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1.  Targeting antigens through blood dendritic cell antigen 2 (BDCA2) on plasmacytoid dendritic cells promotes immunologic tolerance1 
The C-type lectin receptor blood dendritic cell antigen 2 (BDCA2) is expressed exclusively on human plasmacytoid dendritic cells (pDCs) and plays a role in Ag capture, internalization and presentation to T cells. We used transgenic mice that express human BDCA2 and anti-BDCA2 mAbs to deliver Ags directly to BDCA2 on pDCs in vivo. Targeting Ag to pDCs in this manner resulted in significant suppression of Ag-specific CD4+ T cell and Ab responses upon secondary exposure to Ag in the presence of adjuvant. Suppression of Ab responses required both a decrease in effector CD4+ T cells and preservation of Foxp3+ regulatory T cells (Tregs). Reduction in Treg cell numbers following Ag delivery to BDCA2 restored both CD4+ T cell activation and Ab responses, demonstrating that Tregs were required for the observed tolerance. Our results demonstrate that Ag delivery to pDCs through BDCA2 is an effective method to induce immunological tolerance, which may be useful for treating autoimmune diseases or to inhibit unwanted Ab responses.
doi:10.4049/jimmunol.1303259
PMCID: PMC4048786  PMID: 24829416
2.  Systematic Comparison of Gene Expression between Murine Memory and Naive B Cells Demonstrates That Memory B Cells Have Unique Signaling Capabilities1 
Memory B cells play essential roles in the maintenance of long-term immunity and may be important in the pathogenesis of autoimmune disease, but how these cells are distinguished from their naive precursors is poorly understood. To address this, it would be important to understand how gene expression differs between memory and naive B cells to elucidate memory-specific functions. Using model systems that help overcome the lack of murine memory-specific markers and the low frequency of Agspecific memory and naive cells, we undertook a global comparison of gene expression between memory B cells and their naive precursors. We identified genes with differential expression and confirmed the differential expression of many of these by quantitative RT-PCR and of some of these at the protein level. Our initial analysis revealed differential expression patterns of genes that regulate signaling. Memory B cells have increased expression of genes important in regulating adenosine signaling and in modulating cAMP responses. Furthermore, memory B cells up-regulate receptors that are essential for embryonic stem cell self-renewal. We further demonstrate that one of these, leukemia inhibitory factor receptor, can initiate functional signaling in memory B cells whereas it does not in naive B cells. Thus, memory and naive B cells are intrinsically wired to signal differently from one another and express a functional signaling pathway that is known to maintain stem cells in other lineages.
PMCID: PMC4437802  PMID: 18566367
3.  Integrating B cell lineage information into statistical tests for detecting selection in Ig sequences1 
Detecting selection in B cell immunoglobulin (Ig) sequences is critical to understanding affinity maturation, and can provide insights into antigen-driven selection in normal and pathologic immune responses. The most common sequence-based methods for detecting selection analyze the ratio of replacement (R) and silent (S) mutations using a binomial statistical analysis. However, these approaches have been criticized for low sensitivity. An alternative method is based on the analysis of lineage trees constructed from sets of clonally-related Ig sequences. Several tree shape measures have been proposed as indicators of selection that can be statistically compared across cohorts. However, we show that tree shape analysis is confounded by underlying experimental factors that are difficult to control for in practice, including the sequencing depth and number of generations in each clone. Thus, though lineage tree shapes may reflect selection, their analysis alone is an unreliable measure of in vivo selection. To usefully capture the information provided by lineage trees, we propose a new method that applies the binomial statistical method to mutations identified based on lineage tree structure. This hybrid method is able to detect selection with increased sensitivity in both simulated and experimental data sets. We anticipate that this approach will be especially useful in the analysis of large-scale Ig sequencing data sets generated by high-throughput sequencing technologies.
doi:10.4049/jimmunol.1301551
PMCID: PMC4363135  PMID: 24376267
4.  CD80 and PD-L2 define functionally distinct memory B cell subsets that are independent of antibody isotype 
Nature immunology  2014;15(7):631-637.
