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1.  Modulation of Naive CD4+ T-Cell Responses to an Airway Antigen during Pulmonary Mycobacterial Infection▿  
Infection and Immunity  2007;75(5):2260-2268.
During pulmonary mycobacterial infection, there is increased trafficking of dendritic cells from the lungs to the draining lymph nodes. We hypothesized that ongoing mycobacterial infection would modulate recruitment and activation of antigen-specific naive CD4+ T cells after airway antigen challenge. BALB/c mice were infected by aerosol with Mycobacterium bovis BCG. At peak bacterial burden in the lungs (4 to 6 weeks postinfection), carboxy-fluorescein diacetate succinimidyl ester-labeled naive ovalbumin-specific DO11.10 T cells were adoptively transferred into infected and uninfected mice. Recipient mice were challenged intranasally with soluble ovalbumin (OVA), and OVA-specific T-cell responses were measured in the lungs, draining mediastinal lymph nodes (MLN), and spleens. OVA challenge resulted in increased activation and proliferation of OVA-specific T cells in the draining MLN of both infected and uninfected mice. However, only BCG-infected mice had prominent OVA-specific T-cell activation, proliferation, and Th1 differentiation in the lungs. BCG infection caused greater distribution of airway OVA to pulmonary dendritic cells and enhanced presentation of OVA peptide by lung CD11c+ cells. Together, these data suggest that an existing pulmonary mycobacterial infection alters the phenotype of lung dendritic cells so that they can activate antigen-specific naive CD4+ T cells in the lungs in response to airway antigen challenge.
doi:10.1128/IAI.01709-06
PMCID: PMC1865791  PMID: 17296758
2.  Distribution of Primed T Cells and Antigen-Loaded Antigen Presenting Cells Following Intranasal Immunization in Mice 
PLoS ONE  2011;6(4):e19346.
Priming of T cells is a key event in vaccination, since it bears a decisive influence on the type and magnitude of the immune response. T-cell priming after mucosal immunization via the nasal route was studied by investigating the distribution of antigen-loaded antigen presenting cells (APCs) and primed antigen-specific T cells. Nasal immunization studies were conducted using the model protein antigen ovalbumin (OVA) plus CpG oligodeoxynucleotide adjuvant. Trafficking of antigen-specific primed T cells was analyzed in vivo after adoptive transfer of OVA-specific transgenic T cells in the presence or absence of fingolimod, a drug that causes lymphocytes sequestration within lymph nodes. Antigen-loaded APCs were observed in mediastinal lymph nodes, draining the respiratory tract, but not in distal lymph nodes. Antigen-specific proliferating T cells were first observed within draining lymph nodes, and later in distal iliac and mesenteric lymph nodes and in the spleen. The presence at distal sites was due to migration of locally primed T cells as shown by fingolimod treatment that caused a drastic reduction of proliferated T cells in non-draining lymph nodes and an accumulation of extensively divided T cells within draining lymph nodes. Homing of nasally primed T cells in distal iliac lymph nodes was CD62L-dependent, while entry into mesenteric lymph nodes depended on both CD62L and α4β7, as shown by in vivo antibody-mediated inhibition of T-cell trafficking. These data, elucidating the trafficking of antigen-specific primed T cells to non-draining peripheral and mucosa-associated lymph nodes following nasal immunization, provide relevant insights for the design of vaccination strategies based on mucosal priming.
doi:10.1371/journal.pone.0019346
PMCID: PMC3084830  PMID: 21559409
3.  Flt3 Ligand Generates Morphologically Distinct Semimature Dendritic Cells in Ovalbumin-sensitized Mice 
Dendritic cells (DCs) are unique antigen presenting cells that are immature prior to their encounter with an antigen. Exposure to allergens induces the maturation of DCs with changes in morphology and presence of dendrites. Here, we demonstrate that the DCs in the lungs of ovalbumin (OVA)-sensitized and challenged mice are more mature owing to their pronounced dendrites than the DCs in the lungs and spleen of PBS-treated mice, which are immature and possess cytoplasmic veils. Intermediate to these two groups are the DCs in the Flt3 ligand-treated group that exhibit comparatively fewer dendrites and cytoplasmic veils and hence are classified as semimature. Presence of large numbers of well developed mitochondria and rough endoplasmic reticulum in myeloid DCs from both lungs and spleen of OVA-sensitized and challenged mice indicate greater functional activity. Additionally, DCs from the OVA-sensitized and challenged mice also exhibit fat and glycogen stores, which are indicative of a mature population. In addition, treatment of the animals with Flt3 ligand attenuated airway hyperresponsiveness to methacholine in OVA-sensitized and challenged mice. These data suggest that morphological features could be indicative of the maturation and distinct functional state of DCs, and this could be associated with underlying mechanisms of Flt3 ligand-induced immunomodulation in allergic asthma.
doi:10.1016/j.yexmp.2006.09.007
PMCID: PMC2745173  PMID: 17182033
Airway hyperresponsiveness; Allergy; Asthma; Cytoplasmic veils; Dendritic cells; Flt3 ligand; Immunomodulation; Morphology of dendritic cells
4.  TLR3 and TLR7 Modulate IgE Production in Antigen Induced Pulmonary Inflammation via Influencing IL-4 Expression in Immune Organs 
PLoS ONE  2011;6(2):e17252.
