Delayed-type Hypersensitivity Response Research Articles

Preliminary Results from a Phase 1/2 Study of DSP-7888, a Novel WT1 Peptide-Based Vaccine, in Patients with Myelodysplastic Syndrome (MDS)

▪Background: Hypomethylating agents (HMA), including azacitidine (AZA) are currently the first-line treatment option for higher-risk myelodysplastic syndrome (MDS). However, the prognosis of patients after AZA failure is poor with a median overall survival of 5.6 months from the treatment failure (J Clin Onclol 2011;29:3322-7 Prébet T et al.), and currently there are no approved therapeutic options for such patients. Suzuki et al. reported that WT4869, one of the HLA-A*24;02-restricted synthetic peptide vaccine derived from Wilms’ tumor 1 (WT1) protein, demonstrated the sign of prolongation of overall survival (OS) with a median OS of 13.0 months in AZA failure higher-risk population, in the phase 1/2 study with MDS patients (Blood 126:2868, 2015;Suzuki et al.). DSP-7888 is a novel WT1-based peptide vaccine which induces the CTLs that recognize WT1 antigens in HLA-A*02:01/06 and HLA-A*24:02 restricted manner, and also includes a WT1-derived helper peptide applicable for various subtypes of HLA-DRB1. DSP-7888 is currently being investigated in a phase 1/2 study to evaluate the safety and efficacy in HLA-A*24:02+ and/or HLA-A*02:01/:06+ MDS patients, with some exploratory biomarker analyses.Methods: The objectives of this study were to assess the tolerability of DSP-7888 treatment in MDS patients in the phase 1 portion and to evaluate preliminary clinical activity of DSP-7888 in higher-risk MDS patients after AZA failure in the study. In phase 1 portion, higher-risk or transfusion-dependent lower-risk MDS patients including the AZA failure population were enrolled, and DSP-7888 was administered at doses of 3.5 to 10.5 mg/body by intradermal injections every two to four weeks in dose-escalation cohorts according to the 3 + 3 design until discontinuation criteria were met. Overall survival was evaluated for primarily clinical activity and hematological response and time to AML were also examined. Delayed type hypersensitivity (DTH), WT1-specific CTL induction and expression of WT1 mRNA in peripheral blood and bone marrow cells were also evaluated as exploratory biomarkers. The clinical data in phase 1 portion as of May 25th 2016 was presented in this report.Results: In phase 1 portion, enrollment of patients and dose-limiting toxicities (DLTs) evaluation were completed. Twelve patients including 7 higher- and 5 lower-risk patients were enrolled in 3.5 and 10.5 mg/body cohorts of 6 patients each, and safety profiles were evaluated. DLTs were not observed in either cohorts and the most common adverse drug reaction (ADR) was injection site reaction (ISR). ISR in 6 patients worsened to grade 3 with continuous treatment of DSP-7888 (2 patients at 3.5 mg/body, and 4 patients at 10.5 mg/body). Five serious ADR including 3 ISR, 1 pyrexia and 1 myocarditis were reported and dose-dependent toxicity was not observed except for ISR. All 12 patients were analyzed for preliminary clinical activity. Eight patients remained in stable disease (SD) with 2 hematological improvements (HI), and disease control rate (SD + any HI) was 66.6 %. Seven high risk AZA failure patients were enrolled, and the current survival time in this population is 7.3-10.8 months. Though preliminary, CTL induction was observed in 6 patients, and a trend of higher CTL induction was observed in patients with grade 3 ISR. DTH response was observed in 10 patients.Conclusions: In the phase 1 portion, DSP-7888 was well-tolerated in MDS patients although ISR was observed in all patients. CTL induction was detected with clinically observed reactions that may suggest preliminary signs of clinical activity. Further evaluation is needed to confirm the clinical potential of DSP-7888, and phase 2 portion is currently ongoing to evaluate the efficacy of DSP-7888 in higher-risk MDS patients after AZA failure. DisclosuresUsuki:Nippon Shinyaku: Honoraria; MSD: Honoraria; Kyouwa-Kirin: Honoraria; Novartis: Honoraria; Bristol-Myer-Squibb: Honoraria; Sumitomo Dainippon Pharma: Honoraria; SymBio Pharmaceuticals: Research Funding. Matsumura:Bristol-Myers Squibb Company: Honoraria; Novartis Pharma K.K: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Consultancy, Honoraria; Pfizer Japan Inc.: Honoraria. Ueda:Alexion Pharmaceuticals Inc.: Membership on an entity’s Board of Directors or advisory committees. Origuchi:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Tagashira:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Naoi:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Naoe:Bristol-Myers Squibb: Honoraria; Sumitomo Dainippon Pharma Co.,Ltd.: Honoraria, Research Funding; Pfizer Inc.: Research Funding; Astellas Pharma Inc.: Research Funding; Kyowa-Hakko Kirin Co.,Ltd.: Honoraria, Patents & Royalties, Research Funding; Amgen Astellas BioPharma K.K.: Honoraria; Otsuka Pharmaceutical Co.,Ltd.: Honoraria, Research Funding; CMIC Co., Ltd.: Research Funding; Celgene K.K.: Honoraria, Research Funding; TOYAMA CHEMICAL CO.,LTD.: Research Funding; Chugai Pharmaceutical Co.,LTD.: Honoraria, Patents & Royalties; Nippon Boehringer Ingelheim Co., Ltd.: Honoraria, Research Funding; Fujifilm Corporation: Honoraria, Patents & Royalties, Research Funding. Heike:Sumitomo Dainippon Pharma: Consultancy; Chugai Pharma: Consultancy; Otsuka Pharma: Consultancy. Miyazaki:Kyowa Kirin: Honoraria; Nihonshinyaku: Honoraria; Celgene Japan: Honoraria; Sumitomo Dainippon Pharma Co.,Ltd.: Honoraria.

