Levels Of IFN-γ Production Research Articles

VEGF-R2 and TNF-R1 expression and cytokine production by samples of mammary adenocarcinomas and correlations with histopathological parameters of these malignant tumors.

Currently, the role of cytokines in the tumor progression, including breast cancer, is universally recognized. At the same time, there are still many questions concerning the role of individual cytokines and receptors for cytokines in various morphogenetic processes underlying the tumor progression. The objective of this work was to study cytokine production and vascular endothelial growth factor (VEGF)-R2 and VEGF-R1 expression by mammary adenocarcinoma (MAC) and the correlations with histopathological parameters of malignant tumors. The object of the study was cultured tumor biopsy samples from 47 women aged 43–75 years with invasive ductal carcinoma, which was classified as grade II–III adenocarcinoma. It was shown that the cytokine profiles of the supernatants of MAC samples from patients differ greatly. A correlation between the levels of VEGF-R2 and tumor necrosis factor (TNF)-R1 expression was observed. Correlations were also revealed during analysis of the relations of histopathological MAC indicators with KVEGF-R2/VEGF-A and KTNF-R1/TNF-α coefficients, which are equal, respectively, to the ratio of expression values of receptors VEGF-R2 and TNF-R1 to the concentrations of the relevant cytokines (VEGF-A and TNF-α) in the culture supernatants of the same MAC samples. A direct correlation was identified between KVEGF-R/VEGF-A and some histopathological MAC characteristics: proportion of cells undergoing mitosis or pathological mitosis in MAC and poorly differentiated cells. KVEGF-R2/VEGF-A directly correlated with the concentration in supernatant interleukin (IL)-18 and interferon (IFN)-γ. KTNF-R1/TNF-α was inversely correlated with the concentration in supernatant of IL-1Ra, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF). The data obtained show that the high-level production of IL-18 and IL-1β by MAC, overexpression of VEGF-R2 in tumor (at relatively low VEGF-A production), and the high level of IFN-γ production are attributed factors contributing to the formation of a population of low-grade cells in the tumor. The factors regulating the population of moderately differentiated cells in the tumor are referred to as IL-1Ra, IL-8, and GM-CSF.

Downmodulation of Effector Functions in NK Cells upon Toxoplasma gondii Infection.

The obligate intracellular parasite Toxoplasma gondii can actively infect any nucleated cell type, including cells from the immune system. The rapid transfer of T. gondii from infected dendritic cells to effector natural killer (NK) cells may contribute to the parasite's sequestration and shielding from immune recognition shortly after infection. However, subversion of NK cell functions, such as cytotoxicity or production of proinflammatory cytokines, such as gamma interferon (IFN-γ), upon parasite infection might also be beneficial to the parasite. In the present study, we investigated the effects of T. gondii infection on NK cells. In vitro, infected NK cells were found to be poor at killing target cells and had reduced levels of IFN-γ production. This could be attributed in part to the inability of infected cells to form conjugates with their target cells. However, even upon NK1.1 cross-linking of NK cells, the infected NK cells also exhibited poor degranulation and IFN-γ production. Similarly, NK cells infected in vivo were also poor at killing target cells and producing IFN-γ. Increased levels of transforming growth factor β production, as well as increased levels of expression of SHP-1 in the cytosol of infected NK cells upon infection, were observed in infected NK cells. However, the phosphorylation of STAT4 was not altered in infected NK cells, suggesting that transcriptional regulation mediates the reduced IFN-γ production, which was confirmed by quantitative PCR. These data suggest that infection of NK cells by T. gondii impairs NK cell recognition of target cells and cytokine release, two mechanisms that independently could enhance T. gondii survival.

Abstract 1699: Impact of whole blood interferon gamma production for patient selection in vaccination with autologous formalin-fixed tumor vaccine

Abstract Introduction: Immune checkpoint blockade by anti-CTLA-4 antibody and anti-PD-1 antibody led to clinical breakthrough for the treatment of patients with solid tumors. Moreover, recent reports have shown that neoantigens are important functional targets mediating response to immune checkpoint inhibitors. Therefore, sequential combination of personalized cancer vaccines and immune checkpoint blockade has emerged as a potential therapeutic strategy. However, existing drawbacks is that it is difficult to identify robust biomarkers for patient selection in vaccination with cancer vaccine. In this study, we examined whether the development of delayed-type hypersensitivity (DTH) response, measured after vaccination with autologous formalin-fixed tumor vaccine (AFTV), correlates to parameters of immune function such as whole blood cytokine levels and peripheral regulatory T cells (Treg) in patients with solid tumors. Methods: Subjects comprised 52 patients with solid malignancy. AFTV treatment consisted of 3 courses of vaccination performed at intervals of 2 weeks. DTH test was performed before and after vaccination. A positive DTH response was defined as a ≥ 10mm diameter erythema/induration. We evaluated whole blood cytokine production after phytohemagglutinin (PHA) stimulation using the bioplex array system. We also assessed the number of peripheral Tregs as well as routine hematological and biochemical parameters. Results: DTH positive patients and DTH negative patients did not differ with respect to the number of peripheral Tregs, leukocytes, lymphocytes or granulocyte/lymphocyte ratio. Whole blood IFN-γ production levels after phytohemagglutinin (PHA) stimulation at baseline in DTH positive patients were significantly higher than those in DTH negative patients (p=0.0397). Conclusion: These data indicate that whole blood IFN-γ production at baseline is likely to be useful to assess immunologic competence in vaccinated patients with AFTV. Consequently, clinical utility of whole blood IFN-γ production, as immunomonitoring method for patient selection in vaccination with cancer vaccine, is required to investigate in larger clinical studies in the future. Citation Format: Naoyuki Sakamoto, Takeshi Ishikawa, Tetsuya Okayama, Tomoyo Yasuda, Toshifumi Doi, Mari Tanigawa, Yuji Naito, Yoshito Itoh, Toshikazu Yoshikawa. Impact of whole blood interferon gamma production for patient selection in vaccination with autologous formalin-fixed tumor vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1699. doi:10.1158/1538-7445.AM2017-1699