Memory B cells (MBCs) are long-lived sources of rapid, isotype-switched secondary antibody-forming cell (AFC) responses. Whether MBCs homogeneously retain the ability to self-renew and terminally differentiate or if these functions are compartmentalized into MBC subsets has been unclear. It was previously suggested that antibody isotype controls MBC differentiation upon restimulation. Here we demonstrate that subdividing MBCs based on expression of CD80 and PD-L2, independent of isotype, identified MBC subsets with distinct functional behaviors upon rechallenge. CD80+PD-L2+ MBCs differentiated rapidly into AFCs but did not generate germinal centers (GCs); conversely CD80−PD-L2− MBCs generated few early AFCs but robustly seeded GCs. Gene expression patterns of subsets support both the identity and function of these distinct MBC types. Hence, MBC differentiation and regeneration are compartmentalized.
doi:10.1038/ni.2914
PMCID: PMC4105703  PMID: 24880458
5.  Context-specific BAFF-R signaling by the NF-κB and PI3K pathways 
Cell reports  2013;5(4):10.1016/j.celrep.2013.10.022.
Summary
BAFF is a soluble factor required for B cell maturation and survival. BAFF-R signals via the non-canonical NF-κB pathway regulated by the TRAF3/NIK/IKK1 axis. We show that deletion of Ikk1 during early B cell development causes a partial impairment in B cell maturation and BAFF-dependent survival, but inactivation of Ikk1 in mature B cells does not affect survival. We further show that BAFF-R employs CD19 to promote survival via PI3K, and that co-inactivation of Cd19 and Ikk1 causes a profound block in B cell maturation at the transitional stage. Consistent with a role for PI3K in BAFF-R function, inactivation of PTEN mediates a partial rescue of B cell maturation and function in Baff−/− animals. Elevated PI3K signaling also circumvents BAFF-dependent survival in a spontaneous B cell lymphoma model. These findings indicate that the combined activities of PI3K and IKK1 drive peripheral B cell differentiation and survival in a context-dependent manner.
doi:10.1016/j.celrep.2013.10.022
PMCID: PMC3887557  PMID: 24239354
6.  Spontaneous loss of tolerance of autoreative B cell in Act1-deficient AM14 Tg rheumatoid factor (RF) mice 
Self-reactive B cells in BALB/c AM14 transgenic (AM14 Tg) rheumatoid factor (RF) mice are not subject to central or peripheral toleralization. Instead, they remain at a stage of “clonal ignorance”, i.e. they do not proliferate and differentiate into Ab-producing cells. However, the immunoregulatory mechanisms that prevent autoantibody production in these mice remain unclear. In this study, we show that crossing AM14 Tg mice to a mouse strain deficient in Act1, a molecule involved in the regulation of BAFF-R and CD40-signaling in B cells, results in spontaneous activation of AM14 Tg B cells and production of AM14-specific antibodies. Three to five-month old AM14 Tg Act1−/− mice showed significant expansion of AM14 Tg B cells, including a 2–3 fold increase in the spleen and cLNs compared to AM14 Tg Act1+/+ mice. Furthermore, in the presence of endogenous self-Ag (IgHa congenic background), AM14 Tg Act1−/− B cells were spontaneously activated and differentiated into antibody forming cells (AFC). In contrast with previous studies using AM14 Tg MLR.Faslpr mice, we found that a significant number of AM14 Tg cells AM14 Tg Act1−/− mice displayed phenotypic characteristics of GC B cells. Anti-CD40L treatment significantly limited the expansion and activation of AM14 Tg Act1−/− B cells, suggesting that CD40L-mediated signals are required for the retention of these cells. Our results support the important role of Act1 in the regulation of self-reactive B cells and reveal how Act1 functions to prevent the production of autoantibodies.
doi:10.4049/jimmunol.1300152
PMCID: PMC3893933  PMID: 23904159
7.  T Cell-Independent and Toll-like Receptor-Dependent Antigen-Driven Activation of Autoreactive B Cells 
Immunity  2008;29(2):249-260.
