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The early B cell protein λ5 is an essential component of the surrogate light chain and the preB cell receptor (preBCR), which is critical for optimal B cell development. To investigate the effect of λ5 and/or B cells on bone acquisition over time, we developed a panel of JH-/-, λ5-/-, JH-/- λ5-/-, and wild-type (WT) BALB/c mice and then studied postnatal bone development and aging in these mice at one, six, twelve, and twenty-two months of age. The trabecular bone volume over total volume (BV/TV) in JH-/- mice was similar to WT mice at all ages. In contrast, at six months of age and thereafter, λ5-/- and JH-/-λ5-/- mice demonstrated a severe decrease in trabecular bone mass. Surprisingly, bone mass in six-month-old λ5-/- and JH-/-λ5-/- mice was similar to or even lower than in aged (twenty-two-months) WT mice, suggesting accelerated skeletal aging. The postnatal development and the acquisition of cortical bone mass in JH-/-λ5-/- mice were generally comparable to WT. However, JH-/-λ5-/- mice showed a significant decrease in cortical BV/TV at six- and twelve months of age. To examine the contribution of λ5 and B cells to postnatal bone synthesis, we separately transplanted whole bone marrow cells from JH-/-λ5-/- and WT mice into irradiated JH-/-λ5-/- and WT recipients. WT recipients of JH-/-λ5-/- marrow cells failed to show acquisition of trabecular bone mass, whereas transplanting WT marrow cells into JH-/-λ5-/- recipients led to the recovery of trabecular bone mass. Transfer of WT marrow cells into JH-/-λ5-/- mice promoted synthesis of new cortical and trabecular bone. Our findings indicate that λ5 plays a major role in preserving bone mass during postnatal development and skeletal aging which is distinct from its role in B cell development. The absence of both λ5 and B cells in JH-/-λ5-/- mice leads to delayed acquisition of cortical bone during postnatal development. Dissecting the mechanism(s) by which λ5 regulates bone homeostasis may provide new avenues for the treatment of age-related loss of bone mass and osteoporosis.

Background PCV13 (conjugated polysaccharide) and PPSV23 (polysaccharide only) are two licensed vaccines targeting S. pneumoniae. The role of CD4 T-cell responses in pneumococcal vaccines among healthy participants and their impact on antibodies is not yet known. Methods Ten adults (5 old and 5 young) received PCV13 (prime) and a year later PPSV23 (boost). Blood samples were collected prior to and multiple time points after vaccination. CD4 T cells responding to CRM197, polysaccharide (PS), CRM197 conjugated polysaccharide (CPS), PCV13 and PPSV23 vaccines were measured by flow cytometry. Serum antibodies were analyzed via multiplex opsonophagocytosis (MOPA) and pneumococcal IgG assays. Results Vaccine-specific CD4 T cells were induced in all ten vaccinees post PCV13. Older vaccinees mounted higher peak responses and those specific for PCV13 and conjugated PS-1 were more polyfunctional compared to the younger group. Vaccine-elicited peripheral T follicular helper (Tfh) cells were only detected in the younger group who also exhibited a higher fold change in OPA titers post both vaccines. Importantly, Tfh cells following PCV13 correlated only with PCV13 serotype specific OPA titers after PPSV23 vaccination. Conclusions These findings demonstrate age related differences in immune response and the potential importance of Tfh in modulating functional antibody responses following pneumococcal vaccination.

We hypothesized that the scarcity of N-nucleotides might contribute to the inability of the neonate to mount a robust allergic immune response. To test this, we used terminal deoxyribunucleotidyl Transferase deficient (TdT−/−) mice, which express “fetal-like” T cell receptor and immunoglobulin repertoires with largely germline-encoded CDR3 regions. Intraperitoneal sensitization was followed by aerosol provocation with either PBS or the allergen OVA in both TdT−/−mice and wild-type mice to develop allergic respiratory inflammation. The effects of this procedure were investigated by lung function test, immunological analysis of serum and brochoalveolar lavage. The local TH2 cytokine milieu was significantly attenuated in TdT−/−mice. Within this group, the induction of total IgE levels was also significantly reduced after sensitization. TdT−/−mice showed a tendency toward reduced eosinophilic inflow into the bronchial tubes, which was associated with the elimination of respiratory hyperreactivity. In conclusion, in a murine model of allergic airway inflammation, the expression of fetal-like antigen receptors was associated with potent indications of a reduced ability to mount an asthma phenotype. This underlines the importance of somatically-generated antigen-receptor repertoire diversity in type one allergic immune responses and suggests that the fetus may be protected from allergic responses, at least in part, by controlling N addition.

Demodex mites are commensal parasites of hair follicles (HFs). Normally asymptomatic, inflammatory outgrowth of mites can accompany malnutrition, immune dysfunction, and aging, but mechanisms restricting Demodex outgrowth are not defined. Here, we show that control of mite HF colonization in mice required group 2 innate lymphoid cells (ILC2s), interleukin-13 (IL-13), and its receptor, IL-4Ra-IL-13Ra1. HF-associated ILC2s elaborated IL-13 that attenuated HFs and epithelial proliferation at anagen onset; in their absence, Demodex colonization led to increased epithelial proliferation and replacement of gene programs for repair by aberrant inflammation, leading to the loss of barrier function and HF exhaustion. Humans with rhinophymatous acne rosacea, an inflammatory condition associated with Demodex, had increased HF inflammation with decreased type 2 cytokines, consistent with the inverse relationship seen in mice. Our studies uncover a key role for skin ILC2s and IL-13, which comprise an immune checkpoint that sustains cutaneous integrity and restricts pathologic infestation by colonizing HF mites.

Human cytomegalovirus (HCMV) establishes latency in myeloid cells. Using the Kasumi-3 latency model, we previously showed that lytic gene expression is activated prior to the establishment of latency in these cells. The early events in infection may have a critical role in shaping the establishment of latency. Here, we have used an integrative multi-omics approach to investigate dynamic changes in host and HCMV gene expression and epigenomes at early times postinfection. Our results show dynamic changes in viral gene expression and viral chromatin. Analyses of polymerase II (Pol II), histone 3 lysine 27 acetylation (H3K27Ac), and histone 3 lysine 27 trimethylation (H3K27me3) occupancy of the viral genome showed that (i) Pol II occupancy was highest at the major IE promoter (MIEP) at 4 h postinfection. However, it was observed throughout the genome. (ii) At 24 h, H3K27Ac was localized to the major immediate early promoter/enhancer and to a possible second enhancer in the origin of replication oriLyt; (iii) viral chromatin was broadly accessible at 24 hpi. In addition, although HCMV infection activated expression of some host genes, we observed an overall loss of de novo transcription. This was associated with loss of promoter-proximal Pol II and H3K27Ac but not with changes in chromatin accessibility or a switch in modification of H3K27. IMPORTANCE Human cytomegalovirus (HCMV) is an important human pathogen in immunocompromised hosts and developing fetuses. Current antiviral therapies are limited by toxicity and emergence of resistant strains. Our studies highlight emerging concepts that challenge current paradigms of regulation of HCMV gene expression in myeloid cells. In addition, our studies show that HCMV has a profound effect on de novo transcription and the cellular epigenome. These results may have implications for mechanisms of viral pathogenesis.

