Escherichia coli Nissle 1917 protects gnotobiotic pigs against human rotavirus by modulating pDC and NK‐cell responses

Lactobacillus rhamnosus GG (LGG), a gram‐positive lactic acid bacterium, is one of the most widely used probiotics; while fewer gram‐negative probiotics including Escherichia coli Nissle 1917 (EcN) are characterized. A mechanistic understanding of their individual and interactive effects on human rotavirus (HRV) and immunity is lacking. In this study, noncolonized, EcN‐, LGG‐, and EcN + LGG‐colonized neonatal gnotobiotic (Gn) pigs were challenged with HRV. EcN colonization is associated with a greater protection against HRV, and induces the highest frequencies of plasmacytoid dendritic cells (pDCs), significantly increased NK‐cell function and decreased frequencies of apoptotic and TLR4⁺ mononuclear cells (MNCs). Consistent with the highest NK‐cell activity, splenic CD172⁺ MNCs (DC enriched fraction) of EcN‐colonized pigs produced the highest levels of IL‐12 in vitro. LGG colonization has little effect on the above parameters, which are intermediate in EcN + LGG‐colonized pigs, suggesting that probiotics modulate each other's effects. Additionally, in vitro EcN‐treated splenic or intestinal MNCs produce higher levels of innate, immunoregulatory and immunostimulatory cytokines, IFN‐α, IL‐12, and IL‐10, compared to MNCs of pigs treated with LGG. These results indicate that the EcN‐mediated greater protection against HRV is associated with potent stimulation of the innate immune system and activation of the DC‐IL‐12‐NK immune axis.     

Lactobacillus GG has in vitro effects on enhanced interleukin-10 and interferon-γ release of mononuclear cells but no in vivo effects in supplemented mothers and their neonates

Background The value of probiotics for primary prevention is controversial. Moreover, only little is known about the underlying immunological mechanisms of action. Therefore, we assessed the proliferative response and cytokine release in cultures of isolated mononuclear cells from pregnant women and their neonates supplemented with Lactobacillus GG (LGG) or placebo. Methods In a double‐blind, placebo‐controlled prospective trial, pregnant women with at least one first‐degree relative or a partner with an atopic disease were randomly assigned to receive either the probiotic LGG (ATCC 53103; 5 × 10⁹ colony‐forming units LGG twice daily) or placebo 4–6 weeks before expected delivery, followed by a post‐natal period of 6 months. Cord blood mononuclear cells (CBMC) and peripheral blood mononuclear cells (PBMC) of the corresponding mother were isolated from cord blood and peripheral blood (n=68). The proliferative response of CBMC and PBMC was expressed as the stimulation index (SI), which was calculated according to the ratio between the mean counts per minute (c.p.m.) values measured in the wells with stimulated cells and the mean c.p.m. values measured in the wells with unstimulated cells. Additionally, the cytokines IFN‐γ, IL‐10 and IL‐13 in the cell culture supernatants were measured using the ELISA technique. Results No difference was observed between the LGG‐supplemented group and the placebo group in terms of the proliferative capacity of maternal or neonatal cord blood cells in response to IL‐2, β‐lactoglobulin or LGG. In vitro stimulation with LGG resulted in significantly enhanced release of IL‐10 and IFN‐γ, compared with cytokine release in unstimulated controls. However, this phenomenon was observed in supernatants of maternal and neonatal MC in both groups, independent of prior supplementation with LGG. Conclusion LGG has in vitro effects on enhanced IL‐10 and IFN‐γ release of mononuclear cells. However, supplementation with LGG during pregnancy did not alter the proliferative capacity or cytokine pattern in their recipients.     