Background
Toll-like receptors (TLRs) as pattern recognition receptors, participate in both innate and adaptive immune responses, and seem to play an important role in the pathogenesis of asthma. This study aimed to identify key TLRs involved in antigen induced pulmonary inflammation (AIPI), a rat model for asthma, and to explore the role of TLRs in the disease development.
Methods and Findings
E3 rats were sensitized with ovalbumin (OVA)/alum intraperitoneally and intranasally challenged with OVA to induce AIPI model. TLR1-9 and cytokine mRNA expression in spleen, lung and mediastinal lymph node (mLN) tissues were screened by quantitative real-time polymerase chain reaction. TLR7 expression was found to be significantly down-regulated in spleen while TLR3 and TLR8 expression was up-regulated in mLN of AIPI rats. Furthermore, imiquimod (a ligand of TLR7) and TLR3 specific short-hairpin RNA plasmid for RNA interference were administrated, respectively, in vivo to AIPI rats to observe their effects on the disease by assessing various asthmatic parameters. The numbers of total cells, eosinophils, macrophages and lymphocytes were counted according to differential morphology in bronchoalveolar lavage fluid. Serum IgE and OVA specific IgG1 concentration was detected by enzyme-linked immunosorbent assay. The results showed that both TLR7 ligand treatment and TLR3 RNAi in vivo decreased serum IgE level and interleukin-4 mRNA expression.
Conclusion/Significance
TLR3 in mLN and TLR7 in spleen both systemically modulate disease development in AIPI rats via altering serum IgE concentration relevant to Th2 responses. And these findings may provide an important clue for further research in the asthma pathogenesis and suggest a new remedy for asthma treatment.
doi:10.1371/journal.pone.0017252
PMCID: PMC3045401  PMID: 21364926
5.  IL-1 enhances expansion, effector function, tissue localization, and memory response of antigen-specific CD8 T cells 
Here, we show that interleukin-1 (IL-1) enhances antigen-driven CD8 T cell responses. When administered to recipients of OT-I T cell receptor transgenic CD8 T cells specific for an ovalbumin (OVA) peptide, IL-1 results in an increase in the numbers of wild-type but not IL1R1−/− OT-I cells, particularly in spleen, liver, and lung, upon immunization with OVA and lipopolysaccharide. IL-1 administration also results in an enhancement in the frequency of antigen-specific cells that are granzyme B+, have cytotoxic activity, and/ or produce interferon γ (IFN-γ). Cells primed in the presence of IL-1 display enhanced expression of granzyme B and increased capacity to produce IFN-γ when rechallenged 2 mo after priming. In three in vivo models, IL-1 enhances the protective value of weak immunogens. Thus, IL-1 has a marked enhancing effect on antigen-specific CD8 T cell expansion, differentiation, migration to the periphery, and memory.
doi:10.1084/jem.20122006
PMCID: PMC3600912  PMID: 23460726
6.  Primary Activation of Antigen-Specific Naive CD4+ and CD8+ T Cells following Intranasal Vaccination with Recombinant Bacteria▿  
Infection and Immunity  2008;76(12):5817-5825.
The primary activation of T-helper and T-cytotoxic cells following mucosal immunization with recombinant Streptococcus gordonii was studied in vivo by adoptive transfer of ovalbumin (OVA)-specific transgenic CD8+ (OT-I) and CD4+ (OT-II) T cells. A recombinant strain, expressing on the surface the vaccine antigen Ag85B-ESAT-6 from Mycobacterium tuberculosis fused to OVA T-helper and T-cytotoxic epitopes (peptides 323 to 339 and 257 to 264), was constructed and used to immunize C57BL/6 mice by the intranasal route. Recombinant, but not wild-type, bacteria induced OVA-specific CD4+ and CD8+ T-cell clonal expansion in cervical lymph nodes, lung, and spleen. OVA-specific CD4+ and CD8+ T-cell proliferation appeared first in cervical lymph nodes and later in the spleen, suggesting a possible migration of activated cells from the inductive site to the systemic district. A significant correlation between the percentages of CD4+ and CD8+ proliferating T cells was observed for each animal. The expression of CD69, CD44, and CD45RB on proliferating T lymphocytes changed as a function of the cell division number, confirming T-cell activation following the antigen encounter. These data indicate that intranasal immunization with recombinant S. gordonii is capable of inducing primary activation of naive antigen-specific CD4+ and CD8+ T cells, both locally and systemically.
doi:10.1128/IAI.00793-08
PMCID: PMC2583588  PMID: 18838521
7.  CTLA-4 Blockade Enhances the Immune Response Induced by Mycobacterial Infection but Does Not Lead to Increased Protection 
Infection and Immunity  1999;67(8):3786-3792.
