Expression Of Cell Surface Molecules Research Articles

Plasmacytoid dendritic cells and RNA-containing immune complexes drive expansion of peripheral B cell subsets with an SLE-like phenotype.

BackgroundHyperactive B cells and a continuous interferon (IFN)-α production by plasmacytoid dendritic cells (pDCs) play a key role in systemic lupus erythematosus (SLE). We asked whether the interaction between B cells and pDCs stimulated with RNA-containing immune complexes affects peripheral B cell subsets.MethodsB cells and pDCs were isolated from blood of healthy individuals and stimulated with immune complexes consisting of SLE-IgG and U1snRNP (RNA-IC). Expression of cell surface molecules as well as IL-6 and IL-10 production were determined by flow cytometry and immunoassays. Gene expression profiles were determined by a NanoString nCounter expression array.ResultsWe found a remarkable increase of double negative CD27-IgD- B cells, from 7% within fresh CD19+ B cells to 37% in the RNA-IC-stimulated co-cultures of B cells and pDCs, comparable to the frequency of double negative B cells in SLE patients. Gene expression analysis of the double negative CD27-IgD- and the CD27+IgD- memory B cells revealed that twenty-one genes were differentially expressed between the two B cell subsets (≥ 2-fold, p<0.001). The, IL21R, IL4R, CCL4, CCL3, CD83 and the IKAROS Family Zinc Finger 2 (IKZ2) showed higher expression in the double negative CD27-IgD- B cells.ConclusionThe interactions between B cells and pDCs together with RNA-containing IC led to an expansion of B cells with similar phenotype as seen in SLE, suggesting that the pDC-B cell crosstalk contributes to the autoimmune feed-forward loop in SLE.

Ruxolitinib and Polycation Combination Treatment Overcomes Multiple Mechanisms of Resistance of Pancreatic Cancer Cells to Oncolytic Vesicular Stomatitis Virus.

Vesicular stomatitis virus (VSV) is a promising oncolytic virus (OV). Although VSV is effective against a majority of pancreatic ductal adenocarcinoma cell (PDAC) cell lines, some PDAC cell lines are highly resistant to VSV, and the mechanisms of resistance are still unclear. JAK1/2 inhibitors (such as ruxolitinib and JAK inhibitor I) strongly stimulate VSV replication and oncolysis in all resistant cell lines but only partially improve the susceptibility of resistant PDACs to VSV. VSV tumor tropism is generally dependent on the permissiveness of malignant cells to viral replication rather than on receptor specificity, with several ubiquitously expressed cell surface molecules playing a role in VSV attachment to host cells. However, as VSV attachment to PDAC cells has never been tested before, here we examined if it was possibly inhibited in resistant PDAC cells. Our data show a dramatically weaker attachment of VSV to HPAF-II cells, the most resistant human PDAC cell line. Although sequence analysis of low-density lipoprotein (LDL) receptor (LDLR) mRNA did not reveal any amino acid substitutions in this cell line, HPAF-II cells displayed the lowest level of LDLR expression and dramatically lower LDL uptake. Treatment of cells with various statins strongly increased LDLR expression levels but did not improve VSV attachment or LDL uptake in HPAF-II cells. However, LDLR-independent attachment of VSV to HPAF-II cells was dramatically improved by treating cells with Polybrene or DEAE-dextran. Moreover, combining VSV with ruxolitinib and Polybrene or DEAE-dextran successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication.IMPORTANCE Oncolytic virus (OV) therapy is an anticancer approach that uses viruses that selectively infect and kill cancer cells. This study focuses on oncolytic vesicular stomatitis virus (VSV) against pancreatic ductal adenocarcinoma (PDAC) cells. Although VSV is effective against most PDAC cells, some are highly resistant to VSV, and the mechanisms are still unclear. Here we examined if VSV attachment to cells was inhibited in resistant PDAC cells. Our data show very inefficient attachment of VSV to the most resistant human PDAC cell line, HPAF-II. However, VSV attachment to HPAF-II cells was dramatically improved by treating cells with polycations. Moreover, combining VSV with polycations and ruxolitinib (which inhibits antiviral signaling) successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. We envision that this novel triple-combination approach could be used in the future to treat PDAC tumors that are highly resistant to OV therapy.

