Development Of Antigen-specific Immune Responses Research Articles

Black Carbon Content in Airway Macrophages is Associated with Reduced CD80 Expression and Increased Exacerbations in Former Smokers With COPD.

Chronic obstructive pulmonary disease (COPD) is characterized by recurrent exacerbations. Macrophages play a critical role in immune response and tissue repair in COPD. Airway macrophages (AM) are exposed to environmental exposures which are retained in the cytoplasmic material. Both biomass and particulate matter have been linked to higher AM black carbon. It is unknown if AM black carbon is associated with COPD morbidity and macrophage phenotype. Former smokers with COPD were enrolled and sputum induction was performed to obtain airway macrophages. Macrophages underwent black carbon quantification and flow cytometry phenotyping. Health information was obtained the same day as sputum induction and prospective exacerbations were assessed by monthly telephone calls. We studied 30 former smokers with COPD who had a mean age of 67 years and mean forced expiratory volume in 1 second (FEV1)% predicted of 60.8%. Higher AM black carbon content was associated with increased total exacerbations and severe exacerbations and reduced CD80 expression. AM black carbon association with respiratory morbidity is largely unexplored and this is the first study to identify association with prospective exacerbations. Macrophages expressed reduced CD80, a surface marker providing costimulatory signals required for development of antigen-specific immune responses. Our findings suggest that reduced CD80 expression is the pathophysiologic mechanism for the association of AM black carbon content and increased exacerbations. Therefore, beyond solely serving as a marker for increased exposures, AM black carbon content may be a predictor of future exacerbations given a macrophage less equipped to respond to an acute infectious exposure.

Human intestinal dendritic cell and macrophage subsets in coeliac disease.

Dendritic cells (DC) and macrophages (Mϕ) constitute the most abundant antigen presenting cells in the human intestinal mucosa. In resting conditions, they are essential to maintain the mechanisms of immune tolerance toward food antigens and commensals, at the time that they keep the capacity to initiate and maintain antigen-specific pro-inflammatory immune responses toward invading pathogens. Nevertheless, this delicate equilibrium between immunity and tolerance is not perfect, like in coeliac disease (CD), where DC and Mϕ drive the development of antigen-specific immune responses toward dietary gluten peptides. In this review, we provide therefore a comprehensive discussion about CD pathogenesis, the human intestinal immune system and the biology of intestinal DC and Mϕ both in resting conditions and in CD. Last, but not least, we discuss about all the remaining issues pending to be studied regarding DC and Mϕ contribution toward CD pathogenesis. This may allow the identification of unique and specific factors which may be useful in the clinical practice, as well as identify new therapeutic targets in order to reestablish the loss intestinal homeostasis in CD.

Analysis of Anti-Tumor Immune Responses with Radiation Combined with Anti-PD-L1 Immunotherapy in an HPV Specific Head & Neck Cancer Model.

Anti-PD-1 checkpoint blockade immunotherapy (CBI) was recently approved for recurrent or metastatic cisplatin refractory Head and Neck Squamous Cell Carcinoma (HNSCC); however, response rates remain low. Multiple pre-clinical studies and emerging clinical studies have described synergistic interactions between radiation (RT) and CBI. We previously defined the role of RT in enhancing T-cell activation and proliferation via antigen cross-presentation in the draining lymph node (DLN) when combined with anti-PD-1 CBI. However, the ability for RT to enhance local control and anti-tumor immune responses when combined with anti-PD-L1 CBI in HPV specific HNSCC models is understudied. Here we used novel flank and orthotopic HNSCC tumor models including AT-84 expressing HPV E7 to study the effects of RT+CBI. Focused RT (8-12Gyx1) was delivered to AT84-E7 flank tumors and orthotopic tongue tumors via the JL Shepard CS-137. Cohorts of C3H or B6 mice received either no treatment or three IP injections of 200ug anti-PD-L1 Ab immediately before, and at 3 and 6 days post RT. Tumor diameter was measured every 2 to 3 days using electronic caliper and reported as volume using the formula (m1 x m22)/2. Cell surface markers and development of antigen-specific immune responses were analyzed in tumor draining lymph node (DLN) and matched tumor infiltrating lymphocytes (TIL) by flow cytometry on BD FACSAria II. RT + anti-PD-L1 CBI resulted in a statistically significant improvement in local tumor control compared to RT alone in our HNSCC orthotopic tumor models (RT 16.5 +/- 8.9 mm3 vs RT + CBI 1.2 +/- 0.6 mm3, p<0.001). RT + CBI also significantly increased CD8+ and CD4+ activated T-cells in the TIL population (CD8: RT 2.3 +/- 0.7% vs RT + IT 4.1 +/- 1.2%, p < 0.05; CD4: RT 0.7 +/- 0.3% vs RT + IT 4.1 +/- 2.2%, p< 0.05). Additionally, RT + CBI enhanced IFNg production and development of antigen specific anti-tumor immunity in the DLN compared to RT or CBI alone. Mechanistically we observed significant changes in cell adhesion molecules including ICAM-1 and VCAM-1 which may contribute to enhanced T-cell infiltration. Response rates to single agent CBI remain low in HNSCC and combinatorial strategies are required to improve outcomes. Here we used novel HPV specific flank and orthotopic HNSCC models and observed that RT + anti-PD-L1 CBI results in a significant improvement in local control and development of anti-tumor immune responses compared to RT or CBI alone. These findings indicate that combination RT + CBI could be an effective therapy for HPV positive head and neck cancer, and provide additional rationale for clinical trials in this space.

