IFN-gamma Production Research Articles

IT-16 * COMBINATION TREATMENT WITH DELTA24-RGD AND TEMOZOLOMIDE IMPROVES SURVIVAL AND AFFECTS INTRATUMORAL IMMUNE CELL SUBSETS IN A MURINE MALIGNANT GLIOMA MODEL

INTRODUCTION: The oncolytic adenovirus Delta24-RGD specifically lyses tumor cells and is currently tested in clinical phase I/II trials for recurrent glioma. We recently showed that the efficacy of Delta24-RGD is mainly dependent on an induced anti-tumor immune response. As known, TMZ has pronounced effects on the immune system, which could hamper the viral-induced anti-tumor immune response. To assess the combined effects, we tested different treatment regimens in an immune competent glioma mouse model. METHODS: C57BL/6 mice were injected intracranially with GL261 cells. TMZ was given for 5 days before (pre-treatment regimen) or after (post-treatment regimen) intratumoral injection of Delta24-RGD. Mice were followed for survival or sacrificed at earlier time-points to assess intratumoral influx of immune cells. T-cell response directed to the tumor cells was assessed by co-culture of splenocytes with GL261 tumor cells and analysis of IFNgamma production. Furthermore, to assess the type of cell death induced by the treatments, western blotting for LC3 (autophagy) and caspase 3/7 analysis (apoptosis) was performed. RESULTS: In vitro, apoptosis is highly induced in virus and combination treated cells, whereas autophagy is induced by TMZ monotherapy. In vivo, both combination treatment regimens significantly improved survival compared to TMZ alone. The post-treatment regimen also prolonged survival significantly compared to virus only. In the pre-treatment group, influx of dendritic cells is reduced, followed by a diminished influx of CD8+ T-cells. Interestingly, splenocytes from mice of virus and both combination treatment groups recognize GL261 cells, indicating that the diminished influx of immune cells did not hamper T-cell activation. CONCLUSION: The addition of TMZ to Delta24-RGD treatment in immune competent mice improves survival compared to Delta24-RGD or TMZ alone. Contrary to the post-treatment regimen, administering TMZ prior to virus injection hampers the intratumoral influx of immune cells; however, with both regimens an effective anti-tumor immune response is elicited.

PO-0185 Acute Respiratory Tract Infections (arti) In Hospitalised Children: Viruses, Interferon-alpha And Gamma, S-iga Levels

Background Detection of respiratory viruses, IFN-alpha, gamma, s-IgA levels in the first 3 years old hospitalised children within 3 days of symptom onset. Methods The human viruses were detected in nasal wash specimens obtained from 86 children by polymerase chain reaction (PCR), IFN-alpha, gamma, s-IgA plasma and nasal wash specimens levels, IFN-alpha, gamma induced production in vitro were investigated. The IFN-alpha production was induced by Newcastle disease virus, IFN-gamma production was induced by phytohemagglutinin. Results Influenza virus B was detected in 35% investigations, A(H1N1) - 23%, A(H3N2) - 4%, Parainfluenza virus (PIV) - 5%, rhinoviruses (HRV) - 4%, metapneumovirus (HMPV) - 6%, bocavirus (HBoV) - 5%, Adenovirus (AdV) and Respiratory syncytial virus (RSV) - 9%. Seasonal features were found: A(H1N1) dominated in March (66.7%), B - in June (77,8%), PIV was recorded in March only. HRV and RSV were noted as the longest circulation (from January to May), AdV, HBoV, HMPV were detected from March to June. The s-IgA decrease less than 1.5 mg/ml was found in 75.8% children in nasal wash specimens. Serum IFN-alpha, gamma were decreasing below a sensitivity threshold (less 2 pg/ml) in 67.7% and 69.4%, especially in influenza children and in ARTI complications. The nasal wash specimens INF-alpha, gamma levels less than 2 pg/ml were found in 38.7% and 48.4%. The IFN-alpha, gamma induced levels in vitro were lowered in 22.6% and 40.3%. Conclusions Our data demonstrate the IFN-alpha, gamma deficit in children with ARTI. It contributes IFN-alpha, gamma replaceable therapy in infants especially. This is very necessary the ARTI seasonal prevention.