The murine immune response to a pulmonary mycobacterial infection is slow to develop, allowing bacterial numbers to increase in the lung for several weeks after infection. We sought to enhance the protective immune response induced during Mycobacterium bovis BCG infection by administering an antibody that blocks the interaction of CTLA-4 with its ligands, CD80 and CD86. We found that injection of anti-CTLA-4 monoclonal antibody (MAb) greatly enhanced and accelerated the immune response, as measured by increased cellularity of the draining mediastinal lymph nodes, and enhanced antigen-inducible proliferation and gamma interferon production by mediastinal lymphocytes in vitro. However, despite the apparently enhanced immune response in the mediastinal lymph node following treatment with anti-CTLA-4 MAb, there was no improvement in clearance of mycobacteria in the lungs, liver, or spleen. Examination of the primary site of infection, the lung, revealed that CTLA-4 blockade had no effect on the number or function of lymphocytes infiltrating the infected lung tissue. Taken together, these data suggest that in vivo CTLA-4 blockade enhances mycobacterial-infection-induced lymphocyte expansion and effector cell cytokine production in the draining lymph node but does not alter the number or function of lymphocytes at the primary site of infection and therefore does not lead to enhanced clearance of the infection.
PMCID: PMC96655  PMID: 10417139
8.  Periportal and sinusoidal liver dendritic cells suppressing T helper type 1‐mediated hepatitis 
Gut  2007;56(10):1445-1451.
Background
Recently, we found that portal vein tolerance is associated with generation of Th2 cells and apoptosis of Th1 cells in the liver, which is regulated by antigen (Ag)‐presenting dendritic cells (DCs) in the periportal area and sinusoids.
Aim
In this study, we tested whether the periportal and sinusoidal DCs, which were loaded with an Ag in vivo, can inhibit liver injury caused by Th1 cells activated by the Ag administered systemically.
Methods
Ag‐specific hepatitis model was created by adoptively transferring ovalbumin (OVA)‐specific CD4+ T cells to BALB/c mice and venous injection of OVA‐containing liposomes. Liver CD11c+ cells obtained from mice fed OVA were then transferred into these mice.
Results
The transfer of liver CD11c+ cells from OVA‐fed mice completely inhibited hepatic injury, which was associated with apoptosis of OVA‐specific CD4+ T cells and emergence of Th2 cells in the liver. Transfer of CD11c+ cells and subcutaneous OVA challenge led to enhancement of OVA‐specific IgE Ab as well as Th2 cytokine responses in the recipient mice.
Conclusions
Periportal and sinusoidal DCs loaded with an Ag in the portal vein can induce Th2 response in the liver and prevent hepatic injury caused by Th1 cells.
doi:10.1136/gut.2007.121251
PMCID: PMC2000248  PMID: 17872571
9.  A single exposure to iron oxide nanoparticles attenuates antigen-specific antibody production and T-cell reactivity in ovalbumin-sensitized BALB/c mice 
Background:
Superparamagnetic iron oxide nanoparticles have been used in clinical applications as a diagnostic contrasting agent. Previous studies showed that iron oxide nanoparticles deposited in the liver and spleen after systemic administration. The present study investigated the effect of iron oxide nanoparticles on antigen-specific immune responses in mice sensitized with the T cell-dependent antigen ovalbumin (OVA).
Methods:
BALB/c mice were intravenously administered with a single dose of iron oxide nanoparticles (10–60 mg Fe/kg) 1 hour prior to OVA sensitization, and the serum antibody production and splenocyte reactivity were examined 7 days later.
Results:
The serum levels of OVA-specific IgG1 and IgG2a were significantly attenuated by treatment with iron oxide nanoparticles. The production of interferon-γ and interleukin-4 by splenocytes re-stimulated with OVA in culture was robustly suppressed in mice administered with iron oxide nanoparticles. The viability of OVA-stimulated splenocytes was also attenuated. In contrast, treatment with iron oxide nanoparticles did not affect the viability of splenocytes stimulated with concanavalin A, a T-cell mitogen.
Conclusion:
Collectively, these data indicate that systemic exposure to a single dose of iron oxide nanoparticles compromises subsequent antigen-specific immune reactions, including the serum production of antigen-specific antibodies, and the functionality of T cells.
doi:10.2147/IJN.S21019
PMCID: PMC3131189  PMID: 21753874
iron oxide nanoparticle; antigen-specific; immune; ovalbumin
10.  Antigen-sensitized CD4+CD62Llow memory/effector T helper 2 cells can induce airway hyperresponsiveness in an antigen free setting 
Respiratory Research  2005;6(1):46.
Background
Airway hyperresponsiveness (AHR) is one of the most prominent features of asthma, however, precise mechanisms for its induction have not been fully elucidated. We previously reported that systemic antigen sensitization alone directly induces AHR before development of eosinophilic airway inflammation in a mouse model of allergic airway inflammation, which suggests a critical role of antigen-specific systemic immune response itself in the induction of AHR. In the present study, we examined this possibility by cell transfer experiment, and then analyzed which cell source was essential for this process.
Methods
BALB/c mice were immunized with ovalbumin (OVA) twice. Spleen cells were obtained from the mice and were transferred in naive mice. Four days later, AHR was assessed. We carried out bronchoalveolar lavage (BAL) to analyze inflammation and cytokine production in the lung. Fluorescence and immunohistochemical studies were performed to identify T cells recruiting and proliferating in the lung or in the gut of the recipient. To determine the essential phenotype, spleen cells were column purified by antibody-coated microbeads with negative or positive selection, and transferred. Then, AHR was assessed.