Reduced membrane bound CD14 expression in the cord blood of infants with a family history of allergic disease

Background Infants at increased risk of allergic disease have altered immune function at birth, but the specific immune changes described differ between studies and their precise nature is not well defined. Changes affecting innate immune responses may be particularly important in allergic disease pathogenesis. Objective We investigated whether inherited risk of allergic disease is associated with altered markers of innate immunity, T cell regulation or dendritic cell (DC) percentage in human newborns. Methods Cord blood was collected from infants at low risk (no parent affected by allergic disease, n=14), intermediate risk (one affected parent, n=54) or high risk (two affected parents, n=25) of developing allergic disease. Cord blood mononuclear cells were cultured with ovalbumin (OVA), lipopolysaccharide, lipoteichoic acid (LTA), heat‐killed lactobacillus, α‐CD3 or medium alone. Cells were analysed by flow cytometry for expression of CD14, FoxP3 and DC percentage, and by real‐time RT‐PCR for TLR2 and TLR4 expression in infants at intermediate or high risk of allergic disease. Results Infants at high risk of allergic disease had reduced percentage of CD14⁺ monocytes (P=0.01) and reduced CD14 mean fluorescence intensity (P=0.01) in uncultured mononuclear cells. They also had decreased DC percentage in mononuclear cells cultured with OVA (P=0.04), lipopolysaccharide (P=0.01), LTA (P=0.02) and α‐CD3 (P=0.03) as compared with infants at intermediate or low risk of allergic disease. No relationship was seen between risk of allergic disease and TLR2 or TLR4 expression, or FoxP3 expression in cultured cells. Conclusions Infants with a biparental history of allergic disease have altered markers of innate immunity at birth, with reduced expression of membrane bound CD14 and consequently reduced in vitro development of DCs. Further work is needed to understand the role that these alterations play in the pathogenesis of allergic disease, and whether interventions to up‐regulate fetal CD14 expression can prevent allergic disease.     

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.     

Ovalbumin‐specific immunoglobulins A and G levels at age 2 years are associated with the occurrence of atopic disorders

Background Humoral responses to food antigens may reflect the propensity of a child's immune system to develop tolerance to innocuous antigens. Early nutrition as well as probiotics may influence these immunological responses. Objective To study the association of humoral responses to early food antigens with the administration of prebiotics and probiotics, with the occurrence of allergy, and with the length of exclusive breastfeeding. Methods In a randomized double‐blind allergy prevention trial in high‐risk children, 1018 mothers took probiotics or placebo from the 36th week of gestation, and their newborn infants received probiotics and prebiotics or placebo during 6 months. At 2 and 5 years, we evaluated the cumulative incidence of allergic diseases (food allergy, eczema, asthma, rhinitis) and sensitization (skin prick test ?3 mm or serum antigen‐specific IgE>0.7 kU/L). In 688 infants at age 2, we measured in sera‐specific IgA, IgG, IgG1, and IgG4 antibody levels to cow's milk (CM), α‐casein (CAS), β‐lactoglobulin (BLG), and ovalbumin (OVA) with ELISA, and specific IgE levels to CM and hen's egg with UniCap. Results Probiotic treatment (n=342) compared with placebo (n=346) showed no effect on serum food‐specific IgA, IgG, IgG1, or IgG4 concentrations at age 2. Atopic children had higher OVA‐IgA (P<0.001), OVA‐IgG (P=0.001), OVA‐IgG1 (P<0.001), and egg‐IgE but lower OVA‐IgG4/egg‐IgE ratio (P<0.001) than non‐atopic children. Longer duration of exclusive breastfeeding (?4 vs. <4 months) was associated with reduced CM‐ and CAS‐specific serum IgA (P<0.001) and IgG levels (P<0.001; P=0.003). Conclusion and Clinical Relevance Allergy was associated with more intense IgA and IgG responses to OVA. Breastfeeding depressed humoral responses, whereas prebiotics and probiotics supplementation showed no immunomodulatory effect. The effect of probiotics on allergies is not mediated through food‐specific antibody responses. Furthermore, OVA‐specific IgA and IgG antibodies may help in assessing the risk for atopy. [Trial registration: Clinicaltrials.gov NCT00298337] Cite this as: A. K. Kukkonen, E. M. Savilahti, T. Haahtela, E. Savilahti and M. Kuitunen, Clinical & Experimental Allergy, 2011 (41) 1414–1421.     

