IL-21 regulates germinal center B cell differentiation and proliferation through a B cell–intrinsic mechanism

Germinal centers (GCs) are sites of B cell proliferation, somatic hypermutation, and selection of variants with improved affinity for antigen. Long-lived memory B cells and plasma cells are also generated in GCs, although how B cell differentiation in GCs is regulated is unclear. IL-21, secreted by T follicular helper cells, is important for adaptive immune responses, although there are conflicting reports on its target cells and mode of action in vivo. We show that the absence of IL-21 signaling profoundly affects the B cell response to protein antigen, reducing splenic and bone marrow plasma cell formation and GC persistence and function, influencing their proliferation, transition into memory B cells, and affinity maturation. Using bone marrow chimeras, we show that these activities are primarily a result of CD3-expressing cells producing IL-21 that acts directly on B cells. Molecularly, IL-21 maintains expression of Bcl-6 in GC B cells. The absence of IL-21 or IL-21 receptor does not abrogate the appearance of T cells in GCs or the appearance of CD4 T cells with a follicular helper phenotype. IL-21 thus controls fate choices of GC B cells directly.The immunological memory that develops during T cell–dependent (TD) immune responses comprises populations of plasma cells and recirculating antigen-experienced B and T lymphocytes (Tarlinton, 2006). Two compartments of humoral memory, plasma cells and memory B cells, are generated in germinal centers (GCs) that develop within the secondary lymphoid organs during TD responses (Tarlinton, 2006). Although composed primarily of B lymphocytes, GCs contain small numbers of CD4⁺ T cells, dendritic cells, and macrophages and develop in association with antigen localized on the surface of follicular dendritic cells (Haberman and Shlomchik, 2003; Allen et al., 2007). After a period of B cell proliferation, several processes are initiated within the GC that affect affinity maturation whereby the mean binding affinity of antigen-specific antibody increases as a function of time (MacLennan, 1994; Allen et al., 2007). Affinity maturation is driven in large part by the somatic hypermutation (SHM) of the immunoglobulin V genes of proliferating GC B cells, a process which is mediated by the enzyme activation-induced cytidine deaminase (AID). B cells expressing antigen receptors of improved affinity, usually as a result of SHM, are preserved preferentially. Iterations of proliferation, mutation, and avidity-based selection improve the mean affinity of the responding B cell population (MacLennan, 1994; Allen et al., 2007).Normally, in an immune response to a protein antigen the vast majority of memory B cells and bone marrow plasma cells arise from the somatically diversified affinity-matured population of GC B cells (Tarlinton, 2006). It is inferred that avidity for antigen is a major determinant in plasma cell differentiation of GC B cells, whereas memory B cell formation is more influenced by survival (Lanzavecchia and Sallusto, 2002; Phan et al., 2006; Tarlinton, 2006). It also appears that both types of post-GC B cell are produced throughout the GC reaction rather than being released into the circulation in a single event (Blink et al., 2005). The persistence and continued activity of GC, which is indicated by the continued production of plasma cells and memory B cells and the increasing frequency of V gene mutation, implies that a proportion of GC B cells remain within the GC and undergo additional rounds of proliferation, mutation, and selection (MacLennan, 1994; Allen et al., 2007). B cells within GC therefore have several possible fates: death, division with or without SHM, or differentiation into either the memory B cell or plasma cell compartments.GC persistence, development, and function absolutely require CD4⁺ T cells. T cells activated by antigen-presenting dendritic cells migrate into the B cell area in part as a result of their up-regulation of CXCR5, a chemokine receptor normally restricted to B cells (Allen et al., 2007). Indeed, the expression of CXCR5 contributes to the definition of what are now called T follicular helper (Tfh) cells (Vinuesa et al., 2005). In addition to CXCR5, Tfh cells are distinguished from other CD4 T cells by their elevated expression of ICOS and CD40L (Vinuesa et al., 2005), both of which are critical for the initiation and maintenance of the GC (Tarlinton, 2006). Intriguingly, up-regulation