Abstract 589: Hedgehog signaling augments PDL-1 expression in cancer cells under hypoxic condition to inhibit antitumor effects by activated lymphocytes

Abstract [Background] Hedgehog (Hh) signaling is activated under hypoxic condition in cancerous tissue. This is thought to be one of the mechanisms of the induction of malignant phenotype of cancers. We have shown that Hh inhibitor decreases proliferation, invasiveness, and tumorigenesis. We have also revealed that Hh signaling plays pivotal roles for the maintenance of function in activated lymphocytes and dendritic cells under hypoxia. Recently, immune checkpoint inhibitor takes much attention, however, response rate is still limited. The mechanisms regulating PDL-1 expression also remains unclear. In this study, to increase reponse rate of immune checkpoint inhibitor, we investigated whether hypoxia-induced activation of Hh signaling contributes to PDL-1 expression in cancer and whether it affects the antitumor function of activated lymphocytes. [Materials and Methods] Pancreatic cancer cell line, Panc-1, gallbladder cancer cell line, NOZ and GBd, and lung small cell cancer cell line, SBC-5 were used as target cancer cells. Human lymphocytes derived from PBMC were activated by using anti CD3 mAb and IL-2, and were used as effector cells. For normoxic conditions, cells were cultured in 5% CO2 and 95% air. For hypoxic conditions, cells were cultured in 1% O2, 5% CO2, and 94% N2 using a multigas incubator. For Hh signaling inhibition, cyclopamine, polysaccharide-K (PSK), and small interfering RNA targeting Gli1, Smo, MAML3, and RBPJ were used. Cell numbers were counted by light microscope. Expression of cell surface molecules was estimated by FACS. [Results] 1) Hypoxia augmented PDL-1 expression in all 4 cancer cell lines. 2) Inhibition of Hh signaling using MAML3 siRNA, cyclopamine and PSK reduced PDL-1 expression under hypoxia in all 4 cancer cell lines. 3) When activated lymphocytes were cocultured with cancers treated with a Hh inhibitor, activated lymphocyte cell numbers significantly increased under hypoxia. 4) In contrast, this increase was abrogated when cancer cells were treated with a PDL-1 neutralizing antibody. 5) When activated lymphocytes were cocultured with cancers treated with a Hh inhibitor and/or anti-PDL-1 Ab, the percent of CD8+ lymphocytes decreased in both the cyclopamine- and anti-PDL-1-treated groups under hypoxic conditions, while there was no significant change in CD3 expression. 6) NKG2D expression increased on activated lymphocytes in anti PDL-1Ab-treated group. [Conclusion] These results suggest that Hh signaling is one of regulatory pathways of PDL-1 expression under hypoxia and that inhibiting Hh signaling may induce lymphocyte antitumor activity. Therefore, Hh inhibition could be a promising drug target, not only as a direct tumor suppressor agent, but also as a PDL-1 inhibitor. Citation Format: Hideya Onishi, Akiko Fujimura, Yasuhiro Oyama, Makoto Kawamoto, Akio Yamasaki, Takashi Morisaki. Hedgehog signaling augments PDL-1 expression in cancer cells under hypoxic condition to inhibit antitumor effects by activated lymphocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 589. doi:10.1158/1538-7445.AM2017-589

Abstract 5676: Functional pairing of immunomodulatory targets in anaplastic thyroid cancer

Abstract Thyroid cancer is the most common type of endocrine malignancy that has an escalating global frequency. Although most well differentiated thyroid cancers (WDTC) are manageable and respond to current therapeutic modalities, undifferentiated anaplastic thyroid cancers (ATC) exhibit a dramatically different clinical behavior and poor prognosis. With the recent development of immunotherapies, targeted, well-defined treatment plans can demonstrate promising treatment outcomes in ATC patients. Precise immunological targets in ATCs with potential clinical relevance are unknown. Major progress has been made in last 5 years toward development of immune checkpoint inhibitors using anti-CTLA-4 and anti-PD-1/PD-L1 antibodies for cancer treatment which has made immunotherapies one of the mainstream treatment choices. Few additional members of the immunoglobulin superfamily of receptors, like LAG3, TIM3 and VISTA have recently been identified as potential checkpoint targets. Interestingly some of these molecules including TIM3 and PD-L1 promote tumor progression and immune escape. Identification of specific immunotherapeutic targets requirs a better understanding of the immune microenvironment in ATC. To this end we evaluated the expression of prominent co-stimulatory and co-inhibitory cell surface molecules by RT-PCR in three thyroid cancer cell lines - TPC-1 (papillary), CGTH-W-1 (follicular) and 8505C (anaplastic). We observed that many co-inhibitory molecules were upregulated in all three tumor cell lines. CTLA4, interestingly, had the highest expression in 8505C. Additionally we observed differential expression of BTLA, LAIR1, TIM3 and VISTA between TPC-1 and 8505C. LAG3, PD-1 and PD-L1 were also upregulated in 8505C compared to TPC-1. Similar pattern was observed with the expression of co-stimulatory molecules, CD40L and GITR . GITR has been shown to have a tumor suppressor function in multiple myeloma. Another co-stimulatory molecule OX40, which has shown promise in tumor recession when targeted, was upregulated in all three cell lines and 8505C showed the highest expression. Our findings suggest that the aggressive and less immunogenic phenotype of ATC might be attributed to the differential expression of these molecules. Targeting these immunomodulatory molecules in ATC warrants a better understanding of the crosstalk between them and it might provide an efficient means for the disease management. Citation Format: Sanjukta Chakraborty, Rachana R. Maniyar, Neha Y. Tuli, Ghada Ben Rahoma, Cameron Budenz, Sarnath Singh, Jan Geliebter, Raj Tiwari. Functional pairing of immunomodulatory targets in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5676. doi:10.1158/1538-7445.AM2017-5676

