T cell costimulation through CD28 depends on induction of the Bcl-xgamma isoform: analysis of Bcl-xgamma-deficient mice

The molecular basis of CD28-dependent costimulation of T cells is poorly understood. Bcl-xgamma is a member of the Bcl-x family whose expression is restricted to activated T cells and requires CD28-dependent ligation for full expression. We report that Bcl-xgamma-deficient (Bcl-xgamma-/-) T cells display defective proliferative and cytokine responses to CD28-dependent costimulatory signals, impaired memory responses to proteolipid protein peptide (PLP), and do not develop PLP-induced experimental autoimmune encephalomyelitis (EAE). In contrast, enforced expression of Bcl-xgamma largely replaces the requirement for B7-dependent ligation of CD28. These findings identify the Bcl-xgamma cytosolic protein as an essential downstream link in the CD28-dependent signaling pathway that underlies T cell costimulation.     

T cell-dependent and -independent pathways to tissue destruction following herpes simplex virus-1 infection

Viral infections can trigger tissue destruction through innate and/or adaptive immune mechanisms. Here we show that these pathways can be differentially activated after infection by different strains of the herpes simplex virus-1 (HSV-1) virus. Infection of murine corneal tissue by HSV-1 (KOS) triggers an autoreactive clone of CD4 cells that is cross-reactive with an HSV-1 epitope to initiate corneal destruction. In contrast, ocular infection by the HSV-1 (RE) strain induces murine corneal destruction through direct, T cell-independent, activation of the innate immune system. Although the relative role of these two pathways to blindness following clinical HSV-1 ocular infection is unknown, this analysis suggests a general experimental approach to evaluate the relative contribution of adaptive and innate immune mechanisms to virally induced host tissue destruction.     

Suppression of autoimmune disease after vaccination with autoreactive T cells that express Qa-1 peptide complexes

The ability of autoreactive T cells to provoke autoimmune disease is well documented. The finding that immunization with attenuated autoreactive T cells (T cell vaccination, or TCV) can induce T cell-dependent inhibition of autoimmune responses has opened the possibility that regulatory T cells may be harnessed to inhibit autoimmune disease. Progress in the clinical application of TCV, however, has been slow, in part because the underlying mechanism has remained clouded in uncertainty. We have investigated the molecular basis of TCV-induced disease resistance in two murine models of autoimmunity: herpes simplex virus-1 (KOS strain)-induced herpes stromal keratitis and murine autoimmune diabetes in non-obese diabetic (NOD) mice. We find that the therapeutic effects of TCV depend on activation of suppressive CD8 cells that specifically recognize Qa-1-bound peptides expressed by autoreactive CD4 cells. We clarify the molecular interaction between Qa-1 and self peptides that generates biologically active ligands capable of both inducing suppressive CD8 cells and targeting them to autoreactive CD4 cells. These studies suggest that vaccination with peptide-pulsed cells bearing the human equivalent of murine Qa-1 (HLA-E) may represent a convenient and effective clinical approach to cellular therapy of autoimmune disease.     

Analysis of the relationship between viral infection and autoimmune disease

The clinical association between viral infection and onset or exacerbation of autoimmune disorders remains poorly understood. Here, we examine the relative roles of molecular mimicry and nonspecific inflammatory stimuli in progression from infection to autoimmune disease. Murine herpes virus 1 (HSV-1 KOS) infection triggers T cell-dependent autoimmune reactions to corneal tissue. We generated an HSV-1 KOS point mutant containing a single amino acid exchange within the putative mimicry epitope as well as mice expressing a TCR transgene specific for the self-peptide mimic to allow dissection of two pathogenic mechanisms in disease induction. These experiments indicate that viral mimicry is essential for disease induction after low-level viral infection of animals containing limited numbers of autoreactive T cells, while innate immune mechanisms become sufficient to provoke disease in animals containing relatively high numbers of autoreactive T cells.     

