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Generating a broadly protective influenza vaccine is critical to global health. Understanding how immune memory influences influenza immunity is central to this goal. We undertook an in-depth study of the B cell response to the pandemic 2009 H1N1 vaccine over consecutive years. Analysis of monoclonal antibodies generated from vaccine-induced plasmablasts demonstrated that individuals with low preexisting serological titers to the vaccinating strain generated a broadly reactive, hemagglutinin (HA) stalk-biased response. Higher preexisting serum antibody levels correlated with a strain-specific HA head-dominated response. We demonstrate that this HA head immunodominance encompasses poor accessibility of the HA stalk epitopes. Further, we show polyreactivity of HA stalk-reactive antibodies that could cause counterselection of these cells. Thus, preexisting memory B cells against HA head epitopes predominate, inhibiting a broadly protective response against the HA stalk upon revaccination with similar strains. Consideration of influenza exposure history is critical for new vaccine strategies designed to elicit broadly neutralizing antibodies.

We characterized human monoclonal antibodies (MAbs) cloned from influenza virus-infected patients and from influenza vaccine recipients by complement-dependent lysis (CDL) assay. Most MAbs active in CDL were neutralizing, but not all neutralizing MAbs can mediate CDL. Two of the three stalk-specific neutralizing MAbs tested were able to mediate CDL and were more cross-reactive to temporally distant H1N1 strains than the conventional hemagglutination-inhibiting and neutralizing MAbs. One of the stalk-specific MAbs was subtype cross-reactive to H1 and H2 hemagglutinins, suggesting a role for stalk-specific antibodies in protection against influenza illness, especially by a novel viral subtype which can cause pandemics.

Racial differences in school readiness are a form of health disparity. By examining, from the perspective of low-income minority families participating in an Early Head Start study, community and policy environments as they shape and inform lived experiences, we identified several types of social and economic dislocation that undermine the efforts of parents to ready their children for school. The multiple dislocations of community triggered by housing and welfare reform and “urban renewal” are sources of stress for parents and children and affect the health and development of young children. Our findings suggest that racial differences in school readiness result not from race but from poverty and structural racism in American society.

Self-reactive B cells not controlled by receptor editing or clonal deletion may become anergic. We report that fully mature human B cells negative for surface IgM and retaining only IgD are autoreactive and functionally attenuated (referred to as naive IgD+IgM− B cells [BND]). These BND cells typically make up 2.5% of B cells in the peripheral blood, have antibody variable region genes in germline (unmutated) configuration, and, by all current measures, are fully mature. Analysis of 95 recombinant antibodies expressed from the variable genes of single BND cells demonstrated that they are predominantly autoreactive, binding to HEp-2 cell antigens and DNA. Upon B cell receptor cross-linkage, BND cells have a reduced capacity to mobilize intracellular calcium or phosphorylate tyrosines, demonstrating that they are anergic. However, intense stimulation causes BND cells to fully respond, suggesting that these cells could be the precursors of autoantibody secreting plasma cells in autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis. This is the first identification of a distinct mature human B cell subset that is naturally autoreactive and controlled by the tolerizing mechanism of functional anergy.

This article examines a state-sponsored hacienda invasion by indigenous people in Amazonian Ecuador in the context of neoliberal state restructuring and decentralization. Studies have demonstrated how neoliberal reforms limit the delivery of social services by the state, but in the case examined, the municipal government offered increased access to basic infrastructural and social services to residents of the new community and encouraged the land invasion. This may indicate shifts in neoliberal policy in parts of Ecuador, where decentralization is accompanied by promises of enhanced state services. I argue, however, that Ecuador's changing neoliberal agenda may provide new mechanisms for state control in Amazonia while reinforcing enduring racist ideologies of modernizing nationalism.

The B cell–specific enzyme activation-induced cytidine deaminase (AID) has been shown to be essential for isotype switching and affinity maturation of antibody genes during the immune response. Conversely, AID activity has also been linked to autoimmunity and tumorigenesis. Determining how AID expression is regulated in vivo is therefore central to understanding its role in health and disease. Here we use phylogenetic footprinting and high-resolution histone acetylation mapping to accurately demarcate AID gene regulatory boundaries. Based on this strategy, we identify a novel, positive regulatory element required for AID transcription. Furthermore, we generate two AID indicator mouse strains using bacterial artificial chromosomes that faithfully recapitulate endogenous AID expression. The first strain uses a green fluorescent protein reporter to identify B cells that actively express AID during the immune response. In the second strain, AID transcription affects the permanent expression of a yellow fluorescent protein reporter in post–germinal center and terminally differentiated lymphocytes. We demonstrate the usefulness of these novel strains by resolving recent contradictory observations on AID expression during B cell ontogeny.

SummaryImmunoglobulin A (IgA) is prominently secreted at mucosal surfaces and coats a fraction of the intestinal microbiota. However, the commensal bacteria bound by IgA are poorly characterized and the type of humoral immunity they elicit remains elusive. We used bacterial flow cytometry coupled with 16S rRNA gene sequencing (IgA-Seq) in murine models of immunodeficiency to identify IgA-bound bacteria and elucidate mechanisms of commensal IgA targeting. We found that residence in the small intestine, rather than bacterial identity, dictated induction of specific IgA. Most commensals elicited strong T-independent (TI) responses that originated from the orphan B1b lineage and from B2 cells, but excluded natural antibacterial B1a specificities. Atypical commensals including segmented filamentous bacteria and Mucispirillum evaded TI responses but elicited T-dependent IgA. These data demonstrate exquisite targeting of distinct commensal bacteria by multiple layers of humoral immunity and reveal a specialized function of the B1b lineage in TI mucosal IgA responses.