Role of Radiation Therapy in Inducing Antigen Specific Antitumor Immune Responses When Combined With Anti-PD1 Checkpoint Blockade: Mechanism and Clinical Implications

Immunotherapy with anti-CTLA-4 and anti-PD1 antibodies (Ab) has gained significant interest due to activity in multiple tumor types and high rates of systemic control in metastatic melanoma. Preclinical studies and case reports have described potential synergy between radiation therapy (RT) and immunotherapy (IT); however, the direct role that RT may play in augmenting anti-tumor immune responses remains unclear. Here we report a mechanistic role for RT in inducing antigen-specific immune responses when combined with anti-PD1 Ab in melanoma and breast carcinoma. Stereotactic image-guided RT was delivered to B16-OVA and 4T1-HA flank tumors via the small animal radiation research platform (SARRP). Mice received three IP injections of 200 ug anti-PD1 and/or anti-CTLA-4 Ab before, during, and after RT. Cell surface markers and development of antigen-specific immune responses was analyzed by flow cytometry on LSR II. Adoptive transfer studies were performed to validate protective immunity and in vitro studies elucidated a molecular mechanism of action. Stereotactic RT modified the immunophenotype by increasing the surface expression of tumor associated antigens, MHC, and Fas. RT of B16-OVA enhanced tumor associated antigen presentation in-vitro and in the draining lymph node (DLN) in-vivo. Additionally, hypofractionated RT of 18 Gy x 1 or 7 Gy x 3 increased the proliferation and activation of antigen specific CD8 T-cells in the DLN (IFN-gamma positive: mock 20.8 +/- 3.6% vs 18 Gy 45.6 +/- 1.9%, p < 0.01). We observed significant increases in the development of endogenous SIINFEKL Pentamer positive anti-tumor immune responses when RT was combined with anti-PD1 Ab (RT 0.6% vs RT + anti-PD1 1.3%, p < 0.05). Furthermore, combination therapy resulted in primary tumor control, increased survival of treated mice, and elicited the abscopal effect (B16-OVA: IT 372 +/- 138 cc, RT 121 +/- 43 cc, RT + IT 57 +/- 31 cc∗, p < 0.05; 4T1-HA: IT 663 +/- 23 cc, RT 351 +/- 150 cc, RT + IT 99 +/- 51 cc∗, p < 0.05). Mechanistically, we identified that RT upregulates specific chemokine receptors which may drive development of antigen specific immune responses. RT altered the immunophenotype of tumor cells and enhanced tumor associated antigen presentation. Combination RT + IT resulted in significantly increased antigen-specific immune responses, primary tumor control, and abscopal effects outside of the radiation field. These data provide critical evidence supporting the ability of stereotactic RT to induce anti-tumor immune responses and augment the efficacy of anti-PD1 checkpoint blockade in melanoma and breast carcinoma. These findings have important implications for the novel treatment paradigm of RT combined with IT and provide mechanistic rational for clinical trials of combined therapy in the definitive and metastatic setting.

