Indoleamine 2,3-dioxygenase as a modifier of pathogenic inflammation in cancer and other inflammation-associated diseases.

Chronic inflammation underlies the basis for development and progression of cancers and a variety of other disorders, but what specifically defines its pathogenic nature remains largely undefined. Recent genetic and pharmacological studies in the mouse suggest that the immune modulatory enzyme indoleamine 2,3-dioxygenase (IDO), identified as an important mediator of immune escape in cancer, can also contribute to the development of pathology in the context of chronic inflammatory models of arthritis and allergic airway disease. IDO-deficient mice do not display spontaneous disorders of classical inflammation and small molecule inhibitors of IDO do not elicit generalized inflammatory reactions. Rather, in the context of a classical model of skin cancer that is promoted by chronic inflammation, or in models of inflammation-associated arthritis and allergic airway disease, IDO impairment can alleviate disease severity. Here we offer a survey of preclinical literature suggesting that IDO functions as a modifier of inflammatory states rather than simply as a suppressor of immune function. We propose that IDO induction in a chronically inflamed tissue may shape the inflammatory state to support, or in some cases retard, pathogenesis and disease severity.

Signaling through TNF receptor p55 in TNF-alpha-deficient mice alters the CXCL13/CCL19/CCL21 ratio in the spleen and induces maturation and migration of anergic B cells into the B cell follicle.

The organization of secondary lymphoid tissues into distinct T and B cell compartments supports proper regulation of an immune response to foreign Ags. In the splenic white pulp, this compartmentalization is also thought to be important in the maintenance of B cell tolerance. Using lymphotoxin-alpha-(LT-alpha)-, TNF-alpha-, or TNFRp55-deficient mice, all with disrupted splenic architecture, we tested whether normal T/B segregation and/or intact follicular structure are necessary for the maintenance of anti-dsDNA B cell anergy. This study demonstrates that anti-dsDNA B cells remain tolerant in LT-alpha(-/-), TNF-alpha(-/-), and TNFRp55(-/-) mice; however, TNF-alpha or a TNF-alpha-dependent factor is required for their characteristic positioning to the T/B interface. Providing a TNF-alpha signal in TNF-alpha(-/-) mice by systemic administration of an agonist anti-TNFRp55 mAb induces the maturation of the anti-dsDNA B cells and their movement away from the T cell area toward the B cell area. Additionally, the agonist Ab induces changes in the follicular environment, including FDC clustering, up-regulation of the CXC chemokine ligand CXCL13, and down-regulation of the CC chemokine ligands CCL19 and CCL21. Therefore, this study suggests that a balance between B and T cell tropic chemokine signals may be an important mechanism for positioning anergic B cells at the T/B interface of the splenic white pulp.

Alterations in splenic architecture and the localization of anti-double-stranded DNA B cells in aged mice.

Aging is characterized by a decline in humoral immunity and a concommitant increased incidence of anti-DNA and other autoantibodies. To define how the regulation of autoreactive B cells is altered with age, we have used BALB/c mice with an Ig heavy H chain transgene to track the fate of anti-double-stranded (ds) DNA B cells in vivo. In young adult mice, anti-dsDNA B cells are developmentally arrested and excluded from the splenic B cell follicle, whereas in most aged mice they are mature and localize within the B cell follicle. Furthermore, we have detailed global changes in lymphoid architecture that accompany aging: CD4(+) T cells are found not only in the periarteriolar lymphoid sheath, but also in the B cell follicles. Strikingly, these disruptions are similar to those that precede serum anti-dsDNA antibody expression in autoimmune MRL-lpr/lpr mice.

The origin of anti-nuclear antibodies in bcl-2 transgenic mice.

bcl-2 transgenic mice develop anti-double-stranded (ds) DNA antibodies similar to those present in systemic lupus erythematosus. To begin to understand where a breakdown in the regulation of autoreactive lymphocytes is occurring, we have used a bcl-2 transgene (Tg) in conjunction with an Ig Tg that allows us to identify and track anti-dsDNA B cells. Previously, we have shown that anti-dsDNA B cells are actively tolerized in BALB/c mice as manifested by their developmental arrest, follicular exclusion, increased in vivo turnover rate and lack of their antibody in the serum. The bcl-2 Tg mice increased the lifespan of anti-dsDNA B cells, but did not alter the other features of tolerance, indicating that the anergy of the anti-dsDNA B cells is independent of their reduced lifespan. Furthermore, these data suggest that the serum anti-dsDNA antibodies in bcl-2 transgenic mice are not due to a breakdown in the induction or maintenance of B cell anergy; rather they may originate from B cells that have transited through a germinal center.

Functional consequences of the developmental arrest and follicular exclusion of anti-double-stranded DNA B cells.