Induction of Antigen-Specific T-Cell Responses through Dendritic Cell Vaccination in AML: Results of a Phase I/II Trial and Ex Vivo Enhancement By Checkpoint Blockade

Postremission therapy is critical for successful elimination of minimal residual disease (MRD) in acute myeloid leukemia (AML). Innovative treatment options are needed for patients that are not eligible for allogeneic stem cell transplantation. As the intrinsic immune response against leukemia-associated antigens (LAAs) is generally low, the clinical application of checkpoint inhibitors as monotherapy is less promising in AML compared to other hemato-oncological diseases. Therapeutic vaccination with autologous dendritic cells (DCs) loaded with LAAs is a promising treatment strategy to induce anti-leukemic immune responses.We have conducted a phase I/II proof-of-concept study using monocyte-derived next-generation DCs as postremission therapy of AML patients with a non-favorable risk profile in CR/CRi after intensive induction therapy (NCT01734304). These DCs are generated using a GMP-compliant 3-day protocol including a TLR7/8 agonist, loaded with RNA encoding the LAAs WT1 and PRAME as well as CMVpp65 as adjuvant/surrogate antigen, and are applied intradermally up to 10 times within 26 weeks. The primary endpoint of the trial is feasibility and safety of the vaccination. Secondary endpoints are immunological responses and disease control.After the safety and toxicity profile was evaluated within phase I (n=6), the patient cohort was expanded to a total of 13 patients. DCs of sufficient number and quality could be generated from leukapheresis in 11/12 cases. DCs exhibited an immune-stimulatory profile based on high costimulatory molecule expression, IL-12p70 secretion, migration towards a chemokine gradient and processing and presentation of antigen. In 9/9 patients that are currently evaluable, we observed delayed-type hypersensitivity (DTH) responses at the vaccination site, but no grade III/IV toxicities. TCR repertoire analysis by next-generation sequencing revealed an enrichment of particular clonotypes at DTH sites. In the peripheral blood, we detected vaccination-specific T cell responses by multimer staining and by ELISPOT analysis: 7/7 patients showed responses to CMVpp65, including both boosting of pre-existing T cells in CMV+ patients and induction of a primary T cell response in CMV- patients. 2/7 patients exhibited responses to PRAME and WT each. 7/10 vaccinated patients are still alive, and 5/10 are in CR, with an observation period of up to 840 days.In vitro, DC-activated T cells showed an upregulation of PD-1 and LAG-3, while the DCs expressed the respective ligands PD-L1 and HLA-DR. Therefore, we studied the capacity of checkpoint blocking antibodies to further enhance T-cell activation by DCs. We found that blockade of PD-1 and particularly of LAG-3 was highly effective in enhancing both IFN-γ secretion and proliferation of T cells. Both pathways seem to target different T-cell subsets, as PD-1 blockade resulted in increased IFN-γ secretion by TN- and TEM-subsets, while blockade of LAG-3 significantly affected TN- and TCM-subsets.We conclude that vaccination with next-generation LAA-expressing DCs in AML is feasible, safe, and induces anti-leukemic immune responses in vivo. Our in vitro data supports the hypothesis that T-cell activation by means of the vaccine could be further enhanced by blocking PD-1 and/or LAG-3. DisclosuresSubklewe:AMGEN Research Munich: Research Funding.

Diclofenac-Induced Allergic Contact Dermatitis: A Series of Four Patients.

Allergic contact dermatitis is an immune-mediated antigen-specific skin reaction to an allergenic chemical that corresponds to a delayed-type hypersensitivity response (type IV reaction). Allergic contact dermatitis should be suspected when skin lesions are localized to the site of previous applications of the culprit drug. Lesions appear after re-exposure in susceptible persons, with delayed onset (more than 24 h after exposure). The gold standard for diagnosis is patch (epicutaneous) testing; identification and removal of any potential causal agents is crucial. Diclofenac sodium 1% topical gel contains active (diclofenac sodium) and inactive ingredients. It is a widely used non-steroidal anti-inflammatory drug, known to cause allergic contact dermatitis, and especially photoallergic contact reactions. We present four cases of diclofenac-sodium-induced allergic contact dermatitis, diagnosed based on clinical grounds: intensively itchy eczematous lesions on the sites of drug application after several days of treatment. No allergic history and no other drug intake were reported by the patients. The application of diclofenac sodium 1% topical gel was strictly forbidden in all cases; potent topical steroids proved to be effective in all cases within 2 weeks of therapy. Patch tests were performed in all cases with European standard battery, with patients’ own diclofenac sodium 1% topical gels and with diclofenac sodium 1% in petrolatum 3 weeks after completion of local steroid therapy. Readings were done after 48 h (Day 2) and 72 h (Day 3) and proved to be positive only to patients’ diclofenac sodium 1% topical gel and diclofenac sodium 1% in petrolatum. No sun exposure was allowed during the testing, and any other treatments were forbidden.