Enrichment for tyrosine in immunoglobulin CDR-H3 is due in large part to natural selection of germline immunoglobulin DH sequence. We have previously shown that when DH sequence is modified to reduce the contribution of tyrosine codons, epitope recognition is altered and B cell development, antibody production, autoantibody production, and morbidity and mortality following pathogen challenge are adversely affected. TCRβ diversity (Dβ) gene segment sequences are even more highly conserved than DH, with trout Dβ1 identical to human and mouse Dβ1. We hypothesized that natural selection of Dβ sequence also shapes CDR-B3 diversity and influences T cell development and T cell function. To test this, we used a mouse strain that lacked Dβ2 and contained a novel Dβ1 allele (DβYTL) that replaces Dβ1 with an immunoglobulin DH, DSP2.3. Unlike Dβ1, wherein glycine predominates in all three reading frames (RFs), in DSP2.3 there is enrichment for tyrosine in RF1, threonine in RF2, and leucine in RF3. Mature T cells using DβYTL expressed TCRs enriched at particular CDR-B3 positions for tyrosine but depleted of leucine. Changing Dβ sequence altered thymocyte and peripheral T cell numbers and the T cell response to an ovalbumin immunodominant epitope. The differences in tyrosine content might explain, at least in part, why TCRs are more polyspecific and of lower affinity for their cognate antigens than their immunoglobulin counterparts.

We have previously shown that the sequence of the immunoglobulin diversity gene segment (D H ) helps dictate the structure and composition of complementarity determining region 3 of the immunoglobulin heavy chain (CDR-H3). In order to test the role of germline D sequence on the diversity of the preimmune TCRβ repertoire of T cells, we generated a mouse with a mutant TCRβ DJC locus wherein the Dβ2-Jβ2 gene segment cluster was deleted and the remaining diversity gene segment, Dβ1 (IMGT:TRDB1), was replaced with DSP2.3 (IMGT:IGHD2-02), a commonly used B cell immunoglobulin D H gene segment. Crystallographic studies have shown that the length and thus structure of TCR CDR-B3 places amino acids at the tip of CDR-B3 in a position to directly interact with peptide bound to an MHC molecule. The length distribution of complementarity determining region 3 of the T cell receptor beta chain (CDR-B3) has been proposed to be restricted largely by MHC-specific selection, disfavoring CDR-B3 that are too long or too short. Here we show that the mechanism of control of CDR-B3 length depends on the Dβ sequence, which in turn dictates exonucleolytic nibbling. By contrast, the extent of N addition and the variance of created CDR3 lengths are regulated by the cell of origin, the thymocyte. We found that the sequence of the D and control of N addition collaborate to bias the distribution of CDR-B3 lengths in the pre-immune TCR repertoire and to focus the diversity provided by N addition and the sequence of the D on that portion of CDR-B3 that is most likely to interact with the peptide that is bound to the presenting MHC.

Although at first glance the diversity of the immunoglobulin repertoire appears random, there are a number of mechanisms that act to constrain diversity. For example, key mechanisms controlling the diversity of the third complementarity determining region of the immunoglobulin heavy chain (CDR-H3) include natural selection of germline diversity (DH ) gene segment sequence and somatic selection upon passage through successive B-cell developmental checkpoints. To test the role of DH gene segment sequence, we generated a panel of mice limited to the use of a single germline or frameshifted DH gene segment. Specific individual amino acids within core DH gene segment sequence heavily influenced the absolute numbers of developing and mature B-cell subsets, antibody production, epitope recognition, protection against pathogen challenge, and susceptibility to the production of autoreactive antibodies. At the tip of the antigen-binding loop (PDB position 101) in CDR-H3, both natural (germline) and somatic selection favored tyrosine while disfavoring the presence of hydrophobic amino acids. Enrichment for arginine in CDR-H3 appeared to broaden recognition of epitopes of varying hydrophobicity, but led to diminished binding intensity and an increased likelihood of generating potentially pathogenic dsDNA-binding autoreactive antibodies. The phenotype of altering the sequence of the DH was recessive for T-independent antibody production, but dominant for T-cell-dependent responses. Our work suggests that the antibody repertoire is structured, with the sequence of individual DH selected by evolution to preferentially generate an apparently preferred category of antigen-binding sites. The result of this structured approach appears to be a repertoire that has been adapted, or optimized, to produce protective antibodies for a wide range of pathogen epitopes while reducing the likelihood of generating autoreactive specificities.

Systemic lupus erythematosus (SLE) is an autoimmune disease that reflects a failure to block the production of self-reactive antibodies, especially those that bind double-stranded DNA (dsDNA). Backcrossing the lupus-prone NZM2410 genome onto C57BL/6 led to the identification of three genomic intervals, termed sle1, sle2 and sle3, which are associated with lupus susceptibility. We previously generated a C57BL/6 strain congenic for an immunoglobulin DH locus (ΔD-iD) that enriches for arginine at dsDNA-binding positions. We individually introduced the ΔD-iD allele into the three sle strains to test whether one or more of these susceptibility loci could affect the developmental fate of B cells bearing arginine-enriched CDR-H3s, the CDR-H3 repertoire created by the DH and the prevalence of dsDNA-binding antibodies. We found that the combination of the ΔD-iD allele and the sle1 locus led to a decrease in mature, recirculating B cell numbers and an increase in marginal zone cell numbers while maintaining a highly charged CDR-H3 repertoire. ΔD-iD and sle2 had no effect on peripheral B cell numbers, but the CDR-H3 repertoire was partially normalized. ΔD-iD and sle3 led to an increase in marginal zone B cell numbers, with some normalization of hydrophobicity. Mice with ΔD-iD combined with either sle1 or sle3 had increased production of dsDNA-binding IgM and IgG by 12 months of age. These findings indicate that the peripheral CDR-H3 repertoire can be categorically manipulated by the effects of nonimmunoglobulin genes.

In the bone marrow, preB cells are found adjacent to the bone endosteum where bone synthesizing osteoblast and bone resorbing osteoclasts reside. Although there is evidence of interactions between preB and bone cells, the factors that contribute to such interactions are poorly understood. A critical checkpoint for preB cell development assesses the integrity of the nascent immunoglobulin μ heavy chain (HC) by testing whether it can participate in the formation of a preB cell receptor (preBCR), composed of the μ HC and surrogate light chain (LC). In this work, we tested whether loss of preBCR components can affect bone synthesis. A panel of gene targeted mice with sequential blocks in preBCR formation or function [surrogate light chain component lambda 5 deleted (λ5-/-), transmembrane domain of μHC deleted (IgM-mem-/-), and CD19 preBCR co-receptor deleted (CD19-/-)] were evaluated for effects on postnatal bone synthesis. Postnatal bone mass was analyzed in 6 month old mice using μ-CT, histomorphometry and double calcein labeling. Both cortical and trabecular bone mass were significantly decreased in the femurs of the λ5 and IgM-mem deficient mice. Histomorphometric analysis showed a decrease in the numbers of osteoblasts and osteoclasts in all three mutant strains. Double calcein labeling revealed a significant decrease in dynamic synthesis and mineralization of bone in λ5-/- mice. Our data strongly suggest that interference with preBCR formation or function affects bone homeostasis independent of the presence or absence of mature B cells, and that components of the preBCR play important, and potentially distinct, roles in regulating adult bone mass.

Background Analysis of allergen-specific IgE responses in birth cohorts with microarrayed allergens has provided detailed information regarding the evolution of specific IgE responses in children. High-resolution data regarding early development of allergen-specific IgG are needed. Objective We sought to analyze IgG reactivity to microarrayed allergens in mothers during pregnancy, in cord blood samples, in breast milk, and in infants in the first years of life with the aim to investigate whether maternal allergen-specific IgG can protect against IgE sensitization in the offspring. Methods Plasma samples from mothers during the third trimester, cord blood, breast milk collected 2 months after delivery, and plasma samples from children at 6, 12, and 60 months of age were analyzed for IgG reactivity to 164 microarrayed allergens (ImmunoCAP ISAC technology) in 99 families of the Swedish birth cohort Assessment of Lifestyle and Allergic Disease During Infancy (ALADDIN). IgE sensitizations to microarrayed allergens were determined at 5 years of age in the children. Results Allergen-specific IgG reactivity profiles in mothers, cord blood, and breast milk were highly correlated. Maternal allergen-specific IgG persisted in some children at 6 months. Children’s allergen-specific IgG production occurred at 6 months and reflected allergen exposure. Children who were IgE sensitized against an allergen at 5 years of age had significantly higher allergen-specific IgG levels than nonsensitized children. For all 164 tested allergens, children from mothers with increased (>30 ISAC standardized units) specific plasma IgG levels against an allergen had no IgE sensitizations against that allergen at 5 years of age. Conclusion This is the first detailed analysis of the molecular IgG recognition profile in mothers and their children in early life. High allergen-specific IgG reactivity in the mother’s plasma and breast milk and in cord blood seemed to protect against allergic sensitization at 5 years of age.

