Release Of Th2 Cytokines Research Articles

Genipin crosslinking reduced the immunogenicity of xenogeneic decellularized porcine whole-liver matrices through regulation of immune cell proliferation and polarization.

Decellularized xenogeneic whole-liver matrices are plausible biomedical materials for the bioengineering of liver transplantation. A common method to reduce the inflammatory potential of xenogeneic matrices is crosslinking. Nevertheless, a comprehensive analysis of the immunogenic features of cross-linked decellularized tissue is still lacking. We aimed to reduce the immunogenicity of decellularized porcine whole-liver matrix through crosslinking with glutaraldehyde or genipin, a new natural agent, and investigated the mechanism of the immune-mediated responses. The histologic assessment of the host’s immune reaction activated in response to these scaffolds, as well as the M1/M2 phenotypic polarization profile of macrophages, was studied in vivo. The genipin-fixed scaffold elicited a predominantly M2 phenotype response, while the glutaraldehyde-fixed scaffold resulted in disrupted host tissue remodeling and a mixed macrophage polarization profile. The specific subsets of immune cells involved in the responses to the scaffolds were identified in vitro. Crosslinking alleviated the host response by reducing the proliferation of lymphocytes and their subsets, accompanied by a decreased release of both Th1 and Th2 cytokines. Therefore, we conclude that the natural genipin crosslinking could lower the immunogenic potential of xenogeneic decellularized whole-liver scaffolds.

Differential immunomodulatory activity of tumor cell death induced by cancer therapeutic toll-like receptor ligands.

Synthetic toll-like receptor (TLR) ligands stimulate defined immune cell subsets and are currently tested as novel immunotherapeutic agents against cancer with, however, varying clinical efficacy. Recent data showed the expression of TLR receptors also on tumor cells. In this study we investigated immunological events associated with the induction of tumor cell death by poly(I:C) and imiquimod. A human head and neck squamous cell carcinoma (HNSCC) cell line was exposed to poly(I:C) and imiquimod, which were delivered exogenously via culture medium or via electroporation. Cell death and cell biological consequences thereof were analyzed. For in vivo analyses, a human xenograft and a syngeneic immunocompetent mouse model were used. Poly(I:C) induced cell death only if delivered by electroporation into the cytosol. Cell death induced by poly(I:C) resulted in cytokine release and activation of monocytes in vitro. Monocytes activated by the supernatant of cancer cells previously exposed to poly(I:C) recruited significantly more Th1 cells than monocytes exposed to control supernatants. If delivered exogenously, imiquimod also induced tumor cell death and some release of interleukin-6, but cell death was not associated with release of Th1 cytokines, interferons, monocyte activation and Th1 recruitment. Interestingly, intratumoral injection of poly(I:C) triggered tumor cell death in tumor-bearing mice and reduced tumor growth independent of TLR signaling on host cells. Imiquimod did not affect tumor size. Our data suggest that common cancer therapeutic RNA compounds can induce functionally diverse types of cell death in tumor cells with implications for the use of TLR ligands in cancer immunotherapy.

A small molecule inhibitor for ATPase activity of Hsp70 and Hsc70 enhances the immune response to protein antigens.

The ATPase activities of Hsp70 and Hsc70 are known to be responsible for regulation of various biological processes. However, little is known about the roles of Hsp70 and Hsc70 in modulation of immune responses to antigens. In the present study, we investigated the effect of apoptozole (Az), a small molecule inhibitor of Hsp70 and Hsc70, on immune responses to protein antigens. The results show that mice administered with both protein antigen and Az produce more antibodies than those treated with antigen alone, showing that Az enhances immune responses to administered antigens. Treatment of mice with Az elicits production of antibodies with a high IgG2c/IgG1 ratio and stimulates the release of Th1 and Th2-type cytokines, suggesting that Az activates the Th1 and Th2 immune responses. The observations made in the present study suggest that inhibition of Hsp70 and Hsc70 activities could be a novel strategy designing small molecule-based adjuvants in protein vaccines.

Toluene diisocyanate and methylene diphenyl diisocyanate: asthmatic response and cross-reactivity in a mouse model.

