Michael A. Oropallo

Role of type I interferons in the activation of autoreactive B cells.

Type I interferons (IFNs) are a family of cytokines involved in the defense against viral infections that play a key role in the activation of both the innate and adaptive immune system. IFNs both directly and indirectly enhance the capacity of B lymphocytes to respond to viral challenge and produce cytotoxic and neutralizing antibodies. However, prolonged type I IFN exposure is not always beneficial to the host. If not regulated properly IFN can drive autoantibody production as well as other parameters of systemic autoimmune disease. Type I IFNs impact B‐cell function through a variety of mechanisms, including effects on receptor engagement, Toll‐like receptor expression, cell migration, antigen presentation, cytokine responsiveness, cytokine production, survival, differentiation and class‐switch recombination. Type I IFNs are also cytotoxic for a variety of cell types and thereby contribute to the accumulation of cell debris that serves as a potential source for autoantigens. Type I IFN engagement of a variety of accessory cells further promotes B‐cell survival and activation, as exemplified by the capacity of type I IFNs to increase the level of B‐cell survival factors, such as B lymphocyte stimulator, produced by dendritic cells. Therefore, it is not surprising that the loss of expression of the type I IFN receptor can have dramatic effects on the production of autoantibodies and on the clinical features of systemic autoimmune diseases such as systemic lupus erythematosus.

Cell-Intrinsic Expression of TLR9 in Autoreactive B Cells Constrains BCR/TLR7-Dependent Responses

Endosomal TLRs play an important role in systemic autoimmune diseases, such as systemic erythematosus lupus, in which DNA- and RNA-associated autoantigens activate autoreactive B cells through TLR9- and TLR7-dependent pathways. Nevertheless, TLR9-deficient autoimmune-prone mice develop more severe clinical disease, whereas TLR7-deficient and TLR7/9–double deficient autoimmune-prone mice develop less severe disease. To determine whether the regulatory activity of TLR9 is B cell intrinsic, we directly compared the functional properties of autoantigen-activated wild-type, TLR9-deficient, and TLR7-deficient B cells in an experimental system in which proliferation depends on BCR/TLR coengagement. In vitro, TLR9-deficient cells are less dependent on survival factors for a sustained proliferative response than are either wild-type or TLR7-deficient cells. The TLR9-deficient cells also preferentially differentiate toward the plasma cell lineage, as indicated by expression of CD138, sustained expression of IRF4, and other molecular markers of plasma cells. In vivo, autoantigen-activated TLR9-deficient cells give rise to greater numbers of autoantibody-producing cells. Our results identify distinct roles for TLR7 and TLR9 in the differentiation of autoreactive B cells that explain the capacity of TLR9 to limit, as well as TLR7 to promote, the clinical features of systemic erythematosus lupus.

Chronic Spinal Cord Injury Impairs Primary Antibody Responses but Spares Existing Humoral Immunity in Mice

Spinal cord injury (SCI) results in immune depression. To better understand how injury inhibits humoral immunity, the effects of chronic thoracic SCI on B cell development and immune responses to thymus-independent type 2 and thymus-dependent Ags were determined. Mice received complete crush injury or control laminectomy at either thoracic level 3, which disrupts descending autonomic control of the spleen, or at thoracic level 9, which conserves most splenic sympathetic activity. Although mature B cell numbers were only mildly reduced, bone marrow B cell production was transiently but profoundly depressed immediately after injury. Despite the return of normal B cell production 4 wk after SCI, mice receiving thoracic level 3 injury showed a significant reduction in their ability to mount primary thymus-independent type 2 or thymus-dependent immune responses. The latter were marked by decreases in germinal center B cells as well as class-switched high-affinity Ab-secreting cells. Importantly, injury did not affect affinity maturation per se, pre-existing B cell memory, or secondary humoral immune responses. Taken together, these findings show that chronic high thoracic SCI impairs the ability to mount optimal Ab responses to new antigenic challenges, but spares previously established humoral immunity.

The role of B lymphocyte stimulator in B cell biology: implications for the treatment of lupus.

B lymphocyte stimulator (BLyS; also known as B cell activating factor (BAFF)) plays a key role in peripheral B cell tolerance. Mounting evidence indicates that B cell tolerance can be either broken or modulated by deliberately manipulating BLyS levels, and belimumab, a BLyS-neutralizing antibody, was recently approved for the treatment of systemic lupus erythematosus (SLE). Thus, intense investigation has focused on understanding how therapeutics targeting BLyS may work, and accumulating evidence suggests multiple points of action. BLyS signaling, in conjunction with B cell receptor (BCR) signaling, determines the size and quality of the mature primary B cell compartment. Moreover, BLyS family members play roles in antigen-experienced B cell selection and differentiation. Together, these findings have implications for the continued development of novel therapeutics that target BLyS.