In vivo protection by antimicrobial neutralizing Abs can require the contribution of effector functions mediated by Fc-Fcγ receptor (Fc-FcγR) interactions for optimal efficacy. In influenza, broadly neutralizing anti-hemagglutinin (anti-HA) stalk mAbs require Fc-FcγR interactions to mediate in vivo protection, but strain-specific anti-HA head mAbs do not. Whether this rule applies only to anti-stalk Abs or is applicable to any broadly neutralizing Ab (bNAb) against influenza is unknown. Here, we characterized the contribution of Fc-FcγR interactions during in vivo protection for a panel of 13 anti-HA mAbs, including bNAbs and non-neutralizing Abs, against both the stalk and head domains. All classes of broadly binding anti-HA mAbs required Fc-FcγR interactions to provide protection in vivo, including those mAbs that bind the HA head and those that do not neutralize virus in vitro. Further, a broadly neutralizing anti-neuraminidase (anti-NA) mAb also required FcγRs to provide protection in vivo, but a strain-specific anti-NA mAb did not. Thus, these findings suggest that the breadth of reactivity of anti-influenza Abs, regardless of their epitope, necessitates interactions with FcγRs on effector cell populations to mediate in vivo protection. These findings will guide the design of antiviral Ab therapeutics and inform vaccine design to elicit Abs with optimal binding properties and effector functions.

The B-cell immune response is a remarkable evolutionary system found in jawed vertebrates. B-cell receptors, the membrane-bound form of antibodies, are capable of evolving high affinity to almost any foreign protein. High germline diversity and rapid evolution upon encounter with antigen explain the general adaptability of B-cell populations, but the dynamics of repertoires are less well understood. These dynamics are scientifically and clinically important. After highlighting the remarkable characteristics of naive and experienced B-cell repertoires, especially biased usage of genes encoding the B-cell receptors, we contrast methods of sequence analysis and their attempts to explain patterns of B-cell evolution. These phylogenetic approaches are currently unlinked to explicit models of B-cell competition, which analyse repertoire evolution at the level of phenotype, the affinities and specificities to particular antigenic sites. The models, in turn, suggest how chance, infection history and other factors contribute to different patterns of immunodominance and protection between people. Challenges in rational vaccine design, specifically vaccines to induce broadly neutralizing antibodies to HIV, underscore critical gaps in our understanding of B cells' evolutionary and ecological dynamics.

Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus.

Objective. While rheumatoid arthritis (RA) is considered a prototypical autoimmune disease, the specific roles of B-cells in RA pathogenesis is not fully delineated. Methods. We performed microarray expression profiling of peripheral blood B-cells from RA patients and controls. Data were analysed using differential gene expression analysis and ‘gene networking’ analysis (characterizing clusters of functionally inter-relelated genes) to identify both regulatory genes and the pathways in which they participate. Results were confirmed by quantitative real-time polymerase chain reaction and by measuring the levels of 10 serum cytokines involved in the pathways identified. Results. Genes regulating and effecting the cell-cycle, proliferation, apoptosis, autoimmunity, cytokine networks, angiogenesis and neuro-immune regulation were differentially expressed in RA B-cells. Moreover, the serum levels of several soluble factors that modulate these pathways, including IL-1b, IL-5, IL-6, IL-10, IL-12p40, IL-17 and VEGF were significantly increased in this cohort of RA patients. Conclusions. These results outline aspects of the multifaceted role B-cells play in RA pathogenesis in which immune dysregulation in RA modulates B-cell biology and thereby contributes to the induction and perpetuation of a pathogenic humoral immune response.

It is believed that immunoglobulin-variable region gene (IgV) somatic hypermutation (SHM) is initiated by activation-induced cytidine deaminase (AID) upon deamination of cytidine to deoxyuracil. Patch-excision repair of these lesions involving error prone DNA polymerases such as polη causes mutations at all base positions. If not repaired, the deaminated nucleotides on the coding and noncoding strands result in C-to-T and G-to-A exchanges, respectively. Herein it is reported that IgV gene evolution has been considerably influenced by the need to accommodate extensive C deaminations and the resulting accumulation of C-to-T and G-to-A exchanges. Although seemingly counterintuitive, the precise placement of C and G nucleotides causes most C-to-T and G-to-A mutations to be silent or conservative. We hypothesize that without intricate positioning of C and G nucleotides the efficiency of affinity maturation would be significantly reduced due to a dominance of replacements caused by C and G transition mutations. The complexity of these evolved biases in codon use are compounded by the precise concomitant hotspot/coldspot targeting of AID activity and Polη errors to maximize SHM in the CDRs and minimize mutations in the FWRs.