Both 2,4-toluene diisocyanate (TDI) and 4,4-methylene diphenyl diisocyanate (MDI) can cause occupational asthma. In this study, we optimized our mouse model of chemical-induced asthma in the C57Bl/6 mice strain using the model agent TDI. Furthermore, we validated MDI in this mouse model and investigated whether cross-reactivity between TDI and MDI is present. On days 1 and 8, C57Bl/6 mice were dermally treated (20µl/ear) with 3% MDI, 2% TDI or the vehicle acetone olive oil (AOO) (3:2). On day 15, they received a single oropharyngeal challenge with 0.04% MDI, 0.01% TDI or the vehicle AOO (4:1). One day later, airway hyperreactivity (AHR) and pulmonary inflammation in the bronchoalveolar lavage (BAL) were assessed. Furthermore, total serum IgE levels, lymphocyte subpopulations in auricular lymph nodes and cytokine levels in supernatants of lymphocytes were measured. Both dermal sensitization with TDI or MDI resulted in increased total serum IgE levels along with T and B cell proliferation in the auricular lymph nodes. The auricular lymphocytes showed an increased release of both Th2 and Th1 cytokines. Mice sensitized and challenged with either TDI or MDI showed AHR, along with a predominant neutrophil lung inflammation. Mice sensitized with MDI and challenged with TDI or the other way around showed no AHR, nor BAL inflammation. Both TDI and MDI are able to induce an asthma-like response in this mouse model. However, cross-reactivity between both diisocyanates remained absent.

PARP inhibition blocks chronic asthma symptoms in a house dust mite asthma model and differentially modulates human CD4+T cell function (HYP2P.332)

Abstract Asthma incidence is on the rise. In the United States alone, more than 20 million individuals suffer from the disease. A sizable portion of these asthmatics do not respond to the existing drugs. Accordingly, the need for new therapies is an immediate a necessity. One of the most essential goals for treatment is the identification of drugs that block the release or effects of Th2 cytokines. Using HDM-based chronic model, we show that a single administration of olaparib, a PARP inhibitor, was effective in decreasing lung eosinophilia. Two additional administrations of the drug provided better protection and significantly reduced airway hyper-responsiveness. These results were confirmed using similarly exposed PARP-1−/− mice. Such protective effects may be mediated by a significant reduction in Th2 cytokines and a concomitant increase in T-regulatory cells. In addition, we show that PARP is highly activated in lung tissues and PBMCs from asthmatics demonstrating the relevance to the human disease. Using human CD4+T cells, we show that PARP inhibition significantly reduced GATA-3 expression, Th2 cytokine production without affecting production of IFN-γ, T cell proliferation, or the overall numbers of Th1 or Th2 cells. However and similarly to our animal studies, PARP inhibition markedly increased the number of human T-regulatory cells. Our results provide an additional support to the potential of PARP inhibition as a novel strategy for the treatment of asthma

Therapeutic effects of R8, a semi-synthetic analogue of Vasicine, on murine model of allergic airway inflammation via STAT6 inhibition.

This is a follow-up study of our previous work in which we screened a series of Vasicine analogues for their anti-inflammatory activity in a preventive OVA induced murine model of asthma. The study demonstrated that R8, one of the analogues, significantly suppressed the Th2 cytokine production and eosinophil recruitment to the airways. In the present study, we have been using two standard experimental murine models of asthma, where the mice were treated with R8 either during (preventive use) or after (therapeutic use) the development of asthma features. In the preventive model, R8 reduced inflammatory cell infiltration to the airways, OVA specific IgE and Th2 cytokine production. Also, the R8 treatment in the therapeutic model decreased methacholine induced AHR, Th2 cytokine release, serum IgE levels, infiltration of inflammatory cells into the airways, phosphorylation of STAT6 and expression of GATA3. Moreover, R8 not only reduced goblet cell metaplasia in asthmatic mice but also reduced IL-4 induced Muc5AC gene expression in human alveolar basal epithelial cells. Further, R8 attenuated IL-4 induced differentiation of murine splenocytes into Th2 cells in vitro. So, we may deduce that R8 treatment profoundly reduced asthma features by attenuating the differentiation of T cells into Th2 cells by interfering with the binding of IL-4 to its receptor in turn decreasing the phosphorylation of STAT6 and expression of GATA3 in murine model of asthma. These preclinical findings suggest a possible therapeutic role of R8 in allergic asthma.