Abstract 685: GPC2 is an oncogene and immunotherapeutic target in high-risk neuroblastoma

Abstract Background: GD2-directed immunotherapeutic strategies have improved outcomes in neuroblastoma; however, the majority of patients treated suffer relapse and GD2 expression on pain fibers causes dose-limiting toxicities. Methods: To identify alternative cell surface immunotherapeutic targets, we compared high-risk neuroblastoma (n=126 tumors) and normal tissue RNA sequencing data (GTEx; n=7859 samples from 31 normal tissues) and prioritized genes by differential and absolute expression and cell surface prediction. Genes were further surveyed for somatic copy number gain and correlative expression with MYCN amplification. Differential protein expression and localization were confirmed in neuroblastoma primary tumors (n=98), patient-derived xenografts (n=32; PDXs), cell lines (n=23), and normal pediatric tissues (n=36). Cell lines were subjected to candidate gene gain and loss of function studies (n=11). Additional pediatric tumor RNA sequencing data was surveyed followed by confirmatory immunohistochemistry (IHC). Finally, candidate specific antibodies were isolated from a human Fab phage library and utilized for antibody-drug conjugate (ADC) engineering followed by cytotoxicity studies. Results: We identified 33 differentially expressed cell surface molecules from which we prioritized glypican-2 (GPC2) for validation given GPC2’s robust differential expression (log-fold change tumor vs. normal tissue = 1.71-9.22; p=1.99 x 10-9-1.88 x10-300), high-level absolute RNA expression (median FPKM=60), and frequent DNA copy number gain associated with higher GPC2 expression (35%, n=182 tumors; p&amp;lt;0.005). GPC2 expression was also higher in MYCN amplified neuroblastomas (p&amp;lt;0.05), MYCN binds the GPC2 promoter shown by chromatin immunoprecipitation (ChIP) sequencing and reporter assays, and MYCN depletion resulted in decreased GPC2 expression. Immunoblot, flow cytometry, immunofluorescence, and IHC analysis of primary tumors, PDXs, and cell lines confirmed dense cell surface GPC2 expression. Medulloblastomas (n=62) were also found to have high GPC2 expression that positively correlated with MYC, MYCN, and GPC2 loci gain (p&amp;lt;0.0001). Pediatric normal tissues had very restricted cell surface GPC2 expression, with only low levels found in the esophagus and skin. GPC2 depletion in neuroblastoma cell lines resulted in apoptosis and growth inhibition and GPC2 forced over-expression increased neuroblastoma cell proliferation (p&amp;lt;0.001 for all assays). Finally, a human GPC2 antibody, D3-GPC2-Fab, was developed and shown to bind GPC2 with high affinity and specificity. D3-GPC2-IgG1 induced internalization of GPC2 and was conjugated to pyrrolobenzodiazepine (PBD) dimers to form an ADC which induced potent and specific cytotoxicity to GPC2 expressing neuroblastoma cells (IC50 = 1.7-11 pM). Conclusions: GPC2 is an oncogene and immunotherapeutic target in neuroblastoma and potentially other cancers. Citation Format: Kristopher R. Bosse, Pichai Raman, Maria Lane, Robyn T. Sussman, Jo Lynne Harenza, Daniel Martinez, Sabine Heitzeneder, Zhongyu Zhu, Komal Rathi, Michael Randall, Laura Donovan, Sorana Morrissy, Doncho V. Zhelev, Yang Feng, Jennifer Hwang, Yanping Wang, Bruce Pawel, Tricia Bhatti, Mariarita Santi, Javed Khan, Michael Taylor, Dimiter S. Dimitrov, Crystal Mackall, John M. Maris. GPC2 is an oncogene and immunotherapeutic target in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 685. doi:10.1158/1538-7445.AM2017-685

The constitutive protease release by primary human acute myeloid leukemia cells.