Abstract 2561: Yeast vector-encoding multiple MUC1 agonist epitopes (yeast-MUC1) can induce MUC1-specific T-cell immune responses

Abstract The tumor-associated antigen MUC1 is overexpressed on various human hematological and epithelial malignancies. The MUC1 molecule, which has an N-terminal (MUC1-N) and a C-terminal (MUC1-C, which has been shown to act as an oncogene), is an attractive target for cancer immunotherapy. We have identified and reported 10 agonist epitopes (7 in the C-terminus, 2 in the N-terminus VNTR region, and 1 in the non-VNTR region) that enhance production of CD8 cytotoxic T lymphocytes. These epitopes span HLA-A2, HLA-A3, and HLA-A24 MHC class I alleles, thus encompassing the majority of the North American population. Compared to native epitopes, these agonist epitopes more efficiently generate MUC1-specific T cells, induce production of IFN-gamma by MUC1-specific T cells, and lyse human tumor cells expressing MUC1 native epitopes in an MHC-restricted manner. We have previously shown that heat-killed recombinant Saccharomyces cerevisiae yeast genetically modified to express carcinoembryonic antigen can efficiently activate human dendritic cells (DCs) and stimulate CEA-specific CD8+ T cells. Here, we investigated the ability of a Saccharomyces cerevisiae vector containing the MUC1 transgene (yeast-MUC1) and encoding MUC1 with 8 agonist epitopes to activate human DCs, stimulate MUC1-N- and MUC1-C-specific T cells, and generate MUC1-specific T cells. We show here for the first time that human DCs treated with yeast-MUC1 vectors can activate MUC1-N and MUC1-C agonist-specific T-cell lines and can act as antigen-presenting cells to generate MUC1-N- and MUC1-C-specific T cells specific to each agonist epitopes, and that these T cells are capable of lysing MUC1-expressing tumor cells. Together, these findings provide a rationale for further clinical evaluation of yeast-MUC1 constructs encoding MUC1 agonist epitopes in cancer vaccine immunotherapy. Citation Format: Kwong Y. Tsang, Benjamin Boyerinas, Caroline Jochems, Tim Rodell, Thomas King, Jeffey Schlom. Yeast vector-encoding multiple MUC1 agonist epitopes (yeast-MUC1) can induce MUC1-specific T-cell immune responses. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2561. doi:10.1158/1538-7445.AM2014-2561

Leishmania donovani phosphoproteins pp41 and pp29 re-establishes host protective immune response in visceral leishmaniasis

As phospho proteins are reported to be involved in virulence and survival, the ability of Leishmania to inhibit macrophage effector functions may result from a direct interference of leishmanial molecules with macrophage signal transduction pathways. Several such proteins such as pp63, pp41 and pp29 have also been identified as a Th1 stimulatory protein in the Leishmania donovani. In the present study, the immunogenicity of a cocktail of pp63+pp41+pp29 was assessed by estimation of serum antibody titre, nitric oxide(NO) production, estimation of Th1 cytokine(IFN-γ) as well as Th2 cytokines(IL-4), and determination of parasite load in L. donovani infected mice. In the group immunized with antigenic cocktail there was a sharp rise in antibody titer up to Day 20 which reduced considerably by Day 50. Groups of mice vaccinated with pp63, pp41, pp29 and the antigenic cocktail expressed 10-fold, 16-fold, 22-fold and 25-fold increase respectively in NO production by splenocytes. The animal groups immunized with pp63, pp41, pp29 and the antigenic cocktail showed reduced parasite load in the liver and spleen, as well as increased IFN-gamma production in the spleen. Furthermore immunized animals remained with a normal hematological profile, whereas L. donovani in unimmunized mice lead to significant anemia.

Autoreactivity to glucose regulated protein 78 links emphysema and osteoporosis in smokers.