PARP Inhibition Blocks Asthma Manifestation in a Chronic House Dust Mite (HDM) Asthma Model and Differentially Modulates Human CD4+T cell Function

One of the most essential goals for treatment of allergic asthma is the identification of therapeutic drugs that block the release or effects of Th2 cytokines. Here we show that PARP is highly activated in lung tissues and PBMCs from asthmatics demonstrating relevance to the human disease. Using HDM‐based chronic model, we show that a single administration of olaparib, a PARP inhibitor, was effective in decreasing lung eosinophilia and overall cellularity. Two additional administrations of the drug provided better protection and significantly reduced airway hyper‐responsiveness. These results were confirmed using similarly exposed PARP‐1−/− mice. Such protective effects may be mediated by a significant reduction in Th2 cytokines and a concomitant increase in T‐regulatory cells. Using human CD4+T cells, we show that PARP inhibition significantly reduced Th2 cytokine production in response to CD3/CD28 stimulation without affecting production of IFN‐γ, T cell proliferation, or the overall numbers of Th1 or Th2 cells. However and similarly to our animal studies, PARP inhibition markedly increased the number of human T‐regulatory cells suggesting that PARP inhibition may not affect Th1/Th2 differentiation but alters their ability to produce Th2 cytokines. Our findings provide additional support to the potential of PARP inhibition as a novel strategy for the treatment of asthma.Source: NIH, ECEB funds

P11 Induction of immunomodulatory cell death by synthetic RNA mimetics

Background Immune cells detect viral and bacterial DNA/RNA via specific so-called pattern recognition receptors (PRRs). Synthetic RNA and DNA mimetics stimulate defined immune cell subsets and are currently tested as novel immunotherapeutic agents against cancer with, however, varying clinical efficacy. Recent data showed the expression of RNA-recognizing receptors also on tumor cells. In this study we exposed human head and neck squamous cell carcinoma (HNSCC) cells to poly(I:C) and imiquimod, two commercially available immunotherapeutic RNA mimetics, and investigated immunological events associated with the induction of tumor cell death by these compounds. Methods A HNSCC cell line was exposed to poly(I:C) and imiquimod, which were delivered via culture medium or into the cytosol via electroporation. Cell death and cell biological consequences thereof were analyzed in vitro . For in vivo analyses a human HNSCC xenograft model was used. Results Poly(I:C) induced cell death only if delivered into the cytosol. Interestingly, injection of poly(I:C) into the tumor reduced tumor growth and induced tumor cell apoptosis in tumor bearing mice, whereas imiquimod did not affect tumor size. Cell death induced by poly(I:C) resulted in cytokine release (interleukin-6, type I interferon and CXCL-10) and activation of monocytes in vitro . Monocytes activated by the supernatant of cancer cells previously exposed to poly(I:C) recruited significantly more Th1 cells than monocytes exposed to control supernatants. If delivered exogenously, Imiquimod also induced tumor cell death and release of interleukin-6, but cell death was not associated with release of Th1 cytokines, interferons, monocyte activation and Th1 recruitment. Conclusion Our data demonstrate the induction of immunostimulatory HNSCC cell death by intracellular delivery of poly(I:C). Interestingly, imiquimod also induced cancer cell death, but failed to promote additional immunostimulatory mechanisms. These findings have implications for the use of RNA mimetics in cancer immunotherapy and suggest the local delivery of RNA compounds. In addition to the well-known activation of immune cells, local delivery of RNA mimetics to the malignant tissue will induce additional tumor cell death associated with compound-specific promotion of local cellular immunity.