Immunoglobulin replacement therapy (IGRT) can protect against lung function decline in CVID. We tested whether increasing IgG dosage was beneficial in patients who exhibited a decline in forced expiratory flow at 25–75% (FEF25–75%) even though they were receiving IgG doses within the therapeutic range. Of 189 CVID patients seen over 12 years, 38 patients met inclusion criteria, were seen on ≥ 3 visits, and demonstrated a ≥ 10% decrease in FEF25–75% from visits 1 to 2. FEF25–75%, forced expiratory flow at 1 s (FEV1), and FEV1/FVC at visit 3 were compared among those with non-dose adjustment (non-DA) versus additional IgG dose adjustment (DA). Three FEF25–75% tiers were identified: top (> 80% predicted), middle (50–80%), and bottom (

BACKGROUND: Common variable immunodeficiency (CVID) and IgG deficiency are two of the more prevalent primary humoral immune defects. Whereas the former is defined by consensus with criteria for quantitative and qualitative antibody defects, the latter is used to describe patients with reduced IgG, who commonly have recurrent sinopulmonary infections but do not fulfill CVID criteria. However, these patients are often given this diagnosis. OBJECTIVE: We compared immunological findings and clinical manifestations of two large cohorts of patients with CVID or IgG deficient to better delineate differences between those syndromes. METHODS: We extracted clinical and laboratory data from electronic medical records of patients at our institution who had received International Classification of Disease codes for either CVID, or IgG deficiency. We gathered immunoglobulin levels, lymphocyte subpopulation counts, and serological vaccine responses. In some patients, we performed flow cytometry to determine percentages of memory and switched-memory B cells. We compiled and statistically compared clinical data related to infectious manifestations, bronchiectasis, autoimmune diseases, infiltrative inflammatory processes and lymphoid malignancies. RESULTS: In contrast to IgG deficient patients, we found that CVID patients had lower IgG levels, greater unresponsiveness to most vaccines, lower percentages of memory and isotype switched-memory B cells, and lower CD4 T cell counts. Clinically, CVID patients presented similar rates of sinusitis and pneumonias, but a significantly higher prevalence of bronchiectasis and especially non-infectious complications. CONCLUSION: CVID and IgG deficiency do not share the same disease spectrum, the former being associated with immunodysregulative manifestations and markers of a more severe immune defect. These data may allow clinicians to distinguish these conditions and the management differences that these patients pose.

Sequential developmental checkpoints are used to “optimize” the B cell antigen receptor repertoire by minimizing production of autoreactive or useless immunoglobulins and enriching for potentially protective antibodies. The first and apparently most impactful checkpoint requires μHC to form a functional pre-B cell receptor (preBCR) by associating with surrogate light chain, which is composed of VpreB and λ5. Absence of any of the preBCR components causes a block in B cell development that is characterized by severe immature B cell lymphopenia. Previously, we showed that preBCR controls the amino acid content of the third complementary determining region of the H chain (CDR-H3) by using a VpreB amino acid motif (RDR) to select for tyrosine at CDR-H3 position 101 (Y101). In antibodies bound to antigen, Y101 is commonly in direct contact with the antigen, thus preBCR selection impacts the antigen binding characteristics of the repertoire. In this work, we sought to determine the forces that shape the peripheral B cell repertoire when it is denied preBCR selection. Using bromodeoxyuridine incorporation and evaluation of apoptosis, we found that in the absence of preBCR there is increased turnover of B cells due to increased apoptosis. CDR-H3 sequencing revealed that this is accompanied by adjustments to DH identity, DH reading frame, JH, and CDR-H3 amino acid content. These adjustments in the periphery led to wild-type levels of CDR-H3 Y101 content among transitional (T1), mature recirculating, and marginal zone B cells. However, peripheral selection proved incomplete, with failure to restore Y101 levels in follicular B cells and increased production of dsDNA-binding IgM antibodies.

We have developed a very simple method, termed absorbance summation (AS), for comparing protein concentrations between samples in ELISA assays without a standard. This method sums the observed absorbance values from all dilutions to obtain one data point for each sample to be used for comparison. AS is less computationally intensive than fitting sigmoidal curves, and it avoids the difficulty of parameter estimation for samples with absorbance values lying primarily at the lower tail of the curve. Our simulation studies showed that it performs much better than the sigmoidal curve fitting method and the conventional endpoint titer method. The power of this simple method is as high as the formal curve fitting followed by the estimation of area under the curve (AUC).

Complementarity Determining Region 3 of the immunoglobulin (Ig) H chain (CDR-H3) lies at the center of the antigen-binding site where it often plays a decisive role in antigen recognition and binding. Amino acids encoded by the diversity (DH) gene segment are the main component of CDR-H3. Each DH has the potential to rearrange into one of six DH reading frames (RFs), each of which exhibits a characteristic amino acid hydrophobicity signature that has been conserved among jawed vertebrates by natural selection. A preference for use of RF1 promotes the incorporation of tyrosine into CDR-H3 while suppressing the inclusion of hydrophobic or charged amino acids. To test the hypothesis that these evolutionary constraints on DH sequence influence epitope recognition, we used mice with a single DH that has been altered to preferentially use RF2 or inverted RF1. B cells in these mice produce a CDR-H3 repertoire that is enriched for valine or arginine in place of tyrosine. We serially immunized this panel of mice with gp140 from HIV-1 JR-FL isolate and then used enzyme-linked immunosorbent assay (ELISA) or peptide microarray to assess antibody binding to key or overlapping HIV-1 envelope epitopes. By ELISA, serum reactivity to key epitopes varied by DH sequence. By microarray, sera with Ig CDR-H3s enriched for arginine bound to linear peptides with a greater range of hydrophobicity but had a lower intensity of binding than sera containing Ig CDR-H3s enriched for tyrosine or valine. We conclude that patterns of epitope recognition and binding can be heavily influenced by DH germ line sequence. This may help explain why antibodies in HIV-infected patients must undergo extensive somatic mutation in order to bind to specific viral epitopes and achieve neutralization.

Developmental checkpoints eliminate B cells that synthesize defective immunoglobulin (Ig) heavy (HC) and light (LC) chains. The first checkpoint tests μHCs paired with VpreB/λ5 in a pre-B cell receptor (pre-BCR) to determine whether the μHC will be able to bind conventional LCs to form membrane IgM. VpreB and λ5 also create a sensing site that interacts with the μHC antigen-binding region complementarity-determining region (CDR)–H3; however, whether this site contributes to Ig repertoire selection and function is unknown. We analyzed the amino acid content of CDR-H3s from HCs cloned from living and apoptotic pre-B cells and from IgG-antigen structures. Using a panel of D H gene–targeted mice, we showed that progressively reducing CDR-H3 tyrosine content increasingly impaired pre-BCR checkpoint passage. Counting from cysteine at framework 3 position 96, we found that VpreB particularly selected for tyrosine at CDR-H3 position 101 and that Y101 also bound antigen in IgG-antigen structures. Therefore, in addition to its stabilization role in the pre-BCR, VpreB also acts as an early invariant antigen by selecting for particular CDR-H3 amino acids. These interactions shape the specificity of the IgG humoral response and may thus impose limitations on development of certain neutralizing antibodies.

