Extracellular Immunoglobulin Domains Research Articles

The involvement of VEGF and VEGFR in bacterial recognition and regulation of antimicrobial peptides in Eriocheir sinensis

Vascular endothelial growth factor (VEGF) and VEGF reporter (VEGFR) are essential molecules in VEGF signalling pathway. Although the functions of VEGF and VEGFR have been well reported in vertebrates, their functions are still poorly understood in invertebrates. In this study, the open reading frame sequences of EsVEGF1 and EsVEGFR4 were cloned from Eriocheir sinensis, and their corresponding proteins shared typical structure characteristics with their counterparts in other species. EsVEGF1 were predominantly expressed in hepatopancreas and muscle while EsVEGFR4 mainly expressed in hemocytes and intestine. The expression levels of EsVEGF1 in hemocytes were rapidly induced by Staphylococcus aureus and Vibrio parahaemolyticus, and it also increased rapidly in hepatopancreas after being challenged with V. parahaemolyticus. The expression levels of EsVEGFR4 only increased in hepatopancreas of crabs injected with S. aureus. The extracellular immunoglobulin domain of EsVEGFR4 could bind with Gram-negative and Gram-positive bacteria as well as lipopolysaccharide and peptidoglycan. EsVEGF1 could act as the ligand for EsVEGFR4 and Toll-like receptor and regulate the expression of crustins and lysozyme with a tissue-specific manner, while have no regulatory function on that of anti-lipopolysaccharide factors. This study will provide new insights into the immune defense mechanisms mediated by VEGF and VEGFR in crustaceans.

Characterization of CD166 from Nile tilapia (Oreochromis niloticus) displays a broad pathogen recognition spectrum and involved the immune response to microbial aggression

CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules, and its mediated adhesion plays a crucial role in different physiological and pathological phenomena, especially related to leukocyte extravasation, immune synapse stability, T cell activation and proliferation. In this study, CD166 was identified from Nile tilapia (Oreochromis niloticus, OnCD166). OnCD166 contains an open reading frame of 1671 bp that encodes a peptide of 556 amino acids, and contains five consecutive extracellular immunoglobulin domains. It's tissue distribution and expression patterns after S. agalactiae challenge were also investigated. OnCD166 is widely distributed in various tissues of healthy tilapia. After Streptococcus agalactiae challenge, OnCD166 expressions were significantly up-regulated in all tested immune tissues. Meanwhile, the recombinant OnCD166 (rOnCD166E) protein showed strong agglutinating activities against both Gram-negative bacteria and Gram-positive bacteria. Moreover, rOnCD166E could promote phagocytosis of macrophages. Taken together, our results illustrated that OnCD166 might as a receptor involved in the immune recognition and phagocytosis against invading pathogen, which play important roles in the immune responses of Nile tilapia against bacterial pathogens.

Cardiac sodium channel complexes and arrhythmia: structural and functional roles of the β1 and β3 subunits.

In cardiac myocytes, the voltage-gated sodium channel NaV 1.5 opens in response to membrane depolarisation and initiates the action potential. The NaV 1.5 channel is typically associated with regulatory β-subunits that modify gating and trafficking behaviour. These β-subunits contain a single extracellular immunoglobulin (Ig) domain, a single transmembrane α-helix and an intracellular region. Here we focus on the role of the β1 and β3 subunits in regulating NaV 1.5. We catalogue β1 and β3 domain specific mutations that have been associated with inherited cardiac arrhythmia, including Brugada syndrome, long QT syndrome, atrial fibrillation and sudden death. We discuss how new structural insights into these proteins raises new questions about physiological function.

A highly diverse set of novel immunoglobulin-like transcript (NILT) genes in zebrafish indicates a wide range of functions with complex relationships to mammalian receptors.