Functionally impaired B cells play an important role in the pathogenesis of Sjögren's syndrome (SS). The aim of the study was to investigate the apoptosis susceptibility of peripheral blood B cells from patients with SS and the impact of B cell activating factor (BAFF) on the apoptosis capability of these cells in correlation with IgG production. Peripheral blood B cells were isolated and stained for apoptosis markers (Bax, Bcl-2) and members of the TNF-R superfamily, CD95 and CD40. The apoptosis frequency of cells bearing these markers were assessed. Also, the apoptosis capability of cultured B-lymphocytes was investigated in medium alone, with anti-CD95 or with soluble BAFF. Quantitative ELISA was performed to detect plasma levels of sBAFF. Furthermore, the level of circulating B-cell cytokines was measured. BAFF levels were compared between patients with normal and elevated IgG levels. In SS, Bcl-2 positive B cell counts were significantly higher then in controls, also in this population the apoptosis frequency was reduced. Apoptosis within Bax+ and CD40+ B cells were significantly decreased in patients. BAFF induced a significant antiapoptotic effect in SS; also this effect was clearly evident in B cells from SS with hypergammaglobulinaemia. Plasma BAFF levels were significantly higher in SS, mostly in patients with hypergammaglobulinaemia. Plasma B-cell cytokines were raised in SS. In Sjögren's syndrome B cells, a general antiapoptotic tendency might lead to prolonged B-cell survival driven at least partly by elevated levels of BAFF and supposedly by B-cell cytokines. Also, the exaggerated BAFF stimulation might lead to excessive immunoglobulin production. The B-cell apoptosis defects, the increased BAFF levels-correlating with hypergammaglobulinaemia-together with the raised B-cell cytokine levels indicates the disturbed B-cell biology in the disease.

Current paradigms of peripheral B cell selection suggest that autoreactive B cells are controlled by clonal deletion, anergy, and developmental arrest. We report that changes to the human antibody repertoire likely resulting from these mechanisms both for a well-characterized autoreactivity from antibodies encoded by the V(H)4-34 gene and for other hallmarks of an autoreactive repertoire are apparent mainly for class-switched B cells and not for IgM germinal center, IgM memory, or IgM plasma cells. Other possible indicators of autoreactivity found selected with immunoglobulin class include J(H)6 gene segment usage, increased frequency of B cells with long third hypervariable regions, and distal J(kappa) gene segment bias. Of particular interest is the finding that B cells with these same characteristics are selected into the lineage of B cells that have undergone the unusual class switch from constant region C mu to C delta (C delta-CS). The C delta-CS population also displays an increased frequency of charged amino acids localized to the complementarity-determining regions, further suggesting autoreactivity, and evidence is presented that these B cells had undergone extensive receptor editing. Thus, the C delta-CS lineage may be a "sink" for B cells harboring autoreactive specificities in normal humans. A model for a new tolerizing mechanism that could account for the C delta-CS lineage is presented.

This article is based on an in-depth qualitative study of efforts by an Early Head Start program to include fathers in program activities with the aim of supporting and strengthening their involvement in their children's lives. Since this program employs a home-visiting model of service delivery, our focus is on the work and experiences of home-visiting staff. Our key findings concern the evolutionary development of program thinking and practice regarding fathers and father involvement; barriers or challenges to father involvement; and successful strategies for engaging fathers on both a programmatic and an individual family basis. Unlike most previous research on father involvement, this study provides a close-up look at staff experiences as they attempt to involve fathers in programming for infants and toddlers. Although significant barriers to father involvement were identified, certain approaches and strategies proved most effective in encouraging father engagement in key program components and with their youngest children. Language: en

In this issue of Blood , [Bhoj et al][1] explore the longevity of plasma cells (PCs) and humoral immunity in the absence of B cells. This is done by studying subjects experiencing B-cell aplasia after CD19-directed chimeric antigen receptor (CAR) therapy.[1][2] Rapid immune protection against

Endocrine-disrupting chemicals (EDCs) occur in reclaimed water (RW), which may serve as an exposure source for humans. The presence of EDCs in RW used to irrigate turf and in nearby water-retention ponds was determined. In addition, the total dislodgeable mass of each EDC was determined after irrigation (using RW) to simulate exposure of a 3-year-child playing in turf grass recently irrigated with RW. Five EDCs (estrone, 17β-estradiol, 17α-ethynylestradiol, bisphenol A, and 4-n-nonylphenol) were quantified in 28 samples of RWs (wastewater-treatment plant effluents) and 88 samples from residential surface water-retention ponds. St. Augustine variety of turf grass was irrigated with spiked RW to study dislodgement of the five EDCs overtime using a drag-sled method. Grass clippings were analyzed to relate masses of EDC on grass with masses dislodged. EDCs were detected in both RW and ponds at ng/L concentrations. Maximum EDC masses were dislodged immediately after irrigation. Dislodged masses of estrone and 17β-estradiol are two separate EDCs, 17β-estradiol and 17α-ethynylestradiol decreased rapidly and were lower than detection limits 4 h after application. Dislodged bisphenol-A and nonylphenol decreased more slowly but were not detected 6 h after application. Avoiding contact with recently irrigated turf grass should decrease the risks of exposure to these EDCs.