Acute myeloid leukemia (AML) cells show constitutive release of matrix metalloproteases and their inhibitors. We now investigated this constitutive release of protease/protease regulators associated with carcinogenesis (ADAM12, uPA, cystatin B), angiogenesis (serpin E1, uPA, CD147), cancer cell migration (uPA, cystatin C), coagulation (ADAM TS13, serpin C1), inflammation (fetuin A, caspase 1, cystatin C), monocytic differentiation (CFD) or regulation of hematopoiesis (neutrophil elastase). AML blasts from 79 consecutive patients were cultured in serum-free medium and mediator levels determined in culture supernatants. Detectable release of serpin C1 and E1, cystatin B and C, CD147 and uPA was seen for most patients. These mediators together with fetuin A, caspase 1, and CFD were included in a hierarchical clustering analysis and three patient subsets were identified (high, intermediate, and low release). High levels were associated with monocytic differentiation. Global gene expression analyses showed increased levels of several zinc finger proteins for low-release patients and high expression of several cell surface molecules, ATPases, and calcium-binding proteins for high-release patients. Constitutive release of several mediators was also seen for normal hematopoietic cells and mesenchymal stem cells. In cocultures of the latter and AML blasts, the release level for most mediators was altered to resemble the levels of the mesenchymal cells cultured alone. Differences in constitutive release of protease/protease regulators are a part of the disease heterogeneity in AML.

HIV-1 Vpu Downmodulates ICAM-1 Expression, Resulting in Decreased Killing of Infected CD4 + T Cells by NK Cells

HIV-1 Vpu is known to alter the expression of numerous cell surface molecules. Given the ever-increasing list of Vpu targets identified to date, we undertook a proteomic screen to discover novel cell membrane proteins modulated by this viral protein. Plasma membrane proteome isolates from Vpu-inducible T cells were subjected to stable isotope labeling of amino acids in cell culture (SILAC)-based mass spectrometry analysis, and putative targets were validated by infection of primary CD4+ T cells. We report here that while intercellular adhesion molecule 1 (ICAM-1) and ICAM-3 are upregulated by HIV-1 infection, expression of Vpu offsets this increase by downregulating these molecules from the cell surface. Specifically, we show that Vpu is sufficient to downregulate and deplete ICAM-1 in a manner requiring the Vpu transmembrane domain and a dual-serine (S52/S56) motif necessary for recruitment of the beta-transducin repeat-containing E3 ubiquitin protein ligase (β-TrCP) component of the Skp, Cullin, F-box (SCFβ-TrCP) E3 ubiquitin ligase. Vpu interacts with ICAM-1 to induce its proteasomal degradation. Interestingly, the E3 ubiquitin ligase component β-TrCP-1 is dispensable for ICAM-1 surface downregulation yet is necessary for ICAM-1 degradation. Functionally, Vpu-mediated ICAM-1 downregulation lowers packaging of this adhesion molecule into virions, resulting in decreased infectivity. Importantly, while Vpu-mediated downregulation of ICAM-3 has a limited effect on the conjugation of NK cells to HIV-1-infected CD4+ T cells, downregulation of ICAM-1 by Vpu results in a reduced ability of NK cells to bind and kill infected T cells. Vpu-mediated ICAM-1 downregulation may therefore represent an evolutionary compromise in viral fitness by impeding the formation of cell-to-cell contacts between immune cells and infected T cells at the cost of decreased virion infectivity.IMPORTANCE The major barrier to eradicating HIV-1 infection is the establishment of treatment-resistant reservoirs early in infection. Vpu-mediated ICAM-1 downregulation may contribute to the evasion of cell-mediated immunity during acute infection to promote viral dissemination and the development of viral reservoirs. By aiding the immune system to clear infection prior to the development of reservoirs, novel treatments designed to disrupt Vpu-mediated ICAM-1 downregulation may be beneficial during acute infection or as a prophylactic treatment.

Forkhead-box transcription factor 1 affects the apoptosis of natural regulatory T cells by controlling Aven expression

BackgroundRegulatory T (Treg) cells play important roles in autoimmune diseases, cancer, and organ transplantation. Forkhead box protein o1 (Foxo1) and IL-7Rα(CD127) are closely related to the homeostasis of Treg cells. However, the mechanism underlying Treg proliferation and activation remains unclear. Here, we evaluated how the over-expression of Foxo1 affects Treg cell proliferation via intracellular signaling. nTreg cells were transfected separately with Foxo1 and Aven small-interfering RNA (siRNA) or over-expression plasmid. The expression of signaling pathway genes and CD127 was confirmed using RT-qPCR and western blot analysis. The expression of cell surface molecules and apoptosis was confirmed by Flow Cytometry 3-(4, 5-Dimethylthiazol-2-yl) 2,5- diphenyltetrazolium bromide for cell proliferation assays.ResultsFoxo1 strengthened the proliferative ability of Treg cells by activating IL-7/CD127 signaling. In addition, Foxo1 suppressed Treg cell apoptosis by regulating Aven expression.ConclusionsThe results in this study indicated that Foxo1 is a positive regulatory factor for the proliferation and activity of Treg cells. Foxo1 might be a potential target for the activation of nTreg cells in vivo and in vitro.

