Staphylococcus aureus alpha toxin suppresses effective innate and adaptive immune responses in a murine dermonecrosis model.

An optimal host response against Staphylococcus aureus skin and soft tissue infections (SSTI) is dependent on IL-1ß and IL-17 mediated abscess formation. Alpha toxin (AT), an essential virulence factor for SSTI, has been reported to damage tissue integrity; however its effect on the immune response has not been investigated. Here, we demonstrate that infection with USA300 AT isogenic mutant (Δhla), or passive immunization with an AT neutralizing mAb, 2A3, 24 h prior to infection with wild type USA300 (WT), resulted in dermonecrotic lesion size reduction, and robust neutrophil infiltration. Infiltration correlates with increase in proinflammatory cytokines and chemokines, as well as enhanced bacterial clearance relative to immunization with a negative control mAb. In addition, infection with Δhla, or with WT +2A3, resulted in an early influx of innate IL-17(+)γδT cells and a more rapid induction of an adaptive immune response as measured by Th1 and Th17 cell recruitment at the site of infection. These results are the first direct evidence of a role for AT in subverting the innate and adaptive immune responses during a S. aureus SSTI. Further, these effects of AT can be overcome with a high affinity anti-AT mAb resulting in a reduction in disease severity.

Modulation of Treg cells/T effector function by GITR signaling is context-dependent.

Treg cells express high levels of the glucocorticoid-induced tumor necrosis factor-related receptor (GITR), while resting conventional T (Tconv) cells express low levels that are increased upon activation. Manipulation of GITR/GITR-Ligand (GITR-L) interactions results in enhancement of immune responses, but it remains unclear whether this enhancement is secondary to costimulation of Tconv cells or to reversal of Treg-cell-mediated suppression. Here, we used a nondepleting Fc-GITR-L and combinations of WT and GITR KO Treg cells and Tconv cells to reexamine the effects of GITR stimulation on each subpopulation in both unmanipulated mice and mice with inflammatory bowel disease. Treatment of mice with Fc-GITR-L resulted in significant expansion of Treg cells and a modest expansion of Tconv cells. When RAG KO mice were reconstituted with Tconv cells alone, GITR-L resulted in Tconv-cell expansion and severe inflammatory bowel disease. The protective effect of Treg cells was lost in the presence of Fc-GITR-L, secondary to death of the Treg cells. When RAG KO mice were reconstituted with Treg cells alone, the transferred cells expanded normally, and Fc-GITR-L treatment resulted in a loss of Foxp3 expression, but the ex-Treg cells did not cause any pathology. The effects of GITR activation are complex and depend on the host environment and the activation state of the Treg cells and T effector cells.

Kynurenine 3-monooxygenase mediates inhibition of Th17 differentiation via catabolism of endogenous aryl hydrocarbon receptor ligands.

The aryl hydrocarbon receptor (AhR) is a key transcriptional regulator of Th17-cell differentiation. Although endogenous ligands have yet to be identified, evidence suggests that tryptophan metabolites can act as agonists for the AhR. Tryptophan metabolites are abundant in circulation, so we hypothesized that cell intrinsic factors might exist to regulate the exposure of Th17 cells to AhR-dependent activities. Here, we find that Th17 cells preferentially express kynurenine 3-monooxygenase (KMO), which is an enzyme involved in catabolism of the tryptophan metabolite kynurenine. KMO inhibition, either with a specific inhibitor or via siRNA-mediated silencing, markedly increased IL-17 production in vitro, whereas IFN-γ production by Th1 cells was unaffected. Inhibition of KMO significantly exacerbated disease in a Th17-driven model of autoimmune gastritis, suggesting that expression of KMO by Th17 cells serves to limit their continuous exposure to physiological levels of endogenous AhR ligands in vivo.

Mycophenolic acid differentially impacts B cell function depending on the stage of differentiation.

