The role of protease‐activated receptors PAR‐1 and PAR‐2 in the repair of 16HBE 14o− epithelial cell monolayers in vitro

Background We recently reported that repair following mechanical wounding of epithelial cell layers in vitro is dependent on fibrin formation and the activity of locally expressed coagulation cascade proteins. Serine proteases of the coagulation cascade are an important group of protease‐activated receptor (PAR) activators and PAR‐1 to 4 are expressed by the normal bronchial epithelium. Objective We tested the hypothesis that activation of PAR‐1 and PAR‐2 by coagulation cascade proteases stimulates epithelial repair via effects on fibrin formation. Methods Using mechanically wounded 16HBE 14o^? epithelial cell layers in culture, we investigated the effect of PAR‐1 and PAR‐2 agonist peptides, control partially scrambled peptides and PAR‐neutralizing antibodies on the rate of repair and fibrin formation. Coagulation factors in culture supernatants were measured by immunoblot. RT‐PCR was used to investigate PAR‐1, PAR‐2 and PGE2 receptor (EP‐1 to EP‐4) expression in this model and qRT‐PCR to quantify responses to wounding. Additionally, we investigated the effect of exogenously added factor Xa (FXa) and neutrophil elastase and the influence of PGE2 and indomethacin on the repair response. Results PAR‐1 and PAR‐2 peptide agonists stimulated the rate of repair and enhanced the formation of a fibrin provisional matrix to support the repair process. Conversely, PAR‐neutralizing antibodies inhibited repair. Under serum‐free culture conditions, 16HBE 14o^? cells expressed EP‐2 and EP‐3, but not EP‐1 or EP‐4, receptors. Wounding induced an increased expression of EP‐3 but did not alter EP‐2, PAR‐1 or PAR‐2 expression. In the absence of PAR agonists, there was no evidence for a role for PGE2 in fibrin formation or the repair process. Indomethacin attenuated fibrin formation in wounded cultures only in the presence of the PAR‐2 peptide. FXa stimulated epithelial repair while neutrophil elastase reduced the levels of coagulation factors and inhibited repair. Conclusion Locally expressed serine proteases of the coagulation cascade activate PAR‐1 and PAR‐2 to enhance fibrin formation and bronchial epithelial repair. Cite this as: D. Ewen, S.L. Clarke, J.R. Smith, C. Berger, G. Salmon, M. Trevethick and J.K. Shute, Clinical & Experimental Allergy, 2010 (40) 435–449.     

In CD28‐costimulated human naïve CD4+ T cells,I‐κB kinase controls the expression of cell cycle regulatory proteins via interleukin‐2‐independent mechanisms

Stimulation of naïve CD4⁺ T cells through engagement of the T‐cell receptor (TCR) and the CD28 co‐receptor initiates cell proliferation which critically depends on interleukin (IL)‐2 secretion and subsequent autocrine signalling via the IL‐2 receptor. However, several studies indicate that in CD28‐costimulated T cells additional IL‐2‐independent signals are also required for cell proliferation. In this study, using a neutralizing anti‐human IL‐2 antibody and two selective, structurally unrelated, cell‐permeable I‐κB kinase (IKK) inhibitors, BMS‐345541 and PS‐1145, we show that in human naïve CD4⁺ T cells stimulated through a short engagement of the TCR and the CD28 co‐receptor, IKK controls the expression of the cell cycle regulatory proteins cyclin D3, cyclin E and cyclin‐dependent kinase 2 (CDK2) and the stability of the F‐box protein S‐phase kinase‐associated protein 2 (SKP2) and its co‐factor CDC28 protein kinase regulatory subunit 1B (CKS1B), through IL‐2‐independent mechanisms.     