Anti-dsDNA B cells are actively tolerized in nonautoimmune BALB/c mice, as manifested by their developmental arrest, follicular exclusion, and rapid turnover rate. Previously, we have documented changes in the maturation status and follicular localization of anti-dsDNA B cells in autoimmune-prone MRL (+/+ and lpr/lpr) mice. To determine whether these differences in developmental status and follicular localization affect the functional capacity of anti-dsDNA B cells, we have now compared their in vivo life spans and their responses to in vitro stimuli. Our study shows that although anti-dsDNA B cells from both BALB/c and MRL-+/+ mice are localized to the T/B interface, only those in BALB/c mice have a rapid turnover rate. Therefore, the immature status and not the exclusion from the B cell follicle correlates with a shortened life span. Interestingly, apoptotic anti-dsDNA B cells were not detected at the T/B interface in BALB/c mice, suggesting that they are not dying there. This study also demonstrates that anti-dsDNA B cells, regardless of maturation status or follicular localization, are able to proliferate and up-regulate the costimulatory molecule B7-2 in response to CD40 ligand and IL-4. Therefore, one of the critical in vivo differences between anti-dsDNA B cells in BALB/c and MRL-+/+ mice compared with MRL-lpr/lpr mice may be the availability of T cell help.

MRL-lpr/lpr mice exhibit a defect in maintaining developmental arrest and follicular exclusion of anti-double-stranded DNA B cells.

A hallmark of systemic lupus erythematosus and the MRL murine model for lupus is the presence of anti-double-stranded (ds)DNA antibodies (Abs). To identify the steps leading to the production of these Abs in autoimmune mice, we have compared the phenotype and localization of anti-dsDNA B cells in autoimmune (MRL+/+ and lpr/lpr) mice with that in nonautoimmune (BALB/c) mice. Anti-dsDNA B cells are actively regulated in BALB/c mice as indicated by their developmental arrest and accumulation at the T-B interface of the splenic follicle. In the MRL genetic background, anti-dsDNA B cells are no longer developmentally arrested, suggesting an intrinsic B cell defect conferred by MRL background genes. With intact Fas, they continue to exhibit follicular exclusion; however, in the presence of the lpr/lpr mutation, anti-dsDNA B cells are now present in the follicle. Coincident with the altered localization of anti-dsDNA B cells is a follicular infiltration of CD4 T cells. Together, these data suggest that MRL mice are defective in maintaining the developmental arrest of autoreactive B cells and indicate a role for Fas in restricting entry into the follicle.

Characterization of anergic anti-DNA B cells: B cell anergy is a T cell-independent and potentially reversible process.

Anti-single stranded DNA (ssDNA) and anti-double stranded DNA (dsDNA) B cells are regulated in non-autoimmune mice. In this report we show that while both anti-ssDNA and anti-dsDNA B cells are blocked in their ability to differentiate into antibody-secreting cells, other phenotypic and functional characteristics distinguish them from one another. Splenic anti-ssDNA B cells are found distributed throughout the B cell follicle, and are phenotypically mature and long-lived. On the other hand, splenic anti-dsDNA B cells are short-lived, exhibit an immature and antigen-experienced phenotype, and localize to the T-B interface of the splenic follicle. Functionally, anti-ssDNA B cells proliferate, albeit suboptimally, in response to anti-IgM, lipopolysaccharide (LPS) and CD40L/IL-4 + anti-IgM stimulation, and tyrosine phosphorylate intracellular proteins upon mIgM cross-linking. Anti-dsDNA B cells, on the other hand, are functionally unresponsive to anti-IgM and LPS stimulation, and do not phosphorylate intracellular proteins, including Syk, upon mIg stimulation. Importantly, anti-DNA B cell anergy is maintained in the absence of T cells since both anti-ssDNA and anti-dsDNA B cells are as efficiently regulated in RAG2(-/-) mice as in their RAG2(+/+) counterparts. Interestingly, the severely anergic state of anti-dsDNA B cells is partially reversible upon stimulation with CD40 ligand and IL-4. In response to these signals, anti-dsDNA B cells remain viable, up-regulate cell surface expression of B7-2 and IgM, and restore their ability to proliferate and phosphorylate Syk upon mIg cross-linking. Collectively, these data suggest that anti-DNA B cell anergy encompasses distinct phenotypes which, even in its most severe form, may be reversible upon stimulation with T cell-derived factors.

Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: localization to the T-B interface of the splenic follicle.

Systemic lupus erythematosus (SLE) and the MRL-lpr/lpr murine model for SLE are characterized by the presence of serum anti-double-stranded (ds)DNA antibodies (Abs), whereas nonautoimmune individuals have negligible levels of these Abs. To increase the frequency of anti-DNA B cells and identify the mechanisms involved in their regulation in nonautoimmune mice, we have used Ig transgenes (tgs). In the present study, we used the VH3H9 heavy (H) chain tg which expresses an H chain that was repeatedly isolated from anti-dsDNA Abs from MRL-lpr/lpr mice. Because the VH3H9 H chain can pair with endogenous L chains to generate anti-single-stranded DNA, anti-dsDNA, and non-DNA B cells, this allowed us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire. We have identified anti-dsDNA B cells that are located at the T-B interface in the splenic follicle where they have an increased in vivo turnover rate. These anti-dsDNA B cells exhibit a unique surface phenotype suggesting developmental arrest due to antigen exposure.