Withania somnifera chemotype NMITLI 101R significantly increases the efficacy of antileishmanial drugs by generating strong IFN-γ and IL-12 mediated immune responses in Leishmania donovani infected hamsters

BackgroundWithania somnifera (L.) Dunal (Solanaceae), commonly known as Ashwagandha, is one of the most important medicinal plant in the traditional Indian medical systems. Pharmacological studies have established that root extracts of W. somnifera contain several bioactive constituents called withanolides. The plant has long been used for its several beneficial properties and recently as an immunomodulator. Hypothesis/PurposeA combination therapy including a potential and safe immunostimulant with lower doses of effective drug, which can reduce the parasitic burden and simultaneously can produce an enhancement of adaptive immunity, has proven to be significantly a more effective approach than immunotherapy or drug therapy alone. Study designEvaluation of the immunostimulatory effect of W. somnifera chemotype NMITLI 101R when used in combination with ED50 doses of antileishmanial drugs in Leishmania donovani infected hamsters. MethodsInfected animals were administered with chemotype 101R(30mg/kg × 15 days) either alone or in combination with ED50 doses of miltefosine (10mg/kg × 5 days), paromomycin (30mg/kg × 5 days) or amphotericin B (0.5mg/kg × 5 days). The treated animals were euthanized on days 30 and 60 post-treatment (p.t.) and checked for parasite clearance, delayed type hypersensitivity (DTH) response, cytokine and inducible nitric oxide synthase levels by real-time PCR, nitric oxide (NO) production, reactive oxygen species (ROS) generation, lymphoproliferative and antibody responses. ResultsThe group of animals that received 101R and ED50 dose of miltefosine showed optimum inhibition of parasite multiplication (∼98%) by day 60 p.t. followed by the group that received 101R plus paromomycin (∼94%) and 101R plus amphotericin B (∼93%). The efficacy was well supported by the increased inducible NO synthase mRNA transcript, strong IFN-γand IL-12 mediated Th1 immune responses and significantly suppressed levels of Th2 cytokines (IL-4, IL-10 and TGF-β). Additionally, same therapy also induced significant increase in the level of NO production, ROS generation, Leishmania specific IgG2 antibody along with profound DTH and strong T-cell responses as compared with all the other treated groups. ConclusionOur results suggest that combination of chemotype 101R with ED50 doses of antileishmanial drugs may provide a promising alternative for the cure of visceral leishmaniasis with significant restoration of the host immune response.

Abstract B002: TLR7/8-matured dendritic cells for therapeutic vaccination in AML: Results of a clinical Phase I/II trial

Abstract Postremission therapy for acute myeloid leukemia (AML) is critical for elimination of minimal residual disease (MRD). In patients not eligible for allogeneic stem cell transplantation, alternative treatment options are needed. Therapeutic vaccination with autologous dendritic cells (DCs) loaded with leukemia-associated antigens (LAAs) is a promising treatment strategy to induce anti-leukemic immune responses and to eradicate chemorefractory cells. Using a TLR7/8 agonist, we have developed a GMP-compliant 3-day protocol to differentiate monocytes of intensively pretreated AML patients into highly functional, therapeutic DCs. A phase I/II proof-of-concept study has been initiated using TLR7/8-matured DCs as postremission therapy of AML patients with a non-favorable risk profile in CR or CRi after intensive induction therapy (NCT01734304). DCs have been loaded with in vitro transcribed RNA encoding the LAAs WT1 and PRAME as well as CMVpp65 as adjuvant and surrogate antigen. Patients have been vaccinated intradermally with 5×106 DCs of each antigen species up to 10 times within 26 weeks. The primary endpoint of the phase I/II trial is feasibility and safety of the vaccination. Secondary endpoints are immunological responses and disease control. In total, 13 patients have been enrolled into the study. The first 6 patients were analysed in phase I for safety and toxicity of the DC vaccine. No higher grade toxicities were observed during their treatment and hence phase II has been initiated. DCs of sufficient number and quality were generated from leukapheresis in 10/11 cases. DCs exhibited an immune-stimulatory profile based on high surface expression of positive costimulatory molecules, the capacity to secrete IL-12p70, the migration towards a chemokine gradient and processing and presentation of antigen. In 9/9 vaccinated patients, we observed delayed-type hypersensitivity (DTH) responses at the vaccination site, accompanied by slight erythema and indurations at the injection site, but no grade III/IV toxicities. TCR repertoire analysis by NGS revealed an enrichment of particular clonotypes at DTH sites. In addition, we detected vaccine-specific T-cell responses by multimer staining and by Interferon-gamma-ELISPOT analysis: 7/7 patients showed responses to CMVpp65 and 2/7 exhibited responses to PRAME and WT1, respectively. In an individual treatment attempt, an enrolled patient with impending relapse was treated with a combination of DC vaccination and 5-azacytidine, resulting in MRD conversion. Long-term disease control and immunological responses are studied in the ongoing phase II trial. We conclude that vaccination with TLR7/8-matured, LAA-expressing DCs in AML is feasible, safe and induces anti-leukemia-specific immune responses in vivo. Citation Format: Katrin Deiser, Felix S. Lichtenegger, Frauke M. Schnorfeil, Thomas Köhnke, Torben Altmann, Veit Bücklein, Christian Augsberger, Andreas Moosmann, Monika Brüggemann, Mirjam HM Heemskerk, Beate Wagner, Wolfgang Hiddemann, Iris Bigalke, Gunnar Kvalheim, Marion Subklewe. TLR7/8-matured dendritic cells for therapeutic vaccination in AML: Results of a clinical Phase I/II trial [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B002.

