Targeting SIRP‐α protects from type 2‐driven allergic airway inflammation

The interplay between innate and adaptive immune responses is essential for the establishment of allergic diseases. CD47 and its receptor, signal regulatory protein α (SIRP‐α), govern innate cell trafficking. We previously reported that administration of CD47^+/+ but not CD47^−/− SIRP‐α⁺ BM‐derived DC (BMDC) induced airway inflammation and Th2 responses in otherwise resistant CD47‐deficient mice. We show here that early administration of a CD47‐Fc fusion molecule suppressed the accumulation of SIRP‐α⁺ DC in mediastinal LN, the development of systemic and local Th2 responses as well as airway inflammation in sensitized and challenged BALB/c mice. Mechanistic studies highlighted that SIRP‐α ligation by CD47‐Fc on BMDC did not impair Ag uptake, Ag presentation and Ag‐specific DO11.10 Tg Th2 priming and effector function in vitro, whereas in vivo administration of CD47‐Fc or CD47‐Fc‐pretreated BMDC inhibited Tg T‐cell proliferation, pinpointing that altered DC trafficking accounts for defective Th priming. We conclude that the CD47/SIRP‐α axis may be harnessed in vivo to suppress airway SIRP‐α⁺ DC homing to mediastinal LN, Th2 responses and allergic airway inflammation.     

DC therapy induces long‐term NK reactivity to tumors via host DC

We vaccinated mice with DC loaded with or without invariant NKT‐cell ligand α‐galactosylceramide and evaluated long‐term resistance against tumor challenge. When mice had been given either DC or DC/galactosylceramide and were challenged with tumor cells even 6–12 months later, both NK and NKT cells were quickly activated to express CD69 and produce IFN‐γ. The NK cells could resist a challenge with several different tumors in vivo. The activated NK and NKT cells could be depleted with anti‐NK1.1 treatment. In spite of this, the activated cells recovered, indicating that tumor‐responsive NK and NKT cells were being generated continuously as a result of vaccination with DC and were not true memory cells. The NK and NKT antitumor response in DC‐vaccinated mice depended on CD4⁺ T cells, but neither CD8⁺T cells nor CD4⁺CD25⁺ regulatory T cells. However, both vaccine DC and host DC were required for the development of long‐term, tumor reactive innate immunity. These results indicate that DC therapy in mice induces long‐lasting innate NK‐ and NKT‐cell activation through a pathway that requires host DC and CD4⁺ T cells and that the continued generation of active NK cells resists the establishment of metastases in vivo.     

Induction of peripheral CD4+ T‐cell tolerance and CD8+ T‐cell cross‐tolerance by dendritic cells

DC can present and cross‐present self‐antigens to autoreactive CD4⁺ and CD8⁺ T cells, respectively, and incapacitate them by inducing anergy, deletion or converting them into Treg. In this review, we summarize the recent progress in immune tolerance research, which has been achieved by employing antigen‐ and TCR‐transgenic mice. We cover the numerous discoveries that have furthered our knowledge of the DC subsets and maturation pathways involved in tolerance; the signals, such as CD70, TGF‐β, B7‐H1/PD‐L1, which dictate the decision between immunity and tolerance; and the in vivo role of DC in the maintenance of CD4⁺ T‐cell tolerance and CD8⁺ T‐cell cross‐tolerance.     

Fcγ receptor‐mediated antigen uptake by lung DC contributes to allergic airway hyper‐responsiveness and inflammation

During asthma, lung DC capture and process antigens to initiate and maintain allergic Th2 cell responses to inhaled allergens. The aim of the study was to investigate whether allergen‐specific IgG, generated during sensitization, can potentiate the acute airway inflammation through Fcγ receptor (FcγR)‐mediated antigen uptake and enhance antigen presentation resulting in augmented T‐cell proliferation. We examined the impact of antigen presentation and T‐cell stimulation on allergic airway hyperresponsiveness and inflammation using transgenic and gene‐deficient mice. Both airway inflammation and eosinophilia in bronchoalveolar lavage fluid were markedly reduced in sensitized and challenged FcγR‐deficient mice. Lung DC of WT, but not FcγR‐deficient mice, induced increased antigen‐specific CD4⁺ T‐cell proliferation when pulsed with anti‐OVA IgG immune complexes. Intranasal application of anti‐OVA IgG immune complexes resulted in enhanced airway inflammation, eosinophilia and Th2 cytokine release, mediated through enhanced antigen‐specific T‐cell proliferation in vivo. Finally, antigen‐specific IgG in the serum of sensitized mice led to a significant increase of antigen‐specific CD4⁺ T‐cell proliferation induced by WT, but not FcγR‐deficient, lung DC. We conclude that FcγR‐mediated enhanced antigen presentation and T‐cell stimulation by lung DC has a significant impact on inflammatory responses following allergen challenge in asthma.     