Introduction and purpose Among the translational biomarkers, antibodies (Abs) are the best ones and thus key mediators of inflammatory responses to generate the events. Abs against myelin basic protein/MBP endowing with proteolytic activity (Ab-proteases) are to monitor demyelination at either of the stages. Ab-proteases were found in bronchial asthma (BA), hemophilia; autoimmune myocarditis (AIM) and antithyroid autoAbs. Outcome Anti-MBP autoAbs from MS patients and mice with EAE exhibited specific proteolytic cleavage of MBP. The activity of the latter markedly differs between: (i) MS patients and healthy controls; (ii) different clinical MS courses; (iii) EDSS scales of demyelination to correlate with the disability of MS patients to predict the transformations prior to clinical changes. Ab-mediated proteolysis of MBP results in generating a fixed set of peptides with MW ranged in fixed boundaries to suit common principles of the molecular architectonics of MBP. The sequence-specificity of Ab-proteases demonstrates five sites of preferential proteolysis to be located within the immunodominant regions of MBP. Two of them falling inside the sequence covering 81–103 peptide and the 82–98 segment as well, with the highest encephalitogenic properties to be attacked by Ab-proteases in MS patients at the most severe (progradient) courses. Meanwhile, sites localized within the frame of 43–68 and 146–170 segments whilst being less immunogenic happened to be EAE inducers very rare and were shown to be attacked by Ab-proteases at moderate (remittent) courses. The activity of Ab-proteases was first registered at the subclinical stages 1–2 years prior to the clinical illness and demonstrated a variety of sequence specificity to suit the definite stages of the disorder. Conclusions The activity of Ab-proteases in combination with the sequence-specificity would confirm a high subclinical and predictive value of the tools (biomarker-related) as applicable for personalized monitoring protocols. Ab-proteases can be programmed and re-programmed to suit the needs of the body metabolism or could be designed for the development of principally new catalysts with no natural counterparts. Further studies on targeted Ab-mediated proteolysis may provide translational tools for predicting demyelination and thus the disability of the MS patients.

Summary To test whether mechanisms controlling the range of diversity of the developing antibody repertoire in C57BL/6 mice (IgH b ) operate similarly to those identified in BALB/c mice (IgH a ), we compared the sequences of VH7183-containing H-chain transcripts from sorted adult bone marrow C57BL/6 B-cell subsets with those previously obtained from BALB/c mice. Patterns of VDJ gene segment utilization and CDR-H3 amino acid composition, charge, and average length in C57BL/6 pro-B cells were similar, although not identical, to BALB/c pro-B cells. However, C57BL/6 mature, recirculating B cells failed to demonstrate the reduction in the use of VH81X and the narrowing in the range of variance of CDR-H3 hydrophobicity that characterizes B-cell maturation in BALB/c mice. To further test the ability of the C57BL/6 strain to discard B cells expressing highly charged CDR-H3s, we introduced a mutant IgH a DH allele that forces use of arginine, asparagine and histidine. Unlike BALB/c mice, C57BL/6 mice congenic for the charged DH maintained normal numbers of mature, recirculating B cells that were enriched for charged CDR-H3s. Together; these findings indicate that the mature C57BL/6 B-cell pool permits expression of immunoglobulins with antigen binding sites that are typically discarded during late stage bone marrow B-cell development in BALB/c mice.

Natural antibodies (NAbs) are produced in the absence of exogenous antigenic stimulation and circulate in the blood of normal, healthy individuals. These antibodies have been shown to provide one of the first lines of defense against both bacterial and viral pathogens. Conservation of the NAb repertoire reactivity profile is observed both within and across species. One view holds that this conservation of NAb self reactivities reflects the use of germline antibody sequence, whereas the opposing view holds that the self-reactivities reflect selection driven by key conserved self-antigens. In mice, B-1a B cells are a major source of NAbs. A significant fraction of the B-1a antibody repertoire is devoid of N nucleotides in H chain complementarity determining region 3 (CDR-H3) and thus completely germline encoded. To test the role of germline DH sequence on the self-reactivity profile of the NAb repertoire, we examined the composition and self-antigen specificity of NAbs produced by a panel of DH gene targeted BALB/c mice, each strain of which expresses a polyclonal, altered CDR-H3 repertoire that differs from the wild-type norm. We found that in most cases the same key self-antigens were recognized by the NAbs created by each DH altered strain. The differences in reactivity appeared to represent the genetic signature of the NAb repertoire of each mouse strain in of reactivities. These findings suggest that although germline CDR-H3 sequence may facilitate the production of certain NAbs, a core set of self-antigens are likely the main force driving the selection of Nab self-specificities.

PURPOSE: Gain-of-function (GOF) mutations in the signal transducer and activator of transcription 1 (STAT1) result in unbalanced STAT signaling and cause immune dysregulation and immunodeficiency. The latter is often characterized by the susceptibility to recurrent Candida infections, resulting in the clinical picture of chronic mucocutaneous candidiasis (CMC). This study aims to assess the frequency of GOF STAT1 mutations in a large international cohort of CMC patients.METHODS: STAT1 was sequenced in genomic DNA from 57 CMC patients and 35 healthy family members. The functional relevance of nine different STAT1 variants was shown by flow cytometric analysis of STAT1 phosphorylation in patients' peripheral blood cells (PBMC) after stimulation with interferon (IFN)-α, IFN-γ or interleukin-27 respectively. Extended clinical data sets were collected and summarized for 26 patients.RESULTS: Heterozygous mutations within STAT1 were identified in 35 of 57 CMC patients (61%). Out of 39 familial cases from 11 families, 26 patients (67%) from 9 families and out of 18 sporadic cases, 9 patients (50%) were shown to have heterozygous mutations within STAT1. Thirteen distinct STAT1 mutations are reported in this paper. Eight of these mutations are known to cause CMC (p.M202V, p.A267V, p.R274W, p.R274Q, p.T385M, p.K388E, p.N397D, and p.F404Y). However, five STAT1 variants (p.F172L, p.Y287D, p.P293S, p.T385K and p.S466R) have not been reported before in CMC patients.CONCLUSION: STAT1 mutations are frequently observed in patients suffering from CMC. Thus, sequence analysis of STAT1 in CMC patients is advised. Measurement of IFN- or IL-induced STAT1 phosphorylation in PBMC provides a fast and reliable diagnostic tool and should be carried out in addition to genetic testing.

A major diagnostic intervention in the consideration of many patients suspected to have primary immunodeficiency diseases (PIDDs) is the application and interpretation of vaccination. Specifically, the antibody response to antigenic challenge with vaccines can provide substantive insight into the status of human immune function. There are numerous vaccines that are commonly used in healthy individuals, as well as others that are available for specialized applications. Both can potentially be used to facilitate consideration of PIDD. However, the application of vaccines and interpretation of antibody responses in this context are complex. These rely on consideration of numerous existing specific studies, interpolation of data from healthy populations, current diagnostic guidelines, and expert subspecialist practice. This document represents an attempt of a working group of the American Academy of Allergy, Asthma & Immunology to provide further guidance and synthesis in this use of vaccination for diagnostic purposes in consideration of PIDD, as well as to identify key areas for further research.