SUMMARY
On the lupus-prone MRL-lpr/lpr (MRL-lpr) background AM14 rheumatoid factor (RF) B cells are activated, differentiate into plasmablasts, and undergo somatic hypermutation outside of follicles. Using multiple strategies to impair T cells, we found that such AM14 B cell activation did not require T cells, but could be modulated by them. In vitro, the signaling adaptor MyD88 is required for IgG anti-chromatin to stimulate AM14 B cell proliferation when T cells are absent. However the roles of Toll-like receptors (TLRs) in AM14 B cell activation in vivo have not been investigated. We found that activation, expansion and differentiation of AM14 B cells depended on MyD88; however, mice lacking either TLR7 or TLR9 displayed partial defects, indicating complex roles for these receptors. T-independent activation of certain autoreactive B cells, which instead can gain stimuli via endogenous TLR ligands, may be the initial step in the generation of canonical autoantibodies.
doi:10.1016/j.immuni.2008.06.009
PMCID: PMC4106705  PMID: 18691914
8.  Taking Advantage: High Affinity B cells in the Germinal Center Have Lower Death Rates, But Similar Rates of Division Compared to Low Affinity Cells1 
B lymphocytes producing high affinity antibodies (Abs) are critical for protection from extracellular pathogens, such as bacteria and parasites. The process by which high affinity B cells are selected during the immune response has never been elucidated. Though it has been shown that high affinity cells directly outcompete low affinity cells in the germinal center (GC)2, whether there are also intrinsic differences between these cells has not been addressed. It could be that higher affinity cells proliferate more rapidly or are more likely to enter cell cycle, thereby outgrowing lower affinity cells. Alternatively, higher affinity cells could be relatively more resistant to cell death in the germinal center. By comparing high and low affinity B cells for the same antigen, we show here that low affinity cells have an intrinsically higher death rate than cells of higher affinity, even in the absence of competition. This suggests that selection in the GC reaction is due at least in part to the control of survival of higher affinity B cells and not by a proliferative advantage conferred upon these cells compared to lower affinity B cells. Control over survival rather than proliferation of low and high affinity B cells in the GC allows greater diversity not only in the primary response but also in the memory response.
doi:10.4049/jimmunol.0902452
PMCID: PMC4106706  PMID: 19917681
9.  TLR9 Regulates TLR7- and MyD88-Dependent Autoantibody Production and Disease in a Murine Model of Lupus 
Systemic lupus erythematosus is characterized by the production of autoantibodies against nucleic acid-associated Ags. We previously found that Tlr7 was required for anti-Sm and Tlr9 for anti-chromatin autoantibodies. Yet, although Tlr7 deficiency ameliorated disease, Tlr9 deficiency exacerbated it. Despite the mechanistic and clinical implications of this finding, it has yet to be elucidated. In this study, we characterize MRL/lpr lupus-prone mice genetically deficient in Tlr7, Tlr9, both Tlr7 and Tlr9, or Myd88 to test whether Tlr7 and Tlr9 function independently or instead regulate each other. We find that disease that is regulated by Tlr9 (and hence is worse in its absence) depends on Tlr7 for its manifestation. In addition, although Tlr7 and Tlr9 act in parallel pathways on different subsets of autoantibodies, Tlr9 also suppresses the production of Tlr7-dependent RNA-associated autoantibodies, suggesting previously unrecognized cross-regulation of autoantibody production as well. By comparing disease in mice deficient for Tlr7 and/or Tlr9 to those lacking Myd88, we also identify aspects of disease that have Tlr- and Myd88-independent components. These results suggest new models for how Tlr9 regulates and Tlr7 enhances disease and provide insight into aspects of autoimmune disease that are, and are not, influenced by TLR signals.
doi:10.4049/jimmunol.0902592
PMCID: PMC4098568  PMID: 20089701
10.  Selective targeting of B cells with agonistic anti-CD40 is an efficacious strategy for the generation of induced regulatory T2-like B cells and for the suppression of lupus in MRL/lpr mice1 
We have previously reported that IL10+ regulatory B cells, known to play an important role in controlling autoimmunity and inflammatory disorders, are contained within the Transitional-2 immature (T2) B cell pool (T2Bregs). Therapeutic strategies facilitating their enrichment or enhancing their suppressive activity are highly attractive. Here we report that agonistic anti-CD40 specifically targets T2 B cells and enriches B regs upon short term in vitro culture. Whilst transfer of unmanipulated T2 B cells, isolated from mice with established lupus, failed to confer protection to diseased mice, transfer of in vitro anti-CD40-generated T2 B cells (T2-like-Bregs) significantly improved renal disease and survival by an IL-10-dependent mechanism. T2-like-Bregs readily accumulated in the spleen after transfer, suppressed Th1 responses, induced the differentiation of IL-10+CD4+T cells and conveyed regulatory effect to CD4+T cells. In addition, in vivo administration of agonistic anti-CD40, currently on trial for the treatment of cancer, halted and reversed established lupus. Taken together our results suggest a novel cellular approach for the amelioration of experimental lupus.