DC‐induced CD8+ T‐cell response is inhibited by MHC class II‐dependent DX5+CD4+ Treg

CD4⁺ T cells are important for CD8⁺ T‐cell priming by providing cognate signals for DC maturation. We analyzed the capacity of CD4⁺ T cells to influence CD8⁺ T‐cell responses induced by activated DC. Surprisingly, mice depleted for CD4⁺ cells were able to generate stronger antigen‐specific CD8⁺ T‐cell responses after DC vaccination than non‐depleted mice. The same observation was made when mice were vaccinated with MHC class II^?/? DC, indicating the presence of a MHC class II‐dependent CD4⁺ T‐cell population inhibiting CD8⁺ T‐cell responses. Recently we described the expansion of DX5⁺CD4⁺ T cells, a T‐cell population displaying immune regulatory properties, upon vaccination with DC. Intriguingly, we now observe an inverse correlation between CD8⁺ T‐cell induction and expansion of DX5⁺CD4⁺ T cells as the latter cells did not expand after vaccination with MHC class II^?/? DC. In vitro, DX5⁺CD4⁺ T cells were able to limit proliferation, modulate cytokine production and induce Foxp3⁺ expression in OVA‐specific CD8⁺ T cells. Together, our data show an inhibitory role of CD4⁺ T cells on the induction of CD8⁺ T‐cell responses by activated DC and indicate the involvement of DX5⁺CD4⁺, but not CD4⁺CD25⁺, T cells in this process.     

Protease‐activated receptor‐2 signalling by tissue factor on dendritic cells suppresses antigen‐specific CD4+ T‐cell priming

The precise function of tissue factor (TF) expressed by dendritic cells (DC) is uncertain. As well as initiating thrombin generation it can signal through protease‐activated receptor 2 (PAR‐2) when complexed with factor VIIa. We investigated the expression and function of TF on mouse bone marrow (BM) ‐derived DC; 20% of BM‐derived DC expressed TF, which did not vary after incubation with lipopolysaccharide (LPS) or dexamethasone (DEX). However, the pro‐coagulant activity of DEX‐treated DC in recalcified plasma was 30‐fold less than LPS‐treated DC. In antigen‐specific and allogeneic T‐cell culture experiments, the TF on DEX‐treated DC provided a signal through PAR‐2, which contributed to the reduced ability of these cells to stimulate CD4⁺ T‐cell proliferation and cytokine production. In vivo, an inhibitory anti‐TF antibody and a PAR‐2 antagonist enhanced antigen‐specific priming in two models where antigen was given without adjuvant, with an effect approximately 50% that seen with LPS, suggesting that a similar mechanism was operational physiologically. These data suggest a novel TF and PAR‐2‐dependent mechanism on DEX‐DC in vitro and unprimed DC in vivo that contributes to the low immunogenicity of these cells. Targeting this pathway has the potential to influence antigen‐specific CD4⁺ T‐cell activation.     

Influence of arylamine n‐acetyltransferase,sex, and age on 4‐aminobiphenyl‐induced in vivo mutant frequencies and spectra in mouse liver