The emergence of pandemic influenza viruses poses a major public health threat. Therefore, there is a need for a vaccine that can induce broadly cross-reactive antibodies that protect against seasonal as well as pandemic influenza strains. Human broadly neutralizing antibodies directed against highly conserved epitopes in the stem region of influenza virus HA have been recently characterized. However, it remains unknown what the baseline levels are of antibodies and memory B cells that are directed against these conserved epitopes. More importantly, it is also not known to what extent anti-HA stem B-cell responses get boosted in humans after seasonal influenza vaccination. In this study, we have addressed these two outstanding questions. Our data show that: (i) antibodies and memory B cells directed against the conserved HA stem region are prevalent in humans, but their levels are much lower than B-cell responses directed to variable epitopes in the HA head; (ii) current seasonal influenza vaccines are efficient in inducing B-cell responses to the variable HA head region but they fail to boost responses to the conserved HA stem region; and (iii) in striking contrast, immunization of humans with the avian influenza virus H5N1 induced broadly cross-reactive HA stem-specific antibodies. Taken together, our findings provide a potential vaccination strategy where heterologous influenza immunization could be used for increasing the levels of broadly neutralizing antibodies and for priming the human population to respond quickly to emerging pandemic influenza threats.

Coeliac disease (CD), an enteropathy caused by cereal gluten ingestion, is characterized by CD4+ T cells recognizing deamidated gluten and by antibodies reactive to gluten or the self-antigen transglutaminase 2 (TG2). TG2-specific immunoglobulin A (IgA) of plasma cells (PCs) from CD lesions have limited somatic hypermutation (SHM). Here we report that gluten-specific IgA of lesion-resident PCs share this feature. Monoclonal antibodies were expression cloned from single PCs of patients either isolated from cultures with reactivity to complex deamidated gluten antigen or by sorting with gluten peptide tetramers. Typically, the antibodies bind gluten peptides related to T-cell epitopes and many have higher reactivity to deamidated peptides. There is restricted VH and VL combination and usage among the antibodies. Limited SHM suggests that a common factor governs the mutation level in PCs producing TG2- and gluten-specific IgA. The antibodies have potential use for diagnosis of CD and for detection of gluten., Coeliac disease is characterized by an inappropriate immune response to dietary gluten proteins, involving the production of antibodies reactive to gluten. Here, the authors study the intestinal antibody response against gluten and show that gluten-specific antibodies have a low degree of somatic hypermutations.

BackgroundThe recommendation by the American Society of Transplantation for annual trivalent inactivated influenza vaccination greater than 3 to 6 months post-kidney transplantation provides a unique opportunity to test the in vivo impact of immunosuppression on recall T- and B-cell responses to influenza vaccination.MethodsThis study took advantage of recent breakthroughs in the single-cell quantification of human peripheral blood B-cell responses to prospectively evaluate both B- and T-cell responses to the seasonal (2010 and 2011) influenza vaccine in 23 stable renal transplant recipients and 22 healthy controls.Results and conclusionThe results demonstrate that the early B-cell response to influenza vaccination, quantified by the frequency of influenza-specific antibody-secreting cells (ASC) in peripheral blood, was significantly reduced in stable transplant recipients compared to healthy controls. The magnitude of the seroresponse and the rate of seroconversion were also blunted. The influenza-specific interferon-gamma (IFNγ) T-cell response was significantly reduced in transplant recipients; however, there was no correlation between the magnitude of the influenza-specific IgG ASC and IFNγ responses. The induction of memory T- and B-cell responses to influenza vaccination supports the recommendation to vaccinate while the blunted responses demonstrate the efficacy of immunosuppression in controlling memory responses individual transplant recipients.

Background: We had demonstrated immunocompetent cell infiltration and oligoclonal immune response in human cerebral cavernous malformation (CCM) lesions. However, T- and B- cell crosstalk, relationship between antibody and complement, and IgG repertoire in CCMs remain unknown. We hypothesize the synergetic role of T and B cells, antibody- and complement- mediated cytotoxicity, antigen-driven B cell clonal expansion, contributing to the CCM pathogenesis. Methods: This study enrolled a total of 10 patients with CCMs. B- and T- cells were immunostained in two excised human CCM lesion specimens. IgG and complement protein membrane attack complex (MAC) was co-localized in six other CCM lesions by immunofluorescence. Plasma cells in two CCM lesions were laser-microdissected, in which IgG heavy (H) and light (L) chain variable (V) region were amplified via nested PCR, cloned, sequenced, and aligned to a database IMGT®. The over-expressed IgGHV and IgGLV were used to construct the recombinant antibodies (r-abs). Results: In CCM lesions, T- and B- cells were clustered and co-localized, and plasma cells were clustered. IgG and MAC was co-localized in CCMs. IgG repertoire showed in situ B cell clonal expansion and ongoing somatic mutation. Four r-abs were constructed from clonally expanded plasma cells in CCM lesions. Conclusions: we showed the role of immunity in CCM pathogenesis via T- and B- cell crosstalk, antibody- and complement- mediated cytotoxicity, and clonal expansion. We gave the first demonstration that oligoclonal response is antigen-driven. The first synthesized r-abs will be used to identify disease relevant antigens. This would clarify the relationship of immune response to CCM lesion development, and may open novel therapeutic venues in CCMs.

