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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  B Cell-Derived Interleukin 10 Does Not Regulate Spontaneous Systemic Autoimmunity in MRL.Faslpr mice 
B cells contribute to the pathogenesis of chronic autoimmune disorders like systemic lupus erythematosus (SLE) via multiple effector functions. However, B cells are also implicated in regulating SLE and other autoimmune syndromes via release of IL-10. B cells secreting IL-10 have been termed “Breg” and have been proposed as a separate subset of cells, a concept that remains controversial. The balance between pro- and anti-inflammatory effects could determine the success of B cell targeted therapies for autoimmune disorders and it is therefore pivotal to understand the significance of B cell-secreted IL-10 in spontaneous autoimmunity. By lineage specific deletion of Il10 from B cells we demonstrate that B cell-derived IL-10 is ineffective in suppressing the spontaneous activation of self-reactive B and T cells during lupus. Correspondingly, severity of organ disease and survival rates in mice harboring Il10 deficient B cells are unaltered. Genetic marking of cells that transcribe Il10 illustrates that the pool of IL-10 competent cells is dominated by CD4 T cells and macrophages. IL-10 competent cells of the B lineage are rare in vivo and among them short-lived plasmablasts have the highest frequency, suggesting an activation rather than lineage-driven phenotype. Putative Breg phenotypic subsets such as CD1dhiCD5+ and CD21hiCD23hi B cells are not enriched in Il10 transcription. These genetic studies demonstrate that in a spontaneous model of murine lupus, IL-10 dependent B cell regulation does not restrain disease and thus the pathogenic effects of B cells are not detectably counterbalanced by their IL-10 dependent regulatory functions.
doi:10.4049/jimmunol.1102456
PMCID: PMC3253138  PMID: 22156495
14.  Anti-Chromatin Antibodies Drive In Vivo Antigen-Specific Activation and Somatic Hypermutation of Rheumatoid Factor B cells at Extrafollicular Sites 
European journal of immunology  2007;37(12):3339-3351.
A dominant type of spontaneous autoreactive B cell activation in murine lupus is the extrafollicular generation of plasmablasts. The factors governing such activation have been difficult to identify due to the stochastic onset and chronic nature of the response. Thus, the ability to induce a similar autoreactive B cell response with a known autoantigen in vivo would be a powerful tool in deciphering how autoimmune responses are initiated. We report here the establishment and characterization of a system to initiate autoreactive extrafollicular B cell responses that closely mirror the spontaneous response using IgG anti-chromatin Abs. We demonstrate that exogenously administered anti-chromatin Ab, presumably by forming immune complexes (ICs) with released nuclear material, drives activation of RF B cells in AM14 Tg mice. Anti-chromatin elicits autoreactive B cell activation and development into AFCs at the T-zone/red pulp border. Plasmablast generation occurs equally in BALB/c, MRL/+ and MRL/lpr mice, indicating that an autoimmune-prone genetic background is not required for the induced response. Importantly, infused IgG anti-chromatin induces somatic hypermutation (SHM) in the absence of a GC response, thus proving the extrafollicular SHM pathway. This system provides a window on the initiation of an autoantibody response and reveals authentic initiators of it.
doi:10.1002/eji.200737752
PMCID: PMC3516908  PMID: 18034429
B cells; autoantibodies; systemic lupus erythematosus
15.  Enhancing alloreactivity does not restore GVHD induction but augments skin graft rejection by CD4 effector memory T cells 
European journal of immunology  2011;41(9):2782-2792.
Graft-vs.-host disease (GVHD) caused by donor T cells attacking recipient tissues is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (alloSCT). Studies have shown that effector memory T cells (TEM) do not cause GVHD but are capable of immune functions post-transplant, including graft-vs.-leukemia (GVL) effects, but the reasons for this are unclear. In mice, the TEM pool may have a less-diverse T cell receptor (TCR) repertoire than TN with fewer alloreactive clones. We therefore tested whether enhancing the alloreactivity of TEM would restore their ability to cause GVHD. In an MHC-matched system, alloreactive TEM were created by transferring GVHD effector cells into syngeneic recipients and allowing conversion to TEM. Upon retransfer to freshly transplanted recipients, these cells caused only mild GVHD. Similarly, in an MHC-mismatched system, TEM with a proven increased precursor frequency of alloreactive clones only caused limited GVHD. Nonetheless these same cells mounted strong in vitro alloresponses and caused rapid skin graft rejection. TEM created from CD4 cells that had undergone lymphopenia-induced proliferation also caused only mild GVHD. Our findings establish that conversion to TEM significantly reduces GVHD potency, even in cells with a substantially enhanced alloreactive repertoire.