One model for cancer initiation by 4‐aminobiphenyl (ABP) involves N‐oxidation by cytochrome P450 CYP1A2 followed by O‐conjugation by N‐acetyltransferase(s) NAT1 and/or NAT2 and decomposition to a DNA‐binding nitrenium ion. We recently observed that neonatal ABP exposure produced liver tumors in male but not in female mice, and that NAT deficiency reduced liver tumor incidence. However, ABP‐induced liver tumor incidence did not correlate with liver levels of N‐(deoxyguanosin‐8‐yl)‐ABP adducts 24 hr after exposure. In this study, we compared in vivo ABP‐induced DNA mutant frequencies and spectra between male and female wild‐type and NAT‐deficient Muta?Mouse using both the tumor‐inducing neonatal exposure protocol and a 28‐day repetitive dosing adult exposure protocol. ABP produced an increase in liver DNA mutant frequencies in both neonates and adults. However, we observed no sex or strain differences in mutant frequencies in neonatally exposed mice, and higher frequencies in adult females than males. Neonatal ABP exposure of wild‐type mice increased the proportion of G‐T transversions in both males and females, while exposure of Nat1/2(‐/‐) mice produced increased G‐T transversions in males and a decrease in females, even though females had higher levels of N‐(deoxyguanosin‐8‐yl)‐4‐ABP adducts. There was no correlation of mutant frequencies or spectra between mice dosed as neonates or as adults. These results suggest that observed sex‐ and NAT‐dependent differences in ABP‐induced liver tumor incidence in mice are not due to differences in either mutation rates or mutational spectra, and that mechanisms independent of carcinogen bioactivation, covalent DNA binding and mutation may be responsible for these differences. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.     

T‐cell‐specific deletion of gp130 renders the highly susceptible IL‐10‐deficient mouse resistant to intestinal nematode infection

Gp130 is the common receptor of the IL‐6 family of cytokines and is involved in many biological processes, including acute phase response, inflammation and immune reactions. To investigate the role of gp130 under inflammatory conditions, T‐cell‐specific conditional gp130 mice were first bred to the IL‐10‐deficient background and were then infected with the gastrointestinal nematode Trichuris muris. While IL‐10^?/? mice were highly susceptible to T. muris, developed a mixed Th1/Th17 response and displayed severe inflammation of the caecum, infection of mice with an additional T‐cell‐specific deletion of gp130 signalling completely reversed the phenotype. These mice showed an accelerated worm expulsion that was associated with the rapid generation of a strong Th2 immune response and a significant increase in Foxp3‐expressing Treg. Therefore, gp130 signalling in T cells regulates a switch between proinflammatory and pathogenic Th1/Th17 cells and regulatory Th2/Treg in vivo. Taken together, the data demonstrate that gp130 signalling in T cells is a positive regulator of inflammatory processes, favouring the Th1/Th17 axis.     

Effects of Lactobacillus GG treatment during pregnancy on the development of fetal antigen‐specific immune responses

Background Several clinical trials suggest that probiotics may have a role in the prevention of eczema. The optimal timing and mechanisms underlying this intervention are not clear. In particular it is not known whether such treatment works during pregnancy or whether postnatal exposure is important. Objective We investigated whether the probiotic Lactobacillus rhamnosus strain GG (LGG) influences fetal immune responses when administered to pregnant women, as a possible mechanism for its protective effects against the development of eczema. Methods Peripheral blood mononuclear cell from 11 adults treated with LGG, and cord blood mononuclear cells (CBMCs) from 73 women participating in a randomized controlled trial of LGG treatment were cultured with heat‐killed LGG, ovalbumin (OVA) or without stimulus. Cells were analysed by flow cytometry and real‐time PCR for markers of dendritic cell (DC) phenotype, T cell proliferation and regulation. Cytokine secretion was analysed in culture supernatants by multiplex cytokine assay. Results LGG treatment of adults led to systemic immune responses suggestive of antigen‐specific tolerance including reduced CD4⁺ T cell proliferation to heat‐killed LGG (30% reduction; P=0.03). LGG treatment of pregnant women did not influence CD4⁺ T cell proliferation, forkhead box P3 expression, DC phenotype or cytokine secretion in CBMCs cultured with heat‐killed LGG or OVA. Conclusion LGG treatment of pregnant women fails to influence fetal antigen‐specific immune responses. This suggests that modulation of fetal immune responses may not be a major mechanism by which probiotics such as LGG prevent eczema.     