Results
Transfer of spleen cells obtained from OVA-sensitized mice induced a moderate, but significant, AHR without airway antigen challenge in naive mice without airway eosinophilia. Immunization with T helper (Th) 1 elicited antigen (OVA with complete Freund's adjuvant) did not induce the AHR. Transferred cells distributed among organs, and the cells proliferated in an antigen free setting for at least three days in the lung. This transfer-induced AHR persisted for one week. Interleukin-4 and 5 in the BAL fluid increased in the transferred mice. Immunoglobulin E was not involved in this transfer-induced AHR. Transfer of in vitro polarized CD4+ Th2 cells, but not Th1 cells, induced AHR. We finally clarified that CD4+CD62Llow memory/effector T cells recruited in the lung and proliferated, thus induced AHR.
Conclusion
These results suggest that antigen-sensitized memory/effector Th2 cells themselves play an important role for induction of basal AHR in an antigen free, eosinophil-independent setting. Therefore, regulation of CD4+ T cell-mediated immune response itself could be a critical therapeutic target for allergic asthma.
doi:10.1186/1465-9921-6-46
PMCID: PMC1180472  PMID: 15921525
11.  Sinusoidal tumor angiogenesis is a key component in hepatocellular carcinoma metastasis 
Hepatocellular carcinoma (HCC) has a tendency for intravascular dissemination leading to a poor prognosis. The importance of the sinusoidal structure of the tumor vasculature in HCC has been implicated in the metastasis formation. To clarify the role of tumor angiogenesis in HCC metastasis, we morphologically investigated the interaction of HCC cells with blood vessels during the sequential process of metastasis. Autopsy specimens of 80 patients with HCC were examined with immunohistochemistry using a specific antibody against CD31, a marker for endothelial cells. The most frequent sites of metastasis were the liver (82.5%) and lung (43.8%). In most cases, the metastatic process was initiated by vascular involvement where tumor nests surrounded by sinusoidal vessels extend into the portal and hepatic veins. Subsequently, these endothelial-coated tumor emboli enter the circulation, embolize at distant organs, proliferate within the blood vessel and ultimately form metastatic foci. These steps are indicative of an invasion-independent pathway. Our findings in animal models and now in human cases suggest that sinusoidal angiogenesis may represent a novel target for therapeutic strategies to limit HCC metastasis. In combination with primary tumor treatment, perturbation of tumor emboli may reduce dissemination of disease.
doi:10.1007/s10585-008-9199-6
PMCID: PMC3431607  PMID: 18712609
Hepatocellular carcinoma; Metastasis; Angiogenesis; Invasion-independent pathway; Autopsy
12.  Influenza Virus–induced Dendritic Cell Maturation Is Associated with the Induction of Strong T Cell Immunity to a Coadministered, Normally Nonimmunogenic Protein 
We evaluated the proposal that during microbial infection, dendritic cells (DCs) undergo maturation and present a mixture of peptides derived from the microbe as well as harmless environmental antigens. Mice were exposed to an aerosol of endotoxin free ovalbumin (OVA) in the absence or presence of influenza virus. In its absence, OVA failed to induce B and T cell responses and even tolerized, but with influenza, OVA-specific antibodies and CD8+ cytolytic T lymphocytes developed. With or without infection, OVA was presented selectively in the draining mediastinal lymph nodes, as assessed by the comparable proliferation of infused, CD8+ and CD4+, TCR transgenic T cells. In the absence of influenza, these OVA-specific T cells produced little IL-2, IL-4, IL-10, and IFN-γ, but with infection, both CD4+ and CD8+ T cells made high levels of IL-2 and IFN-γ. The OVA plus influenza-treated mice also showed accelerated recovery to a challenge with recombinant vaccinia OVA virus. CD11c+ DCs from the mediastinal lymph nodes of infected mice selectively stimulated both OVA- and influenza-specific T cells and underwent maturation, with higher levels of MHC class II, CD80, and CD86 molecules. The relatively slow (2–3 d) kinetics of maturation correlated closely to the time at which OVA inhalation elicited specific antibodies. Therefore respiratory infection can induce DC maturation and simultaneously B and T cell immunity to an innocuous antigen inhaled concurrently.
doi:10.1084/jem.20030266
PMCID: PMC2196079  PMID: 12847140
influenza virus; dendritic cell; maturation; endotoxin free ovalbumin; airway
13.  Growth of Mycobacterium marinum in the footpads of T-cell-depleted mice. 
Infection and Immunity  1975;11(5):1088-1093.
Mycobacterium marinum strains 1218 and 1219 were inoculated into the hind footpads of T-cell-depleted specific pathogen-free C57B1/6 mice, and the growth and survival of the organisms at the site of injection, the draining popliteal lymph node, and the spleen and lung were quantitated for up to 70 days. T-cell depletion largely ablated the normal cell-mediated antituberculous response to the M. marinum population. The mice were able to control the further growth of the inoculum within the footpad only after it had reached 5 to 10 times that present in the normal controls. The high temperature-adapted strain (37 C; strain no. 1218) induced an increasing infection in the liver, spleen, and lungs of the THXB mice, and the infection eventually spread to the opposite footpad and to the tail skin. Strain 1219 gave rise to considerable systemic involvement in the THXB host despite its inability to survive at 37 C, but the size of the splenic and lung populations was considerably lower than in the 1218-infected animals. Both M. marinum infections persisted in the tissues of the T-cell-depleted mice with no indication of a cell-mediated immune response. Footpad swelling in the M. marinum-infected mice was not greatly reduced by T-cell depletion, and, if anything, tended to persist at high levels long after the swelling of the control feet had gone into a decline. On the other hand, incorporation of tritiated thymidine by cells within the infected footpads, the draining lymph node, and the spleen was considerably reduced in the T-cell-depleted host compared with control values. Late in the infection, there was a significant increase in the amount of label taken up by the cells in the footpads of the T-cell-depleted host.