Production of pathogenic Abs contributes to disease progression in many autoimmune disorders. The immunosuppressant agent mycophenolic acid (MPA) has shown clinical efficacy for patients with autoimmunity. The goal of these studies was to elucidate the mechanisms of action of MPA on B cells isolated from healthy individuals and autoimmune patients. In this study, we show that MPA significantly inhibited both proliferation and differentiation of primary human B cells stimulated under various conditions. Importantly, MPA did not globally suppress B cell responsiveness or simply induce cell death, but rather selectively inhibited early activation events and arrested cells in the G0/G1 phase of the cell cycle. Furthermore, MPA blocked expansion of both naive and memory B cells and prevented plasma cell (PC) differentiation and Ab production from healthy controls and individuals with rheumatoid arthritis. Finally, whereas MPA potently suppressed Ig secretion from activated primary B cells, terminally differentiated PCs were not susceptible to inhibition by MPA. The target of MPA, IMPDH2, was found to be downregulated in PCs, likely explaining the resistance of these cells to MPA. These results suggest that MPA provides benefit in settings of autoimmunity by directly preventing activation and PC differentiation of B cells; however, MPA is unlikely to impact autoantibody production by preexisting, long-lived PCs.

IL-9 is a Th17-derived cytokine that limits pathogenic activity in organ-specific autoimmune disease.

IL-9 is a pleiotropic cytokine with key functions in tolerance and inflammation, and its expression is considered a hallmark of Th2-lineage cells. Here, we report that human and mouse Th17 cells are a significant source of IL-9. The expression of IL-9 by Th17 cells was strictly dependent on the presence of TGF-ß and IL-1ß, and inhibited by IL-4. IL-9-deficient Th17 cells induced more severe autoimmune gastritis following transfer to nu/nu recipient mice. Th17 cells did not appear to be the target of IL-9 bioactivity as Th17 expansion and differentiation was comparable using IL-9-deficient CD4(+) cells or when IL-9 was neutralized with antibodies in vitro. However, reduced mast cell activity was associated with the increased pathogenicity of IL-9-deficient Th17 cells. Together, these results demonstrate a previously unappreciated role for IL-9 in dampening the pathogenic activities of Th17 cells.

MEDI-563, a humanized anti-IL-5 receptor alpha mAb with enhanced antibody-dependent cell-mediated cytotoxicity function.

BACKGROUND: Peripheral blood eosinophilia and lung mucosal eosinophil infiltration are hallmarks of bronchial asthma. IL-5 is a critical cytokine for eosinophil maturation, survival, and mobilization. Attempts to target eosinophils for the treatment of asthma by means of IL-5 neutralization have only resulted in partial removal of airway eosinophils, and this warrants the development of more effective interventions to further explore the role of eosinophils in the clinical expression of asthma. OBJECTIVE: We sought to develop a novel humanized anti-IL-5 receptor alpha (IL-5Ralpha) mAb with enhanced effector function (MEDI-563) that potently depletes circulating and tissue-resident eosinophils and basophils for the treatment of asthma. METHODS: We used surface plasmon resonance to determine the binding affinity of MEDI-563 to FcgammaRIIIa. Primary human eosinophils and basophils were used to demonstrate antibody-dependent cell-mediated cytotoxicity. The binding epitope of MEDI-563 on IL-5Ralpha was determined by using site-directed mutagenesis. The consequences of MEDI-563 administration on peripheral blood and bone marrow eosinophil depletion was investigated in nonhuman primates. RESULTS: MEDI-563 binds to an epitope on IL-5Ralpha that is in close proximity to the IL-5 binding site, and it inhibits IL-5-mediated cell proliferation. MEDI-563 potently induces antibody-dependent cell-mediated cytotoxicity of both eosinophils (half-maximal effective concentration = 0.9 pmol/L) and basophils (half-maximal effective concentration = 0.5 pmol/L) in vitro. In nonhuman primates MEDI-563 depletes blood eosinophils and eosinophil precursors in the bone marrow. CONCLUSIONS: MEDI-563 might provide a novel approach for the treatment of asthma through active antibody-dependent cell-mediated depletion of eosinophils and basophils rather than through passive removal of IL-5.