An Optimized CFSE‐based T‐cell Suppression Assay to Evaluate the Suppressive Capacity of Regulatory T‐Cells Induced by Human Tolerogenic Dendritic Cells

In autoimmune diseases or transplant graft rejection, a therapy that will prevent or reduce the present immune activation is highly desired. Ex vivo generated tolerogenic dendritic cells (DC) are considered to have a strong potential as cellular therapy for these diseases. One of the mechanisms of immune suppression mediated by tolerogenic DC is the induction of regulatory T‐cells (Treg). Consequently, the efficacy of such DC to induce Treg will reflect their tolerogenic capacity. Because no specific markers have been described for human induced (i)Treg yet, the Treg can only be appreciated by functionality. Therefore, we have optimized an in vitro suppression assay to screen for human DC‐induced‐Treg activity. IL‐10‐generated tolerogenic DC were used to induce Treg that were previously shown to effectively suppress the proliferation of responder T‐cells stimulated with allogeneic mature DC (mDC). Our results show that the suppressive capacity of IL‐10 DC‐induced Treg measured in the suppression assay increases with the iTreg dose and decreases with higher numbers of antigen‐presenting cells (APC) as T‐cell stimulation. Lowering the ratio between responder T‐cells and stimulator mDC present in the coculture clearly improved the read‐out of the suppression assay. Furthermore, mDC‐primed T‐cells in the suppression assay were shown to be an essential control condition. In conclusion, we recommend titrations of both APC and iTreg in the suppression assay and to include a negative control condition with T‐cells primed by mDC, to distinguish specific and functional suppression by iTreg from possible generalized suppressive activity.     

HIV gag protein is efficiently cross‐presented when targeted with an antibody towards the DEC‐205 receptor in Flt3 ligand‐mobilized murine DC

DC present exogenous proteins to MHC class I‐restricted CD8⁺ T cells. This function does not require endogenous antigen synthesis within DC, providing the potential to elicit CD8⁺ T‐cell responses to immune complexes, inactivated microbes, dying cells, and proteins such as OVA. In mice, the CD8⁺ or DEC‐205⁺ DC are specialized for cross‐presentation, and this subset can be increased 10‐fold in numbers following Fms‐like tyrosine kinase 3 ligand (Flt3L) treatment in vivo. Therefore, we studied cross‐presentation by abundant Flt3L DC using HIV gag protein. When enriched by positive selection with anti‐CD11c beads, cells from Flt3L mice are not only more abundant but are also more highly enriched in CD11c^high DC, particularly the DEC‐205⁺ subset. DC cross‐present HIV gag to primed CD8⁺ T cells, but when the antigen is delivered within an antibody to DEC‐205 receptor, cross‐presentation becomes 100‐fold more efficient than non‐targeted antigen. This finding requires gag to be engineered into anti‐DEC antibody, not just mixed with antibody. Flt3L DC are a valuable tool to study cross‐presentation, since their use overcomes the obstacle posed by the low number of cross‐presenting DC in the steady state. These findings support future experiments to use Flt3L to enhance presentation of DC‐targeted vaccines.     

Increased salt exposure affects both lymphoid and myeloid effector functions,influencing innate‐associated disease but not T‐cell‐associated autoimmunity

High salt consumption has since long been associated with elevated blood pressure and cardiovascular disease. Recently, mouse studies suggested that a high dietary salt intake exacerbates the clinical manifestations of autoimmunity. Using naïve cells ex vivo after pre‐exposure of mice to high salt intake, we showed that increased salt exposure affects the viability and effector functions of immune cells. CD4⁺ T‐cells evidenced a pro‐inflammatory phenotype characterized by increased secretion of IFNγ and IL‐17A, when exposed to high salt concentrations in vitro. Interestingly, this phenotype was associated with osmotic pressure, as replacing salt for d ‐mannitol resulted in similar observations. However, high salt intake did not alter the development of T‐cell‐dependent autoimmunity. Instead, recruitment of peritoneal macrophages was increased in mice pre‐exposed to high salt concentrations. These cells had an increased production of both TNFα and IL‐10, suggesting that salt stimulates expansion and differentiation of different subsets of macrophages. Moreover, mice pre‐exposed to high salt intake developed exacerbated symptoms of colitis, when induced by dextran sulphate sodium. The aggravated colitis in salt‐exposed animals was associated with a higher frequency of CD4⁺ T‐cells and CD11b⁺ CD64⁺ macrophages producing TNFα. These phenotypes correlated with elevated titres of faecal IgA and higher lymphocytic cellularity in the colon, mesenteric lymph nodes and spleen. In conclusion, we report here that high salt intake affects both lymphoid and myeloid cells ex vivo. However, the effects of high salt intake in vivo seem less pronounced in terms of CD4⁺ T‐cell responses, whereas macrophage‐dependent pathologies are significantly influenced.     