IL-18/IL-15/IL-12 synergy induces elevated and prolonged IFN-γ production by ex vivo expanded NK cells which is not due to enhanced STAT4 activation

The synergistic effect of IL-18/IL-15/IL-12 stimulation potently activates NK cells, inducing high levels of IFN-γ production. As a result of this potent stimulatory effect, NK cell pre-activation with IL-18/IL-15/IL-12 is being developed as a cancer immunotherapy. Ex vivo expansion of NK cells enables the efficient generation of large numbers of NK cells for wide-scale and repeated therapeutic use, and is thus an important source of NK cells for clinical application. However, the effects of IL-18/IL-15/IL-12 stimulation on ex vivo expanded NK cells have not yet been assessed. Thus, the present study assessed the effects of IL-18/IL-15/IL-12 stimulation on NK cells expanded ex vivo using K562-based artificial antigen presenting cells expressing membrane-bound IL-21. We report that ex vivo expanded NK cells stimulated with IL-18/IL-15/IL-12 produce high levels of IFN-γ and TNFα, have potent cytotoxicity, and maintain prolonged IFN-γ production following removal of stimulation. IL-18/IL-15/IL-12 stimulation induces a phenotypically unique IFN-γ-producing population with reduced CD16 expression and greater CD25 expression as compared to stimulated IFN-γ- NK cells and unstimulated NK cells. We elucidate that the mechanism of synergy for induction and maintenance of IFN-γ production is not due to a further enhancement of STAT4 activation compared to stimulation with IL-12 alone. Furthermore, we demonstrate that the synergistic increase in IFN-γ is not solely under translational regulation, as elevated levels of IFN-γ mRNA contribute to the synergistic increase in IFN-γ. Overall, this study characterizes the response of ex vivo expanded NK cells to IL-18/IL-15/IL-12 stimulation and supports the use of ex vivo expanded NK cells as a feasible and efficient source of IL-18/IL-15/IL-12 pre-activated NK cells for adoptive transfer in cancer immunotherapies.

Hypervirulent Mycobacterium tuberculosis strain triggers necrotic lung pathology associated with enhanced recruitment of neutrophils in resistant C57BL/6 mice.

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) that in most cases induces irreversible necrosis of lung tissue as a result of excessive inflammatory reactions. The murine model of TB in resistant C57BL/6 mice infected with reference Mtb strains is widely used in TB studies; however, these mice do not show a necrotic pathology, which restricts their use in studies of irreversible tissue damage. Recently, we demonstrated that necrotic lung lesions could be induced in the C57BL/6 mice by highly virulent Mtb strains belonging to the modern Beijing sublineage. However, the pathogenic mechanisms leading to necrosis in this model were not elucidated. In this study, we investigated the dynamics of lung lesions in mice infected with highly virulent Beijing Mtb strain M299, compared with those infected with laboratory Mtb strain H37Rv. The data demonstrate that necrotic lung lesions in mice infected by the strain M299 were associated with enhanced recruitment of myeloid cells, especially neutrophils, and increased levels of proinflammatory cytokines, consistent with exacerbated inflammation. High levels of IFN-γ production contributed to the control of bacterial growth. Further progression to chronic disease was associated with a reduction in the levels of inflammatory mediators in the lungs, the accumulation of foamy macrophages and partial healing of the necrotic tissue by fibrosis. At a late stage of disease, degradation of foamy cells resulted in the liberation of accumulated lipids and persisting bacilli and further activation of inflammation, which promoted lung consolidation. Overall, our studies show that C57BL/6 mice infected with highly virulent Mtb strain may serve as a TB model reproducing an exacerbated inflammatory response in a resistant host to hypervirulent mycobacteria, leading to irreversible necrotic lung lesions.

Regulation of Gastric Carcinogenesis by InflammatoryCytokines.