Induced regulatory T cells are phenotypically unstable and do not protect mice from rapidly progressive glomerulonephritis.

Regulatory T (Treg) cells are a suppressive CD4+ T-cell subset. We generated induced Treg (iTreg) cells and explored their therapeutic potential in a murine model of rapidly progressive glomerulonephritis. Polyclonal naive CD4+ T cells were cultured in vitro with interleukin-2 (IL-2), transforming growth factor-β1, all-trans-retinoic acid and monoclonal antibodies against interferon-γ and IL-4, generating Foxp3+ iTreg cells. To enhance their suppressive phenotype, iTreg cultures were modified with the addition of a monoclonal antibody against IL-12p40 or by using RORγt-/- CD4+ T cells. Induced Treg cells were transferred into models of delayed-type hypersensitivity and experimental glomerulonephritis. The iTreg cells exhibited comparable surface receptor expression and in vitro suppressive ability to natural Treg cells, but did not regulate antigen-specific delayed-type hypersensitivity or systemic inflammatory immune responses, losing Foxp3 expression in vivo. In glomerulonephritis, transferred iTreg cells did not prevent renal injury or modulate systemic T helper type 1 immune responses. Induced Treg cells cultured with anti-IL-12p40 had an enhanced suppressive phenotype in vitro and regulated dermal delayed-type hypersensitivity in vivo, but were not protective against renal injury, losing Foxp3 expression, especially in the transferred cells recruited to the kidney. Use of RORγt-/- CD4+ T cells or iTreg cells generated from sensitized CD4+ Foxp3- cells did not regulate renal or systemic inflammatory responses in vivo. In conclusion, iTreg cells suppress T-cell proliferation in vitro, but do not regulate experimental glomerulonephritis, being unstable in this inflammatory milieu in vivo.

Allogeneic Sensitization and Tolerance Induction After Corneal Endothelial Cell Transplantation in Mice.

We evaluated the allogeneic response after corneal endothelial cell transplantation in the anterior chamber (AC) in a new mouse model by examining the acquisition of a delayed-type hypersensitivity (DTH) response, induction of allogeneic AC-associated immune deviation (ACAID), and acquisition of delayed transplantation tolerance. The corneal eyecups from C57BL/6 mice were prepared. The epithelial layer was detached with EDTA solution and treated with trypsin to release mouse-derived primary corneal endothelial cells (mpCECs). The mpCECs (1 × 104 cells) were transplanted into the AC of the eye or subcutaneously (SC) into the neck of BALB/c mice. In the mouse model of endothelial cell transplantation, the endothelial cells in a 2-mm central area of the cornea were eliminated by cryoinjury. The mpCEC transplant model was evaluated by measuring allogeneic cell survival and corneal thickness. The allospecific DTH response and ACAID induction were evaluated 1 week after transplantation. The long-term transplantation tolerance was evaluated by observing a secondary penetrating keratoplasty (PKP) performed on the same donor C57BL/6 mice. The SC injection of mpCECs induced a DTH response, whereas the AC injection induced ACAID. However, eyes inflamed by cryoinjury showed neither the DTH response nor ACAID following AC injection. The mpCECs survived for at least 1 week after injection. Penetrating keratoplasty allografts at 8 weeks after mpCEC transplantation survived indefinitely (100%). The mpCECs display low allogenicity in the AC and are capable of inducing allogeneic tolerance. Corneal endothelial cell transplantation into the AC may represent a safe technique for allogeneic transplantation.

The leishmanicidal activity of oleuropein is selectively regulated through inflammation- and oxidative stress-related genes.

