Receptor Ligation Research Articles

AKT/mTORC2 Inhibition Activates FOXO1 Function in CLL Cells Reducing B-Cell Receptor-Mediated Survival.

To determine whether inhibition of mTOR kinase-mediated signaling represents a valid therapeutic approach for chronic lymphocytic leukemia (CLL). Stratification of mTOR activity was carried out in patients with primary CLL samples and an aggressive CLL-like mouse model. The potency of dual mTOR inhibitor AZD8055 to induce apoptosis in primary CLL cells was assessed in the presence/absence of B-cell receptor (BCR) ligation. Furthermore, we addressed the molecular and functional impact of dual mTOR inhibition in combination with BTK inhibitor ibrutinib. Differential regulation of basal mTORC1 activity was observed in poor prognostic CLL samples, with elevated p4EBP1T37/46 and decreased p70S6 kinase activity, suggesting that dual mTORC1/2 inhibitors may exhibit improved response in poor prognostic CLL compared with rapalogs. AZD8055 treatment of primary CLL cells significantly reduced CLL survival in vitro compared with rapamycin, preferentially targeting poor prognostic subsets and overcoming BCR-mediated survival advantages. Furthermore, AZD8055, and clinical analog AZD2014, significantly reduced CLL tumor load in mice. AKT substrate FOXO1, while overexpressed in CLL cells of poor prognostic patients in LN biopsies, peripheral CLL cells, and mouse-derived CLL-like cells, appeared to be inactive. AZD8055 treatment partially reversed FOXO1 inactivation downstream of BCR crosslinking, significantly inhibiting FOXO1T24 phosphorylation in an mTORC2-AKT-dependent manner, to promote FOXO1 nuclear localization, activity, and FOXO1-mediated gene regulation. FOXO1 activity was further significantly enhanced on combining AZD8055 with ibrutinib. Our studies demonstrate that dual mTOR inhibitors show promise as future CLL therapies, particularly in combination with ibrutinib.

LC3-associated phagocytosis at a glance.

Classically, canonical autophagy has been considered a survival mechanism initiated in response to nutrient insufficiency. We now understand that autophagy functions in multiple scenarios where it is necessary to maintain homeostasis. Recent evidence has established that a variety of non-canonical functions for autophagy proteins are mechanistically and functionally distinct from autophagy. LC3-associated phagocytosis (LAP) is one such novel function for autophagy proteins and is a contributor to immune regulation and inflammatory responses across various cell and tissue types. Characterized by the conjugation of LC3 family proteins to phagosome membranes, LAP uses a portion of the canonical autophagy machinery, following ligation of surface receptors that recognize a variety of cargos including pathogens, dying cells, soluble ligands and protein aggregates. However, instead of affecting canonical autophagy, manipulation of the LAP pathway in vivo alters immune activation and inflammatory responses. In this Cell Science at a Glance article and the accompanying poster, we detail the divergence of this distinctive mechanism from that of canonical autophagy by comparing and contrasting shared and unique components of each pathway.

The Evolving Role of TRAFs in Mediating Inflammatory Responses.

TRAFs [tumor necrosis factor (TNF) receptor associated factors] are a family of signaling molecules that function downstream of multiple receptor signaling pathways and play a pivotal role in the biology of innate, and adaptive immune cells. Following receptor ligation, TRAFs generally function as adapter proteins to mediate the activation of intracellular signaling cascades. With the exception of TRAF1 that lacks a Ring domain, TRAFs have an E3 ubiquitin ligase activity which also contributes to their ability to activate downstream signaling pathways. TRAF-mediated signaling pathways culminate in the activation of several transcription factors, including nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPKs; e.g., ERK-1 and ERK-2, JNK, and p38), and interferon-regulatory factors (IRF; e.g., IRF3 and IRF7). The biological role of TRAFs is largely due to their ability to positively or negatively regulate canonical and non-canonical NF-κB signaling. While TRAF-mediated signaling regulates various immune cell functions, this review is focused on the recent advances in our knowledge regarding the molecular mechanisms through which TRAF proteins regulate, positively and negatively, inflammatory signaling pathways, including Toll–IL-1 receptors, RIG-I like receptors, and Nod-like receptors. The review also offers a perspective on the unanswered questions that need to be addressed to fully understand how TRAFs regulate inflammation.