Chronic inflammation caused by infection with Helicobacter pylori and autoimmune gastritis increases an individual’s risk of developing gastric cancer. More than 90% of gastric cancers are adenocarcinomas, which originate from epithelial cells in the chronically inflamed gastric mucosa. However, only a small subset of chronic gastritis patients develops gastric cancer, implying a role for genetic and environmental factors in cancer development. A number of DNA polymorphisms that increase gastric cancer risk have mapped to genes encoding cytokines. Many different cytokines secreted by immune cells and epithelial cells during chronic gastritis have been identified, but a better understanding of how cytokines regulate the severity of gastritis, epithelial cell changes, and neoplastic transformation is needed. This review summarizes studies in both human and mouse models, describing a number of different findings that implicate various cytokines in regulating the development of gastric cancer.

Anti-inflammatory effect and mechanisms of Huangqi glycoprotein in treating experimental autoimmune encephalomyelitis

<i>Huangqi glycoprotein (HQGP) is prepared from Astragalus membranaceus by ammonium sulfate precipitation. It was indicated that HQGP has an immunoregulatory effect. In this study, we established a chronic experimental autoimmune encephalomyelitis (EAE) model and observed the therapeutic effect and possible mechanisms of HQGP (intraperitoneally at 1 mg/kg/day) on EAE. The results showed that HQGP delayed onset and ameliorated severity of EAE, and reduced the infiltration and accumulation of pathogenic T cells in the central nerves system (CNS). HQGP also reduced the production of IL-6, IL-17 and TNF-α and increased the level of IL-10. However, the level of IFN-γ production was also increased in HQGP-treated mice compared with EAE control mice. In brain, chemokines such as CCL2 and CCL5 were inhibited in HQGP-treated EAE compared with control mice. These results demonstrate that HQGP alleviates the pathogenesis of EAE possibly by suppressing the neuroinflammation and decreasing the secretion of chemokines and cell adhesion.</i>.

Decoction of Dictamnus Dasycarpus Turcz. Root Bark Ameliorates Skin Lesions and Inhibits Inflammatory Reactions in Mice with Contact Dermatitis.

Background:The root bark of Dictamnus dasycarpus Turcz. (Dictamni Radicis Cortex) has been widely used to treat skin diseases in Korea, and its anti-inflammatory efficacies were recently reported.Objective:The paper aims to investigate the inhibitory effects of decoction of Dictamni Radicis Cortex (DDRC) in mice with contact dermatitis (CD).Materials and Methods:We investigated the effects of DDRC on skin lesion characteristics such as crust, scales, incrustation and petechiae, the erythema and melanin indexes, skin thickness, histopathologic changes, and cytokine production in 1-fluoro-2,4-dinitrofluorobenzene (DNFB)-induced CD mice.Results:Topical application of DDRC ameliorated crust, scales, incrustation, and induced by DNFB. In addition, DDRC lowered the erythema index significantly (P < 0.05). DDRC effectively inhibited enlargement of skin thickness (P < 0.05). Histopathologic observation showed that DDRC inhibited epidermal hyperplasia, hyperkeratosis, and spongiotic changes. Finally, DDRC decreased production levels of IFN-γ, TNF-α and IL-6 induced by repeated application of DNFB (P < 0.05).Conclusion:These data suggest that DDRC can be used in the treatment of inflammatory skin diseases including CD. Moreover, these results are closely related to the decreasing production of TNF-α IFN-γ and IL-6 in inflamed tissues.SUMMARY DDRC ameliorated skin lesions such as crust, scales, incrustation and petechiae, and lowered erythema index on skin surface in CD miceDDRC inhibited enlargement of dorsal skin and prevented epidermal hyperplasia, hyperkeratosis, and spongiotic changes in inflamed tissuesDDRC reduced the levels of TNF-α, IFN-γ, and IL-6 in inflamed tissues of CD miceDDRC did not affect spleen/body weight ratio in CD mice. Abbreviations used: DDRC: decoction of Dictamni Radicis Cortex, CD: contact dermatitis, DNFB: 1-fluoro-2,4-dinitrofluorobenzene, AOO: acetone and olive oil, DEX: dexamethasone, CBA: cytometric bead array

Prevalence of Latent Tuberculosis Infection (LTBI) among House Hold Contacts of Sudanese Patients with Pulmonary Tuberculosis in Eastern Sudan: Revisiting the Tuberculin Skin Test