Multiple novel immunoglobulin-like transcripts (NILTs) have been identified from salmon, trout, and carp. NILTs typically encode activating or inhibitory transmembrane receptors with extracellular immunoglobulin (Ig) domains. Although predicted to provide immune recognition in ray-finned fish, we currently lack a definitive framework of NILT diversity, thereby limiting our predictions for their evolutionary origin and function. In order to better understand the diversity of NILTs and their possible roles in immune function, we identified five NILT loci in the Atlantic salmon (Salmo salar) genome, defined 86 NILT Ig domains within a 3-Mbp region of zebrafish (Danio rerio) chromosome 1, and described 41 NILT Ig domains as part of an alternative haplotype for this same genomic region. We then identified transcripts encoded by 43 different NILT genes which reflect an unprecedented diversity of Ig domain sequences and combinations for a family of non-recombining receptors within a single species. Zebrafish NILTs include a sole putative activating receptor but extensive inhibitory and secreted forms as well as membrane-bound forms with no known signaling motifs. These results reveal a higher level of genetic complexity, interindividual variation, and sequence diversity for NILTs than previously described, suggesting that this gene family likely plays multiple roles in host immunity.

FLT4 Mutations Are Associated with Segmental Lymphatic Dysfunction and Initial Lymphatic Aplasia in Patients with Milroy Disease.

This study explored mutations in the Fms-related tyrosine kinase 4/vascular endothelial growth factor receptor 3 gene (FLT4) and lymphatic defects in patients with Milroy disease (MD). Twenty-nine patients with lower limb lymphedema were enrolled. Sixteen patients had a familial history of MD, while 13 patients exhibited sporadic MD. Clinical signs, FLT4 mutations, indocyanine green (ICG) lymphography findings, and skin tissue immunohistochemical staining results were evaluated. Twenty-eight variants in FLT4 were identified. Twelve of these have previously been reported, while 16 are novel. Of the 28 variants, 26 are missense mutations, and the remaining two comprise a splicing mutation and a non-frame shift mutation. Twenty-five variants are located in the intracellular protein tyrosine kinase domain; three are located in the extracellular immunoglobulin domain. Substantially delayed contrast-enhanced tortuous lymphatic vessels were visualized to the ankle or knee level in 15 of 23 patients who underwent ICG lymphography. No initial lymphatic vessels were visualized in skin specimens from four patients who did not exhibit lymphatic vessels during imaging analyses. No specific variant was identified in relation to the unique clinical phenotype. Segmental dysfunction of lymphatic vessels and initial lymphatic aplasia are present in MD patients with FLT4 mutations.

Ptk7 Is Dynamically Localized at Neural Crest Cell–Cell Contact Sites and Functions in Contact Inhibition of Locomotion

Neural crest (NC) cells are highly migratory cells that contribute to various vertebrate tissues, and whose migratory behaviors resemble cancer cell migration and invasion. Information exchange via dynamic NC cell–cell contact is one mechanism by which the directionality of migrating NC cells is controlled. One transmembrane protein that is most likely involved in this process is protein tyrosine kinase 7 (PTK7), an evolutionary conserved Wnt co-receptor that is expressed in cranial NC cells and several tumor cells. In Xenopus, Ptk7 is required for NC migration. In this study, we show that the Ptk7 protein is dynamically localized at cell–cell contact zones of migrating Xenopus NC cells and required for contact inhibition of locomotion (CIL). Using deletion constructs of Ptk7, we determined that the extracellular immunoglobulin domains of Ptk7 are important for its transient accumulation and that they mediate homophilic binding. Conversely, we found that ectopic expression of Ptk7 in non-NC cells was able to prevent NC cell invasion. However, deletion of the extracellular domains of Ptk7 abolished this effect. Thus, Ptk7 is sufficient at protecting non-NC tissue from NC cell invasion, suggesting a common role of PTK7 in contact inhibition, cell invasion, and tissue integrity.

Comprehensive analysis of BTN3A1 in cancers: mining of omics data and validation in patient samples and cellular models.

