Immune Response In Murine Model Research Articles

The impact of size for liposomes modified with pH-responsive β-glucan derivatives on the initiation of cellular and humoral immune responses in murine models

Size is one of the important factors for antigen carriers because it influences cellular interaction, pharmacokinetics, immune cell activation and the types of immune responses. We developed pH-sensitive polysaccharide derivative-modified liposomes as antigen carriers for induction of antigen-specific immune responses. For an earlier study, the size of these liposomes was controlled to within 100–200 nm considering the suitable size for endocytosis. However, since most antigen-presenting cells can engulf larger particles, the proper liposome size and its effect on the induction of cellular versus humoral immune responses remains unclear. Here, we examined the effects of polysaccharide derivative-modified liposome size on immune responses. Liposome size was controlled by using an extrusion method by passing liposomes through a polycarbonate membrane of different pore sizes. Small liposomes encapsulating model antigenic protein suppressed antigen-expressing tumour growth effectively and increased the IgG2a/IgG1 ratio early after liposome administration, suggesting that Th1 response was mainly induced. Large liposomes increased antigen-specific IgG1 and IgG2a production for 56 days compared with small liposomes. These results suggest that small liposomes modified with pH-responsive polysaccharide derivatives could be effective for cancer immunotherapy whereas large liposomes could be suitable for induction of antibody production towards vaccination.

Combination of vitamin D and photodynamic therapy enhances immune responses in murine models of squamous cell skin cancer

Improved treatment outcomes for non-melanoma skin cancers can be achieved if Vitamin D (Vit D) is used as a neoadjuvant prior to photodynamic therapy (PDT). However, the mechanisms for this effect are unclear. Vit D elevates protoporphyrin (PpIX) levels within tumor cells, but also exerts immune-modulatory effects. Here, two murine models, UVB-induced actinic keratoses (AK) and human squamous cell carcinoma (A431) xenografts, were used to analyze the time course of local and systemic immune responses after PDT ± Vit D. Fluorescence immunohistochemistry of tissues and flow analysis (FACS) of blood were employed. In tissue, damage-associated molecular patterns (DAMPs) were increased, and infiltration of neutrophils (Ly6G+), macrophages (F4/80+), and dendritic cells (CD11c+) were observed. In most cases, Vit D alone or PDT alone increased cell recruitment, but Vit D + PDT showed even greater recruitment effects. Similarly for T cells, increased infiltration of total (CD3+), cytotoxic (CD8+) and regulatory (FoxP3+) T-cells was observed after Vit D or PDT, but the increase was even greater with the combination. FACS analysis revealed a variety of interesting changes in circulating immune cell levels. In particular, neutrophils decreased in the blood after Vit D, consistent with migration of neutrophils into AK lesions. Levels of cells expressing the PD-1+ checkpoint receptor were reduced in AKs following Vit D, potentially counteracting PD-1+ elevations seen after PDT alone. In summary, Vit D and ALA-PDT, two treatments with individual immunogenic effects, may be advantageous in combination to improve treatment efficacy and management of AK in the dermatology clinic.

Light-enhanced VEGF121/rGel induce immunogenic cell death and increase the antitumor activity of αCTLA4 treatment.

Immune-checkpoint inhibitors (ICIs) represent a revolution in cancer therapy and are currently implemented as standard therapy within several cancer indications. Nevertheless, the treatment is only effective in a subset of patients, and immune-related adverse effects complicate the improved survival. Adjuvant treatments that can improve the efficacy of ICIs are highly warranted, not only to increase the response rate, but also to reduce the therapeutic ICI dosage. Several treatment modalities have been suggested as ICI adjuvants including vascular targeted treatments and photodynamic therapy (PDT). Photochemical internalization (PCI) is a drug delivery system, based on PDT. PCI is long known to generate an immune response in murine models and was recently shown to enhance the cellular immune response of a vaccine in a clinical study. In the present work we evaluated PCI in combination with the vascular targeting toxin VEGF121/rGel with respect to induction of immune-mediated cell death as well as in vitro ICI enhancement. DAMP signaling post VEGF121/rGel-PCI was assessed in CT26 and MC38 murine colon cancer cell lines. Hypericin-PDT, previously indicated as an highly efficient DAMP inducer (but difficult to utilize clinically), was used as a control. ATP release was detected by a bioluminescent kit while HMGB1 and HSP90 relocalization and secretion was detected by fluorescence microscopy and western blotting. VEGF121/rGel-PCI was further investigated as an αCTLA enhancer in CT26 and MC38 tumors by measurement of tumor growth delay. CD8+ Dependent efficacy was evaluated in vivo using a CD8+ antibody. VEGF121/rGel-PCI was shown to induce increased DAMP signaling as compared to PDT and VEGF121/rGel alone and the magnitude was found similar to that induced by Hypericin-PDT. Furthermore, a significant CD8+ dependent enhanced αCTLA-4 treatment effect was observed when VEGF121/rGel-PCI was used as an adjuvant in both tumor models. VEGF121/rGel-PCI describes a novel concept for ICI enhancement which induces a rapid CD8+ dependent tumor eradication in both CT26 and MC38 tumors. The concept is based on the combination of intracellular ROS generation and vascular targeting using a plant derived toxin and will be developed towards clinical utilization.