PMCID: PMC415183  PMID: 1079017
14.  Enhancement of In Vivo and In Vitro Immune Functions by a Conformationally-Biased, Response-Selective Agonist of Human C5a: Implications for a Novel Adjuvant in Vaccine Design 
Vaccine  2009;28(2):463-469.
A conformationally-biased, agonist of human C5a65–74 (EP67) was assessed for its adjuvant activities in vitro and in vivo. EP67 induced the release of the inflammatory (Th1) type cytokines from C5a receptor (CD88)-bearing antigen presenting cells (APC). Serum from mice immunized with EP67-ovalbumin (OVA) contained high OVA-specific antibody (Ab) titers [IgG1, IgG2a (IGg2c), IgG2b]. Mice receiving OVA alone produced only IgG1 Abs, indicating the ability of EP67 to induce a Th1-like antibody (A)b class switch. Spleen cell cultures from wild type mice but not CD88−/− mice showed an enhanced OVA-specific proliferative response in vitro. These results indicate the ability of EP67 to drive a Th1-mediated immune response and its potential use as a unique adjuvant
doi:10.1016/j.vaccine.2009.10.029
PMCID: PMC2789185  PMID: 19836478
15.  IL-10R Blockade during Chronic Schistosomiasis Mansoni Results in the Loss of B Cells from the Liver and the Development of Severe Pulmonary Disease 
PLoS Pathogens  2012;8(1):e1002490.
In schistosomiasis patients, parasite eggs trapped in hepatic sinusoids become foci for CD4+ T cell-orchestrated granulomatous cellular infiltrates. Since the immune response is unable to clear the infection, the liver is subjected to ongoing cycles of focal inflammation and healing that lead to vascular obstruction and tissue fibrosis. This is mitigated by regulatory mechanisms that develop over time and which minimize the inflammatory response to newly deposited eggs. Exploring changes in the hepatic inflammatory infiltrate over time in infected mice, we found an accumulation of schistosome egg antigen-specific IgG1-secreting plasma cells during chronic infection. This population was significantly diminished by blockade of the receptor for IL-10, a cytokine implicated in plasma cell development. Strikingly, IL-10R blockade precipitated the development of portal hypertension and the accumulation of parasite eggs in the lungs and heart. This did not reflect more aggressive Th2 cell responsiveness, increased hepatic fibrosis, or the emergence of Th1 or Th17 responses. Rather, a role for antibody in the prevention of severe disease was suggested by the finding that pulmonary involvement was also apparent in mice unable to secrete class switched antibody. A major effect of anti-IL-10R treatment was the loss of a myeloid population that stained positively for surface IgG1, and which exhibited characteristics of regulatory/anti-inflammatory macrophages. This finding suggests that antibody may promote protective effects within the liver through local interactions with macrophages. In summary, our data describe a role for IL-10-dependent B cell responses in the regulation of tissue damage during a chronic helminth infection.
Author Summary
Schistosomiasis is a chronic disease that affects approximately 200 million people. Immune modulation is a hallmark of chronic disease and serves to protect the host from severe pathology. A significant percentage of people infected with schistosomiasis fail to undergo this protective modulation and can develop portal hypertension with resulting pulmonary complications. Here we show that schistosome-specific antibody-secreting B cells accumulate in the liver as the infection progresses to the chronic state and that this accumulation is dependent on the cytokine Interleukin-10. Blocking the IL-10R results in not only the loss of B cells from the liver, but also the development of severe pulmonary pathology. We found similar changes in disease progression in mice genetically unable to mount normal antibody responses. We believe that antibody is important for triggering the production of anti-inflammatory factors, including IL-10 itself, by other immune cells called macrophages. Our data suggest that during chronic schistosomiasis IL-10 promotes the development of a population of B cells within the liver that is responsible for minimizing inflammation and preventing the development of disease in the lungs. Our findings provide a mouse model that may be of use for studying the development of pulmonary complications due to chronic schistosomiasis.
doi:10.1371/journal.ppat.1002490
PMCID: PMC3266936  PMID: 22291593
16.  Airborne lipid antigens mobilize resident intravascular NKT cells to induce allergic airway inflammation 
The Journal of Experimental Medicine  2011;208(10):2113-2124.
Resident intravascular NKT cells exacerbate airway hyperreactivity in mice dependent on dendritic cell co-presentation of glycolipid and peptide antigens.