doi:10.4049/jimmunol.0803052
PMCID: PMC4082659  PMID: 19265127
B cells; tolerance; IL-10; antibodies; autoimmunity
11.  MHC class II–dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies 
The Journal of Experimental Medicine  2013;210(13):2921-2937.
Antigen presentation, but not antibody secretion, by B cells drives CNS autoimmunity induced by immunization with human MOG.
Whether B cells serve as antigen-presenting cells (APCs) for activation of pathogenic T cells in the multiple sclerosis model experimental autoimmune encephalomyelitis (EAE) is unclear. To evaluate their role as APCs, we engineered mice selectively deficient in MHC II on B cells (B–MHC II−/−), and to distinguish this function from antibody production, we created transgenic (Tg) mice that express the myelin oligodendrocyte glycoprotein (MOG)–specific B cell receptor (BCR; IgHMOG-mem) but cannot secrete antibodies. B–MHC II−/− mice were resistant to EAE induced by recombinant human MOG (rhMOG), a T cell– and B cell–dependent autoantigen, and exhibited diminished Th1 and Th17 responses, suggesting a role for B cell APC function. In comparison, selective B cell IL-6 deficiency reduced EAE susceptibility and Th17 responses alone. Administration of MOG-specific antibodies only partially restored EAE susceptibility in B–MHC II−/− mice. In the absence of antibodies, IgHMOG-mem mice, but not mice expressing a BCR of irrelevant specificity, were fully susceptible to acute rhMOG-induced EAE, also demonstrating the importance of BCR specificity. Spontaneous opticospinal EAE and meningeal follicle–like structures were observed in IgHMOG-mem mice crossed with MOG-specific TCR Tg mice. Thus, B cells provide a critical cellular function in pathogenesis of central nervous system autoimmunity independent of their humoral involvement, findings which may be relevant to B cell–targeted therapies.
doi:10.1084/jem.20130699
PMCID: PMC3865476  PMID: 24323356
12.  IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression and activity 
The Journal of Experimental Medicine  2013;210(13):2887-2902.
The transcription factor IRF4 limits the retention of B cells in the marginal zone by inhibiting NOTCH2 signaling.
The transcription factor interferon regulatory factor-4 (IRF4) is expressed in B cells at most developmental stages. In antigen-activated B cells, IRF4 controls germinal center formation, class-switch recombination, and the generation of plasma cells. Here we describe a novel function for IRF4 in the homeostasis of mature B cells. Inducible deletion of irf4 specifically in B cells in vivo led to the aberrant accumulation of irf4-deleted follicular B cells in the marginal zone (MZ) area. IRF4-deficient B cells showed elevated protein expression and activation of NOTCH2, a transmembrane receptor and transcriptional regulator known to be required for MZ B cell development. Administration of a NOTCH2-inhibitory antibody abolished nuclear translocation of NOTCH2 in B cells within 12 h and caused a rapid and progressive disintegration of the MZ that was virtually complete 48 h after injection. The disappearance of the MZ was accompanied by a transient increase of MZ-like B cells in the blood rather than increased B cell apoptosis, demonstrating that continued NOTCH2 activation is critical for the retention of B cells in the MZ. Our results suggest that IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression. These findings may have implications for the understanding of B cell malignancies with dysregulated IRF4 and NOTCH2 activity.