Emergence of a distinct HIV‐specific IL‐10‐producing CD8+ T‐cell subset with immunomodulatory functions during chronic HIV‐1 infection

Interleukin‐10 (IL‐10) plays a key role in regulating proinflammatory immune responses to infection but can interfere with pathogen clearance. Although IL‐10 is upregulated throughout HIV‐1 infection in multiple cell subsets, whether this is a viral immune evasion strategy or an appropriate response to immune activation is unresolved. Analysis of IL‐10 production at the single cell level in 51 chronically infected subjects (31 antiretroviral (ART) naïve and 20 ART treated) showed that a subset of CD8⁺ T cells with a CD25^neg FoxP3^neg phenotype contributes substantially to IL‐10 production in response to HIV‐1 gag stimulation. The frequencies of gag‐specific IL‐10‐ and IFN‐γ‐producing T cells in ART‐naïve subjects were strongly correlated and the majority of these IL‐10⁺ CD8⁺ T cells co‐produced IFN‐γ; however, patients with a predominant IL‐10⁺/IFN‐γ^neg profile showed better control of viraemia. Depletion of HIV‐specific CD8⁺ IL‐10⁺ cells from PBMCs led to upregulation of CD38 on CD14⁺ monocytes together with increased IL‐6 production, in response to gag stimulation. Increased CD38 expression was positively correlated with the frequency of the IL‐10⁺ population and was also induced by exposure of monocytes to HIV‐1 in vitro. Production of IL‐10 by HIV‐specific CD8⁺ T cells may represent an adaptive regulatory response to monocyte activation during chronic infection.     

Abnormalities in iNKT cells are associated with impaired ability of monocytes to produce IL‐10 and suppress T‐cell proliferation in sarcoidosis

Sarcoidosis is a multisystem granulomatous disorder characterized by marked T‐cell expansion of T helper 1 (Th1) cells. The cause of T‐cell overactivity is unknown. We hypothesized that interleukin‐10 (IL‐10) production by a yet undefined cell type might be defective, resulting in loss of regulation of T‐cell activity. Focusing on IL‐10‐producing monocytes, we first showed that monocytes isolated from the peripheral blood of corticosteroid‐naïve sarcoidosis patients (n = 51) produced less IL‐10 compared to controls, and were less able to suppress T‐cell proliferation. In addition, monocytic IL‐10 production correlated negatively with disease activity score. As invariant natural killer T (iNKT) cells are known to both interact with monocytes and be reduced in sarcoidosis patients, we then asked whether iNKT‐specific defects might be responsible for this reduced IL‐10 production. We found that greater numbers of circulating iNKT cells was associated with higher IL‐10 production. Moreover, iNKT cells enhanced monocytic IL‐10 production in vitro. Defective IL‐10 production and T‐cell suppression by sarcoidosis monocytes could be restored following their coculture with iNKT cells, in a CD1d‐ and cell contact‐dependent process. We suggest that reduced iNKT‐cell numbers in sarcoidosis may lead to impaired monocytic IL‐10 production and unchecked T‐cell expansion in sarcoidosis. These findings provide fresh insight into the mechanism of sarcoidosis disease, and interaction between iNKT cells and monocytes.     

TSST-1 induces Th1 or Th2 differentiation in naïve CD4+ T cells in a dose- and APC-dependent manner

Superantigens are potent activators of the immune system, causing a variety of diseases, ranging from food poisoning to septic shock. Here, we examined the effects of different toxic shock syndrome toxin 1 (TSST‐1) concentrations on the activation, proliferation and synthesis of interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4) in purified naïve human CD4⁺ T cells in a serum‐free in vitro system. TSST‐1 given in low doses (1–10 pg/ml) generates a pronounced T helper 2 (Th2)‐like cytokine profile, characterized by elevated IL‐4‐expressing T‐cell populations and reduced IFN‐γ‐producing populations, whereas higher doses (100 pg/ml) induce a Th1‐like profile, with increased expression of IFN‐γ and reduced expression of IL‐4. These patterns were even more pronounced by adding exogenous cytokines like IL‐12 and IL‐4 and by the type of antigen‐presenting cells (APCs). Thus, B cells induced Th2 shifts, whereas monocytes favoured Th1 induction. Moreover, IL‐12 in conditions with B cells counteracted their Th2 bias. Interestingly, in purified naïve T‐cell cultures, containing a small population of HLA‐DR⁺ T cells, Th1/Th2 differentiation can be induced by TSST‐1 too. There, Th‐cell polarization is strongly dependent on TSST‐1 concentration, indicating that this is a key parameter in regulating the differentiation of T cells. In conclusion, our data show that Th1/Th2 differentiation of TSST‐1‐stimulated naïve T cells is controlled by the type of APCs, and in APC‐depleted cultures, it depends on the presence of HLA‐DR⁺ cells and TSST‐1 concentration.     