Is There a Positive Side to T Cell Exhaustion?

T cell “exhaustion” describes a state of late-stage differentiation usually associated with active prevention of functionality via ligation of negative signaling receptors on the cell surface, and which can be reversed by blocking these interactions. This contrasts with T cell “senescence,” which has been defined as a state that is maintained by intrinsic internal cell signaling (caused by DNA damage or other stresses) and which can be reversed pharmacologically. Interventions to alleviate these two different categories of inhibitory pathways may be desirable in immunotherapy for cancer and possibly certain infectious diseases, but reciprocally inducing and maintaining these states, or some properties thereof, may be beneficial in organ transplantation and autoimmunity. Even under physiological non-pathological conditions, T cell exhaustion and senescence may play a role in the retention of T cell clones required for immunosurveillance, and prevent their loss via elimination at the Hayflick limit. This essay briefly reviews T cell exhaustion in contrast to replicative senescence, and circumstances under which their modulation may be beneficial.

Role of Complement Activation in Allograft Inflammation.

Novel paradigms have broadened our understanding of mechanisms through which complement mediates allograft inflammation/injury. Herein we review advances in the field and highlight therapeutic implications. Pre-clinical and translational human trials have elucidated complement-dependent mechanisms of post-transplant ischemia-reperfusion (I/R) injury. Immune cell-derived, and intracellular, complement activation are newly linked to proinflammatory T cell immunity relevant to allograft rejection. Complement-induced immune regulation, including C5a ligation of C5a receptor 2 on T cells, C5a/C5a receptor 1 interactions on regulatory myeloid cells, and C1q binding to CD8+ T cells can inhibit proinflammatory T cells and/or prolong murine allograft survival. Pilot trials of complement inhibition to treat/prevent human I/R- or antibody-initiated allograft injury show promise. The complement system participates in allograft injury through multiple context- dependent mechanisms involving various components and receptors. These new insights along with development and implementation of individualized complement inhibitory strategies have potential to improve transplant outcomes.

Extra-Nuclear and Nuclear Rarα Reciprocally Control Tcr-Induced Proliferation and Differentiation

Ligation of nuclear retinoic acid receptors (RARs) by retinoic acid (RA) activates transcription and promotes cell differentiation. RARs can also participate in non-genomic functions in extra-nuclear spaces. Here we identify an alternative isoform of RARα expressed in the cytoplasm of T lymphocytes. Extranuclear RARα functions in T cell receptor (TCR) proximal signaling and NOTCH/c-MYC-driven proliferation. In contrast to nuclear RARα, RA negatively affects extra-nuclear RARα function in T cells. Upon activation, TCR signaling induces expression of the cellular retinoic acid binding protein 2 (CRABP2), which transports RA from the cytoplasm to the nucleus to activate nuclear RARα and at the same time sequesters RA away from extra-nuclear RARα thereby promoting TCR-driven proliferation. These data show that T cell proliferation and differentiation are reciprocally regulated by extra-nuclear and nuclear RARα and that activation-induced CRABP2 functions as a rheostat of TCR signaling via its dual control over the non-genomic and genomic actions of cytoplasmic and nuclear RARα.

Quinacrine-mediated detection of intracellular ATP.

Several antineoplastic agents are endowed with the ability to induce immunogenic cell death (ICD), a modality of cellular demise that is accompanied by the release of danger associated molecular patterns such as adenosine triphosphate (ATP) into the tumor microenvironment. ATP-mediated ligation of purinergic P2R receptors then facilitates the chemotactic recruitment and activation of innate immune effectors, thus favoring the induction of anticancer immunity. Here, we provide a protocol for the fluorescence microscopy-based quantification of ICD-associated ATP secretion that is amenable to high-throughput screening. As compared to the traditional luciferase-based detection of ATP in cell culture supernatants, the analysis presented here is cost-efficient and can be combined with the parallel assessment of cellular morphology.

