Nonspecific Cells Research Articles

HISTOLOGIC AND RADIOGRAPHIC STUDY OF PATHOLOGIC CHANGE IN COMPLETE IMPACTED THIRD MOLARS DENTAL FOLLICLES

Background: Prophylactic extraction of the asymptomatic impacted third molar is routinely practiced in Europe and the United States. The justification for prophylactic extraction includes the need to reduce the risk of pathologic changes such as cysts and tumors. 
 Objectives: This study aimed to study the histological and radiological changes in the tooth follicles of upper and lower complete impacted 3rd molars -which appeared radiologically normal.
 Material and method: A prospective study included fifty patients aged 20 years and above who were referred to the Oral Surgery Clinic, Faculty of Dentistry, University of Sana'a. Patients had follicular space between (2.5mm -3mm) as measured by the panoramic X-ray. These teeth were removed surgically and the follicle was sent for histopathological examination.
 Results: Most histopathological changes were in dental follicles with a size of <2.5 mm (86%), and only 14% with 2.5 mm - 3 mm. There was statistical significance between the smallest size of dental follicles with the incidence of pathological histological changes indicating a high probability of developing neoplasm (p =0.008). Of the 50 follicular patients, 28% showed HC, nine (64%) had ameloblastoma, four (29%) had a dentigerous cyst, and only one case (7%) had a multicalcified focus with islands of odontogenic epithelium. While 72% of the samples had normal follicles and non-specific chronic inflammatory cells. There is an association between female sex and pathological histological changes (12 females: 2 males, p =0.008), age group 21-25 years (93% HC), with mandibles (65% HC). Regarding angle and histopathological changes, 36% were vertical, 29% mesioangular, 14.2% horizontal and destioangular, and 7.1% buccoangular.
 Conclusion: High incidence of HC occurred in patients with DF, and it was associated with smaller dental follicle size, most HC was ameloblastoma, followed by dentigerous cyst, while 72% of samples had normal follicles and non-specific chronic inflammatory cells. There is a correlation between female gender, younger age group, and jaw position with HC. Prophylactic extraction of the asymptomatic impacted third molar should be routinely practiced in Yemen, to reduce the risk of pathological changes, especially in females and younger age groups.
 
 Peer Review History: 
 Received 11 January 2021; Revised 8 February; Accepted 28 February, Available online 15 March 2021
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 Received file: Reviewer's Comments:
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 Reviewer(s) detail:
 Dr. A.A. Mgbahurike, University of Port Harcourt, Nigeria, amaka_mgbahurike@yahoo.com
 Dr. Alfonso Alexander Aguileral, University of Veracruz, Mexico, aalexander_2000@yahoo.com
 
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 RADIOGRAPHIC ASSESSMENT OF THE COURSE AND VISIBILITY OF THE MANDIBULAR CANAL BY PANORAMIC RADIOGRAPHY

Selective capture of circulating tumor cells by antifouling nanostructure substrate made of hydrogel nanoparticles

The detection and analysis of circulating tumor cells (CTCs) from cancer patients' blood samples present a powerful means to monitor cancer progression. In this work, an antifouling nanostructure substrate made of hydrogel nanoparticles was fabricated for an effective capture of CTCs from the blood samples. The hydrogel nanoparticles were synthesized by zwitterionic sulfobetaine methacrylate (SBMA), methacrylic acid (MAA) and N, N’-methylene bisacrylamide (MBA) through a simple polymerization. SBMA could provide an effective antifouling layer for the substrate to prevent nonspecific cell adhesion, MAA could afford active carboxyl groups for the immobilization of antibody to achieve specific CTC capture, and the nanostructured surface could improve the interaction of the target cells with the antibody modified substrate surface to enhance the capture efficiency of CTCs. Moreover, it was not necessary to further modify the antifouling molecules on the hydrogel nanoparticle substrate's surface, reducing the complexity and difficulty of the substrate preparation. The results showed that about 87 % of target cells (MCF-7 cells) were captured on the antibody modified hydrogel nanoparticle substrate. In contrast, the substrate showed little adhesive capacity for the nonspecific cells (K562 cells), and only 0.15 % of cells were captured. And 98 % of the captured cells kept good cell viability. Finally, 1–32 CTCs/mL were detected from the blood samples of five cancer patients, while no CTC was found in five healthy samples. It is envisaged that the new hydrogel nanostructure substrate is capable of capturing CTCs efficiently and specifically from patient blood samples to be used in cancer treatment.

Exopolysaccharide from Porphyridium cruentum (purpureum) is Not Toxic and Stimulates Immune Response against Vibriosis: The Assessment Using Zebrafish and White Shrimp Litopenaeus vannamei.