RationaleEmphysema and osteoporosis are epidemiologically associated diseases of cigarette smokers. The causal mechanism(s) linking these illnesses is unknown. We hypothesized autoimmune responses may be involved in both disorders.ObjectivesTo discover an antigen-specific autoimmune response associated with both emphysema and osteoporosis among smokers.MethodsReplicate nonbiased discovery assays indicated that autoimmunity to glucose regulated protein 78 (GRP78), an endoplasmic reticulum chaperone and cell surface signaling receptor, is present in many smokers. Subject assessments included spirometry, chest CT scans, dual x-ray absorptiometry, and immunoblots for anti-GRP78 IgG. Anti-GRP78 autoantibodies were isolated from patient plasma by affinity chromatography, leukocyte functions assessed by flow cytometry, and soluble metabolites and mediators measured by immunoassays.Measurements and Main ResultsCirculating anti-GRP78 IgG autoantibodies were detected in plasma specimens from 86 (32%) of the 265 smoking subjects. Anti-GRP78 autoantibodies were singularly prevalent among subjects with radiographic emphysema (OR 3.1, 95%CI 1.7–5.7, p = 0.003). Anti-GRP78 autoantibodies were also associated with osteoporosis (OR 4.7, 95%CI 1.7–13.3, p = 0.002), and increased circulating bone metabolites (p = 0.006). Among emphysematous subjects, GRP78 protein was an autoantigen of CD4 T-cells, stimulating lymphocyte proliferation (p = 0.0002) and IFN-gamma production (p = 0.03). Patient-derived anti-GRP78 autoantibodies had avidities for osteoclasts and macrophages, and increased macrophage NFkB phosphorylation (p = 0.005) and productions of IL-8, CCL-2, and MMP9 (p = 0.005, 0.007, 0.03, respectively).ConclusionsHumoral and cellular GRP78 autoimmune responses in smokers have numerous biologically-relevant pro-inflammatory and other deleterious actions, and are associated with emphysema and osteoporosis. These findings may have relevance for the pathogenesis of smoking-associated diseases, and development of biomarker immunoassays and/or novel treatments for these disorders.

Abstract 354: Vascular Oxidative Stress Promotes Aortic Stiffening In Hypertension

We have previously shown that T cells and the cytokine IL-17a contribute to aortic stiffening in hypertension. Because oxidative stress also contributes to hypertension, we sought to determine if chronic oxidative stress causes aortic stiffening and to determine if T cells are involved. We studied tgsm/p22phox mice, which have an overexpression of the NADPH oxidase subunit p22phox in the vascular smooth muscle and approximately 2-fold increase vascular reactive oxygen species (ROS). Six month-old tgsm/p22phox mice were normotensive at baseline but exhibited increased aortic stiffness as indicated by the leftward shift of stress-strain curves (p<0.05). Aortic collagen content, as determined by hydroxyproline assay, was also increased in these mice compared to age-matched C57Bl/6 mice (76.3±11.9 vs. 43.2±11.9 ng/μg total protein, p<0.01). At nine months of age, aortic stiffening did not further increase but tgsm/p22phox mice developed vascular inflammation and mild hypertension (137±9 systolic and 100±7 diastolic, p<0.05), indicating that aortic stiffening precedes hypertension. Surprisingly, T cells isolated from the spleen of 9 month-old Tgsm/p22phox mice demonstrated marked production of IL-17a and IFN-gamma. To determine if inflammation contributes to these pathological events, we crossed the tgsm/p22phox mice with RAG-1-/- mice. The resultant mice were protected from vascular inflammation, aortic stiffening and hypertension. Tempol, a superoxide dismutase mimetic, normalized blood pressure (109±4 systolic and 95±4 diastolic, p<0.05), reduced vascular collagen deposition (37.5±9.5 ng/μg total protein, p<0.01) and prevented aortic stiffening in tgsm/p22phox mice. We have recently shown that isoketals, which are oxidation products of arachidonic acid, promote T cell activation. Treatment with the isoketal scavenger 2-hydroxybenzylamine also normalized blood pressure, prevented collagen deposition and aortic stiffening in 9 month-old tgsm/p22phox mice. These data are compatible with a pathway in which vascular oxidative stress leads to formation of isoketals, which promote T cell activation, production of cytokines such as IL-17a and IFN-gamma, which in turn promote collagen deposition and aortic stiffening.

Immunoregulatory networks in human Chagas disease

Chagas disease, caused by the infection with Trypanosoma cruzi, is endemic in all Latin America. Due to the increase in population migration, Chagas disease has spread worldwide and is now considered a health issue not only in endemic countries. While most chronically infected individuals remain asymptomatic, approximately 30% of the patients develop a potentially deadly cardiomyopathy. The exact mechanisms that underlie the establishment and maintenance of the cardiac pathology are not clear. However, there is consistent evidence that immunoregulatory cytokines are critical for orchestrating the immune response and thus influence disease development or control. While the asymptomatic (indeterminate) form represents a state of balance between the host and the parasite, the establishment of the cardiac form represents the loss of this balance. Analysis of data obtained from several studies has led to the hypothesis that the indeterminate form is associated with an anti-inflammatory cytokine profile, represented by high expression of IL-10, while cardiac form is associated with a high production of IFN-gamma and TNF-alpha in relation to IL-10, leading to an inflammatory profile. Here, we discuss the immunoregulatory events that might influence disease outcome, as well as the mechanisms that influence the establishment of these complex immunoregulatory networks.