Butyrophilin 3A1 (BTN3A1), a major histocompatibility complex‐associated gene that encodes a membrane protein with two extracellular immunoglobulin domains and an intracellular B30.2 domain, is critical in T‐cell activation and adaptive immune response. Here, the expression of BTN3A1 in cancers was analyzed in eight databases comprising 86 733 patients of 33 cancers, and the findings were validated in patient samples and cell models. We showed that BTN3A1 was expressed in most cancers, and its expression level was strongly correlated with clinical outcome of 13 cancers. Mutations of BTN3A1 were detected, and the mutations were distributed throughout the entire gene. Gene set enrichment analysis showed that BTN3A1 co‐expression genes and interacting proteins were enriched in immune regulation‐related pathways. BTN3A1 was associated with tumor‐infiltrating immune cells and was co‐expressed with multiple immune checkpoints in patients with breast cancer (BRCA) and non‐small cell lung cancer (NSCLC). We reported that BTN3A1 was downregulated in 46 of 65 (70.8%) NSCLCs, and its expression level was inversely associated with clinical outcome of the patients. BTN3A1 in tumor samples was lower than in counterpart normal tissues in 31 of 38 (81.6%) BRCAs. Bioinformatics analyses showed that BTN3A1 could be a target gene of transcription factor Spi‐1 proto‐oncogene (SPI1), and our ‘wet’ experiments showed that ectopic expression of SPI1 upregulated, whereas silencing of SPI1 downregulated, BTN3A1 expression in cells. These results suggest that BTN3A1 may function as a tumor suppressor and may serve as a potential prognostic biomarker in NSCLCs and BRCAs.

A highly diverse set of novel immunoglobulin-like transcript (NILT) genes in zebrafish indicates a wide range of functions

Abstract Multiple novel immunoglobulin-like transcripts (NILTs) have been identified from salmon, trout and carp. NILTs typically encode activating or inhibitory transmembrane receptors with extracellular immunoglobulin (Ig) domains. Although predicted to provide some level of immune recognition in ray-finned fish, we currently lack a definitive framework of NILT diversity, thereby challenging our ability to understand the evolutionary origin and specific function of these genes. In order to better understand the diversity of NILT genes and their possible roles in immune function, we identified 5 NILT loci in the Atlantic salmon (Salmo salar) genome, defined 86 NILT Ig domains within a 3 Mbp region of zebrafish (Danio rerio) chromosome 1, and described 41 NILT Ig domains as part of an alternate haplotype for this same genomic region. We then identified transcripts that define 43 different NILT genes and reflect an unprecedented range of sequence diversity and combinatorial diversity of Ig domains for a single family of non-recombining receptors. Zebrafish NILTs include a single putative activating receptor but extensive inhibitory and secreted forms as well as membrane-bound forms with no known signaling motifs. Whole genome sequences from different genetic backgrounds reveal at least three alternative haplotypes of the zebrafish NILT gene cluster that display gene content variation. This observation indicates that different individual fish can encode different combinations of NILTs which could impact their immune function. Furthermore, these results reveal a higher level of genetic complexity and sequence diversity for NILTs than previously described, suggesting that this gene family likely plays multiple roles in host immunity.

Development of Anti-human T Cell Immunoreceptor with Ig and ITIM Domains (TIGIT) Monoclonal Antibodies for Flow Cytometry.

Immune checkpoint inhibitors targeting programmed cell death-ligand 1 (PD-L1), programmed cell death-1 (PD-1), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) recently made a significant survival rate improvement in cancer treatment. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is expressed in T and NK cells related to their activities. It has a single extracellular immunoglobulin domain, a type 1 transmembrane domain, and a single intracellular ITIM. TIGIT binds with poliovirus receptor (PVR) or PVR2, resulting in suppressing T and NK cell activities. Some studies showed that the combined use of a TIGIT inhibitor with another immune checkpoint inhibitor enhanced antitumor activities more strongly than their single use. Therefore, TIGIT should be a new target for immunotherapy. In this study, we developed new anti-human TIGIT (hTIGIT) monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. Mice were immunized with hTIGIT-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/hTIGIT), and hybridomas were screened by flow cytometry. One of the mAbs, TgMab-2 (IgG1, kappa), specifically and sensitively detects hTIGIT in CHO/hTIGIT and NK cells. The dissociation constants (KD) of TgMab-2 for CHO/hTIGIT cells were determined to be 3.5 × 10-9 M. These results suggest that TgMab-2, which was developed by CBIS method, is useful for analyzing the function of hTIGIT by flow cytometry.