C5a Complement Depletion Suppresses In Vivo B Lymphoma Progression and Enhances Development Of Cellular Anti-Tumor Immune Response

BackgroundProteins within the complement system have complex effects on cellular immune responses. In previous studies, we found that active complement components, especially C5a, can dampen the development of antigen-specific immune responses following vaccination with a model antigen, in part by promoting generation of APC-induced T regulatory (Treg) cells. These studies also demonstrated that B lymphoma cell lines exposed to complement can induce Treg generation in vitro. The current study was designed to address whether depletion of C5a could enhance development of a cellular anti-lymphoma immune response in vivo. MethodsImmunocompetent Balb/C mice were inoculated subcutaneously with syngeneic A20 B lymphoma cells mixed with either 10 μg of rat anti-mouse C5a monoclonal antibody (mAb) or 10 μg of isotype-matched Rat IgG2a control mAb. Tumor growth was followed. In select experiments, mice were sacrificed and analyzed for the percentage and activity of tumor-infiltrating T cells and A20-specific splenic T cell responses. Results1. Tumor progression. Lymphoma grew more slowly in mice treated with anti-C5a mAb compared to mice treated with control mAb (p<0.05) {Fig. 1). [Display omitted] 2. Intratumoral T cells. Tumors from mice treated with anti-C5a mAb had higher CD8+ T cell infiltration compared to mice treated with control mAb (p=0.002) (Fig. 2). Tumor-infiltrating CD8+ T cells showed a trend towards higher intracellular IFNg production in mice treated with anti-C5a mAb compared to control mAb (p=0.051). [Display omitted] 3. Splenic T cells. Splenic T cells from mice treated with anti-C5a mAb produced IFNg to a greater degree than did splenic T cells from control mice when splenocytes were cultured with irradiated A20 cells in vitro (p=0.041) (Fig. 3). There was a trend towards decreased numbers of splenic CD4+CD25highFoxp3+ Tregs in C5a-depleted mice compared to control mice. [Display omitted] ConclusionsDepletion of C5a at the site of tumor inoculation slows tumor growth and increases the number of tumor infiltrating CD8 T cells in a syngenic immunocompetent model of lymphoma. A trend towards enhanced production of IFNg in the tumor infiltrating T cells, increased numbers of tumor-specific splenic T cells, and reduced numbers of splenic Tregs, suggests intratumoral C5a depletion can enhance tumor-specific immune responses both within the tumor and systemically. Ongoing studies are exploring the molecular mechanisms involved in C5a-promoted tumor progression and the use of C5a depletion as a novel strategy to improve anti-tumor immunity. Disclosures:No relevant conflicts of interest to declare.

CT Antigen-Specific Immune Response From DAC/DC Vaccine Correlates With Clinical Outcome In Patients With Relapsed Neuroblastoma