Natural Products Induce Different Anti-Tumor Immune Responses in Murine Models of 4T1 Mammary Carcinoma and B16-F10 Melanoma

Natural products obtained from Petiveria alliacea (Anamu-SC) and Caesalpinia spinosa (P2Et) have been used for cancer treatment, but the mechanisms by which they exert their antitumor activity appear to be different. In the present work, we show that the Anamu-SC extract reduces tumor growth in the 4T1 murine mammary carcinoma model but not in the B16-F10 melanoma model, unlike the standardized P2Et extract. Both extracts decreased the levels of interleukin-10 (IL-10) in the B16-F10 model, but only P2Et increased the levels of tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). Likewise, co-treatment of P2Et and doxorubicin (Dox) significantly reduced tumor size by 70% compared to the control group, but co-treatment of Anamu-SC with Dox had no additive effect. Analysis of intratumoral immune infiltrates showed that Anamu-SC decreased CD4+ T cell frequency more than P2Et but increased CD8+ T cell frequency more significantly. Both extracts reduced intratumoral monocytic myeloid-derived suppressor-like cell (M-MDSC-LC) migration, but the effect was lost when co-treated with doxorubicin. The use of P2Et alone or in co-treatment with Anamu-SC reduced the frequency of regulatory T cells and increased the CD8+/Treg ratio. In addition, Anamu-SC reduced glucose consumption in tumor cells, but this apparently has no effect on IFNγ- and TNFα-producing T cells, although it did reduce the frequency of IL-2-producing T cells. The efficacy of these herbal preparations is increasingly clear, as is the specificity conditioned by tumor heterogeneity as well as the different chemical complexity of each preparation. Although these results contribute to the understanding of specificity and its future benefits, they also underline the fact that the development of each of these standardized extracts called polymolecular drugs must follow a rigorous path to elucidate their biological activity.

Piper nigrum extract suppresses tumor growth and enhances the antitumor immune response in murine models of breast cancer and melanoma

Although the antitumor effect of P. nigrum has been widely studied, research related to its possible immunomodulatory effects is relatively scarce. Here, the antitumor and immunomodulatory activity of an ethanolic extract of P. nigrum were evaluated in the murine models of 4T1 breast cancer and B16-F10 melanoma. In vitro evaluations showed that the P. nigrum extract has cytotoxic activity, induces apoptotic cell death, and has a pro-oxidant effect in both cell lines, but it regulates glucose uptake differently in both lines, decreasing it in 4T1 but not in B16-F10. P. nigrum extract significantly reduced tumor size in both models and decreased the occurrence of macrometastases in 4T1 model. Evaluation of immune subpopulations by flow cytometry revealed that the P. nigrum extract significantly increases the frequency of dendritic cells and activated CD8+ T cells and decreases the frequency of myeloid-derived suppressor like cells and Tregs in the tumor microenvironment of both models but with different dynamics. Our findings strongly suggest that the P. nigrum extract exerts immunomodulatory functions, slightly related to the modulation of cellular energy metabolism, which could ultimately contribute to the promising antitumor effect of P. nigrum.

Effective adjuvantation of nanograms of influenza vaccine and induction of cross-protective immunity by physical radiofrequency adjuvant.

Novel adjuvants are highly demanded to aid in development of improved or new vaccines against existing or emerging infectious diseases. Considering commonly used Alum and MF59 adjuvants induce tissue stress and release of endogenous danger signals to mediate their adjuvant effects, physical modalities may be used to induce tissue stress and endogenous danger signal release to enhance vaccine-induced immune responses. Furthermore, physical adjuvants are less likely to induce significant systemic adverse reactions due to their localized effects. Recently we found non-invasive radiofrequency (RF) pretreatment of the skin could significantly enhance intradermal vaccine-induced immune responses in murine models that included pandemic influenza vaccine, pre-pandemic vaccine, and influenza internal antigen vaccine. It remained to be explored whether the physical RF adjuvant (RFA) could be used to boost seasonal influenza vaccination, spare vaccine doses, and induce cross-protective immunity. This study found the physical RFA could significantly enhance seasonal influenza vaccine-induced immune responses against each viral strain and robustly enhance low-dose (nanograms) H3N2 vaccine-induced immune responses and protection in murine models. RFA also induced cross-protective immunity against heterologous and heterosubtypic influenza viruses. Further studies found heat shock protein 70 (inducible endogenous danger signal) and myeloid differentiation primary response 88 adaptor played a crucial role in dose-sparing effects of RFA. These data strongly support further development of the physical RFA to boost influenza vaccination.