of many of the molecules that define the Tfh phenotype appears to be mediated by Bcl-6, which is required for their development in a cell-intrinsic manner (Johnston et al., 2009). Tfh cells are also enriched for secretion of IL-21 (Chtanova et al., 2004; Nurieva et al., 2008) and IL-4 (Reinhardt et al., 2009). IL-21 is associated with growth and differentiation of many types of lymphocytes, including B and T cells (Ettinger et al., 2008). The effects of IL-21 on B cells vary depending on the context. In vivo, IL-21R deficiency leads to a state of pan-hypogammaglobulinaemia while promoting high titers of IgE (Ozaki et al., 2002). In vitro, IL-21 has been shown to increase both Blimp-1 and Bcl-6 in B cells (Ozaki et al., 2004; Arguni et al., 2006), suggesting an ability for IL-21 to influence multiple aspects of B cell differentiation. Recent data support the notion that IL-21 has a critical, possibly obligatory, role in the development of Tfh cells and, through this, on the formation of GC (Nurieva et al., 2008; Vogelzang et al., 2008), whereas other data suggest a less universal association (Linterman et al., 2009). IL-21 has also been shown to augment the formation of Th17 cells (Korn et al., 2007; Nurieva et al., 2007; Zhou et al., 2007), which have been shown to both secrete IL-21 and promote GC formation in a mouse model of autoimmunity (Hsu et al., 2008), strengthening the view that the effects of IL-21 on GC activity are T cell mediated. An earlier study, however, using IL-21R–deficient mice reported GC and memory development to be normal (Ozaki et al., 2002), raising uncertainty as to exactly what the requirement of IL-21 may be in the GC reaction, on which cell types it may act, and what its activities might be. This uncertainty has been heightened by recent publications suggesting that IL-4 is a key mediator of Tfh activity (King and Mohrs, 2009; Reinhardt et al., 2009).Given the multitude of potential roles for IL-21 on lymphocyte behavior (Ettinger et al., 2008), we wished to assess the development of a humoral immune response in mice lacking IL-21 or the IL-21R. These experiments confirmed a role for IL-21 in the formation of plasma cells, contradicted a mandatory autocrine role for IL-21 in Tfh development or function, and revealed a previously undefined role for this cytokine in the GC reaction and the regulation of their output. These actions of IL-21 on B cells were direct, as they were replicated by the selective absence of the IL-21R on B cells and not on T cells, suggesting that the major activity of IL-21 in the GC is on B cells and is not to establish or maintain cells of a Tfh phenotype.     

Dual-reactive B cells are autoreactive and highly enriched in the plasmablast and memory B cell subsets of autoimmune mice

Rare dual-reactive B cells expressing two types of Ig light or heavy chains have been shown to participate in immune responses and differentiate into IgG⁺ cells in healthy mice. These cells are generated more often in autoreactive mice, leading us to hypothesize they might be relevant in autoimmunity. Using mice bearing Igk allotypic markers and a wild-type Ig repertoire, we demonstrate that the generation of dual-κ B cells increases with age and disease progression in autoimmune-prone MRL and MRL/lpr mice. These dual-reactive cells express markers of activation and are more frequently autoreactive than single-reactive B cells. Moreover, dual-κ B cells represent up to half of plasmablasts and memory B cells in autoimmune mice, whereas they remain infrequent in healthy mice. Differentiation of dual-κ B cells into plasmablasts is driven by MRL genes, whereas the maintenance of IgG⁺ cells is partly dependent on Fas inactivation. Furthermore, dual-κ B cells that differentiate into plasmablasts retain the capacity to secrete autoantibodies. Overall, our study indicates that dual-reactive B cells significantly contribute to the plasmablast and memory B cell populations of autoimmune-prone mice suggesting a role in autoimmunity.While developing in the BM, B cells undergo stochastic rearrangement of Ig heavy (IgH) and Ig light (IgL) chain V(D)J gene segments resulting in the random expression of Ig H and L (κ and λ) chains in the emerging B cell population (Schlissel, 2003; Nemazee, 2006). During V(D)J recombination, allelic and isotypic exclusion at the Ig loci are also established, leading to the expression of a unique H and L chain pair and, therefore, of BCRs with unique specificity in each B cell (Langman and Cohn, 2002; Nemazee, 2006; Vettermann