Protective antiviral antibody responses in a mouse model of influenza virus infection require TACI.

Antiviral Abs, for example those produced in response to influenza virus infection, are critical for virus neutralization and defense against secondary infection. While the half-life of Abs is short, Ab titers can last a lifetime due to a subset of the Ab-secreting cells (ASCs) that is long lived. However, the mechanisms governing ASC longevity are poorly understood. Here, we have identified a critical role for extrinsic cytokine signals in the survival of respiratory tract ASCs in a mouse model of influenza infection. Irradiation of mice at various time points after influenza virus infection markedly diminished numbers of lung ASCs, suggesting that they are short-lived and require extrinsic factors in order to persist. Neutralization of the TNF superfamily cytokines B lymphocyte stimulator (BLyS; also known as BAFF) and a proliferation-inducing ligand (APRIL) reduced numbers of antiviral ASCs in the lungs and bone marrow, whereas ASCs in the spleen and lung-draining lymph node were surprisingly unaffected. Mice deficient in transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), a receptor for BLyS and APRIL, mounted an initial antiviral B cell response similar to that generated in WT mice but failed to sustain protective Ab titers in the airways and serum, leading to increased susceptibility to secondary viral challenge. These studies highlight the importance of TACI signaling for the maintenance of ASCs and protection against influenza virus infection.

ICOS expression by effector T cells influences the ability of regulatory T cells to inhibit anti-chromatin B cell responses in recipient mice.

T regulatory cells are critical for the prevention of autoimmunity. Specifically, Treg cells can control anti-chromatin antibody production in vivo, and this correlates with decreased ICOS expression on CD4(+) T helper cells. Here we test the significance of high ICOS expression by T effector cells, firstly in terms of the anti-chromatin B cell response, and secondly on the ability of Treg cells to suppress T cell help. We bred CD4(+) T cell receptor transgenic mice with mice that carry the Roquin(san/san) mutation. The Roquin gene functions to limit ICOS mRNA such that CD4 T cells from mutant mice express elevated ICOS. Using an in vivo model, TS1.Roquin(san/san) Th cells were compared with wild-type TS1 Th cells with regard to their ability to help anti-chromatin B cells in the presence or absence of Treg cells. Both TS1 and TS1.Roquin(san/san) Th cells induced anti-chromatin IgM(a) antibodies, but the TS1.Roquin(san/san) Th cells resulted in the recovery of more class-switched and germinal center B cells. Neither source of Th cells were capable of inducing long-lived autoantibodies. Treg cells completely suppressed anti-chromatin IgM(a) antibody production and reduced anti-chromatin B cell recovery induced by TS1 Th cells. Importantly, this suppression was less effective when TS1.Roquin(san/san) Th cells were used. Thus, high ICOS levels on effector T cells results in autoimmunity by augmenting the autoreactive B cell response and by dampening the effect of Treg cell suppression.

Efficient help for autoreactive B-cell activation requires CD4+ T-cell recognition of an agonist peptide at the effector stage.

T-cell recognition of peptide/MHC complexes is flexible and can lead to differential activation, but how interactions with agonist (full activation) or partial agonist (suboptimal activation) peptides can shape immune responses in vivo is not well characterized. We investigated the effect of stimulation by agonist or partial agonist ligands during initial CD4(+) T-cell priming, and subsequent T-B-cell cognate interactions, on antibody production by anti-chromatin B cells. We found that autoantibody production required TCR recognition of an agonist peptide at the effector stage of B-cell activation. However, interaction with a weak agonist ligand at this effector stage failed to promote efficient autoantibody production, even if the CD4(+) T cells were fully primed by an agonist peptide. These studies suggest that the reactivity of the TCR for a target self-peptide during CD4(+) T-B-cell interaction can be a critical determinant in restraining anti-chromatin autoantibody production.

B cell activator PAX5 promotes lymphomagenesis through stimulation of B cell receptor signaling.

The presumed involvement of paired box gene 5 (PAX5) in B-lymphomagenesis is based largely on the discovery of Pax5-specific translocations and somatic hypermutations in non-Hodgkin lymphomas. Yet mechanistically, the contribution of Pax5 to neoplastic growth remains undeciphered. Here we used 2 Myc-induced mouse B lymphoma cell lines, Myc5-M5 and Myc5-M12, which spontaneously silence Pax5. Reconstitution of these cells with Pax5-tamoxifen receptor fusion protein (Pax5ER(TAM)) increased neoplastic growth in a hormone-dependent manner. Conversely, expression of dominant-negative Pax5 in murine lymphomas and Pax5 knockdown in human lymphomas negatively affected cell expansion. Expression profiling revealed that Pax5 was required to maintain mRNA levels of several crucial components of B cell receptor (BCR) signaling, including CD79a, a protein with the immunoreceptor tyrosine-based activation motif (ITAM). In contrast, expression of 2 known ITAM antagonists, CD22 and PIR-B, was suppressed. The key role of BCR/ITAM signaling in Pax5-dependent lymphomagenesis was corroborated in Syk, an ITAM-associated tyrosine kinase. Moreover, we observed consistent expression of phosphorylated BLNK, an activated BCR adaptor protein, in human B cell lymphomas. Thus, stimulation of neoplastic growth by Pax5 occurs through BCR and is sensitive to genetic and pharmacological inhibitors of this pathway.

The role of BLyS/BLyS receptors in anti-chromatin B cell regulation.