Dendritic cell immunotherapy with miR-155 enriched tumor-derived exosome suppressed cancer growth and induced antitumor immune responses in murine model of colorectal cancer induced by CT26 cell line

Nowadays, various strategies are considered to prime Dendritic cells (DCs) with tumor antigens. The tumor cell-derived exosomes are recognized as one of the most efficient strategies for achieving this purpose. In this regard, MicroRNA 155 (miR-155) is employed as one of the most prominent miRNAs, which play substantial roles in DCs maturation and IL-12 production. This study investigates the tumor growth suppression and antitumor effects of DCs primed with miR-155-enriched exosome on the BALB/c murine model of colorectal cancer induced by CT-26 cell lines.Therefore, a holistic framework is proposed for the analysis procedure. In the first stage, miRNA-155 was electroporated into texosomes. In the second stage, bonemarrow-derived DCs were treated with miRNA-155 enriched texosomes. Then, antitumor properties of manipulated DC have been evaluated in the BALB/c mice model of colorectal cancer. After DC immunotherapy, several features have been assessed for each animal, including survival, body weight, tumor volume/size, histopathology, and serum cytokine levels. Also, flow cytometric evaluation has been performed for the spleen and the tumor tissue T-cell subsets.The findings demonstrated that the primed DCs could significantly increase IL-12p70 and IFN-γ in serum and accelerate the differentiation, proliferation, and cytotoxicity effects on the Th and CTL cells. Also, the treatment also increased the infiltration of Th and CTL cells into the tumor microenvironment while decreasing Tregs. This situation causes tumor growth control, and survival improvement.Therefore, DC immunotherapywith miR-155-enriched texosomes can be employed as a the desired approach for inducing antitumor immune responses, controlling tumor growth, and improving survival in mice with colorectal cancer. However, it is essential to perform more investigations to confirm the clinical application of this approach in humans and other types of tumors.

Activating Mucosal-Associated Invariant T Cells Induces a Broad Antitumor Response.

Mucosal-associated invariant T (MAIT) cells are MR1-restricted innate-like T cells that recognize non-peptide antigens including riboflavin derivates. Although in vitro-activated MAIT cells show antitumor activity, the in vivo role of MAIT cells in cancer is still unclear. Here, we have shown that MAIT cells have antitumor function in vivo when activated by a combination of the synthetic riboflavin synthesis pathway-derived antigen 5-OP-RU [5-(2-oxopropylideneamino)-6-D-ribitylaminouracil] and the Toll-like receptor 9 (TLR9) agonist CpG. Coadministration of 5-OP-RU and CpG induced strong systemic in vivo expansion and activation of MAIT cells with high CD69 expression, pronounced effector memory phenotype, and upregulated levels of effector molecules including IFNγ, granzyme B, and perforin. Activated and expanded MAITs induced a potent and broad antitumor immune response in murine models of liver metastasis and hepatocellular carcinoma, lung metastasis, and subcutaneous tumors in two different mouse strains. Such tumor inhibition was absent in MAIT-deficient Mr1 -/- mice. CRISPR/Cas9-mediated MR1 knockout in tumor cells did not affect efficacy of this MAIT-directed immunotherapy, pointing toward an indirect mechanism of action. Our findings suggest that MAIT cells are an attractive target for cancer immunotherapy.See related Spotlight by Lantz, p. 996.

Optimization of Ex Vivo Activation and Expansion of T Cells for Therapy of Lymphoma Via Antagonism of PI3K δ and Vasoactive Intestinal Peptide Signaling