A third of the world’s population is latently infected with TB with an increased risk of developing active TB. Household contacts (HHCs) of pulmonary TB cases are at a greater risk of developing disease. Early identification and treatment of latent TB infected individuals may reduce progression to active TB. This study aimed to determine latent TB infections (LTBI) point prevalence among HHCs and community contacts (CCs) using Tuberculin Skin Test (TST) and whole blood IFN-γ release assay in an area of high TB prevalence. In a prospective, longitudinal and community-based study and following informed consent, 768 volunteers (HHCs n = 245; CCs n = 523) were enrolled. Tuberculin Skin Test (TST), whole blood stimulation with PPD and IFN-γ levels determination using ELISA were performed. Mean ages of HHCs and the CCs were not significantly different (HHCs 35.6 ± 15.7 and CCs 30.6 ± 11.7 years; p = 0.99), with a Male:Female ratio of 1:2. Mean recruitment TST indurations were 4.6 ± 5.5 mm and 2.8 ± 3 mm for HHCs and CCs respectively (p = 0.000). Follow-up (Day 614) mean TST indurations increased significantly to 9.1 ± 7.2 mm and 4.4 ± 3.2 mm for HHCs and CCs respectively (p = 0.001). Using TST indurations ≥ 10 mm, LTBI point prevalence for HHCs and CCs was significantly different (HHCs 461/1000 and 367/1000 individuals, p = 0.03). The mean IFN-γ levels for HHCs and CCs at recruitment day (Day 0) were 0.66 ± 0.17 IU/ml and 0.06 ± 0.04 respectively. The mean of IFN-γ production levels dropped significantly at Day 614 for HHCs and CCs to 0.66 ± 0.15 IU/ml and 0.02 ± 0.02 respectively (p = 0.03) (p = 0.00001). Recruitment LTBI point prevalence using IFN-γ level ≥ 0.35 IU/ml for HHCs and CCs was 440/1000 and 203/1000 respectively (p = 0.000000001). No correlations between TST indurations and IFN-γ levels were detected among HHCs or CCs (p > 0.05). TST is a simple, efficient and cheap technique for LTBI diagnosis and triaging individuals for treatment.

Mass Cytometry As a Modality to Identify Candidates for Immune Checkpoint Inhibitor Therapy within Acute Myeloid Leukemia

Introduction: Immune checkpoint inhibitors (ICIs) have shown significant efficacy in solid malignancies and recently in Hodgkin's disease. However, there are presently no biomarkers that predict which cancer types and/or individual patients will benefit from ICIs. Despite intensification of chemotherapeutic regimens, survival rates in acute myeloid leukemia (AML) have plateaued. We are therefore, performing studies to characterize the immune phenotype and genetic profile of AML patients who may respond to ICI therapy. Specifically, we are identifying cell subsets present in the marrow of patients with AML, evaluating their differentiation and activation status, and quantifying the density of immune modifiers. We are also performing functional assays to define the status of T cells in the AML microenvironment by evaluating the responsiveness of these cells to stimulation. Additionally, these studies assess the impact of several ICIs on the proliferative potential and cytokine production capacity of T cells resident in the bone marrow of AML patients.Methods: Immunophenotyping was performed on the diagnostic bone marrow (Dx BM), diagnostic peripheral blood (Dx PB), and remission bone marrow (CR BM) for a single patient with AML. These studies used a time-of-flight mass cytometer (CyTOF) by staining samples with a panel containing 35 antibodies to surface markers and cytokines (Table 1). T cells were defined and then differentiated based on the presence of CD3 and CD4 or CD8. T cells were further classified into naive, central memory (CM), effector memory (EM), and effector cells. Functional assays measured proliferative capacity and cytokine production profile of bone marrow T cells in response to CD3 stimulation for the Dx BM and Dx PB. Changes to the baseline T cell functional profile in the presence of antibodies against PD1, CTLA4, TIM3, and TIGIT were measured in the Dx BM.Results: T cell percentages were 3.14%, 5.46%, and 17.1% for the Dx BM, Dx, PB, and CR BM, respectively. CD4:CD8 ratios were 3.5, 2.9, and 4.5, respectively. EM cells made up the largest percentage of CD8+ T cells in the Dx BM (40.99%), Dx PB (57.06%), and CR BM (53.55%). Naïve (52.49%), EM (37.63%) and CM (44.3%) cells made up the largest percentages of CD4+ T cells in the Dx BM, Dx PB, and CR BM, respectively (Figure 1). Checkpoint expression was absent (median intensity < 1) on all T cells except for some CD8+ subsets (Figure 2). Within the Dx BM, the effector (1.93) and EM (4.34) cells expressed TIGIT and the EM cells expressed PD1 (1.92). Within the Dx PB, the effector (1.81) and EM (4.0) cells expressed TIGIT. Within the CR BM, the effector cells (2.3) expressed TIGIT. In response to CD3 stimulation, 57.5% of the Dx PB T cells divided while there was no proliferation observed in the Dx BM. In response to CD3 stimulation and PD1 blockade, 28.2% of the Dx BM T cells divided and there was a 4-fold increase in IFNγ production. There was no division in the Dx BM T cells in response to TIGIT blockade but there was a 10-fold and 121-fold increase in IL-2 and IFNγ production, respectively. There was no significant proliferation or cytokine production in response to TIM3 or CTLA4 blockade.Conclusion: We observed differential T cell responses to stimulation in the presence of ICIs in this patient. The Dx PB T cells were capable of proliferating in the presence of CD3 stimulation alone whereas the Dx BM T cells were only able to divide in the presence of PD1 blocking antibody. PD1 blockade elicited proliferation and low levels of IFNγ production whereas TIGIT blockade elicited negligible proliferation but high levels of IFNγ production. There was low but differential checkpoint expression on T cells, with PD1 and TIGIT having the highest expression, specifically on CD8+ T cell subsets. The predominant CD8+ T cell subset, along with differential checkpoint expression, persisted into the CR BM but there was a shift in the predominant CD4+ T cell subset. These data suggest the presence of immune suppression in the bone marrow microenvironment that may be amenable to checkpoint inhibition. [Display omitted] [Display omitted] [Display omitted] DisclosuresHuang:Janssen Research & Development, LLC: Employment, Equity Ownership. Sasser:Janssen Research & Development, LLC: Employment. Adams:Janssen Research & Development, LLC: Employment. Druker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy. Lind:Janssen: Research Funding; Fluidigm: Honoraria.

