CXCR3 blockade protects against Listeria monocytogenes infection-induced fetal wastage.

Mammalian pregnancy requires protection against immunological rejection of the developing fetus bearing discordant paternal antigens. Immune evasion in this developmental context entails silenced expression of chemoattractant proteins (chemokines), thereby preventing harmful immune cells from penetrating the maternal-fetal interface. Here, we demonstrate that fetal wastage triggered by prenatal Listeria monocytogenes infection is driven by placental recruitment of CXCL9-producing inflammatory neutrophils and macrophages that promote infiltration of fetal-specific T cells into the decidua. Maternal CD8+ T cells with fetal specificity upregulated expression of the chemokine receptor CXCR3 and, together with neutrophils and macrophages, were essential for L. monocytogenes-induced fetal resorption. Conversely, decidual accumulation of maternal T cells with fetal specificity and fetal wastage were extinguished by CXCR3 blockade or in CXCR3-deficient mice. Remarkably, protection against fetal wastage and in utero L. monocytogenes invasion was maintained even when CXCR3 neutralization was initiated after infection, and this protective effect extended to fetal resorption triggered by partial ablation of immune-suppressive maternal Tregs, which expand during pregnancy to sustain fetal tolerance. Together, our results indicate that functionally overriding chemokine silencing at the maternal-fetal interface promotes the pathogenesis of prenatal infection and suggest that therapeutically reinforcing this pathway represents a universal approach for mitigating immune-mediated pregnancy complications.

Commensal microbes drive intestinal inflammation by IL-17-producing CD4+ T cells through ICOSL and OX40L costimulation in the absence of B7-1 and B7-2.

The costimulatory B7-1 (CD80)/B7-2 (CD86) molecules, along with T-cell receptor stimulation, together facilitate T-cell activation. This explains why in vivo B7 costimulation neutralization efficiently silences a variety of human autoimmune disorders. Paradoxically, however, B7 blockade also potently moderates accumulation of immune-suppressive regulatory T cells (Tregs) essential for protection against multiorgan systemic autoimmunity. Here we show that B7 deprivation in mice overrides the necessity for Tregs in averting systemic autoimmunity and inflammation in extraintestinal tissues, whereas peripherally induced Tregs retained in the absence of B7 selectively mitigate intestinal inflammation caused by Th17 effector CD4(+) T cells. The need for additional immune suppression in the intestine reflects commensal microbe-driven T-cell activation through the accessory costimulation molecules ICOSL and OX40L. Eradication of commensal enteric bacteria mitigates intestinal inflammation and IL-17 production triggered by Treg depletion in B7-deficient mice, whereas re-establishing intestinal colonization with Candida albicans primes expansion of Th17 cells with commensal specificity. Thus, neutralizing B7 costimulation uncovers an essential role for Tregs in selectively averting intestinal inflammation by Th17 CD4(+) T cells with commensal microbe specificity.

Immunosuppressive CD71+ erythroid cells compromise neonatal host defence against infection.

Newborn infants are highly susceptible to infection. This defect in host defence has generally been ascribed to the immaturity of neonatal immune cells; however, the degree of hyporesponsiveness is highly variable and depends on the stimulation conditions. These discordant responses illustrate the need for a more unified explanation for why immunity is compromised in neonates. Here we show that physiologically enriched CD71(+) erythroid cells in neonatal mice and human cord blood have distinctive immunosuppressive properties. The production of innate immune protective cytokines by adult cells is diminished after transfer to neonatal mice or after co-culture with neonatal splenocytes. Neonatal CD71(+) cells express the enzyme arginase-2, and arginase activity is essential for the immunosuppressive properties of these cells because molecular inhibition of this enzyme or supplementation with L-arginine overrides immunosuppression. In addition, the ablation of CD71(+) cells in neonatal mice, or the decline in number of these cells as postnatal development progresses parallels the loss of suppression, and restored resistance to the perinatal pathogens Listeria monocytogenes and Escherichia coli. However, CD71(+) cell-mediated susceptibility to infection is counterbalanced by CD71(+) cell-mediated protection against aberrant immune cell activation in the intestine, where colonization with commensal microorganisms occurs swiftly after parturition. Conversely, circumventing such colonization by using antimicrobials or gnotobiotic germ-free mice overrides these protective benefits. Thus, CD71(+) cells quench the excessive inflammation induced by abrupt colonization with commensal microorganisms after parturition. This finding challenges the idea that the susceptibility of neonates to infection reflects immune-cell-intrinsic defects and instead highlights processes that are developmentally more essential and inadvertently mitigate innate immune protection. We anticipate that these results will spark renewed investigation into the need for immunosuppression in neonates, as well as improved strategies for augmenting host defence in this vulnerable population.