doi:10.1084/jem.20131026
PMCID: PMC3865479  PMID: 24323359
13.  Exacerbated Autoimmunity in the Absence of TLR9 in MRLFaslpr Mice Depends on Ifnar11 
TLR9 suppresses TLR7-driven pathogenesis in the MRL.Faslpr murine model of systemic lupus erythematosus, but the mechanisms by which TLR7 promotes and TLR9 prevents disease in this and other lupus models remain unclear. Type I interferons have also been implicated in the pathogenesis of lupus both in patients and in several murine models of disease, but their role in MRL.Faslpr mice is controversial. Using MRL.Faslpr mice genetically deficient in a subunit of the receptor for type I interferon, Ifnar1, we show that type I interferons contribute significantly to renal disease in this model. Ifnar1 had no effect on anti-nucleosome or anti-Sm autoantibody titers, but instead regulated anti-cytoplasmic and anti-RNA specificities. Moreover, Ifnar1 deficiency prevented the exacerbation of clinical disease observed in Tlr9-deficient animals in this lupus model. Thus, type I interferon signaling is an important mediator of lupus pathogenesis and anti-RNA antibody production that is dysregulated in the absence of Tlr9.
doi:10.4049/jimmunol.1203525
PMCID: PMC3622185  PMID: 23467932
autoimmunity; systemic lupus erythematosus; TLR9; IFN–I
14.  CD73 Expression Is Dynamically Regulated in the Germinal Center and Bone Marrow Plasma Cells Are Diminished in Its Absence 
PLoS ONE  2014;9(3):e92009.
CD73 catalyzes the conversion of extracellular nucleosides to adenosine, modulating inflammatory and T cell responses. Elevated expression of CD73 marks subpopulations of murine memory B cells (MBC), but its role in memory development or function is unknown. Here, we demonstrate that CD73 is progressively upregulated on germinal center (GC) B cells following immunization, is expressed at even higher levels among T follicular helper cells, but is absent among plasma cells (PC) and plasmablasts (PB). We analyzed the T-dependent B cell response in CD73 knockout mice (CD73KO). During the early response, CD73KO and wild type (WT) mice formed GCs, MBCs and splenic PBs and PCs similarly, and MBCs functioned similarly in the early secondary response. Late in the primary response, however, bone marrow (BM) PCs were markedly decreased in CD73KO animals. Tracking this phenotype, we found that CD73 expression was required on BM-derived cells for optimal BM PC responses. However, deletion of CD73 from either B or T lymphocytes alone did not recapitulate the phenotype. This suggests that CD73 expression is sufficient on either cell type, consistent with its function as an ectoenzyme. Together, these findings suggest that CD73-dependent adenosine signaling is prominent in the mature GC and required for establishment of the long-lived PC compartment, thus identifying a novel role for CD73 in humoral immunity.
doi:10.1371/journal.pone.0092009
PMCID: PMC3963874  PMID: 24664100
15.  Signals via the Adaptor MyD88 in B cells and DCs Make Distinct and Synergistic Contributions to Immune Activation and Tissue Damage in Lupus 
Immunity  2013;38(3):528-540.
Summary
Detection of self nucleic acids by Toll-like receptors (TLR) preciptates autoimmune diseases, including systemic lupus erythematosus (SLE). It remains unknown how TLR signals in specific cell types contribute to distinct manifestations of SLE. Here, we demonstrate that formation of anti-nuclear antibodies in MRL.Faslpr mice entirely depends on the TLR signaling adaptor MyD88 in B cells. Further, MyD88 deficiency in B cells ameliorated nephritis, including antibody-independent interstitial T cell infiltrates, suggesting that nucleic acid-specific B cells activate nephrotoxic T cells. Surprisingly, MyD88 deletion in dendritic cells (DCs) did not affect nephritis, despite the importance of DCs in renal inflammation. In contrast, MyD88 in DCs was critical for dermatitis, revealing a separate pathogenetic mechanism. DC-expressed MyD88 promoted interferon-α production by plasmacytoid DCs, which was associated with Death domain-associated protein 6 upregulation and B lymphopenia. Our findings thus reveal unique immunopathological consequences of MyD88 signaling in B cells and DCs in lupus.