Human platelet antigen (HPA)‐1a peptides do not reliably suppress anti‐HPA‐1a responses using a humanized severe combined immunodeficiency (SCID) mouse model

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) occurs most frequently when human platelet antigen (HPA)‐1a‐positive fetal platelets are destroyed by maternal HPA‐1a immunoglobulin (Ig)G antibodies. Pregnancies at risk are treated by administration of high‐dose intravenous Ig (IVIG) to women, but this is expensive and often not well tolerated. Peptide immunotherapy may be effective for ameliorating some allergic and autoimmune diseases. The HPA‐1a/1b polymorphism is Leu/Pro33 on β3 integrin (CD61), and the anti‐HPA‐1a response is restricted to HPA‐1b1b and HLA‐DRB3*0101‐positive pregnant women with an HPA‐1a‐positive fetus. We investigated whether or not HPA‐1a antigen‐specific peptides that formed the T cell epitope could reduce IgG anti‐HPA‐1a responses, using a mouse model we had developed previously. Peripheral blood mononuclear cells (PBMC) in blood donations from HPA‐1a‐immunized women were injected intraperitoneally (i.p.) into severe combined immunodeficient (SCID) mice with peptides and HPA‐1a‐positive platelets. Human anti‐HPA‐1a in murine plasma was quantitated at intervals up to 15 weeks. HPA‐1a‐specific T cells in PBMC were identified by proliferation assays. Using PBMC of three donors who had little T cell reactivity to HPA‐1a peptides in vitro, stimulation of anti‐HPA‐1a responses by these peptides occurred in vivo. However, with a second donation from one of these women which, uniquely, had high HPA‐1a‐specific T cell proliferation in vitro, marked suppression of the anti‐HPA‐1a response by HPA‐1a peptides occurred in vivo. HPA‐1a peptide immunotherapy in this model depended upon reactivation of HPA‐1a T cell responses in the donor. For FNAIT, we suggest that administration of antigen‐specific peptides to pregnant women might cause either enhancement or reduction of pathogenic antibodies.     

Sublingual tolerance induction with antigen conjugated to cholera toxin B subunit induces Foxp3+CD25+CD4+ regulatory T cells and suppresses delayed-type hypersensitivity reactions

Although sublingual (s.l.) immunotherapy with selected allergens is safe and often effective for treating patients with allergies, knowledge of the immunological mechanisms involved remains limited. Can s.l. administration of antigen (Ag) induce peripheral immunological tolerance and also suppress delayed‐type hypersensitivity (DTH) responses? To what extent can s.l.‐induced tolerance be explained by the generation of Foxp3⁺CD25⁺CD4⁺ regulatory T cells (T_reg)? This study addressed these questions in mice and compared the relative efficacy of administering ovalbumin (OVA) conjugated to cholera toxin B (CTB) subunit with administration of the same Ag alone. We found that s.l. administration of a single or even more efficiently three repeated 40‐μg doses of OVA/CTB conjugate suppressed T‐cell proliferative responses to OVA by cervical lymph node (CLN), mesenteric lymph node (MLN) and spleen cells and concurrently strongly increased the frequency of Ag‐specific T_reg in CLN, MLN and spleen and also transforming growth factor‐β (TGF‐β) levels in serum. The CLN and splenic cells from OVA/CTB‐treated BALB/c mice efficiently suppressed OVA‐specific T‐cell receptor (TCR) transgenic (DO11.10) CD25^?CD4⁺ effector T‐cell proliferation in vitro. Further, s.l. treatment with OVA/CTB completely suppressed OVA‐specific DTH responses in vivo and T‐cell proliferative responses in mice immunized subcutaneously with OVA in Freund's complete adjuvant. The intracellular expression of Foxp3 was strongly increased in OVA‐specific (KJ1‐26⁺) CD4⁺ T cells from OVA/CTB‐treated mice. Thus, s.l. administration of CTB‐conjugated Ag can efficiently induce peripheral T‐cell tolerance associated with strong increases in serum TGF‐β levels and in Ag‐specific Foxp3⁺CD25⁺CD4⁺ T_reg cells.     