Mechanisms of Pin1 Regulation of IRAKM Stability in Toll-Like Receptor/Interleukin-1 Receptor Signaling

The molecular mechanisms of asthma, a chronic inflammatory sickness that involves activation of the innate immunity signaling pathways, are not yet fully understood. Our studies focus on the peptidyl-prolyl isomerase enzyme Pin1 and its substrates in innate immunity signaling. Prolyl peptide bonds, such as in pS/T-P motifs, can exist in two distinct isomer conformations, cis and trans, that exchange on a slow time scale (several minutes). Isomerization of pS/T-P motifs can be accelerated by Pin1. Pin1 has recently been shown to be activated by IL-33 which activates IL-1R signaling in Th2 cells, inducing asthma. Pin1 knockout (KO -/-) abolishes Th2 cytokine release thereby preventing asthma symptoms. Pin1 also prevents degradation of IRAKM, a negative regulator of TLR/IL-1R signaling, but promotes the nuclear localization of IRAKM. IRAKM is phosphorylated upon IL-33 treatment. The phosphomimetic mutation IRAKM S110E is dramatically stabilized in a Pin1-dependent manner, even more than wild type IRAKM. We test the model that Pin1 regulation of IRAKM stability and nuclear localization is mediated by the direct interaction of Pin1 with phosphorylated IRAKM. The dissociation constants, KD, for Pin1 binding to phosphorylated IRAK-M (pIRAKM) and to the phosphomimetic mutation IRAKM S110E were determined using the 1H −15N HSQC NMR experiment. Furthermore, the Pin1 catalyzed isomerization of wild type pIRAKM and IRAKM S110E were measured using the ROESY NMR experiment. This study is expected to contribute to our understanding of the novel mechanism of Pin1 regulation of the TLR/IL-1R signaling pathway in terms of specific domains of IRAKM.

Expression of DP2 (CRTh2), a prostaglandin D₂ receptor, in human mast cells.

PGD2 has long been implicated in allergic diseases. Recent cloning of a second PGD2 receptor, DP2 (also known as CRTh2), led to a greater understanding of the physiological and pathophysiological implications of PGD2. PGD2 signaling through DP1 and DP2 mediates different and often opposite effects in many cell types of the immune system. Although mast cells (MC) are the largest source of PGD2 in the body, there is little information about their potential expression of DP2 and its functional significance. In this study, we show that tissue MC in human nasal polyps express DP2 protein, and that human MC lines and primary cultured human MC express mRNA as well as protein of DP2. By immunohistochemistry, we detected that 34% of MC in human nasal polyps expressed DP2. In addition, flow cytometry showed that 87% of the LAD2 human MC line and 98% of primary cultured human MC contained intracellular DP2. However, we could not detect surface expression of DP2 on human MC by single cell analysis using imaging flow cytometry. Blocking of endogenous PGD2 production with aspirin did not induce surface expression of DP2 in human MC. Two DP2 selective agonists, DK-PGD2 and 15R-15-methyl PGD2 induced dose-dependent intracellular calcium mobilization that was abrogated by pertussis toxin, but not by three DP2 selective antagonists. MC mediator release including degranulation was not affected by DP2 selective agonists. Thus, human MC express DP2 intracellularly rather than on their surface, and the function of DP2 in human MC is different than in other immune cells such as Th2 cells, eosinophils and basophils where it is expressed on the cell surface and induces Th2 cytokine and/or granule associated mediator release. Further studies to elucidate the role of intracellular DP2 in human MC may expand our understanding of this molecule and provide novel therapeutic opportunities.

CD1d-mediated Presentation of Endogenous Lipid Antigens by Adipocytes Requires Microsomal Triglyceride Transfer Protein