The cytokine production of peripheral blood mononuclear cells reflects the autoantibody profile of patients suffering from type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disorder characterised by the immune-mediated destruction of insulin-producing pancreatic beta cells. The inflammatory process appears to be primarily mediated by pro-inflammatory Th1 lymphocytes, while the role Th17 cells in T1D is currently being investigated. T1D is characterised by the presence of autoantigen-specific autoantibodies.This study was conducted using patients with confirmed T1D and healthy control subjects. We examined the effect of the patient’s autoantibody profile on peripheral blood mononuclear cell (PBMC) cytokine production following stimulation with the major diabetogenic autoantigens GAD65 and IA2. IFN-gamma and IL17 production was detected by ELISPOT and the ratio of basic cellular populations in PBMCs was measured by flow cytometry.We demonstrated a significant interaction between the patient’s autoantibody profile and mode of stimulation. This suggests that autoantigen stimulation has a different effect on different groups of patients depending on their autoantibody profile. An increased production of IL17 was found in patients with high IA2 autoantibodies compared to patients with low levels of autoantibodies and healthy controls regardless of the mode of stimulation. The titre of IA2 autoantibodies positively correlates with the proportion of Tc lymphocytes and negatively correlates with the proportion of Th lymphocytes.Our results show that a patient’s autoantibody profile reflects the type of cellular immune responses. It seems that the high titre of IA2 autoantibodies is related to increased production of IL17 and an increased proportion of Tc lymphocytes. This finding may be useful in designing immunointervention studies to prevent T1D.

NK CELLS FROM GLIOMA-BEARING MICE TREATED WITH TEMOZOLOMIDE ARE ENRICHED FOR GENES RELATED TO MULTI-DRUG RESISTANCE AND CHEMOTAXIS

BACKGROUND: Chemotherapy can influence the immune response by inducing immunogenic death of tumor cells or modulating tumor microenvironment (Galluzzi et al. 2012). The limitations of chemotherapy and immunotherapy as single therapeutic modalities have generated considerable interest in combinatorial strategies (Zitvogel L et al 2008). Here we focused on understanding the molecular mechanisms directly induced by temozolomide (TMZ), the standard chemotherapeutic agent for glioblastoma, that can activate immune cells. METHODS: We studied the in vivo effects of TMZ on immune cells by treating mice 9 days after intracranial implantation of GL261 gliomas with intraperitoneal injections of TMZ (5 mg/kg) or vehicle (control mice) for 5 days. Mice were sacrificed 20 hours after treatment on days 1 to 5: tumor and peripheral blood were harvested and analyzed by flow cytometry. RESULTS: Trafficking of NK1.1+ CD3- NK cells in blood, but not of CD8+ T cells, and their homing ability into the brain significantly increased in TMZ-treated mice on day 12 after the second administration of TMZ. At this early time point TMZ led to a significant enrichment of CD11b(low) CD27(high) and CD11b(high) CD27(high) subsets. Microarray analysis revealed differentially expressed genes represented in different clusters of which chemotaxis, cell cycle, multidrug resistance and anti-apoptosis were predominantly up-regulated in blood-derived NK cells isolated from TMZ-treated compared to control mice. We focused validation experiments on genes related to multidrug resistance and chemotaxis. Chemotaxis was assayed using the in vitro transwell system confirming that the migration ability of NK cells from treated mice significantly increased by 3-fold compared with controls. Increased expression of three ATP transporter genes was confirmed by real time PCR (2.6-fold higher than controls, P = 0.006). Five days after the end of the treatment we found an enrichment of CD11(high) CD27(low), the most potent effector subset of NK cells (Chiossone et al 2009). In agreement, IFN-gamma production evaluated by flow cytometry and the in vitro cytotoxic activity of NK cells from TMZ-treated mice were higher than that of NK cells from control mice. CONCLUSIONS: Our results support the idea that NK cells can be resistant to chemotherapy (Lowdell 2003; MacDonald HR et al 1995). In particular at early time points TMZ leads to an enrichment of NK cells with migratory function, in the later phase TMZ leads to an increase of NK cells promoting cytotoxic ability. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

HPV-targeted tumor-infiltrating lymphocytes for cervical cancer.