Position-Dependent Silencing of Germline Vβ Segments on TCRβ Alleles Containing Preassembled VβDJβCβ1 Genes

The genomic organization of TCRβ loci enables Vβ-to-DJβ2 rearrangements on alleles with assembled VβDJβCβ1 genes, which could have deleterious physiologic consequences. To determine whether such Vβ rearrangements occur and, if so, how they might be regulated, we analyzed mice with TCRβ alleles containing preassembled functional VβDJβCβ1 genes. Vβ10 segments were transcribed, rearranged, and expressed in thymocytes when located immediately upstream of a Vβ1DJβCβ1 gene, but not on alleles with a Vβ14DJβCβ1 gene. Germline Vβ10 transcription was silenced in mature αβ T cells. This allele-dependent and developmental stage-specific silencing of Vβ10 correlated with increased CpG methylation and decreased histone acetylation over the Vβ10 promoter and coding region. Transcription, rearrangement, and expression of the Vβ4 and Vβ16 segments located upstream of Vβ10 were silenced on alleles containing either VβDJβCβ1 gene; sequences within Vβ4, Vβ16, and the Vβ4/Vβ16-Vβ10 intergenic region exhibited constitutive high CpG methylation and low histone acetylation. Collectively, our data indicate that the position of Vβ segments relative to assembled VβDJβCβ1 genes influences their rearrangement and suggest that DNA sequences between Vβ segments may form boundaries between active and inactive Vβ chromatin domains upstream of VβDJβCβ genes.

Beyond transitional selection: New roles for BLyS in peripheral tolerance

B‐cell targeted therapies have enjoyed recent success in the treatment of systemic autoimmune diseases. Among these, Belimumab, which blocks the B‐cell survival cytokine BLyS, was recently approved for the treatment of systemic lupus erythematosus. It is therefore important to consider the roles BLyS plays in B‐cell tolerance. Herein, we review how BLyS contributes to the negative selection of autoreactive B‐cell clones from the preimmune repertoire as well as its role in regulating both germinal center and extrafollicular peripheral B‐cell responses. We further examine the complex role of Toll‐like receptors (TLRs) in humoral autoimmunity, pointing out potential crosstalk between BLyS and TLR pathways. Drug Dev Res 72:779–787, 2011.

A TLR9-dependent checkpoint governs B cell responses to DNA-containing antigens

Mature B cell pools retain a substantial proportion of polyreactive and self-reactive clonotypes, suggesting that activation checkpoints exist to reduce the initiation of autoreactive B cell responses. Here, we have described a relationship among the B cell receptor (BCR), TLR9, and cytokine signals that regulate B cell responses to DNA-containing antigens. In both mouse and human B cells, BCR ligands that deliver a TLR9 agonist induce an initial proliferative burst that is followed by apoptotic death. The latter mechanism involves p38-dependent G1 cell-cycle arrest and subsequent intrinsic mitochondrial apoptosis and is shared by all preimmune murine B cell subsets and CD27– human B cells. Survival or costimulatory signals rescue B cells from this fate, but the outcome varies depending on the signals involved. B lymphocyte stimulator (BLyS) engenders survival and antibody secretion, whereas CD40 costimulation with IL-21 or IFN-&ggr; promotes a T-bet+ B cell phenotype. Finally, in vivo immunization studies revealed that when protein antigens are conjugated with DNA, the humoral immune response is blunted and acquires features associated with T-bet+ B cell differentiation. We propose that this mechanism integrating BCR, TLR9, and cytokine signals provides a peripheral checkpoint for DNA-containing antigens that, if circumvented by survival and differentiative cues, yields B cells with the autoimmune-associated T-bet+ phenotype.

Spinal cord injury impacts B cell production, homeostasis, and activation

Complex interactions govern the interplay of central nervous and immune systems, including the generation, homeostatic maintenance, and activation of B cells. Accordingly, spinal cord injury will likely impact all of these processes. Several laboratories have recently explored this possibility, and their observations in aggregate reveal both acute and chronic consequences that can vary based on the injury location. Acute effects include a transient cessation of bone marrow B lymphopoiesis, with a corresponding drop in the peripheral follicular and transitional B cell subsets, whereas the marginal zone subset is preserved. Despite recovery of B lymphopoiesis by 28 days post injury, follicular B cell numbers remain depressed; this may reflect reduced levels of the homeostatic cytokine BLyS. In general, the ability to mount T dependent antibody responses after injury are intact, as are pre-existing memory B cell pools and antibody levels. In contrast, T-independent responses are chronically compromised. Both glucocorticoid-dependent and -independent processes mediate these effects, but a detailed understanding of the mechanisms involved awaits further study. Nonetheless, these observations in toto strengthen the growing appreciation for bidirectional interactions between the CNS and immune system, highlighting the need for further basic and translational efforts.

Understanding B Cell Biology

Humoral autoimmunity reflects failures in B cell tolerance and regulation. Accordingly, B cells have long been proposed as targets for treating autoimmune disease. The last decade has witnessed substantial growth in the number of therapeutic agents that target B cells themselves, or molecules key to B cell survival or function. In order to understand, develop, and eventually predict the outcomes of B cell targeted therapies, a thorough understating of the mechanisms underlying B cell development, activation, and regulation is necessary. Here we summarize B cell genesis, differentiation, and tolerance, and illustrate how an understanding of basic B cell biology can afford insight into the design and action of therapeutic agents.