Cytokine profiles in Opisthorchis viverrini stimulated peripheral blood mononuclear cells from cholangiocarcinoma patients

The carcinogenic liver fluke Opisthorchis viverrini causes chronic inflammation in the bile duct and resulting in unremitting tissue damage that lead to hepatobiliary diseases, including cholangiocarcinoma (CCA). Despite inflammatory cytokine expression having been studied in the animal model, so far no studies have been carried out on cytokines in human CCA cases. Here we report the profile of cytokine production in peripheral blood mononuclear cells (PBMCs) collected from O. viverrini-associated human CCA and uninfected normal controls after stimulation with O. viverrini-excretory secretory (ES) product. Eleven cytokine profiles including IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNF-α and LT-α measured by flow cytometry revealed both pro-inflammatory and anti-inflammatory cytokines were increased in the O. viverrini-associated CCA compared to uninfected normal controls. Specifically, net production levels of IFN-γ, IL-10, and LT-α were 40 to >320 times higher in CCA than those of controls. These results suggest dysregulation of the immune response in the liver fluke associated CCA.

Cysteine mutagenesis improves the production without abrogating antigenicity of a recombinant protein vaccine candidate for human chagas disease

ABSTRACTA therapeutic vaccine for human Chagas disease is under development by the Sabin Vaccine Institute Product Development Partnership. The aim of the vaccine is to significantly reduce the parasite burden of Trypanosoma cruzi in humans, either as a standalone product or in combination with conventional chemotherapy. Vaccination of mice with Tc24 formulated with monophosphoryl-lipid A (MPLA) adjuvant results in a Th1 skewed immune response with elevated IgG2a and IFNγ levels and a statistically significant decrease in parasitemia following T. cruzi challenge. Tc24 was therefore selected for scale-up and further evaluation. During scale up and downstream process development, significant protein aggregation was observed due to intermolecular disulfide bond formation. To prevent protein aggregation, cysteine codons were replaced with serine codons which resulted in the production of a non-aggregated and soluble recombinant protein, Tc24-C4. No changes to the secondary structure of the modified molecule were detected by circular dichroism. Immunization of mice with wild-type Tc24 or Tc24-C4, formulated with E6020 (TLR4 agonist analog to MPLA) emulsified in a squalene-oil-in-water emulsion, resulted in IgG2a and antigen specific IFNγ production levels from splenocytes that were not significantly different, indicating that eliminating putative intermolecular disulfide bonds had no significant impact on the immunogenicity of the molecule. In addition, vaccination with either formulated wild type Tc24 or Tc24-C4 antigen also significantly increased survival and reduced cardiac parasite burden in mice. Investigations are now underway to examine the efficacy of Tc24-C4 formulated with other adjuvants to reduce parasite burden and increase survival in pre-clinical studies.

NK Cell Activation in the Antitumor Response Induced by IFN-α Dendritic Cells Loaded with Apoptotic Cells from Follicular Lymphoma Patients.

Follicular lymphoma (FL) is the most common form of indolent non-Hodgkin lymphoma. This malignancy is considered virtually incurable, with high response rates to therapy but frequent relapses. We investigated the ability of monocyte-derived dendritic cells generated in the presence of IFN-α and GM-CSF (IFN-DC) and loaded with apoptotic lymphoma cells to activate immune responses against FL cells, with the ultimate goal of designing novel patient-specific vaccination strategies for the treatment of FL. In this article, we show that apoptotic tumor cell-loaded IFN-DC from FL patients, which were cultured for 2 wk with autologous lymphocytes, led to Th1 response skewing, based on significantly higher levels of IFN-γ production and a remarkable increase in CD8(+) and NK cell frequency, consistent with the detection of enhanced cytotoxic effector function toward autologous FL cells. IFN-DC were found to promote efficient NK cell activation, increased expression of cytotoxicity receptors, and extensive IFN-γ production in the virtual absence of IL-10. Moreover, direct recognition and killing of primary autologous lymphoma cells by activated NK cells from FL patients was also demonstrated. A critical role was demonstrated for MHC class I-related chain A and B and membrane-bound IL-15 in IFN-DC-mediated NK cell activation and early IFN-γ production. The overall results indicate that IFN-DC loaded with autologous apoptotic FL cells represent a valuable tool for improving the potency of therapeutic cancer vaccines through the efficient induction of NK cell activation and promotion of CD8(+) T cell antitumor immunity.

