Jaime L. Cullen

Exacerbated autoimmunity in the absence of TLR9 in MRL.Fas(lpr) mice depends on Ifnar1.

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 IFNs (IFN-I) 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 IFN-I, Ifnar1, we show that IFN-I contribute significantly to renal disease in this model. Ifnar1 had no effect on anti-nucleosome or anti-Sm autoantibody titers, but instead regulated anticytoplasmic and anti-RNA specificities. Moreover, Ifnar1 deficiency prevented the exacerbation of clinical disease observed in Tlr9-deficient animals in this lupus model. Thus, IFN-I signaling is an important mediator of lupus pathogenesis and anti-RNA Ab production that is dysregulated in the absence of Tlr9.

TLR9 Promotes Tolerance by Restricting Survival of Anergic Anti-DNA B Cells, Yet Is Also Required for Their Activation

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. In this study, 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 L chain editing during B cell development in the bone marrow, TLR9 limits anti-DNA B cell life span in the periphery and is thus tolerogenic. In the absence of TLR9, anti-DNA B cells have much longer life spans 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, whereas 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 Ab-forming cells 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.

Facultative role for T cells in extrafollicular Toll-like receptor-dependent autoreactive B-cell responses in vivo

Extrafollicular (EF) B-cell responses are increasingly being recognized as an alternative pathway of B-cell activation, particularly in autoimmunity. Critical cellular interactions required for the EF B-cell response are unclear. A key question in autoimmunity, in which Toll-like receptor (TLR) signals are costimulatory and could be sufficient for B-cell activation, is whether T cells are required for the response. This is pivotal, because autoreactive B cells are considered antigen-presenting cells for autoreactive T cells, but where such interactions occur has not been identified. Here, using AM14 site-directed transgenic rheumatoid factor (RF) mice, we report that B cells can be activated, differentiate, and isotype-switch independent of antigen-specific T-cell help, αβ T cells, CD40L signaling, and IL-21 signaling to B cells. However, T cells do dramatically enhance the response, and this occurs via CD40L and IL-21 signals. Surprisingly, the response is completely inducible T-cell costimulator ligand independent. These results establish that, although not required, T cells substantially amplify EF autoantibody production and thereby implicate T-independent autoreactive B cells as a potential vector for breaking T-cell tolerance. We suggest that these findings explain why autoreactivity first focuses on self-components for which B cells carry TLR ligands, because these will uniquely be able to activate B cells independently of T cells, with subsequent T–B interactions activating autoreactive T cells, resulting in chronic autoimmunity.

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 (MBCs), which are typically generated via germinal centers. The development and functions of autoreactive MBCs 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 corecognition, raising the question of whether MBCs are generated in this context. In this study, we investigated autoreactive MBC generation associated with this type of response. We transferred B cells from AM14 site-directed BCR transgenic mice into nontransgenic 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 wk. 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 plasmablast 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 MBCs that share some, but not all, of the characteristics of traditional MBCs. Such cells could play a role in chronic or flaring autoimmune disease.

Local Triggering of the ICOS Coreceptor by CD11c+ Myeloid Cells Drives Organ Inflammation in Lupus

The inducible T cell costimulator (ICOS) is a potent promoter of organ inflammation in murine lupus. ICOS stimulates T follicular helper cell differentiation in lymphoid tissue, suggesting that it might drive autoimmunity by enhancing autoantibody production. Yet the pathogenic relevance of this mechanism remains unclear. It is also unknown whether other ICOS-induced processes might contribute to lupus pathology. Here we show that selective ablation of ICOS ligand (ICOSL) in CD11c(+) cells, but not in B cells, dramatically ameliorates kidney and lung inflammation in lupus-prone MRL.Fas(lpr) mice. Autoantibody formation was largely unaffected by ICOSL deficiency in CD11c(+) cells. However, ICOSL display by CD11c(+) cells in inflamed organs had a nonredundant role in protecting invading T cells from apoptosis by elevating activity of the PI3K-Akt signaling pathway, thereby facilitating T cell accrual. These findings reveal a mechanism that locally sustains organ inflammation in lupus.