Natural Killer Cells Integrate Signals Received from Tumour Interactions and IL2 to Induce Robust and Prolonged Anti-Tumour and Metabolic Responses.

Natural Killer (NK) cells are lymphocytes with an important role in anti-tumour responses. NK cells bridge the innate and adaptive arms of the immune system; they are primed for immediate anti-tumour function but can also have prolonged actions alongside the adaptive T cell response. However, the key signals and cellular processes that are required for extended NK cell responses are not fully known. Herein we show that murine NK cell interaction with tumour cells induces the expression of CD25, the high affinity IL2 receptor, rendering these NK cells highly sensitive to the T cell-derived cytokine IL2. In response to IL2, CD25high NK cells show robust increases in metabolic signalling pathways (mTORC1, cMyc), nutrient transporter expression (CD71, CD98), cellular growth and in NK cell effector functions (IFNγ, granzyme B). Specific ligation of an individual activating NK cell receptor, NK1.1, showed similar increases in CD25 expression and IL2-induced responses. NK cell receptor ligation and IL2 collaborate to induce mTORC1/cMyc signalling leading to high rates of glycolysis and oxidative phosphorylation (OXPHOS) and prolonged NK cell survival. Disrupting mTORC1 and cMyc signalling in CD25high tumour interacting NK cells prevents IL2-induced cell growth and function and compromises NK cell viability. This study reveals that tumour cell interactions and T cell-derived IL2 cooperate to promote robust and prolonged NK cell anti-tumour metabolic responses.

Neutrophil recruitment to inflamed joints can occur without cellular priming.

Recruitment of neutrophils from blood to tissues is a cardinal event in inflammation during which neutrophils switch from a resting, naive state to a preactivated, primed phenotype; the priming process is characterized by alterations in the composition of cell surface adhesins, for example, shedding of l-selectin and mobilization of granule-stored integrins to the cell surface. Ligation of chemotactic receptors and interactions with the endothelial lining are established triggers of neutrophil priming and in line with this, in vivo transmigrated neutrophils obtained from tissues are typically highly primed. We here characterize the priming of neutrophils brought about by in vivo recruitment from blood to inflamed joints by the analyses of synovial fluid and blood from patients with inflammatory arthritis. For comparisons, we used controlled in vivo models of neutrophil transmigration to skin of healthy subjects. In contrast to the residing view and in vivo transmigrated neutrophils from skin models, neutrophils from synovial fluid were often surprisingly resting and phenotypically very similar to naive cells isolated from peripheral blood; synovial fluid cells often retained l-selectin and had undergone minimal up-regulation of integrin receptors. In complete agreement with our in vivo findings, cell-free synovial fluid was potently chemotactic without triggering alteration of surface receptors also in vitro. We conclude that tissue recruitment of neutrophils does not by default trigger l-selectin shedding and granule mobilization, and the chemoattractant(s) guiding neutrophils to synovial fluid apparently operate without inducing cellular priming.

Induced PD-1 Expression on Bone Marrow CD34+ Cells from MDS Patients Treated with 5-Azacitadine in Combination with Nivolumab and/or Ipilimumab