A truncation mutation in the <i>L1CAM</i> gene in a child with hydrocephalus

<abstract> <p>Hydrocephalus is a neurodevelopmental, X-linked recessive disorder caused by mutations in the <italic>L1CAM</italic> gene. The <italic>L1CAM</italic> gene encodes for L1CAM protein which is essential for the nervous system development including adhesion between neurons, Myelination, Synaptogenesis etc. Herein, the present study has reported mutations in L1 syndrome patient with Hydrocephalus and Adducted thumb. Genomic DNA was extracted from patients whole blood (n = 18). The 11 exons of the <italic>L1CAM</italic> gene were amplified using specific PCR primers. The sequenced data was analysed and the pathogenicity of the mutation was predicted using the various bioinformatics programs: PROVEAN, PolyPhen2, and MUpro. The results revealed that the proband described here had nonsense mutation G1120→T at position 1120 in exon 9 which is in extracellular immunoglobulin domain (Ig4) of the <italic>L1CAM</italic> gene. This nonsense mutation is found to be truncated with a deleterious effect on developing brain of the child, and this is the first report of this novel mutation in patient with X-linked Hydrocephalus in India.</p> </abstract>

Dissecting the Interaction of FGF8 with Receptor FGFRL1.

In mammals, the novel protein fibroblast growth factor receptor-like 1 (FGFRL1) is involved in the development of metanephric kidneys. It appears that this receptor controls a crucial transition of the induced metanephric mesenchyme to epithelial renal vesicles, which further develop into functional nephrons. FGFRL1 knockout mice lack metanephric kidneys and do not express any fibroblast growth factor (FGF) 8 in the metanephric mesenchyme, suggesting that FGFRL1 and FGF8 play a decisive role during kidney formation. FGFRL1 consists of three extracellular immunoglobulin (Ig) domains (Ig1-Ig2-Ig3), a transmembrane domain and a short intracellular domain. We have prepared the extracellular domain (Ig123), the three individual Ig domains (Ig1, Ig2, Ig3) as well as all combinations containing two Ig domains (Ig12, Ig23, Ig13) in recombinant form in human cells. All polypeptides that contain the Ig2 domain (Ig123, Ig12, Ig23, Ig2) were found to interact with FGF8 with very high affinity, whereas all constructs that lack the Ig2 domain (Ig1, Ig3, Ig13) poorly interacted with FGF8 as shown by ELISA and surface plasmon resonance. It is therefore likely that FGFRL1 represents a physiological receptor for FGF8 in the kidney and that the ligand primarily binds to the Ig2 domain of the receptor. With Biacore experiments, we also measured the affinity of FGF8 for the different constructs. All constructs containing the Ig2 domain showed a rapid association and a slow dissociation phase, from which a KD of 2–3 × 10−9 M was calculated. Our data support the hypothesis that binding of FGF8 to FGFRL1 could play an important role in driving the formation of nephrons in the developing kidney.

The divergent evolution of cytomegalovirus-encoded CD48 homologs contributes to the expansion of the immunevasin repertoire

