Fc Chimera Research Articles

The HCoV-HKU1 N-Terminal Domain Binds a Wide Range of 9-O-Acetylated Sialic Acids Presented on Different Glycan Cores.

Coronaviruses (CoVs) recognize a wide array of protein and glycan receptors by using the S1 subunit of the spike (S) glycoprotein. The S1 subunit contains two functional domains: the N-terminal domain (S1-NTD) and the C-terminal domain (S1-CTD). The S1-NTD of SARS-CoV-2, MERS-CoV, and HCoV-HKU1 possesses an evolutionarily conserved glycan binding cleft that facilitates weak interactions with sialic acids on cell surfaces. HCoV-HKU1 employs 9-O-acetylated α2-8-linked disialylated structures for initial binding, followed by TMPRSS2 receptor binding and virus-cell fusion. Here, we demonstrate that the HCoV-HKU1 NTD has a broader receptor binding repertoire than previously recognized. We presented HCoV-HKU1 NTD Fc chimeras on a nanoparticle system to mimic the densely decorated surface of HCoV-HKU1. These proteins were expressed by HEK293S GnTI- cells, generating species carrying Man-5 structures, often observed near the receptor binding site of CoVs. This multivalent presentation of high mannose-containing NTD proteins revealed a much broader receptor binding profile compared to that of its fully glycosylated counterpart. Using glycan microarrays, we observed that 9-O-acetylated α2-3-linked sialylated LacNAc structures are also bound, comparable to OC43 NTD, suggesting an evolutionarily conserved glycan-binding modality. Further characterization of receptor specificity indicated promiscuous binding toward 9-O-acetylated sialoglycans, independent of the glycan core (glycolipids, N- or O-glycans). We demonstrate that HCoV-HKU1 may employ additional sialoglycan receptors to trigger conformational changes in the spike glycoprotein to expose the S1-CTD for proteinaceous receptor binding.

The HCoV-HKU1 N-terminal domain binds a wide range of 9-O-acetylated sialic acids presented on different glycan cores.

Coronaviruses recognize a wide array of protein and glycan receptors using the S1 subunit of the spike (S) glycoprotein. The S1 subunit contains two functional domains: the N-terminal (S1-NTD) and C-terminal (S1-CTD). The S1-NTD of SARS-CoV-2, MERS-CoV, and HCoV-HKU1 possess an evolutionarily conserved glycan binding cleft that facilitates weak interactions with sialic acids on cell surfaces. HCoV-HKU1 employs 9-O-acetylated α2-8-linked disialylated structures for initial binding, followed by TMPRSS2 receptor binding and virus-cell fusion. Here, we demonstrate that HCoV-HKU1 NTD has a broader receptor binding repertoire than previously recognized. We presented HCoV-HKU1 NTD Fc chimeras on a nanoparticle system to mimic the densely decorated surface of HCoV-HKU1. These proteins were expressed by HEK293S GNTI- cells, generating species carrying Man-5 structures, often observed near the receptor binding site of CoVs. This multivalent presentation of high-mannose-containing NTD proteins revealed a much broader receptor binding profile compared to its fully glycosylated counterpart. Using glycan microarrays, we observed that 9-O-acetylated α2-3 linked sialylated LacNAc structures are also bound, comparable to OC43 NTD, suggesting an evolutionarily conserved glycan-binding modality. Further characterization of receptor specificity indicated promiscuous binding towards 9-O-acetylated sialoglycans, independent of the glycan core (glycolipids, N- or O-glycans). We demonstrate that HCoV-HKU1 may employ additional sialoglycan receptors to trigger conformational changes in the spike glycoprotein to expose the S1-CTD for proteinaceous receptor binding.

Let's Get Rolling: Precise Control of Microfluidic Assay Conditions to Recapitulate Selectin-Mediated Rolling Interactions of the Leukocyte Adhesion Cascade.

The leukocyte adhesion cascade governs the recruitment of circulating immune cells from the vasculature to distal sites. The initial adhesive interactions between cell surface ligands displaying sialyl-LewisX (sLeX) and endothelial E- and P-selectins serve to slow the cells down enough to interact more closely with the surface, polarize, and exit into the tissues. Therefore, precise microfluidic assays are critical in modeling how well immune cells can interact and "roll" on selectins to slow down enough to complete further steps of the cascade. Here, we present a systematic protocol for selectin mediated rolling on recombinant surfaces and endothelial cell monolayers on polyacrylamide gels of varying stiffness. We also describe step-by-step the protocol for setting up and performing the experiment and how to analyze and present the data collected. This protocol serves to simplify and detail the procedure needed to investigate the initial selectin-mediated interactions of immune cells with the vasculature. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing dishes for cell rolling experiments Basic Protocol 2: Fabrication of polyacrylamide gels for cell rolling experiments Alternate Protocol 1: Protein conjugation with N6 linker Alternate Protocol 2: HUVEC culturing for monolayers Basic Protocol 3: Conducting cell rolling experiments on polyacrylamide gels Basic Protocol 4: ImageJ analysis of cell rolling movies Basic Protocol 5: Quantification of Fc site density on a surface (e.g., for Fc chimeras).