▪Neuroblastoma is the most common solid tumor in children in the first year of life. Despite high-dose chemotherapy, irradiation and autologous stem cell transplantation, nearly half of these patients relapse, a group for whom there are limited treatment options. The cancer-testis (CT) antigens MAGE-A1, MAGE-A3 and NY-ESO-1 are expressed on neuroblastoma cells in low levels and we have previously shown that the demethylating chemotherapy drug decitabine (DAC) can upregulate the expression of CT antigens in neuroblastoma. We developed a clinical study combining DAC to upregulate CT antigens followed by a dendritic cell (DC) vaccine targeting CT antigens MAGE-A1, MAGE-A3 and NY-ESO-1. Here we report the effects of DAC/DC vaccine in generating antigen-specific immune response and evaluate if there exists a correlation between development of antigen-specific immune responses and clinical responses. The treatment regimen includes 4 cycles of therapy, each consisting of DAC 10mg/m2/day for 5 days, followed by 2 weekly vaccinations consisting of autologous DC pulsed with overlapping peptide mixes derived from full length MAGE-A1, MAGE-A3 and NY-ESO-1. The number of DC administered in the vaccine was based on patient weight, and ranged from 3 to 10 x106 cells. The topical TLR agonist imiquimod was used at the site of vaccination to facilitate immune responses to the vaccine. Peripheral blood was collected weekly to assess antigen-specific immune response. Peripheral blood mononuclear cells were archived at various time points, stimulated for 24 h with MAGE-A1, MAGE-A3 and NY-ESO-1 peptide mixes and studied for the presence of CD137+ antigen-specific cells by flow cytometry. The regimen was well tolerated and highly feasible. We were able to culture DC for 10/10 neuroblastoma patients enrolled on the study. Development of an antibody or a T cell response to the vaccine was defined as either new onset or a two fold increase in the level of antibodies or number of MAGE-A1, MAGE-A3 and NY-ESO-1 specific, CD137+ T cells over baseline levels. The clinical and immunological outcomes of seven neuroblastoma patients treated so far with the DAC/CT antigen vaccine is summarized in table 1. Two patients are in complete remission, one of whom is two years from completing therapy, and another patient is 9 months from therapy. Both these patients demonstrated an increase in the number of circulating CD3+CD8+CD137+ and CD3+CD4+CD137+ T cells against one of the CT antigens in the vaccine. Of the five patients who had disease progression, one had a partial response to his chemotherapy and radiation resistant tumor 2 months post-vaccine. This patient had an antibody response to these antigens post-vaccination but no CD8+ or CD4+ T cell response. Another patient who had no evidence of disease for 8 months following the last vaccine prior to disease recurrence had an antigen-specific CD8+ T cell response against MAGE-A1, MAGE-A3 and NY-ESO-1 antigens but no CD4+ T cell response. These data indicate that DAC/DC vaccine targeting MAGE-A1, MAGE-A3 and NY-ESO-1 are efficient in generating an antigen-specific immune response in four of seven patients studied and there exist a correlation between the presence of immune response and positive clinical outcome.Table 1Relapsed Neuroblastoma Patient Summary#Age (yr)Site of Relapse/Extent of Disease at TreatmentTherapy StatusClinical Outcome after VaccineAntibody ResponseT cell Response015.5Recurrence in marrow; microscopic tumor clumpsCompleted 3 CyclesComplete ResponseNot determinedYes023Left maxiallary sinus (causing left proptosis & compression of optic nerve)Completed 4 CyclesPartial response followed by progressionYesNo034.5Refractory metastatic Stage IV neuroblastoma (Isolated to the brain)Completed 4 CyclesProgressionNoNo042.5Right proximal humerus NED at time of treatmentCompleted 4 CyclesRelapsed 8 months post-therapyNoYes056.5Chest wall mass, multiple lung nodulesCompleted 2 CyclesProgressionNot determinedNo064Lung and paraspinal disease; multiple bony metastases NED at time of treatmentCompleted 1 CycleDeveloped Urticaria multiforme post-vaccine but remains disease free at 9 months post-vaccineAnalysis in processYes076Widespread leptomeningeal disease with masses compressing the spinal cordCompleted 1 CycleProgressionAnalysis in processNot determined Disclosures:No relevant conflicts of interest to declare.

Accumulation of Myeloid-Derived Suppressor Cells in Bone Marrow in Multiple Myeloma Induces Tumor-Specific Immune Suppression and Promotes Tumor Growth

Abstract 3954Multiple myeloma (MM) is an incurable malignancy of clonal plasma cells in bone marrow (BM). Accumulation of MM cells in BM is associated with a corresponding dysfunction of local immune responses. The mechanisms involved in the immune suppression remains poorly understood. Myeloid-derived suppressor cells (MDSC) are heterogeneous group of relatively immature myeloid cells characterized by potent immune suppressive activity. However, their role in regulation of immune responses in BM in MM remains unclear.To determine MDSC accumulation in MM patients, BM and blood samples were obtained from newly diagnosed patients and healthy donors. We observed a significant increase in CD11b+ CD33+ CD14− HLA-DRlo MDSCs, but not in CD11b+ CD33+ CD14+ HLA-DRhi monocytes, in the blood and BM of patients as compared to cells with the same phenotype in healthy donors. MDSC isolated from patient BM suppressed T cell responses as measured by IFN-g Elispot assays of mixed leukocyte reactions. Immature myeloid cells with the same phenotype from BM of healthy donor did not inhibit T cells.The possible role of MDSC in MM progression was further evaluated in mouse model of MM. Three different MM cell lines (BCM, DP42 and ATLN) obtained from Dr Van Ness (University of Minnesota) were used. MM tumors were established by i.v. injection of syngeneic mice. We observed an expansion of CD11b+ GR1+ MDSCs (up to 70%) in the BM of tumor-bearing (TB) mice within one week after injection of tumor cells. MDSC purified from BM of TB mice at that time point potently suppressed antigen-specific T cell proliferation and IFN-g production. As tumor progressed, CD138+ MM cells expanded (up to 90%) in the BM replacing normal BM cells including MDSC. However, at later time points (2–3 weeks), MDSC expansion was seen in spleen and lymph nodes of TB mice.To evaluate the possible role of MDSC in MM progression, we used S100A9 knockout (KO) mice. S100A9 plays an important role in MDSC accumulation in cancer and S100A9 KO TB mice had reduced accumulation of these cells. When wild-type (WT) and S100A9 KO mice were inoculated with DP42 cells no differences in tumor growth and survival were observed. S100A9 KO mice had a decrease in MDSC accumulation in the BM of TB mice compared to WT mice. Additionally, MDSC from S100A9 KO mice failed to suppress antigen-specific T cells ex vivo, whereas MDSC from WT TB mice did. To evaluate the development of antigen-specific immune responses in MM, we generated a MM cell line over-expressing the chicken ovalbumin protein (DP42-OVA). S100A9 KO mice injected with the immunogenic DP42-OVA cell line showed significantly delayed tumor growth and improved survival as compared with WT mice. Further analysis of T cell responses in vivo showed increased accumulation of antigen-specific T cells within the BM of S100A9 KO mice, but not WT mice, as early as one week post-tumor inoculation. Antigen-specific T cells in spleens were not detected at that time point. These cells were seen in spleens on S100A9 KO mice only 2–3 weeks after tumor injection.Thus, our study demonstrated that MM is associated with rapid accumulation of MDSC with potent immune suppressive activity against tumor-specific T cells. Our data directly suggest that targeting MDSC in MM may improve antitumor immune response and clinical outcome of the disease. Disclosures:No relevant conflicts of interest to declare.