Chitin induces upregulation of B7‐H1 on macrophages and inhibits T‐cell proliferation

Chitin is a highly abundant glycopolymer, which serves as structural component in fungi, arthropods and crustaceans but is not synthesized by vertebrates. However, vertebrates express chitinases and chitinase‐like proteins, some of which are induced by infection with helminths suggesting that chitinous structures may be targets of the immune system. The chitin‐induced modulations of the innate and adaptive immune responses are not well understood. Here, we demonstrate that intranasal administration of OVA and chitin resulted in diminished T‐cell expansion and Th2 polarization as compared with OVA administration alone. Chitin did not promote nor attenuate Th2 polarization in vitro. Chitin‐exposed macrophages inhibited proliferation of CD4⁺ T cells in a cell–cell contact‐dependent manner. Chitin induced upregulation of the inhibitory ligand B7‐H1 (PD‐L1) on macrophages independently of MyD88, TRIF, TLR2, TLR3, TLR4 and Stat6. Inhibition of T‐cell proliferation was largely dependent on B7‐H1, as the effect was not observed in cocultures with cells from B7‐H1‐deficient mice.     

EGF receptor activation during allergic sensitization affects IL‐6‐induced T‐cell influx to airways in a rat model of asthma

EGF receptor (EGFR) is involved in cell differentiation and proliferation in airways and may trigger cytokine production by T cells. We hypothesized that EGFR inhibition at the time of allergic sensitization may affect subsequent immune reactions. Brown Norway rats were sensitized with OVA, received the EGFR tyrosine kinase inhibitor, AG1478 from days 0 to 7 and OVA challenge on day 14. OVA‐specific IgE in serum and cytokines and chemokines in BAL were measured 24 h after challenge. To evaluate effects on airway hyperresponsiveness (AHR), rats were sensitized, treated with AG1478, intranasally challenged, and then AHR was assessed. Furthermore chemotactic activity of BALF for CD4⁺ T cells was examined. The eosinophils, neutrophils and lymphocytes in BAL were increased by OVA and only the lymphocytes were reduced by AG1478. OVA significantly enhanced IL‐6 concentration in BAL, which was inhibited by AG1478. However AHR, OVA‐specific IgE and IL‐4 mRNA expression in CD4⁺ T cells were not affected by AG1478. BALF from OVA‐sensitized/challenged rats induced CD4⁺ T‐cell migration, which was inhibited by both AG1478 treatment in vivo and neutralization of IL‐6 in vitro. EGFR activation during sensitization may affect the subsequent influx of CD4⁺ T cells to airways, mainly mediated through IL‐6.     

Imprinted DC mediate the immune‐educating effect of early‐life microbial exposure

It has been long proposed that exposure to environmental factors early in life may have an educating effect on the development of immune regulatory functions. However, experimental studies on this issue are limited and the related molecular and cellular basis remains unclear. Here we report that neonatal exposure to killed bacteria (Chlamydia muridarum, originally called Chlamydia trachomatis mouse pneumonitis (MoPn)) changed the pattern of the hosts' immune responses to a model allergen (OVA) in adulthood. This was associated with altered phenotype and function of DC. We found that DC from adult mice treated neonatally with UV‐killed MoPn exhibited distinct patterns of surface marker and TLR expression and cytokine production from control mice (DC from adult mice neonatally treated with vehicle, (Sham‐DC)). More importantly, DC from adult mice treated neonatally with UV‐killed MoPn induced significantly lower type‐2 antigen‐specific T‐cell responses than Sham‐DC shown in DC:T co‐culture experiments in vitro and in adoptive transfer experiments in vivo. In addition, depletion of T cells in vivo largely abolished the phenotypic and functional alterations of DC caused by bacterial exposure, suggesting the involvement of T cell in this process. Our study demonstrates a central role of DC in linking the early‐life exposure to microbial products and the balanced development of immune regulatory functions and the involvement of T cells in imprinting of the DC function.     

Defective T‐cell receptor‐induced apoptosis of T cells and rejection of transplanted immunogenic tumors in p53−/− mice

Mice lacking the tumor suppressor gene p53 spontaneously develop T‐cell lymphomas at a high rate, suggesting that in these mice lymphomas arise due to defective apoptosis mechanisms in T cells mediated by p53. However, a role of p53 in regulation of T‐cell responses or apoptosis has been poorly defined. TCR‐mediated signaling in the absence of CD28 costimulation induces both apoptosis and proliferation of naïve T cells from WT mice. In this report we show that, in response to TCR stimulation, T cells from naïve p53‐deficient mice exhibited higher proliferation and drastically reduced apoptosis than WT T cells. CD28 costimulation enhanced the proliferation of TCR‐stimulated WT and p53^?/? T cells, suggesting that p53 uncouples CD28‐mediated antiapoptotic and proliferative signals. To evaluate the physiological significance of these findings, we transplanted OVA expressing‐EG.7 tumor cells into WT and p53^?/? mice. Unlike WT mice, p53^?/? mice exhibited a robust tumor‐resistant phenotype and developed cytotoxic T‐cell responses against OVA. Collectively, these data support the hypothesis that p53 is an essential factor in negative regulation of T‐cell responses and have implication for immunomodulation during treatment of cancers and other inflammatory conditions.     