B lymphocyte stimulator (BLyS), also known as B cell-activating factor, is a key positive regulator of B cell homeostasis, and elevated levels of BLyS have been observed in systemic lupus erythematosus (SLE) patients. Given that anti-chromatin auto-antibodies are one of the hallmarks of SLE, we examined the role of BLyS and its receptors in the regulation of anti-chromatin B cells. We demonstrate that exogenous BLyS treatment leads to an increase in B cell numbers, particularly anti-chromatin B cells; yet, their localization in the spleen and auto-antibody production remain unaffected. We also examined transmembrane activator and CAML interactor (TACI), BLyS receptor 3 (BR3) and B cell maturation antigen expression on anti-chromatin B cells before and after receiving T cell help. Interestingly, in the absence of T cell help, TACI expression is greater on immature anti-chromatin B cells compared with immature Tg(-) B cells, whereas BR3 levels are comparable. After receiving T cell help, the anti-chromatin B cells that have differentiated into short-lived plasma cells no longer express BR3 but retain TACI. These data suggest a novel role for TACI in anti-chromatin B cell homeostasis and differentiation.

Exogenous and endogenous TLR ligands activate anti-chromatin and polyreactive B cells.

Autoreactive B cells may become activated in a T-independent manner via synergistic engagement of the BCR and TLRs. Using the VH3H9 Ig H chain transgene to track anti-chromatin B cells, we demonstrate that VH3H9/Vlambda1 anti-chromatin B cells proliferate in response to stimulatory oligodeoxynucleotides containing CpG motifs, suggesting that these autoreactive B cells are responsive to TLR9 signaling. Strikingly, some VH3H9 B cells, but not the well-characterized VH3H9/Vlambda1 B cells, proliferate spontaneously in culture medium. This proliferation is blocked by inhibitory CpG oligodeoxynucleotides, implicating the TLR9 (or possibly TLR7) pathway. Most hybridomas generated from the proliferating cells are polyreactive, and one exhibits binding to nuclear Ags but not to the other Ags tested. Thus, B cells carrying autoreactive and/or polyreactive specificities may be susceptible to T cell-independent activation via dual engagement of the BCR and TLRs.

CD4+ CD25+ regulatory T cells inhibit the maturation but not the initiation of an autoantibody response.

To investigate the mechanism by which T regulatory (Treg) cells may control the early onset of autoimmunity, we have used an adoptive transfer model to track Treg, Th, and anti-chromatin B cell interactions in vivo. We show that anti-chromatin B cells secrete Abs by day 8 in vivo upon provision of undeviated, Th1- or Th2-type CD4+ T cell help, but this secretion is blocked by the coinjection of CD4+ CD25+ Treg cells. Although Treg cells do not interfere with the initial follicular entry or activation of Th or B cells at day 3, ICOS levels on Th cells are decreased. Furthermore, Treg cells must be administered during the initial phases of the Ab response to exert full suppression of autoantibody production. These studies indicate that CD25+ Treg cells act to inhibit the maturation, rather than the initiation, of autoantibody responses.

The influence of effector T cells and Fas ligand on lupus-associated B cells.

Circulating autoantibodies against dsDNA and chromatin are a characteristic of systemic lupus erythematosus in humans and many mouse models of this disease. B cells expressing these autoantibodies are normally regulated in nonautoimmune-prone mice but are induced to secrete Abs following T cell help. Likewise, anti-chromatin autoantibody production is T cell-dependent in Fas/Fas ligand (FasL)-deficient (lpr/lpr or gld/gld) mice. In this study, we demonstrate that Th2 cells promote anti-chromatin B cell survival and autoantibody production in vivo. FasL influences the ability of Th2 cells to help B cells, as Th2-gld/gld cells support higher titers of anti-chromatin Abs than their FasL-sufficient counterparts and promote anti-chromatin B cell participation in germinal centers. Th1 cells induce anti-chromatin B cell germinal centers regardless of FasL status; however, their ability to stimulate anti-chromatin Ab production positively correlates with their level of IFN-gamma production. This distinction is lost if FasL-deficient T cells are used: Th1-gld/gld cells promote significant titers of anti-chromatin Abs regardless of IFN-gamma production levels. Thus, FasL from effector T cells plays an important role in determining the fate of anti-chromatin B cells.

The regulation and activation of lupus-associated B cells.

Anti-double-stranded DNA (anti-dsDNA) B cells are regulated in non-autoimmune mice. While some are deleted or undergo receptor editing, a population of anti-dsDNA (VH3H9/V lambda 1) B cells that emigrate into the periphery has also been identified. These cells have an altered phenotype relative to normal B cells in that they have a reduced lifespan, appear developmentally arrested, and localize primarily to the T/B-cell interface in the spleen. This phenotype may be the consequence of immature B cells encountering antigen in the absence of T-cell help. When provided with T-cell help, the anti-dsDNA B cells differentiate into antibody-forming cells. In the context of the autoimmune-prone lpr/lpr or gld/gld mutations, the VH3H9/V lambda 1 anti-dsDNA B cells populate the B-cell follicle and by 12 weeks of age produce serum autoantibodies. The early event of anti-dsDNA B-cell follicular entry, in the absence of autoantibody production, is dependent upon CD4(+) T cells. We hypothesize that control of autoantibody production in young autoimmune-prone mice may be regulated by the counterbalancing effect of T-regulatory (T(reg)) cells. Consistent with this model, we have demonstrated that T(reg) cells are able to prevent autoantibody production induced by T-cell help. Additional studies are aimed at investigating the mechanisms of this suppression as well as probing the impact of distinct forms of T-cell-dependent and -independent activation on anti-dsDNA B cells.