BackgroundMuch research effort has been focused on investigating new compounds derived from low-cost sources, such as natural products, for treating leishmaniasis. Oleuropein derived from numerous plants, particularly from the olive tree, Olea europaea L. (Oleaceae), is a biophenol with many biological activities. Our previous findings showed that oleuropein exhibits leishmanicidal effects against three Leishmania spp. in vitro, and minimizes the parasite burden in L. donovani-infected BALB/c mice. The aim of the present study is to investigate the possible mechanism(s) that mediate this leishmanicidal activity.MethodsWe determined the efficacy of oleuropein in elevating ROS and NO production in L. donovani-infected J774A.1 macrophages and in explanted splenocytes and hepatocytes obtained from L. donovani-infected BALB/c mice. We also assessed the expression of genes that are related to inflammation, T-cell polarization and antioxidant defense, in splenocytes. Finally, we determined the ratios of specific IgG2a/IgG1 antibodies and DTH reactions in L. donovani-infected BALB/c mice treated with oleuropein.ResultsOleuropein was able to elevate ROS production in both in vitro and in vivo models of visceral leishmaniasis and raised NO production in ex vivo cultures of splenocytes and hepatocytes. The extensive oxidative stress found in oleuropein-treated mice was obviated by the upregulation of the host’s antioxidant enzyme (mGCLC) and the simultaneous downregulation of the corresponding enzyme of the parasite (LdGCLC). Moreover, oleuropein was able to mount a significant Th1 polarization characterized by the expression of immune genes (IL-12β, IL-10, TGF-β1, IFN-γ) and transcription factors (Tbx21 and GATA3). Moreover, this immunomodulatory effect was also correlated with an inhibitory effect on IL-1β gene expression, rather than with the expression of IL-1α, IL-1rn and TNF-α. Furthermore, oleuropein-treated BALB/c mice mounted a delayed-type hypersensitivity (DTH) response and an elevated Leishmania-specific IgG2a/IgG1 ratio that clearly demonstrated an in vivo protective mechanism.ConclusionThe ability of Oleuropein to promote a Th1 type immune response in L. donovani-infected BALB/c mice points towards the candidacy of this bioactive compound as an immunomodulatory agent that may complement therapeutic approaches to leishmaniasis.

Maternal feed restriction during pregnancy in Wistar rats: Evaluation of offspring using classical and immunoteratology protocols

Studies have revealed that impairment of the pregnant body weight reduces the fetal body weight and causes minor changes in skeletal development. The aim of the present study was to assess the effects of maternal feed restriction during pregnancy in offspring immune system development. Pregnant Wistar rats were distributed into 5 groups: 1 control in which dams received food ad libitum and 4 experimental groups in which dams were fed restricted amounts of rodent ration (16, 12, 9, or 6 g/rat/day) from the 6th to 17th gestation day. Teratogenicity was assessed using classical teratological evaluation and developmental immunotoxicology protocols. Maternal body weight gain, fetus weight, and placenta weight were reduced for feed-restricted females from the groups fed 12, 9, and 6 g/rat/day ( p < 0.05). No pup mortality was observed immediately after cesarean sections among the groups, and no visceral or skeletal malformations were detected. An immunoteratological study revealed an increase in the relative weight of the thymus and an increase in the phorbol myristate-acetate solution-induced hydrogen peroxide release by inflammatory cells in 21-day-old pups. Alterations in the delayed-type hypersensitivity response and the humoral immune response against sheep red blood cells were observed in pups from feed-restricted mothers. Feed restriction in Wistar rats during organogenesis did not promote structural malformations but resulted in offspring with lower birth weights and promoted significant changes in the immune responses of the rat pups.

Effect of Astragali Radix with different sulfur fumigation technologies on immune function in mice

To investigate the effect of Astragali Radix with different sulfur fumigation technologies on immune function in mice, and observe the effect of different Astragali Radix samples on carbon clearance in cyclophosphamide induced immunosuppressed mice, on immune organ weight in immunosuppressed mice and on delayed type hypersensitivity response (DTH) induced by 2,4 dinitrochlorobenzene. Carbon clearance index, phagocytic index, organ index and ear swelling rate were taken as the indexes. The results showed that, all of the Astragali Radix with different sulfur fumigation technologies markedly increased the carbon clearance index K, phagocytic index α, immune organ weight and improved the ability of DTH response in immunosuppressed mice. As compared with the model group, combined hot air-microwave group had the most significant difference, but when other groups were compared with and combined hot air-microwave group, only carbon clearance test had significant difference. From the perspective of pharmacodynamics, the effect of Astragali Radix with different sulfur fumigationon technologies on the immune function of mice was investigated, which provided a reference for the selection of appropriate alternative technology, and also provided guidance for clinical medication.

Effects of thymol and carvacrol on T-helper cell subset cytokines and their main transcription factors in ovalbumin-immunized mice