Intravenous immunoglobulin modulates the expansion and cytotoxicity of CD8+ T cells

Intravenous immunoglobulin (IVIg) is successfully used in the treatment of autoimmune diseases involving self‐reactive CD8⁺ T cells. However, its direct influence on the cytotoxic response remains unknown. Using an antigen cross‐presentation assay and a mouse model of ovalbumin (OVA) immunization, we showed that IVIg decreases the in vitro activation, proliferation and cytokine secretion of OVA‐specific CD8⁺ T cells (OT‐I), as well as the in vivo generation of OVA‐specific CD8⁺ T cells. In addition, IVIg significantly decreases the proportion of perforin‐ and CD107a‐expressing CD8⁺ T cells, and inhibits the cytotoxic activity of OVA‐activated OT‐I cells. The interference of IVIg with the CD8⁺ T‐cell response is associated with T‐cell receptor blockade, therefore reducing the interaction between effector and target cells. A similar blockade is observed on human CD8⁺ T cells, suggesting that the observations reported here could apply to the IVIg‐mediated improvement of CD8⁺ T‐cell‐mediated autoimmune conditions in human patients.     

UV inhibits allergic airways disease in mice by reducing effector CD4+ T cells

Background In human asthma, and experimental allergic airways disease in mice, antigen‐presenting cells and CD4⁺ effector cells at the airway mucosa orchestrate, and CD4⁺CD25⁺ regulatory T cells attenuate, allergen immunity. UV irradiation of skin before sensitization with ovalbumin (OVA) causes significantly reduced asthma‐like responses in respiratory tissues. Objective To determine whether UV‐induced changes in CD11c⁺ cells, CD4⁺CD25⁺ effector cells or CD4⁺CD25⁺ regulatory cells in the trachea and airway draining lymph nodes (ADLNs) were responsible for reduced allergic airways disease. Methods The phenotype and function of CD11c⁺ cells and CD4⁺CD25⁺ cells in the trachea and ADLNs of UV‐ and non‐irradiated, OVA‐sensitized mice was examined 24 h after a single exposure to aerosolized OVA. Results No changes in the function of CD11c⁺ cells from UV‐irradiated mice were observed. CD4⁺CD25⁺ cells from UV‐irradiated, OVA‐sensitized mice harvested 24 h after OVA aerosol proliferated less in response to OVA in vitro and were unable to suppress the proliferation of OVA‐sensitized responder cells. This result suggested reduced activation of effector T cells in the airway mucosa of UV‐irradiated, OVA‐sensitized mice. To exclude regulatory cells of any type, there was similar proliferation in vivo to aerosolized OVA by CFSE‐loaded, OVA‐TCR‐specific CD4⁺ cells adoptively transferred into UV‐ and non‐irradiated, OVA‐sensitized mice. In addition, there was no difference in the expression of regulatory T cell markers (Foxp3, IL‐10, TGF‐β mRNA). To examine effector T cells, ADLN cells from UV‐irradiated, OVA‐sensitized and ‐challenged mice were cultured with OVA. There was reduced expression of the early activation marker CD69 by CD4⁺CD25⁺ cells, and reduced proliferation in the absence of the regulatory cytokine, IL‐10. Conclusion Reduced allergic airways disease in UV‐irradiated mice is due to fewer effector CD4⁺CD25⁺ cells in the trachea and ADLNs, and not due to UV‐induced regulatory cells. Cite this as: J. P. McGlade, D. H. Strickland, M. J. M. Lambert, S. Gorman, J. A. Thomas, M. A. Judge, J. T. Burchell, G. R. Zosky and P. H. Hart, Clinical & Experimental Allergy, 2010 (40) 772–785.     