LBA3008 Background: Adoptive T-cell therapy (ACT) is a promising cancer treatment modality with potentially broad application. It is not known if ACT can mediate regression of carcinomas, the most common solid tumors in humans. We studied carcinoma of the uterine cervix, a virally induced malignancy that constitutively expresses the HPV E6 and E7 oncoproteins, as a model cancer to test if ACT can mediate regression of an epithelial malignancy. Methods: We initiated a clinical trial to treat metastatic HPV+ cancers with tumor-infiltrating lymphocytes (TIL) selected for HPV E6- and E7-reactivity (HPV-TIL). Patients from the cervical cancer cohort are reported here. HPV-TIL infusion was preceded b y non-myeloablative conditioning and followed by high-dose bolus aldesleukin. HPV-reactivity was assessed by ELISPOT, IFN-gamma production, and CD137 expression assays. Results: Nine patients were treated on the study. They received a median of 81 x 109 T cells (range 33 to 159 x 109) as a single infusion. The infused cells possessed reactivity against high-risk HPV E6 and/or E7 in 6/8 patients. The two patients with no HPV reactivity did not respond to treatment. 3/6 patients with HPV reactivity demonstrated objective tumor responses by RECIST (1 PR and 2 CR). One patient had a 39% best response. Two patients with widespread metastases had complete tumor responses that are ongoing 18 and 11 months after treatment. One patient with a complete response had a chemotherapy-refractory HPV-16+ squamous cell carcinoma and the other a chemoradiation-refractory HPV-18+ adenocarcinoma. Both patients demonstrated prolonged repopulation with HPV-reactive T cells following treatment. Increased frequencies of HPV-specific T cells were detectable after 13 months in one patient and 6 months in the other. Two patients with HPV-reactive TIL that did not respond to treatment did not display repopulation with HPV-reactive T cells. Conclusions: HPV-TIL can mediate durable, complete regression of metastatic cervical cancer. Continued investigation of HPV-TIL for cervical cancer, and possibly other HPV+ malignancies, is warranted. Cellular therapy can mediate complete regression of an epithelial malignancy. Clinical trial information: NCT01585428.

Vaccination with PSA146-154 peptide for advanced prostate cancer: A 10-year follow-up.

e14024 Background: Prostate cancer is the second leading cause of cancer-related death among men in the United States. Although localized disease may be definitively treated with prostatectomy or radiotherapy, advanced metastatic disease can only be managed transiently with androgen-deprivation therapy (ADT) and other systemic therapies. Given the prevalence and lack of curative options for advanced disease, novel treatments warrant investigation. Methods: A study begun in 2002 at UIC examined whether vaccination against PSA146-154 peptide elicited specific T cell immunity in 28 patients with advanced disease. In order to measure immune responses, three distinct readouts were used: induction of responses to DTH skin testing for PSA146-154 peptide, detection of CD8+ PSA146-154 peptide-tetramer +T cells, and via PSA146-154 peptide-induced release of IFN-γ in pre versus post-vaccine peripheral blood mononuclear cell samples. All three assays were completed by 26 and 52 weeks following vaccination. The current study examines the long-term effects of the vaccine ten years post immunization and determines survival differences between immune responders and non-responders. Results: Individuals who demonstrated a DTH response against PSA146-154 peptide have improved survival compared to individuals who did not develop a DTH response against the peptide (80.154 months ± 7.849 v. 72.69 months ± 10.358, p = .12). By contrast, specific responses to the PSA peptide detected by IFN gamma production or tetramer staining did not correlate with improved survival. Analysis of mRNA from peripheral blood mononuclear cells of DTH responders revealed increased expression of genes involved in plasmacytoid dendritic cell function (7 genes, with .5 fold to 4.25 fold increased expression in DTH responders). Conclusions: Together, these findings suggest that vaccination against the PSA146-154 peptide may lead to improved survival in patients who effectively mount a peptide-specific DTH response. Furthermore, the RNA expression profile of responders implicates an antitumor role for plasmacytoid dendritic cells.