Abstract The genesis of gene families by capture of host genes and their subsequent duplication is a crucial process in the evolution of large DNA viruses. CD48 is a GPI-linked cell surface protein containing two extracellular immunoglobulin (Ig) domains. Via its N-terminal Ig domain, CD48 interacts with the cell surface receptor 2B4, modulating leukocyte cytotoxicity. We previously reported the presence of five CD48 homologs (vCD48s) in two related cytomegaloviruses, derived from a common host CD48 ancestor gene acquired by retrotranscription. Recently, we examined one member of this family, A43, showing that it acts as a functional viral decoy receptor, impairing 2B4-mediated NK cell cytotoxicity. Here, we have characterized the rest of the vCD48s. We show that they are highly glycosylated type I transmembrane proteins which display remarkably distinct structures, biochemical properties, locations, and temporal kinetic classes. Molecular modeling of the N-terminal Ig domains of these vCD48s evidence significant changes in the number and length of their β-strands and inter-sheet loops that participate in the interaction of CD48 with 2B4, and accordingly we found that none of these molecules binds to 2B4. Interestingly, we determined that two of them, S30 and S31, have new targets in T or B lymphocytes. In particular, S30 tightly interacts with CD2, a T- and NK-cell adhesion and costimulatory molecule whose primary ligand is CD58. Thus, altogether our results show how a key host immune receptor captured by a virus can be subsequently remodeled during viral evolution to yield new molecules with shifted binding specificities to additional immune targets, expanding the repertoire of viral immunevasins.

The discovery of potent and stable short peptide FGFR1 antagonist for cancer therapy

Fibroblast growth factor receptor 1 (FGFR1) is one of the attractive pharmaceutical targets for cancer therapy. The FGFR1 targeting antagonist peptides, especially of the short peptides harbouring only coding amino acid might highlights promising aspects for their higher affinity, specificity and lower adverse reactions. However, most of peptides inhibitors remain in preclinical research, likely associating with their instability and short half-life. In this study, we found a stable short peptide inhibitor P48 and speculated that its stability might be related to its non-linear spatial structure. In addition, P48 could target the extracellular immunoglobulin domain of FGFR1, and effectively block the particular signaling pathways of FGFR1, which lead to the inhibition of cancer proliferation, invasion in vitro and restraint of tumor growth in vivo. Together, this study provided a promising FGFR1 inhibitor with the potential to be developed as an antitumor drug.

Abstract 5007: Preclinical evaluation of JTX-8064, an anti-LILRB2 antagonist antibody, for reprogramming tumor-associated macrophages

Abstract Introduction: Jounce has generated cell type-specific gene signatures as a means of probing The Cancer Genome Atlas and other large datasets to identify targets that may be important immune checkpoints. Using a tumor-associated macrophage (TAM) gene signature, we have found a strong correlation and coherence between TAMs and LILRB2 (leukocyte immunoglobulin like receptor B2; ILT4) across multiple tumors types. LILRB2 is a myeloid cell surface receptor containing four extracellular immunoglobulin domains, a transmembrane domain, and three cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Ligation of LILRB2 on myeloid cells, via its endogenous ligands (classical MHC I molecules [e.g. HLA-A, HLA-B] and non-classical MHC I molecules [e.g. HLA-G]), provides a negative signal that inhibits stimulation of an immune response. HLA-G is recognized as an important immunosuppressive molecule playing a role in maternal-fetal tolerance and being overexpressed in cancer - often associated with advanced disease stage and poor prognosis. As tumor-associated macrophages are known to suppress the anti-cancer immune response, these findings provide rationale for targeting LILRB2. Methods and Results: We have generated a panel of monoclonal antibodies that bind specifically to LILRB2, but not other LILR family members, and can block binding of LILRB2 to MHC I molecules (i.e. HLA-A and HLA-G). In vitro differentiated monocyte-derived macrophages (MDMs) cultured for 24h in the presence of anti-LILRB2 antibodies and lipopolysaccharide (LPS) show polarization toward a more inflammatory phenotype - secreting higher levels of TNF-α and IL-6 with decreased amounts of IL-10 and CCL2 as compared to an isotype control antibody. NanoString mRNA analysis revealed that, in the absence of LPS or any additional stimuli, MDMs cultured with anti-LILRB2 antibodies showed gene changes consistent with inflammatory or M1-like polarization of macrophages. Anti-LILRB2 antibodies were also evaluated in human tumor histoculture and induced pharmacodynamic responses consistent with macrophage and T cell activation in a variety of tumor types. While mice do not express LILRB2 specifically, they do express a LILRB-like molecule known as Pirb. Mice that are deficient in Pirb display resistance to mouse colon 38 (MC-38) tumor growth suggesting this pathway functions as immune checkpoint in cancer. Conclusions: Based on these preclinical data, JTX-8064, a high affinity LILRB2-specific humanized antagonist monoclonal antibody, is being developed as an immunotherapeutic to reprogram suppressive macrophages within the tumor microenvironment. Citation Format: Heather Cohen, Yasmin Hashambhoy-Ramsay, Lauren R. Pepper, Jeffrey Y. Smith, Margaret Willer, Kevin Guay, Vikki Spaulding, Kristin O'Malley, Monica Gostissa, Abha Dhaneshwar, Edward C. Stack, Alessandro Mora, Donald R. Shaffer. Preclinical evaluation of JTX-8064, an anti-LILRB2 antagonist antibody, for reprogramming tumor-associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5007.