Human decidua mesenchymal stem cells regulate decidual natural killer cell function via interactions between collagen and leukocyte‑associated immunoglobulin‑like receptor 1.

The development of maternal tolerance to the fetal allograft in critical for the maintenance of the pregnancy, and it is accompanied by the development of a special decidual natural killer (dNK) cell tolerance phenotype. To understand the factors that influence dNK cells during early pregnancy, the present study aimed to identify mesenchymal stem cells (MSCs) from human first‑trimester deciduas, termed decidual MSCs (DMSCs), and to investigate the effect of DMSCs on the regulation of dNK cells via collagen. Decidual samples were collected from women with normal pregnancy that had undergone elective vaginal surgical terminations at 6‑9weeks gestation. DMSCs derived from human decidual tissues were cultured under differentiation conditions to examine their multipotent differentiation capacities, and the expression of MSC‑specific markers, including cluster of differentiation (CD)44, CD73, CD105, CD90, CD34, CD31, CD14, CD45, CD11b and human leukocyte antigen‑antigen D related, was determined. dNK cells were co‑cultured with DMSCs in order to examine the effect of DMSCs on the tolerance phenotype of dNK cells. The expression of cell surface molecules, natural cytotoxicity triggering receptor 3 and killer cell immunoglobulin‑like receptor (KIR) 2DL1, and the secretion of cytokines, including interferon‑γ, tumor necrosis factor (TNF)‑α, interleukin (IL)‑10, IL‑4 and perforin, were examined by flow cytometry analysis. To determine whether the regulation of dNK cells by DMSCs was mediated by collagen, DMSCs were pre‑treated with human recombinant leukocyte‑associated immunoglobulin‑like receptor (LAIR)‑2 and transfected with pScoR‑GFP‑hP4H to inhibit the interaction between LAIR‑1 and collagen. The present results demonstrated that collagen produced by DMSCs increased the expression of KIR2DL1 and IL‑4, decrease the expression of NKp30 and TNF‑α. In conclusion, the results of the present study demonstrated that DMSCs may be cultured invitro for prolonged periods, whilst retaining the ability to differentiate into different cell lineages. In addition, DMSCs may modulate the function of dNK cells via the interaction between collagen and LAIR‑1.

Polysaccharide purified from Ganoderma atrum induced activation and maturation of murine myeloid-derived dendritic cells

Ganoderma atrum (G. atrum), a member of the genus Ganoderma, is an edible and medicinal fungus. In this study, we investigated the direct and indirect effects of G. atrum polysaccharide (PSG-1) on dendritic cells (DCs). Firstly, flow cytometric and ELISA analysis showed that PSG-1 increased cell surface molecule expression of MHC-II, CD80 and CD86, and enhanced the production of IL-12 p70, IL-6, IL-10, RANTES, MIP-1α and MCP-1 in DCs. PSG-1-treated DCs promoted the proliferation of splenic T lymphocyte of mouse in mixed lymphocyte reaction. The above results demonstrated that PSG-1 induced the maturation of DCs. Secondly, PSG-1 increased the phosphorylation of p38, ERK and JNK determined by western blot. Inhibitors of p38, ERK and JNK decreased PSG-1-induced expression of MHC-II, CD80 and CD86 and production of IL-6 and IL-10 by DCs. These results suggested that PSG-1 induced mitogen-activated protein kinase (MAPK) activation was involved in the regulation of maturation markers and cytokines expression in DCs. Finally, PSG-1 increased expression of MHC-II of DCs in a DCs-Caco-2 co-culture model, suggesting that PSG-1 could indirectly influence DCs. In summary, our data suggested that PSG-1 directly induced DCs maturation via activating MAPK pathways, and indirectly stimulated DCs separated by intestinal epithelial cells.

Viral epitope-specific T cell responses induced in chronic, hepatitis C virus-infected patients

BackgroundApproximate 180 million people worldwide are infected with hepatitis C virus (HCV). Historically, vaccination has been the most effective strategy for controlling infections of such major health concern. Therapeutic vaccine strategies for HCV, however, have demonstrated negligible success. AimDemonstrate the ability of highly-conserved viral epitopes to overcome the immune dysfunction often associated with chronic HCV infections. MethodsT cells from five chronic, HCV-infected patients were immunophenotyped by flow cytometry. The ex vivo T cell responses to highly-conserved viral epitopes were assessed by ELISpot assay and cytokine bead array analysis. ResultsBoth HLA-DRB-1- and HLA-A2-restricted viral epitopes induced specific, TH1-type cytokine production by T cells derived from the patients. Induction occurred despite expression of cell-surface inhibitory molecules and the presence of regulatory T cells. ConclusionThese findings support the potential ability of a broad, multi-epitope-based therapeutic vaccine to elicit virus-specific immune responses in chronic hepatitis C virus-infected patients.