Activated dendritic cells delivered in tissue compatible biomatrices induce in-situ anti-tumor CTL responses leading to tumor regression.

Dendritic cell (DC) based anti-cancer immunotherapy is well tolerated in patients with advanced cancers. However, the clinical responses seen after adoptive DC therapy have been suboptimal. Several factors including scarce DC numbers in tumors and immunosuppressive tumor microenvironments contribute to the inefficacy of DCs as cellular vaccines. Hence DC based vaccines can benefit from novel methods of cell delivery that would prevent the direct exposure of immune cells to suppressive tumor microenvironments. Here we evaluated the ability of DCs harbored in biocompatible scaffolds (referred to as biomatrix entrapped DCs; beDCs) in activating specific anti-tumor immune responses against primary and post-surgery secondary tumors. Using a preclinical cervical cancer and a melanoma model in mice, we show that single treatment of primary and post-surgery secondary tumors using beDCs resulted in significant tumor growth retardation while multiple inoculations were required to achieve a significant anti-tumor effect when DCs were given in free form. Additionally, we found that, compared to the tumor specific E6/E7 peptide vaccine, total tumor lysate induced higher expression of CD80 and CD40 on DCs that induced increased levels of IFNγ production upon interaction with host lymphocytes. Remarkably, a strong immunocyte infiltration into the host-implanted DC-scaffold was observed. Importantly, the host-implanted beDCs induced the anti-tumor immune responses in the absence of any stromal cell support, and the biomatrix structure was eventually absorbed into the surrounding host tissue. Collectively, these data indicate that the scaffold-based DC delivery may provide an efficient and safe way of delivering cell-based vaccines for treatment of primary and post-surgery secondary tumors.

CXCL16-positive dendritic cells enhance invariant natural killer T cell-dependent IFNγ production and tumor control

ABSTRACTCrosstalk interactions between dendritic cells (DCs) and invariant natural killer T (iNKT) cells are important in regulating antitumor responses elicited by glycolipid antigens. iNKT cells constitutively express the chemokine receptor CXCR6, while cytokine-activated DCs upregulate the transmembrane chemokine ligand, CXCL16. This study examined the co-stimulatory role of CXCR6/CXCL16 interactions in glycolipid-dependent iNKT cell activation and tumor control. Spleen and liver DCs in wild-type mice, but not iNKT cell deficient (Jα18−/−) mice, transiently upregulated surface CXCL16 following in vivo administration of the glycolipid antigen α-galactosylceramide. Recombinant CXCL16 did not directly induce iNKT cell activation in vitro but enhanced interferon (IFN)-γ production when mouse or human iNKT cells were stimulated with plate-bound anti-CD3. Compared with glycolipid-loaded CXCL16neg DCs, CXCL16hi DCs induced higher levels of IFNγ production in iNKT cell cultures and following adoptive transfer in vivo. The number of IFNγ+ iNKT cells and expansion of T-bet+ iNKT cells were reduced in vivo when CXCL16−/− DCs were used to activate iNKT cells. Enhanced IFNγ production in vivo was not dependent on CXCR6 expression on natural killer (NK) cells. Adoptive transfer of glycolipid-loaded CXCL16hi DCs provided superior protection against tumor metastasis compared to CXCL16neg DC transfers. Similarly, wild-type DCs provided superior protection against metastasis compared with CXCL16−/− DCs. These experiments implicate an important role for CXCR6/CXCL16 interactions in regulating iNKT cell IFNγ production and tumor control. The selective use of CXCL16hi DCs in adoptive transfer immunotherapies may prove useful for enhancing T helper (Th) type 1 responses and clinical outcomes in cancer patients.

Abstract A066: Histone deacetylase 11 is an epigenetic regulator of T-cell pro-inflammatory function and novel target for enhancing T-cell anti-tumor activity