Mucins are gel-forming proteins that are responsible for the characteristic viscoelastic properties of mucus. Mucin overproduction is a hallmark of asthma, but the cellular requirements for airway mucin production are poorly understood. The endoplasmic reticulum (ER) protein anterior gradient homolog 2 (AGR2) is required for production of the intestinal mucin MUC2, but its role in the production of the airway mucins MUC5AC and MUC5B is not established. Microarray data were analyzed to examine the relationship between AGR2 and MUC5AC expression in asthma. Immunofluorescence was used to localize AGR2 in airway cells. Coimmunoprecipitation was used to identify AGR2-immature MUC5AC complexes. Agr2(-/-) mice were used to determine the role of AGR2 in allergic airway disease. AGR2 localized to the ER of MUC5AC- and MUC5B-producing airway cells and formed a complex with immature MUC5AC. AGR2 expression increased together with MUC5AC expression in airway epithelium from "Th2-high" asthmatics. Allergen-challenged Agr2(-/-) mice had greater than 50% reductions in MUC5AC and MUC5B proteins compared with allergen-challenged wild-type mice. Impaired mucin production in Agr2(-/-) mice was accompanied by an increase in the proportion of mucins contained within the ER and by evidence of ER stress in airway epithelium. This study shows that AGR2 increases with mucin overproduction in individuals with asthma and in mouse models of allergic airway disease. AGR2 interacts with immature mucin in the ER and loss of AGR2 impairs allergen-induced MUC5AC and MUC5B overproduction.

For the past 20 years, the standard of care for systemic sclerosis (SSc) with lung involvement has been oral or intravenous (IV) cyclophosphamide (CYC). To date, there have been 2 randomized trials and 2 metaanalyses of prospective studies using oral or IV CYC in SSc-related interstitial pneumonitis (interstitial lung disease; ILD) and none have reported improvement in lung function1,2,3,4,5. The Scleroderma Lung Research Study Group’s study in The New England Journal of Medicine reported that oral CYC daily for 1 year is of “modest benefit” compared to placebo1. However, the term “modest benefit” does not mean that lung function improved. In fact, the forced vital capacity (FVC) and DLCO declined in both placebo and CYC-treated patients1. “Modest benefit” means that, after 1 year, the rate of decline in FVC was less in those receiving CYC compared to placebo, but the lung function still worsened on CYC1. Further, at 2-year followup there was no difference in loss of lung function between oral daily CYC and placebo2. Due to a lack of effective standard therapy, SSc — a lethal disease that involves vital organs — needs a new and effective approach. An approach that began in patients about 14 years ago, hematopoietic stem cell transplantation (HSCT), has been demonstrated to improve both skin and lung function as well as quality of life in patients with SSc6,7,8. Transplantation has been performed safely in some studies8, but results have been complicated by high treatment-related mortality in others9. Mortality of HSCT for SSc can be markedly reduced, however, if the reasons for mortality are properly recognized. The safety of HSCT is determined by 3 variables: (1) … Address correspondence to Dr. Burt; E-mail: rburt{at}northwestern.edu

The quantitative simultaneous description of both variable region gene usage and antigen specificity of immunoglobulin repertoires is a major goal in immunology. Current quantitative assays are labor intensive and depend on extensive gene expression cloning prior to screening for antigen specificity. Here we describe an alternative method based on high efficiency single B cell cultures coupled with RT-PCR that can be used for rapid characterization of immunoglobulin gene segment usage, clonal size and antigen specificity. This simplified approach should facilitate the study of antibody repertoires expressed by defined B cell subpopulations, the analysis of immune responses to self and nonself-antigens, the development and screening of synthetic antibodies and the accelerated study and screening of neutralizing antibodies to pathogenic threats.

Background: We have previously shown that the allergic sensitization to ovalbumin does not represent a superantigen-like immune response. In gene-targeted mice (ΔD-iD) with a single modified Diversity gene segment (DH) of the immunoglobulin heavy chain, enriched for charged amino acids, the asthma phenotype in a murine model was markedly alleviated compared to wild-type animals. Objective: We now sought to determine whether the confinement to a single DH gene segment alone leads to a reduced allergic phenotype. Methods: We examined another gene-targeted mouse strain (ΔD-DFL) with a single DH gene segment which encodes for neutral amino acids, thus reflecting the preferential repertoire in wild-type mice. Mice were sensitized intraperitoneally to ovalbumin. Results: Despite the constraint to a single DH gene segment, ΔD-DFL mice mounted high total and allergen-specific IgG1 and IgE serum levels after sensitization to ovalbumin. The affinity constants of allergen-specific IgG1 antibodies did not differ between ΔD-DFL and wild type. Following challenge with aerosolized allergen, a marked local TH2 cytokine response and an eosinophilic airway inflammation developed. Quantitative histology revealed increased mucus production and intense goblet cell metaplasia which were identical to those in wild type. Moreover, ΔD-DFL mice developed an airway hyperreactivity to methacholine and to the specific allergen, which both did not differ from those in wild-type animals. Conclusion: A single DH gene segment is sufficient for the establishment of the asthma phenotype in a murine model of allergic airway inflammation. Thus, the allergic phenotype depends on the amino acid composition and not on the diversity of the classical antigen-binding site.

Compared with adult bone marrow (BM), the composition of the perinatal liver CDR-3 of the Ig H chain (CDR-H3) repertoire is marked by a paucity of N nucleotides and by enrichment for use of JH proximal DQ52 and DH proximal VH and JH gene segments. To test the extent to which these differences reflect limited perinatal TdT activity versus differences in the fetal/adult environment, we used the Hardy scheme to sort fractions B–F B lineage cells from TdT-deficient BALB/c adult BM. VH7183-containing VDJCμ transcripts from these cells were amplified, cloned, sequenced, and compared with transcripts from wild-type perinatal liver and adult BM. The pattern of VHDJH usage in TdT-deficient BM largely matched that of TdT-sufficient adult cells. What minor differences were detected in the pro-B cell stage tended to diminish with B cell maturation, suggesting strong environmental or Ag-driven pressure to achieve a specific range of VHDJH usage regardless of the extent of N nucleotide addition. However, although the patterns of VHDJH usage in the TdT-deficient B lineage cells paralleled that of wild-type adult cells, the length distribution, global amino acid composition, and charge distribution of the CDR-H3 repertoire proved to be a close, although not exact, homologue of the CDR-H3 repertoire first expressed by late pre-B cells in the TdT-insufficient perinatal liver. Thus, although differing in VH content, TdT-deficient mice appear to represent a good, although not perfect, model for testing the role of perinatal CDR-H3 limitations on late B cell development and Ab responses.

In mouse and human, the regulated development of antibody repertoire diversity during ontogeny proceeds in parallel with the development of the ability to generate antibodies to an array of specific antigens. Compared to adult, the human fetal antibody repertoire limits N addition and uses specifically positioned VDJ gene segments more frequently, including V6-1 the most DH-proximal VH, DQ52, the most JH-proximal DH, and JH2, which is DH-proximal. The murine fetal antibody repertoire also limits the incorporation of N nucleotides and uses its most DH proximal VH, VH81X, more frequently. To test whether DH and JH also follow the pattern observed in human, we used the scheme of Hardy to sort B lineage cells from BALB/c fetal and neonatal liver, RT-PCR cloned and sequenced VH7183-containing VDJCμ transcripts, and then assessed VH7183-DH-JH and complementary determining region 3 of the immunoglobulin heavy chain (CDR-H3) content in comparison to the previously studied adult BALB/c mouse repertoire. Due to the deficiency in N nucleotide addition, perinatal CDR-H3s manifested a distinct pattern of amino acid usage and predicted loop structures. As in the case of adult bone marrow, we observed a focusing of CDR-H3 length and CDR-H3 loop hydrophobicity, especially in the transition from the early to late pre-B cell stage, a developmental checkpoint associated with expression of the pre-B cell receptor. However, fetal liver usage of JH-proximal DHQ52 and DH-proximal JH2 was markedly greater than that of adult bone marrow. Thus, the early pattern of DH and JH usage in mouse feta liver mirrors that of human. Electronic supplementary material The online version of this article (doi:10.1007/s00251-010-0469-5) contains supplementary material, which is available to authorized users.