Effects of repeated injections of fibroblast‐stimulating lipopeptide‐1 on fever,formation of cytokines,and on the responsiveness to endotoxin in guinea‐pigs

Aims: We investigated, whether the Toll‐like receptors (TLRs)‐2/6‐agonist fibroblast‐stimulating lipopeptide‐1 (FSL‐1), like the TLR‐4 agonist lipopolysaccharide (LPS), induces a state of tolerance. We further tested the influence of repeated pre‐treatment with FSL‐1 on the animals’ responsiveness to LPS. Methods: Abdominal temperature was recorded in unrestrained guinea‐pigs with intra‐abdominally implanted radiotransmitters. Circulating concentrations of tumour necrosis factor (TNF) and interleukin‐6 (IL‐6) were measured with specific bioassays. We tested the effects of intra‐arterial (i.a.) or intraperitoneal (i.p.) injections of 100 μg kg^?1 FSL‐1, repeated five times at intervals of 3 days. The animals’ responses to i.a. or i.p. injections of 10 μg kg^?1 LPS were determined another 3 days later and compared to those of naïve guinea‐pigs. Results: The FSL‐1‐induced TNF peak was significantly attenuated starting with the third i.a. administration, while fever was unimpaired and the IL‐6‐peak just tended to decrease. Fever and IL‐6 in response to i.a. injections of LPS were identical in both groups, while circulating TNF was higher in naïve compared to FSL‐1 pre‐treated animals. The effects of repeated i.p. injections of FSL‐1 were more pronounced resulting in attenuation of fever as well as circulating TNF and IL‐6, the strongest reduction observed after the third stimulation with FSL‐1. Repeated i.p. pre‐treatment with FSL‐1 induced hyporesponsiveness to i.p. administration of LPS compared to naïve animals with regard to fever and especially with regard to LPS‐induced formation of cytokines. Conclusions: There is a development of tolerance to FSL‐1 and cross‐tolerance between FSL‐1 and LPS depending on the route of administration of the respective TLR‐2/6 and TLR‐4 agonists.     

Suppressive and pro‐inflammatory roles for IL‐4 in the pathogenesis of experimental drug‐induced liver injury

The pathogenesis of immune‐mediated drug‐induced liver injury (DILI) following halogenated anesthetics, carbamazepine or alcohol has not been fully elucidated. Detecting cytochrome P450 2E1 (CYP2E1) IgG4 auto‐antibodies in anesthetic DILI patients suggests a role for IL‐4 in this hapten‐mediated process. We investigated IL‐4‐mediated mechanisms using our model of experimental DILI induced by immunizing BALB/c (WT) and IL‐4^?/? (KO) mice with S100 liver proteins covalently modified by a trifluoroacetyl chloride (TFA) hapten formed following halogenated anesthetic metabolism by CYP2E1. WT mice developed more hepatitis, TFA and S100 antibodies (p<0.01), as well as T‐cell proliferation to CYP2E1 and TFA (p<0.01) than KO mice. Additionally, WT CD4⁺ T cells adoptively transferred hepatitis to naïve Rag^?/? mice (p<0.01). Pro‐inflammatory cytokines were expectedly decreased in TFA hapten‐stimulated KO splenocyte supernatants (p<0.001); however, IL‐2 and IFN‐γ (p<0.05), as well as IL‐6 and IL‐10 (p<0.001) levels were elevated in CYP2E1‐stimulated KO splenocyte supernatants, suggesting dual IL‐4‐mediated pro‐inflammatory and regulatory responses. Anti‐IL‐10 administered to KO mice increased hepatitis, TFA and CYP2E1 antibodies in KO mice confirming a critical role for IL‐4. This is the first demonstration of dual roles for IL‐4 in the pathogenesis of immune‐mediated DILI by suppressing auto‐antigen‐induced regulatory responses while promoting hapten‐induced pro‐inflammatory responses.     