and Schlissel, 2010). These mechanisms ensure that developing B cells expressing BCRs reactive with self-antigens (i.e., autoreactive B cells) undergo tolerance induction, whereas those expressing BCRs specific for a foreign antigen or a peripheral self-antigen proceed in differentiation and selection into the periphery (Burnet, 1959). Autoreactive B cells are silenced by central tolerance in the BM via receptor editing and, less frequently, clonal deletion (Halverson et al., 2004; Ait-Azzouzene et al., 2005), whereas peripheral B cell tolerance proceeds via anergy and clonal deletion (Goodnow et al., 2005; Pelanda and Torres, 2006, 2012; Shlomchik, 2008). Despite these tolerance mechanisms, small numbers of autoreactive B cells are detected in peripheral tissues of healthy mice and humans (Grandien et al., 1994; Wardemann et al., 2003) and their numbers are increased in autoimmunity (Andrews et al., 1978; Izui et al., 1984; Warren et al., 1984; Samuels et al., 2005; Yurasov et al., 2005, 2006; Liang et al., 2009).A small population of dual-reactive B cells expressing two types of L chains (or more rarely H chains) has been observed both in mice and humans (Nossal and Makela, 1962; Pauza et al., 1993; Giachino et al., 1995; Gerdes and Wabl, 2004; Rezanka et al., 2005; Casellas et al., 2007; Velez et al., 2007; Kalinina et al., 2011). These allelically and isotypically (overall haplotype) included B cells are <5% of all peripheral B cells in normal mice (Barreto and Cumano, 2000; Rezanka et al., 2005; Casellas et al., 2007; Velez et al., 2007), but they are more frequent in Ig knockin mice in which newly generated B cells are autoreactive and actively undergo receptor editing (Li et al., 2002a,b; Liu et al., 2005; Huang et al., 2006; Casellas et al., 2007). B cells that coexpress autoreactive and nonautoreactive antibodies can escape at least some of the mechanisms of central and peripheral B cell tolerance and be selected into the mature peripheral B cell population (Kenny et al., 2000; Li et al., 2002a,b; Gerdes and Wabl, 2004; Liu et al., 2005; Huang et al., 2006), sometimes with a preference for the marginal zone (MZ) B cell subset (Li et al., 2002b).Furthermore, dual-reactive B cells observed within a normal polyclonal Ig repertoire exhibit characteristics of cells that develop through the receptor editing process, including delayed kinetics of differentiation and more frequent binding to self-antigens (Casellas et al., 2007). Hence, dual-reactive B cells might play a role in autoantibody generation and autoimmunity. However, the contribution of these B cells to autoimmunity has not yet been established. Our hypothesis is that haplotype-included autoreactive B cells are positively selected within the context of genetic backgrounds that manifest defects in immunological tolerance and contribute to the development of autoimmunity.Until recently, the analysis of dual-reactive B cells was impaired by the inability to detect dual-κ cells, which are the most frequent among haplotype-included B cells (Casellas et al., 2007; Velez et al., 2007). To overcome this issue, we took advantage of Igk^h mice that bear a gene-targeted human Ig Ck allele in the context of a wild-type Ig repertoire (Casellas et al., 2001) and crossed these to MRL-Fas^lpr/lpr (MRL/lpr) and MRL mice that develop an autoimmune pathology with characteristics similar to human lupus (Izui et al., 1984; Rordorf-Adam et al., 1985; Theofilopoulos and Dixon, 1985; Cohen and Eisenberg, 1991; Watanabe-Fukunaga et al., 1992). MRL/lpr mice, moreover, display defects in receptor editing (Li et al., 2002a; Lamoureux et al., 2007; Panigrahi et al., 2008) and reduced tolerance induction (Li et al., 2002a), which could potentially contribute to higher frequency of haplotype-included autoreactive B cells.We found that the frequency of dual-κ cells increased with age and progression of disease in autoimmune-prone mice and independent of the expression of Fas. Dual-κ B cells exhibited higher prevalence of autoreactivity than single-κ B cells and were frequently selected into the antigen-activated cell subsets in MRL/lpr and MRL mice where up to half of the plasmablasts and memory B cells were dual-κ B cells. Moreover, disruption of Fas expression appeared to mediate increased survival of dual-reactive memory B cells. Overall, these data indicate that dual-reactive B cells significantly contribute to the plasmablast and memory B cell populations of autoimmune-prone mice suggesting a role in the development of autoimmunity.     