VISTA Is the Predominant 2nd Generation of ICI (Immune Check-Point Inhibitor) in Ph(-) myeloproliferative Neoplasm

VISTA Is the Predominant 2nd Generation of ICI (Immune Check-Point Inhibitor) in Ph(-) myeloproliferative Neoplasm

Delivery of a novel membrane-anchored Fc chimera enhances NK cell-mediated killing of tumor cells and persistently virus-infected cells.

Antibody-dependent cellular cytotoxicity (ADCC) is one of the most powerful mechanisms for Natural Killer (NK) cells to kill cancer cells or virus-infected cells. A novel chimeric protein (NA-Fc) was created, which when expressed in cells, positions an IgG Fc domain on the plasma membrane, mimicking the orientation of IgG bound to the cell surface. This NA-Fc chimera was tested with PM21-NK cells, produced through a previously developed particle-based method which yields superior NK cells for immunotherapeutic applications. Real time viability assays revealed higher PM21-NK killing of both ovarian and lung cancer cells expressing NA-Fc, which correlated with increased release of TNF-α and IFN-γ cytokines from NK cells and was dependent on CD16-Fc interactions. Lentivirus delivery of NA-Fc to target cells increased the rate of PM21-NK cell killing of A549 and H1299 lung, SKOV3 ovarian and A375 melanoma cancer cells. This NA-Fc-directed killing was extended to virus infected cells, where delivery of NA-Fc to lung cells that were persistently infected with Parainfluenza virus resulted in increased killing by PM21-NK cells. In contrast to its effect on PM21-NK cells, the NA-Fc molecule did not enhance complement mediated lysis of lung cancer cells. Our study lays the foundation for application of the novel NA-Fc chimera that could be delivered specifically to tumors during oncolytic virotherapy to mark target cells for ADCC by co-treatment with adoptive NK cells. This strategy would potentially eliminate the need to search for unique cancer specific antigens for development of new antibody therapeutics.

Xiaotansanjiefang inhibits the viability of colorectal cancer cells via Jagged 1/Notch 3/Snail signaling pathway.

The purpose of this study is to explore the anti-colorectal cancer of Xiaotansanjiefang, a famous traditional Chinese medicine, and its potential anti-cancer mechanism. In this study, the HCT116 cell spheres were prepared as in vitro study model. We found the Xiaotansanjiefang medication was able to inhibit the proliferation of HCT116 cell spheres in a dose-dependent manner, especially in 3 and 6mg/ml Xiaotansanjiefang medication treated groups. We also found the high concentration of Xiaotansanjiefang medication could suppress the migration and promote the apoptosis of HCT116 cell spheres. Moreover, we found the expression of Jagged 1, Notch 3, Snail, and Hes 1 were decreased in HCT116 cell spheres treated with Xiaotansanjiefang medication. Furthermore, the proliferation and apoptosis behaviors of HCT116 cell spheres treated with Xiaotansanjiefang medication were reversed with the addition of Jagged 1 Fc chimera protein. The expression of Jagged 1, Notch 3, Snail, and Hes 1 were also increased again in HCT116 cells treated with Xiaotansanjiefang medication plus with Jagged 1 Fc chimera protein. The presented study may provide a promising strategy to treat and prevent colorectal cancer.

TIGIT Signaling Pathway Regulates Natural Killer Cell Function in Chronic Hepatitis B Virus Infection

Background and ObjectivePersistent infection of hepatitis B virus (HBV) and liver damage in immune active chronic hepatitis B (CHB) could be partly due to the overreaction of natural killer (NK) cells, including pro-inflammatory cytokine secretion and cytotoxicity. An immunosuppressive receptor, T-cell immunoglobulin and immunoreceptor tyrosine–based inhibitory motif (ITIM) domain (TIGIT) is specifically expressed in NK cells. This study aims to investigate the role of the TIGIT signaling pathway in regulating NK cell functions in patients with CHB.MethodWe comparatively assessed the expression of TIGIT in NK cells of patients with immune active CHB (CHB-IA), carriers of immune control chronic HBV (CHB-IC), and healthy controls (HCs), and then explored mechanisms of the TIGIT signaling pathway in regulating NK cell-mediated liver injury by different molecular assessments.ResultThe expression of TIGIT in NK cells was enhanced in CHB-IC but was reduced in CHB-IA compared with the HC group. In patients with CHB-IA, the expression of TIGIT was inversely correlated with intensity of the liver damage. Moreover, TIGIT-NK cells show higher IFN-γ secretion capability, degranulation activity, and cytotoxicity but lower apoptosis than TIGIT+ NK cells. Blockade of the TIGIT pathway with anti-TIGIT antibody increased NK cell function, while activation of the TIGIT pathway with TIGIT Fc and CD155 Fc chimera protein down-regulated NK cell function.ConclusionOur data showed that the TIGIT signaling pathway participates in NK cell impairment, which could be used as a new therapeutic target to protect patients with chronic HBV infection from severe liver injury.

