the peripheral B-cell repertoire. Central B-cell tolerance, in fact, refers to the process that negatively selects newly generated immature B cells that react with a self-antigen in the bone marrow environment. This is considered the first checkpoint of B-cell tolerance, and the results of this checkpoint are fundamental to the generation of a naïve repertoire that contains foreign reactive antibodies and is largely devoid of self-reactive specificities.Open in a separate windowFigure 1.Schematic representation of B-cell development and Ig loci in mice. Large pro-B cells initiate Ig gene rearrangement at the IgH locus. Expression of a H chain following a productive V_HD_HJ_H recombination event promotes the differentiation of large pre-B cells in which the expression of pre-BCR (H chain pairing with surrogate light chains) results in the clonal expansion of H chain-positive pre-B cells and the development of small pre-B cells. Expression of conventional L chains following productive rearrangements at the IgL chain loci in small pre-B cells promotes the development of a diverse population of IgM⁺ immature B cells, which then differentiate into IgM⁺IgD⁺ transitional B cells. The scheme of mouse Ig H, κ, and λ loci (not to scale) indicate the presence of V (white rectangles), D (black vertical lines), J (brown vertical lines; a dashed line indicates a nonfunctional element), and C (black rectangles; a gray rectangle indicates a nonfunctional element) gene segments. The scheme does not represent the number of V_H, D_H, and Vκ gene segments in the actual Ig loci.On passing this central checkpoint, immature B cells continue to differentiate into transitional and mature B cells before and after they travel to the spleen (Loder et al. 1999; Allman et al. 2001; Su and Rawlings 2002; Tarlinton et al. 2003). Analysis of the bone marrow early immature B-cell repertoire indicates that a staggering 50%–75% of these cells express BCRs that are specific for self-antigens, both in mice and humans (Grandien et al. 1994; Wardemann et al. 2003). Similar studies performed on cell populations at the other end of this central checkpoint, namely, transitional and naïve mature B cells in spleen and blood, show a much lower frequency (20%–40%) of cells expressing autoreactive antibodies (Grandien et al. 1994; Wardemann et al. 2003), demonstrating the stringency and limitation of this initial selection step. Moreover, individuals affected by autoimmune disease such as lupus erythematosus or rheumatoid arthritis bear many more autoreactive cells in their new emigrant and naïve B-cell populations (Samuels et al. 2005; Yurasov et al. 2005), indicating a defect in central (and/or peripheral) B-cell selection. Thus, it seems important that the development of autoreactive immature B cells be constrained to prevent the potential occurrence of autoimmunity. However, there are also reasons to believe that the high frequency of autoreactive specificities generated during primary Ig gene rearrangements may be necessary for the generation of the peripheral B-cell repertoire (Pelanda et al. 1997; Kohler et al. 2008). Indeed, a fraction of autoreactive immature B cells, those manifesting a low level of self-reactivity, do bypass the central checkpoint of tolerance and differentiate into mature B cells (Hayakawa et al. 2003; Wardemann et al. 2003; Wen et al. 2005). The inclusion of these weakly self-reactive B cells in the peripheral B-cell repertoire may allow recognition of a broader spectrum of foreign molecules, potentially decreasing the negative impact of infections, especially at early stages (Mouquet et al. 2010).What are the rules that govern the selection of immature B cells? Most studies of central tolerance have been conducted by following the selection of B cells expressing BCRs displaying well-defined reactivity for natural or synthetic self-antigens. This has been accomplished through the use of Ig transgenic mice in which developing B cells have been altered to carry prerearranged Ig H and L chain genes encoding antibodies of defined antigen specificity and reactivity. Here we review some of these studies, what we have learned from them, and open questions that still await answers.     

Resistance to Bemisia tabaci biotype B of Solanum pimpinellifolium is associated with higher densities of type IV glandular trichomes and acylsugar accumulation