Initiation and regulation of T-cell responses in tuberculosis.

Tuberculosis (TB) poses a great challenge to immunologists, as it represents a chronic infection characterized by persistence of the pathogen despite development of antigen-specific immune responses. Among the characteristics of adaptive immune responses to Mycobacterium tuberculosis is a delay in the onset of detectable T-cell responses, in both humans and experimental animals. Recent studies have revealed mechanisms that contribute to this delay, including pathogen inhibition of apoptosis, delayed migration of dendritic cells from the lungs to the local lymph node, and influence of regulatory T cells. In addition, novel features of M. tuberculosis antigen-specific T-cell differentiation have been discovered, which reveal pathways that limit and promote immune control of infection. Taken together, these results highlight the need for additional basic research and provide optimism for the development of TB vaccines with greater efficacy.

Autoimmune Hepatitis Induced by Syngeneic Liver Cytosolic Proteins Biotransformed by Alcohol Metabolites

Aldehydes that are produced following the breakdown of ethanol (acetaldehyde) and lipid peroxidation of membranes (malondialdehyde) have been shown to bind (adduct) proteins. Additionally, these two aldehydes can combine (MAA) on nonsyngeneic and syngeneic proteins to initiate numerous immune responses to the unmodified part of the protein in the absence of an adjuvant. Therefore, these studies provide a potential mechanism for the development of antigen-specific immune responses resulting in liver damage should syngeneic liver proteins be adducted with MAA. This study sought to test whether MAA-modified syngeneic liver cytosolic proteins administered daily in the absence of adjuvant into C57BL/6 mice abrogates tolerance to initiate a MAA-induced autoimmune-like hepatitis. In mice immunized with MAA-modified cytosols, there was an increase in liver damage as assessed by aspartate aminotransferase/alanine aminotransferase levels that correlated with liver pathology scores and the presence of the pro-fibrotic factors, smooth muscle actin, TGF-β, and collagen. IgG antibodies and T-cell proliferative responses specific for cytosolic proteins were also detected. Pro-inflammatory cytokines were produced in the livers of animals exposed to MAA-modified cytosols. Finally, transfer of immunized T cells to naïve animals caused biochemical and histological evidence of liver damage. These data demonstrate that a disease with an autoimmune-like pathophysiology can be generated in this animal model using soluble MAA-modified syngeneic liver cytosols as the immunogen. These studies provide insight into potential mechanism(s) that the metabolites of alcohol may play in contributing to the onset of an autoimmune-like disease in patients with alcoholic liver disease.