Antigen‐loaded ER microsomes from APC induce potent immune responses against viral infection

Although matured DC are capable of inducing effective primary and secondary immune responses in vivo, it is difficult to control the maturation and antigen loading in vitro. In this study, we show that ER‐enriched microsomal membranes (microsomes) isolated from DC contain more peptide‐receptive MHC I and II molecules than, and a similar level of costimulatory molecules to, their parental DC. After loading with defined antigenic peptides, the microsomes deliver antigenic peptide–MHC complexes (pMHC) to both CD4 and CD8 T cells effectively in vivo. The peptide‐loaded microsomes accumulate in peripheral lymphoid organs and induce stronger immune responses than peptide‐pulsed DC. The microsomal vaccines protect against acute viral infection. Our data demonstrate that peptide–MHC complexes armed microsomes from DC can be an important alternative to DC‐based vaccines for protection from viral infection.     

Infusion of ex‐vivo expanded human TCR‐αβ+ double‐negative regulatory T cells delays onset of xenogeneic graft‐versus‐host disease

Despite the demonstration of potent immunosuppressive function of T cell receptor (TCR)‐αβ⁺ double‐negative regulatory T cells (DN T_regs), scarce numbers and lack of effective expansion method limit their clinical applications. Here we describe an approach that allows for ～3500‐fold ex‐vivo expansion of human DN T_regs within 3 weeks with > 97% purity. Ex‐vivo‐expanded DN T_regs suppress proliferation of polyclonally stimulated autologous T and B cells in vitro through direct cell‐to‐cell contact. In vivo, we demonstrate for the first time that infusion of human DN T_regs delayed an onset of xenogeneic graft‐versus‐host disease (GVHD) significantly in a humanized mouse model. Furthermore, preincubation of ex‐vivo‐expanded DN T_regs with a mechanistic target of rapamycin (mTOR) inhibitor rapamycin enhanced their immune regulatory function further. Taken together, this study demonstrates that human DN T_regs can be expanded ex vivo to therapeutic numbers. The expanded DN T_regs can suppress proliferation of T and B cells and attenuate GVHD, highlighting the potential clinical use of DN T_regs to mitigate GVHD.     

Macrophages play an essential role in antigen‐specific immune suppression mediated by T CD8+ cell‐derived exosomes

Murine contact sensitivity (CS) reaction could be antigen‐specifically regulated by T CD8⁺ suppressor (Ts) lymphocytes releasing microRNA‐150 in antibody light‐chain‐coated exosomes that were formerly suggested to suppress CS through action on macrophages (Mφ). The present studies investigated the role of Mφ in Ts cell‐exosome‐mediated antigen‐specific suppression as well as modulation of Mφ antigen‐presenting function in humoral and cellular immunity by suppressive exosomes. Mice depleted of Mφ by clodronate liposomes could not be tolerized and did not produce suppressive exosomes. Moreover, isolated T effector lymphocytes transferring CS were suppressed by exosomes only in the presence of Mφ, demonstrating the substantial role of Mφ in the generation and action of Ts cell regulatory exosomes. Further, significant decrease of number of splenic B cells producing trinitrophenyl (TNP) ‐specific antibodies with the alteration of the ratio of serum titres of IgM to IgG was observed in recipients of exosome‐treated, antigen‐pulsed Mφ and the significant suppression of CS was demonstrated in recipients of exosome‐treated, TNP‐conjugated Mφ. Additionally, exosome‐pulsed, TNP‐conjugated Mφ mediated suppression of CS in mice pre‐treated with a low‐dose of cyclophosphamide, suggesting de novo induction of T regulatory (Treg) lymphocytes. Treg cell involvement in the effector phase of the studied suppression mechanism was proved by unsuccessful tolerization of DEREG mice depleted of Treg lymphocytes. Furthermore, the inhibition of proliferation of CS effector cells cultured with exosome‐treated Mφ in a transmembrane manner was observed. Our results demonstrated the essential role of Mφ in antigen‐specific immune suppression mediated by Ts cell‐derived exosomes and realized by induction of Treg lymphocytes and inhibition of T effector cell proliferation.     