Airborne exposure to microbial cell wall lipids such as lipopolysaccharide triggers innate immune responses that regulate susceptibility to allergic airway inflammation. α-Glycosylceramides represent another widespread class of microbial lipids that directly stimulate innate-like, IL-4– and IL-13–producing, CD1d-restricted NKT cells. In this study, we demonstrate that NKT cells constitutively accumulate and reside in the microvasculature of the mouse lung. After a single airborne exposure to lipid antigen, they promptly extravasate to orchestrate the formation of peribronchiolar and interstitial lymphohistiocytic granulomas containing numerous eosinophils. Concomitant airborne exposure to ovalbumin (OVA) induces the priming of OVA-specific Th2 cells and IgE antibodies by the same dendritic cell coexpressing CD1d and MHC class II. Although NKT cell activation remains confined to the lipid-exposed lung and draining lymph nodes, Th2 cells recirculate and seed the lung of a parabiotic partner, conferring susceptibility to OVA challenge months after the initial exposure, in a manner independent of NKT cells and CD1d. Thus, transient recruitment and activation of lung-resident intravascular NKT cells can trigger long-term susceptibility to allergic airway inflammation.
doi:10.1084/jem.20110522
PMCID: PMC3182052  PMID: 21930768
17.  Langerhans cells and dermal dendritic cells capture protein antigens in the skin: Possible targets for vaccination through the skin 
Immunobiology  2010;215(9-10):770-779.
Dendritic cells capture and process antigen and present it to T lymphocytes in the lymphoid organs. Dendritic cells of the skin, including epidermal Langerhans cells, langerin+ and langerinnegative dermal dendritic cells are ideally positioned to take up pathogens that enter the body through the skin or vaccines that are administered into (intradermal) or onto (epicutaneous) the skin. The antigen uptake properties of skin dendritic cells have not thoroughly been studied yet. We therefore investigated the uptake of the fluorochrome-conjugated model antigen ovalbumin (OVA) by skin dendritic cells in the mouse. OVA was readily taken up by immature Langerhans cells both in situ and in cell suspensions. When offered to Langerhans cells in situ either by “bathing” skin explants in OVA-containing culture medium or by intradermal injection they retained the captured OVA for at least 2–3 days when migrating into the culture medium and, importantly, into the draining lymph nodes. Also langerin+ and – to a larger extent – langerinnegative skin dendritic cells took up and transported OVA to the lymph nodes. Interestingly, mature Langerhans cells were still capable of ingesting substantial amounts of OVA, indicating that predominantly receptor-mediated endocytosis is operative in these cells. Unlike macropinocytosis, this pathway of endocytosis is not shut down upon dendritic cell maturation. These observations indicate that in intradermal vaccination schemes, Langerhans cells from the epidermis are prominently involved. They were recently shown to possess the capacity to induce functional cytotoxic T lymphocytes. Furthermore, the potential to markedly enhance antigen uptake and processing by targeting antigen to c-type lectin receptors on Langerhans cells was also recently demonstrated. Our data provide a rationale and an incentive to explore in more detail antigen targeting to Langerhans cells with the aim of harnessing it for immunotherapy.
doi:10.1016/j.imbio.2010.05.014
PMCID: PMC2939980  PMID: 20599290
Dendritic cells; Langerhans cells; Antigen
18.  Sublingual Vaccination Induces Mucosal and Systemic Adaptive Immunity for Protection against Lung Tumor Challenge 
PLoS ONE  2014;9(3):e90001.
Sublingual route offers a safer and more practical approach for delivering vaccines relative to other systemic and mucosal immunization strategies. Here we present evidence demonstrating protection against ovalbumin expressing B16 (B16-OVA) metastatic melanoma lung tumor formation by sublingual vaccination with the model tumor antigen OVA plus synthetic glycolipid alpha-galactosylceramide (aGalCer) for harnessing the adjuvant potential of natural killer T (NKT) cells, which effectively bridge innate and adaptive arms of the immune system. The protective efficacy of immunization with OVA plus aGalCer was antigen-specific as immunized mice challenged with parental B16 tumors lacking OVA expression were not protected. Multiple sublingual immunizations in the presence, but not in the absence of aGalCer, resulted in repeated activation of NKT cells in the draining lymph nodes, spleens, and lungs of immunized animals concurrent with progressively increasing OVA-specific CD8+ T cell responses as well as serum IgG and vaginal IgA levels. Furthermore, sublingual administration of the antigen only in the presence of the aGalCer adjuvant effectively boosted the OVA-specific immune responses. These results support potential clinical utility of sublingual route of vaccination with aGalCer-for prevention of pulmonary metastases.