The emergence of avian H7N9 viruses in humans in China has renewed concerns about influenza pandemics emerging from Asia. Vaccines are still the best countermeasure against emerging influenza virus infections, but the process from the identification of vaccine seed strains to the distribution of the final product can take several months. In the case of the 2009 H1N1 pandemic, a vaccine was not available before the first pandemic wave hit and therefore came too late to reduce influenza morbidity. H7 vaccines based on divergent isolates of the Eurasian and North American lineages have been tested in clinical trials, and seed strains and reagents are already available and can potentially be used initially to curtail influenza-induced disease until a more appropriately matched H7N9 vaccine is ready. In a challenge experiment in the mouse model, we assessed the efficacy of both inactivated virus and recombinant hemagglutinin vaccines made from seed strains that are divergent from H7N9 from each of the two major H7 lineages. Furthermore, we analyzed the cross-reactive responses of sera from human subjects vaccinated with heterologous North American and Eurasian lineage H7 vaccines to H7N9. Vaccinations with inactivated virus and recombinant hemagglutinin protein preparations from both lineages raised hemagglutination-inhibiting antibodies against H7N9 viruses and protected mice from stringent viral challenges. Similar cross-reactivity was observed in sera of human subjects from a clinical trial with a divergent H7 vaccine. Existing H7 vaccine candidates based on divergent strains could be used as a first line of defense against an H7N9 pandemic. In addition, this also suggests that H7N9 vaccines that are currently under development might be stockpiled and used for divergent avian H7 strains that emerge in the future. IMPORTANCE Sporadic human infections with H7N9 viruses started being reported in China in the early spring of 2013. Despite a significant drop in the number of infections during the summer months of 2013, an increased number of cases has already been reported for the 2013-2014 winter season. The high case fatality rate, the ability to bind to receptors in the human upper respiratory tract in combination with several family clusters, and the emergence of neuraminidase inhibitor-resistant variants that show no loss of pathogenicity and the ability to transmit in animal models have raised concerns about a potential pandemic and have spurred efforts to produce vaccine candidates. Here we show that antigen preparations from divergent H7 strains are able to induce protective immunity against H7N9 infection.

A chironomid midge, Cricotopus lebetis (Sublette) (Diptera: Chironomidae), was discovered attacking the apical meristems of Hydrilla verticillata (L.f. Royle) in Crystal River, Citrus Co., Florida in 1992. The larvae mine the stems of H. verticillata and cause basal branching and stunting of the plant. Temperature-dependent development, cold tolerance, and the potential distribution of the midge were investigated. The results of the temperature-dependent development study showed that optimal temperatures for larval development were between 20 and 30°C, and these data were used to construct a map of the potential number of generations per year of C. lebetis in Florida. Data from the cold tolerance study, in conjunction with historical weather data, were used to generate a predicted distribution of C. lebetis in the United States. A distribution was also predicted using an ecological niche modeling approach by characterizing the climate at locations where C. lebetis is known to occur and then finding other locations with similar climate. The distributions predicted using the two modeling approaches were not significantly different and suggested that much of the southeastern United States was climatically suitable for C. lebetis .

Class switch recombination (CSR) is a region-specific DNA recombination reaction that replaces one immunoglobulin heavy-chain constant region (Ch) gene with another. This enables a single variable (V) region gene to be used in conjunction with different downstream Ch genes, each having a unique biological activity. The molecular mechanisms that mediate CSR have not been defined, but activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is required for this reaction. Here we report that the Nijmegen breakage syndrome protein (Nbs1) and phosphorylated H2A histone family member X (gamma-H2AX, also known as gamma-H2afx), which facilitate DNA double-strand break (DSB) repair, form nuclear foci at the Ch region in the G1 phase of the cell cycle in cells undergoing CSR, and that switching is impaired in H2AX-/- mice. Localization of Nbs1 and gamma-H2AX to the Igh locus during CSR is dependent on AID. In addition, AID is required for induction of switch region (S mu)-specific DNA lesions that precede CSR. These results place AID function upstream of the DNA modifications that initiate CSR.

Assembly of T cell receptor (TCR)alpha/beta genes by variable/diversity/joining (V[D]J) rearrangement is an ordered process beginning with recombination activating gene (RAG) expression and TCRbeta recombination in CD4(-)CD8(-)CD25(+) thymocytes. In these cells, TCRbeta expression leads to clonal expansion, RAG downregulation, and TCRbeta allelic exclusion. At the subsequent CD4(+)CD8(+) stage, RAG expression is reinduced and V(D)J recombination is initiated at the TCRalpha locus. This second wave of RAG expression is terminated upon expression of a positively selected alpha/beta TCR. To examine the physiologic role of the second wave of RAG expression, we analyzed mice that cannot reinduce RAG expression in CD4(+)CD8(+) T cells because the transgenic locus that directs RAG1 and RAG2 expression in these mice is missing a distal regulatory element essential for reinduction. In the absence of RAG reinduction we find normal numbers of CD4(+)CD8(+) cells but a 50-70% reduction in the number of mature CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes. TCRalpha rearrangement is restricted to the 5' end of the Jalpha cluster and there is little apparent secondary TCRalpha recombination. Comparison of the TCRalpha genes expressed in wild-type or mutant mice shows that 65% of all alpha/beta T cells carry receptors that are normally assembled by secondary TCRalpha rearrangement. We conclude that RAG reinduction in CD4(+)CD8(+) thymocytes is not required for initial TCRalpha recombination but is essential for secondary TCRalpha recombination and that the majority of TCRalpha chains expressed in mature T cells are products of secondary recombination.