doi:10.1002/eji.201141678
PMCID: PMC3517133  PMID: 21660940
16.  An acquired defect in IgG-dependent phagocytosis explains the impairment in Ab-mediated cellular depletion in lupus 
B cells play important roles in autoimmune diseases ranging from multiple sclerosis to rheumatoid arthritis. B cells have long been considered central players in systemic lupus erythematosus. However, anti-CD20 mediated B cell depletion was not effective in two clinical lupus studies, while anti-BLyS, which inhibits B cell survival, was effective. Others and we previously found that anti-CD20 based depletion was surprisingly ineffective in tissues of lupus-prone mice, but that persistent high doses eventually led to depletion and ameliorated lupus. Lupus patients might also have incomplete depletion, as suggested in several studies, and which could have led to therapeutic failure. Here we investigated the mechanism of resistance to Ab-mediated cellular depletion in murine lupus. B cells from lupus-prone mice were easily depleted when transferred into normal environments or in lupus-prone mice that lacked serum Ig. Serum from lupus-prone mice transferred depletion resistance, with the active component being IgG. Because depletion is FcγR-dependent, we assayed macrophages and neutrophils exposed to lupus mouse serum, showing they are impaired in IgG-mediated phagocytosis. We conclude that depletion resistance is an acquired, reversible phagocytic defect depending on exposure to lupus serum IgG. These results have implications for optimizing and monitoring cellular depletion therapy.
doi:10.4049/jimmunol.1101629
PMCID: PMC3178685  PMID: 21873531
17.  Differential Cytokine Production and Bystander Activation of Autoreactive B Cells in Response to CpG-A and CpG-B ODNs1 
Synthetic oligonucleotides containing CpG motifs (CpG ODNs) have been shown to induce proliferation, differentiation and cytokine production in B cells, macrophages and DCs through a TLR9-dependent mechanism. A class (CpG-A) and B class (CpG-B) ODNs display distinct physical properties. CpG-A, but not CpG-B, can multimerize to form exceedingly large lattices. CpG-A cannot effectively activate B cells but does induce pDCs to produce high levels of IFNα, while CpG-B is a potent B cell mitogen. Here we report that CpG-A is internalized by B cells, and CpG-A and CpG-B accumulate to distinct intracellular compartments. When present in the form of an immune complex (CpG-A IC), CpG-A is taken up more efficiently by AM14 IgG2a-specific B cells, and elicits a robust TLR9-dependent B cell proliferative response. B cells proliferating comparably and in a TLR9-dependent fashion in response to CpG-A IC and CpG-B exhibited distinct cytokine profiles. CpG-A IC induced enhanced production of RANTES and markedly reduced levels of IL-6 when compared to CpG-B. We also found that engagement of the AM14 BCR by a protein IC, which cannot by itself induce proliferation, promoted TLR9-dependent but BCR-independent proliferation by bystander CpG-A or fragments of mammalian dsDNA. These data identify direct and indirect mechanisms by which BCR engagement facilitates access of exogenous ligands to TLR9-associated compartments and subsequent B cell activation.
doi:10.4049/jimmunol.0901941
PMCID: PMC3426913  PMID: 19864612
18.  Cutting Edge: Hierarchy of Maturity of Murine Memory B Cell Subsets 
The paucity of murine memory B cell markers has been a significant impediment to the study of memory. The most commonly used marker is IgG, which is neither sensitive nor specific, because activated nonmemory cells can be IgG+, and memory cells can be IgM+. In this article, we show that, together, PD-L2 (CD273), CD80, and CD73 define at least five phenotypic subsets of murine memory B cells. These subsets are generated from naive cells bearing a single BCR in response to a single T-dependent Ag. This diversity is independent of class switch, because IgG1- and IgM-bearing memory cells are found within each compartment. Memory subsets defined by PD-L2, CD80, and CD73 are biologically distinct from one another, because they differ in ontogeny and selection. Together, these distinctions suggest that there is a spectrum of memory B cells and progressive acquisition from more naive-like to more memory-like properties.
doi:10.4049/jimmunol.1002163
PMCID: PMC3133669  PMID: 21078902
19.  Dendritic Cells in Lupus are not Required for Activation of T and B Cells but Promote their Expansion Resulting in Tissue Damage 
Immunity  2010;33(6):967-978.