Decoration of T-independent antigen with ligands for CD22 and Siglec-G can suppress immunity and induce B cell tolerance in vivo

Autoreactive B lymphocytes first encountering self-antigens in peripheral tissues are normally regulated by induction of anergy or apoptosis. According to the “two-signal” model, antigen recognition alone should render B cells tolerant unless T cell help or inflammatory signals such as lipopolysaccharide are provided. However, no such signals seem necessary for responses to T-independent type 2 (TI-2) antigens, which are multimeric antigens lacking T cell epitopes and Toll-like receptor ligands. How then do mature B cells avoid making a TI-2–like response to multimeric self-antigens? We present evidence that TI-2 antigens decorated with ligands of inhibitory sialic acid–binding Ig-like lectins (siglecs) are poorly immunogenic and can induce tolerance to subsequent challenge with immunogenic antigen. Two siglecs, CD22 and Siglec-G, contributed to tolerance induction, preventing plasma cell differentiation or survival. Although mutations in CD22 and its signaling machinery have been associated with dysregulated B cell development and autoantibody production, previous analyses failed to identify a tolerance defect in antigen-specific mutant B cells. Our results support a role for siglecs in B cell self-/nonself-discrimination, namely suppressing responses to self-associated antigens while permitting rapid “missing self”–responses to unsialylated multimeric antigens. The results suggest use of siglec ligand antigen constructs as an approach for inducing tolerance.B lymphocytes can respond rapidly to nonself-antigens, yet even at mature stages of development can be rendered tolerant if they encounter self-antigen (Goodnow et al., 2005). How B cells distinguish self from nonself has been explained in part by Bretscher and Cohn’s associative recognition (“two-signal”) hypothesis (Bretscher and Cohn, 1970), which posits that B cells can only achieve activation after a second signal is delivered, the first being recognition of antigen by the BCR. Without this second signal, tolerance is induced. In response to T-dependent antigens, activated helper T cells provide this second signal. In a T-independent type 1 response, the second signal might come from the B cells’ Toll-like receptors (TLRs) recognizing conserved microbial motifs attached to the antigen (e.g., lipopolysaccharide; Coutinho et al., 1974). This model, however, fails to explain how T-independent type 2 (TI-2) responses occur, as TI-2 antigens require neither T cells (Mond et al., 1995) nor recognition by known innate immune receptors (Gavin et al., 2006), and can elicit antibody responses in cultures of single B cells (Nossal and Pike, 1984). Although we do not dispute contributory roles of innate immune receptors, cytokines, or accessory cells in amplifying their responses (Mond et al., 1995; Vos et al., 2000; Hinton et al., 2008), TI-2 antigens appear to have only two surprisingly simple properties, high molecular weight and ≥20 closely spaced BCR epitopes (Dintzis et al., 1976), and are thus unlikely to have innate receptors specialized for their recognition.Alternatively, B cells might be capable of “missing self”–recognition (Parish, 1996; Nemazee and Gavin, 2003) similar to that originally observed in NK cells (Kärre et al., 1986). In NK cell recognition, the decision to lyse a target cell depends on integration of opposing signals from activating and inhibitory receptors (Lanier, 2008). Activating receptors trigger recruitment of tyrosine kinases to immunotyrosine activating motifs of associated adapter molecules but are kept in check by inhibitory receptors recognizing classical MHC I molecules expressed on target cells (Lanier, 2008). Inhibitory receptors carry immunotyrosine inhibitory motifs (ITIMs), which serve as docking sites for phosphatases, such as SHP-1, that counteract activation (Ravetch and Lanier, 2000). Target cells that down-regulate MHC I are lysed owing to unopposed activation, hence missing self–recognition.Extrapolating from this model, we hypothesize that besides their BCR epitopes, self-antigens carry self-markers that can engage inhibitory receptors on B cells, preventing antiself TI-2–like responses and rendering activation dependent on second signals. The concept