Increased antigen presentation and Th1 polarization in genetically histamine-free mice

Histamine is a well-known inflammatory mediator exerting various immunomodulatory effects and affecting the development of antigen-specific immune responses. Dendritic cells (DCs) are the most potent antigen-presenting cells specialized for capture, uptake, transport, processing and presentation of antigens to T cells. Using a genetically histamine-free [histidine decarboxylase knockout (HDC-/-)] mouse model, we examined the effects of histamine on DC-mediated antigen presentation. Applying an in vitro antigen presentation assay, we found that spleen DCs, derived from HDC-/- mice, display a higher efficiency in antigen presentation compared with wild-type cells. Flow cytometric characterization of DCs disclosed that this difference was not due to an altered distribution of DCs between or within the major functional sub-populations (assessed by CD11b and CD4 as myeloid and CD8alpha and DEC205 as lymphoid DC markers) or major changes in the co-stimulatory molecule profile (CD40, CD80, CD86). However, real-time PCR analysis of in vivo CFA-induced IL-12p35, IFNgamma, IL-10 and IL-4 expression showed that DCs matured in a histamine-free environment exhibit significantly elevated levels of IL-12p35 and IFNgamma mRNA. In vitro investigations confirmed that isolated DCs, developed in the absence of histamine, exhibit indeed a predominantly T(h)1-polarized cytokine pattern, as they show elevated levels of IFNgamma mRNA upon LPS stimulation. Similar difference was found at the protein level by ELISA, as well. Our study demonstrates that histamine interferes with antigen presentation and alters the cytokine profile of DCs.

131. Design and Construction of an Adenovirus Vector Containing Fiber Fibritin/Canine CD40L Fibers Directed to Canine CD40-Expressing Cells

Top of pageAbstract Dendritic cells (DCs) are a central element in the development of antigen-specific immune responses. On this basis, antigen loading of dendritic cells via transduction with viral vectors in vivo has been proposed as a standardized and easily clinically applicable approach for tumor vaccination strategies. For genetic vaccine approaches to be effective, it is of vital importance to achieve a selective and high transduction efficiency of the DC present in situ, while also ensuring their effective activation and migration. Towards this goal, we have reported that an adenovirus (Ad) serotype 5 vector targeted to human (hCD40) via genetic capsid incorporation of human CD40 ligand (hCD40L) achieves selective transduction of DC in vitro (Belousova et al. Journal of Virology, 2003). We have sought further evaluation of this CD40-targeted vector system in vivo. Of note, certain dog breeds are subject to spontaneous cancers such as osteosarcoma and lymphoma, thereby providing large animal models as stringent in vivo models of human immunotherapy. We have therefore adapted our CD40 targeting system for use in canine systems. We have constructed an E1-deleted, luciferase transgene Ad5 vector encoding a genetically modified fiber that displays canine CD40L, utilizing similar technique as our hCD40L-incorporating Ad5 vector. Viral particles were harvested from Ad5 fiber included- 293F28 cells and 293 cells, purified by standard cesium chloride gradient centrifugation and yielded titers in excess of 3.0 |[times]| 10e12 vp/ mL. Characterization of AdFFcCD40L vector infectivity in canine CD40-positive 293 cells (293cCD40) showed 6.9-to-14.6 fold greater luciferase reporter gene activities versus cCD40-negative 293 cells. Herein, we have demonstrated the construction, purification, and initial infectivity characterization of a novel Ad vector that exploits the native canine CD40-CD40L pathway. These data support our use of our cCD40-targeted Ad vectors in novel canine models of immunotherapy.

Investigation of the kinetics of histidine-rich protein 2 and of the antibody responses to this antigen, in a group of malaria patients from India