Minor Histocompatibility Antigen UTY as Target for Graft‐versus‐Leukemia and Graft‐versus‐Haematopoiesis in the Canine Model

Male patients with female‐stem‐cell donors have better prognosis compared to female‐to‐male combinations due to Y‐encoded minor histocompatibility antigens recognized by female‐alloimmune‐effector lymphocytes in the context of a graft‐versus‐leukemia (GvL) effect. We provide data in a dog‐model that the minor histocompatibility antigen UTY might be a promising target to further improve GvL‐immune reactions after allogeneic‐stem‐cell transplantations. Female‐canine‐UTY‐specific T cells (CTLs) were stimulated in vitro using autologous‐DCs loaded with three HLA‐A2‐restricted‐UTY‐derived peptides (3‐fold‐expansion), and specific T cell responses were determined in 3/6 female dogs. CTLs specifically recognized/lysed autologous‐female‐peptide‐loaded DCs, but not naïve‐autologous‐female DCs and monocytes. They mainly recognized bone‐marrow (BM) and to a lower extent DCs, monocytes, PBMCs and B‐cells from DLA‐identical‐male littermates and peptide‐loaded T2‐cells in an MHC‐I‐restricted manner. A UTY‐/male‐specific reactivity was also obtained in vivo after stimulation of a female dog with DLA‐identical‐male PBMCs. In summary, we demonstrated natural UTY processing and presentation in dogs. We showed that female‐dog CTLs were specifically stimulated by HLA‐A2‐restricted‐UTY peptides, thereby enabling recognition of DLA‐identical‐male cells, mainly BM cells. These observations suggest UTY as a promising candidate‐antigen to improve GvL‐reactions in the course of immunotherapy.     

Distinct IL‐7 signaling in recent thymic emigrants versus mature naïve T cells controls T‐cell homeostasis

IL‐7 is essential for T‐cell survival but its availability is limited in vivo. Consequently, all peripheral T cells, including recent thymic emigrants (RTEs) are constantly competing for IL‐7 to survive. RTEs are required to replenish TCR diversity and rejuvenate the peripheral T‐cell pool. However, it remains unknown how RTEs successfully compete with resident mature T cells for IL‐7. Moreover, RTEs express low levels of IL‐7 receptors, presumably rendering them even less competitive. Here, we show that, surprisingly, RTEs are more responsive to IL‐7 than mature naïve T cells as demonstrated by markedly increased STAT5 phosphorylation upon IL‐7 stimulation. Nonetheless, adoptive transfer of RTE cells into lymphopenic host mice resulted in slower IL‐7‐induced homeostatic proliferation and diminished expansion compared to naïve donor T cells. Mechanistically, we found that IL‐7 signaling in RTEs preferentially upregulated expression of Bcl‐2, which is anti‐apoptotic but also anti‐proliferative. In contrast, naïve T cells showed diminished Bcl‐2 induction but greater proliferative response to IL‐7. Collectively, these data indicate that IL‐7 responsiveness in RTE is designed to maximize survival at the expense of reduced proliferation, consistent with RTE serving as a subpopulation of T cells rich in diversity but not in frequency.     

Mycobacterium tuberculosis RpfE promotes simultaneous Th1‐ and Th17‐type T‐cell immunity via TLR4‐dependent maturation of dendritic cells