that self-markers might facilitate self-tolerance was first suggested many years ago by Burnet and Fenner (1949) but has garnered little experimental support with respect to lymphocyte tolerance. According to our model, antigens that simultaneously cross-link the BCRs and inhibitory receptors should prevent or blunt B cell responses. Conversely, antigens that bind only the BCR and not inhibitory receptors are predicted to elicit a TI-2 response, provided that they carry the appropriate number and spacing of epitopes. This missing self–model of self-/nonself-discrimination would explain why B cells constitutively express so many inhibitory receptors that recognize ubiquitous self-components, and why null mutations in those receptors or their signaling machinery can lead to autoantibody formation (Nishimura et al., 1998; Pan et al., 1999; Ravetch and Lanier, 2000; Nemazee and Gavin, 2003).In this study, we chose to test if self-/nonself-discrimination is regulated by self-markers through the roles of the sialic acid–binding Ig-like lectins (siglecs) CD22 and Siglec-G in B cells. The siglec family consists of 9 members in mice and 13 members in humans (for review see Crocker et al., 2007). In mice, mature B cells express CD22 (Siglec 2) and Siglec-G, which bind to host sialic acids carried on glycans of glycoproteins and glycolipids and have properties of inhibitory receptors. They carry ITIMs capable of recruiting the tyrosine phosphatase SHP-1 and attenuating BCR signaling (Campbell and Klinman, 1995; Doody et al., 1995; Cornall et al., 1998). Mice carrying null mutations in either CD22 or Siglecg exhibit B cell hyperactivity, variable responses to T-independent antigens, and a tendency toward autoantibody formation (O’Keefe et al., 1996; Otipoby et al., 1996; Sato et al., 1996; Nitschke et al., 1997; Cornall et al., 1998; O’Keefe et al., 1999; Ding et al., 2007; Hoffmann et al., 2007). Mouse CD22 exhibits a strong preference for sialoside ligands with the disaccharide sequence NeuGcα2-6Gal (Collins et al., 2006a; Crocker et al., 2007), whereas Siglec-G, before this study, has had an unknown ligand specificity. Their disaccharide ligands represent terminal sugars commonly carried on N- and O-linked glycans of glycoproteins and are found on virtually all cells, including B cells (Crocker et al., 2007). It is well documented that CD22 binds to glycans on endogenous B cell glycoproteins in cis, and masks the ligand binding site from binding synthetic polymeric ligands (Hanasaki et al., 1995; Razi and Varki, 1998; Razi and Varki, 1999; Collins et al., 2002; Han et al., 2005). Yet, CD22 is able to recognize native ligands on glycoproteins of apposing cells in trans, causing it to redistribute to the site of cell contact (Lanoue et al., 2002; Collins et al., 2004). Although mutations of the ligand binding domain of CD22 (Jin et al., 2002; Poe et al., 2004) and ablation of enzymes involved in the synthesis of its glycan ligands (Hennet et al., 1998; Poe et al., 2004; Collins et al., 2006b; Ghosh et al., 2006; Grewal et al., 2006; Naito et al., 2007; Cariappa et al., 2009) document the importance of siglec ligands in the regulation of CD22 function, a unifying role for CD22 ligand interactions in B cell biology has not yet emerged (Crocker et al., 2007; Walker and Smith, 2008). Although less is known about the specificity of Siglec-G and its interaction with ligands, it is assumed that similar concepts regarding cis and trans ligands will apply to the modulation of Siglec-G function (Hoffmann et al., 2007; Chen et al., 2009).Because siglecs see sialylated glycans that are usually absent from microbes, with the notable exceptions of some pathogenic microbes (Crocker et al., 2007; Carlin et al., 2009a), one possible role of siglecs is to discriminate self from nonself. Though CD22 and Siglec-G have been implicated to play roles in B cell tolerance, evidence has been indirect, inferred from the facts that they possess ITIMs able to recruit SHP-1 and dampen Ca²⁺ signaling (Otipoby et al., 1996; Sato et al., 1996; Nitschke et al., 1997; O’Keefe et al., 1999; Ding et al., 2007; Hoffmann et al., 2007). Hypomorphic or null alleles of CD22 and SHP-1 (Ptpn6) have been correlated with anti-DNA production and development of lupus erythematosus (Shultz et al., 1993; O’Keefe et al., 1999; Mary et al., 