Introduction: Success of T cell therapy is restricted by the variable success of expanding patients9 T cells ex vivo and the limited persistence of cultured T cells in T cell-treated patients. These problems may be particularly relevant to lymphoma patients previously treated with numerous cycles of chemotherapy, and motivate the development of novel strategies to enhance ex vivo T cell expansion and their persistence in vivo . Recently we have shown that antagonism of vasoactive intestinal peptide (VIP) signaling on lymphocytes augments in vivo T cell activation and growth leading to significant and durable T cell-dependent anti-cancer immune response in murine models of lymphoma and leukemia (Li Cancer Research 2016; Petersen Oncoimmunology 2017). We tested the hypothesis that VIP antagonists would synergize with phosphatidyl inositol-3 kinase inhibitors (PI3Ki) to augment T-cells ex vivo expansion of T cells with anti-cancer immune functions. Methods: Peripheral blood mononuclear cells were obtained from consenting DLBCL patients and healthy controls. Frozen PBMC were thawed and rested overnight in RPMI 1640 supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 50 μM 2-mercaptoethanol then cultured in 96 well flat-bottom plates with 30 U/mL IL-2 and anti-CD3/CD28 beads at a 1:1 bead to cell ratio in the presence of idelalisib, duvelisib, a peptide antagonist to VIP (VIPhyb, KPRRPYTDNYRELRKQMAVKKYLNSILN), or mast cell chymase, a VIP-degrading enzyme. Flow cytometric analysis of T cells from patients, healthy volunteers, and mice prior to and following ex vivo cell expansion measured expression of CD3, CD4, CD8, CD27, CD28 and intracellular cytokines (IL2, IFN-gamma). In vivo persistence of cultured human T -cells was monitored in following ex vivo expansion and transfer into RAG mice. Anti-lymphoma activity of cultured murine T cells was assessed by culturing a mixture of wild-type, OT1, and OT2 murine T-cells and transfer into immune-competent mice bearing subcutaneous E.G7 OVA murine B cell lymphoma. Results: T cells from heavily treated DLBCL patients had a senescent phenotype with loss of CD27 and CD28 expression, with a median of 36.8% (range 4.58-77.3) CD27-CD28- T cells compared with median values of 5.8%, range 1.7-12.4% in healthy controls and 3.9%, range 0.5-23.1% in untreated DLBCL patients. FACS-purified senescent CD27- CD28- T cells failed to expand ex vivo and had fratricide-mediated inhibition of non-senescent T cells expansion. Blockade of PI3K δ and antagonism of vasoactive intestinal peptide (VIP) signaling during T-cell culture with low dose IL2 and anti- CD3/CD28 beads partially inhibited terminal differentiation (mean frequency 54.4% CD27+CD28+ T cells vs 27.4% in control cultures, p Discussion: Taken together, these data demonstrate that synergistic blockade of the PI3K and VIP pathways is an attractive strategy to enhance the expansion and functional capacity of T cells during clinical manufacturing, and that optimization of culture expansion conditions can improve the magnitude and durability of anti-lymphoma responses in vivo . Disclosures Waller: Coulter Foundation: Research Funding; PRA: Consultancy; Chimerix: Equity Ownership; Katz Foundation: Research Funding; Celldex: Consultancy; Helocyte: Consultancy; AMGEN: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; Cambium Medical Technologies: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees, Patents & Royalties; Cerus: Equity Ownership; National Institutes of Health: Research Funding. Jagirdar: Kite: Consultancy. Flowers: Research to Practice: Research Funding; Burroughs Welcome Fund: Research Funding; National Institutes Of Health: Research Funding; Educational Concepts: Research Funding; Prime Oncology: Research Funding; Acerta: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Spectrum: Consultancy; Seattle Genetics: Consultancy; Millennium/Takeda: Research Funding; Genentech/Roche: Consultancy, Research Funding; Eastern Cooperative Oncology Group: Research Funding; OptumRx: Consultancy; TG Therapeutics: Research Funding; Infinity: Research Funding; Onyx: Research Funding; Clinical Care Options: Research Funding; Janssen Pharmaceutical: Research Funding; National Cancer Institute: Research Funding; Celgene: Consultancy, Research Funding; V Foundation: Research Funding; Gilead: Consultancy; Bayer: Consultancy; Abbvie: Consultancy, Research Funding.

Three-Dimensional Light Sheet Fluorescence Microscopy of Lungs To Dissect Local Host Immune-Aspergillus fumigatus Interactions.

Aspergillus fumigatus is an opportunistic fungal pathogen that can cause life-threatening invasive lung infections in immunodeficient patients. The cellular and molecular processes of infection during onset, establishment, and progression of A. fumigatus infections are highly complex and depend on both fungal attributes and the immune status of the host. Therefore, preclinical animal models are of paramount importance to investigate and gain better insight into the infection process. Yet, despite their extensive use, commonly employed murine models of invasive pulmonary aspergillosis are not well understood due to analytical limitations. Here, we present quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the local immune response in whole lungs at cellular resolution within its anatomical context. We analyzed three very common murine models of pulmonary aspergillosis based on immunosuppression with corticosteroids, chemotherapy-induced leukopenia, or myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial distribution, interaction, and activation of two key immune cell populations in antifungal defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the patterns of fungal growth correlated with the detected effects of the immunosuppressive regimens on the local immune cell populations. Moreover, LSFM demonstrates that the commonly used intranasal route of spore administration did not result in complete intra-alveolar deposition, as about 80% of fungal growth occurred outside the alveolar space. Hence, characterization by LSFM is more rigorous than by previously used methods employing murine models of invasive pulmonary aspergillosis and pinpoints their strengths and limitations.IMPORTANCE The use of animal models of infection is essential to advance our understanding of the complex host-pathogen interactions that take place during Aspergillus fumigatus lung infections. As in the case of humans, mice need to suffer an immune imbalance in order to become susceptible to invasive pulmonary aspergillosis (IPA), the most serious infection caused by A. fumigatus There are several immunosuppressive regimens that are routinely used to investigate fungal growth and/or immune responses in murine models of invasive pulmonary aspergillosis. However, the precise consequences of the use of each immunosuppressive model for the local immune populations and for fungal growth are not completely understood. Here, to pin down the scenarios involving commonly used IPA models, we employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular resolution. Our results will be valuable to optimize and refine animal models to maximize their use in future research.