Molecular characterization and expression analysis of Turtle protein in silkworm that is associated with Nosema bombycis infection

In this report, we describe the cloning and characterization of a member of the immunoglobulin superfamily (IgSF); i.e., Turtle. The cDNA of Turtle was cloned from the silkworm Bombyx mori using the rapid amplification of cDNA ends (RACE) technique. Three isoforms of Bombyx Turtle were obtained, including Bmtutl-464, Bmtutl-519, and Bmtutl-810. The three isoforms had identical 27-amino acid signal peptides and four extracellular immunoglobulin (Ig) domains (IgI–IgIV). Sequence similarity and phylogenic analysis indicated that Bmtutl-810 belongs to the group of insect Turtle isoforms and shares 76.2% identity with Drosophila Turtle. Quantitative real-time PCR analysis revealed that the Bombyx Turtle isoforms were expressed throughout the entire development period, the highest levels of expression of Bmtutl-464 and Bmtutl-519 were observed at the second instar larvae stage, whereas that of Bmtutl-810 peaked at the embryonic stage. The ubiquitous expression of Bmtutl-464, Bmtutl-519, and Bmtutl-810 were observed in all studied tissues, except for Bmtutl-519 in the silk gland. The expression level of Bmtutl-464 was highest in the ovary, whereas that of Bmtutl-519 and Bmtutl-810 was highest in the hemolymph. Bmtutl-519 was upregulated in BmN cells infected by Nosema bombycis, We speculated that Bombyx Turtle was not only involved in neural development in silkworm, as well as Drosophila Turtle, but was also involved in the regulation of other biological functions. For example, Bmtutl-519 might be involved in N. bombycis infection and may play an important role in the immune response of silkworms to N. bombycis infection.

The polymorphic proteins TgrB1 and TgrC1 function as a ligand-receptor pair in Dictyostelium allorecognition.

Allorecognition is a key factor in Dictyostelium development and sociality. It is mediated by two polymorphic transmembrane proteins, TgrB1 and TgrC1, which contain extracellular immunoglobulin domains. TgrB1 and TgrC1 are necessary and sufficient for allorecognition, and they carry out separate albeit overlapping functions in development, but their mechanism of action is unknown. Here, we show that TgrB1 acts as a receptor with TgrC1 as its ligand in cooperative aggregation and differentiation. The proteins bind each other in a sequence-specific manner; TgrB1 exhibits a cell-autonomous function and TgrC1 acts non-cell-autonomously. The TgrB1 cytoplasmic tail is essential for its function and it becomes phosphorylated upon association with TgrC1. Dominant mutations in TgrB1 activate the receptor function and confer partial ligand independence. These roles in development and sociality suggest that allorecognition is crucial in the integration of individual cells into a coherent organism.

Butyrophilins: an important new element of resistance.