Abstract 3034: Proteomic profiling identifies PTK7 as a novel immunotherapeutic candidate for neuroblastoma

Abstract Anti-GD2 antibody therapy significantly improved high-risk neuroblastoma (NB) patient survival yet is associated with toxicity due to GD2 expression on pain fibers. Also, GD2 may be downregulated in NB following chemotherapy and relapse. It is paramount to identify novel tumor-associated antigens that are upregulated on the cell surface throughout therapy with minimal normal tissue expression. We utilized a novel cell surface capturing technology to define the NB cell surfaceome before and after chemotherapy and nominate potential targets for cell-mediated immunotherapy. Two NB patient derived xenografts (PDXs) and one cell line xenograft (IMR5) were treated with topotecan/cyclophosphamide in vivo. Treated and untreated tumors were disaggregated and N-glycoproteins on intact cells were oxidized, selectively labeled with biocytin-hydrazide, lysed, proteins digested, and cell surface peptides enriched using NeutrAvidin beads, followed by LC-MS analysis. We prioritized cell surface proteins that were abundant on all NB models and did not change under the selective pressure of chemotherapy. Our top hit was confirmed for cell surface expression by flow, evaluated for primary tumor and normal cell expression using gene expression datasets, and evaluated for functional relevance using CRISPR/CAS9 and immunotherapy targeting via chimeric antigen receptor (CAR). In two replicates, 292 cell surface proteins were identified as abundant and unchanged following chemotherapy. 34/292 proteins had high abundance of ≥70 a.u. across all xenografts, including NB surface proteins ALK and NCAM1. One protein identified was PTK7, a member of the WNT signaling pathway that is overexpressed in adult tumors. PTK7 is highly expressed in primary NB tumors with very low normal tissue expression. PTK7 protein is present on the cell surface in 17/17 NB cell lines and PDXs. While high PTK7 expression in primary NB is associated with poor patient prognosis, CRISPR/CAS9 knockout of PTK7 shows no functional effect on cell survival, proliferation or chemotherapy response. We developed a codon-optimized, second-generation CAR construct specific to PTK7. Our CAR-engineered T cells specifically bound to recombinant human-PTK7 Fc chimera. PTK7 CAR Jurkat T cells became activated in the presence of PTK7-expressing KELLY, IMR-5, and NB-1643 NB cells, and activation was not seen in the presence of PTK7 negative CMK leukemia cells or NB-1643 PTK7 knockout cells, confirming activation is specific to the CAR recognizing PTK7. Primary T cell PTK7 CAR studies are ongoing to assess cytotoxicity against NB. Multiple surface proteins remain stable on NB tumors after chemotherapy treatment. PTK7 is a potential cell surface antigen that could be utilized for immunotherapy approaches in NB while minimizing damage to normal tissues. PTK7 is robustly expressed in other pediatric and adult cancers, suggesting broader clinical relevance. Citation Format: Arhanti Sadanand, Victor Maximov, Jasmine Lee, Suttipong Suttapitugsakul, Jenny Shim, Chris Doering, Ronghu Wu, Robert Schnepp, Trent Spencer, Kelly Goldsmith. Proteomic profiling identifies PTK7 as a novel immunotherapeutic candidate for neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3034.