Foxp3+ regulatory T cells: selfishness under scrutiny.

The Foxp3+ T cell lineage is thought to arise from self-specific precursors during development. By using a unique mouse model, Pacholczyk et al. (2007) present compelling evidence that self-specific cells are exceedingly rare among Foxp3(-) and, surprisingly, Foxp3+ subsets.

Distinct subsets of FoxP3+ regulatory T cells participate in the control of immune responses.

Expression of the transcription factor FoxP3 is the hallmark of regulatory T cells that play a crucial role in dampening immune responses. A comparison of the development and phenotype of FoxP3(+) T cells in relation to the expression of conventional MHC molecules facilitated the identification of several distinct lineages of naive and effector/memory populations of Foxp3(+) T cells. One subpopulation of effector/memory Foxp3(+) T cells develops in the thymic medulla, whereas the second is thymic independent. Both lineages display a distinct activated phenotype, undergo extensive steady-state proliferation, home to sites of acute inflammation, and are unique in their capacity to mediate Ag-nonspecific suppression of T cell activation directly ex vivo. Effector FoxP3(+) T cells may act as a sentinel of tolerance, providing a first line of defense against potentially harmful responses by rapidly suppressing immunity to peripheral self-Ags.

CD4+CD25+ regulatory T cells are activated in vivo by recognition of self.

Naturally occurring CD4(+)CD25(+) regulatory T cells (nT(R)) comprise a separate lineage of T cells that are essential for maintaining immunological tolerance to self. Here we demonstrate that the level of phosphorylation of the TCR zeta-chain is approximately 1.5- to 4-fold higher in nT(R) as compared with CD4(+)CD25(-) T cells. The increased level of TCR zeta-chain phosphorylation is presumably secondary to their higher affinity for self, resulting in a stronger TCR signal as it was completely blocked by treatment with anti-MHC class II. The enhanced level of TCR zeta-chain phosphorylation was correlated with the capacity of nT(R) to develop non-specific suppressor effector function following culture with IL-2 or IL-4 in the absence of TCR stimulus. Thus, a sub-population of nT(R) is activated by recognition of self-peptide-MHC class II ligands in vivo, resulting in their capacity to be induced to mediate suppressor function in vitro in the absence of TCR stimulation.

Measurement of T and B cell turnover with bromodeoxyuridine.

This unit describes two methods used to determine the rate of turnover of T and B cells in vivo. In each method, this is accomplished by administering 5-bromo-2'-deoxyuridine (BrdU), a thymidine analog, and measuring the rate at which cells become labeled with this DNA precursor during the S phase of the cell cycle. Cells that contain BrdU are then detected in vitro using fluorochrome-labelled anti-BrdU monoclonal antibody by flow cytometry. The unit includes an Alternate Protocol that may be useful in situations where the Basic Protocol proves incompatible with particular antibody-fluorochrome conjugates or other downstream staining procedures. Using this procedure, a wide range of antibody-fluorochrome combinations can be used.

The lifestyle of naturally occurring CD4+ CD25+ Foxp3+ regulatory T cells.

Numerous studies over the past 10 years have demonstrated the importance of naturally occurring CD4+ CD25+ Foxp3+ regulatory T cells (nTregs) in immune regulation. We analyzed the mechanism of action of nTregs in a well-characterized model of autoimmune gastritis and demonstrated that nTregs act at an early stage of disease progression to inhibit the differentiation of naïve T cells to pathogenic T-helper 1 effectors. The effects of nTregs in this model are not antigen-specific but are mediated by activation of the nTregs by ubiquitous self-peptide major histocompatibility complex class II complexes together with cytokines released by activated effector cells. Studies in vitro confirmed that some nTregs exist in an activated state in vivo and can be activated to exert non-specific suppressor effector function by stimulation with interleukin-2 in the absence of engagement of their T-cell receptor. Natural Tregs can differentiate in vitro to exhibit potent granzyme B-dependent, partially perforin-independent cytotoxic cells that are capable of specifically killing antigen-presenting B cells. Natural Treg-mediated killing of antigen-presenting cells may represent one pathway by which they can induce long-lasting suppression of autoimmune disease.