Butyrophilins belonging to the immunoglobulin superfamily are new immune system regulators because they are present on lymphocytes, dendritic cells, monocytes, macrophages, neutrophils and eosinophils, and they exert a stimulatory and (or) inhibitory effect on them. The role of butyrophilins is associated and results from their similarity to the regulatory B7 protein family involved in the modulation of immune phenomena. Butyrophilins are glycoproteins built of two extracellular immunoglobulin domains, stabilized with disulfide bonds: constant IgC, and variable IgV and a transmembrane region. Most of these proteins contain a conserved domain encoded by a single exon – B30.2, also referred to as PRYSPRY. In humans, the family of butyrophilins includes 7 butyrophilin proteins, 5 butyrophilin-like proteins and the SKINT-like factor. Butyrophilins have been also demonstrated to play a role in various infections, e.g. tuberculosis or diseases that include sarcoidosis, systemic lupus erythematosus, rheumatoid arthritis, genetic metabolic diseases, ulcerative colitis, cancer and kidney disease.

A Novel NKp44-Based Chimeric Antigen Receptor That Targets Multiple Types of Cancer

Genetic modification of T cells with an artificial tumor-targeting chimeric antigen receptor (CAR) is a new approach for adoptive cell therapy for cancer. Defining cell surface molecules that are both selectively expressed on cancer cells and can be safely targeted with T cells or NK cells is a significant challenge in this research field.NKp44 is a member of the natural cytotoxicity receptor (NCR) families and also known as NCR2. Expression of NKp44 is limited to activating NK cells, which leads to a marked increase in cytotoxicity against tumors. The receptor contains one extracellular immunoglobulin domain, type I transmembrane (TM) domain, and intracellular (IC) domain, and its surface expression seems to require binding of the TM domain to adaptor molecules of DAP12 accessory protein that contains ITAMs. The ligand for NKp44 is considered damage-associated molecular pattern molecules, which have been reported to be expressed by various types of cancer cells but not by healthy cells. Therefore, a wide range of cancer cells may be safely targeted if the ligand-binding domain of this receptor is used in a construction of a chimeric antigen receptor (CAR) as an antigen recognition site, instead of using single chain variable domains derived from monoclonal antibody.We created several NKp44-based CAR constructs, which shares the extracellular NKp44 IG domain as a ligand-binding domain. Surface expression levels and subsequent functional properties can differ among T cells or NK cells transduced with novel CARs with different structural characteristics. We thus tested whether swapping the domains other than the antigen-binding domain affected expression and function. The CAR genes were retrovirally transduced into human primary T cells according to a standard method. We also transduced human primary NK cells with NKp44-based CARs, by a previously reported method (Imai C, et al. Blood 2005), to compare the expression pattern of the CAR in NK cells with that in human T cells.Retroviral transfer of wild type NKp44 gene and a construct harboring IC(p44) both did not induce NKp44 surface expression (Fig 1A,B). By sharp contrast, primary NK cells were able to express the CAR protein on the cell surface after transfer of these two genes. Removal of the IC(p44) [EH(p44)-TM(p44)-IC(CD3z)] allowed slight surface expression in T cells (Fig1C). The replacement of TM(p44) with TM(CD8a) resulted in higher surface expression in T cells (Fig 1D). These observations indicated the presence of IC(p44) as well as TM(p44) in the CAR constructs hampered surface expression in T cells most likely due to the lack of DAP12 expression. In addition to TM replacement, replacement of EH(p44) with EH(CD8a) markedly increased surface expression of the CAR (Fig 1E). Similarly, we tested use of CD28 domains instead of CD8a. Surprisingly, as different from the case of CD8a, the construct EH(p44)-TM(CD28)-IC(CD3z) yielded highest surface expression among the all CAR constructs created in this study in T cells as well as in NK cells (Fig1F), while the replacement of EH(p44) of the abovementioned CAR with EH(CD28) resulted in marked reduction of the CAR expression (Fig 1G).We confirmed surface expression of NKp44 ligand with flow cytometric analysis using recombinant human NKp44 Fc chimera protein (R&D Systems, McKinley Place, Minneapolis, USA) on various tumor cell lines including myeloid leukemia (K562, THP-1, U937, KY821, HL60), T-cell leukemia (PEER, MOLT4, HSB2), Burkitt lymphoma (Raji), BCR-ABL-positive B-ALL (OP-1), osteosarcoma (MG63, NOS1, NOS2, NOS10, U2OS, SaOS2), rhabdomyosarcoma (Rh28, RMS-YM), neuroblastoma (SK-N-SH, NB1, NB16, IMR32), and cervical carcinoma (Hela). Function of the best construct [EH(p44)-TM(CD28)-IC(CD3z)] was further evaluated. Primary T cells transduced with this NKp44-based CAR exerted powerful cytotoxicity against tumor cell lines tested and produced interferon-g and granzyme B, while GFP-transduced T cells and control T cells transduced with truncated NKp44-based CAR did not.In conclusion, we have created a novel CAR based on the antigen-binding property derived from NKp44 receptor immunoglobulin domain. This CAR should be effective to redirect T cells as well as NK cells against various types of cancer including hematological malignancies. [Display omitted] DisclosuresImai:Juno Therapeutics: Patents & Royalties.