Lipopolysaccharide (LPS) from gram-negative bacteria activates B cells, enabling them to proliferate and differentiate into plasma cells. This response is critically dependent on the expression of TLR4, but other genes, such as RP105 and MHC class II, have also been shown to contribute to B cell LPS response. Here we have evaluated the role of genetic control of the B cell response to LPS at the single cell level by using limiting dilution analysis. This approach avoided the co-stimulatory and feeder-cell dependent effects that occur in bulk cultured B lymphocytes. We compared the response to LPS of peritoneal cavity (PEC) and splenic B cells on the BALB/c genetic background (LPS-low responder) to those on the C57BL/6J background (LPS-high responder) and their F1 progeny (B6xBc). Both PEC and splenic B cells from B6 exhibited 100% clonal growth in the presence of LPS, whereas BALB/c PEC and splenic B cells achieved only 50% and 23% clonal growth, respectively. Surprisingly, PEC B cells on the F1 (B6xBc) background behaved as high responders (close to 100 %, as in B6), while splenic B cells displayed a response that was closer to that of the low responder BALB/c strain (≈ 30 %), revealing a differential genetic control. Splenic follicular B cells on both the F1 and BALB/c backgrounds were also low responders, averaging 18 % and 25 % growth, respectively. Marginal zone B cells on the F1 and BALB/c backgrounds were moderately higher, achieving 45–50% growth. Adding CpG (the TLR9 ligand) to the LPS stimulus promoted pushed splenic B cell clonal growth in the low responder strain BALB/c up to 90%, showing that the non-response to LPS is a specific effect that cannot be attributed to a general deficiency of in vitro growth by the BALB/c cells. The data presented here reveals a previous unsuspected behavior in the genetic control of the B cell response to LPS, with an opposing impact in splenic versus peritoneal cavity B cells. These results suggest the existence of an as yet unidentified genetic factor exclusively expressed by coelomic B cells that contributes to the control the LPS signaling pathway in the B lymphocyte.

Recombinant DNA technology has the potential to produce allergen-specific immunotherapy vaccines with defined composition.To evaluate the effectiveness of a new recombinant birch pollen allergen vaccine in patients with birch pollen allergy.A multicenter, randomized, double-blind, placebo-controlled trial was undertaken to compare the following 3 vaccines in 134 adults with birch pollen allergy: recombinant birch pollen allergen vaccine (rBet v 1a), licensed birch pollen extract, natural purified birch pollen allergen (nBet v 1), and placebo. Patients received 12 weekly injections followed by monthly injections of the maintenance dose containing 15 microg Bet v 1 for 2 years.Significant reductions (about 50%) in rhinoconjunctivitis symptoms (rBet v 1, P = .0002; nBet v 1, P = .0006; birch extract, P = .0024), rescue medication (rBet v 1, P = .0011; nBet v 1, P = .0025; birch extract, P = .0063), and skin sensitivities (P.0001) were observed in the 3 actively treated groups compared with placebo during 2 consecutive pollen seasons. Clinical improvement was accompanied by marked increases in Bet v 1-specific IgG levels, which were higher in the rBet v 1-treated group than in the birch and nBet v 1-treated groups. New IgE specificities were induced in 3 of 29 patients treated with birch pollen extract, but in none of the 32 rBet v 1-treated or 29 nBet v 1-treated patients. No severe systemic adverse events were observed in the rBet v 1-treated group.The rBet v 1-based vaccine was safe and effective in treating birch pollen allergy, and induced a highly specific immune response.

Heterosubtypic immunity (HSI) is defined as cross-protection to infection with an influenza A virus serotype other than the one used for primary infection. Although HSI has been thought to be mediated by serotype cross-reactive cytotoxic T lymphocytes (CTL) that recognize conserved epitopes of structural proteins, recent studies suggest that antibodies (Abs) may make a significant contribution. In this study, we provide further evidence for the role of Abs in HSI using transgenic mice lacking terminal deoxyribonucleotidyltransferase (TdT), which adds N nucleotides to V-D and D-J junctions of the complementary determining region 3 (CDR3) (TdT −/− ) and mice with altered Ab repertoires due to replacement of the complete locus of heavy chain diversity segments (D H ) with an altered D H segment (namely, ΔD-iD). Both types of mice failed to generate complete HSI, although they were able to mount protective immunity to a homologous challenge. Lower levels of virus-specific antibodies along with more severely impaired HSI were observed in TdT −/− mice compared to those in ΔD-iD mice, while CTL activity remained unchanged in both types of mice. These findings indicate that a properly diversified antibody repertoire is required for HSI and that N addition by TdT is a more effective mechanism in the induction of a properly diversified antibody repertoire and, therefore, complete HSI. The results suggest that the diversity of the antibody repertoire as determined by the composition of the D region of HCDR3 and by N addition are among the mechanisms selected for in evolution to create a favorable environment to resolve infections with mutated viruses.

OBJECTIVE To assess the effects of psychological stress on the antibody response to tetanus vaccine adjusting for cytokine gene polymorphisms and other nongenetic factors in caregivers of patients with Alzheimer's disease (AD). METHODS A family-based follow-up study was conducted in 119 spouses and offspring of community-dwelling patients with AD. Psychological stress was measured by the Perceived Stress Scale (PSS) and the Center for Epidemiologic Studies Depression (CES-D) scale at baseline and 1 month after the vaccination. Nutritional status, health behaviors, comorbidity, and stress-buffering factors were assessed by self-administered questionnaires, 10 single nucleotide polymorphisms (SNP) from six selected cytokines genotyped, and anti-tetanus toxoid immunoglobulin G (IgG) concentrations tested using enzyme-linked immunosorbent assays. The effects of stress and other potential confounders were assessed by mixed models that account for familial correlations. RESULTS The baseline PSS score, the baseline CES-D score, the interleukin-10-1082 A>G SNP GG genotype, and the baseline anti-tetanus IgG were inversely associated with antibody fold increase. CONCLUSION Both psychological stress and cytokine gene polymorphisms affected antibody fold increase. The study provided additional support for the detrimental effects of psychological stress on the antibody response to tetanus vaccine.

Twenty cull dairy cows (645 ± 83 kg) were treated with 2.2 mg/kg bw flunixin by intravenous (IV) or intramuscular (IM) administration with, or without, exposure to lipopolysaccharide in a two factor balanced design. The usefulness of screening assays to identify violative flunixin levels in a variety of easily accessible ante-mortem fluids in cattle was explored. Two animals with violative flunixin liver residue and/or violative 5-hydroxy flunixin milk residues were correctly identified by a flunixin liver ELISA screen. Oral fluid did not produce anticipated flunixin concentration profiles using ELISA determination. One cow that had liver and milk violative residues, and one cow that had a milk violation at the prescribed withdrawal period were correctly identified by flunixin milk lateral flow analyses. The ratio of urinary flunixin and 5-hydroxy flunixin may be useful for predicting disruption of metabolism caused by disease or other factors potentially leading to violative liver flunixin residues.