The GITR-GITRL interaction: co-stimulation or contrasuppression of regulatory activity?

Stimulation of T cells through GITR (glucocorticoid-induced tumour-necrosis-factor-receptor-related protein) has been shown to enhance immunity to tumours and viral pathogens, and to exacerbate autoimmune disease. The effects of stimulation through GITR are generally thought to be caused by attenuation of the effector activity of immunosuppressive CD4+ CD25+ regulatory T (T(Reg)) cells. Here we propose a model in which GITR-GITR-ligand interactions co-stimulate both responder T-cell functions and the suppressive functions of T(Reg) cells.

Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells.

Nonactivated CD4+CD25+ regulatory T cells constitutively express glucocorticoid-induced TNFR family-related receptor (GITR), a TNFR family member whose engagement was presumed to abrogate regulatory T cell-mediated suppression. Using GITR-/- mice, we report that GITR engagement on CD25-, not CD25+ T cells abrogates T cell-mediated suppression. Mouse APCs constitutively express GITR ligand (GITR-L), which is down-regulated following TLR signaling in vivo. Although GITR-/-CD25- T cells were capable of mounting proliferative responses, they were incapable of proliferation in the presence of physiological numbers of CD25+ T cells. Thus, GITR-L provides an important signal for CD25- T cells, rendering them resistant to CD25+ -mediated regulation at the initiation of the immune response. The down-regulation of GITR-L by inflammatory stimuli may enhance the susceptibility of effector T cells to suppressor activity during the course of an infectious insult.

Decreasing the threshold for thymocyte activation biases CD4+ T cells toward a regulatory (CD4+CD25+) lineage.

Thymus-derived CD4(+)CD25(+) regulatory T (T(r)) cells play a critical role in suppressing aberrant responses to self in vivo. The factors that influence a CD4(+) T cell's decision to commit to an immunoregulatory T(r) cell lineage are currently unknown. In the present study, we found that in mice, abundantly expressing a few or one peptide(s) bound to MHC class II molecules, a large portion of conventional CD4(+) T cells could be biased towards the commitment to a T(r) lineage by reducing the threshold required for thymocyte activation. This occurred in the presence of either an antisense glucocorticoid receptor transgene or a pharmacological inhibitor of glucocorticoid synthesis. These results demonstrate a novel in vivo pathway for the generation of T(r) cells, and raise the possibility that therapeutic enhancement of the T(r) cell repertoire through pharmacological manipulation of TCR signaling thresholds may provide a feasible means of ameliorating autoimmunity.

Mutually antagonistic signals regulate selection of the T cell repertoire.

The sensitivity of T cells to agonist-induced death during development contrasts with their proliferative responses after agonist challenge in the periphery. The means by which TCR engagement results in these distinct outcomes is incompletely understood. It has been previously hypothesized that glucocorticoids (GC) modulate the threshold for thymocyte activation by blunting the consequences of TCR engagement. In support of this possibility, inhibition of GC production in fetal thymic organ culture was shown to result in CD4(+)CD8(+) thymocyte apoptosis. This was dependent upon MHC diversity, implying that endogenous GC might regulate antigen-specific selection. Similarly, mice expressing reduced GC receptor (GR) levels due to the presence of an antisense transgene have fewer CD4(+)CD8(+) thymocytes, which was due to an impaired transition from CD4(-)CD8(-) precursors and increased apoptosis. Here we ask how manipulating peptide diversity in the context of reduced GC signaling might affect T cell development and function. In mice with impaired GR expression there was a rescue of thymocyte cellularity and proportions as the diversity of peptides presented by self-MHC was reduced. Furthermore, whereas more CD4(+) T cells survived the selection process in mice expressing single-peptide-MHC class II complexes and reduced GR levels, these cells largely failed to recognize the same MHC molecules bound with foreign peptides. Together, these results support a role for endogenous GC in balancing TCR-mediated signals during thymic selection.