Introduction PD-1 is a negative costimulatory receptor on activated T lymphocytes which counters the activation signal provided by T cell receptor ligation. The expression of PD-1 on T lymphocytes can be dynamically modulated by DNA methylation. A recent study in solid tumors (Kleffel S et al, Cell 2015) suggested that PD-1 was not only expressed by immune cells, but also by a subpopulation of cancer cells, and the cancer intrinsic PD-1 can promote tumorigenesis. Our previous study (Yang H et al, Leukemia 2014) demonstrated an increased PD-1 mRNA expression in peripheral blood mononuclear cells from MDS patients under hypomethylating agent (HMA) treatment, but little is known about detailed PD-1 expression in MDS. PD-1 signaling has been reported to be involved in MDS pathogenesis and resistance mechanisms to HMAs (Yang H et al, Leukemia 2014). Therefore, the PD-1 pathway represents an attractive target and anti-PD-1 monoclonal antibodies are being increasingly used to study MDS. Precise understanding of PD-1 expression in MDS patients may allow for effective treatment.Methods and Human Specimens With this purpose, using multicolor flow cytometry analysis, we studied the PD-1 expression on bone marrow Lin-CD34+ cells (Lineage cells: CD2, CD3, CD4, CD7, CD10, CD11b, CD14, CD19, CD20, CD33, CD56 and CD235a) from 51 patients enrolled in a clinical trial 2014-0930. Based on 5-azacytidine treatment, these patients can be divided into two groups: group A included 29 patients (57%) (22 MDS, 5 CMML, 1 MDS/MPD and 1 AML) which were treated with 5-azacitidine in combination with ipilimumab (N=10), nivolumab (N=16) or ipilimumab + nivolumab (N=2), one patient treated with 5-azacytidine only was also included; group B included 22 patients (43%) (16 MDS, 4 CMML and 2 MDS/MPD) which did not receive 5-azacytidine treatment and were treated with ipilimumab (N=12), nivolumab (N=5) or ipilimumab + nivolumab (N=5) alone. Positive PD-1 expression was considered when the median fluorescence intensity (MFI) ratio between sample and FMO negative control was more than 2.Results In group A, 26 patients (90%) were previously untreated at the time of enrollment. PD-1 expression was not observed in 8 untreated baseline samples collected. We then performed the analysis in samples under different time points of treatment. PD-1 expression was observed in 11 patients (38%, 9 responders, 1 stable disease and 1 non-responder) in at least one time point during treatment. Interestingly, 9 (82%) of them were treated with 5-azacytidine in combination with nivolumab, the other two were treated with 5-azacytidine in combination ipilimumab and ipilimumab + nivolumab separately. PD-1 expression was observed in 56% of the patients under 5-azacytidine in combination with nivolumab treatment. Figure 1 is an example of PD-1 expression in Lin-CD34+ cells from patient treated with 5-azacytidine in combination with nivolumab.In group B, all patients except one (95%) were previously treated at the time of enrollment. PD-1 expression was observed in 2 out of 13 baseline samples collected, and the PD-1 expression was not detected on these two patients after follow-up treatment with ipilimumab. PD-1 expression was observed in two patients (9%, 1 responder, 1 stable disease) in at least one time point during treatment (1 ipilimumab, 1 ipilimumab + nivolumab).Taken together, PD-1 expression was significantly induced in patients treated with 5-azacytidine when compare to patients without 5-azacytidine treatment (38% vs 9%, p=0.023), especially in the group of patients treated with 5-azacytidine in combination with nivolumab (56% vs 10%, p=0.037, compare with 5-azacytidine in combination with ipilimumab). Induction of PD-1 expression may contribute to the response of the treatment (77% vs 46%, p=0.058).Conclusion Our study demonstrates that induced expression of PD-1 on MDS Lin-CD34+ cells may contribute to the response of MDS patients to treatment with 5-azacytidine in combination with nivolumab. PD-1 methylation as well as other biological functions related to this treatment need to be further studied. [Display omitted] DisclosuresColla:Abbvie: Research Funding. Daver:Novartis: Research Funding; Incyte: Research Funding; Otsuka: Consultancy; Daiichi-Sankyo: Research Funding; Sunesis: Consultancy; ImmunoGen: Consultancy; Karyopharm: Consultancy; Alexion: Consultancy; Incyte: Consultancy; Kiromic: Research Funding; Pfizer: Consultancy; ARIAD: Research Funding; Pfizer: Research Funding; Novartis: Consultancy; BMS: Research Funding; Karyopharm: Research Funding; Sunesis: Research Funding.