Summary
Dendritic cells (DCs) initiate and control the adaptive immune response against infections. However, their contributions to the anti-self adaptive immune response in autoimmune disorders like systemic lupus erythematosus are uncertain. By constitutively deleting DCs in MRL.Faslpr mice we show that they have complex roles in murine lupus. The net effect of DC deletion was to ameliorate disease. DCs were crucial for the expansion and differentiation of T cells but, surprisingly, not required for their initial activation. Correspondingly, kidney interstitial infiltrates developed in the absence of DCs, but failed to progress. DC deletion concomitantly decreased inflammatory and regulatory T cell numbers. Unexpectedly, plasmablast numbers and autoantibody concentrations depended on DCs, in contrast to total serum immunoglobulin concentrations, suggesting an effect of DCs on extrafollicular humoral responses. These findings reveal that DCs operate in unanticipated ways in murine lupus and validate them as a potential therapeutic target in autoimmunity.
doi:10.1016/j.immuni.2010.11.025
PMCID: PMC3010763  PMID: 21167752
20.  Recipient B Cells Are Not Required For GVHD Induction 
Recipient antigen presenting cells (APCs) are required for CD8-mediated GVHD and have an important and nonredundant role in CD4-mediated GVHD in mouse MHC-matched allogeneic bone marrow transplantation (alloBMT). However, the precise roles of specific recipient APCs — dendritic cells, macrophages, and B cells — are not well defined. If recipient B cells are important APCs they could be depleted with Rituximab, an anti-CD20 monoclonal antibody. On the other hand, B cells can downregulate T cell responses and consequently B cell depletion could exacerbate GVHD. Patients with B cell lymphomas undergo allogeneic hematopoietic stem cell transplantation (alloSCT) and many are B-cell-deficient due to prior Rituximab. We therefore studied the role of recipient B cells in MHC-matched murine models of CD8- and CD4-mediated GVHD by using recipients genetically deficient in B cells and with antibody-mediated depletion of host B cells. In both CD4-and CD8-dependent models, B cell deficient recipients developed clinical and pathologic GVHD. However, although CD8-mediated GVHD was clinically less severe in hosts genetically deficient in B cells, it was unaffected in anti-CD20-treated recipients. These data indicate that recipient B cells are not important initiators of GVHD and that efforts to prevent GVHD by APC depletion should focus on other APC subsets.
doi:10.1016/j.bbmt.2010.03.015
PMCID: PMC3135976  PMID: 20338255
21.  Langerhans cells suppress CHS responses via cognate CD4 interaction and LC-derived IL-10 
SUMMARY
Mice lacking epidermal Langerhans cells (LC) develop exaggerated contact-hypersensitivity (CHS) responses due to the absence of LC during sensitization/initiation. Examination of T cell responses reveals that the absence of LC leads to increased numbers of hapten-specific CD4 and CD8 T cells but does not alter cytokine expression or development of Treg. CHS responses and antigen-specific T cells are increased in mice in which MHC-II is ablated specifically in LC suggesting that direct cognate interaction between LC and CD4 cells is required for LC suppression. LC-derived IL-10 is also required for optimal inhibition of CHS. Both LC-derived IL-10 mediated suppression and full LC activation require LC expression of MHC-II. These data support a model in which cognate interaction of LC with CD4 T cells enables LC to inhibit expansion of antigen-specific responses via elaboration of IL-10.
doi:10.4049/jimmunol.0901884
PMCID: PMC3131075  PMID: 19801524
22.  Detecting selection in immunoglobulin sequences 
Nucleic Acids Research  2011;39(Web Server issue):W499-W504.