2000). CD22 mutations also lead to increased in vivo B cell proliferation and turnover (Otipoby et al., 1996; Nitschke et al., 1997; Poe et al., 2004; Haas et al., 2006; Onodera et al., 2008). However, studies designed to directly assess tolerance induction in antigen-specific CD22^−/− or SHP-1 mutant B cells found, paradoxically, a more robust tolerance relative to unmutated controls (Cyster and Goodnow, 1995; Cornall et al., 1998; Ferry et al., 2005). This suggests that the autoimmune phenotypes of siglec and SHP-1 null mutants could be caused by abnormal B cell selection and development rather than failure of tolerance. It is generally assumed that physical association of CD22 with the BCR will allow CD22 to exert a maximal inhibitory response (Pezzutto et al., 1987; Doody et al., 1995; Lanoue et al., 2002; Courtney et al., 2009), but evidence to support this has been garnered only from in vitro experiments (Ravetch and Lanier, 2000; Lanoue et al., 2002; Tedder et al., 2005; Courtney et al., 2009). In this paper, we show in wild-type mice with unaltered B cell selection and development that decorating a TI-2 antigen with siglec ligands not only prevents its immunogenicity but can also tolerize B cells to subsequent challenges with the unsialylated, immunogenic form. The results suggest that one function of B cell inhibitory receptors like siglecs is to assist B cells in distinguishing self from nonself.     

B cell depletion reduces the development of atherosclerosis in mice

B cell depletion significantly reduces the burden of several immune-mediated diseases. However, B cell activation has been until now associated with a protection against atherosclerosis, suggesting that B cell–depleting therapies would enhance cardiovascular risk. We unexpectedly show that mature B cell depletion using a CD20-specific monoclonal antibody induces a significant reduction of atherosclerosis in various mouse models of the disease. This treatment preserves the production of natural and potentially protective anti–oxidized low-density lipoprotein (oxLDL) IgM autoantibodies over IgG type anti-oxLDL antibodies, and markedly reduces pathogenic T cell activation. B cell depletion diminished T cell–derived IFN-γ secretion and enhanced production of IL-17; neutralization of the latter abrogated CD20 antibody–mediated atheroprotection. These results challenge the current paradigm that B cell activation plays an overall protective role in atherogenesis and identify new antiatherogenic strategies based on B cell modulation.Atherosclerosis-related cardiovascular diseases are the leading cause of mortality worldwide. Immune-mediated reactions initiated in response to multiple potential antigens, including oxidatively modified lipoproteins and phospholipids, play prominent roles in atherosclerotic lesion development, progression, and complications (Binder et al., 2002; Hansson and Libby, 2006; Tedgui and Mallat, 2006). Besides the critical requirement for monocytes/macrophages (Smith et al., 1995), adaptive immunity substantially contributes to the perpetuation of the immunoinflammatory response, further promoting vascular inflammation and lesion development (Binder et al., 2002; Hansson and Libby, 2006; Tedgui and Mallat, 2006). Mice on a severe combined immunodeficiency or Rag-deficient background show reduced susceptibility to atherosclerosis under moderate cholesterol overload (Dansky et al., 1997; Daugherty et al., 1997; Zhou et al., 2000). Resupplementation of these mice with purified T lymphocytes accelerates lesion development (Zhou et al., 2000), even though it does not fully recapitulate lesion development of the immunocompetent mice. The proatherogenic T cells are related to the Th1 lineage (Gupta et al., 1997; Buono et al., 2005), and are counterregulated by both Th2 (Binder et al., 2004; Miller et al., 2008) and T reg cell responses (Ait-Oufella et al., 2006; Tedgui and Mallat, 2006).The development of atherosclerosis is also associated with signs of B cell activation, particularly manifested by enhanced production of natural IgM type and adaptive IgG type anti–oxidized low-density lipoprotein (oxLDL) autoantibodies (Shaw et al., 2000; Caligiuri et al., 2002). However, in contrast to other immune-mediated diseases, i.e., rheumatoid arthritis and systemic lupus erythematosus, B cells have