Abstract The efficacy of immunotherapy is dependent on the ability of antigen-specific T-cells to mount a proper anti-tumor response while avoiding a plethora of immunosuppressive mechanisms. Unfortunately, these immunosuppressive mechanisms often overwhelm the T-cell response, resulting in patients failing to respond. Histone deacetylases (HDACs) are a family of epigenetic modifiers named for their ability to remove acetyl groups from lysine residues of histone tails, thereby regulating the chromatin structure and gene expression. Here we report that HDAC11, the most recently discovered HDAC, is a regulator of T-cell pro-inflammatory function. Previous reports have shown HDAC11 expression to be tissue restricted. Consequently, we compared expression levels of HDAC11 using an eGFP reporter mouse, in various immune cells. It was found that T-cells expressed relatively high levels of HDAC11. However, while resting T-cells, both central memory and naïve, had high levels of HDAC11 expression, effector or effector memory T-cells displayed lower levels. Additionally, T-cells experienced rapid down-regulation of HDAC11 upon activation. To expand upon these observations, the function of T-cells from HDAC11KO mice were investigated. Initial experiments revealed no gross changes in the numbers or maturation of T-cells. However, HDAC11KO mice displayed an increased percentage of central memory CD8+ T-cells (p&amp;lt;0.05). Additionally, upon activation these T-cells were more apt to acquire effector status (p&amp;lt;0.001). Upon activation, HDAC11KO T-cells demonstrated a robust increase in IL-2, TNF and IFNγ production (p&amp;lt;0.01), but showed no differences in Th2 or Th17 cytokine production. These T-cells also expressed higher levels of the effector molecules granzyme B and perforin (p&amp;lt;0.01). Moreover, post-activation, CD8+ T-cells lacking HDAC11 also displayed a higher percentage of proliferating cells, and more divisions of proliferating cells (p&amp;lt;0.01). HDAC11KO T-cells displayed higher resistance to proliferative suppression by Tregs and failed to succumb to anergy in a high dose antigen model. Indeed, while OTII T-cells from mice injected with high dose OVA peptide displayed reduced levels of IFNγ production compared to cells from non-injected mice after rechallenge, HDAC11KO/OTII T-cells had an increase in IFNγ production. Mechanistically, T-cells devoid of HDAC11 did not appear to have differences in TCR signaling, but did express higher levels of the transcription factors EOMES and T-bet. Chromatin immunoprecipitation (ChIP) revealed higher basal levels of acetylation of these genes in HDAC11KO mice. ChIP analysis also revealed interactions of HDAC11 at the promoters of both EOMES and T-bet. In a graft vs. host disease (GvHD) model, HDAC11KO T-cells produced a more rapid onset of GvHD, and were able to do so with transfers of cell numbers below that able to induce GvHD using WT T-cells (p&amp;lt;0.01). This GvHD was characterized by greater levels of IFNγ and TNF as well as increased T-cell expansion (p&amp;lt;0.05). In a B16 melanoma model, adoptive transfer of HDAC11KO T-cells resulted in delayed tumor progression (p&amp;lt;0.05) compared to WT T-cells. To begin investigating the clinical potential of these data, the expression levels of HDAC11 were investigated in melanoma tumor sections utilized to grow tumor infiltrating lymphocytes (TIL) for clinical trials of adoptive cell therapy. A reduced level of HDAC11 was found in tumor sections from those patients responding to TIL therapy compared to progressing patients (p&amp;lt;0.01). Collectively, beyond demonstrating HDAC11 as a novel epigenetic regulator of T-cell pro-inflammatory function, these results highlight HDAC11 as a novel target for enhancing the efficacy of immunotherapy. Citation Format: David M. Woods, Andressa L. Sodre, Karrune Woan, Alejandro Villagra, Amod Sarnaik, Jeffrey Weber, Eduardo M. Sotomayor. Histone deacetylase 11 is an epigenetic regulator of T-cell pro-inflammatory function and novel target for enhancing T-cell anti-tumor activity. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A066.

Anti-fibrotic effects of a novel small compound on the regulation of cytokine production in a mouse model of colorectal fibrosis

Intestinal fibrotic stricture is a major complication of inflammatory bowel disease. Despite its clinical importance, anti-fibrotic therapy has not been implemented. Transforming growth factor-β (TGF-β) is considered to be a major factor contributing to tissue fibrosis. We have previously shown that the administration of a small compound, HSc025, which promotes the nuclear translocation of YB-1 as a downstream effector of IFN-γ and antagonizes TGF-β/Smad signaling, improves fibrosis in several murine tissues. In this study, we evaluated the anti-fibrotic effect of HSc025 on colorectal fibrosis in TNBS-induced murine chronic colitis. Daily oral administration of HSc025 (3, 15 and 75 mg/kg) suppressed collagen production and decreased the severity of colorectal fibrosis in a dose-dependent manner. In addition, the local production of TGF-β was decreased after HSc025 treatment, whereas that of IL-13 and TNF-α was not affected. HSc025 administration maintained the level of IFN-γ production, even at a late stage when IFN-γ production was lost without the drug treatment. These results demonstrate that HSc025 could be a therapeutic candidate for intestinal fibrosis in inflammatory bowel disease that acts by altering the local production of cytokines, as well as by directly suppressing collagen production.

NK cell compartment in the peripheral blood and spleen in adult patients with primary immune thrombocytopenia

Immune thrombocytopenic purpura (ITP) is a disease characterized by antibody-mediated platelet destruction. The T- and B-cell subsets have been extensively studied in primary ITP, but the NK cell compartment has been less thoroughly explored. We investigated the NK cell receptor repertoire and the functionality of NK cells in the peripheral blood and spleen in patients with primary ITP. An immunophenotypic analysis of peripheral blood lymphocytes from patients revealed that the numbers of CD19+ B lymphocytes, CD4+ and CD8+ T lymphocytes and CD3−CD56+ NK cells were within the normal range. No major alteration to the expression of distinct inhibitory or activating NK cell receptors was observed. The functionality of NK cells, as evaluated by their ability to degranulate in conditions of natural cytotoxicity or antibody-dependent cell cytotoxicity (ADCC), was preserved in these patients. By contrast, these stimuli induced lower levels of IFNγ production by the NK cells of ITP patients than by those of healthy controls. We then compared the splenic NK cell functions of ITP patients with those of cadaveric heart-beating donors (CHBD) as controls. The splenic NK cells of ITP patients tended to be less efficient in natural cytotoxicity conditions and more efficient in ADCC conditions than control splenic NK cells. Finally, we found that infusions of intravenous immunoglobulin led to the inhibition of NK cell activation through the modulation of the interface between target cells and NK cells.