Production of specific monoclonal antibody against CD3ε in olive flounder

T cell activation is initiated by the binding of antigen to the specific T cell receptor (TCR) that triggers the formation of CD3-signal transduction complex and ends in proliferation and differentiation of antigenspecific T cells. CD3, an essential component of the CD3-TCR complex, has been classified into γ, δ, e, and ζ chains. The CD3γ, CD3δ, and CD3e chains are highly related to cell surface proteins of the immunoglobulin superfamily containing a single extracellular immunoglobulin domain. In mammals, T cells were characterized by detecting one of the CD3 molecules through the use of specific antibody for CD3e. This suggests that CD3 molecules can be used as markers in identifying T cells in teleost fish; hence, it is essential to produce CD3-specific antibodies that could serve as T cell markers in fish. Here, we produced a CD3e-specific monoclonal antibody. Western blot result showed a distinct band at approximately 15 kDa detecting the CD3e antibody while MALDI-TOF analysis, clearly identified the band as olive flounder CD3e. Once a monoclonal antibody has been produced, it can be used to detect the presence of a substance specific to it. The results from this study revealed that the CD3e monoclonal antibody produced could detect CD3e in olive flounder. However, cell markers for T cell differentiation in olive flounder were not fully investigated yet so we hope that this antibody could be useful in elucidating T cell differentiation in teleosts.

Apert Syndrome: A Case Report and Review of Literature

Apert syndrome, also known as acrocephalosyndactyly, is one of the causes of craniofacial syndrome or deformity. It is a rare congenital disorder characterized by premature fusion of cranial sutures (craniosynostosis), malformation of skull, hands, face and feet. This congenital deformity has incidence of 1/50,000 to 1/80,000 live births and is an autosomal dominant in inheritance. Apert syndrome, fibroblast growth factor receptor 2 (FGFR2) and the missense substitution mutations occur at adjacent amino acids (i.e. Ser252Trp, Ser 252Phe, Pro253Arg) between the second and third extra cellular immunoglobulin domain of FGFR2, which maps to chromosome bands 10q26. Increased paternal age has been implicated in the development of Apert syndrome. The syndrome has to be thoroughly evaluated as early definitive diagnosis is important in order to distinguish Apert syndrome from other forms of craniosynostosis like Carpenter syndrome, Crouzon disease, Pfeiffer and Saethre-Chotzen syndrome. It is generally accepted that management of Apert syndrome is multidisciplinary in approach, which should compose of neonatologists, neurosurgeons, craniofacial surgeons, plastic surgeons, otolaryngologists, orthodontists, orthopaedic surgeons, ophthalmologists, radiologists, geneticists, clinical psychologists and speech and language pathologists for the effective management of this condition. Early diagnosis and treatment is important because Apert syndrome when treated early has good prognosis in adult life.