Thymol and carvacrol, two main components of thyme, have several valuable effects on the immune system. This study aims to evaluate the effects of these components on T-helper (TH) cell responses and their subsets in mice immunized with ovalbumin. The effects of these components on: a specific in vivo immune response were evaluated by assessing changes in delayed-type hypersensitivity (DTH); ex vivo splenocyte proliferative responses were evaluated using a BrdU assay gene expression of cytokines and key transcription factors involved in T-cells subset differentiation among the mouse splenocytes were assessed using real-time polymerase chain reaction (PCR); and splenocyte cytokine formation (ex vivo) and levels of the cytokines in mouse sera were measured by ELISA. Mice treated with thymol or carvacrol had reduced DTH responses (26% and 50%, respectively) compared with control mice. Thymol and carvacrol each diminished splenocyte proliferation to nearly 65–72% of control levels (p < 0.01). These agents also led to decreased TH1 [interleukin (IL)-2, interferon (IFN)-γ)], TH2 (IL-4) and TH17 (IL-17A) levels in the splenocyte cultures and in the sera of mice but increased levels of IL-10 and transforming growth factor (TGF)-β. Treated immunized mice showed significantly reduced T-box 21 (T-bet) expression from 3.8 [± 0.3]-fold in untreated ovalbumin-immunized mice to 0.9 [± 0.4]-(thymol) and 0.8 [± 0.2]-fold (carvacrol) (p < 0.01). GATA binding protein 3 (GATA-3) expression declined from 3.4 [± 0.4]- to 0.5 [± 0.3]-fold (thymol) and 0.6 [± 0.4]-fold (carvacrol), whereas RORγc decreased from 13.4 [± 1.6]- to 1.5 [± 0.6]-fold (thymol) and 0.8 [± 0.4]-fold (carvacrol) (p < 0.001). As carvacrol and thymol each suppressed the antigen-specific immune response by reducing TH cell-related cytokines\\specific transcription factors, this indicated their potential to modulate destructive immune responses attributed to T-cells over-activation.

Immuno-pathological studies on broiler chicken experimentally infected with Escherichia coli and supplemented with neem (Azadirachta indica) leaf extract

Aim:The present study was conducted to evaluate the effects of neem leaf extract (NLE) supplementation on immunological response and pathology of different lymphoid organs in experimentally Escherichia coli challenged broiler chickens.Materials and Methods:For this study, we procured 192-day-old broiler chicks from local hatchery and divided them into Groups A and Group B containing 96 birds each on the first day. Chicks of Group A were supplemented with 10% NLE in water, whereas chicks of Group B were not supplemented with NLE throughout the experiment. At 7th day of age, chicks of Group A were divided into A1 and A2 and Group B into B1 and B2 with 54 and 42 chicks, respectively, and chicks of Groups A1 and B1 were injected with E. coli O78 at 107 colony-forming units/0.5 ml intraperitoneally. Six chicks from each group were sacrificed at 0, 2, 4, 7, 14, 21, and 28 days post infection; blood was collected and thorough post-mortem examination was conducted. Tissue pieces of spleen and bursa of Fabricius were collected in 10% buffered formalin for histopathological examination. Serum was separated for immunological studies.Result:E. coli specific antibody titer was significantly higher in Group A1 in comparison to Group B1. Delayed-type hypersensitivity response against 2,4 dinirochlorobenzene (DNCB) antigen was significantly higher in Group A1 as compared to Group B1. Pathological studies revealed that E. coli infection caused depletion of lymphocytes in bursa of Fabricius and spleen. Severity of lesions in Group A1 was significantly lower in comparison to Group B1.Conclusion:10% NLE supplementation enhanced the humoral as well as cellular immune responses attributed to its immunomodulatory property in experimentally E. coli infected broiler chicken.

Vaccination with Irradiated Autologous Tumor Cells Mixed with Irradiated GM-K562 Cells Stimulates Antitumor Immunity and T Lymphocyte Activation in Patients with Recurrent Malignant Glioma

Recurrent malignant glioma carries a dismal prognosis, and novel therapies are needed. We examined the feasibility and safety of vaccination with irradiated autologous glioma cells mixed with irradiated GM-K562 cells in patients undergoing craniotomy for recurrent malignant glioma. We initiated a phase I study examining the safety of 2 doses of GM-K562 cells mixed with autologous cells. Primary endpoints were feasibility and safety. Feasibility was defined as the ability for 60% of enrolled subjects to initiate vaccination. Dose-limiting toxicity was assessed via a 3+3 dose-escalation format, examining irradiated tumor cells mixed with 5 × 10(6) GM-K562 cells or 1 × 10(7) GM-K562 cells. Eligibility required a priori indication for resection of a recurrent high-grade glioma. We measured biological activity by measuring delayed type hypersensitivity (DTH) responses, humoral immunity against tumor-associated antigens, and T-lymphocyte activation. Eleven patients were enrolled. Sufficient numbers of autologous tumor cells were harvested in 10 patients, all of whom went on to receive vaccine. There were no dose-limiting toxicities. Vaccination strengthened DTH responses to irradiated autologous tumor cells in most patients, and vigorous humoral responses to tumor-associated angiogenic cytokines were seen as well. T-lymphocyte activation was seen with significantly increased expression of CTLA-4, PD-1, 4-1BB, and OX40 by CD4(+) cells and PD-1 and 4-1BB by CD8(+) cells. Activation was coupled with vaccine-associated increase in the frequency of regulatory CD4(+) T lymphocytes. Vaccination with irradiated autologous tumor cells mixed with GM-K562 cells is feasible, well tolerated, and active in patients with recurrent malignant glioma. Clin Cancer Res; 22(12); 2885-96. ©2016 AACR.

Production of endocannabinoids by activated Tcells and Bcells modulates inflammation associated with delayed-type hypersensitivity.