Activation of Rheumatoid Factor–Specific B Cells Is Antigen Dependent and Occurs Preferentially Outside of Germinal Centers in the Lupus-Prone NZM2410 Mouse Model

AM14 rheumatoid factor (RF) B cells in the MRL/lpr mice are activated by dual BCR and TLR7/9 ligation and differentiate into plasmablasts via an extrafollicular (EF) route. It was not known whether this mechanism of activation of RF B cells applied to other lupus-prone mouse models. We investigated the mechanisms by which RF B cells break tolerance in the NZM2410-derived B6.Sle1.Sle2.Sle3 (TC) strain in comparison with C57BL/6 (B6) controls, each expressing the AM14 H chain transgene in the presence or absence of the IgG2aa autoantigen. The TC, but not B6, genetic background promotes the differentiation of RF B cells into Ab-forming cells (AFCs) in the presence of the autoantigen. Activated RF B cells preferentially differentiated into plasmablasts in EF zones. Contrary to the MRL/lpr strain, TC RF B cells were also located within germinal centers, but only the formation of EF foci was positively correlated with the production of RF AFCs. Immunization of young TC.AM14 H chain transgenic mice with IgG2aa anti-chromatin immune complexes (ICs) activated RF B cells in a BCR- and TLR9-dependent manner. However, these IC immunizations did not result in the production of RF AFCs. These results show that RF B cells break tolerance with the same general mechanisms in the TC and the MRL/lpr lupus-prone genetic backgrounds, namely the dual activation of the BCR and TLR9 pathways. There are also distinct differences, such as the presence of RF B cells in GCs and the requirement of chronic IgG2aa anti-chromatin ICs for full differentiation of RF AFCs.

Requirement for Transcription Factor Ets1 in B Cell Tolerance to Self-Antigens

The differentiation and survival of autoreactive B cells is normally limited by a variety of self-tolerance mechanisms, including clonal deletion, anergy, and clonal ignorance. The transcription factor c-ets-1 (encoded by the Ets1 gene) has B cell–intrinsic roles in regulating formation of Ab-secreting cells by controlling the activity of Blimp1 and Pax5 and may be required for B cell tolerance to self-antigen. To test this, we crossed Ets1−/− mice to two different transgenic models of B cell self-reactivity, the anti–hen egg lysozyme BCR transgenic strain and the AM14 rheumatoid factor transgenic strain. BCR transgenic Ets1−/− mice were subsequently crossed to mice either carrying or lacking relevant autoantigens. We found that B cells lacking c-ets-1 are generally hyperresponsive in terms of Ab secretion and form large numbers of Ab-secreting cells even in the absence of cognate Ags. When in the presence of cognate Ag, different responses were noted depending on the physical characteristics of the Ag. We found that clonal deletion of highly autoreactive B cells in the bone marrow was intact in the absence of c-ets-1. However, peripheral B cells lacking c-ets-1 failed to become tolerant in response to stimuli that normally induce B cell anergy or B cell clonal ignorance. Interestingly, high-affinity soluble self-antigen did cause B cells to adopt many of the classical features of anergic B cells, although such cells still secreted Ab. Therefore, maintenance of appropriate c-ets-1 levels is essential to prevent loss of self-tolerance in the B cell compartment.

Spontaneous Loss of Tolerance of Autoreactive B Cells in Act1-Deficient Rheumatoid Factor Transgenic Mice

Self-reactive B cells in BALB/c AM14 transgenic (Tg) rheumatoid factor mice are not subject to central or peripheral tolerization. Instead, they remain at a stage of “clonal ignorance”; that is, they do not proliferate and differentiate into Ab-forming 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 Abs. Three- to 5-mo-old AM14 Tg Act1−/− mice showed significant expansion of AM14 Tg B cells, including a 2- to 3-fold increase in the spleen and cervical lymph nodes compared with 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 Ab-forming cells. 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 germinal center 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.

B Cell–Extrinsic Myd88 and Fcer1g Negatively Regulate Autoreactive and Normal B Cell Immune Responses

MyD88 and FcR common γ-chain (Fcer1g, FcRγ) elicit proinflammatory responses to exogenous Ags. Deletion of these receptors in autoimmune models has generally led to reduced overall disease. In B cells, Myd88 is required for anti-DNA and anti-RNA autoantibody responses, whereas Fcer1g is not expressed in these cells. The roles of these receptors in myeloid cells during B cell autoimmune activation remain less clear. To investigate the roles of Myd88 and Fcer1g in non-B cells, we transferred anti–self-IgG (rheumatoid factor) B cells and their physiologic target Ag, anti-chromatin Ab, into mice lacking Fcer1g, Myd88, or both and studied the extrafollicular plasmablast response. Surprisingly, we found a markedly higher and more prolonged response in the absence of either molecule; this effect was accentuated in doubly deficient recipients, with a 40-fold increase compared with wild-type recipients at day 10. This enhancement was dependent on CD40L, indicating that Myd88 and FcRγ, presumably on myeloid APCs, were required to downregulate T cell help for the extrafollicular response. To extend the generality, we then investigated a classic T cell–dependent response to (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken γ globulin and found a similar effect. Thus, these results reveal novel regulatory roles in the B cell response for receptors that are typically proinflammatory.