Obesity-induced adipose tissue (AT) dysfunction results in a chronic low-grade inflammation that predisposes to the development of insulin resistance and type 2 diabetes. During the development of obesity, the AT-resident immune cell profile alters to create a pro-inflammatory state. Very recently, CD1d-restricted invariant (i) natural killer T (NKT) cells, a unique subset of lymphocytes that are reactive to so called lipid antigens, were implicated in AT homeostasis. Interestingly, recent data also suggest that human and mouse adipocytes can present such lipid antigens to iNKT cells in a CD1d-dependent fashion, but little is known about the lipid antigen presentation machinery in adipocytes. Here we show that CD1d, as well as the lipid antigen loading machinery genes pro-saposin (Psap), Niemann Pick type C2 (Npc2), α-galactosidase (Gla), are up-regulated in early adipogenesis, and are transcriptionally controlled by CCAAT/enhancer-binding protein (C/EBP)-β and -δ. Moreover, adipocyte-induced Th1 and Th2 cytokine release by iNKT cells also occurred in the absence of exogenous ligands, suggesting the display of endogenous lipid antigen-D1d complexes by 3T3-L1 adipocytes. Furthermore, we identified microsomal triglyceride transfer protein, which we show is also under the transcriptional regulation of C/EBPβ and -δ, as a novel player in the presentation of endogenous lipid antigens by adipocytes. Overall, our findings indicate that adipocytes can function as non-professional lipid antigen presenting cells, which may present an important aspect of adipocyte-immune cell communication in the regulation of whole body energy metabolism and immune homeostasis.

Mycobacterium tuberculosis multidrug resistant strain M induces an altered activation of cytotoxic CD8+ T cells.

In human tuberculosis (TB), CD8+ T cells contribute to host defense by the release of Th1 cytokines and the direct killing of Mycobacterium tuberculosis (Mtb)-infected macrophages via granule exocytosis pathway or the engagement of receptors on target cells. Previously we demonstrated that strain M, the most prevalent multidrug-resistant (MDR) Mtb strain in Argentine, is a weak inducer of IFN-γ and elicits a remarkably low CD8-dependent cytotoxic T cell activity (CTL). In contrast, the closely related strain 410, which caused a unique case of MDR-TB, elicits a CTL response similar to H37Rv. In this work we extend our previous study investigating some parameters that can account for this discrepancy. We evaluated the expressions of the lytic molecules perforin, granzyme B and granulysin and the chemokine CCL5 in CD8+ T cells as well as activation markers CD69 and CD25 and IL-2 expression in CD4+ and CD8+ T cells stimulated with strains H37Rv, M and 410. Our results demonstrate that M-stimulated CD8+ T cells from purified protein derivative positive healthy donors show low intracellular expression of perforin, granzyme B, granulysin and CCL5 together with an impaired ability to form conjugates with autologous M-pulsed macrophages. Besides, M induces low CD69 and IL-2 expression in CD4+ and CD8+ T cells, being CD69 and IL-2 expression closely associated. Furthermore, IL-2 addition enhanced perforin and granulysin expression as well as the degranulation marker CD107 in M-stimulated CD8+ T cells, making no differences with cells stimulated with strains H37Rv or 410. Thus, our results highlight the role of IL-2 in M-induced CTL activity that drives the proper activation of CD8+ T cells as well as CD4+ T cells collaboration.

Heme oxygenase-1/CO protect against post-traumatic suppression of adaptive immune responses (IRC8P.475)

Abstract Traumatic injury induces a systemic dysregulated immuno-inflammatory response that contributes to mortality. This response includes an upregulation of pro-inflammatory cytokines by innate cells and an independent suppression of adaptive immunity characterized by reduced Th1 responses. Heme oxygenase-1 (HO-1) and its byproduct, carbon monoxide (CO), are protective against injury-induced inflammation and end-organ damage, but their beneficial role in the dysregulation of adaptive responses following trauma is unknown. To investigate this, B6 mice received multiple-trauma or no manipulation, and were given either vehicle or a HO-1 inducer (Cobalt protoporphyrin, CoPP; 5mg/kg) prior to injury. Another cohort received either room air or CO (250ppm) for 4 hours directly post-injury. At 48hr post-trauma, Tcell functions were assessed by ex-vivo mitogen-induced Tcell proliferation and Th1 cytokine release (IL-2 and IFN-γ). As expected after injury, a near 50% reduction in Tcell proliferation was seen in vehicle- or air-treated trauma mice, compared to uninjured controls (p=0.007). A parallel reduction in Tcell IL-2 and IFN-γ release was observed. In clear contrast, CoPP or CO treatment prevented trauma-induced reductions in Tcell proliferation and cytokine release (fold difference 0.90±0.10 and 1.21±0.16 to uninjured controls, respectively). Overall, HO-1 represents an endogenous protective pathway that can be exploited following trauma to preserve normal immune functions & survival.