Although immunological tests based on the detection of histidine-rich protein 2 (HRP2) from the parasites permit the rapid diagnosis of Plasmodium falciparum malaria, such tests are not yet sufficiently sensitive to detect every bloodsmear-positive case. Some individuals infected with P. falciparum may appear test-negative because of the presence of anti-HRP2 antibodies in their sera. A longitudinal follow-up of HRP2 antigenaemia and antibody responses to this antigen has now been conducted in a group of 45, bloodsmear-positive malaria cases of various ages, both during acute infection with P. falciparum and after antimalarial treatment. Pre-treatment, 'day-0' samples of fingerprick blood were tested for HRP2 (in antigen-capture ELISA) and for antigen-specific IgM and IgG (in indirect ELISA). The patients were then treated, with standard doses of chloroquine, before being retested, for HRP2 and anti-HRP2 antibodies, on days 7, 15 and 28. The level of antigenaemia, which on day 0 was found to be positively correlated with parasitaemia (r = 0.741; P < 0.001), had only fallen by an insignificant amount by day 7 but showed further, significant falls between days 7 and 15 (P < 0.001) and between days 15 and 28 (P < 0.01). Although no significant relationship was observed between the blood concentrations of HRP2 and anti-HRP2 IgM or IgG on days 0 or 7, the level of HRP2 antigenaemia was found to be positively correlated with the concurrent titre of anti-HRP2 IgM on day 15 (r = 0.612; P < 0.001) and day 28 (r = 0.501; P < 0.001). The titres of HRP2-specific IgG gradually increased over the 28 days of follow-up but were not found to be significantly correlated with the decreasing levels of HRP2 antigenaemia. When the 45 day-0 samples of blood were tested for HRP2 in a rapid diagnostic test (RDT), three appeared negative, probably because of interference from the circulating, free, anti-HRP2 antibodies in the plasma. The three RDT-negative samples were significantly different from the 42 RDT-positive, having relatively low HRP2 antigenaemias (P < 0.001) and relatively high titres of anti-HRP2 IgM (P < 0.05) and IgG (P < 0.001). Control samples of blood, from four patients infected with P. vivax and five healthy, normal individuals, were considered ELISA-negative for HRP2 and anti-HRP2 IgM or IgG. It appears that, during human infection with P. falciparum, serum levels of HRP2 antigen remain elevated for at least 7 days post-treatment, despite the host's development of antigen-specific immune responses both before and after treatment.

High efficiency transduction of dendritic cells by adenoviral vectors targeted to DC-SIGN

Dendritic cells (DCs) are a central element in the development of antigen-specific immune responses. The lack of a specific and efficient technique for the in vivo delivery of antigens to DCs remains a major obstacle limiting a vaccine’s ability to induce an effective immune response. The efficacy of adenoviral (Ad) vectors in this regard can be enhanced through alterations in vector tropism such that DC-targeted transduction is achieved. Here, the efficiency of DC transduction by Ad vectors retargeted to DC-specific ICAM-3 grabbing non-integrin (DC-SIGN) was studied and compared to that of Ad vectors retargeted through CD40. A comparable and significant enhancement of gene transfer to monocyte derived DCs (MDDCs) was accomplished by means of an Ad vector harboring the Fc-binding domain of Staphylococcus aureus protein A in combination with antibodies to DC-SIGN or to CD40 or with fused complexes of human Ig-Fc with their natural ligands, i.e. ICAM-3 or CD40L, respectively. Whereas CD40-targeted Ad transduction resulted in a more profound phenotypic DC maturation, DC-SIGN- and CD40-targeted Ad both induced similar levels of IL-12 secretion. These data demonstrate the usefulness of DC-SIGN as a DC-restricted targeting motif for Ad-mediated vaccination strategies.

124. Efficient Transduction and Activation of Dendritic Cells by Adenoviral Vectors Targeted to DC-SIGN

Dendritic cells (DCs) are a central element of the development of antigen-specific immune responses. The lack of a specific and efficient technique for delivery of antigens to DCs remains the major obstacle, limiting the vaccine's ability to induce an effective immune response. In this regard, adenoviral (Ad) vectors offer a number of significant advantages for this application. Further gains in the utility of Ad would accrue to the ability to alter tropism such that DC-selective transduction could be achieved. Immature DCs monitor the environment via antigen recognition receptors including the Toll-like receptors and the C-type lectins. The C-type lectin, DC-SIGN, is an internalizing receptor, and, due to high level expression by monocyte-derived DCs is an attractive receptor for targeting Ad vectors for DC-based tumor vaccination approaches.

Attenuation of Th1 Effector Cell Responses and Susceptibility to Experimental Allergic Encephalomyelitis in Histamine H 2 Receptor Knockout Mice Is Due to Dysregulation of Cytokine Production by Antigen-Presenting Cells