Abstract 1638: Discovery and preclinical characterization of anti-Siglec-15 antibodies that rescue T cells from macrophage-mediated immune suppression

Abstract Background: Siglec-15 is an immunosuppressive sialic acid-binding Ig-like lectin expressed by myeloid cells, tumor associated macrophages (TAMs) and some human tumors. Siglec-15 expressed by TAMs inhibits anti-tumor immune responses by engaging unknown immune checkpoint(s) on T cells. Interactions between Siglec-15 on TAMs and sialy Tn (sTn) antigen found on tumor cells contributes to the immunosuppressive tumor microenvironment. Notably, the mutually exclusive expression of Siglec-15 and the checkpoint ligand PD-L1 by cancer cells emphasizes Siglec-15 as an attractive target for cancer immunotherapy. Methods: Anti-Siglec-15 antibodies were cloned from B cells derived from rabbits immunized with human Siglec-15. Cells were cultured at clonal density, and IgG antibodies in supernatants were evaluated for binding to human, murine and cynomolgus Siglec-15. Variable-regions from positive hits were sequenced, cloned, and expressed as recombinant rabbit human IgG1 Fc chimeras. Anti-Siglec-15 chimeric antibodies were evaluated in a panel of functional and phenotypic assays using primary human macrophages and T cells, and then prioritized for evaluation in murine tumor models. Results: Thirty-one rabbit anti-Siglec-15 clones were expressed as rabbit-human IgG1 chimeras based on binding to recombinant human, cynomolgus and murine Siglec-15 proteins, binding to Siglec-15 expressing cell lines, and lack of binding to other Siglec family members. A subset of these clones inhibited the binding of Siglec-15 to sTn. The top 5 clones were identified in functional screens modeling Siglec-15 mediated immune suppression. The clones rescued the NFAT promotor activity of a T cell reporter cell line as well as the proliferative and IFN-γ response of anti-CD3 activated human T cells from the inhibitory activity of recombinant Siglec-15-Fc protein. Selected clones also relieved M2c-macrophage-mediated immune suppression in M2c/T cell coculture assays by restoring T-cell proliferation and IFN-γ secretion. The prioritized clones are currently under evaluation in in vivo tumor models for PK and efficacy. Conclusions: We identified novel anti-Siglec-15 antibodies that restore T cell effector function following suppression by recombinant and cell-expressed Siglec-15. These data support further development of these anti-Siglec 15 antibodies as an anti-cancer immunotherapy. Citation Format: Myriam Bouchlaka, Huyen Dinh, Sam Lam, Valerie Wall, Darbie Whitman, Ramya Chandrasekaran, Johanna Harshman, Texia Loh, Lauren Loh, Tom Graddis, Kamal D. Puri, Peter Probst. Discovery and preclinical characterization of anti-Siglec-15 antibodies that rescue T cells from macrophage-mediated immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1638.

Targeting Siglec-7: A Novel Immunotherapeutic Approach to Potentiate the Cytotoxic Functions of Natural Killer Cells Against Multiple Myeloma

Background: MultipleMyeloma (MM) is a malignant plasma cell disorder, accounting for approximately 10% of all haematological malignancies. The intrinsic ability of the malignant cells to evade the host immune system is one of the key hallmarks of the disease, and is associated with poor overall survival of MM patients. The immune-suppressive functions of sialylated glycans, which are abundantly expressed on the surface of myeloma cell, has not previously been addressed. We hypothesize that hypersialylation of MM cells is a key mechanism enabling evasion of cytotoxic Natural Killer (NK) cells within the MM bone-marrow (BM) niche. Furthermore, we hypothesize that disrupting the immune-suppressive interactions between sialylated glycans on MM cells, and their cognate sialic acid-binding lectin (Siglec) receptors on NK cells could pave the way for the development of novel immunotherapeutic strategies for treating MM patients.Methods and Results: Using a recombinant Siglec-7 Fc chimera, we observed that Siglec-7 ligands (Siglec-7L) are highly expressed (>85%) across a panel of MM cell lines (RPMI 8226, H929 and MM1S). Furthermore, Siglec-7L expression was also observed on CD38+ CD138+ primary MM cells isolated from BM aspirate of MM patient. To determine if Siglec-7L expression on MM cells plays a role in immune-evasion, we next analysed the cognate Siglec-7L receptor (Siglec-7R) on primary BM NK cells from MM patients (n=5). Interestingly, immunophenotyping analysis revealed that CD3- NKp46+ NK cells circulating within the BM of MM patients have significant expression of Siglec-7R on their cell surface (78 ±27%).To investigate the immune-suppressive effects of Siglec-7L and Siglec-7R interaction in dampening the cytotoxic functions of NK cells, we treated MM cell lines RPMI-8226, H929 and MM1S with a sialyltransferase inhibitor (SIA) (3Fax-Peracetyl Neu5Ac; dose-200µM) for 5 days. As expected, this treatment resulted in a complete eradication of Siglec-7L from the surface of MM cells. Subsequently, SIA pre-treated cells were co-cultured with the human cytotoxic NK cell line KHYG-1 at E:T ratios of 1:1, 2.5:1 and 5:1 for 12 hours. Flow cytometry based analysis of cell viability using Annexin V and Sytox Green revealed that abolishing the expression of sialylated glycans from the cell surface of MM cells results in a 1.3-1.6 fold increase (p<0.05) in NK cell induced cell death in MM cell lines RPMI 8226 and H929 (n=3) (Figure 1). However, a significant increase in NK cell mediated killing was not observed for MM1S cells (n=3) pre-treated with SIA, suggesting a role of “classical” NK cell inhibitory signals in NK cell anti-tumor immunity in the case of MM1S.Furthermore to validate our observations we knocked-down (KD) Siglec-7R in KHYG-1 cells using an siRNA approach. KHYG-1 has relatively lower expression of Siglec-7R (20-25%), as compared to peripheral blood NK cells (>90%), and other NK cell lines such as NK-92 (>60%). siRNA targeting Siglec-7R reduced the expression of Siglec-7R from 25% in the scrambled siRNA control to 15% in the Siglec-7R siRNA experimental condition. Upon co-culture of the scrambled siRNA or Siglec-7R siRNA KD KHYG-1 cells with RPMI-8226 at E:T ratio of 0.25:1 and 0.5:1, we observed a 7% increase in NK cell mediated cytotoxicity towards the myeloma cells (n=2) .Conclusion: We observed that sialylated Siglec-7L is abundantly expressed on the surface of MM cells. The cognate receptors of the ligands are significantly expressed on primary NK cells circulating within the BM of MM patients. Functional assays revealed that Siglec-7L and Siglec-7R receptor interactions may have significant immune-suppressive effects on the cytotoxicity of NK cells. Thus, we can leverage the findings of this study to develop NK cell based cellular therapies to target Siglec-7 ligand- receptor interaction in MM. This could be achieved by knocking down Siglec-7R in the setting of an autologous or allogeneic blood derived NK cell therapy. A more efficient approach, both therapeutically and logistically, would be the use of Siglec-7R deficient NK cell lines, such as KHYG-1 or NK-92 for the treatment of patients with MM.Conflict Of Interest: Michael O'Dwyer: Founder, Board of Directors, Equity in Onkimmune. Subhashis Sarkar is supported by a fellowship from Onkimmune. [Display omitted] DisclosuresSarkar:Onkimmune: Other: Fellowship Funding. O'Dwyer:Onkimmune Ltd: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Interleukin-6 sensitizes TNF-α and TRAIL/Apo2L dependent cell death through upregulation of death receptors in human cancer cells