doi:10.1016/j.immuni.2012.11.017
PMCID: PMC3638041  PMID: 23499488
16.  Rheumatoid Factor B Cell Memory Leads to Rapid, Switched Antibody Forming Cell Responses 
B cells are critical in the initiation and maintenance of lupus. Autoreactive B cells clonally expand, isotype switch and mutate—properties associated with memory B cells (MBC), which are typically generated via germinal centers (GCs). The development and functions of autoreactive MBC in lupus are poorly understood. Moreover, mounting evidence implicates the extrafollicular (EF) response in the generation of switched and mutated autoantibodies that are driven by BCR and TLR co-recognition, raising the question of whether MBC are generated in this context. Here we investigated autoreactive MBC generation associated with this type of response. We transferred B cells from AM14 site directed BCR transgenic mice into non-transgenic normal recipients and elicited an EF response with anti-chromatin Ab, as in prior studies. By following the fate of the stimulated cells at late time points we found that AM14 B cells persisted at increased frequency for up to 7 weeks. Furthermore, these cells had divided in response to Ag, but were subsequently quiescent, with a subset expressing the memory marker CD73. These cells engendered rapid, isotype switched secondary plamablast responses upon restimulation. Both memory and rapid secondary responses required T cell help to develop, emphasizing the need for T-B collaboration for long-term self-reactivity. Thus, using this model system, we show that the EF response generated persistent and functional MBC that share some but not all of the characteristics of traditional MBC. Such cells could play a role in chronic or flaring autoimmune disease.
doi:10.4049/jimmunol.1202816
PMCID: PMC3578004  PMID: 23365079
17.  TLR9 Promotes Tolerance by Restricting Survival of Anergic Anti-DNA B Cells Yet is Also Required for Their Activation1 
Nucleic acid reactive B cells frequently arise in the bone marrow but are tolerized by mechanisms including receptor editing, functional anergy, and/or deletion. TLR9, a sensor of endosomal dsDNA, both promotes and regulates systemic autoimmunity in vivo, but the precise nature of its apparently contradictory roles in autoimmunity remained unclear. Here, using the 3H9 anti-DNA BCR transgene in the autoimmune-prone MRL.Faslpr mouse model of systemic lupus erythematosus, we identify the stages at which TLR9 contributes to establishing and breaking B cell tolerance. Although TLR9 is dispensable for light chain editing during B cell development in the bone marrow, TLR9 limits anti-DNA B cell lifespan in the periphery and is thus tolerogenic. In the absence of TLR9, anti-DNA B cells have much longer lifespans and accumulate in the follicle, neither activated nor deleted. These cells retain some characteristics of anergic cells, in that they have elevated basal BCR signaling but impaired induced responses and downregulate their cell surface BCR expression. In contrast, while TLR9-intact anergic B cells accumulate near the T/B border, TLR9-deficient anti-DNA B cells are somewhat more dispersed throughout the follicle. Nonetheless, in older autoimmune-prone animals, TLR9 expression specifically within the B cell compartment is required for spontaneous peripheral activation of anti-DNA B cells and their differentiation into AFCs via an extrafollicular pathway. Thus, TLR9 has paradoxical roles in regulating anti-DNA B cells: it helps purge the peripheral repertoire of autoreactive cells yet is also required for their activation.
doi:10.4049/jimmunol.1202115
PMCID: PMC3563726  PMID: 23296704
18.  B cells are essential for Protective Immunity against Salmonella Independent of Antibody Secretion1 
Typhoid fever and non-typhoidal bacteremia caused by Salmonella remain critical human health problems. B cells are required for protective immunity to Salmonella but the mechanism of protection remains unclear. Here, we immunized WildType, B cell deficient, antibody-deficient and class-switched antibody-deficient mice with attenuated Salmonella and examined protection against secondary infection. As expected, WildType mice were protected and B cell deficient mice succumbed to secondary infection. Interestingly, mice with B cells but lacking secreted antibody or class-switched antibody had little deficiency in resistance to Salmonella infection. The susceptibility of B cell deficient mice correlated with marked reductions in CD4 T cell IFN-γ production after secondary infection. Together, these data suggest that the primary role of B cells in acquired immunity to Salmonella is via the development of protective T cell immunity.
doi:10.4049/jimmunol.1201413
PMCID: PMC3518619  PMID: 23150714
19.  A new site directed transgenic Rheumatoid Factor mouse model demonstrates extrafollicular class switch and plasmablast formation 
Autoimmunity  2010;43(8):10.3109/08916930903567500.