Sa1756 Co-Infection With an Intestinal Helminth Exacerbates Salmonella Enterocolitis in Mice

Background and Aim:Murine norovirus (MNV) infection was shown to induce histopathological changes in the intestine of conventional housed mice or lethal systemic infection in immunocompromised mice. The mechanism which triggered the inflammation is not well understood. In the present study, the influence of MNV infection on histological and immunological characteristics of mucosal inflammation in germfree and Schaedler flora colonized IL10-deficient (Il10-/-) mouse model was examined. Methods: Germfree (GF) C57Bl/6J-Il10-/mice and GF mice colonized with Schaedler Flora were monitored after MNV infection for structural and functional intestinal barrier changes using histology, RTqPCR, ELISA and TUNEL assay. Results: No inflammatory lesions were observed in GF B6Il10-/and MNV-infected mice whereas in MNV-infected GF mice colonized with Schaedler Flora inflammatory lesions were detected comparable to SPF mice infected with MNV. Interferon inducible genes were increased in the small intestine of GFmice 48h after infection. However, four weeks after infection GF mice showed no increased IFNgamma production, whereas in Schaedler Flora colonized mice IFNgamma production was increased similar to SPF infected mice. Although there is no reduction of gene expression of tight junction molecules and an enhanced rate of epithelial apoptosis 48h after infection of GF mice the expression level changed within 4 weeks in MNV infected Schaedler Flora. Conclusions: MNV induces inflammation and epithelial barrier disruption that depends on the presence of the bacteria. Thus, MNV and enteric microbiota together represent a potent colitogenic stimulus and inflammatory trigger.

Suppression of B cell responsiveness by LP-BM5 retrovirus-induced myeloid derived suppressor cells generated during a murine acquired immunodeficiency syndrome: a role for negative checkpoint regulator expression on the MDSCs (VIR7P.1059)

Abstract The inhibitory activities of myeloid derived suppressor cells (MDSC) against T cell responses are well established in tumor microenvironments. We published (2013, J. Virol. 87:2058-2071) on the induction of monocytic MDSCs during infection of susceptible B6 mice by LP-BM5 retrovirus, which causes a profound immunodeficiency. These MDSCs inhibited not only T, but also B cell, responsiveness to polyclonal stimulation in ex vivo suppression assays. Whereas MDSC inhibition of stimulated T cell proliferation and IFN-gamma production was largely iNOS/NO dependent, MDSC suppression of B cell responses was only partially (~50%) due to iNOS/NO - as shown by using iNOS inhibitors and LP-BM5 infected iNOS k.o. mice as a source of MDSCs. Here, we further study additional suppressive mechanism(s) in the MDSC inhibition of B cell responsiveness. Using MDSCs from LP-BM5 infected mice of negative checkpoint regulator knock-out origin, and specific blocking antibody, MDSC suppression of B cell responses was partly dependent on MDSC expression of certain checkpoint regulators. Combining the use of reagents to interrupt both iNOS/NO and checkpoint regulation lead to a synergistic blocking of MDSC suppression of B cell responsiveness. These results were compatible with a role for MDSC in LP-BM5 induced immunodeficiency and highlight the involvement of multiple and unique suppressive pathways in the under-studied area of MDSC suppression of B cell responses.

Induction of memory immunity to Mycobacterium tuberculosis is independent of Toll-like receptor 9 signaling (IRC9P.706)

Abstract Mycobacterium tuberculosis (Mtb) is known to engage Toll-like receptors (TLR) 2 and 9 on macrophages and dendritic cells to initiate an inflammatory response. Nonetheless, studies in the murine model of tuberculosis revealed minimal involvement of TLR2 and TLR9 in host resistance during acute infection. We have published that the induction of memory immunity to Mtb is not compromised in the absence of TLR2. Given that the inclusion of TLR9 ligands in vaccine preparations enhances their efficacy, we hypothesized that TLR9 adjuvanticity is necessary for the induction of memory immunity to Mtb. Mice were immunized with an Mtb auxotroph. Upon challenge with Mtb, WT and TLR9-/- immunized mice demonstrated similarly enhanced control of bacterial burden in the lungs compared to their unimmunized counterparts. The granulomatous response, IFN-gamma production and the frequency of immune cells recruited to the lungs was similar in the two genotypes. These findings indicate that the absence of TLR9 signaling during priming does not affect T cell memory response to Mtb infection. Together, our studies point that induction of effector and memory T cell responses and resistance to acute Mtb infection are independent of TLR2 and TLR9. In a recent study we showed that TLR2 signaling, although nonessential in acute infection, was critical to host resistance during chronic Mtb infection. In ongoing studies we are investigating whether TLR9, like TLR2, has a role in chronic Mtb infection.