doi:10.1371/journal.pone.0090001
PMCID: PMC3943861  PMID: 24599269
19.  Calcium-Activated Potassium Channel KCa3.1 in Lung Dendritic Cell Migration 
Migration to draining lymph nodes is a critical requirement for dendritic cells (DCs) to control T-cell–mediated immunity. The calcium-activated potassium channel KCa3.1 has been shown to be involved in regulating cell migration in multiple cell types. In this study, KCa3.1 expression and its functional role in lung DC migration were examined. Fluorescence-labeled antigen was intranasally delivered into mouse lungs to label lung Ag-carrying DCs. Lung CD11chighCD11blow and CD11clowCD11bhigh DCs from PBS-treated and ovalbumin (OVA)-sensitized mice were sorted using MACS and FACS. Indo-1 and DiBAC4(3) were used to measure intracellular Ca2+ and membrane potential, respectively. The mRNA expression of KCa3.1 was examined using real-time PCR. Expression of KCa3.1 protein and CCR7 was measured using flow cytometry. Migration of two lung DC subsets to lymphatic chemokines was examined using TransWell in the absence or presence of the KCa3.1 blocker TRAM-34. OVA sensitization up-regulated mRNA and protein expression of KCa3.1 in lung DCs, with a greater response by the CD11chighCD11blow than CD11clowCD11bhigh DCs. Although KCa3.1 expression in Ag-carrying DCs was higher than that in non–Ag-carrying DCs in OVA-sensitized mice, the difference was not as prominent. However, Ag-carrying lung DCs expressed significantly higher CCR7 than non–Ag-carrying DCs. CCL19, CCL21, and KCa3.1 activator 1-EBIO induced an increase in intracellular calcium in both DC subsets. In addition, 1-EBIO–induced calcium increase was suppressed by TRAM-34. In vitro blockade of KCa3.1 with TRAM-34 impaired CCL19/CCL21-induced transmigration. In conclusion, KCa3.1 expression in lung DCs is up-regulated by OVA sensitization in both lung DC subsets, and KCa3.1 is involved in lung DC migration to lymphatic chemokines.
doi:10.1165/rcmb.2010-0514OC
PMCID: PMC3262686  PMID: 21493782
allergic airway inflammation; antigen uptake; asthma; calcium-activated potassium channel; dendritic cell
20.  Inflammatory response following intranasal infection with Mycobacterium avium complex: role of T-cell subsets and gamma interferon. 
Infection and Immunity  1995;63(6):2282-2287.
The role of CD4+ and CD8+ T cells in the response to intranasal infection with a Mycobacterium avium complex isolate (MAC) was investigated. Depletion of CD4+ T cells by injected antibody exacerbated infection in the lung, spleen, and liver. There were decreased numbers of inflammatory cells in the lungs of CD4-depleted mice and a significant decrease in lung cytotoxic activity. The neutrophil response was unaffected, and in CD4-depleted mice, unlike intact infected mice, these cells were found with large numbers of associated MAC. Purified CD4+ splenic T cells produced gamma interferon (IFN-gamma) in vitro in response to MAC antigen. IFN-gamma production by cultured spleen, lung, or mediastinal lymph node cells was markedly reduced in CD4-depleted mice. In contrast, CD8+ T cells did not produce IFN-gamma in vitro, and depletion of CD8+ T cells from infected mice had no effect on bacterial growth or lung cell activation. Depletion of IFN-gamma by injected monoclonal antibody had effects similar to those of CD4 depletion, namely, exacerbation of infection and decreased lung cell cytotoxicity. We conclude that CD4+ T cells are the main T cells involved in the lung response to MAC infection and that this response is at least partially dependent on the production of IFN-gamma.
PMCID: PMC173298  PMID: 7768610
21.  Constitutive versus Activation-dependent Cross-Presentation of Immune Complexes by CD8+ and CD8− Dendritic Cells In Vivo 
Murine splenic dendritic cells (DCs) can be divided into two subsets based on CD8α expression, but the specific role of each subset in stimulation of T cells is largely unknown. An important function of DCs is the ability to take up exogenous antigens and cross-present them in the context of major histocompatibility complex (MHC) class I molecules to CD8+ T cells. We previously demonstrated that, when cell-associated ovalbumin (OVA) is injected into mice, only the CD8+ DC subset cross-presents OVA in the context of MHC class I. In contrast to this selectivity with cell-associated antigen, we show here that both DC subsets isolated from mice injected with OVA/anti-OVA immune complexes (OVA-IC) cross-present OVA to CD8+ T cells. The use of immunoglobulin G Fc receptor (FcγR) common γ-chain–deficient mice revealed that the cross-presentation by CD8− DCs depended on the expression of γ-chain–containing activating FcγRs, whereas cross-presentation by CD8+ DCs was not reduced in γ-chain–deficient mice. These results suggest that although CD8+ DCs constitutively cross-present exogenous antigens in the context of MHC class I molecules, CD8− DCs only do so after activation, such as via ligation of FcγRs. Cross-presentation of immune complexes may play an important role in autoimmune diseases and the therapeutic effect of antitumor antibodies.
doi:10.1084/jem.20020295
PMCID: PMC2194052  PMID: 12235214
antigen presentation; cytotoxic T lymphocyte; cross-priming; dendritic cell; Fc receptors
22.  Requirement for T cells in the production of migration inhibitory factor 
The Journal of Experimental Medicine  1975;142(5):1306-1311.
The question whether B lymphocytes are capable of being activated by antigen in the absence of functional T cells was investigated in a model that excludes participation of T cells by virtue of an immune response gene restriction. Strain 2 guinea pigs are capable of responding to immunization with DNP-PLL, whereas strain 13 animals are not. In the present experiments, animals of both strains were immunized with DNP-PLL complexed to ovalbumin (DNP-PLL-Ova) under conditions in which equal titers of antibodies to DNP were produced by both strains. The failure of T cells of strain 13 animals to respond to DNP-PLL was confirmed by the virus plaque assay. While spleen cells from both strains produced MIF after stimulation with DNP-PLL-Ova, in response to DNP-PLL only strain 2 spleens were able to produce MIF. Cells from neither strain could be activated by DNP-guinea pig albumin to produce MIF. We conclude that B lymphocytes are incapable of being stimulated by antigen in the absence of T cells, and that MIF production is a thymus-dependent response. While the results indicate that MIF production is a valid qualitative assay for T-cell competence, since MIF can be produced by B and T cells, the degree of migration inhibition cannot be regarded as a quantitative measure of T-cell function.