Natural killer: dendritic cell activation using anti-CD137 mAb improves cetuximab mediated innate and adaptive immune responses in head and neck cancer (TUM7P.948)

Abstract EGFR-specific mAb cetuximab mediated activation of NK by FcγRIIIa cross-linking is important for anti-tumor immunity.Following cetuximab activation,NK express costimulatory receptor CD137, which can be ligated to improve activity of cetuximab-activated NK,which promote DC maturation and CD8+ T-cell priming, leading to tumor antigen spreading and Th1 cytokine release through NK-DC cross-talk. We show that the clinically available CD137 agonist mAb augments cetuximab-mediated ADCC in FcγRIIIa-V/F donors,but not in poor binding FcγRIIIa-F/F.We show that cetuximab-activated NK induce CD137 expression on DC, which can be further stimulated by CD137 mAb, leading to enhanced DC maturation and cross-presentation capacity.In a prospective phase II trial of neoadjuvant cetuximab in stage III/IV HNC patients, intratumoral activated NK cells also upregulated CD137, which was influenced by FcγRIIIa-V/F polymorphism. Moreover, cetuximab treatment induces CD137 expression on intratumoral activated CTL.We show that FcγRIIIaV/F polymorphism on NK cells greatly influences their induction of CD137 expression, as does the EGFR receptor density on HNC.Thus, careful selection of HNC patients in combination immunotherapy using cetuximab plus CD137 mAb may be necessary, using EGFR level of expression and FcgammaRIIIa genotype, to optimize NK:DC priming of tumor antigen specific CTL.Results suggest a beneficial effect of combination of cetuximab and CD137 mAb in currently designed clinical trials.

Signaling via neurokinin 1 and 2 receptors exerts opposing effects on mast cell pro-inflammatory and Th2-biasing functions. (HYP3P.347)

Abstract Following activation via FcεRI, peripheral tissue mast cells (MCs) initiate and sustain atopic diseases, by secreting proinflammatory and Th2 biasing cytokines. MC express neuropeptide receptors and are exposed to neuroinflammatory mediators. However, the role of neuropeptides in FcεRI-activated MCs remains mainly unknown. Two of the most potent pro-inflammatory neuropeptides, substance-P (SP) and hemokinin-1 (HK-1), bind with high affinity to the neurokinin 1 receptor (NK1R) and with lower affinity to the NK2R. We addressed in vitro and in vivo the role of the NK1R and NK2R in MC responses initiated via FcεRI. In vitro, FcεRI activation of bone marrow derived (BM)MCs from wild type (WT) mice increased NK1R and NK2R expression and HK-1 release, but not release of SP. Utilizing NK1R-/- BMMCs, we determined that the NK1R was necessary for Akt-NFκB signaling and for the secretion of the pro-inflammatory TNF-α, IL-6 and Th2 biasing IL-4 and IL-13. Conversely, inhibition of NK2R signaling in WT BMMCs with GR159897 decreased the release of proinflammatory and Th2 cytokines. In vivo, NK1R signaling was necessary for amplifying IgE-Ag mediated cutaneous and systemic anaphylaxis and chronic asthma. We conclude that NK1R and the NK2R display opposing effects on the inflammatory function of FcεRI-activated MCs relevant for the control of MC derived atopic disorders.

Immunological derangement in hypocellular myelodysplastic syndromes.