B7-1/B7-2 blockade overrides the activation of protective CD8 T cells stimulated in the absence of Foxp3+ regulatory T cells.

Although T cell activation has been classically described to require distinct, positive stimulation signals that include B7-1 (CD80) and B7-2 (CD86) costimulation, overriding suppression signals that avert immune-mediated host injury are equally important. How these opposing stimulation and suppression signals work together remains incompletely defined. Our recent studies demonstrate that CD8 Teff activation in response to cognate peptide stimulation is actively suppressed by the Foxp3(+) subset of CD4 cells, called Tregs. Here, we show that the elimination of Treg suppression does not bypass the requirement for positive B7-1/B7-2 costimulation. The expansion, IFN-γ cytokine production, cytolytic, and protective features of antigen-specific CD8 T cells stimulated with purified cognate peptide in Treg-ablated mice were each neutralized effectively by CTLA-4-Ig that blocks B7-1/B7-2. In turn, given the efficiency whereby CTLA-4-Ig overrides the effects of Treg ablation, the role of Foxp3(+) cell-intrinsic CTLA-4 in mitigating CD8 Teff activation was also investigated. With the use of mixed chimera mice that contain CTLA-4-deficient Tregs exclusively after the ablation of WT Foxp3(+) cells, a critical role for Treg CTLA-4 in suppressing the expansion, cytokine production, cytotoxicity, and protective features of peptide-stimulated CD8 T cells is revealed. Thus, the activation of protective CD8 T cells requires positive B7-1/B7-2 costimulation even when suppression by Tregs and in particular, Treg-intrinsic CTLA-4 is circumvented.

Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ.

Protein N-myristoylation is a 14-carbon fatty-acid modification that is conserved across eukaryotic species and occurs on nearly 1% of the cellular proteome. The ability of the myristoyl group to facilitate dynamic protein-protein and protein-membrane interactions (known as the myristoyl switch) makes it an essential feature of many signal transduction systems. Thus pathogenic strategies that facilitate protein demyristoylation would markedly alter the signalling landscape of infected host cells. Here we describe an irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a previously uncharacterized Shigella flexneri type III effector protein with cysteine protease activity. A yeast genetic screen for IpaJ substrates identified ADP-ribosylation factor (ARF)1p and ARF2p, small molecular mass GTPases that regulate cargo transport through the Golgi apparatus. Mass spectrometry showed that IpaJ cleaved the peptide bond between N-myristoylated glycine-2 and asparagine-3 of human ARF1, thereby providing a new mechanism for host secretory inhibition by a bacterial pathogen. We further demonstrate that IpaJ cleaves an array of N-myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. Taken together, these findings show a previously unrecognized pathogenic mechanism for the site-specific elimination of N-myristoyl protein modification.

Naturally occurring altered peptide ligands control Salmonella-specific CD4+ T cell proliferation, IFN-gamma production, and protective potency.

T cell activation required for host defense against infection is an intricately regulated and precisely controlled process. Although in vitro studies indicate that three distinct stimulatory signals are required for T cell activation, the precise contribution of each signal in regulating T cell proliferation and differentiation after in vivo infection is unknown. In this study, altered peptide ligands (APLs) derived from the protective Salmonella-specific FliC Ag and CD4+ T cells specific for the immune-dominant FliC(431-439) peptide within this Ag were used to determine how changes in TCR stimulation impact CD4+ T cell proliferation, differentiation, and protective potency. To explore the prevalence and potential use of altered TCR stimulation by bacterial pathogens, naturally occurring APLs containing single amino acid substitutions in putative TCR contact residues within the FliC(431-439) peptide were identified and used for stimulation under both noninfection and infection conditions. On the basis of this analysis, naturally-occurring APLs that prime proliferation of FliC-specific CD4+ T cells either more potently or less potently compared with the wild-type FliC(431-439) peptide were identified. Remarkably, despite these differences in proliferation, all of the APLs primed reduced IFN-gamma production by FliC(431-439)-specific CD4+ T cells after stimulation in vivo. Moreover, after expression of the parental FliC(431-439) peptide or each APL in recombinant Listeria monocytogenes, only CD4+ T cells stimulated with the wild-type FliC(431-439) peptide conferred significant protection against challenge with virulent Salmonella. These results reveal important and unanticipated roles for TCR stimulation in controlling pathogen-specific CD4+ T cell proliferation, differentiation, and protective potency.