Neuropilin-1 Is Expressed on Lymphoid Tissue Residing LTi-like Group 3 Innate Lymphoid Cells and Associated with Ectopic Lymphoid Aggregates

SummaryHere, we characterize a subset of ILC3s that express Neuropilin1 (NRP1) and are present in lymphoid tissues, but not in the peripheral blood or skin. NRP1+ group 3 innate lymphoid cells (ILC3s) display in vitro lymphoid tissue inducer (LTi) activity. In agreement with this, NRP1+ ILC3s are mainly located in proximity to high endothelial venules (HEVs) and express cell surface molecules involved in lymphocyte migration in secondary lymphoid tissues via HEVs. NRP1 was also expressed on mouse fetal LTi cells, indicating that NRP1 is a conserved marker for LTi cells. Human NRP1+ ILC3s are primed cells because they express CD45RO and produce higher amounts of cytokines than NRP1− cells, which express CD45RA. The NRP1 ligand vascular endothelial growth factor A (VEGF-A) served as a chemotactic factor for NRP1+ ILC3s. NRP1+ ILC3s are present in lung tissues from smokers and patients with chronic obstructive pulmonary disease, suggesting a role in angiogenesis and/or the initiation of ectopic pulmonary lymphoid aggregates.

Relevance of meniscal cell regional phenotype to tissue engineering

ABSTRACTPurpose: Meniscus contains heterogeneous populations of cells that have not been fully characterized. Cell phenotype is often lost during culture; however, culture expansion is typically required for tissue engineering. We examined and compared cell-surface molecule expression levels on human meniscus cells from the vascular and avascular regions and articular chondrocytes while documenting changes during culture-induced dedifferentiation. Materials and Methods: Expressions of 16 different surface molecules were examined by flow cytometry after monolayer culture for 24 h, 1 week, and 2 weeks. Menisci were also immunostained to document the spatial distributions of selected surface molecules. Results: Meniscus cells and chondrocytes exhibited several similarities in surface molecule profiles with dynamic changes during culture. A greater percentage of meniscal cells were positive for CD14, CD26, CD49c, and CD49f compared to articular chondrocytes. Initially, more meniscal cells from the vascular region were positive for CD90 compared to cells from the avascular region or chondrocytes. Cells from the vascular region also expressed higher levels of CD166 and CD271 compared to cells from the avascular region. CD90, CD166, and CD271-positive cells were predominately perivascular in location. However, CD166-positive cells were also located in the superficial layer and in the adjacent synovial and adipose tissue. Conclusions: These surface marker profiles provide a target phenotype for differentiation of progenitors in tissue engineering. The spatial location of progenitor cells in meniscus is consistent with higher regenerative capacity of the vascular region, while the surface progenitor subpopulations have potential to be utilized in tears created in the avascular region.

\u03b1-1 Antitrypsin Inhibits RANKL-induced Osteoclast Formation and Functions

Osteoporosis is a global public health problem affecting more than 200 million people worldwide. We previously showed that treatment with α-1 antitrypsin (AAT), a multifunctional protein with antiinflammatory properties, mitigated bone loss in an ovariectomized mouse model. However, the underlying mechanisms of the protective effect of AAT on bone tissue are largely unknown. In this study, we investigated the effect of AAT on osteoclast formation and function in vitro. Our results showed that AAT dose-dependently inhibited the formation of receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclasts derived from mouse bone marrow macrophage/monocyte (BMM) lineage cells and the RAW 264.7 murine macrophage cell line. To elucidate the possible mechanisms underlying this inhibition, we tested the effect of AAT on the gene expression of cell surface molecules, transcription factors and cytokines associated with osteoclast formation. We showed that AAT inhibited macrophage colony-stimulating factor (M-CSF)-induced cell surface RANK expression in osteoclast precursor cells. In addition, AAT inhibited RANKL-induced TNF-α production, cell surface CD9 expression and dendritic cell-specific transmembrane protein (DC-STAMP) gene expression. Importantly, AAT treatment significantly inhibited osteoclast-associated mineral resorption. Together, these results uncover new mechanisms for the protective effects of AAT and strongly support the notion that AAT has therapeutic potential for the treatment of osteoporosis.

GR-independent down-modulation on GM-CSF bone marrow-derived dendritic cells by the selective glucocorticoid receptor modulator Compound A.