Endocannabinoids are endogenous ligands for the cannabinoid (CB) receptors which include anandamide (AEA) and 2-arachidonyl glycerol (2-AG). 2-AG has been linked to inflammation due to its elevated expression in animal models of autoimmunity and hypersensitivity. However, administration of exogenous 2-AG has been shown to suppress inflammation making its precise role unclear. In the current study, we investigated the role of 2-AG following immunization of C57BL/6 (BL6) mice with methylated BSA (mBSA) antigen, which triggers both delayed-type hypersensitivity (DTH) and antibody response. We found that while naïve Tcells and Bcells expressed low levels of 2-AG, expression significantly increased upon activation. Furthermore, mBSA-immunized mice exhibited higher 2-AG concentration than naïve mice. Exogenous 2-AG treatment (40mg/kg) in mBSA-immunized mice led to reduced DTH response, and decreased Th1 and Th17-associated cytokines including IL-6, IL-2, TNF-α, and the IgG response. Addition of 2-AG to activated popliteal lymph node (PopLN) cell cultures also inhibited lymphocyte proliferation. Together, these data show for the first time that activated T and Bcells produce 2-AG, which plays a negative regulatory role to decrease DTH via inhibition of T-cell activation and proliferation. Moreover, these findings suggest that exogenous 2-AG treatment can be used therapeutically in Th1- or Th17-driven disease.

Effects of combined exposure to formaldehyde and benzene on immune cells in the blood and spleen in Balb/c mice.

Formaldehyde and benzene are the two major indoor air pollutants due to their prevalence and toxicity. This study aimed to explore the toxic effect on the spleen and relevant immune responses of Balb/c mice caused by exposure to a combination of formaldehyde and benzene. Balb/c mice were divided randomly into five groups (n=9/group): blank control group (Ctrl); solvent ([corn] Oil) control; formaldehyde only (FA, 3mg/m(3)); benzene only (BZ, 150mg/kg BW); and, formaldehyde+benzene group (FA+BZ). Exposures were performed for 8h/day, 5 day/week, for 2 weeks. Tail blood was collected after the final exposure; 24-h later, the mice were euthanized to permit assessment of a variety of immune endpoints. The endpoints' three areas were: (1) in living mice, body weight and delayed-type hypersensitivity (DTH) responses; (2) in blood, immune cell counts and serum antibody levels (serum hemagglutination); and, (3) in spleen samples, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), caspase-3 (cell apoptosis) levels and lymphocyte proliferation. In this study we fund (1) BZ and FA+BZ exposure can lead to the reduction in the number of some immune cells in peripheral blood; (2) Formaldehyde has certain synergistic effects on benzene-induced cytotoxicity in peripheral blood, (3) FA, BZ and FA+BZ exposure can lead to ROS and GSH depletion in spleen cells, and spleen cell apoptosis (caspase-3 increased) may be one of the downstream events, decreased splenic lymphocyte proliferation; and (4) the FA+BZ combined exposure can lead to the decreased body weight, serum antibody level (by serum hemagglutination assay).

IL-6R signaling inhibits generation of Th3 cells and is a promising therapeutic target for enhancing oral tolerance induction

Abstract Oral antigen administration results in systemic hyporesponsiveness to a subsequent challenge with the fed antigen (oral tolerance) and has been shown to inhibit autoimmune disease in multiple animal models. However, transfer of this strategy to the clinics has not yet been achieved. Our aim is to better understand mechanisms implicated in the generation of Th3 cells and oral tolerance in order to enhance oral tolerance induction for a use in the clinics. Low dose oral tolerance depends on the presence of Th3 type Tregs, characterized by surface expression of latent TGF-beta (LAP and TGF-beta) and its adapter protein GARP (LRRC32). We established an in vitro system to induce Th3 like cells that express LAP and GARP on their surface. In vitro induced CD4+LAP+ T cells suppress proliferation of naïve T cells and production of IL-17A by Th17 cells. RT- PCR showed that membrane bound TGF-beta correlated with mRNA transcription levels of GARP but not TGF-beta, suggesting that the availability of GARP directly limits the expression of membrane bound TGF-beta. We used this in vitro system to screen for molecules that modulate generation of Th3 like cells and identified IL-6 as a potent inhibitor of membrane TGF-beta. IL-6 inhibited transcription of GARP in a STAT3 dependent manner. To test the therapeutic potential of targeting IL-6R signaling additionally to oral tolerance induction we assessed the DTH (delayed-type hypersensitivity) response in wild-type or CD4CreIL6Rafl/fl mice after feeding with ovalbumin for 5 consecutive days. Importantly, oral tolerance was significantly improved in mice that lack IL-6R signaling in CD4+ T cells. This demonstrates that blocking of IL-6R signaling is a promising approach to enhance oral tolerance induction.

Immunoprotective responses of T helper type 1 stimulatory protein-S-adenosyl-L-homocysteine hydrolase against experimental visceral leishmaniasis.