Reciprocal induction of the Th1 and Th17 immune responses is essential for optimal protection against Mycobacterium tuberculosis (Mtb); however, only a few Mtb antigens are known to fulfill this task. A functional role for resuscitation‐promoting factor (Rpf) E, a latency‐associated member of the Rpf family, in promoting naïve CD4⁺ T‐cell differentiation toward both Th1 and Th17 cell fates through interaction with dendritic cells (DCs) was identified in this study. RpfE induces DC maturation by increasing expression of surface molecules and the production of IL‐6, IL‐1β, IL‐23p19, IL‐12p70, and TNF‐α but not IL‐10. This induction is mediated through TLR4 binding and subsequent activation of ERK, p38 MAPKs, and NF‐κB signaling. RpfE‐treated DCs effectively caused naïve CD4⁺ T cells to secrete IFN‐γ, IL‐2, and IL‐17A, which resulted in reciprocal expansions of the Th1 and Th17 cell response along with activation of T‐bet and RORγt but not GATA‐3. Furthermore, lung and spleen cells from Mtb‐infected WT mice but not from TLR4^?/? mice exhibited Th1 and Th17 polarization upon RpfE stimulation. Taken together, our data suggest that RpfE has the potential to be an effective Mtb vaccine because of its ability to activate DCs that simultaneously induce both Th1‐ and Th17‐polarized T‐cell expansion.     

Production of endocannabinoids by activated T cells and B cells modulates inflammation associated with delayed‐type hypersensitivity

Endocannabinoids are endogenous ligands for the cannabinoid (CB) receptors which include anandamide (AEA) and 2‐arachidonyl glycerol (2‐AG). 2‐AG has been linked to inflammation due to its elevated expression in animal models of autoimmunity and hypersensitivity. However, administration of exogenous 2‐AG has been shown to suppress inflammation making its precise role unclear. In the current study, we investigated the role of 2‐AG following immunization of C57BL/6 (BL6) mice with methylated BSA (mBSA) antigen, which triggers both delayed‐type hypersensitivity (DTH) and antibody response. We found that while naïve T cells and B cells expressed low levels of 2‐AG, expression significantly increased upon activation. Furthermore, mBSA‐immunized mice exhibited higher 2‐AG concentration than naïve mice. Exogenous 2‐AG treatment (40 mg/kg) in mBSA‐immunized mice led to reduced DTH response, and decreased Th1 and Th17‐associated cytokines including IL‐6, IL‐2, TNF‐α, and the IgG response. Addition of 2‐AG to activated popliteal lymph node (PopLN) cell cultures also inhibited lymphocyte proliferation. Together, these data show for the first time that activated T and B cells produce 2‐AG, which plays a negative regulatory role to decrease DTH via inhibition of T‐cell activation and proliferation. Moreover, these findings suggest that exogenous 2‐AG treatment can be used therapeutically in Th1‐ or Th17‐driven disease.     

Tim‐3 regulates inflammatory cytokine expression and Th17 cell response induced by monocytes from patients with chronic hepatitis B

Tim‐3 is expressed on monocytes/macrophages and is involved in the regulation of inflammatory responses. The aim of this study was to determine the effect of Tim‐3 on inflammatory response triggered by peripheral monocytes from patients with chronic hepatitis B (CHB). Tim‐3 expression on peripheral monocytes and frequency of Th17 cells in peripheral blood mononuclear cells (PBMCs) derived from CHB patients were detected. Followed by lipopolysaccharides (LPS) activation of circulating monocytes from CHB patients, expression of inflammatory cytokines including TNF‐α，IL‐1β and IL‐6 were examined in the presence and absence of Galectin‐9 which is the ligand for Tim‐3. Subsequently, after purified CD4+T cells were cocultured with LPS‐activated monocytes from CHB patients in the presence of anti‐Tim‐3 antibody, percentage of Th17 cells and production of IL‐17 were measured. Tim‐3 expression was significantly upregulated and closely correlated to the frequency of Th17 cells in patients with CHB. Expression of TNF‐α，IL‐1β and IL‐6 increased significantly in monocytes stimulated with LPS and Galectin‐9, compared to LPS stimulation alone. LPS‐activated monocytes from CHB patients could drive differentiation of memory CD4+T cells to Th17 cells. However, under the blockade of Tim‐3 signalling by anti‐Tim‐3 antibody, percentage of Th17 cells and production of IL‐17 decreased significantly. Our results demonstrate that upregulated expression of Tim‐3 on circulating monocytes accelerates inflammatory response by promoting production of inflammatory cytokines and Th17 responses in CHB.     