Dendritic cells (DC) initiate the adaptive immune response. Glucocorticoids (GCs) down-modulate the function of DC. Compound A (CpdA, (2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride) is a plant-derived GR-ligand with marked dissociative properties. We investigated the effects of CpdA on in vitro generated GM-CSF-conditioned bone marrow-derived DC (BMDC). CpdA-exposed BMDC exhibited low expression of cell-surface molecules and diminution of the release of proinflammatory cytokines upon LPS stimulation; processes associated with BMDC maturation and activation. CpdA-treated BMDC were inefficient at Ag capture via mannose receptor-mediated endocytosis and displayed reduced T-cell priming. CpdA prevented the LPS-induced rise in pErk1/2 and pP38, kinases involved in TLR4 signaling. CpdA fully inhibited LPS-induced pAktSer473, a marker associated with the generation of tolerogenic DC. We used pharmacological blockade and selective genetic loss-of-function tools and demonstrated GR-independent inhibitory effects of CpdA in BMDC. Mechanistically, CpdA-mediated inactivation of the NF-κB intracellular signaling pathway was associated with a short-circuiting of pErk1/2 and pP38 upstream signaling. Assessment of the in vivo function of CpdA-treated BMDC pulsed with the hapten trinitrobenzenesulfonic acid showed impaired cell-mediated contact hypersensitivity. Collectively, we provide evidence that CpdA is an effective BMDC modulator that might have a benefit for immune disorders, even when GR is not directly targeted.

Abstract A042: Targeting of alpha-enolase (ENO1) as a novel immunotherapeutical strategy for pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness, and resistance to treatment. By using a proteome approach, we have found that protein recognized with the highest frequency by autoantibodies in PDAC sera was the alpha-enolase (ENO1), a glycolytic enzyme that also acts as a plasminogen-binding receptor. We have found that either ENO1 silencing or treatment of the PDAC cells with single administration of Adeno-Associated Virus-expressing monoclonal antibody against human ENO1 inhibited lung metastasis in immunosuppressed mice injected with PDAC cells. In addition, PDAC ENO1-silenced cells displayed a down modulated expression of cell surface molecules that regulate the adhesion to the extracellular matrix as confirmed by their reduced binding to laminin, collagen and fibronectin. ENO1 silencing in PDAC cells increased reactive oxygen species mainly generated through the sorbitol and NADPH oxidase pathways, as well as autophagy and catabolic pathway adaptations, which together affect PDAC cell growth and induced senescence. This data indicated that ENO1 plays a critical role in PDAC progression and invasion. ENO1 possesses interesting antigenic properties as it is able to elicit T cell proliferation, activation and cytotoxic specific T lymphocytes (CTL) differentiation both in vitro and in vivo. Tumor infiltrating T lymphocytes specific for ENO1 have been identified in PDAC patients and the number of ENO1-specific T cell clones generated from peripheral blood of PDAC positively correlated with a better survival. Antibody and T cell responses to ENO1 are increase in PDAC patient underwent chemotherapy. DNA vaccination to ENO1 elicits an integrated humoral and cellular immune response that was accompanied by a reduced and regulatory T cells and MDSC that significantly extends survival of Genetically Engineered Mice that spontaneously develop PDAC. Notably, mouse anti-human ENO1 monoclonal antibody inhibits MDSC adhesion to pancreatic endothelial cells and in vitro and in vivo migration and decreases MDSC arginase activity and secretion of IL-6. As whole these data demonstrate that the self-antigen ENO1 is a promising target suitable for therapeutic purposes in PDAC. In particular, the antibodies anti-ENO1 may inhibit PDA cell and myeloid cell invasion and positively modulate T cell response, making the immunotherapy more effective. Citation Format: Francesco Novelli, Michela Capello, Michelle Chattaragada, Sammy Ferri Borgogno, Giorgia Mandili, Moitza Principe, Emanuela Mazza, Sara Bulfamante, Roberta Curto, Daniele Giordano, Paola Cappello. Targeting of alpha-enolase (ENO1) as a novel immunotherapeutical strategy for pancreatic cancer [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 A042.

Effects of Macrolide Antibiotics on Th1 Cell and Th2 Cell Development Mediated by Langerhans Cells.

LC-like dendritic cells (LDCs) were generated from mouse bone marrow cells and used as substitutes for LCs. Mice were primed with ovalbumin (OVA) peptide-pulsed LDCs, which had been treated with each macrolide antibiotic, via the hind footpad. After 5 days, the cytokine response in the popliteal lymph nodes was investigated by enzyme-linked immunosorbent assay. The expression of cell surface molecules on LDCs was investigated using reverse transcriptase polymerase chain reaction. Injection of OVA peptide-pulsed LDCs, which had been treated with josamycin or spiramycin, inhibited Th2 cell development as represented by down-regulation of interleukin (IL)-4 production as well as Th1 cell development as represented by down-regulation of interferon (IFN)-g production. This inhibition of Th1 cell and Th2 cell development was associated with suppression of CD86 and T-cell immunoglobulin and mucin domain-containing protein (TIM)-4 expression, respectively, in LDCs. Furthermore, Staphylococcus aureus strains isolated from skin lesions of patients with atopic dermatitis (AD) were more susceptible to josamycin than to spiramycin. These results suggest that topical application of josamycin to AD lesions colonized with S. aureus would be beneficial for control of AD by acting on both superficial S. aureus and epidermal LCs, and inhibiting the development of Th2 cells.