PMCID: PMC2189982  PMID: 1104742
23.  Ectopic activation of M. tuberculosis-specific CD4+ T cells in lungs of CCR7−/− mice 
Initiation of an adaptive cellular immune response depends on intimate interactions with antigen-presenting cells and naive T lymphocytes. We have previously reported that activation of naive Mycobacterium tuberculosis-specific CD4+ T cells depends on dendritic cell (DC) transport of live bacteria from the lungs to the mediastinal lymph node (MDLN). Since the migratory paths of DCs are largely governed by the chemokine receptor CCR7, which is expressed on DCs upon maturation by proinflammatory stimuli, we examined the quantitative contribution of CCR7-dependent DC migration in the context of tuberculosis, and found that early trafficking of DCs from the lungs to the MDLN depended on CCR7-mediated signaling, but alternative mechanism(s) are employed later in infection. Impaired migration of DCs in CCR7−/− mice resulted in delayed dissemination of bacteria to MDLN and spleen, and in delayed kinetics of activation of adoptively-transferred Ag85B-specific CD4+ T cells. Furthermore, in contrast to control mice, we found that naive Ag85B-specific CD4+ T cells are activated to proliferate in the lungs of CCR7−/− mice and, when infected with higher doses of bacteria, resistance to M. tuberculosis infection in CCR7−/− mice is compromised compared to wild type mice.
doi:10.4049/jimmunol.0901230
PMCID: PMC2879893  PMID: 20007536
dendritic cells; bacterial; chemokines; cell activation; cell trafficking
24.  Homing of Antigen-Presenting Cells in Head Kidney and Spleen – Salmon Head Kidney Hosts Diverse APC Types 
Lymph nodes and spleen are major organs where mammalian antigen-presenting cells (APCs) initiate and orchestrate Ag-specific immune responses. Unlike mammals, teleosts lack lymph nodes and an interesting question is whether alternative organs may serve as sites for antigen presentation in teleosts. In the current study, fluorescent ovalbumin (Ova) and CpG oligonucleotides (ODNs) injected intra-abdominally were detected in significant numbers of salmon head kidney (HK) MHCII+ cells over a period of 2 weeks while in spleen the percentage of these was transient and declined from day 1 post injection. In vitro studies further shed light on the properties of the diverse MHCII+ cell types found in HK. The ultrastructure of a subpopulation of MHCII+ cells with a high capacity to endocytose and process Ova indicated that these were able to perform constitutive macropinocytosis. Upon stimulation with CpG ODNs these cells upregulated CD86 and gave very high levels of TNF mRNA indicating that these are professional APCs, related to macrophages and dendritic cells (DCs). A subpopulation of HK granulocytes expressed high levels of surface MHCII and upon CpG stimulation upregulated most of the tested APC marker genes. Although these granulocytes expressed TNF weakly, they had relatively high basal levels of IL-1β mRNA and the CpG stimulation upregulated IL-1β, along with its signaling and decoy receptors, to the highest levels as compared to other HK cell types. Interestingly, the high expression of IL-1β mRNA in the granulocytes correlated with a high autophagy flux as demonstrated by LC3-II conversion. Autophagy has recently been found to be implicated in IL-1β processing and secretion and the presented data suggests that granulocytes of salmon, and perhaps other teleost species, may serve as a valuable model to study the involvement of autophagy in regulation of the vertebrate immune response.
doi:10.3389/fimmu.2013.00137
PMCID: PMC3674399  PMID: 23761795
MHCII; APC; granulocytes; Atlantic salmon; CpG oligonucleotides; IL-1β; TNF; autophagy
25.  In Vivo Activation of Naive CD4+ T Cells in Nasal Mucosa-Associated Lymphoid Tissue following Intranasal Immunization with Recombinant Streptococcus gordonii  
Infection and Immunity  2006;74(5):2760-2766.
The antigen-specific primary activation of CD4+ T cells was studied in vivo by adoptive transfer of ovalbumin-specific transgenic T cells (KJ1-26+ CD4+) following intranasal immunization with recombinant Streptococcus gordonii. A strain of S. gordonii expressing on its surface a model vaccine antigen fused to the ovalbumin (OVA) peptide from position 323 to 339 was constructed and used to study the OVA-specific T-cell activation in nasal mucosa-associated lymphoid tissue (NALT), lymph nodes, and spleens of mice immunized by the intranasal route. The recombinant strain, but not the wild type, activated the OVA-specific CD4+ T-cell population in the NALT (89% of KJ1-26+ CD4+ T cells) just 3 days following immunization. In the cervical lymph nodes and in the spleen, the percentage of proliferating cells was initially low, but it reached the peak of activation at day 5 (90%). This antigen-specific clonal expansion of KJ1-26+ CD4+ T cells after intranasal immunization was obtained with live and inactivated recombinant bacteria, and it indicates that the NALT is the site of antigen-specific T-cell priming.
doi:10.1128/IAI.74.5.2760-2766.2006
PMCID: PMC1459748  PMID: 16622213

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