Interleukin-6 (IL-6) enhanced TNF-α and TRAIL/Apo2L induced cell death in various human cancer cells derived from malignant glioma, melanoma, breast cancer and leukemia, although the effect was not detected with IL-6 alone. The effects of IL-6 using SKBR3 cells were associated with the generation of apoptotic cells as analyzed by fluorescence microscopy and flow cytometry. IL-6 activated p53 and upregulated TRAIL death receptors (DR-4 and DR-5) and stimulated the TNF-α and TRAIL dependent extrinsic apoptotic pathway without activation of the p53 mediated intrinsic apoptotic pathway. TNF-α and TRAIL induced cleavage of caspase-8 and caspase-3 was more enhanced by IL-6, although these caspases were not cleaved by IL-6 alone. The dead cell generation elicited by the combination with IL-6 was blocked by anti-human TRAIL R2/TNFRSF10B Fc chimera antibody which can neutralize the DR-5 mediated death signal. These findings indicate that IL-6 could contribute to the enhancement of TNF-α or TRAIL induced apoptosis through p53 dependent upregulation of DR-4 and DR-5. The data suggest that a favorable therapeutic interaction could occur between TNF-α or TRAIL and IL-6, and provide an experimental basis for rational clinical treatments in various cancers.

Inhibition of Activin A suppressed tumor necrosis factor-α secretion and improved histopathological conditions in malarial mice.