Advances in tomato breeding for pest resistance have been achieved via gene introgression from wild Solanum (section Lycopersicon) species (Solanaceae). Ninety‐nine F₃ families derived from an interspecific cross using as parental lines Solanum lycopersicum L. ‘LAM‐148' (susceptible standard) and Solanum pimpinellifolium L. ‘TO‐937‐15’ (multiple pest resistance accession with type IV glandular trichomes and acylsugar accumulation) were evaluated for their resistance against the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype B in free‐choice and no‐choice tests for oviposition and adult colonization. The parental lines and eight F₃ families with contrasting levels of resistance against the whitefly were selected and investigated in additional assays, which included the estimation of trichome densities and foliar acylsugar levels. The F₃ families BTR‐302 and BTR‐331 exhibited low amounts of eggs of whitefly and transgressive segregation for type IV glandular trichome density with values greater than that of TO‐937‐15 plants. However, the tested families did not surpass the total foliar acylsugar content found in TO‐937‐15. BTR‐331 exhibited low colonization in the free‐choice test and it was the least preferred F₃ family in the no‐choice test. The higher resistance levels of BTR‐331 were associated with a positive combination of higher type IV trichome density and higher acylsugar levels. Some F₃ families displayed reduced fruit set due to the presence of flowers with style exertion of the antheridial‐cone. Fruit weight at harvest stage of the selected families (from 4.9 to 14.5 g) was lower than that of LAM‐148 (139.5 g) but higher than that of TO‐937‐15 plants (1.3 g). Therefore, although difficult to reach due to the simultaneous segregation of many polygenic traits, the combination of high B. tabaci resistance levels with superior horticultural traits is feasible. These results confirm TO‐937‐15 as a source of biotype B resistance. From the breeding standpoint, the genetic similarity between S. lycopersicum and S. pimpinellifolium would allow a more efficient resistance introgression by facilitating recombination and minimizing the potentially undesirable linkage drag associated with this trait.     

Increasing calling accuracy,coverage, and read-depth in sequence data by the use of haplotype blocks

High-throughput genotyping of large numbers of lines remains a key challenge in plant genetics, requiring geneticists and breeders to find a balance between data quality and the number of genotyped lines under a variety of different existing genotyping technologies when resources are limited. In this work, we are proposing a new imputation pipeline (“HBimpute”) that can be used to generate high-quality genomic data from low read-depth whole-genome-sequence data. The key idea of the pipeline is the use of haplotype blocks from the software HaploBlocker to identify locally similar lines and subsequently use the reads of all locally similar lines in the variant calling for a specific line. The effectiveness of the pipeline is showcased on a dataset of 321 doubled haploid lines of a European maize landrace, which were sequenced at 0.5X read-depth. The overall imputing error rates are cut in half compared to state-of-the-art software like BEAGLE and STITCH, while the average read-depth is increased to 83X, thus enabling the calling of copy number variation. The usefulness of the obtained imputed data panel is further evaluated by comparing the performance of sequence data in common breeding applications to that of genomic data generated with a genotyping array. For both genome-wide association studies and genomic prediction, results are on par or even slightly better than results obtained with high-density array data (600k). In particular for genomic prediction, we observe slightly higher data quality for the sequence data compared to the 600k array in the form of higher prediction accuracies. This occurred specifically when reducing the data panel to the set of overlapping markers between sequence and array, indicating that sequencing data can benefit from the same marker ascertainment as used in the array process to increase the quality and usability of genomic data.     

Graph Based Study of Allergen Cross-Reactivity of Plant Lipid Transfer Proteins (LTPs) Using Microarray in a Multicenter Study

The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.     

Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria

The ability to transport and use haemin as an iron source is frequently observed in clinical isolates of Shigella spp. and pathogenic Escherichia coli . We found that many of these haem-utilizing E. coli strains contain a gene that hybridizes at high stringency to the S. dysenteriae type 1 haem receptor gene, shuA . These shuA -positive strains belong to multiple phylogenetic groups and include clinical isolates from enteric, urinary tract and systemic infections. The distribution of shuA in these strains suggests horizontal transfer of the haem transport locus. Some haem-utilizing pathogenic E. coli strains did not hybridize with shuA , so at least one other haem transport system is present in this group. We also characterized the chromosomal region containing shuA in S. dysenteriae . The shuA gene is present in a discrete locus, designated the haem transport locus, containing eight open reading frames. Several of the proteins encoded in this locus participate with ShuA in haem transport, as a Salmonella typhimurium strain containing the entire haem transport locus used haem much more efficiently than the same strain containing only shuA . The haem transport locus is not present in E. coli K-12 strains, but the sequences flanking the haem transport locus in S. dysenteriae matched those at the 78.7 minute region of E. coli K-12. The junctions and flanking sequences in the shuA -positive pathogenic E. coli strains tested were nearly identical to those in S. dysenteriae , indicating that, in these strains, the haem transport locus has an organization similar to that in S. dysenteriae , and it is located in the same relative position on the chromosome.     

Assembly of active zone precursor vesicles: obligatory trafficking of presynaptic cytomatrix proteins Bassoon and Piccolo via a trans-Golgi compartment

Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles, little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking. We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins, or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking of distinct cytoplasmic scaffolding proteins.     

Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish

The zebrafish heart has the capacity to regenerate after ventricular resection. Although this regeneration model has proved useful for the elucidation of certain regeneration mechanisms, it is based on the removal of heart tissue rather than its damage. Here, we characterize the cellular response and regenerative capacity of the zebrafish heart after cryoinjury, an alternative procedure that more closely models the pathophysiological process undergone by the human heart after myocardial infarction (MI). Localized damage was induced in 25% of the ventricle by cryocauterization (CC). During the first 24 hours post-injury, CC leads to cardiomyocyte death within the injured area and the near coronary vasculature. Cell death is followed by a rapid proliferative response in endocardium, epicardium and myocardium. During the first 3 weeks post-injury cell debris was cleared and the injured area replaced by a massive scar. The fibrotic tissue was subsequently degraded and replaced by cardiac tissue. Although animals survived CC, their hearts showed nonhomogeneous ventricular contraction and had a thickened ventricular wall, suggesting that regeneration is associated with processes resembling mammalian ventricular remodeling after acute MI. Our results provide the first evidence that, like mammalian hearts, teleost hearts undergo massive fibrosis after cardiac damage. Unlike mammals, however, the fish heart can progressively eliminate the scar and regenerate the lost myocardium, indicating that scar formation is compatible with myocardial regeneration and the existence of endogenous mechanisms of scar regression. This finding suggests that CC-induced damage in zebrafish could provide a valuable model for the study of the mechanisms of scar removal post-MI.     

A study of the anti‐plasmodium activity of angiotensin II analogs

Controlling the dissemination of malaria requires the development of new drugs against its etiological agent, a protozoan of the Plasmodium genus. Angiotensin II and its analog peptides exhibit activity against the development of immature and mature sporozoites of Plasmodium gallinaceum. In this study, we report the synthesis and characterization of angiotensin II linear and cyclic analogs with anti‐plasmodium activity. The peptides were synthesized by a conventional solid‐phase method on Merrifield's resin using the t‐Boc strategy, purified by RP‐HPLC and characterized by liquid chromatography/ESI (+) MS (LC‐ESI(+)/MS), amino acid analysis, and capillary electrophoresis. Anti‐plasmodium activity was measured in vitro by fluorescence microscopy using propidium iodine uptake as an indicator of cellular damage. The activities of the linear and cyclic peptides are not significantly different (p < 0.05). Kinetics studies indicate that the effects of these peptides on plasmodium viability overtime exhibit a sigmoidal profile and that the system stabilizes after a period of 1 h for all peptides examined. The results were rationalized by partial least‐square analysis, assessing the position‐wise contribution of each amino acid. The highest contribution of polar amino acids and a Lys residue proximal to the C‐terminus, as well as that of hydrophobic amino acids in the N‐terminus, suggests that the mechanism underlying the anti‐malarial activity of these peptides is attributed to its amphiphilic character. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Microbial population dynamics on Golden Delicious apples from bud to harvest and effect of fungicide applications

The microbial population dynamics on apples cv. Golden Delicious were analysed every 15 days between bud and harvest in a fully replicated experiment in northern Spain in 1994 and 1995. The total microbial populations varied with developmental stage, and with prevailing climatic conditions. The predominant mycroflora were the filamentous fungi Cladosporium and Alternaria spp. and white and pink yeasts. Other genera isolated included mainly species of Epicoccum, Fusarium and Acremonium. However, the most important post-harvest pathogens Penicillium expansum and Botrytis cinerea were seldom isolated from ripening apples. Maximum total filamentous fungal populations occurred after fruit set and during early ripening [2 × 10⁴cfu (colony-forming units) g^-1 approximately] while those of bacteria were maximum at bud stage (3.5 × 10⁵and 3.0 × 10⁴ cfu g^-1 in 1994 and 1995 respectively). White yeasts were more numerous than pink yeasts. Endophytic infection of apple buds by Alternaria spp., responsible for core rot, was found in almost all bud tissue. By contrast, Cladosporium spp. were initially isolated later from 12.5–50% of tissue samples during blooming and fruit set. The impact of a four-spray fungicide regime during apple development significantly decreased the total filamentous fungal populations in both years, and that of Cladosporium spp. in 1994. However, bacterial populations were often higher on apples from fungicide-treated plots. Fungicide sprays decreased populations of Cladosporium, Alternaria and white yeasts for a maximum of up to 15–30 days after application. Fungicide application had little effect on endophytic infection of apples by Alternaria spp. between bud and harvest.