Cellular and Molecular Events in the Airway Epithelium Defining the Interaction Between House Dust Mite Group 1 Allergens and Innate Defences.

Serodominant group 1 allergens of house dust mites (HDMs) are cysteine protease digestive enzymes. By increasing the detection of any allergen by dendritic antigen presenting cells, upregulating inflammatory signalling molecules, and activating cells crucial to the transition from innate to acquired immune responses, the proteolytic activity of these HDM allergens also underlies their behaviour as inhalant allergens. The significance of this property is underlined by the attenuation of allergic responses to HDMs by novel inhibitors in experimental models. The group 1 HDM allergens act as prothrombinases, enabling them to operate the canonical stimulation of protease activated receptors 1 and 4. This leads to the ligation of Toll-like receptor 4, which is an indispensable component in HDM allergy development, and reactive oxidant-regulated gene expression. Intermediate steps involve epidermal growth factor receptor ligation, activation of a disintegrin and metalloproteases, and the opening of pannexons. Elements of this transduction pathway are shared with downstream signalling from biosensors which bind viral RNA, suggesting a mechanistic linkage between allergens and respiratory viruses in disease exacerbations. This review describes recent progress in the characterisation of an arterial route which links innate responses to inhaled allergens to events underpinning the progression of allergy to unrelated allergens.

The T Cell Antigen Receptor α Transmembrane Domain Coordinates Triggering through Regulation of Bilayer Immersion and CD3 Subunit Associations

Initial molecular details of cellular activation following αβT cell antigen receptor (TCR) ligation by peptide-major histocompatibility complexes (pMHC) remain unexplored. We determined the nuclear magnetic resonance (NMR) structure of the TCRα subunit transmembrane (TM) domain revealing a bipartite helix whose segmentation fosters dynamic movement. Positively charged TM residues Arg251 and Lys256 project from opposite faces of the helix, with Lys256 controlling immersion depth. Their modification caused stepwise reduction in TCR associations with CD3ζζ homodimers and CD3εγ plus CD3εδ heterodimers, respectively, leading to an activated transcriptome. Optical tweezers revealed that Arg251 and Lys256 mutations altered αβTCR-pMHC bond lifetimes, while mutations within interacting TCRα connecting peptide and CD3δ CxxC motif juxtamembrane elements selectively attenuated signal transduction. Our findings suggest that mechanical forces applied during pMHC ligation initiate Tcell activation via a dissociative mechanism, shifting disposition of those basic sidechains to rearrange TCR complex membrane topology and weaken TCRαβ and CD3 associations.

C-27-carboxylated oleanane triterpenoids up-regulate TRAIL DISC assembly via p38 MAPK and CHOP-mediated DR5 expression in human glioblastoma cells

Despite recent tremendous progress, targeting of TNF-related apoptosis-inducing ligand (TRAIL) as a cancer therapy has limited success in many clinical trials, in part due to inactivation of death inducing signaling complex (DISC)-mediated caspase-8 signaling cascade in highly malignant tumors such as glioblastoma. In this study, screening of constituents derived from Astilbe rivularis for TRAIL-sensitizing activity identified C-27-carboxylated oleanolic acid derivatives (C27OAs) including 3β-hydroxyolean-12-en-27-oic acid (C27OA-1), 3β,6β,7α-trihydroxyolean-12-en-27-oic acid (C27OA-2), and 3β-trans-p-coumaroyloxy-olean-12-en-27-oic acid (C27OA-3) as novel TRAIL sensitizers. Interestingly, these C27OAs did not affect apoptotic cell death induced by either ligation of other death receptor (DR) types, such as TNF and Fas or DNA damaging agents, which suggests that C27OAs effectively and selectively sensitize TRAIL-mediated caspase-8 activation. Mechanistically, C27OAs upregulate the expression of cell surface DR5 and DISC formation without affecting downstream intracellular apoptosis-related proteins. The upregulation of DR5 expression by C27OAs strictly depends on transactivation of C/EBP homology protein, which is regulated through the p38 MAPK pathway, rather than p53 and intracellular reactive oxygen species status. Taken together, our results identify the novel C27OAs as TRAIL sensitizers targeting the upstream DISC assembly of DR5, and provide a rationale for further development of C27OAs for facilitating TRAIL-based chemotherapy in glioblastoma patients.