CD4+CD25+ T cell depletion impairs tolerance induction in a murine model of asthma

Background Regulatory T cells (Tregs) are key players in controlling the development of airway inflammation. However, their role in the mechanisms leading to tolerance in established allergic asthma is unclear. Objective To examine the role of Tregs in tolerance induction in a murine model of asthma. Methods Ovalbumin (OVA) sensitized asthmatic mice were depleted or not of CD25⁺ T cells by anti‐CD25 PC61 monoclonal antibody (mAb) before intranasal treatment (INT) with OVA, then challenged with OVA aerosol. To further evaluate the respective regulatory activity of CD4⁺CD25⁺ and CD4⁺CD25^? T cells, both T cell subsets were transferred from tolerized or non‐tolerized animals to asthmatic recipients. Bronchoalveolar lavage fluid (BALF), T cell proliferation and cytokine secretion were examined. Results Intranasal treatment with OVA led to increased levels of IL‐10, TGF‐β and IL‐17 in lung homogenates, inhibition of eosinophil recruitment into the BALF and antigen specific T cell hyporesponsiveness. CD4⁺CD25⁺Foxp3⁺ T cells were markedly upregulated in lungs and suppressed in vitro and in vivo OVA‐specific T cell responses. Depletion of CD25⁺ cells before OVA INT severely hampered tolerance induction as indicated by a strong recruitment of eosinophils into BALF and a vigorous T cell response to OVA upon challenge. However, the transfer of CD4⁺CD25^? T cells not only suppressed antigen specific T cell responsiveness but also significantly reduced eosinophil recruitment as opposed to CD4⁺CD25⁺ T cells. As compared with control mice, a significantly higher proportion of CD4⁺CD25^? T cells from OVA treated mice expressed mTGF‐β. Conclusion Both CD4⁺CD25⁺ and CD4⁺CD25^? T cells appear to be essential to tolerance induction. The relationship between both subsets and the mechanisms of their regulatory activity will have to be further analyzed.     

Direct visualization of endogenous Salmonella‐specific B cells reveals a marked delay in clonal expansion and germinal center development

CD4⁺ T cells and B cells are both essential for acquired immunity to Salmonella infection. It is well established that Salmonella inhibit host CD4⁺ T‐cell responses, but a corresponding inhibitory effect on B cells is less well defined. Here, we utilize an Ag tetramer and pull‐down enrichment strategy to directly visualize OVA‐specific B cells in mice, as they respond to infection with Salmonella‐OVA. Surprisingly, OVA‐specific B‐cell expansion and germinal center formation was not detected until bacteria were cleared from the host. Furthermore, Salmonella infection also actively inhibited both B‐ and T‐cell responses to the same coinjected Ag but this did not require the presence of iNOS. The Salmonella Pathogenicity Island 2 (SPI2) locus has been shown to be responsible for inhibition of Salmonella‐specific CD4⁺ T‐cell responses, and an examination of SPI2‐deficient bacteria demonstrated a recovery in B‐cell expansion in infected mice. Together, these data suggest that Salmonella can simultaneously inhibit host B‐ and T‐cell responses using SPI2‐dependent mechanisms.     

Antigen-specific B cells are required for the secondary response of T cells but not for their priming

We studied the potential role of B cells in T cell responses using severe-combined immunodeficient (SCID) mice grafted with the thymus from fetal C.B-17 mice (TG mice). These mice developed both CD4⁺ and CD8⁺ T cells, but not B cells within 2 months after transplantation. TG mice showed normal delayed-type hypersensitivity responses against the immunizing antigen ovalbumin (OVA). Lymph node (LN) cells of TG mice proliferated well in response to concanavalin A (Con A). Further, Con A stimulation induced the production of interleukin (IL)-2, IL-6 and interferon (IFN)-γ and the expression of IL-4 mRNA. Thus, TG mice were reconstituted without remarkable immunodeficiency. However, these T cells failed to proliferate to OVA stimulation. Response to OVA was also inhibited in SCID mice grafted with fetal C.B-17 liver cells when B cells were depleted in the proliferation assay. Unresponsiveness against immunizing antigen was restored by the addition of antigen-primed B cells, but not by naive B cells, lipopolysaccharide-activated B cells or B cells primed with sheep red blood cells. Next, we examined whether antigen-primed B cells could induce T cell responses without professional antigen-presenting cells (APC). T and B cells were purified from OVA-immunized mice by cell sorter. These T cells proliferated in response to OVA and produced IFN-γ in the absence of non-B APC. When anti-CD80 or anti-CD86 was added in the assay, proliferation and IFN-γ production was inhibited. These results indicate that B cells activated specifically with antigen are required for the secondary response of T cells, but not for their priming.     