In this issue of Blood , Isnardi and colleagues describe a phenotypically distinct population of autoreactive B cells that have become functionally limited upon stimulation, or “anergic.”[1][1] Importantly, these cells are found at increased frequency in some rheumatoid arthritis patients and in

Summary: The sequence analysis of Ig variable region genes transcribed within different B-cell subpopulations from human tonsil led us to identify a rare DNA sequence modification event consisting of Lip insertions and/or deletions (I/D). Although these events were previously reported, they had never been formally associated with the somatic hypermutation process. I/D events share with more convemtional somatic hypermutation events their localization within hypervariable regions and, most particularly, within DNA motifs known to be mutational hoc spots, Repetitive; DNA tracts or DNA elements capable of forming DNA loop intermediates seem to be the preferred substrate for I/D to occur. These characteristics suggest a model for somatic hypermutation reminiscent of the “polymerase slippage” model involved in replication and repair mutations in prokaryotes, yeast, and mammals.

During a germinal center reaction, random mutations are introduced into immunoglobulin V genes to increase the affinity of antibody molecules and to further diversify the B cell repertoire. Antigen-directed selection of B cell clones that generate high affinity surface Ig results in the affinity maturation of the antibody response. The mutations of Ig genes are typically basepair substitutions, although DNA insertions and deletions have been reported to occur at a low frequency. In this study, we describe five insertion and four deletion events in otherwise somatically mutated VH gene cDNA molecules. Two of these insertions and all four deletions were obtained through the sequencing of 395 cDNA clones (∼110,000 nucleotides) from CD38+IgD− germinal center, and CD38−IgD− memory B cell populations from a single human tonsil. No germline genes that could have encoded these six cDNA clones were found after an extensive characterization of the genomic VH4 repertoire of the tonsil donor. These six insertions or deletions and three additional insertion events isolated from other sources occurred as triplets or multiples thereof, leaving the transcripts in frame. Additionally, 8 of 9 of these events occurred in the CDR1 or CDR2, following a pattern consistent with selection, and making it unlikely that these events were artifacts of the experimental system. The lack of similar instances in unmutated IgD+CD38− follicular mantle cDNA clones statistically associates these events to the somatic hypermutation process (P = 0.014). Close scrutiny of the 9 insertion/deletion events reported here, and of 25 additional insertions or deletions collected from the literature, suggest that secondary structural elements in the DNA sequences capable of producing loop intermediates may be a prerequisite in most instances. Furthermore, these events most frequently involve sequence motifs resembling known intrinsic hotspots of somatic hypermutation. These insertion/deletion events are consistent with models of somatic hypermutation involving an unstable polymerase enzyme complex lacking proofreading capabilities, and suggest a downregulation or alteration of DNA repair at the V locus during the hypermutation process.

Influenza remains one of the most important causes of respiratory infection despite the widespread availability of vaccines. As effective vaccines against influenza principally rely on the induction of strain-specific neutralizing antibodies for their protective efficacy, drifted escape mutants and genetically reassortant pandemic strains can rapidly overcome them. Several groups have recently described cross-reactive influenza antibodies in humans, some of which bind to the conserved hemagglutinin stem. If such antibodies could be consistently induced at high levels by vaccines, they might protect against both seasonal and pandemic influenza strains. Here we discuss the humoral responses to influenza infection and vaccination, with particular reference to the pandemic H1N1 2009 virus and induction of broadly cross-reactive stem-binding antibodies. Having shown that cross-reactive antibodies are preferentially induced by a pandemic hemagglutinin, the challenge is now to design a vaccine that applies these principles to the induction of heterosubtypic immunity.

The human antibody repertoire is one of the most important defenses against infectious disease, and the development of vaccines has enabled the conferral of targeted protection to specific pathogens. However, there are many challenges to measuring and analyzing the immunoglobulin sequence repertoire, such as the fact that each B cell contains a distinct antibody sequence encoded in its genome, that the antibody repertoire is not constant but changes over time, and the high similarity between antibody sequences. We have addressed this challenge by using high-throughput long read sequencing to perform immunogenomic characterization of expressed human antibody repertoires in the context of influenza vaccination. Informatic analysis of 5 million antibody heavy chain sequences from healthy individuals allowed us to perform global characterizations of isotype distributions, determine the lineage structure of the repertoire and measure age and antigen related mutational activity. Our analysis of the clonal structure and mutational distribution of individuals’ repertoires shows that elderly subjects have a decreased number of lineages but an increased pre-vaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. We have thus shown that global analysis of the immune system’s clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.

