Mutations in the Bruton's tyrosine kinase (Btk) gene have been linked to severe early B cell developmental blocks in human X-linked agammaglobulinemia (XLA), and to milder B cell activation deficiencies in murine X-linked immune deficiency (Xid). To elucidate unequivocally potential Btk functions in mice, we generated mutations in embryonic stem cells, which eliminated the ability to encode Btk pleckstrin homology or kinase domains, and assayed their effects by RAG2-deficient blastocyst complementation or introduction into the germline. Both mutations block expression of Btk protein and lead to reduced numbers of mature conventional B cells, severe B1 cell deficiency, serum IgM and IgG3 deficiency, and defective responses in vitro to various B cell activators and in vivo to immunization with thymus-independent type II antigens. These results prove that lack of Btk function results in an Xid phenotype and further suggest a differential requirement for Btk during the early stages of murine versus human B lymphocyte development.
The long-standing hypothesis that tolerance to self antigens is mediated by either elimination or functional inactivation (anergy) or self-reactive lymphocytes is now accepted, but little is known about the factors responsible for initiating one process rather than the other. In the B-cell lineage, tolerant self-reactive cells persist in the peripheral lymphoid organs of transgenic mice expressing lysozyme and anti-lysozyme immunoglobulin genes, but are eliminated in similar transgenic mice expressing anti-major histocompatibility complex immunoglobulin genes. By modifying the structure of the lysozyme transgene and the isotype of the anti-lysozyme immunoglobulin genes, we demonstrate here that induction of anergy or deletion is not due to differences in antibody affinity or isotype, but to recognition of monomeric or oligomeric soluble antigen versus highly multivalent membrane-bound antigen. Our findings indicate that the degree of receptor crosslinking can have qualitatively distinct signalling consequences for lymphocyte development.
Cell-transfer studies presented here distinguish three murine B cell lineages: conventional B cells, which develop late and are continually replenished from progenitors in adult bone marrow; Ly-l B cells (B-la), which develop early and maintain their numbers by self-replenishment; and Ly-1 B "sister" (B-lb) cells, which share many of the properties of Ly-1 B cells, including self-replenishment and feedback regulation of development but can also readily develop from progenitors in adult bone marrow. The sequential emergence of these lineages, the time at which their progenitors function during ontogeny, and the distinctions among their repertoires and functions suggest that evolution has created a layered immune system in which-the immune response potential ofeach successive lineage is adapted to its particular niche. (5) and B-cell neoplasms (6). They tend to use a restricted set of variable-region (V) genes (7-10) and to use N-region insertions less often than conventional B cells (11). These repertoire differences may arise from differences in the diversity-generating mechanisms in individual lineages (12), to the different times that lineages develop (3), and/or to selection by particular antigens (13)(14)(15)(16) (17).In this study, we characterize the progenitor capacity of FL and adult bone marrow (BM) for three kinds of mature B We show here that significant progenitor activity for B-lb cells is present in fetal and adult animals, whereas progenitor activity for B-la cells is readily detectable in FL but is largely missing or nonfunctional in adult BM. We further show (i) that the failure to detect progenitor activity for B-la cells in adult BM is not due to the presence of inhibitors or the absence of inducers that regulate B-la development, (ii) that the progenitors for conventional B cells are already distinct from progenitors for the B-1 subsets in 14-day FL, and (iii) that the distinctive development of the three B-cell populations depends on properties inherent in their progenitors and cannot be explained solely by differential selection.Taken together, these studies indicate that B-ia and B-lb cells belong to separate developmental lineages and that both lineages are distinct from the conventional B lineage. We discuss these findings in the context of our recent hypothesis that these B-cell lineages reflect the existence of an evolutionarily layered immune system in which the immune response potential of each successive lineage is adapted to particular challenges (22). MATERIALS AND METHODS
Processes that define immunoglobulin repertoires are commonly presumed to be the same for all murine B cells. However, studies here that couple high-dimensional FACS sorting with large-scale quantitative IgH deep-sequencing demonstrate that B-1a IgH repertoire differs dramatically from the follicular and marginal zone B cells repertoires and is defined by distinct mechanisms. We track B-1a cells from their early appearance in neonatal spleen to their long-term residence in adult peritoneum and spleen. We show that de novo B-1a IgH rearrangement mainly occurs during the first few weeks of life, after which their repertoire continues to evolve profoundly, including convergent selection of certain V(D)J rearrangements encoding specific CDR3 peptides in all adults and progressive introduction of hypermutation and class-switching as animals age. This V(D)J selection and AID-mediated diversification operate comparably in germ-free and conventional mice, indicating these unique B-1a repertoire-defining mechanisms are driven by antigens that are not derived from microbiota.DOI: http://dx.doi.org/10.7554/eLife.09083.001
Modern methods that use systematic, quantitative and unbiased approaches are making it possible to discover proteins altered by a disease. To identify proteins that might be differentially expressed in autism, serum proteins from blood were subjected to trypsin digestion followed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) on time-of-flight (TOF) instruments to identify differentially expressed peptides. Children with autism 4-6 years of age (n = 69) were compared to typically developing children (n = 35) with similar age and gender distributions. A total of 6348 peptide components were quantified. Of these, five peptide components corresponding to four known proteins had an effect size > 0.99 with a P < 0.05 and a Mascot identification score of 30 or greater for autism compared to controls. The four proteins were: Apolipoprotein (apo) B-100, Complement Factor H Related Protein (FHR1), Complement C1q and Fibronectin 1 (FN1). In addition, apo B-100 and apo A-IV were higher in children with high compared to low functioning autism. Apos are involved in the transport of lipids, cholesterol and vitamin E. The complement system is involved in the lysis and removal of infectious organisms in blood, and may be involved in cellular apoptosis in brain. Despite limitations of the study, including the low fold changes and variable detection rates for the peptide components, the data support possible differences of circulating proteins in autism, and should help stimulate the continued search for causes and treatments of autism by examining peripheral blood.
BackgroundAutism is a neurodevelopmental disorder characterized by impairments in social behavior, communication difficulties and the occurrence of repetitive or stereotyped behaviors. There has been substantial evidence for dysregulation of the immune system in autism.MethodsWe evaluated differences in the number and phenotype of circulating blood cells in young children with autism (n = 70) compared with age-matched controls (n = 35). Children with a confirmed diagnosis of autism (4–6 years of age) were further subdivided into low (IQ<68, n = 35) or high functioning (IQ≥68, n = 35) groups. Age- and gender-matched typically developing children constituted the control group. Six hundred and forty four primary and secondary variables, including cell counts and the abundance of cell surface antigens, were assessed using microvolume laser scanning cytometry.ResultsThere were multiple differences in immune cell populations between the autism and control groups. The absolute number of B cells per volume of blood was over 20% higher for children with autism and the absolute number of NK cells was about 40% higher. Neither of these variables showed significant difference between the low and high functioning autism groups. While the absolute number of T cells was not different across groups, a number of cellular activation markers, including HLA-DR and CD26 on T cells, and CD38 on B cells, were significantly higher in the autism group compared to controls.ConclusionsThese results support previous findings that immune dysfunction may occur in some children with autism. Further evaluation of the nature of the dysfunction and how it may play a role in the etiology of autism or in facets of autism neuropathology and/or behavior are needed.
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