Sa1758 NLRP3 Inflammasome Is Activated in Mice by Helicobacter pylori Infection

Background and Aim:Murine norovirus (MNV) infection was shown to induce histopathological changes in the intestine of conventional housed mice or lethal systemic infection in immunocompromised mice. The mechanism which triggered the inflammation is not well understood. In the present study, the influence of MNV infection on histological and immunological characteristics of mucosal inflammation in germfree and Schaedler flora colonized IL10-deficient (Il10-/-) mouse model was examined. Methods: Germfree (GF) C57Bl/6J-Il10-/mice and GF mice colonized with Schaedler Flora were monitored after MNV infection for structural and functional intestinal barrier changes using histology, RTqPCR, ELISA and TUNEL assay. Results: No inflammatory lesions were observed in GF B6Il10-/and MNV-infected mice whereas in MNV-infected GF mice colonized with Schaedler Flora inflammatory lesions were detected comparable to SPF mice infected with MNV. Interferon inducible genes were increased in the small intestine of GFmice 48h after infection. However, four weeks after infection GF mice showed no increased IFNgamma production, whereas in Schaedler Flora colonized mice IFNgamma production was increased similar to SPF infected mice. Although there is no reduction of gene expression of tight junction molecules and an enhanced rate of epithelial apoptosis 48h after infection of GF mice the expression level changed within 4 weeks in MNV infected Schaedler Flora. Conclusions: MNV induces inflammation and epithelial barrier disruption that depends on the presence of the bacteria. Thus, MNV and enteric microbiota together represent a potent colitogenic stimulus and inflammatory trigger.

Exosomes in human semen impair antigen-presenting cell function and decrease antigen-specific T cell responses (MUC7P.767)

Abstract Exposure to semen is the primary route of transmission for many sexually transmitted infections. Mounting evidence suggests that components in semen directly influence leukocytes, which could help to explain the difficulty of inducing vaccination-based protection in the genital mucosa. Exosomes, small microvesicles with demonstrated immunomodulatory functions, are present at an average concentration of 2.2 x 1013 particles per ejaculate (n = 18 healthy semen donors). These seminal exosomes (SE) efficiently and rapidly entered peripheral and vaginal dendritic cells (DCs), whereas T cell uptake of SE was poor. SE impaired the immune responses of DCs stimulated by bacterial lipopolysaccharide, where cytokine production was reduced at both the mRNA level in the cells and the protein level in supernatants. Preliminary experiments also indicated that SE down-regulate CD80 and CD83 expression on DCs. In PBMC cultures, SE impaired memory T cell function, reducing the production of TNF-alpha and/or IFN-gamma in response to virus-derived peptides an average of 73% for CD4+ T cells and 55% for CD8+ T cells, in a dose-responsive manner (n = 4 PBMC donors). Thus, SE likely inhibit antigen-specific T cell responses by affecting the co-stimulatory capacity of antigen-presenting cells. Understanding how programmed immune responses are altered by the presence of semen is important to developing the next generation of vaccine and preventative treatments against sexually transmitted disease.

Liver sulfatide-reactive type II NKT cells recognize endogenous phospholipids. (INC9P.445)

Abstract NKT cells are T cells recognizing lipids presented by CD1d. Type I and II subsets recognize distinct antigens. Type I NKT cells recognize alpha-galactosylceramide (aGC). aGC-loaded CD1d tetramers are widely used to study them. Type II NKT cells and their antigens are much less well characterized than type I due to lack of widely available tools to study them. Sulfatide (3-o-sulfo-beta-D-galactosylceramide), an endogenous lipid, is the only lipid proven to be recognized by type II NKT cells in vivo. Recently, phosphatidylglycerol (PG) and phosphatidylinositol (PI) were reported to be recognized by type II NKT cell hybridomas. Here, PG-or PI-loaded CD1d-tetramer-reactive cells were detected in liver mononuclear cells as a distinct population from aGC-loaded CD1d-tetramer-reactive type I NKT cells, consistent with type II NKT cells. Simultaneous staining with PG-, PI-, and sulfatide-loaded CD1d-tetramers indicated, surprisingly, that these three populations were largely identical, even though the phospholipids have structures quite distinct from that of sulfatide. IFN-gamma and IL-4 production by spleen cells was detected after stimulation with sulfatide, but not with PG or PI. These findings suggest that although PG and PI, when loaded onto CD1d, bind the same cells as do sulfatide-CD1d tetramers, their ability to induce cytokine production was distinct. Whether these phospholipids are weak agonists or antagonists or induce other functional activities is under investigation.