CD8α+ dendritic cells prime TCR‐peptide‐reactive regulatory CD4+FOXP3− T cells

CD4⁺ T cells with immune regulatory function can be either FOXP3⁺ or FOXP3^?. We have previously shown that priming of naturally occurring TCR‐peptide‐reactive CD4⁺FOXP3^? Treg specifically controls Vβ8.2⁺CD4⁺ T cells mediating EAE. However, the mechanism by which these Treg are primed to recognize their cognate antigenic determinant, which is derived from the TCRVβ8.2‐chain, is not known. In this study we show that APC derived from splenocytes of naïve mice are able to stimulate cloned CD4⁺ Treg in the absence of exogenous antigen, and their stimulation capacity is augmented during EAE. Among the APC populations, DC were the most efficient in stimulating the Treg. Stimulation of CD4⁺ Treg was dependent upon processing and presentation of TCR peptides from ingested Vβ8.2TCR⁺CD4⁺ T cells. Additionally, DC pulsed with TCR peptide or apoptotic Vβ8.2⁺ T cells were able to prime Treg in vivo and mediate protection from disease in a CD8‐dependent fashion. These data highlight a novel mechanism for the priming of CD4⁺ Treg by CD8α⁺ DC and suggest a pathway that can be exploited to prime antigen‐specific regulation of T‐cell‐mediated inflammatory disease.     

Regulatory role of DC‐derived osteopontin in systemic allergen sensitization

Osteopontin (OPN) is a secreted phosphoglycoprotein with a wide range of functions, and is involved in various pathophysiological conditions. However, the role of OPN in IgE and Th2‐associated allergic responses remains incompletely defined. The aim of this study was to elucidate the role of OPN in systemic allergen sensitization in mice. When compared with OPN^+/+ mice, significantly increased levels of OVA‐induced IgE were found in OPN^?/? mice. OPN^?/? DC demonstrated an increased capacity to enhance Th2 cytokine production in CD4⁺ T cells from sensitized OPN^+/+ mice. Furthermore, significantly reduced levels of IL‐12p70 expression were seen in LPS‐stimulated OPN^?/? DC as compared with the WT DC, and the reduction was reversible by the addition of recombinant OPN (rOPN). rOPN was able to suppress OVA‐induced IL‐13 production in the cultures of CD4 and OPN^?/? DC, but this inhibitory activity was neutralized by the addition of anti‐IL‐12 Ab. In addition, administration of rOPN in vivo suppressed OVA‐specific IgE production; however, this suppressive effect was abrogated in IL‐12‐deficient mice. These results indicate that DC‐derived OPN plays a regulatory role in the development of systemic allergen sensitization, which is mediated, at least in part, through the production of endogenous IL‐12.     

IFN‐γ‐STAT1 signal regulates the differentiation of inducible Treg: Potential role for ROS‐mediated apoptosis

Regulatory CD4⁺ T cells are important for the homeostasis of immune cells, and their absence correlates with autoimmune disorders. However, how the immune system regulates Treg homeostasis remains unclear. We found that IFN‐γ‐deficient‐mice had more forkhead box P3 (FOXP3⁺) cells than WT mice in all secondary lymphoid organs except the thymus. However, T‐bet‐ or IL‐4Rα‐deficient mice did not show a similar increase. In vitro differentiation studies showed that conversion of naïve T cells into FOXP3⁺ cells (neo‐generated inducible Treg (iTreg)) by TGF‐β was significantly inhibited by IFN‐γ in a STAT‐1‐dependent manner. Moreover, an in vivo adoptive transfer study showed that inhibition of FOXP3⁺ iTreg generation by IFN‐γ was a T‐cell autocrine effect. This inhibitory effect of IFN‐γ on iTreg generation was significantly abrogated after N‐acetyl‐L ‐cysteine treatment both in vitro and in vivo, indicating that IFN‐γ regulation of iTreg generation is dependent on ROS‐mediated apoptosis. Therefore, our results suggest that autocrine IFN‐γ can negatively regulate the neo‐generation of FOXP3⁺ iTreg through ROS‐mediated apoptosis in the periphery.