PO-322 Investigating co-expression of the immune checkpoints heme oxygenase-1 and PD-L in breast cancer

IntroductionUnprecedented clinical outcomes have been observed in a variety of cancers by targeting the immune checkpoint molecules PD-1/PD-L and CTLA-4. However, a significant proportion of patients fail to respond to these therapies, indicating the potential existence of further significant immune regulatory pathways. Heme Oxygenase-1 (HO-1) is an enzyme responsible for the breakdown of heme to biliverdin, iron and the immune suppressive molecule carbon monoxide. We recently demonstrated that HO-1 acts as an immune checkpoint by suppressing chemotherapy-elicited anti-tumour immune responses in murine models of breast cancer. In the present study we investigate the co-expression of HO-1 with the clinically targeted immune checkpoints PD-L1/2 to explore the immune checkpoint landscape in breast cancer.Material and methodsWe investigate the expression of checkpoint molecules PD-L1/2 and HO-1 (HMOX1) in a microarray dataset of tumour tissue from breast cancer patients. We then analyse a single-cell RNAseq dataset, grouping tumoral cells according to their expression profiles using t-distributed stochastic neighbour embedding (tSNE) followed by density clustering, and probe for the expression of genes of interest. We utilise the aggressive spontaneous murine model of mammary adenocarcinoma (MMTV-PyMT) to investigate the therapeutic efficacy of targeting PD-1 (the receptor for PD-L1/2) and/or HO-1 activity.Results and discussionsAnalysis of microarray data from patients with breast cancer demonstrated that the expression of HO-1 and PD-L1/2 were associated within the tumour microenvironment. Analysis of unbiased human breast cancer single-cell RNAseq data indicated that myeloid cells were a significant source of HO-1 and PD-L1/2 in the tumour microenvironment. The MMTV-PyMT mouse model paralleled these clinical observations where both HO-1 and PD-L1/2 were expressed by the tumour associated macrophages. Using a chemotherapy-elicited anti-tumour immune response we pharmacologically inhibited HO-1 activity and/or neutralised PD-1 receptors using blocking antibodies to evaluate the therapeutic efficacy of targeting these respective pathways. In this model, targeting HO-1 alongside chemotherapy resulted in a greater regression of tumour growth than targeting PD-1.ConclusionThese data prompt the need to further investigate the redundancy and hierarchy of immune checkpoints in the tumour microenvironment to effectively harness the anti-tumour immune response and efficiently implement immune checkpoint blockade therapy.

Enhancement of Hepatitis E Virus DNA Vaccine Immunity by Beclin-1- Mediated Autophagy

Background: The Hepatitis E virus (HEV) infection is a serious public health problem, with a mortality rate of approximately 20-25% among pregnant women. DNA vaccination was reported to induce humoral and cellular immune responses in murine models; however, a major problem of the strategy is its limited potency. Methods: In this study, we have investigated whether Beclin1, as an autophagy-inducing based plasmid, can serve as an immunostimulatory agent and promote the HEV specific protective immunity of naked DNA vaccine encoding truncated ORF-2 (HEV239). Plasmids encoding HEV239 with or without Beclin1 were used for the BALB/c mice immunization. Results: The results demonstrated a significant increase microtubule-associated protein light chain-3 II (LC3 II) as a marker of autophagy in the HEK293 cell, which were transfected by pVITRO-Beclin1 plasmid. The immunological effects of adding Beclin1 to HEV239 ORF-2 DNA vaccines were associated with the induction of the antigen-specific lymphocyte proliferation and the Th1-type cytokine (gamma-interferon (IFN-γ)), while there were no differences in IL-4 levels between the groups. Examination of humoral immune responses in vaccinated mice represented that immunization with pVITRO-HEV239- Beclin1 notably increased serum level of IgG2a and total IgG against HEV in comparison to the HEV239 plasmid alone. Conclusions: The strong Th1 immune response induced by the Beclin1 suggest that induction of autophagy can be an efficient approach to enhance the immunogenicity of DNA vaccine. This promising procedure could be further exploited as a potential therapeutic vaccine candidate in future studies.