The AM14 rheumatoid factor (RF) transgenic (Tg) mouse has been valuable for studying how self-reactive B cells are regulated beyond central tolerance, because they remain ignorant in normal mice. AM14 B cell activation can be studied on autoimmune-prone strains or by inducing activation with IgG2a anti-chromatin antibodies. Despite the utility of conventional Ig-Tg mice, site-directed Ig Tg (sd-Tg) mice provide a more physiological model for B cell responses, allowing class switch and somatic hypermutation. We report here the creation of an AM14 sd-Tg mouse and describe its phenotype on both normal and autoimmune-prone backgrounds. AM14 sd-Tg B cells develop normally but remain unactivated on the BALB/c background, even after significant aging. In contrast, on the autoimmune prone strain MRL/lpr, AM14 sd-Tg B cells become activated and secrete large amounts of IgG RF antibody into the serum. Class-switched antibody forming cells were found in the spleen and bone marrow. IgG RF plasmablasts were also observed in extrafollicular clusters in the spleens of aged AM14 sd-Tg MRL/lpr mice. Class switch and antibody secretion were observed additionally in AM14 sd-Tg BALB/c B cells activated in vivo using IgG2a anti-chromatin antibodies. Development of IgG autoantibodies is a hallmark of severe autoimmunity, and is related to pathogenesis. Using the AM14 sd-Tg, we now show that switched autoantibody-forming cells develop robustly outside germinal centers, further confirming the extrafollicular expression of AID. This model will allow more physiological studies of B cell biology in the future, including memory responses marked by class switch.
doi:10.3109/08916930903567500
PMCID: PMC3818904  PMID: 20370572
plasmablast; isotype switch; lupus; extrafollicular response
20.  Multiple transcription factor binding sites predict AID targeting in non-immunoglobulin genes 
Aberrant targeting of the enzyme Activation Induced Cytidine Deaminase (AID) results in the accumulation of somatic mutations in approximately 25% of expressed genes in germinal center B cells. Observations in Ung−/− Msh2−/− mice suggest that many other genes efficiently repair AID-induced lesions, so that up to 45% of genes may actually be targeted by AID. It is important to understand the mechanisms that recruit AID to certain genes, as this mis-targeting represents an important risk for genome instability. We hypothesize that several mechanisms will combine to target AID to each locus. In order to resolve which mechanisms affect AID targeting, we analyze 7.3Mb of sequence data, along with the regulatory context, from 83 genes in Ung−/− Msh2−/− mice to identify common properties of AID targets. This analysis identifies the involvement of three transcription factor binding sites (E-box motifs, along with YY1 and C/EBP-beta binding sites) that may work together to recruit AID. Based on previous knowledge and these newly discovered features, a classification tree model was built to predict genome-wide AID targeting. Using this predictive model we were able to identify a set of 101 high-interest genes that are likely targets of AID.
doi:10.4049/jimmunol.1202547
PMCID: PMC3689293  PMID: 23514741
21.  B Cell Signal Transduction in Germinal Center B Cells is Short-Circuited by Increased Phosphatase Activity 
Science (New York, N.Y.)  2012;336(6085):1178-1181.
Germinal centers (GCs) generate memory B and plasma cells, essential for long-lived humoral immunity. GC B cells with high affinity B cell receptors (BCRs) are selectively expanded. To enable this selection, BCRs of such cells are thought to signal differently from those with lower affinity. We show that, surprisingly, most proliferating GC B cells did not demonstrate active BCR signaling. Rather, spontaneous and induced signaling was limited by increased phosphatase activity. Accordingly, both SHP-1 and SHIP-1 were hyperphosphorylated in GC cells and remained colocalized with BCRs after ligation. Furthermore, SHP-1 was required for GC maintenance. Intriguingly, GC B cells in the cell cycle G2 period regained responsiveness to BCR stimulation. These data have implications for how higher affinity B cells are selected in the GC.
doi:10.1126/science.1213368
PMCID: PMC3777391  PMID: 22555432
22.  Langerhans Cells Facilitate Epithelial DNA Damage and Squamous Cell Carcinoma 
Science (New York, N.Y.)  2012;335(6064):104-108.