ICOS cooperates with CD28, IL-2, and IFN-gamma and modulates activation of human naïve CD4+ T cells

Several sets of data indicate that ICOS regulates cytokine production in activated T cells, but is less effective on naïve T cells. This work evaluates ICOS function in human naïve CD4⁺ T cells through an assessment of the effect of soluble forms of the ICOS and CD28 physiological ligands on activation driven by anti‐CD3 mAb. ICOS strikingly potentiated secretion of IL‐2, IFN‐γ, IL‐10, and TNF‐α, but not IL‐4, promoted by optimal stimulation of CD3+CD28, and it was the key switching‐factor of activation when cells received suboptimal stimulation of CD3+CD28 or stimulation of CD3 alone in the presence of exogenous IL‐2. In these conditions, blockade of IL‐2 and IFN‐γ showed that ICOS builds up a positive feedback loop with IFN‐γ, which required IL‐2 and was inhibited by IL‐4. By contrast, in the absence of CD28 triggering or exogenous IL‐2, ICOS‐induced costimulation mainly supported expression of TGF‐β1 and FoxP3 and differentiation of regulatory T cells capable to inhibit proliferation of naïve CD4⁺ T cells driven by allogeneic cells. These data suggest that ICOS favors differentiation of Th effector cells when cooperates with appropriate activation stimuli such as CD3+CD28 or CD3+IL‐2, whereas it supports differentiation of regulatory T cells when costimulatory signals are insufficient.     

Allergen‐specific naïve and memory CD4+ T cells exhibit functional and phenotypic differences between individuals with or without allergy

Although allergen‐specific CD4⁺ T cells are detectable in the peripheral blood of both individuals with or without allergy, their frequencies and phenotypes within the memory as well as naïve repertoires are incompletely known. Here, we analyzed the DRB1*0401‐restricted responses of peripheral blood‐derived memory (CD4⁺CD45RO⁺) and naïve (CD4⁺CD45RA⁺) T cells from subjects with or without allergy against the immunodominant epitope of the major cow dander allergen Bos d 2 by HLA class II tetramers in vitro. The frequency of Bos d 2_127–142‐specific memory T cells in the peripheral blood‐derived cultures appeared to be higher in subjects with allergy than those without, whereas naïve Bos d 2_127–142‐specific T cells were detectable in the cultures of both groups at nearly the same frequency. Surprisingly, the TCR avidity of Bos d 2_127–142‐specific T cells of naïve origin, as assessed by the intensity of HLA class II tetramer staining, was found to be higher in individuals with allergy. Upon restimulation, long‐term Bos d 2_127–142‐specific T‐cell lines generated from both memory and naïve T‐cell pools from individuals with allergy proliferated more strongly, produced more IL‐4 and IL‐10, and expressed higher levels of CD25 but lower levels of CXCR3 than the T‐cell lines from individuals without allergy, demonstrating differences also at the functional level. Collectively, our current results suggest that not only the memory but also the naïve allergen‐specific T‐cell repertoires differ between individuals with or without allergy.     

TGF‐β interactions with IL‐1 family members trigger IL‐4‐independent IL‐9 production by mouse CD4+ T cells

TGF‐β and IL‐4 were recently shown to selectively upregulate IL‐9 production by naïve CD4⁺ T cells. We report here that TGF‐β interactions with IL‐1α, IL‐1β, IL‐18, and IL‐33 have equivalent IL‐9‐stimulating activities that function even in IL‐4‐deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T‐cell activation. Based on intracellular IL‐9 staining, all IL‐9‐producing cells were CD4⁺ and 80–90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL‐9, IL‐13 and IL‐17 were strongly stimulated by IL‐1 and either inhibited (IL‐13) or were unaffected (IL‐17) by addition of TGF‐β. IL‐9 and IL‐17 production also differed in their dependence on IL‐2 and regulation by IL‐1/IL‐23. As IL‐9 levels were much lower in Th2 and Th17 cultures, our results identify TGF‐β/IL‐1 and TGF‐β/IL‐4 as the main control points of IL‐9 synthesis.     