Approximately 500 million years ago (1), vertebrates developed the ability to generate a highly diverse repertoire of immunoglobulins (Igs). These highly versatile proteins serve as both effector molecules and as receptors for antigen ligands. As soluble effectors, Igs can activate and fix complement and they can bind Fc receptors on the surfaces of granulocytes, monocytes, platelets, and other components of the immune response. V(D)J gene segment rearrangement and somatic hypermutation (SHM) create a population of diverse ligand binding sites that allow recognition of an almost unlimited array of self and non-self antigens. Above and beyond the time-honored practice of vaccination, the power of Igs as biotherapeutic agents is changing the face of medicine. In this research topic, we collected several exciting articles that highlight the diversity and similarity of antibody repertoires. We also highlight new bioinformatics approaches for the analysis of this data. We open the research topic with a review of antibody repertoires in fish by Fillatreau and colleagues (2). This review provides a description of the organization of fish Ig loci, with a particular emphasis on their heterogeneity between species, and presents recent data on the structure of the expressed Ig repertoire in healthy and infected fish. This is followed by a review of antibody repertoires in pigs (3). In pigs, the fetal repertoire develops without maternal influences and the precocial nature of multiple offspring provides investigators with the opportunity to study the influence of environmental and maternal factors on repertoire development. Next, we take a closer look at the human repertoire. Vas et al. (4) discuss the role of natural antibodies (Nabs). Mostly, IgM antibodies are produced in the absence of exogenous antigen challenge. The composition of the early immune repertoire is highly enriched for NAbs, which are polyreactive and often autoreactive. Included in Nabs are antibodies that recognize damaged and senescent cells, often via oxidation-associated neo-determinants. Clinical surveys have suggested that anti-apoptotic cell (AC) IgM NAbs may modulate disease activity in some patients with autoimmune disease. This review is followed by a comparative study by Mroczek and colleagues (5) of the antibody repertoire expressed by immature, transitional, mature, memory IgD+, memory IgD−, and plasmacytes isolated from the blood of a single individual. Differences observed between the Igs produced by these cells indicate that studies designed to correlate repertoire expression with diseases of immune function will likely require deep sequencing of B cells sorted by subset. The next paper highlights secondary mechanisms of antibody diversification that act in addition to V(D)J recombination and SHM of the complementary determining regions (CDRs) of the antibody that create the antigen-binding site (6). These secondary mechanisms include V(DD)J recombination (or D–D fusion), SHM-associated insertions and deletions, and affinity maturation and antigen contact by non-CDR regions of the antibody. Next is an analysis of age-related changes in the antibody repertoire following vaccination by Wu et al. (7). Clustering analysis of high-throughput sequencing data enables us to visualize the response in terms of expansions of clonotypes, changes in CDR-H3 characteristics, and SHM as well as identifying the commonly used IgH genes. This study highlights a number of areas for future consideration in vaccine studies of the elderly. Finlay and Almagro (8) pull all of these strands together in the final research based article, which reviews the structural studies and fundamental principles that define how antibodies interact with diverse targets. They compare the antibody repertoires and affinity maturation mechanisms of humans, mice, and chickens, as well as the use of novel single-domain antibodies in camelids and sharks. These species utilize a plethora of evolutionary solutions to generate specific and high-affinity antibodies. The various solutions used by these species illustrate the plasticity of natural antibody repertoires. They end their article by discussing man-made antibody repertoires that have been designed and validated in the last two decades. Together, these comparative studies of natural and man-made repertoires served as tools to explore how the size, diversity, and composition of a repertoire impact the antibody discovery process. High-throughput sequencing is tailor made for the study of antibody repertoires. However, the diversity of the sequences that is obtained from these studies is immense, and thus requires the development of new and friendly bioinformatics techniques to analyze and interpret the data. The final two articles are devoted to methods that can be used for these purposes. The first issue is quality control. Michaeli et al. (9) present a method for automated cleaning and pre-processing of immunoglobulin gene sequences from high-throughput sequencing. Their paper describes Ig high-throughput sequencing cleaner (Ig-HTS-cleaner), a program containing a simple cleaning procedure that successfully deals with pre-processing of Ig sequences derived from HTS, and Ig insertion–deletion identifier (Ig-Indel-identifier), a program for identifying legitimate and artifact insertions and/or deletions (indels). These programs were designed for analyzing Ig gene sequences obtained by 454 sequencing, but they are applicable to all types of sequences and sequencing platforms. Finally, Rogosch et al. (10) present an easy-to-use Microsoft® Excel® based software, named immunoglobulin analysis tool (IgAT), for the summary, interrogation, and further processing of IMGT/HighV-QUEST output files. IgAT generates descriptive statistics and high-quality figures for collections of murine or human Ig heavy or light chain transcripts ranging from 1 to 150,000 sequences. In addition to traditionally studied properties of Ig transcripts – such as the usage of germline gene segments, or the length and composition of the CDR-3 region – IgAT also uses published algorithms to calculate the probability of antigen selection based on somatic mutational patterns, the average hydrophobicity of the antigen-binding sites, and predictable structural properties of the CDR-H3 loop according to Shirai’s H3-rules. The authors that contributed to this volume hope that the reader will find this research topic interesting, thought-providing, and informative. We invite you to read the following articles and immerse yourself in the fascinating world of Igs. In the near term future, this world is likely to continue to provide new venues for the diagnosis, treatment, or prevention of disease.

In the adult mouse, three major subsets of mature B-lymphocytes are recognized: B-1, marginal zone (MZ), and follicular (FO) B cells. These B lymphocytes are the products of B-cell precursors that follow developmental programs that require passage through successive checkpoints to produce appropriately matured B-cell clonotypes. Comparative studies of human subjects found patterns of development and subset composition that are similar, but not identical, to those observed in mice. This chapter focuses on the derivation, development, compartmentalization, and function of these three major B-cell subsets: B-1, MZ and FO. Discussion of the developmental fate of these cells after exposure to antigen, including germinal center formation, class switching, memory B cell, and plasma cell development, can be found in other chapters of this textbook.

Tyrosine and glycine constitute 40% of complementarity determining region 3 of the immunoglobulin heavy chain (CDR-H3), the center of the classic antigen-binding site. To assess the role of DH RF1-encoded tyrosine and glycine in regulating CDR-H3 content and potentially influencing B cell function, we created mice limited to a single DH encoding asparagine, histidine, and arginines in RF1. Tyrosine and glycine content in CDR-H3 was halved. Bone marrow and spleen mature B cell and peritoneal cavity B-1 cell numbers were also halved, whereas marginal zone B cell numbers increased. Serum immunoglobulin G subclass levels and antibody titers to T-dependent and T-independent antigens all declined. Thus, violation of the conserved preference for tyrosine and glycine in DH RF1 alters CDR-H3 content and impairs B cell development and antibody production.

To test whether MHC alleles associated with common variable immune deficiency (CVID) might also be over-represented in patients with normal serum immunoglobulin levels who suffer with recurrent sinopulmonary infections (RESPI), we identified 62 consecutive RESPI patients and compared their HLA-B and HLA-DR antigen frequencies to those of 60 consecutive patients with CVID, 1627 Alabama Caucasian bone marrow donors, and 997,230 published US Caucasians. Either HLA-B44, -B8, -DR3(17), or -DR7 was present in 74% of the RESPI and 85% of the CVID patients. HLA-B44 prevalence in particular proved identical between RESPI and CVID. When compared to US Caucasians, the increased prevalence of the four HLA alleles proved significant at P < 0.0001, P < 0.0001, P = 0.0005, and P = 0.02, respectively. When compared to Alabama Caucasians, only the increased prevalence of HLA-B44 achieved statistical significance (P = 0.0001). Inheritance of HLA-B44 may yield susceptibility to recurrent sinopulmonary infection even in the presence of normal serum immunoglobulin levels.