Exopolysaccharides, or extracellular polysaccharides (EPS, sPS), represent a valuable metabolite compound synthesized from red microalgae. It is a non-toxic natural agent and can be applied as an immunostimulant. The toxicity test of exopolysaccharides from Porphyridium has been done in vivo using zebrafish (Danio rerio) embryonic model, or the ZET (zebrafish embryotoxicity test). The administration of extracellular polysaccharides or exopolysaccharides (EPS) from microalgae Porphyridium cruentum (synonym: P. purpureum) to shrimps Litopenaeus vannamei was investigated to determine the effect of this immunostimulant on their non-specific immune response and to test if this compound can be used as a protective agent for shrimps in relation to Vibrio infection. For immune response, exopolysaccharides were given to shrimps via the immersion method on day 1 and booster on day 8. Shrimp hemocytes were taken on day 1 (EPS administration), day 7 (no treatment), day 8 (EPS booster) and day 9 (Vibrio infection) and tested for their immune response on each treatment. The result shows that the EPS is not toxic, as represented by the normal embryonic development and the mortality data. In the Pacific white shrimps, an increase in the values of all immune parameters was shown, in line with the increasing EPS concentration, except for the differential hemocyte count (DHC). In detail, an increase was noted in total hemocytes (THC) value, phagocytotic activity (PA) and respiratory burst (RB) in line with the EPS concentration increase. These results and other previous studies indicate that EPS from Porphyridium is safe, enhances immune parameters in shrimp rapidly, and has the ability to act as an immunostimulant or an immunomodulator. It is a good modulator for the non-specific immune cells of Pacific white shrimps, and it can be used as a preventive agent against vibriosis.

Highly antifouling, biocompatible and tough double network hydrogel based on carboxybetaine-type zwitterionic polymer and alginate

Because of resistance to bio-macromolecular adhesion, antifouling hydrogels have attracted great attention in biomedical field. But traditional antifouling hydrogels made by hydrophilic polymers are always poor of mechanical properties. Herein, a new hybrid ionic-covalent cross-linked double network (DN) hydrogel was prepared by a simple one-pot method based on sodium alginate and the zwitterionic material carboxybetaine acrylamide (CBAA). The DN hydrogel has good mechanical properties, including high elastic modulus (0.28 MPa), high tensile strength (0.69 MPa), as well as good self-recovery capability. More importantly, the DN hydrogel is highly resistance to the adsorption of non-specific protein, cells, bacteria and algae, exhibiting an outstanding antifouling property. The in vitro and in vivo experiments prove that the DN hydrogel is highly biocompatible. This study provides a new strategy for the preparation of antifouling DN hydrogels with good mechanical properties for different needs, such as tissue scaffolds, wound dressings, implantable devices, and other fields.

Вплив величини угрупування курей на неспецифічну реактивність їх організму

The nonspecific reactivity of the hens under the influence group size with the same stoking density in cages-analogues by design was studied. It was found that with a decrease in the hens group size there is a shift of leukocyte formula to the left, the predominance of nonspecific protective cells, which occurs due to functional increase in bone marrow proliferative activity and is expressed in increased heterophiles and increased activity in the microphage-macrophage immune response system.

CAR-modified memory-like NK cells exhibit potent responses to NK-resistant lymphomas

AbstractNatural killer (NK) cells are a promising cellular immunotherapy for cancer. Cytokine-induced memory-like (ML) NK cells differentiate after activation with interleukin-12 (IL-12), IL-15, and IL-18, exhibit potent antitumor responses, and safely induce complete remissions in patients with leukemia. However, many cancers are not fully recognized via NK cell receptors. Chimeric antigen receptors (CARs) have been used to enhance tumor-specific recognition by effector lymphocytes. We hypothesized that ML differentiation and CAR engineering would result in complementary improvements in NK cell responses against NK-resistant cancers. To test this idea, peripheral blood ML NK cells were modified to express an anti-CD19 CAR (19-CAR-ML), which displayed significantly increased interferon γ production, degranulation, and specific killing against NK-resistant lymphoma lines and primary targets compared with nonspecific control CAR-ML NK cells or conventional CAR NK cells. The 19-CAR and ML responses were synergistic and CAR specific and required immunoreceptor tyrosine-based activation motif signaling. Furthermore, 19-CAR-ML NK cells generated from lymphoma patients exhibited improved responses against their autologous lymphomas. 19-CAR-ML NK cells controlled lymphoma burden in vivo and improved survival in human xenograft models. Thus, CAR engineering of ML NK cells enhanced responses against resistant cancers and warrants further investigation, with the potential to broaden ML NK cell recognition against a variety of NK cell–resistant tumors.