Reverse correlations of collagen-dependent platelet aggregation and adhesion with GPVI shedding during storage.

Platelet receptor GPVI plays an important role in platelet firm adhesion to site of vascular injury. Receptor ligation with collagen, in company with other agonist/receptor interactions, augments inside out signaling pathways leading to platelet aggregation and thrombus formation. As GPVI expression is significantly modulated by ectodomain shedding, this study aimed to examine whether GPVI shedding functionally affects collagen-mediated platelet activation during storage. 6 PRP-platelet concentrates were subjected to adhesion analysis on collagen matrix under mild stirring condition as well as collagen-induced aggregation on day 1, 3 and 5 post-storage. Concurrently, platelet supernatants of same samples were fractionated by ultra-centrifugation and obtained micro-particle-free samples were subjected to western blot analysis for the evaluation of GPVI shedding. We showed a direct correlation between collagen-dependent platelet aggregation and adhesion (r = 0.8, p = 0.0001). The increasing levels of GPVI shedding during storage were in reverse correlation with collagen-induced platelet aggregation (r = - 0.82, p = 0.0004) which was significantly reducing during storage. Platelet adhesion to collagen matrix significantly decreased post-storage while it was also reversely correlated with the levels of GPVI shedding during 5 days storage of platelets (r = - 0.69, p = 0.002). Data presented here demonstrated that progressive shedding of surface adhesion receptor GPVI can affect its functional activities in stored platelets. Thereby considering the crucial role of GPVI in platelet adhesion to the site of injury, whether the therapeutic efficacy of banked platelet products could be influenced by storage-dependent shedding of this receptor, remains to be answered in future studies.

Innate Immune Signals Induce Anterograde Endosome Transport Promoting MHC Class I Cross-Presentation

Both cross-presentation of antigens by dendritic cells, a key pathway triggering Tcell immunity and immune tolerance, and survival of several pathogens residing in intracellular vacuoles are intimately linked to delayed maturation of vesicles containing internalized antigens and microbes. However, how early endosome or phagosome identity is maintained is incompletely understood. We show that Toll-like receptor 4 (TLR4) and Fc receptor ligation induces interaction of the GTPase Rab14 with the kinesin KIF16b mediating plus-end-directed microtubule transport of endosomes. As a result, Rab14 recruitment to phagosomes delays their maturation and killing of an internalized pathogen. Enhancing anterograde transport by overexpressing Rab14, promoting the GTP-bound Rab14 state, or inhibiting retrograde transport upregulates cross-presentation. Conversely, reducing Rab14 expression, destabilizing Rab14 endosomes, and inhibiting anterograde microtubule transport by Kif16b knockdown compromise cross-presentation. Therefore, regulation of early endosome trafficking by innate immune signals is a critical parameter in cross-presentation by dendritic cells.

Reprogramming of Th1 cells into regulatory T cells through rewiring of the metabolic status.

T helper type 1 (Th1) cells form one of the most stable CD4 T-cell subsets, and direct conversion of fully differentiated Th1 to regulatory T (Treg) cells has been poorly investigated. Here, we established a culture method for inducing Foxp3 from Th1 cells of mice and humans. This is achieved simply by resting Th1 cells without T-cell receptor ligation before stimulation in the presence of transforming growth factor-beta (TGF-β). We named the resulting Th1-derived Foxp3+ cells Th1reg cells. Mouse Th1reg cells showed an inducible Treg-like phenotype and suppressive ability both in vitro and in vivo. Th1reg cells could also be induced from in vivo-developed mouse Th1 cells. Unexpectedly, the resting process enabled Foxp3 expression not through epigenetic changes at the locus, but through metabolic change resulting from reduced mammalian target of rapamycin complex 1 (mTORC1) activity. mTORC1 suppressed TGF-β-induced phosphorylation of Smad2/3 in Th1 cells, which was restored in rested cells. Our study warrants future research aiming at development of immunotherapy with Th1reg cells.