Malaria infection still remains as one of the most prominent parasitic diseases afflicting mankind in tropical and subtropical regions. The severity of malaria infection has often been associated to exuberant host immune inflammatory responses that could possibly lead to severe immunopathological conditions and subsequent death of host tissues. Activin A is a protein belonging to the transforming growth factor-beta (TGF-β) family that regulates multiple physiological processes and pathological-associated diseases. The biological roles of activin A have been associated with manipulation of inflammation-related processes and modulation of host immune responses. This implies that activin A protein could play a role in malaria pathogenesis since malaria infection has been closely linked to severe immune responses leading to death, However, the actual in vivo role of activin A in malaria infection remains elusive. Hence, this study was undertaken to investigate the involvement of activin A in malaria infection as well as to assess the modulating effects of activin A on the cytokine releases (TNF-α, IFN-γ and IL-10) and histopathological changes in major affected organs (kidney, liver, lung, brain and spleen) in malarial mice infected with Plasmodium berghei ANKA. Our results showed that the concentrations of plasma activin A were significantly increased in malarial mice throughout the study periods. Also. the systemic activin A level was positively correlated with malaria parasitemia. This indicates that activin A could play a role in malaria pathogenesis and malaria parasitemia development. Plasma TNF-α, IFN-γ and IL-10 cytokine levels were significantly increased in malarial mice at day-5 post infection, suggesting that these cytokines attributed to severe malaria pathogenesis. Histopathological features such as sequestration of parasitized red blood cells (pRBCs) and hemozoin formation were amongst the most common pathological conditions observed in tissues of major affected organs (kidney, liver, lung, brain and spleen) in malarial mice. Neutralization of activin A production via recombinant mouse activin RIIA Fc chimera (rmActivin RIIA Fc chimera) had significantly reduced the parasitemia levels in malarial mice. The release of TNF-α cytokine was significantly reduced as well as the sequestration of parasitized pRBCs and hemozoin formation in major affected organs in malarial mice were also alleviated following inhibition of activin A production. Overall, this preliminary study suggests that activin A could play an immune modulation role in malaria pathogenesis through modulation of TNF-α release that benefits host from severe pathological destructions provoked by intensified inflammatory responses. Further studies are warranted to elucidate the precise mechanism of immune modulation mediated by activin A and its associated immune-modulation mediators in regulating the inflammatory responses elicited during the course of malaria infection.

Immunosuppressive Regulation of Dendritic Cells and T Cells in Allergic Rhinitis by Semaphorin 3A.

Semaphrin3A (Sema3A) was found to play a major role in immune regulation in autoimmune diseases and to be of importance in allergic disease. However, the effect of Sema3A on allergic rhinitis (AR) is not fully clear. We sought to elucidate the effects of Sema3A on the regulation of dendritic cells (DCs) and naive CD4+ T cells in AR. The expression of Sema3A in nasal mucosa was measured by immunohistochemical staining and western blotting. Human peripheral blood mononuclear cells were separated by the Ficoll-Hypaque method. DCs and naive CD4+ T cells were purified by magnetic selection. A human Sema3A Fc chimera was added to DCs and naive CD4+ T cells in vitro to evaluate the effect of Sema3A on the function of DCs and T cells. Labeling T cells with CFSE was used to determine cell proliferation. Flow cytometry was used to detect the DC maturation markers (CD40 and CD83) and T helper 17 (Th17) and regulatory T cell (Treg) percentages. ELISA was used to detect the IL10, IL17, IL4, and IFNγ cytokine levels. The expression of Sema3A in AR inferior turbinate tissue was lower than that in healthy control tissue. Compared with healthy control DCs, AR DCs showed decreased levels of the DC maturation markers CD40 and CD83 after Sema3A treatment. Furthermore, Sema3A decreased naive CD4+ T cell proliferation in AR. In addition, Sema3A increased the percentage of Tregs but had no obvious effect on Th17 cells. Moreover, Sema3A significantly increased levels of IL10 and IFNγ, and decreased level of IL4, but had no obvious effect on level of IL17. AR presented with low expression of Sema3A in nasal mucosa, and Sema3A could decrease DC maturation, T cell proliferation, and Treg polarization.

Integrin Crosstalk Modulates Stiffness-independent Motility in CD4+ T Lymphocytes

CD4+ T lymphocytes interact with various cell types and tissue substrates in response to inflammation.Mechanotransduction in T lymphocytes is frequently studied at immunological synapses (IS) during T cell activation, after engagement with T cell receptors (TCR) and costimulatory receptors (i.e. CD3/CD28) and LFA-1 integrins. Recently, in vitro and in vivo studies suggest T cells can migrate upstream (i.e. in the opposite direction of shear flow) on surfaces expressing ICAM-1 through interaction with LFA-1 integrins. LFA-1 likely interacts as a mechanosensor, and thus we hypothesized that substrate mechanics might affect the ability of LFA-1 to support upstream migration of T cells under flow. To test the hypothesis, we measured motility of activated primary human CD4+ T lymphocytes on polyacrylamide gels with pre-determined stiffnesses, functionalized with protein A/G and ICAM-1, VCAM-1, or 1:1 mixture of ICAM-1/VCAM-1 Fc chimeras. We found that CD4+ T cells exhibit stiffness-dependent motility on ICAM-1 surfaces, with a maximum random motility appearing at an elasticity of about 60 kPa. in contrast, motility on VCAM-1 or 1:1 mixture of VCAM-1/ICAM-1 mixed surfaces was independent of elasticity. This mechanosensitivity on ICAM-1 surfaces is overcome when VLA-4 is ligated with soluble VCAM-1. Lastly, we observed that CD4+ T cells can migrate upstream under flow on hydrogels functionalized with ICAM-1, independent of substrate stiffness. In this study, we show that CD4+ T cells can sense stiffness with integrins alone under static conditions and that integrin crosstalk can activate an elasticity-independent motility. interestingly, CD4+ T lymphocytes migrated upstream under flow on ICAM-1 substrates regardless of the substrate stiffness.

ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia

BackgroundCharacterized by abnormal lung growth or maturation, congenital diaphragmatic hernia (CDH) affects 1:3000 live births. Cellular studies report proximal (SOX2+) and distal (SOX9+) progenitor cells as key modulators of branching morphogenesis and epithelial differentiation, whereas transcriptome studies demonstrate ROBO/SLIT as potential therapeutic targets for diaphragm defect repair in CDH. In this study, we tested the hypothesis that (a) experimental-CDH could changes the expression profile of ROBO1, ROBO2, SOX2 and SOX9; and (b) ROBO1 or ROBO2 receptors are regulators of branching morphogenesis and SOX2/SOX9 balance.MethodsThe expression profile for receptors and epithelial progenitor markers were assessed by Western blot and immunohistochemistry in a nitrofen-induced CDH rat model. Immunohistochemistry signals by pulmonary structure were also quantified from embryonic-to-saccular stages in normal and hypoplastic lungs. Ex vivo lung explant cultures were harvested at E13.5, cultures during 4 days and treated with increasing doses of recombinant rat ROBO1 or human ROBO2 Fc Chimera proteins for ROBO1 and ROBO2 inhibition, respectively. The lung explants were analyzed morphometrically and ROBO1, ROBO2, SOX2, SOX9, BMP4, and β-Catenin were quantified by Western blot.ResultsExperimental-CDH induces distinct expression profiles by pulmonary structure and developmental stage for both receptors (ROBO1 and ROBO2) and epithelial progenitor markers (SOX2 and SOX9) that provide evidence of the impairment of proximodistal patterning in experimental-CDH. Ex vivo functional studies showed unchanged branching morphogenesis after ROBO1 inhibition; increased fetal lung growth after ROBO2 inhibition in a mechanism-dependent on SOX2 depletion and overexpression of SOX9, non-phospho β-Catenin, and BMP4.ConclusionsThese studies provided evidence of receptors and epithelial progenitor cells which are severely affected by CDH-induction from embryonic-to-saccular stages and established the ROBO2 inhibition as promoter of branching morphogenesis through SOX2/SOX9 balance.

The development of in vitro potency assay for immune checkpoint regulators

Background & Aim Cancer immunotherapy by aiding the immune system in recognition and destruction of tumor cells by enhancing its ability to react to tumor antigens is now been demonstrated to be effective in many instances. Genomic modifications of multiple gene loci to generate potent effector T cells resistant to multiple inhibitory pathways such as PD-1, CTLA-4 is one of major attractive strategies for cell-based immunotherapy. Both PD-1 and CTLA-4 are type I transmembrane T cell inhibitory molecules that express or being recruited on activated T cells. They interact with their ligands, such as PD-L1 and B7-1 respectively to induce co-inhibitory signals on T cells and suppress T-cell mediated immune response against tumors. Thus, the PD-1: PD-L1/PD-L2 and CTL-4: B7-1/B7-2 interactions are key regulators of the threshold of immune response and a consistent, quantitative in vitro potency assay would benefit the development of immune checkpoint-mediated cancer immunotherapy. Methods, Results & Conclusion Recombinant PD-1, CTLA-4 and their specific ligands were expressed in either HEK293 or CHO cells and purified. Purified molecules' biological activities were initially examined and confirmed using T cell-based proliferation assay where applicable. In this study, we would demonstrate how we used Octet instrument to determine the affinities and kinetics of recombinant PD-1 and CTLA-4 Fc chimera molecules’ bindings to their correspondent ligands, and further identified quantitatively different affinities and kinetics of PD-1 and CTLA-4 when bound to their different ligands. Our results indicate the Octet-based assay can serve as a rapid, label-free, quantitatively suitable in vitro potency analysis with very minimal interference for qualifying and studying immune checkpoint regulators.

Selectin-Binding Assay by Flow Cytometry.

Cell-cell interactions mediated by selectins and their ligand glycans play pivotal roles in a variety of biological processes represented by leukocyte recruitment to inflammatory sites, lymphocyte homing, and extravasation of cancer cells. The interactions are enhanced at least partly through the upregulation of the selectin-ligand glycan expression, which is observed, for instance, during the activation of leukocytes or epithelial-mesenchymal transition of cancer cells. Selectin-binding assays such as cell adhesion assay or rolling assay have long been used to directly evaluate the activity of these cells in the selectin-mediated processes. In this chapter, we introduce a highly quantitative assay by flow cytometry using recombinant selectin-Ig(Fc) chimera proteins, showing our procedure and tips for E-selectin-binding assay of colon cancer cells undergoing epithelial-mesenchymal transition.