Single-cell RNA-seq reveals different subsets of non-specific cytotoxic cells in teleost

Non-specific cytotoxic cells (NCC) are important cytotoxic leukocytes in teleost immune system. However, the NCC subsets have not been clarified. Thus, we create a comprehensive cell map of ~24,062 head kidney-derived leukocytes from Nile tilapia post poly I:C stimulation using single-cell RNA sequencing (scRNA-seq). Based on cell heterogeneity and known markers, the cells were classified into four cell types including B cell, T cell, NCC and monocytes/macrophages (Mo/MΦ). In the meantime, the regulatory network of NCC population was predicted by WGCNA and four hub genes (Stbd1, VWF, PGP, and GRN) and one transcription factor (Hvcn1) were identified. To further study the differentiation of NCC, four subsets including memory-like NCC, mature NCC, immature NCC, and pre-NCC were revealed in NCC population for the first time. Our data will provide new insight into the biology of NCC and enable more accurate functional and developmental analysis of NCC in immune system of lower vertebrates

Aptamer-based electrochemical biosensing strategy toward human non-small cell lung cancer using polyacrylonitrile/polypyrrole nanofibers.

In the present study, a sensitive electrochemical aptamer-based biosensing strategy for human non-small cell lung cancer (NSCLC) detection was proposed using nanofiber-modified disposable pencil graphite electrodes (PGEs). The composite nanofiber was comprised of polyacrylonitrile (PAN) and polypyrrole (PPy) polymers, and fabrication of the nanofibers was accomplished using electrospinning process onto PGEs. Development of the nanofibers was confirmed using scanning electron microscopy (SEM). The high-affinity 5'-aminohexyl-linked aptamer was immobilized onto a PAN/PPy composite nanofiber-modified sensor surface via covalent bonding strategy. After incubation with NSCLC living cells (A549 cell line) at 37.5°C, the recognition between aptamer and target cells was monitored by electrochemical impedance spectroscopy (EIS). The selectivity of the aptasensor was evaluated using nonspecific human cervical cancer cells (HeLa) and a nonspecific aptamer sequence. The proposed electrochemical aptasensor showed high sensitivity toward A549 cells with a detection limit of 1.2 × 103 cells/mL. The results indicate that our label-free electrochemical aptasensor has great potential in the design of aptasensors for the diagnostics of other types of cancer cells with broad detection capability in clinical analysis. Graphical abstract.

AGGRESSIVE CENTRAL GIANT CELL LESION IN THE YOUNG PATIENT: A CASE REPORT

The giant cell central lesion (LCCG) is considered to be rare and benign, and its etiology is uncertain. Clinically and radiographically, it can be differentiated between aggressive and nonaggressive lesions. A female patient with leukoderma, 13 years old, presented with discrete asymmetry in the right hemiface and increased volume in the anterior region of the mandible, of indurated consistency, and painless palpation. In the imaging examinations, the presence of a radiolucent, multilocular, well-delimited image in the anterior region of the mandible crossing the midline, with tooth dislocation was observed. Histopathology revealed a loose, richly vascularized fibrous connective tissue fragment exhibiting intense proliferation of spindle and oval cells with large numbers of nonspecific multinucleated giant cells. Hemorrhagic areas and deposits of brownish pigmentation were compatible with hemosiderin. The diagnosis was central giant cell lesion. The conservative drug therapy was performed, which was not successful, and the surgical intervention was performed. Postoperative control is 6 months, without recurrence.

Abstract 1076: Hexavalent HERA-CD40L induces a productive T cell-mediated anti-tumor immune response and shows superior activity in comparison to benchmark CD40 agonistic antibodies