Abstract 3808: Interrogation and modulation of the immunosuppressive activity of human TAM-like macrophages using in vitro cultures

Abstract Tumor Associated Macrophages (TAMs), characteristic of an M2-like immune-suppressive phenotype, can induce proliferation and survival of tumor cells, facilitate angiogenesis, and suppress anti-tumor immune responses via expression of co-inhibitory molecules (e.g. PD-L1) and cytokines (e.g. IL-10, TGF-β). TAMs are therefore a highly attractive target of innovative cancer immunotherapies. Understanding the ability of pre-clinical candidate compounds to reverse TAM (M2-like)-mediated immune suppression and the potential for reprogramming of M2-like macrophages to a M1-like phenotype is key in the development of effective TAM-targeted cancer immunotherapies. Here we outline development of an assay to assess the capabilities of pre-clinical compounds to reverse M2 macrophage-mediated immune suppression. Monocytes were isolated from whole blood obtained from healthy volunteers and cultured under M2-polarising conditions. The resulting macrophages were phenotypically characterized and then used in co-culture with autologous PBMC, stimulated through T cell receptor ligation. Resulting cytokine production was assessed, alongside CD4+ and CD8+ T cell viability and cell cycle status (flow cytometry). M2-like macrophages polarized with M-CSF displayed an immune suppressive phenotype as shown by their production of IL-10 and their inhibition of IFN-γ production by, and cell cycle status of, T cells in co-culture assays. This suppressive activity was only partially reversed by PD-1-blockade. Modifications to the macrophage polarization protocol were seen to alter the resulting macrophage cell surface phenotype (e.g. expression levels of PD-L1, TIM-3 and CD200R) and their suppressive activity in the co-culture assay. These cell surface phenotypes broadly reflected those seen amongst tumor-derived macrophages from renal and ovarian carcinoma patients (CD14+ CD163+ with expression of TIM-3 and LAG-3). Moreover, compound-mediated changes in functionality seen with macrophages polarized from healthy PBMC monocytes could also be seen using patient-derived material. The assay outlined here therefore provides a M-o-A human in vitro system to test novel compound activity (either singly or in combination) upon TAM-like macrophage generation, phenotype and suppressive function. This allows selection of the most efficacious compounds for further investigation using patient-derived immune cells. Citation Format: Laura E. Gallagher, Andrew Hall, Lauren A. Patience, Lucia Janicova, Stephen Anderton. Interrogation and modulation of the immunosuppressive activity of human TAM-like macrophages using in vitro cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3808.

Local delivery of adenosine receptor agonists to promote bone regeneration and defect healing.

Adenosine receptor activation has been investigated as a potential therapeutic approach to heal bone. Bone has enhanced regenerative potential when influenced by either direct or indirect adenosine receptor agonism. As investigators continue to elucidate how adenosine influences bone cell homeostasis at the cellular and molecular levels, a small but growing body of literature has reported successful in vivo applications of adenosine delivery. This review summarizes the role adenosine receptor ligation plays in osteoblast and osteoclast biology and remodeling/regeneration. It also reports on all the modalities described in the literature at this point for delivery of adenosine through in vivo models for bone healing and regeneration.

Toll-like receptor 4 plays an important role to enhance bacterial clearance from the nose in synergy with triggering receptor expressed on myeloid cells (TREM)-1 expression on polymorphonuclear neutrophils