Defining the role of the alarmin IL-33 during the CD8 T cell effector response (VAC3P.958)

Abstract ST2, the receptor for the alarmin IL-33, is expressed on CD8 T cells and both IL-33 and ST2 can optimize anti-viral CD8 T cell immune responses. Further, signaling through ST2 in combination with innate-derived cytokines such as IL-12 can induce TCR-independent production of IFN-gamma. We sought to understand the role of the IL-33/ST2 pathway during the effector CD8 T cell response elicited after immunization with CD134 and CD137 costimulatory agonists. ST2 is rapidly up-regulated on CD27+ effector CD8 T cells during the earliest phase of T cell re-activation, even before the up-regulation of other well-known T cell activation markers. Also, CD27+ST2+ CD8 T cells expressed increased cytokines compared to CD27-veST2+ CD8 T cells, and blockade of ST2 impeded the expression of those cytokines. Moreover, CD27+ST2+ CD8 T cells can kill EL4 tumor targets. Thus, ST2 seems to be marking effector CD8 T cells with the best capacity to exert effector functions. Upon TCR engagement, viable effector CD8 T cells rapidly up-regulate ST2, which facilitates the delivery of additional activation signals through IL-33 that further augment T cell activation. Based on these findings, we hypothesize that IL-33 may be one of the first signals that link T cell activation with the local stromal environment. Thus, direct targeting of the more highly functional ST2+ CD8 T cells may improve the outcome of T cell-based cancer immunotherapies.

Activation-specific metabolic regulation of NK cell IFN-gamma production (INC6P.334)

Abstract Many factors that stimulate NK cell functional responses have been described; however metabolic fuels that drive NK cell function are unknown. Metabolism is significantly altered in disease states and metabolic regulation of T cell function is well-described. We hypothesize that NK cells require distinct metabolic fuels for survival, proliferation, and functional responses. NK cell metabolic phenotype was determined with an extracellular flux analyzer to determine basal oxidative phosphorylation (oxphos) and glycolysis of resting and activated NK cells. NK cells were then cultured under a variety of conditions using inhibitors and nutrient deprivation to determine cellular requirements for glucose, fatty acids, and oxphos for NK cell survival, proliferation, and function. NK cells primarily utilize oxphos at baseline and, in contrast to T cells, upon proliferation. However, there is significant plasticity in the metabolic requirements for NK cell IFN-gamma production. NK cell IFN-gamma production in response to cytokine stimulation is not dependent on either glycolysis or oxphos. By contrast, NK cell IFN-gamma production in response to receptor stimulation requires oxphos and is partially dependent on glycolysis. The metabolic requirements for NK cell IFN-gamma production are not altered by priming in vitro or in vivo, but do change with proliferation. These findings suggest that the metabolic microenvironment in vivo may significantly alter NK cell function.

ATF4 is induced by ROS and regulates Th1 and Th17 responses (IRM6P.718)

Abstract Reactive oxygen species (ROS) play a major role in the pathogenesis of chronic inflammatory and autoimmune diseases. Little is known about underlying molecular mechanisms regulating T cell immune responses to ROS. Here, we demonstrated that ROS induced ATF4 in activated T cells. We found, in the high ROS condition, ATF4-/- T cells failed to proliferate and produced much less IFN-γ when cultured in the Th1 polarization condition. Inhibition of ROS by addition of antioxidants significantly recovered proliferation and IFN-γ production by ATF4-/- CD4 T cells in the Th1 condition, suggesting a crucial role of ATF4 in preventing oxidative stress during T cell activation and Th1 differentiation. In addition, we demonstrated that ATF4-/- mice develop exacerbated experimental autoimmune encephalomyelitis (EAE), which is associated with elevated levels of IL17 but reduced IFN-gamma production. The in-depth molecular mechanisms are further studied and the result will be presented. Our study establishes that the ROS/ATF4 axis promotes Th1 and suppresses Th17 immune responses and likely plays an important role in autoimmunity and cancer immunology.