In 2009, a novel H1N1 influenza A virus (2009 pH1N1) emerged and caused a pandemic. A human monoclonal antibody (hMAb; EM4C04), highly specific for the 2009 pH1N1 virus hemagglutinin (HA), was isolated from a severely ill 2009 pH1N1 virus-infected patient. We postulated that under immune pressure with EM4C04, the 2009 pH1N1 virus would undergo antigenic drift and mutate at sites that would identify the antibody binding site. To do so, we infected MDCK cells in the presence of EM4C04 and generated 11 escape mutants, displaying 7 distinct amino acid substitutions in the HA. Six substitutions greatly reduced MAb binding (K123N, D131E, K133T, G134S, K157N, and G158E). Residues 131, 133, and 134 are contiguous with residues 157 and 158 in the globular domain structure and contribute to a novel pH1N1 antibody epitope. One mutation near the receptor binding site, S186P, increased the binding affinity of the HA to the receptor. 186P and 131E are present in the highly virulent 1918 virus HA and were recently identified as virulence determinants in a mouse-passaged pH1N1 virus. We found that pH1N1 escape variants expressing these substitutions enhanced replication and lethality in mice compared to wild-type 2009 pH1N1 virus. The increased virulence of these viruses was associated with an increased affinity for α2,3 sialic acid receptors. Our study demonstrates that antibody pressure by an hMAb targeting a novel epitope in the Sa region of 2009 pH1N1 HA is able to inadvertently drive the development of a more virulent virus with altered receptor binding properties. This broadens our understanding of antigenic drift., IMPORTANCE Influenza viruses accumulate amino acid substitutions to evade the antibody response in a process known as antigenic drift, making it necessary to vaccinate against influenza annually. Mapping human monoclonal antibody (hMAb) epitopes is a necessary step towards understanding antigenic drift in humans. We defined the specificity of an hMAb that specifically targeted the 2009 pH1N1 virus and describe a novel epitope. In addition, we identified a previously unappreciated potential for antibody escape to enhance the pathogenicity of a virus. The escape mutation that we identified with in vitro immune pressure was independently reported by other investigators using in vivo selection in nonimmune mice. Although in vitro generation of escape mutants is unlikely to recapitulate antigenic drift in its entirety, the data demonstrate that pressure by a human monoclonal antibody targeting a novel epitope in the hemagglutinin of the 2009 pandemic H1N1 virus can inadvertently drive the development of escape mutants, of which a subset have increased virulence and altered receptor binding properties.

We have previously shown that broadly neutralizing antibodies reactive to the conserved stem region of the influenza virus hemagglutinin (HA) were generated in people infected with the 2009 pandemic H1N1 strain. Such antibodies are rarely seen in humans following infection or vaccination with seasonal influenza virus strains. However, the important question remained whether the inactivated 2009 pandemic H1N1 vaccine, like the infection, could also induce these broadly neutralizing antibodies. To address this question, we analyzed B-cell responses in 24 healthy adults immunized with the pandemic vaccine in 2009. In all cases, we found a rapid, predominantly IgG-producing vaccine-specific plasmablast response. Strikingly, the majority (25 of 28) of HA-specific monoclonal antibodies generated from the vaccine-specific plasmablasts neutralized more than one influenza strain and exhibited high levels of somatic hypermutation, suggesting they were derived from recall of B-cell memory. Indeed, memory B cells that recognized the 2009 pandemic H1N1 HA were detectable before vaccination not only in this cohort but also in samples obtained before the emergence of the pandemic strain. Three antibodies demonstrated extremely broad cross-reactivity and were found to bind the HA stem. Furthermore, one stem-reactive antibody recognized not only H1 and H5, but also H3 influenza viruses. This exceptional cross-reactivity indicates that antibodies capable of neutralizing most influenza subtypes might indeed be elicited by vaccination. The challenge now is to improve upon this result and design influenza vaccines that can elicit these broadly cross-reactive antibodies at sufficiently high levels to provide heterosubtypic protection.

Humoral immune responses are thought to play a major role in dengue virus-induced immunopathology; however, little is known about the plasmablasts producing these antibodies during an ongoing infection. Herein we present an analysis of plasmablast responses in patients with acute dengue virus infection. We found very potent plasmablast responses that often increased more than 1,000-fold over the baseline levels in healthy volunteers. In many patients, these responses made up as much 30% of the peripheral lymphocyte population. These responses were largely dengue virus specific and almost entirely made up of IgG-secreting cells, and plasmablasts reached very high numbers at a time after fever onset that generally coincided with the window where the most serious dengue virus-induced pathology is observed. The presence of these large, rapid, and virus-specific plasmablast responses raises the question as to whether these cells might have a role in dengue immunopathology during the ongoing infection. These findings clearly illustrate the need for a detailed understanding of the repertoire and specificity of the antibodies that these plasmablasts produce.

Triclosan (TCS) is an antimicrobial compound commonly found in biosolids. Thus, plants grown in biosolids-amended soil may be exposed to TCS. We evaluated the plant toxicity and accumulation potential of biosolids-borne TCS in two vegetables (lettuce and radish) and a pasture grass (bahia grass). Vegetables were grown in growth chambers and grass in a greenhouse. Biosolids-amended soil had TCS concentrations of 0.99, 5.9, and 11 mg/kg amended soil. These TCS concentrations represent typical biosolids containing concentrations of 16 mg TCS/kg applied at agronomic rates for 6 to 70 consecutive years, assuming no TCS loss. Plant yields (dry wt) were not reduced at any TCS concentration and the no observed effect concentration was 11 mg TCS/kg soil for all plants. Significantly greater TCS accumulated in the below-ground biomass than in the above-ground biomass. The average bioaccumulation factors (BAFs) were 0.43 ± 0.38 in radish root, 0.04 ± 0.04 in lettuce leaves, 0.004 ± 0.002 in radish leaves, and

The major goal in vaccination is establishment of long-term, prophylactic humoral memory to a pathogen. Two major components to long-lived humoral memory are plasma cells for the production of specific immunoglobulin and memory B cells that survey for their specific antigen in the periphery for later affinity maturation, proliferation, and differentiation. The study of human B cell memory has been aided by the discovery of a general marker for B cell memory, expression of CD27; however, new data suggests the existence of CD27- memory B cells as well. These recently described non-canonical memory populations have increasingly pointed to the heterogeneity of the memory compartment. The novel B memory subsets in humans appear to have unique origins, localization, and functions compared to what was considered to be a classical memory B cell. In this article, we review the known B cell memory subsets, the establishment of B cell memory in vaccination and infection, and how understanding these newly described subsets can inform vaccine design and disease treatment.