BAFF is involved in the pathogenesis of IgA nephropathy by activating the TRAF6/NF‑κB signaling pathway in glomerular mesangial cells.

The aim of the present study was to investigate the involvement of B cell-activating factor (BAFF) in the pathogenesis of IgA nephropathy by activating the tumor necrosis factor receptor-associated factor 6 (TRAF6)/NF-κB signaling pathway in glomerular mesangial cells. For the clinical analysis, blood, urine and kidney tissue samples were collected from 58 patients diagnosed with primary IgA nephropathy by renal biopsy. For the in vitro study, glomerular mesangial cells were divided into five groups: Control (con)-short hairpin RNA (shRNA) (control group); con-shRNA + BAFF (20 ng/ml); con-shRNA + BAFF + BAFF-RFc chimera protein (500 µg/ml); TRAF6-shRNA; and TRAF6-shRNA + BAFF (20 ng/ml). For the in vivo experiments, 60 Sprague-Dawley rats were randomly divided into four groups: Con-small interfering RNA (siRNA) (control group); con-siRNA + IgA (IgA nephropathy group), BAFF-RFc chimera protein (2 µg/ml) + IgA, and TRAF6-siRNA (0.2 µM) + IgA. Reverse transcription-quantitative PCR was performed to evaluate the mRNA expression levels of TRAF6, connective tissue growth factor (CTGF), fibronectin (FN) and NF-κBP65. Western blot analysis was used to detect the protein expression levels of TRAF6, FN, CTGF and phosphorylated-NF-κBP65 in glomerular mesangial cells and kidney tissues. The results revealed that plasma BAFF levels were positively correlated with the severity of pathological damage in patients with IgA nephropathy. In vitro, BAFF induced the mRNA and protein expression of TRAF6, CTGF, FN and NF-κBP65 in glomerular mesangial cells. After the BAFF-RFc chimera protein was added to inhibit the binding of BAFF and BAFF-receptor (-R), this effect was reduced. In vivo, inhibition of the effects of BAFF via injection with the BAFF-R Fc chimera protein reduced kidney damage in rats suffering from IgA nephropathy. The effect on the expression of signaling pathway-associated proteins was also alleviated. In conclusion, BAFF enhanced the expression of fibroblast factors in the kidneys by activating the TRAF6/NF-κB signaling pathway.

A Novel Recurrent Gain-of-Function Mutation of Rltpr Q575E in Adult T Cell Leukemia/Lymphoma

A Novel Recurrent Gain-of-Function Mutation of Rltpr Q575E in Adult T Cell Leukemia/Lymphoma

In Vitro Monolayer Culture of Dispersed Neural Stem Cells on the E-Cadherin-Based Substrate with Long-Term Stemness Maintenance.

Neural stem cells (NSCs) play an important role in neural tissue engineering because of their capacity of self-renewal and differentiation to multiple cell lineages. The in vitro conventional neurosphere culture protocol has some limitations such as limited nutrition and oxygen penetration and distribution causing the heterogeneity of cells inside, inaccessibility of internal cells, and inhomogeneous cellular morphology and properties. As a result, cultivation as a monolayer is a better way to study NSCs and obtain a homogeneous cell population. The cadherins are a classical family of homophilic cell adhesion molecules mediating cell-cell adhesion. Here, we used a recombinant human E-cadherin mouse IgG Fc chimera protein that self-assembles on a hydrophobic polystyrene surface via hydrophobic interaction to obtain an E-cadherin-coated culture plate (ECP). The rat fetal NSCs were cultured on the ECP and routine tissue culture plate (TCP) from passage 2 to passage 5. NSCs on TCP formed uniform floating neurospheres and grew up over time, while cells on the ECP adhered on the bottom of the plate and exhibited individual cells with scattering morphology, forming intercellular connections between cells. The cell proliferation and differentiation behaviors that were evaluated by Cell Counting Kit-8 assay (CCK-8), immunofluorescence staining, and real-time quantitative polymerase chain reaction showed NSCs could maintain the capacity for self-renewal and ability to differentiate into neurons, oligodendrocytes, and astrocytes after the long-term in vitro cell culture and passaging. Therefore, our study indicated that hE-cad-Fc could provide a homogeneous environment for individual cells in monolayer conditions to maintain the capacity of self-renewal and differentiation by mimicking the cell–cell interaction.