Hypocellular or hypoplastic myelodysplastic syndromes (HMDS) are a distinct subgroup accounting for 10-15% of all MDS patients, that are characterized by the presence of bone marrow (BM) hypocellularity, various degree of dysmyelopoiesis and sometimes abnormal karyotype. Laboratory and clinical evidence suggest that HMDS share several immune-mediated pathogenic mechanisms with acquired idiopathic aplastic anemia (AA). Different immune-mediated mechanisms have been documented in the damage of marrow hematopoietic progenitors occurring in HMDS; they include oligoclonal expansion of cytotoxic T lymphocytes (CTLs), polyclonal expansion of various subtypes of T helper lymphocytes, overexpression of FAS-L and of the TNF-related apoptosis-inducing ligand (TRAIL), underexpression of Flice-like inhibitory protein long isoform (FLIPL) in marrow cells as well as higher release of Th1 cytokines, such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). It has also been documented that some HMDS patients have higher frequency of polymorphisms linked both to high production of proinflammatory cytokines such as TNF-α and transforming growth factor-β and to the inhibition of T-cell mediated immune responses such as interleukin-10, further suggesting that immune-mediated mechanisms similar to those seen in AA patients may also operate in HMDS. Clinically, the strongest evidence for immune-mediated hematopoietic suppression in some HMDS is the response to immunosuppression including mainly cyclosporine, anti-thymocyte globulin and/or cyclosporine, or alemtuzumab. Here we review all these immune mechanisms as well as the influence of this deranged cellular and humoral immunologic mileau on the initiation and possible progression of MDS. All these observations are pivotal not only for a better understanding of MDS pathophysiology, but also for their immediate clinical implications, eventually leading to the identification of MDS patients who may benefit from immunosuppression.

An In Silico Approach for Modelling T-Helper Polarizing iNKT Cell Agonists

Many analogues of the glycolipid alpha-galactosylceramide (α-GalCer) are known to activate iNKT cells through their interaction with CD1d-expressing antigen-presenting cells, inducing the release of Th1 and Th2 cytokines. Because of iNKT cell involvement and associated Th1/Th2 cytokine changes in a broad spectrum of human diseases, the design of iNKT cell ligands with selective Th1 and Th2 properties has been the subject of extensive research. This search for novel iNKT cell ligands requires refined structural insights. Here we will visualize the chemical space of 333 currently known iNKT cell activators, including several newly tested analogues, by more than 3000 chemical descriptors which were calculated for each individual analogue. To evaluate the immunological responses we analyzed five different cytokines in five different test-systems. We linked the chemical space to the immunological space using a system biology computational approach resulting in highly sensitive and specific predictive models. Moreover, these models correspond with the current insights of iNKT cell activation by α-GalCer analogues, explaining the Th1 and Th2 biased responses, downstream of iNKT cell activation. We anticipate that such models will be of great value for the future design of iNKT cell agonists.

Induction Of Anti-Myeloma Cellular and Humoral Immunity By Pre-Targeting Clonogenic Myeloma Cells Prior To Stem Cell Transplant With T Cells Armed With Anti-CD3 x Anti-CD20 Bispecific Antibody Leads To Transfer Of Cellular and Humoral Anti-Myeloma Immunity

▪A phase Ib trial was conducted in standard and high risk multiple myeloma (MM) patients to determine whether infusions of anti-CD3 x anti-CD20 bispecific antibody (CD20Bi) armed activated T cells (aATC) prior to autologous stem cell transplant (ASCT) were safe and would induce anti-myeloma (anti-MM) immunity that could be detected after ASCT. We hypothesized that targeting CD20+/CD138- myeloma clonogenic cells with aATC would release “MM antigens” including anti-Sry-HMG-box 2 (SOX-2). Engagement of target cells by aATC also induces release of Th1 cytokines by aATC creating an environment conducive for in situ immunization against MM antigens. Twelve patients received two infusions of 1010 one week apart and within 4 weeks prior to ASCT. Four patients received a 10 x 109 aATC booster infusion between 6 and 18 months after ASCT. There were significant increases in the fraction of patients who developed IFN-g EliSpots directed at CD20+ Daudi lymphoma cells (p<0.06), CD20 negative RPMI 8226 multiple myeloma cells (p<0.02), and K562 natural killer cell targets (p<0.01) from 6 to 12 months after ASCT. Specific cytotoxicity directed at Daudi (p<0.03), K562 (p<0.009), and RPMI 8226 (p<0.02) targets increased significantly after aATC infusions and post ASCT compared to pre infusion baseline cytotoxicity. SOX-2 IgG antibody levels increased above baseline after aATC infusions (p<0.002) and post ASCT (p<0.02) (Fig. 1A). There was no significant difference observed between pre and 1 month post ASCT in anti-SOX-2 levels or between pre and 3 month post ASCT serum samples from control ASCT patients who did not receive IT (Fig. 1B). In order to determine whether there was an association between the level of anti-SOX-2 antibodies and clinical outcome, anti-SOX-2 antibody levels were plotted in patients who progressed and those who did not progress. The box plots show the clear separation between two groups. The median anti-SOX-2 antibody levels at 3 months after ASCT in patients who relapsed was 19.7 ng/ml (25-75%, 8.6 - 40.3) and in patients who remained in remission was 48.1 ng/ml (37.5 - 73.3), these data suggest that anti-SOX-2 levels may predict clinical outcomes (Fig. 1C). There were no dose-limiting toxicities. In summary, aATC infusions pre ASCT induced anti-myeloma IFN-g EliSpot and anti-SOX-2 IgG responses that could be detected at 6-12 months after ASCT and these responses were boosted in 4 of 4 patients after ASCT. Data suggest that infusions of targeted T cells given prior to ASCT could induce cellular and humoral anti-myeloma immunity that could be transferred, detected, and boosted after ASCT. These results have implications for the design of immunotherapy in combination with ASCT. [Display omitted] Disclosures:Lum:Transtarget Inc: Equity Ownership, In situ Immunization Patent, In situ Immunization Patent Patents & Royalties.