Since its first administration in the 1940s, the influenza vaccine has provided tremendous relief against influenza infections. However, time has revealed the ultimate limit of the vaccine and the call for its reinvention has now come, just as we are beginning to appreciate the antibody immune responses vital in preventing infections. New strategies to design the influenza vaccine rely on selectively inducing broadly neutralizing antibodies that are specific for highly conserved viral epitopes. Such approaches take us away from the limited range of protection provided by current seasonal influenza vaccines and towards a future with a pan-influenza vaccine capable of providing universal strain coverage.

Monoclonal antibody technology has undergone rapid and innovative reinvention over the last 30 years. Application of these technologies to human samples revealed valuable therapeutic and experimental insights. These technologies, each with their own benefits and flaws, have proven indispensable for immunological research and in our fight to provide new treatments and improved vaccines for infectious disease.

During seasonal influenza epidemics, disease burden is shouldered predominantly by the very young and the elderly. Elderly individuals are particularly affected, in part because vaccine efficacy wanes with age. This has been linked to a reduced ability to induce a robust serum antibody response. Here, we show that this is due to reduced quantities of vaccine-specific antibodies, rather than a lack of antibody avidity or affinity. We measured levels of vaccine-specific plasmablasts by ELISPOT 1 week after immunization of young and elderly adults with inactivated seasonal influenza vaccine. Plasmablast-derived polyclonal antibodies (PPAbs) were generated from bulk-cultured B cells, while recombinant monoclonal antibodies (re-mAbs) were produced from single plasmablasts. The frequency of vaccine-specific plasmablasts and the concentration of PPAbs were lower in the elderly than in young adults, whereas the yields of secreted IgG per plasmablast were not different. Differences were not detected in the overall vaccine-specific avidity or affinity of PPAbs and re-mAbs between the 2 age groups. In contrast, reactivity of the antibodies induced by the inactivated seasonal influenza vaccine toward the 2009 pandemic H1N1 virus, which was not present in the vaccine, was higher in the elderly than in the young. These results indicate that the inferior antibody response to influenza vaccination in the elderly is primarily due to reduced quantities of vaccine-specific antibodies. They also suggest that exposure history affects the cross-reactivity of vaccination-induced antibodies.

The gut mucosal surface is efficiently protected by Abs, and this site represents one of the richest compartments of Ab-secreting cells in the body. A simple and effective method to generate Ag-specific human monoclonal Abs (hmAbs) from such cells is lacking. In this paper, we describe a method to generate hmAbs from single Ag-specific IgA- or IgM-secreting cells of the intestinal mucosa. We found that CD138-positive plasma cells from the duodenum expressed surface IgA or IgM. Using eGFP-labeled virus-like particles, we harnessed the surface Ig expression to detect rotavirus-specific plasma cells at low frequency (0.03–0.35%) in 9 of 10 adult subjects. Single cells were isolated by FACS, and as they were viable, further testing of secreted Abs by ELISPOT and ELISA indicated a highly specific selection procedure. Ab genes from single cells of three donors were cloned, sequenced, and expressed as recombinant hmAbs. Of 26 cloned H chain Ab genes, 22 were IgA and 4 were IgM. The genes were highly mutated, and there was an overrepresentation of the VH4 family. Of 10 expressed hmAbs, 8 were rotavirus-reactive (6 with Kd < 1 × 10−10). Importantly, our method allows generation of hmAbs from cells implicated in the protection of mucosal surfaces, and it can potentially be used in passive vaccination efforts and for discovery of epitopes directly relevant to human immunity.

During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV disfavours homotypic bivalent antibody binding1–3. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development4, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients5 with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV.

The development of B-cell lymphomas is an intricate interplay among various pathogenic factors, leading to a multi-step process, encompassing various stages of B-cell maturation. Besides genetic abnormalities, a variety of environmental and microbial factors, as well as disproportional immune-regulatory processes lead to the malignant transformation. Yet, little is known about the exact chain of events, which lead from the physiological polyclonal B-cell activation as a response to exogenous antigens through oligoclonality to a monoclonal, uncontrolled, malignant B-cell proliferation. The aim of the present review was to summarize the potential harmful steps in the development of B-cell lymphomas, according to conventional and novel theories, and to depict therapeutic regimens presently in use as well as to envision future drug developments, beneficial in the battle against this lymphoid malignancy.

Methicillin-resistant Staphylococcus aureus (MRSA) has become increasingly prevalent in hand infections. Traditionally, the empiric treatment of hand infections has involved beta-lactam antibiotics, which are ineffective against MRSA. Centers for Disease Control recommends empiric coverage of MRSA infections if the local rate of MRSA exceeds 10% to 15%. A retrospective review was performed on all patients admitted for community-acquired soft tissue infections of the hand between 2004 and 2007 at a single institution. The overall incidence of MRSA was 60%. The incidence of MRSA in healthy adults was 64%, healthy pediatric patients was 100%, immunocompromised patients was 45%, and diabetic patients was 20%. The current rates of MRSA would imply that all patients presenting with hand infections should be treated empirically for MRSA. Linezolid is the only oral antibiotic approved by the Food and Drug Administration for treating MRSA, but many studies have reported that trimethoprim-sulfamethoxazole is an effective antibiotic for outpatient treatment of MRSA.