Polyaromatic hydrocarbons (PAHs) are prevalent, potent carcinogens, and 7,12-dimethylbenz[a]anthracene (DMBA) is a model PAH widely used to study tumorigenesis. Mice lacking Langerhans cells (LCs), a signatory epidermal dendritic cell (DC), are protected from cutaneous chemical carcinogenesis, independent of T cell immunity. Investigation of the underlying mechanism revealed that LC-deficient skin was relatively resistant to DMBA-induced DNA damage. LCs efficiently metabolized DMBA to DMBA-trans-3,4-diol, an intermediate proximal to oncogenic Hras mutation, and DMBA-treated LC-deficient skin contained significantly fewer Hras mutations. Moreover, DMBA-trans-3,4-diol application bypassed tumor resistance in LC-deficient mice. Additionally, the genotoxic impact of DMBA on human keratinocytes was significantly increased by prior incubation with human-derived LC. Thus, tissue-associated DC can enhance chemical carcinogenesis via PAH metabolism, highlighting the complex relation between immune cells and carcinogenesis.
doi:10.1126/science.1211600
PMCID: PMC3753811  PMID: 22223807
23.  Langerhans Cells Are Not Required for Efficient Skin Graft Rejection 
The mechanism of skin allograft rejection has been thought to require presentation of graft antigen by resident epidermal Langerhans cells (LCs). We have previously engineered mice that have a selective and constitutive absence of epidermal LCs. By using donor skin from these LC-deficient mice, we show that LCs are not required for rejection of major (FVB→B6) or minor (H-Y, male→female on B6 background) antigen-mismatched skin grafts. On the FVB background, where H-Y mismatched grafts are normally maintained indefinitely, grafts lacking LCs are efficiently rejected. Thus, LCs in the donor graft are required for long-term skin engraftment, which supports a regulatory role for LCs in skin graft acceptance.
doi:10.1038/jid.2008.52
PMCID: PMC3668350  PMID: 18337832
24.  CD80 expression on B cells regulates murine T follicular helper development, germinal center B cell survival and plasma cell generation1 
Germinal center (GC) B cells and T follicular helper (TFH) cells interact in the production of high-affinity long-lived plasma cells (PCs) and memory B cells, though the mechanisms regulating the formation of these long-lived populations remain unclear. Because CD80 is one of the few markers shared by human and murine memory B cells, we investigated its role in the development of GCs, memory cells and PCs. In CD80-deficient mice, fewer long-lived PCs were generated upon immunization, compared to B6 controls. In concert, the absence of CD80 resulted in an increase in apoptotic GC B cells during the contraction phase of the GC. CD80−/− mice had fewer TFH compared to B6, and residual TFH cells failed to mature, with decreased ICOS and PD-1 expression and decreased synthesis of IL-21 mRNA. Mixed bone marrow chimeras demonstrated a B cell-intrinsic requirement for CD80 expression for normal TFH and PC development. Therefore, B cell expression of CD80 plays a critical role in regulating B-T interactions in both early and late GC responses. This, in turn, results in impaired ability to produce long-lived PCs. These data provide new insights into the development of GCs and AFCs and the functions of CD80 in humoral immunity.
doi:10.4049/jimmunol.1102885
PMCID: PMC3331930  PMID: 22450810
25.  Single-round of antigen receptor signaling programs naïve B cells to receive T cell help 
Immunity  2010;32(3):355-366.
SUMMARY
To simulate transient B cell activation that is the likely initiator of T-dependent responses, we examined the molecular and functional consequences of a single-round of immunoglobulin M (IgM) signaling. This form of activation triggered early cytosolic signaling and the transcription factor NF-κB activation indistinguishably from conventional continuous IgM cross-linking, but did not induce G1 progression. However, single-round IgM signaling changed the expression of chemokine and chemokine receptor genes implicated in initiating T-dependent responses, as well as accentuated responsiveness to CD40 signaling. Several features of single-round IgM signaling in vitro were recapitulated in B cells after short-term exposure to antigen in vivo. We propose that transient BCR signals prime B cells to receive T cell help by increasing the probability of B-T encounter and creating a cellular environment that is hyper-responsive to CD40 signaling.
doi:10.1016/j.immuni.2010.02.013
PMCID: PMC3607434  PMID: 20226693

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