Hepatic Cytochrome P450s, Phase II Enzymes and Nuclear Receptors Are Downregulated in a Th2 Environment during Schistosoma mansoni Infection

Inflammation and infection downregulate the activity and expression of cytochrome P450s (P450s) and other drug metabolizing enzymes (DMEs) involved in hepatic drug clearance. Schistosoma mansoni infection was reported to cause a downregulation of hepatic P450-dependent activities in mouse liver, but little is known about the specific enzymes affected or whether phase II DMEs are also affected. Here we describe the effect of murine schistosomiasis on the expression of hepatic P450s, NADPH-cytochrome P450 reductase (Cpr), phase II drug metabolizing enzymes, and nuclear receptors at 30 and 45 days postinfection (dpi). Although the hepatic expression of some of these genes was altered at 30 dpi, we observed substantial changes in the expression of the majority of P450 mRNAs and proteins measured, Cpr protein, as well as many of the UDP-glucuronosyltransferases and sulfotransferases at 45 dpi. S. mansoni infection also altered nuclear receptor expression, inducing mRNA levels at 30 dpi and depressing levels at 45 dpi. S. mansoni evoked a T helper 2 (Th2) inflammatory response at 45 dpi, as indicated by the induction of hepatic Th2 cytokine mRNAs [interleukins 4, 5, and 13], whereas the hepatic proinflammatory response was relatively weak. Thus, chronic schistosomiasis markedly and selectively alters the expression of multiple DMEs, which may be associated with Th2 cytokine release. This would represent a novel mechanism of DME regulation in disease states. These findings have important implications for drug testing in infected mice, whereas the relevance to humans with schistosomiasis needs to be determined.

Effects of montelukast on subepithelial/peribronchial fibrosis in a murine model of ovalbumin induced chronic asthma

Montelukast, a leukotriene receptor antagonist, is used commercially as a maintenance treatment for asthma and to relieve allergic symptoms. In this study, we evaluated the protective effects of montelukast against the airway inflammation and fibrosis using a murine model of ovalbumin (OVA) induced chronic asthma. The animals received OVA challenge three times a week for 4 weeks. Montelukast (30 mg/kg) was administrated orally once a day for 4 weeks. The administration of montelukast caused a reduction in elevated interleukin (IL)-4, IL-13, eotaxin, immunoglobulin (Ig), inflammatory cell infiltration into the airways, and mucus production after repeated OVA challenges. To investigate the antifibrotic mechanism of montelukast, we examined the expression of profibrotic mediators, including vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β1, and Smad3 proteins in the lung tissue using western blotting and immunohistochemistry. The administration of montelukast reduced the overexpression of profibrotic proteins in the lung tissue, which was confirmed by immnunohistochemistry. These results are consistent with a histopathological examination of lung tissue with Masson's trichrome stain. In conclusion, the administration of montelukast reduced airway inflammation and pulmonary fibrosis by reducing the release of Th2 cytokines and the expression of VEGF, TGF-β1/Smad3 in the lung tissue.