Abstract 1336: Novel immunotherapeutic activity of JAK and PI3Kδ inhibitors in a model of pancreatic cancer

Abstract Immunotherapeutic agents are emerging as key components of efficacious multi-agent regimens in cancer. The majority of immunotherapeutic agents developed thus far either attempt to stimulate a more productive anti-tumoral immune response or to inhibit key proteins in the immunosuppressive tumoral milieu. In contrast, agents targeting signal transduction molecules have been largely developed for their ability to impact the proliferative potential of tumor cells directly. Notably, the JAK/STAT and PI3Kδ signaling pathways have been shown to contribute not only to tumor cell proliferation and survival but also to play a crucial role in regulating stromal cells, including immune cells, which are recruited to the tumor microenvironment. Activation of these pathways has been shown to result in the recruitment and expansion of predominantly negative regulatory cells such as myeloid derived suppressor cells and regulatory T cells, suggesting that inhibition of JAK/STAT and PI3Kδ signaling may promote antitumor immunity. Therefore, we have examined the immunotherapeutic potential of selective inhibitors of either JAK or PI3Kδ. We demonstrate that inhibitors of either JAK or PI3Kδ block tumor growth as single agents in the immuno-competent syngeneic PAN02 pancreatic model, which is not driven by oncogenic JAK or PI3K signaling. Tumor growth inhibition is not observed in immunocompromised mice, demonstrating that the anti-tumor effects of these agents require an intact immune system. These agents were tested pairwise with each other and in combination with other immune checkpoint modulators, including an anti-PD-L1 antibody and the IDO1 inhibitor INCB24360. The combinations of JAK inhibition with IDO1 inhibition, PI3Kδ inhibition or PD-1/PD-L1 blockade resulted in enhanced efficacy. Mechanistic studies revealed that the combination of JAK and IDO1 inhibition did not alter the number of infiltrating T cells within the tumor, but instead resulted in a more activated phenotype of the infiltrating T cells, leading to higher levels of IFNγ production. In contrast, combination treatment with regimens that included an inhibitor to PI3Kδ led to a marked increase in the numbers of T cell infiltrates, although the cells were not maximally activated. Further studies to understand the complex cellular responses elicited by these inhibitors may provide the mechanistic rationale to explore JAK or PI3Kδ inhibitor-based immunotherapy combinations in the clinic. Citation Format: Holly K. Koblish, Michael Hansbury, Liang-Chuan S. Wang, Gengjie Yang, Taisheng Huang, Chu-Biao Xue, Yun-Long Li, Eddy Yue, Andrew Combs, Wenqing Yao, Reid Huber, Peggy Scherle. Novel immunotherapeutic activity of JAK and PI3Kδ inhibitors in a model of pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1336. doi:10.1158/1538-7445.AM2015-1336

Regulatory Rebound in IL-12-Treated Tumors Is Driven by Uncommitted Peripheral Regulatory T Cells.

IL-12 promotes a rapid reversal of immune suppression in the tumor microenvironment. However, the adjuvant activity of IL-12 is short-lived due to regulatory T cell (Treg) reinfiltration. Quantitative analysis of Treg kinetics in IL-12-treated tumors and tumor-draining lymph nodes revealed a transient loss followed by a rapid 4-fold expansion of tumor Treg between days 3 and 10. Subset-specific analysis demonstrated that the posttreatment rebound was driven by the CD4(+)CD25(+)Foxp3(+) neuropilin-1(low) peripheral Treg (pTreg), resulting in a 3-5-fold increase in the pTreg to CD4(+)CD25(+)Foxp3(+) neuropilin-1(high) thymic Treg ratio by day 10. The expanding pTreg displayed hypermethylation of the CpG islands in Treg-specific demethylated region, CTLA-4 exon 2, and glucocorticoid-induced TNFR exon 5, were phenotypically unstable, and exhibited diminished suppressive function consistent with an uncommitted in vitro-induced Treg-like phenotype. In vitro culture of posttherapy Treg populations under Th1-promoting conditions resulted in higher levels of IFN-γ production by pTreg compared with thymic Treg, confirming their transitional state. Blockade of selected molecular mechanisms that are known to promote Treg expansion identified IDO-positive dendritic cells as the primary mediator of post-IL-12 pTreg expansion. Clinical implications of these findings are discussed.