been assigned a protective role in atherosclerosis (Caligiuri et al., 2002; Major et al., 2002; Binder et al., 2004; Miller et al., 2008). Although IgG type anti-oxLDL antibodies show variable association with vascular risk, circulating levels of IgM type anti-oxLDL antibodies have been more frequently linked with reduced vascular risk in humans (Karvonen et al., 2003; Tsimikas et al., 2007). In mice, IL-5– and IL-33–mediated atheroprotective effects have been indirectly associated with specific B1 cell activation and enhanced production of natural IgM type anti-oxLDL antibodies (Binder et al., 2004; Miller et al., 2008). On the other hand, splenectomy (Caligiuri et al., 2002) or transfer of μMT-deficient (B cell–deficient) bone marrow (Major et al., 2002) into lethally irradiated atherosclerosis-susceptible mice resulted in profound reduction of IgG (Caligiuri et al., 2002) or total (Major et al., 2002) anti-oxLDL antibody production, and was associated with acceleration of lesion development. These studies led to the current paradigm that overall B cell activation is atheroprotective. Surprisingly, however, whether mature B cell depletion accelerates atherosclerotic lesion development in immunocompetent mice, as expected from previous studies, is still unexplored. This is a critical question given the potentially important risk of cardiovascular complications that might arise from the clinical use of B cell–depleting CD20-targeted immune therapy in patients with severe rheumatoid arthritis or systemic lupus erythematosus, who are at particularly high risk of cardiovascular diseases (for review see Roman et al., 2001). We have therefore designed a series of experiment to address this important question.     

Co-inhibition of NF-κB and JNK is synergistic in TNF-expressing human AML

Leukemic stem cells (LSCs) isolated from acute myeloid leukemia (AML) patients are more sensitive to nuclear factor κB (NF-κB) inhibition-induced cell death when compared with hematopoietic stem and progenitor cells (HSPCs) in in vitro culture. However, inadequate anti-leukemic activity of NF-κB inhibition in vivo suggests the presence of additional survival/proliferative signals that can compensate for NF-κB inhibition. AML subtypes M3, M4, and M5 cells produce endogenous tumor necrosis factor α (TNF). Although stimulating HSPC with TNF promotes necroptosis and apoptosis, similar treatment with AML cells (leukemic cells, LCs) results in an increase in survival and proliferation. We determined that TNF stimulation drives the JNK–AP1 pathway in a manner parallel to NF-κB, leading to the up-regulation of anti-apoptotic genes in LC. We found that we can significantly sensitize LC to NF-κB inhibitor treatment by blocking the TNF–JNK–AP1 signaling pathway. Our data suggest that co-inhibition of both TNF–JNK–AP1 and NF-κB signals may provide a more comprehensive treatment paradigm for AML patients with TNF-expressing LC.NF-κB is a major mediator of immunity, inflammation, tissue regeneration, and cancer promotion signaling. It regulates multiple cell behaviors such as proliferation, survival, differentiation, and migration (Naugler and Karin, 2008; DiDonato et al., 2012; Perkins, 2012). Leukemic cells (LCs), including leukemic stem cells (LSCs), demonstrate increased NF-κB activity, which provides a critical survival signal (Kuo et al., 2013). In vitro studies demonstrated that NF-κB inhibition can largely eliminate LSC with minimal effects on normal hematopoietic stem and progenitor cells (HSPCs), suggesting the potential for NF-κB inhibition as an anti-leukemia therapy (Guzman et al., 2001). However, the use of NF-κB inhibitors alone in vivo does not effectively eliminate the acute myeloid leukemia (AML) cells, indicating that additional survival signals might be compensating for the effects of NF-κB inhibition. In addition, the clinical use of NF-κB inhibitors is also limited by potential side effects, including compromised T/B cell immunity, inflammatory tissue damage, and skin/liver cancer development (Chen et al., 2001; Zhang et al., 2004, 2007; Maeda et al., 2005; Sakurai et al., 2006; Luedde et al., 2007; Bettermann et al., 2010; Ke et al., 2010).TNF, a pro-inflammatory cytokine, has been shown to be a key mediator of inflammatory reactions in tumor tissues and is responsible for elevated NF-κB