Osteopontin expression by CD103? dendritic cells drives intestinal inflammation

Intestinal CD103⁻ dendritic cells (DCs) are pathogenic for colitis. Unveiling molecular mechanisms that render these cells proinflammatory is important for the design of specific immunotherapies. In this report, we demonstrated that mesenteric lymph node CD103⁻ DCs express, among other proinflammatory cytokines, high levels of osteopontin (Opn) during experimental colitis. Opn expression by CD103⁻ DCs was crucial for their immune profile and pathogenicity, including induction of T helper (Th) 1 and Th17 cell responses. Adoptive transfer of Opn-deficient CD103⁻ DCs resulted in attenuated colitis in comparison to transfer of WT CD103⁻ DCs, whereas transgenic CD103⁻ DCs that overexpress Opn were highly pathogenic in vivo. Neutralization of secreted Opn expressed exclusively by CD103⁻ DCs restrained disease severity. Also, Opn deficiency resulted in milder disease, whereas systemic neutralization of secreted Opn was therapeutic. We determined a specific domain of the Opn protein responsible for its CD103⁻ DC-mediated proinflammatory effect. We demonstrated that disrupting the interaction of this Opn domain with integrin α9, overexpressed on colitic CD103⁻ DCs, suppressed the inflammatory potential of these cells in vitro and in vivo. These results add unique insight into the biology of CD103⁻ DCs and their function during inflammatory bowel disease.Inflammatory bowel diseases (IBDs), including Crohn disease (CD) and ulcerative colitis (UC), are caused by excessive inflammatory responses to commensal microflora and other antigens present in the intestinal lumen (1). Intestinal dendritic cells (DCs) contribute to these inflammatory responses during human IBD, as well as in murine colitis models (2). DCs that reside in draining mesenteric lymph nodes (MLNs) are also crucial mediators of colitis induction (3) and may be grouped based on their surface CD103 (integrin αE) expression as CD11c^highCD103⁺ (CD103⁺ DCs) and CD11c^highCD103⁻ (CD103⁻ DCs) (4–6). CD103⁺ DCs are considered important mediators of gut homeostasis in steady state (4, 5, 7–9), and their tolerogenic properties are conserved between mice and humans (5). However, their role during intestinal inflammation is not well defined. Instead, CD103⁻ DC function has been described mostly during chronic experimental colitis (10–12). These cells secrete IL-23, IL-6, and IL-12 (10–12), contributing to the development of T helper (Th) 17 and Th1 cells, and are highly inflammatory during CD4⁺ T-cell transfer colitis (12) and during 2,4,6 trinitrobenzene sulfonic acid (TNBS)-induced chronic colitis (11). MLN CD103⁻ DCs cultured in the presence of LPS, a Toll-like receptor (TLR) 4 agonist, or R848, a TLR7 agonist, express higher levels of TNF-α and IL-6 (7, 12). In fact, these cells secrete IL-23 and IL-12 even in the absence of TLR stimulation (10). Both MLN CD103⁻ and CD103⁺ DC subsets are present in acute colitis (11, 13); however, their function, as well as their cytokine profile, during this phase of disease, reflecting colitis initiation, remains unknown.Recent studies suggest a proinflammatory role for the cytokine osteopontin (Opn) in TNBS- and dextran sulfate sodium (DSS)-induced colitis (14, 15), which are the models for CD and UC, respectively. Opn is expressed by DCs and other immune cell types, such as lymphocytes, during autoimmune responses (16–22), and its expression by DCs during autoimmunity contributes to disease severity (17–19, 21, 23). In addition, Opn expression is highly up-regulated in intestinal immune and nonimmune cells and in the plasma of patients with CD and UC (24–29), as well as in the colon and plasma of mice with experimental colitis (14, 15, 27, 30). Increased plasma Opn levels are related to the severity of CD inflammation (29), and certain Opn gene (Spp1) haplotypes are modifiers of CD susceptibility (31), indicating that Opn could be used as an IBD biomarker (27). In general, Opn affects DC biology during several inflammatory conditions (17–21, 32–37) and could be a potential therapeutic target in IBD.In this study, we initially asked whether Opn was expressed by MLN CD103⁻ and CD103⁺ DCs during colitis. We found that CD103⁻ DCs express excessive levels of Opn in addition to other proinflammatory cytokines. Conversely, CD103⁺ DCs express profoundly lower levels of Opn and are noninflammatory. Using adoptive transfer of purified specific DC subsets, we determined that MLN CD103⁻ DCs are critical mediators of acute intestinal inflammation and that their Opn expression is essential for their proinflammatory properties in both acute and chronic colitis. Furthermore, Opn-deficient and Opn-neutralized mice developed significantly milder disease. In addition, we constructed transgenic (Tg) mice overexpressing Opn only in DCs. These mice developed exaggerated colitis, and adoptive transfer of their CD103⁻ DCs into recipient mice dramatically exacerbated disease. Because Opn protein contains several domains interacting with various receptors, we defined a specific Opn domain significant for inducing proinflammatory properties in CD103⁻ DCs. Blockade of the interaction of this Opn domain [containing functional Ser-Leu-Ala-Tyr-Gly-Leu-Arg (SLAYGLR) sequence] with integrin α9 expressed on CD103⁻ DCs abrogated their proinflammatory profile and colitogenic effects in vivo.     