Stigmasterol inhibits lipopolysaccharide-induced innate immune responses in murine models

Stigmasterol is a naturally occurring steroid alcohol which occurs in vegetables, soya and a large variety of medicinal plants. Stigmasterol and other phytosterols have been documented as immunomodulators with huge therapeutic potential. We assessed the mitigating effect of stigmasterol on non-fatal and fatal innate immune responses in murine models after intraperitoneal challenge with an endotoxin, lipopolysaccharide, LPS. The effect of stigmasterol on LPS-induced febrile response, inflammatory cell proliferation, multiple organ damage and mortality were respectively investigated. Pretreatment with stigmasterol 10, 50 and 100mg/kg reduced total LPS-induced fever response by 39.93±10.52%, 53.05±5.84% and 77.27±6.25% respectively. Neutrophil proliferation both in blood and recovered peritoneal fluid was significantly reversed by stigmasterol at 50 and 100mg/kg. Lung and liver histopathology showed stigmasterol effectively controlled organ damage. The lung inflammation score of 9.20±0.73 for the polyethylene glycol, PEG-treated disease control mice was reduced respectively to 6.50±0.54, 4.60±0.40 and 4.10±0.42 with 10, 50 and 100mg/kg of stigmasterol. Serum levels of liver enzyme markers, alanine transaminase, ALT and aspartate transaminase, AST were consistent with the observed histological changes. Stigmasterol at 50 and 100mg/kg significantly protected mice from LPS-induced mortality with 40% survival. Overall, stigmasterol inhibits LPS-induced innate immune responses in murine models.

Adiponectin confers protection from acute colitis and restricts a B cell immune response

Adiponectin demonstrates beneficial effects in various metabolic diseases, including diabetes, and in bowel cancer. Recent data also suggest a protective role in colitis. However, the precise molecular mechanisms by which adiponectin and its receptors modulate colitis and the nature of the adaptive immune response in murine models are yet to be elucidated. Adiponectin knock-out mice were orally administered dextran sulfate sodium for 7 days and were compared with wild-type mice. The severity of disease was analyzed histopathologically and through cytokine profiling. HCT116 colonic epithelial cells were employed to analyze the in vitro effects of adiponectin and AdipoR1 interactions in colonic injury following dextran sulfate sodium treatment. Adiponectin knock-out mice receiving dextran sulfate sodium exhibited severe colitis, had greater inflammatory cell infiltration, and an increased presence of activated B cells compared with controls. This was accompanied by an exaggerated proinflammatory cytokine profile and increased STAT3 signaling. Adiponectin knock-out mouse colons had markedly reduced proliferation and increased epithelial apoptosis and cellular stress. In vitro, adiponectin reduced apoptotic, anti-proliferative, and stress signals and restored STAT3 signaling. Following the abrogation of AdipoR1 in vitro, these protective effects of adiponectin were abolished. In summary, adiponectin maintains intestinal homeostasis and protects against murine colitis through interactions with its receptor AdipoR1 and by modulating adaptive immunity.

Beneficial effects of ursodeoxycholic acid via inhibition of airway remodelling, apoptosis of airway epithelial cells, and Th2 immune response in murine model of chronic asthma

Background and aimsIn previous studies, anti-inflammatory, anti-apoptotic and immunomodulatory effects of ursodeoxycholic acid (UDCA) on liver diseases have been shown. In this study, we aimed to investigate the effects of UDCA on airway remodelling, epithelial apoptosis, and T Helper (Th)-2 derived cytokine levels in a murine model of chronic asthma. MethodsTwenty-seven BALB/c mice were divided into five groups; PBS-Control, OVA-Placebo, OVA-50mg/kg UDCA, OVA-150mg/kg UDCA, OVA-Dexamethasone. Mice in groups OVA-50mg/kg UDCA, OVA-150mg/kg UDCA, OVA-Dexamethasone received the UDCA (50mg/kg), UDCA (150mg/kg), and dexamethasone, respectively. Epithelium thickness, sub-epithelial smooth muscle thickness, number of mast and goblet cells of samples isolated from the lung were measured. Immunohistochemical scorings of the lung tissue for matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEG-F), transforming growth factor-beta (TGF-β), terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL) and cysteine-dependent aspartate-specific proteases (caspase)-3 were determined. IL-4, IL-5, IL-13, Nitric oxide, ovalbumin-specific immunoglobulin (Ig) E levels were quantified. ResultsThe dose of 150mg/kg UDCA treatment led to lower epithelial thickness, sub-epithelial smooth muscle thickness, goblet and mast cell numbers compared to placebo. Except for MMP-9 and TUNEL all immunohistochemical scores were similar in both UDCA treated groups and the placebo. All cytokine levels were significantly lower in group IV compared to the placebo. ConclusionsThese findings suggested that the dose of 150mg/kg UDCA improved all histopathological changes of airway remodelling and its beneficial effects might be related to modulating Th-2 derived cytokines and the inhibition of apoptosis of airway epithelial cells.