A novel method of large-scale chromatography has been developed to improve recovery and purity of immunoglobulin G (IgG) from pooled plasma. The current study compares safety, toxicity and efficacy of two intravenous immunoglobulin products: a novel formulation, IGIV caprylate/chromatography (IGIV-C; Gamunex™, 10%) and a licensed solvent/detergent-treated product, Gamimune®N, 10% (IGIV-SD). The study, a randomized, double-blind, parallel group, therapeutic equivalence trial, was conducted at 25 treatment centers in Canada and the United States. Patients (n=172) having confirmed chronic primary immunodeficiency (PID), aged 1–75 years, and receiving IGIV therapy were enrolled. For 9 months, patients were treated with IGIV-C or IGIV-SD in accordance with the patient's individualized treatment regimen utilized before study entry. The primary endpoint was the proportion of patients with ≥1 validated acute sinopulmonary infection during the treatment period. Secondary endpoints included the proportion of patients with all infections, time to first infection, annual infection rates, lung function parameters, infusion-related safety and viral safety. The annual validated infection rate in the IGIV-C group was 0.18 compared to 0.43 in the IGIV-SD group (p=0.023). Nine patients receiving IGIV-C experienced validated infections, compared to 17 patients in IGIV-SD group (p=0.06). Acute sinusitis (validated plus clinically defined) was less frequent in the IGIV-C group (p=0.012). Presence of bronchiectasis did not affect efficacy. Adverse reactions were similar in frequency and severity in both groups. No evidence of viral transmission was observed. IGIV-C appears to be superior to IGIV-SD in preventing validated sinopulmonary infections, especially acute sinusitis, in patients with PID.

Objective A number of non-HLA loci that have shown evidence (P < 0.05) for linkage with rheumatoid arthritis (RA) have been previously identified. The present study attempts to confirm these findings. Methods We performed a second genome-wide screen of 256 new multicase RA families recruited from across the United States by the North American Rheumatoid Arthritis Consortium. Affected sibling pair analysis on the new data set was performed using SIBPAL. We subsequently combined our first and second data sets in an attempt to enhance the evidence for linkages in a larger sample size. We also evaluated the impact of covariates on the support for linkage, using LODPAL. Results Evidence of linkage at 1p13 (D1S1631), 6p21.3 (the HLA complex), and 18q21 (D18S858) (P < 0.05) was replicated in this independent data set. In addition, there was new evidence for linkage at 9p22 (D9S1121 [P = 0.001]) and 10q21 (D10S1221 [P = 0.0002] and D10S1225 [P = 0.0038]) in the current data set. The combined analysis of both data sets (512 families) showed evidence for linkage at the level of P < 0.005 at 1p13 (D1S1631), 1q43 (D1S235), 6q21 (D6S2410), 10q21 (D10S1221), 12q12 (D12S398), 17p13 (D17S1298), and 18q21 (D18S858). Linkage at HLA was also confirmed (P < 5 × 10−12). Inclusion of DRB1∗04 as a covariate significantly increased the probability of linkage on chromosome 6. In addition, some linkages on chromosome 1 showed improved significance when modeling DRB1∗04 or rheumatoid factor positivity as covariates. Conclusion These results provide a rational basis for pursuing high-density linkage and association studies of RA in several regions outside of the HLA region, particularly on chromosomes 1p, 1q, and 18q.

In order to facilitate molecular analysis of antibody responses in Rhesus monkeys ( Macaca mulatta), we used PCR techniques to clone and sequence the germline IGHD gene repertoire and the IGHD7- IGHJ6 locus in its entirety. We identified 30 distinct Rhesus DH genes belonging to seven subgroups and their recombination signal sequences that together share an average of 91% identity with their human counterparts, six potentially functional IGHJ genes and their recombination signal sequences that together share 93% identity with their human counterparts, as well as a novel IGHJ gene, IGHJ5 beta, which is a duplicated variant of IGHJ5. The presence, on average, of one additional IGHD gene in Rhesus IGHD subgroups when compared with human and one additional IGHJ gene suggests Rhesus has undergone at least two independent duplications beyond those that mark the human IGHD/IGHJ locus. Amino acid sequence composition is highly conserved between Rhesus and human, with IGHD insertions and deletions limited to three-nucleotide multiples, which serve to preserve enrichment for tyrosine, glycine, and serine residues in IGHD reading frame 1. The high degree of conservation between human and Rhesus IGHD and IGHJ genes supports the hypothesis that the germline repertoire encodes evolutionarily preferred antibody sequence as a result of selection for function.

The adaptive immune system has to economically generate a large array of T and B cell antigen receptors (T cell receptors [TCRs], B cell receptors [BCRs]) that eliminate both longstanding and novel antigens from the host while preventing the production of deleterious (e.g., autoreactive) antigen receptors. Our studies focus on the mechanisms that shape the development of these antigen receptor repertoires during human ontogeny. The key to BCR and TCR diversity is the third complementarity determining region (CDR3) of the variable domain, which in the immunoglobulin heavy chain and TCR beta chain, is created by the junction between the variable, diversity, and joining gene segments. The CDR3 diversity is constrained by overrepresentation of gene segments and lack of N regions during the first trimester of gestation and then increases exponentially during ontogeny until it reaches adult levels months after birth. This process parallels, and may contribute to, the stepwise acquisition of the ability to respond to specific antigens. Recent studies indicate that maturation of the CDR3 repertoire is not accelerated by premature exposition to extrauterine antigen and thus appears to follow a strictly developmentally regulated program whose pacemaker(s) is still unknown.

The diversity of the third complementarity determining region of the Ig H chain is constrained by natural selection of immunoglobulin diversity (DH) sequence. To test the functional significance of this constraint in the context of thymus-dependent (TD) immune responses, we immunized BALB/c mice with WT or altered DH sequence with 2-phenyloxazolone-coupled chicken serum albumin (phOx-CSA). We chose this antigen because studies of the humoral immune response to the hapten phOx were instrumental in the development of the current theoretical framework on which our understanding of the forces driving TD responses is based. To allow direct comparison, we used the classic approach of generating monoclonal Ab (mAb) from various stages of the immune response to phOx to assess the effect of changing the sequence of the DH on clonal expansion, class switching and affinity maturation, which are hallmarks of TD responses. Compared to WT, TD-induced humoral IgM as well as IgG antibody production in the D-altered D-DFS and D-iD strains were significantly reduced. An increased prevalence of IgM producing hybridomas from late primary, secondary, and tertiary memory responses suggested either impaired class switch recombination (CSR) or impaired clonal expansion of class switched B cells with phOx reactivity. Neither of the D-altered strains demonstrated the restriction in the VH/VL repertoire, the elimination of VH1 family-encoded antibodies, the focusing of the distribution of CDR-H3 lengths, or the selection for the normally dominant Ox1 clonotype which all are hallmarks of the anti-phOx response in WT mice. These changes in clonal selection and expansion as well as class switch recombination indicate that the genetic constitution of the DH locus, which has been selected by evolution, can strongly influence the functional outcome of a TD humoral response.

The vast initial diversity of the antibody repertoire is generated centrally by means of a complex series of V (D) J gene rearrangement events, variation in the site of gene segment joining, and TdT catalyzed N- region addition. Although the diversity is great, close inspection has revealed distinct and unique characteristics in the antibody repertoires expressed by different B cell developmental subsets. In order to illustrate our approach to repertoire analysis, we present an in-depth comparison of V (D) J gene usage, hydrophobicity, length, DH reading frame, and amino acid usage between heavy chain repertoires expressed by immature, transitional, mature, memory IgD+, memory IgD-, and plasmacytes isolated from the blood of a single individual. Our results support the view that in both human and mouse the H chain repertoires expressed by individual, developmental B cell subsets appear to differ in sequence content. Sequencing of unsorted B cells from the blood is thus likely to yield an incomplete or compressed view of what is actually happening in the immune response of the individual. Our findings support the view that studies designed to correlate repertoire expression with diseases of immune function will likely require deep sequencing of B cells sorted by subset.