In vivo targeting of human DC‐SIGN drastically enhances CD8+ T‐cell‐mediated protective immunity

Vaccination is one of the oldest yet still most effective methods to prevent infectious diseases. However, eradication of intracellular pathogens and treatment of certain diseases like cancer requiring efficient cytotoxic immune responses remain a medical challenge. In mice, a successful approach to induce strong cytotoxic CD8⁺ T‐cell (CTL) reactions is to target antigens to DCs using specific antibodies against surface receptors in combination with adjuvants. A major drawback for translating this strategy into one for the clinic is the lack of analogous targets in human DCs. DC‐SIGN (DC‐specific‐ICAM3‐grabbing‐nonintegrin/CD209) is a C‐type lectin receptor with potent endocytic capacity and a highly restricted expression on human immature DCs. Therefore, DC‐SIGN represents an ideal candidate for DC targeting. Using transgenic mice that express human DC‐SIGN under the control of the murine CD11c promoter (hSIGN mice), we explored the efficacy of anti‐DC‐SIGN antibodies to target antigens to DCs and induce protective immune responses in vivo. We show that anti‐DC‐SIGN antibodies conjugated to OVA induced strong and persistent antigen‐specific CD4⁺ and CD8⁺ T‐cell responses, which efficiently protected from infection with OVA‐expressing Listeria monocytogenes. Thus, we propose DC targeting via DC‐SIGN as a promising strategy for novel vaccination protocols against intracellular pathogens.     

Correlations of the expression of γδ T cells and their co‐stimulatory molecules TIGIT,PD‐1, ICOS and BTLA with PR and PIBF in the peripheral blood and decidual tissues of women with unexplained recurrent spontaneous abortion

Semi‐allogeneic embryos are not rejected by the maternal immune system due to maternal–fetal immune tolerance. Progesterone (P) receptor (PR)‐expressing γδ T cells are present in healthy pregnant women. In the presence of P, these cells secrete an immunomodulatory protein called progesterone‐induced blocking factor (PIBF), which can facilitate immune escape and is important in preventing embryonic rejection. This work investigated the correlations of the expression of γδ T cells and their co‐stimulatory molecules T cell immunoglobulin and ITIM domain (TIGIT), programmed cell death 1 (PD‐1), inducible co‐stimulator (ICOS) and B and T lymphocyte attenuator (BTLA) with progesterone receptor (PR) and progesterone‐induced blocking factor (PIBF) in peripheral blood and decidual tissue in women with unexplained recurrent spontaneous abortion (URSA) and normal pregnant (NP) women. We confirmed that γδ T cell proportions and PIBF expression in the peripheral blood and decidua of URSA women decreased significantly, while PR expression in decidua decreased. However, TIGIT, PD‐1, ICOS and BTLA expression in γδ T cells in peripheral blood did not change, while TIGIT and PD‐1 expression in γδ T cells in decidua increased significantly. Under the action of PHA‐P (10 µg/ml), co‐blocking of TIGIT (15 µg/ml) and PD‐1 (10 µg/ml) antibodies further induced γδ T cell proliferation, but PIBF levels in the culture medium supernatant did not change. At 10^?10 M P, γδ T cells proliferated significantly, and PIBF concentrations in the culture medium supernatant increased. γδ T cells co‐cultured with P, TIGIT and PD‐1 blocking antibodies showed the most significant proliferation, and PIBF concentrations in the culture medium supernatant were the highest. These results confirm that P is necessary for PIBF production. The TIGIT and PD‐1 pathways participate in γδ T cell proliferation and activation and PIBF expression and play important roles in maintaining pregnancy.