Ibrutinib Represents a Novel Class of Immune Modulating Therapeutics That Enhances the Survival of Activated T Cells in Vitro and In Vivo through a Non-BTK Mediated Mechanism

Background: Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) that induces durable remissions in chronic lymphocytic leukemia (CLL). In other systems, ibrutinib has also been shown to have an immune modulating effect on T-cells through inhibition of interleukin-associated tyrosine kinase (ITK). Our group and then others have reported that ibrutinib enhances antibacterial and antitumor T cell immune response in murine models. ITK has been demonstrated to be involved in TCR-induced up-regulation of FAS Ligand (FAS-L) and activation induced cell death (AICD) of T cells. In murine models, ITK deficiency or inhibition by specific kinase inhibitors lead to impaired AICD and a much greater persistence and accumulation of activated T cells. We therefore postulate that ibrutinib, by its capability to irreversibly inhibit ITK, may have similar effect on recusing activated T cells from AICD. To date, a comprehensive analysis of the effects on cellular immunity in humans treated with ibrutinib, or more selective BTK inhibitors has not been reported.Methods: PBMCs from previously treated CLL patients enrolled on clinical trials (NCT01589302 and NCT02029443) of ibrutinib (a BTK and ITK inhibitor) and acalabrutinib (ACP-196, a selective BTK inhibitor) were collected at baseline and during treatment at the completion of 2 months( cycle 3) and 5 months (cycle 6). Patients with persistent lymphocytosis post treatment were chosen to allow comparable tumor burden before and after therapy. T cell immune function and the immunosuppressive capacity of CLL cells were evaluated with detailed immunologic and functional studies. Mechanistic studies examining AICD with ibrutinib and acalabrutinib treatment were performed.Results: A total of 25 patients treated with ibrutinib (n=17) and acalabrutinib (n=8) were studied. Ibrutinib, but not acalabrtinib treatment promoted a significant increase (3-5 fold) in absolute CD4(P<0.01) and CD8(p=0.001) T cell numbers in CLL patients that was more prominent in effector/effector memory subsets and less in naïve and central memory subsets (Figure 1). While the number of regulatory T cells (Treg cells) remained unchanged, the ratio of Treg cells to CD4 T cells was reduced by approximately two fold (p<0.001) after ibrutinib treatment. Detailed ex vivo studies demonstrated significantly diminished activation induced cell death of activated T-cells when pre-treated with ibrutinib, but not acalabrutinib (Figure 2). Treatment with ibrutinib or acalabrutinib significantly reduced T cell PD-1 (p<0.01 for CD8 T cells, p<0.05 for CD4 T cells and CTLA-4 (p< 0.05 for CD8 T cells, p<0.001 for CD4 T cells) expression, most prominent in central memory T cell compartment. Ibrutinib and acalabrutinib both significantly decreased CD200 (p<0.001) and BTLA (p<0.001) surface expression as well as IL-10 production by CLL cells after prolonged "B10-Pro" conditioning (p<0.001 for ibrutinib, p<0.05 for acalabrutinib).Conclusions:These findings collectively differentiate ibrutinib as a therapeutic that can both expand activated T-cells and also ameliorate T-cell exhaustion thorough reversal of CLL tumor cell mediated immune suppression. While acalabrutinib reverses CLL cell mediated immune suppression, it lacks ability to expand activated T-cells. Collectively, these data provide support for dual BTK/ITK inhibitor therapeutics when applied to enhance the therapeutic efficacy of adoptive immunotherapy (e.g.chimeric antigen receptor T cells) or tumor vaccines for CLL and potentially other types of hematologic and solid cancers. [Display omitted] [Display omitted] DisclosuresJones:AbbVie: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Andritsos:Hairy Cell Leukemia Foundation: Research Funding. Awan:Novartis Oncology: Consultancy; Innate Pharma: Research Funding; Pharmacyclics: Consultancy. Fraietta:Novartis: Patents & Royalties: Novartis, Research Funding. June:Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Tmunity: Equity Ownership, Other: Founder, stockholder ; University of Pennsylvania: Patents & Royalties; Immune Design: Consultancy, Equity Ownership; Pfizer: Honoraria; Johnson & Johnson: Research Funding; Celldex: Consultancy, Equity Ownership. Maus:Novartis: Patents & Royalties: inventor on patents related to ibrutinib use in T cell therapies. Woyach:Karyopharm: Research Funding; Morphosys: Research Funding; Acerta: Research Funding.

CryJ-LAMP DNA Vaccines for Japanese Red Cedar Allergy Induce Robust Th1-Type Immune Responses in Murine Model.