It is well known that a patient in clinical remission of visceral leishmaniasis (VL) remains immune to reinfection, which provides a rationale for the feasibility of a vaccine against this deadly disease. In earlier studies, observation of significant cellular responses in treated Leishmania patients as well as in hamsters against leishmanial antigens from different fractions led to its further proteomic characterization, wherein S-adenosyl-L-homocysteine hydrolase (AdoHcy) was identified as a helper type 1 (Th1) stimulatory protein. The present study includes immunological characterization of this protein, its cellular responses [lymphoproliferation, nitric oxide (NO) production and cytokine responses] in treated Leishmania-infected hamsters and patients as well as prophylactic efficacy against Leishmania challenge in hamsters and the immune responses generated thereof. Significantly higher cellular responses were noticed against recombinant L. donovani S-adenosyl-L-homocysteine hydrolase (rLdAdoHcy) compared to soluble L. donovani antigen in treated samples. Moreover, stimulation of peripheral blood mononuclear cells with rLdAdoHcy up-regulated the levels of interferon (IFN)-γ, interleukin (IL)-12 and down-regulated IL-10. Furthermore, vaccination with rLdAdoHcy generated perceptible delayed-type hypersensitivity response and exerted considerably good prophylactic efficacy (∼70% inhibition) against L. donovani challenge. The efficacy was confirmed by the increased expression levels of inducible NO synthase and Th1-type cytokines, IFN-γ and IL-12 and down-regulation of IL-4, IL-10 and transforming growth factor (TGF)-β. The results indicate the potentiality of rLdAdoHcy protein as a suitable vaccine candidate against VL.

026 Boosting of the delayed-type hypersensitivity response to varicella-zoster virus antigen following zoster vaccination in ageing individuals is associated with a local reduction in regulatory T cells

026 Boosting of the delayed-type hypersensitivity response to varicella-zoster virus antigen following zoster vaccination in ageing individuals is associated with a local reduction in regulatory T cells

The Scorpion Toxin Analogue BmKTX-D33H as a Potential Kv1.3 Channel-Selective Immunomodulator for Autoimmune Diseases.

The Kv1.3 channel-acting scorpion toxins usually adopt the conserved anti-parallel β-sheet domain as the binding interface, but it remains challenging to discover some highly selective Kv1.3 channel-acting toxins. In this work, we investigated the pharmacological profile of the Kv1.3 channel-acting BmKTX-D33H, a structural analogue of the BmKTX scorpion toxin. Interestingly, BmKTX-D33H, with its conserved anti-parallel β-sheet domain as a Kv1.3 channel-interacting interface, exhibited more than 1000-fold selectivity towards the Kv1.3 channel as compared to other K+ channels (including Kv1.1, Kv1.2, Kv1.7, Kv11.1, KCa2.2, KCa2.3, and KCa3.1). As expected, BmKTX-D33H was found to inhibit the cytokine production and proliferation of both Jurkat cells and human T cells in vitro. It also significantly improved the delayed-type hypersensitivity (DTH) responses, an autoreactive T cell-mediated inflammation in rats. Amino acid sequence alignment and structural analysis strongly suggest that the “evolutionary” Gly11 residue of BmKTX-D33H interacts with the turret domain of Kv1 channels; it appears to be a pivotal amino acid residue with regard to the selectivity of BmKTX-D33H towards the Kv1.3 channel (in comparison with the highly homologous scorpion toxins). Together, our data indicate that BmKTX-D33H is a Kv1.3 channel–specific blocker. Finally, the remarkable selectivity of BmKTX-D33H highlights the great potential of evolutionary-guided peptide drug design in future studies.

Marijuana-derived Δ-9-tetrahydrocannabinol suppresses Th1/Th17 cell-mediated delayed-type hypersensitivity through microRNA regulation.

∆(9)-Tetrahydrocannabinol (THC) is one of the major bioactive cannabinoids derived from the Cannabis sativa plant and is known for its anti-inflammatory properties. Delayed-type hypersensitivity (DTH) is driven by proinflammatory T helper cells including the classic inflammatory Th1 lineage as well as the more recently discovered Th17 lineage. In the current study, we investigated whether THC can alter the induction of Th1/Th17 cells involved in mBSA-induced DTH response. THC treatment (20mg/kg) of C57BL/6 mice with DTH caused decreased swelling and infiltration of immune cells at the site of antigen rechallenge. Additionally, THC treatment decreased lymphocyte activation as well as Th1/Th17 lineage commitment, including reduced lineage-specific transcription factors and cytokines. Interestingly, while DTH caused an overexpression of miR-21, which increases Th17 differentiation via SMAD7 inhibition, and downregulation of miR-29b, an IFN-γ inhibitor, THC treatment reversed this microRNA (miR) dysregulation. Furthermore, when we transfected primary cells from DTH mice with miR-21 inhibitor or miR-29b mimic, as seen with THC treatment, the expression of target gene message was directly impacted increasing SMAD7 and decreasing IFN-γ expression, respectively. In summary, the current study suggests that THC treatment during DTH response can simultaneously inhibit Th1/Th17 activation via regulation of microRNA (miRNA) expression. • THC treatment inhibits simultaneous Th1/Th17 driven inflammation. • THC treatment corrects DTH-mediated microRNA dysregulation. • THC treatment regulates proinflammatory cytokines and transcription factors.