ObjectiveAcute rhinosinusitis (ARS) is among the most common infectious diseases. Neutrophils play a major role in innate host defenses against pathogenic microorganisms such as fungi and bacteria. Recently, in neutrophils, ligation of the triggering receptor expressed on myeloid cells (TREM)-1 was found to activate the full spectrum of neutrophil effector mechanisms, including the release of inflammatory mediators, degranulation, phagocytosis, and oxidative burst in synergy with Toll-like receptors (TLRs). In this study, we investigated the effect of TREM-1 on the functions of neutrophils in relation to TLR4 in a nasal and nasopharyngeal inflammation mouse model via nontypeable Haemophilus influenzae (NTHi) intranasal inoculation. MethodsWe used C3H/HeJ (TLR4-deficient) mice, which arose spontaneously and have non-functional TLR4 protein, and normal wild-type (WT) C3H/HeN mice. Mice were inoculated intranasally with NTHi (107 cfu/mouse) to investigate the effects of TLR4 on the function of Neutrophils. We examined the kinetics of bacterial clearance and inflammatory cell infiltration in nasal washes at 6, 12, 24, and 72 h after inoculation. The expression of TREM-1 on neutrophils, and TREM-1 mRNA expression in neutrophils in the nasal washes were examined by flow cytometric analysis and RT-PCR. ResultsBacterial counts of NTHi from nasal washes were significantly lower in WT mice than in TLR4-mutant mice after inoculation. The numbers of inflammatory cells in nasal washes were significantly higher in WT mice at 6 h, 12 h, and 24 h after inoculation than in TLR4-deficient mice. The expression of TREM-1 protein on neutrophils and the mRNA levels were greater in WT mice than in TLR4-mutant mice. The concentrations of soluble TREM-1 in WT nasal washes were also significantly higher than in those of TLR4-deficient mice. ConclusionTREM-1 may play an important role together with TLR4 in the nasopharyngeal clearance of NTHi by neutrophils. Further studies will need to clarify the innate immune responses of neutrophils via TLR4 to prevent NTHi infection.

Differing Outcome of Experimental Autoimmune Encephalitis in Macrophage/Neutrophil- and T Cell-Specific gp130-Deficient Mice.

gp130 cytokines are differentially involved in regulating the T helper (H) 17-driven pathogenesis of experimental autoimmune encephalomyelitis (EAE), the animal model of human multiple sclerosis. Interleukin (IL)-6 directly promotes the development of TH17 cells through the gp130/IL-6R complex. By contrast, IL-27 has been shown to suppress a TH17 immune response by gp130/IL-27R-alpha (α) receptor ligation. The IL-27-dependent regulation of a TH17 development could be mediated on the level of CD4 T cells. However, because IL-27 also suppresses the secretion of the TH17-driving cytokines IL-6 and IL-12/23p40 in accessory cells, TH17 immune responses may also be controlled by IL-27 on the level of macrophages and/or neutrophils. To analyze these opposing effects of gp130 engagement on the pathogenesis of EAE, we immunized CD4+ T cell-specific gp130-deficient (CD4creposgp130loxP/loxP) and macrophage/neutrophil-specific gp130-deficient (LysMcreposgp130loxP/loxP) mice with the myelin-oligodendrocyte-glycoprotein peptide MOG35–55. Whereas inflammatory immune responses, TH17 differentiation, and pathology in CD4creposgp130loxP/loxP mice were mitigated, disease progression was eventually enhanced in LysMcreposgp130loxP/loxP mice. Exacerbated disease in MOG35–55-immunized LysMcreposgp130loxP/loxP mice was associated with an elevated development of TH17 cells and increased infiltration of the central nervous system with leukocytes indicating a suppressive role of macrophage/neutrophil-gp130. To further prove IL-6 to be responsible for the control of inflammation during EAE through gp130 on macrophages/neutrophils, we immunized LysMcreposIL-6RloxP/loxP mice. In contrast to LysMcreposgp130loxP/loxP mice, neuropathology in MOG35–55-immunized macrophage/neutrophil-specific IL-6R-deficient mice was not enhanced indicating that the alleviation of EAE through macrophage/neutrophil-gp130 is mediated independently of IL-6. Together, this different pathology in macrophage/neutrophil- and CD4 T cell-specific gp130-deficient mice suggests that gp130 cytokines modulate TH17 inflammation differentially by targeting distinct cell types.