Allergies caused by Japanese Red Cedar (JRC) pollen affect up to a third of Japanese people, necessitating development of an effective therapeutic. We utilized the lysosomal targeting property of lysosomal-associated membrane protein-1 (LAMP-1) to make DNA vaccines that encode LAMP-1 and the sequences of immunodominant allergen CryJ1 or CryJ2 from the JRC pollen. This novel strategy is designed to skew the CD4 T cell responses to the target allergens towards a nonallergenic Th1 response. CryJ1-LAMP and CryJ2-LAMP were administrated to BALB/c mice and antigen-specific Th1-type IgG2a and Th2-type IgG1 antibodies, as well as IgE antibodies, were assayed longitudinally. We also isolated different T cell populations from immunized mice and adoptively transferred them into naïve mice followed by CryJ1/CryJ2 protein boosts. We demonstrated that CryJ-LAMP immunized mice produce high levels of IFN-γ and anti-CryJ1 or anti-CryJ2 IgG2a antibodies and low levels of IgE antibodies, suggesting that a Th1 response was induced. In addition, we found that CD4+ T cells are the immunological effectors of DNA vaccination in this allergy model. Together, our results suggest the CryJ-LAMP Vaccine has a potential as an effective therapeutic for JRC induced allergy by skewing Th1/Th2 responses.

Abstract 3136: Adoptive transfer of bone marrow T cells induces antitumor immune responses in murine models of melanoma and neuroblastoma

Abstract Bone marrow (BM) serves as a reservoir for a unique population of memory T cells. Tumor-specific T cells have been identified in the bone marrow of cancer patients. However, few direct functional analyses of tumor-specific responses in T cells isolated from BM have been performed. In this study, we investigated the ability of bone marrow T cells to induce anti-tumor immunity in mice bearing neuro2A neuroblastoma or B16 melanoma. To demonstrate the presence of antigen-specific T cells in BM, we first examined the percent of antigen-specific CD8+ T cells in vaccinated mice. The overall percent of antigen-specific CD8+ T cells was much higher in BM compared to spleen and lymph nodes of vaccinated mice. To investigate the ability of antigen-specific BM T cells to induce cytotoxic function, BM T cells were isolated from mice vaccinated with OVASIINFEKL peptide pulsed dendritic cells (DC). Expanded BM T cells from DC vaccinated mice co-cultured with M05 or irrelevant tumor cells led to tumor-specific cytotoxicity (p&amp;lt;0.001). In B16 tumor-bearing mice, T cells isolated from BM produced IFN-gamma in response to gp100 peptide (1400+210 pg/ml). Adoptive transfer of BM T cells isolated from B16-bearing mice led to a delay in tumor growth compared to control groups (p&amp;lt;0.001). In the neuroblastoma model, T cells isolated from BM of neuro2A-bearing mice produced higher levels of IFN-gamma and were tumor-specific (250+90 pg/ml, p&amp;lt;0.01 compared to T cells cultured with irrelevant tumor cells). Adoptive transfer of expanded BM T cells and DC vaccination in the setting of BM transplant led to a significant delay in tumor growth and enhanced survival in neuro2A bearing mice compared to BM transplant + DC vaccination alone group (p&amp;lt;0.02). These studies demonstrate that tumor-specific T cells can be isolated from the BM of tumor-bearing mice. Adoptive transfer of T cells expanded from the BM can delay tumor growth and improve survival. These studies support the isolation and expansion of T cells from the BM for use in adoptive cell therapy strategies in patients with neuroblastoma and melanoma. Citation Format: Krithika N. Kodumudi, Amy Weber, Ellen Moore, Amod Sarnaik, Shari Pilon-Thomas. Adoptive transfer of bone marrow T cells induces antitumor immune responses in murine models of melanoma and neuroblastoma. [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 3136. doi:10.1158/1538-7445.AM2015-3136

Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors.

The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10(24)-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10(68)-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.

Identification of linear B-cell epitopes within Tarp of Chlamydia trachomatis.

Chlamydia trachomatis is one of the most prevalent sexually transmitted pathogens. There is currently no commercially available vaccine against C. trachomatis. Chlamydial translocated actin-recruiting phosphoprotein (Tarp) can induce cellular and humoral immune responses in murine models and has been regarded as a potential vaccine candidate. In this report, the amino acid sequence of Tarp was analyzed using computer-assisted techniques to scan B-cell epitopes, and six possible linear B-cell epitopes peptides (aa80-95, aa107-123, aa152-170, aa171-186, aa239-253 and aa497-513) with high predicted antigenicity and high conservation were investigated. Sera from mice immunized with these potential immunodominant peptides was analyzed by ELISA, which showed that epitope 152-170 elicited serum immunoglobulin G (IgG) response and epitope 171-186 elicited both serum IgG and mucosal secretory immunoglobulin A response. The response of immune sera of epitope 171-186 to endogenous Tarp antigen obtained from the Hela229 cells infected with C. trachomatis was confirmed by Western blot and indirect fluorescence assay. In addition, binding of the antibodies against epitope 171-186 to endogenous Tarp was further confirmed by competitive ELISA. Our results demonstrated that the putative epitope (aa171-186) was an immunodominant B-cell epitope of Tarp. If proven protective and safe, this epitope, in combination with other well-documented epitopes, might be included into a candidate epitope-based vaccine against C. trachomatis.