Activin-A induces regulatory T cells that suppress T helper cell immune responses and protect from allergic airway disease

Activin-A is a pleiotropic cytokine that participates in developmental, inflammatory, and tissue repair processes. Still, its effects on T helper (Th) cell–mediated immunity, critical for allergic and autoimmune diseases, are elusive. We provide evidence that endogenously produced activin-A suppresses antigen-specific Th2 responses and protects against airway hyperresponsiveness and allergic airway disease in mice. Importantly, we reveal that activin-A exerts suppressive function through induction of antigen-specific regulatory T cells that suppress Th2 responses in vitro and upon transfer in vivo. In fact, activin-A also suppresses Th1-driven responses, pointing to a broader immunoregulatory function. Blockade of interleukin 10 and transforming growth factor β1 reverses activin-A–induced suppression. Remarkably, transfer of activin-A–induced antigen-specific regulatory T cells confers protection against allergic airway disease. This beneficial effect is associated with dramatically decreased maturation of draining lymph node dendritic cells. Therapeutic administration of recombinant activin-A during pulmonary allergen challenge suppresses Th2 responses and protects from allergic disease. Finally, we demonstrate that immune cells infiltrating the lungs from individuals with active allergic asthma, and thus nonregulated inflammatory response, exhibit significantly decreased expression of activin-A''s responsive elements. Our results uncover activin-A as a novel suppressive factor for Th immunity and a critical controller of allergic airway disease.Immune responses by differentiated effector Th1, Th2, and Th17 cell subsets provide protection against pathogens but can also lead to chronic inflammation, autoimmunity, or allergy if not tightly controlled (Reiner, 2007; Steinman, 2007). Critical controllers of these responses are immunosuppressive cytokines, such as IL-10 and TGF-β1, and regulatory T lymphocytes. Subsets of regulatory T cells suppress responses by other effector Th cells mainly via cell-to-cell interactions (Nakamura et al., 2001) or the release of immunosuppressive cytokines (Asseman et al., 1999; Chen et al., 2003; Hawrylowicz and O''Garra, 2005; Ostroukhova et al., 2006; Li et al., 2007). However, blockade of these cytokines does not completely inhibit immune regulation (von Boehmer, 2005; Tang and Bluestone, 2008; Vignali et al., 2008), suggesting that other, as yet unidentified, cytokines are also involved.The cytokine activin-A, a member of the TGF-β superfamily, participates in essential biological processes, such as development, hematopoiesis, wound repair, and fibrosis (Vale et al., 1988; Werner and Alzheimer, 2006). Activin-A^−/− mice are embryonic lethal (Matzuk et al., 1995b), whereas activin receptor type IIA (act-RIIA)^−/− mice reach adulthood but have major deficiencies in their reproductive systems (Matzuk et al., 1995a). Although most studies have focused on the role of activin-A in developmental and fibrotic processes, certain reports demonstrate elevated levels of this cytokine in immune-mediated diseases such as rheumatoid arthritis (Ota et al., 2003) and inflammatory bowel disease (Hubner et al., 1997; Dohi et al., 2005). Activin-A is also increased in the sera of individuals with allergic asthma (Karagiannidis et al., 2006), and in the lung and bronchoalveolar lavage (BAL) of mice during acute (Rosendahl et al., 2001; Hardy et al., 2006) and chronic allergic airway inflammation and remodeling (Le et al., 2007). In addition, activin-A is induced in human (Karagiannidis et al., 2006) and mouse effector Th2 lymphocytes (Ogawa et al., 2006), which are key players in allergic responses. However, whether activin-A has enhancing or suppressive actions during Th immune responses and subsequent disease remains unclear.Certain studies indicate that recombinant activin-A (r-activin-A) reduces in vitro nonspecific proliferation of human Th (Karagiannidis et al., 2006) and mouse B cells (Yu et al., 1998; Werner and Alzheimer, 2006), and inhibits certain functions of human natural killer cells (Robson et al., 2009). In other reports, r-activin-A attenuates in vitro endotoxin-induced maturation and phagocytosis of mouse macrophages (Wang et al., 2008; Zhou et al., 2009) and CD40 ligand–dependent cytokine and chemokine release by human monocytes and DCs (Robson et al., 2008). Nevertheless, a few reports have suggested that activin-A has proinflammatory effects, i.e., during in vivo endotoxin administration and allergen challenge in mice (Hardy et al., 2006; Jones et al., 2007). Collectively, these studies indicate a dual nature of this cytokine, a characteristic feature of certain immune mediators (Veldhoen et al., 2006; Zenewicz et al., 2008). Of note, our previous studies revealed a dual role for another cytokine, osteopontin, in allergic airway inflammation (Xanthou et al., 2007).In the present study, we have investigated the in vivo role of activin-A in Th-mediated immune responses and, more specifically, during Th2-associated allergic airway inflammation. We demonstrate that antibody-mediated depletion of activin-A during pulmonary allergen challenge resulted in significant exacerbation of Th2-mediated allergic airway disease, indicating that endogenous activin-A is suppressive. In fact, our findings reveal that activin-A induces the generation of antigen-specific regulatory T cells that suppress both primary and effector Th responses in vitro and upon adoptive transfer in vivo. Functional in vitro analysis shows that activin-A–mediated suppressive effects on Th responses are dependent on both IL-10 and TGF-β1. Importantly, activin-A–induced antigen-specific regulatory T cells transfer protection against allergic airway disease correlated with decreased DC maturation. Collectively, our data reveal that activin-A can suppress Th responses and represents a critical therapeutic target for allergic asthma.