Cytotoxic T-lymphocyte antigen 4 blockade augments the T-cell response primed by attenuated Listeria monocytogenes resulting in more rapid clearance of virulent bacterial challenge.

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) uniformly suppresses antigen-specific T cells during chronic infection with bacterial, parasitic or viral pathogens. However, the importance of CTLA-4 in controlling the T-cell response during acute infection or after priming with live attenuated vaccine vectors has not been well characterized. Since strategies aimed at blocking CTLA-4 are being actively developed to therapeutically augment T-cell-mediated immunity, the effects of CTLA-4 blockade on T-cell activation during these conditions need to be more clearly defined. We have examined the role of CTLA-4 in a prime-challenge model of acute bacterial infection using both attenuated and virulent strains of the intracellular bacterium Listeria monocytogenes. Although Foxp3(+) CD4(+) T cells are the predominant CTLA-4-expressing cell type in naïve mice, antigen-specific Foxp3(-) CD4(+) cells upregulate CTLA-4 expression after primary L. monocytogenes infection. Blockade of CTLA-4 results in increased numbers of L. monocytogenes-specific CD4 and CD8 T cells after primary infection with attenuated L. monocytogenes, and confers more rapid bacterial clearance after secondary challenge with virulent L. monocytogenes. Accordingly, CTLA-4 plays an important suppressive role in T-cell priming and protective immunity in a prime-challenge model of acute bacterial infection.

PDL-1 blockade impedes T cell expansion and protective immunity primed by attenuated Listeria monocytogenes.

Infection with attenuated Listeria monocytogenes (Lm) is a robust in vivo model for examining how Ag-specific T cells are primed, and subsequent challenge with virulent Lm allows for the protective effects of T cell priming to be quantified. Herein, we investigated the role of programmed death ligand 1 (PDL-1) in T cell priming and immunity conferred after primary infection with Lm DeltaactA followed by virulent Lm challenge. In striking contrast to the inhibitory role of PDL-1 on T cell immunity in other infection models, marked reductions in the magnitude of T cell expansion and the kinetics of T cell proliferation were observed with PDL-1 blockade after primary Lm DeltaactA infection. More importantly, PDL-1 blockade beginning before primary infection and maintained throughout the experiment resulted in delayed bacterial clearance and T cell expansion after secondary challenge with virulent Lm. These results indicate that for immunity to intracellular bacterial infection, PDL-1 plays an important stimulatory role for priming and expansion of protective T cells.

Salmonella flagellin induces bystander activation of splenic dendritic cells and hinders bacterial replication in vivo.

Bacterial flagellin is a target of innate and adaptive immune responses during Salmonella infection. Intravenous injection of Salmonella flagellin into C57BL/6 mice induced rapid IL-6 production and increased expression of activation markers by splenic dendritic cells. CD11b(+), CD8alpha(+), and plasmacytoid dendritic cells each increased expression of CD86 and CD40 in response to flagellin stimulation, although CD11b(+) dendritic cells were more sensitive than the other subsets. In addition, flagellin caused the rapid redistribution of dendritic cells from the red pulp and marginal zone of the spleen into the T cell area of the white pulp. Purified splenic dendritic cells did not respond directly to flagellin, indicating that flagellin-mediated activation of splenic dendritic cells occurs via bystander activation. IL-6 production, increased expression of activation markers, and dendritic cell redistribution in the spleen were dependent on MyD88 expression by bone marrow-derived cells. Avoiding this innate immune response to flagellin is important for bacterial survival, because Salmonella-overexpressing recombinant flagellin was highly attenuated in vivo. These data indicate that flagellin-mediated activation of dendritic cells is rapid, mediated by bystander activation, and highly deleterious to bacterial survival.