Biological factor related to Asian sand dust particles contributes to the exacerbation of asthma.

Epidemiologic studies have revealed that Asian sand dust particles (ASDs) can affect respiratory and immune health represented by asthma. Factors responsible for the exacerbation of asthma remain unclear. The fungus Bjerkandera adusta (B.ad) and polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) have been identified in ASDs collected from the atmosphere when an ASD event occurred. We investigated the effects of B.ad and BaP related to ASDs on respiratory and immune systems. Bone marrow-derived antigen-presenting cells (APCs) and splenocytes from atopic prone NC/Nga mice and human airway epithelial cells were exposed to the B.ad or to BaP in the presence and absence of heated-ASDs (H-ASDs). B.ad and BaP in both the presence and absence of H-ASDs increased the expression of cell surface molecules on APCs. H-ASDs alone slightly activated APCs. The expressions induced by B.ad were higher than those induced by BaP in the presence and absence of H-ASDs. There were no remarkable effects on the activation of splenocytes or the proinflammatory responses in airway epithelial cells. These results suggest that B.ad rather than BaP contributes to the exacerbation of asthma regardless of the presence or absence of sand particles, particularly by the activation of the immune system via APCs. Copyright © 2016 John Wiley & Sons, Ltd.

Periodontal Involvement in Leukocyte Adhesion Deficiency: Review of the Literature and a Case Report

Objective: Leukocyte adhesion deficiency (LAD) is a rare, autosomal recessive inherited disorder; LAD-I which is the most common type, occurs due to mutations in the CD18 gene. This mutation down-regulates the expression of s2 integrin leukocyte cell surface molecules, which are necessary for the adhesion of leukocytes to endothelial cells, transendothelial migration, and chemotaxis. The major symptoms are recurrent severe bacterial infections without pus formation, recurrent or progressive necrotizing soft tissue infections, marked leukocytosis and severe progressive periodontitis accompanied by alveolar bone loss, periodontal pockets, and partial or total early loss of primary and permanent teeth. Cases: Herein, we report a case of moderate LAD-I in a nine year-old boy with severe alveolar bone loss and aggressive periodontitis. For several years, approximately every month, the patient was followed up to evaluate the status of primary and permanent dentition. During these visits, scaling and root planing were performed and severely mobile teeth were extracted. The patient is now under regular follow up. Conclusion: This case confirms the need for interactions between the medical groups to identify and manage medically compromised children with rare diseases. It is important to include LAD in the differential diagnoses of children presenting with periodontal disease. Early correct diagnosis of LAD has various benefits for patients.

Microglia are less pro-inflammatory than myeloid infiltrates in the hippocampus of mice exposed to status epilepticus.

Activated microglia, astrogliosis, expression of pro-inflammatory cytokines, blood brain barrier (BBB) leakage and peripheral immune cell infiltration are features of mesial temporal lobe epilepsy. Numerous studies correlated the expression of pro-inflammatory cytokines with the activated morphology of microglia, attributing them a pro-epileptogenic role. However, microglia and myeloid cells such as macrophages have always been difficult to distinguish due to an overlap in expressed cell surface molecules. Thus, the detrimental role in epilepsy that is attributed to microglia might be shared with myeloid infiltrates. Here, we used a FACS-based approach to discriminate between microglia and myeloid infiltrates isolated from the hippocampus 24 h and 96 h after status epilepticus (SE) in pilocarpine-treated CD1 mice. We observed that microglia do not express MHCII whereas myeloid infiltrates express high levels of MHCII and CD40 96 h after SE. This antigen-presenting cell phenotype correlated with the presence of CD4(pos) T cells. Moreover, microglia only expressed TNFα 24 h after SE while myeloid infiltrates expressed high levels of IL-1β and TNFα. Immunofluorescence showed that astrocytes but not microglia expressed IL-1β. Myeloid infiltrates also expressed matrix metalloproteinase (MMP)-9 and 12 while microglia only expressed MMP-12, suggesting the involvement of both cell types in the BBB leakage that follows SE. Finally, both cell types expressed the phagocytosis receptor Axl, pointing to phagocytosis of apoptotic cells as one of the main functions of microglia. Our data suggests that, during early epileptogenesis, microglia from the hippocampus remain rather immune supressed whereas myeloid infiltrates display a strong inflammatory profile. GLIA 2016 GLIA 2016;64:1350-1362.