Histamine, a biogenic amine with both neurotransmitter and vasoactive properties, is well recognized as an immunomodulatory agent in allergic and inflammatory reactions. It also plays a regulatory role in the development of antigen-specific immune responses. CD4+ T-cells from histamine H1 receptor (H1R)-deficient (H1RKO) mice produce significantly less interferon-gamma and more interleukin (IL)-4 in in vitro recall assays compared to wild-type controls. H1RKO mice are also less susceptible to acute early-phase experimental allergic encephalomyelitis indicating that H1R signaling in CD4+ T cells plays a central role in regulating pathogenic T-cell responses. In this study, we show that mice lacking histamine H2 receptor (H2RKO) are similar to H1RKO mice in that they develop encephalitogen-specific T-cell responses as assessed by proliferation and IL-2 production and present with less severe acute early-phase experimental allergic encephalomyelitis. However, unlike T cells from H1RKO mice, which exhibit a strong Th2 bias, T cells from H2RKO mice do not. Rather, they are uniquely characterized by a significant inhibition of Th1 effector cell responses. Given that both histamine and adjuvants such as pertussis toxin modulate antigen-presenting cell (APC) maturation and function, including T-cell-polarizing activity, we analyzed the cytokines/chemokines secreted by APCs from wild-type, H1RKO, and H2RKO mice. Significant differences in cytokine/chemokine production by APCs from unimmunized and immunized mice were delineated. APCs from H2RKO mice produce significantly less IL-12 and IL-6 and markedly greater amounts of MCP-1 compared to wild-type and H1RKO mice. Because MCP-1 is known to inhibit IL-12 production, the failure of H2RKO mice to generate encephalitogenic Th1 effector cell responses is consistent with inhibition of negative regulation of MCP-1 secretion by H2R signaling in APCs.

Role of B7 costimulatory molecules in immune responses and T-helper cell differentiation in response to recombinant HagB from Porphyromonas gingivalis.

In addition to antigen-specific signals mediated through the T-cell receptor, T cells also require antigen nonspecific costimulation for activation. The B7 family of molecules on antigen-presenting cells, which include B7-1 (CD80) and B7-2 (CD86), play important roles in providing costimulatory signals required for development of antigen-specific immune responses. Hemagglutinin B (HagB) is a nonfimbrial adhesin of the periodontopathic microorganism Porphyromonas gingivalis and is thought to be involved in the attachment of the bacterium to host tissues. However, the immune mechanisms involved in responses to HagB and their roles in pathogenesis have yet to be elucidated. Therefore, the purpose of this study was to determine the role of B7 costimulatory molecules on T-helper-cell differentiation for the induction of immune responses to HagB. Mice deficient in either or both of the costimulatory molecules B7-1 and B7-2 were used to explore their role in immune responses to HagB after subcutaneous immunization. B7-1(-/-) mice had levels of immunoglobulin G (IgG) anti-HagB antibody activity in serum similar to those of wild-type mice, whereas lower serum IgG anti-HagB antibody responses were seen in B7-2(-/-) mice. Moreover, significantly lower numbers of IgG antibody-secreting cells and lower levels of CD4(+)-T-cell proliferation were observed in B7-2(-/-) mice compared to wild-type mice. No serum IgG response to HagB was detected in B7-1/B7-2(-/-) mice. Analysis of the subclass of the serum IgG responses and the cytokines induced in response to HagB revealed that B7-2(-/-) mice had significantly lower IgG1 and higher IgG2a anti-HagB antibody responses compared to wild-type mice. The B7-2(-/-) mice also had significantly reduced levels of interleukin-4 (IL-4) and IL-5 and enhanced level of gamma interferon. Furthermore, assessment of B7-1 and B7-2 expression on B cells and macrophages derived from wild-type BALB/c mice after in vitro stimulation with HagB revealed a predominant upregulation in the expression of the B7-2 costimulatory molecule on B cells and macrophages. Essentially no change was seen in the expression of B7-1. Taken together, these results suggest a critical role for B7, especially B7-2, for the preferential induction of a Th2-like response to HagB.

Dendritic cells presenting tumor antigen.

Since the first identification of dendritic cells by Steinman and Cohn in 1973, progress in understanding their biology has included the development of novel methods of cell culture, recognition of critical aspects of migration and maturation, and appreciation of their major role as antigen-presenting cells (APC), and how this activity is regulated by cytokines and expression of accessory molecules. Dendritic cells are the major APC involved in the initiation of the immune response and the development of tolerance. There is considerable evidence that they can acquire antigen in the peripheral tissues and process, transport, and present it to T cells in secondary lymphoid tissue. A number of studies show that, in vitro or in vivo, antigen-pulsed dendritic cells can directly sensitize T cells and stimulate the development of antigen-specific immune responses, including both protective and therapeutic antitumor responses. In this paper, several important aspects of dendritic cell biology are discussed and a number of studies confirming the role of these professional APC in antitumor immunity are reviewed.