Human milk oligosaccharides promote immune tolerance via direct interactions with human dendritic cells

Human milk oligosaccharides (HMOS) are a complex mixture of bioactive components supporting the immune development of breastfed‐infants. Dendritic cells (DCs) play a central role in the regulation of immune responses, being specialized in antigen presentation and driving T‐cell priming as well as differentiation. However, little is known about the direct effects of HMOS on human DC phenotypes and functions. Here, we report that HMOS mixture isolated from pooled human milk, induced semi‐maturation of human monocytes‐derived DCs (moDCs), and elevated levels of IL‐10, IL‐27 and IL‐6 but not IL‐12p70 and TNF‐α. Consistently, HMOS‐conditioned human moDCs promoted Treg generation from naïve CD4⁺ T cells. Interestingly, HMOS limited LPS‐induced maturation of human moDCs, while maintained IL‐10 and IL‐27 secretion and reduced LPS‐induced production of IL‐12p70, IL‐6 and TNF‐α. Furthermore, HMOS+LPS‐stimulated DCs induced a higher frequency of Tregs and increased IL‐10 production, while a reduction in Tbet+Th1 frequency and IFN‐γ production was detected as compared to LPS‐DCs. The regulatory effects of HMOS seemed to be mediated by interactions of HMOS with receptors, including but not limited to TLR4 and DC‐SIGN on human moDCs. In conclusion, HMOS contain tolerogenic factors influencing human moDCs and thereby modulating the development of the neonatal immune system.     

Adoptive transfer of dendritic cells from allergic mice induces specific immunoglobulin E antibody in naïve recipients in absence of antigen challenge without altering the T helper 1/T helper 2 balance

Dendritic cells (DCs) are important in the regulation of immune responses and it has been proposed that these cells play an important role in asthma; however, their role in food allergy is still largely unknown. Our aim was to study specific immunoglobulin E (IgE) and immunoglobulin G (IgG) responses in naïve recipients following adoptive transfer of myeloid DCs from allergic and control mice. The phenotypic features and lymphokine production of DCs were also investigated. CD11c ^{+ /hi} B220^? DCs isolated from spleen and Peyer's patches (PP) of cow's milk (CM) allergic and control mice were transferred intravenously (i.v.) into naïve syngeneic recipients, and IgE‐ and IgG‐specific responses were evaluated. Experiments were also carried out to determine the levels of interferon‐γ (IFN‐γ) and interleukin (IL)‐4 produced by splenocytes from naïve recipients following the adoptive transfer, and CD40 ligand (CD40L)‐mediated IL‐10 production by DCs from allergic and control mice. DCs isolated from spleen and PP of allergic mice, but not control groups, induced CM‐specific IgG and IgE antibody production in naïve recipients in the absence of previous immunization, but did not modify the T helper 1 (Th1) and T helper 2 (Th2) balance. Furthermore, although no difference was observed in the expression of canonical DC surface markers, PP DCs from allergic mice produced less IL‐10 than DCs from controls. We interpret these data as showing that DCs play a pivotal role in allergen‐specific IgE responses and that a Th2‐skewed response may not be involved in the early phase of allergic responses. The identification of the mechanisms underlying these events may help to design novel strategies of therapeutic intervention in food allergy.     

PKC‐θ exists in an oxidized inactive form in naive human T cells

PKC‐θ plays a central role in TCR‐induced IL‐2 production and T‐cell proliferation. The aim of the present study was to analyse how PKC‐θ is regulated in human T cells during T‐cell activation and differentiation. We show that PKC‐θ is found in a high‐molecular disulfide‐linked complex in naïve T cells, and that PKC‐θ most likely is inactive in this form. In parallel with the accumulation of the major redox regulators, glutathione and thioredoxin, PKC‐θ is gradually reduced to the 82 kDa active form during T‐cell activation. We demonstrate that PKC‐θ is recruited to the plasma membrane in the disulfide‐linked form in naïve T cells, and that activation of PKC‐θ is redox dependent and requires de novo synthesis of glutathione. This is the first study that shows that the activity of PKC‐θ is regulated by the intracellular redox state, and that PKC‐θ is recruited to the plasma membrane in an inactive form in naïve T cells. Our observations underscore the existence of major differences in TCR signaling in naïve versus primed T cells.