activity in many tumors. NF-κB levels are significantly increased in most tumor tissues, being produced by tumor-infiltrating hematopoietic cells, tumor cells, and/or tumor stromal cells (Anderson et al., 2004; Balkwill, 2006; Mantovani et al., 2008; Grivennikov and Karin, 2011; Ren et al., 2012). Animal model studies demonstrate that TNF plays an essential role in the pathogenesis of many types of cancer such as skin, liver, and colon cancers by directly stimulating tumor cell proliferation/survival or by inducing a tumor-promoting environment (Moore et al., 1999; Knight et al., 2000; Sethi et al., 2008; Balkwill, 2009; Oguma et al., 2010). Also, supportive care for some cancers includes inhibition of TNF signaling through use of soluble receptors and neutralizing antibodies (Egberts et al., 2008; Popivanova et al., 2008).Elevated serum TNF levels have been identified in patients with BM failure, including aplastic anemia and myelodysplastic syndromes (MDSs), suggesting that the hematopoietic-repressive activity of TNF might contribute to the cytopenic phenotype of such patients (Molnár et al., 2000; Dybedal et al., 2001; Dufour et al., 2003; Lv et al., 2007). The observed increased levels of TNF during disease progression in MDS patients imply that TNF might also be involved in the leukemic transformation of mutant HSPC (Tsimberidou and Giles, 2002; Stifter et al., 2005; Li et al., 2007; Fleischman et al., 2011). Increased levels of TNF are detected in the peripheral blood (PB) and BM of most human leukemia patients and are correlated to higher white blood cell counts and poorer prognosis (Tsimberidou et al., 2008). In fact, the importance of TNF in leukemogenesis is further documented in Fancc knockout mice and Bcr-Abl–transduced chronic myelogenous leukemia (CML) animal models. In these animals, TNF is required for inducing the leukemic transformation of Fancc mutant cells and promotes the proliferation of CML stem cells (Gallipoli et al., 2013).TNF can stimulate both survival and death signals within the same type of cells in a context-dependent fashion. TNF-dependent survival signals are mediated primarily by canonical NF-κB signaling (Sakurai et al., 2003; Skaug et al., 2009; Vallabhapurapu and Karin, 2009), whereas the TNF-induced death signal is driven by caspase-8–dependent apoptosis or RIP1/3-dependent necroptosis (Wang et al., 2008; He et al., 2009; Zhang et al., 2009, 2011; Feoktistova et al., 2011; Günther et al., 2011; Kaiser et al., 2011; Oberst et al., 2011; Tenev et al., 2011; Xiao et al., 2011). In addition, TNF can also stimulate the activation of MKK4/7-JNK signaling, although the role of the MKK4/7-JNK signaling pathway is also cell context–dependent (Liu and Lin, 2005; Bode and Dong, 2007; Kim et al., 2007; Chen, 2012). Many studies suggest that TNF-induced MKK4/7-JNK signaling is responsible for most of the side effects associated with NF-κB signal inactivation (Chen et al., 2001; Zhang et al., 2004, 2007; Maeda et al., 2005; Sakurai et al., 2006; Luedde et al., 2007; Ke et al., 2010).The role of MKK4/7-JNK signaling in the regulation of hematopoiesis is not entirely clear. Sustained JNK activation has been reported in many types of AML cells, coordinating with AKT/FOXO signaling to maintain an undifferentiated state (Sykes et al., 2011). In Bcr/Abl-induced CML, JNK1-AP1 signaling is required for the development of leukemia by mediating key survival signals (Hess et al., 2002). In Fanconi anemia, JNK is required for the TNF-induced leukemic clonal evolution of Fancc mutant HSPC (Li et al., 2007). These studies suggest that the JNK signal promotes the development and progression of leukemia by inducing proliferative and survival activities (Chen et al., 2001; Zhang et al., 2004, 2007; Maeda et al., 2005; Sakurai et al., 2006; Luedde et al., 2007; Bettermann et al., 2010; Ke et al., 2010).In this study, we searched for the survival signals which compensate for the inhibition of NF-κB signaling in AML stem and progenitor cells. We found that TNF stimulates JNK and NF-κB, which act as parallel survival signals in LC, whereas TNF acts through JNK to induce a death signal in HSPC. Inhibition of TNF-JNK signaling not only significantly sensitizes AML stem and progenitor cells to NF-κB inhibitor treatment but also protects HSPC from the toxicity of such treatment.