The ability to detect selection by analyzing mutation patterns in experimentally derived immunoglobulin (Ig) sequences is a critical part of many studies. Such techniques are useful not only for understanding the response to pathogens, but also to determine the role of antigen-driven selection in autoimmunity, B cell cancers and the diversification of pre-immune repertoires in certain species. Despite its importance, quantifying selection in experimentally derived sequences is fraught with difficulties. The necessary parameters for statistical tests (such as the expected frequency of replacement mutations in the absence of selection) are non-trivial to calculate, and results are not easily interpretable when analyzing more than a handful of sequences. We have developed a web server that implements our previously proposed Focused binomial test for detecting selection. Several features are integrated into the web site in order to facilitate analysis, including V(D)J germline segment identification with IMGT alignment, batch submission of sequences and integration of additional test statistics proposed by other groups. We also implement a Z-score-based statistic that increases the power of detecting selection while maintaining specificity, and further allows for the combined analysis of sequences from different germlines. The tool is freely available at http://clip.med.yale.edu/selection.
doi:10.1093/nar/gkr413
PMCID: PMC3125793  PMID: 21665923
23.  Rituximab Therapy Reduces Organ-Specific T Cell Responses and Ameliorates Experimental Autoimmune Encephalomyelitis 
PLoS ONE  2011;6(2):e17103.
Recent clinical trials have established B cell depletion by the anti-CD20 chimeric antibody Rituximab as a beneficial therapy for patients with relapsing-remitting multiple sclerosis (MS). The impact of Rituximab on T cell responses remains largely unexplored. In the experimental autoimmune encephalomyelitis (EAE) model of MS in mice that express human CD20, Rituximab administration rapidly depleted peripheral B cells and strongly reduced EAE severity. B cell depletion was also associated with diminished Delayed Type Hypersensitivity (DTH) and a reduction in T cell proliferation and IL-17 production during recall immune response experiments. While Rituximab is not considered a broad immunosuppressant, our results indicate a role for B cells as a therapeutic cellular target in regulating encephalitogenic T cell responses in specific tissues.
doi:10.1371/journal.pone.0017103
PMCID: PMC3040191  PMID: 21359213
24.  RAGE-Independent Autoreactive B Cell Activation In Response To Chromatin And HMGB1/DNA Immune Complexes 
Autoimmunity  2010;43(1):103-110.
Increasing evidence suggests that the excessive accumulation of apoptotic or necrotic cellular debris may contribute to the pathology of systemic autoimmune disease. HMGB1 is a nuclear DNA-associated protein, which can be released from dying cells thereby triggering inflammatory processes. We have previously shown that IgG2a-reactive BCR transgenic AM14 B cells proliferate in response to endogenous chromatin immune complexes (ICs), in the form of the anti-nucleosome antibody PL2-3 and cell debris, in a TLR9-dependent manner, and that these ICs contain HMGB1. Activation of AM14 B cells by these chromatin ICs was inhibited by a soluble form of the HMGB1 receptor, RAGE-Fc, suggesting HMGB1/RAGE interaction was important for this response [1]. To further explore the role of HMGB1 in autoreactive B cell activation, we assessed the capacity of purified calf thymus HMGB1 to bind dsDNA fragments and found that HMGB1 bound both CG-rich and CG-poor DNA. However, HMGB1/DNA complexes could not activate AM14 B cells unless HMGB1 was bound by IgG2a and thereby able to engage the BCR. To ascertain the role of RAGE in autoreactive B cell responses to chromatin ICs, we intercrossed AM14 and RAGE-deficient mice. We found that spontaneous and defined DNA ICs activated RAGE+ and RAGE− AM14 B cells to a comparable extent. These results suggest that HMGB1 promotes B cell responses to endogenous TLR9 ligands through a RAGE-independent mechanism.
doi:10.3109/08916930903384591
PMCID: PMC2929824  PMID: 20014975
HMGB1; RAGE; AM14 B cells; TLR9; Systemic Lupus Erythematosus; autoreactive B cell activation
25.  Activating Systemic Autoimmunity: B’s, T’s and Tolls 
Current opinion in immunology  2009;21(6):626-633.
A recent advance in the treatment and understanding of autoimmune disease has been the efficacy of B cell targeted therapy. Such therapies are effective for several such diseases, with systemic autoimmunity being a prototypical example. The mechanism of action is not fully defined, but blocking B cell Ag presentation to T cells is likely to be important. T-B interactions probably engender a positive feedback loop that amplifies and sustains autoimmunity. But how is self-tolerance first broken to initiate this loop? I propose, based on recent data, a model in which autoreactive B cells are activated first, independent of T cells, but dependent upon BCR and TLR signals. These activated B cells then break T celltolerance, initiating full-blown autoimmunity.
doi:10.1016/j.coi.2009.08.005
PMCID: PMC2787881  PMID: 19800208

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