Abstract TNF Receptor Superfamily members (TNF-R-SF), including CD40, are key regulators of the immune system and have been immunotherapeutic targets for over 20 years. CD40 signaling serves as an important co-stimulatory signal for antigen-presenting cells (APC). For the induction of a CD40 driven anti-tumor immune response multiple approaches, - most of them antibody based - are currently under investigation. However, the structural necessity of trimerization within the TNF-R-SF, defines bivalent antibodies generally as unfeasible inducers of signaling events within this protein family. To overcome the known inadequacies of antibodies, we developed HERA-CD40L, composed of two trivalent CD40L-receptor-binding domains, fused to a silenced human IgG1 Fc-domain. This hexavalent CD40 agonist mimics the natural ligand and enables efficient receptor clustering and superior signaling. HERA-CD40L treatment increased the pro-inflammatory state of all CD40-expressing cells examined. It promoted the licensing of dendritic cells (DC), macrophages, B cells and other APC. Comparison to benchmark antibodies revealed that HERA-CD40L elicited a stronger and more rapid activation of NFκB signaling in primary B cells. HERA-CD40L treatment, but not clinical benchmark antibodies triggered immediate NFκB, MAPK, PI3K and STAT-1 signaling in primary monocyte-derived immature DC. As a result, HERA-CD40L induced upregulation of activation markers and co-stimulatory molecules in B cells and DC. Using SEC fractionation followed by a CD40 reporter assay, we could furthermore demonstrate that the activity of a clinical benchmark antibody derived exclusively from antibody aggregates. In vitro HERA-CD40L treatment converted immature phagocytic macrophages into mature/professional APC and induced repolarization of M2- to M1-like macrophages. These findings were confirmed in vivo using a mouse surrogate (mmHERA-CD40L). Upon treatment of MC38-CEA and CT26wt syngeneic mouse models, we observed single agent anti-tumor efficacy. In the CT26wt model, mmHERA-CD40L treatment converted cold into hot tumors by increasing T cell infiltration. Furthermore, mmHERA-CD40L induced a strong dose dependent decrease of tumor associated pro-tumorigenic M2-macrophages indicating a profound reorganization of the tumor microenvironment. Both in vitro (human) and in vivo (mouse), HERA-CD40L increased antigen-specific immune system activation without affecting the non-specific immune cells. These data, together with pilot PD/safety results, demonstrate that the activity of HERA-CD40L is both potent and safe. In conclusion, HERA-CD40L is a potent agonist able to show single agent anti-tumor activity. The biological activity is distinct from and superior to clinical benchmark “agonistic” antibodies. HERA-CD40L has a well-defined mechanism of action, does not depend on Fc gamma receptor-mediated crosslinking and hence functions as a true agonist. Citation Format: Christian Gieffers, David M. Richards, Jaromir Sykora, Christian Merz, Julian P. Sefrin, Katharina Billian-Frey, Karl Heinonen, Mauricio Redondo Müller, Matthias Schröder, Meinolf Thiemann, Oliver Hill. Hexavalent HERA-CD40L induces a productive T cell-mediated anti-tumor immune response and shows superior activity in comparison to benchmark CD40 agonistic antibodies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1076.

Could an Unrelated Live Attenuated Vaccine Serve as a Preventive Measure To Dampen Septic Inflammation Associated with COVID-19 Infection?

We propose the concept that administration of an unrelated live attenuated vaccine, such as MMR (measles, mumps, rubella), could serve as a preventive measure against the worst sequelae of coronavirus disease 2019 (COVID-19). There is mounting evidence that live attenuated vaccines provide nonspecific protection against lethal infections unrelated to the target pathogen of the vaccine by inducing "trained" nonspecific innate immune cells for improved host responses against subsequent infections. Mortality in COVID-19 cases is strongly associated with progressive lung inflammation and eventual sepsis. Vaccination with MMR in immunocompetent individuals has no contraindications and may be especially effective for health care workers who can easily be exposed to COVID-19. Following the lead of other countries conducting clinical trials with the live attenuated Mycobacterium bovis BCG (BCG) vaccine under a similar concept, a clinical trial with MMR in high-risk populations may provide a "low-risk-high-reward" preventive measure in saving lives during this unprecedented COVID-19 pandemic.

Inhibition of CSF-1R curtails inflammation during experimental autoimmune encephalomyelitis

Abstract Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are autoimmune diseases characterized by extensive infiltration of T cells and myeloid cells into the CNS. Treatments for MS are typically either non-specific immunosuppressive drugs or target adaptive immune cells. However, mounting evidence shows that myeloid cells have essential roles in MS/EAE pathology, but few, if any, MS drugs specifically target myeloid cells. A promising strategy for bridging this gap in treatment modalities for MS may be targeting CSF-1R, a receptor tyrosine kinase with important roles in myeloid cells, including survival and differentiation. We found that blocking CSF-1R signaling attenuated EAE and our analysis of the CNS these mice revealed substantially diminished numbers of inflammatory myeloid cells. These data demonstrate that inhibiting CSF-1R signaling is a viable strategy for attenuating myeloid cell responses during autoimmune neuroinflammation. These studies also suggest that targeting CSF-1R signaling may be an effective approach for treating MS.

Natural Biointerface Based on Cancer Cell Membranes for Specific Capture and Release of Circulating Tumor Cells.

Circulating tumor cells (CTCs) are an important part of liquid biopsy as they represent a potentially rich source of information for cancer diagnosis, monitoring, prognosis, and treatment guidance. It has been proved that the nanotopography interaction between cells and the surface of CTC detection platforms can significantly improve the capture efficiency of CTCs, whereas many mature nanostructure substrates have been developed based on chemistry materials. In this work, a natural biointerface with unique biological properties is fabricated for efficient isolation and nondestructive release of CTCs from blood samples using the cancer cell membranes. The cell membrane interfaces are proved to have a good antiadhesion property for nonspecific cells because of their own electronegativity. A natural surface nanostructure is provided by the cancer cell membrane to nicely match with the surface nanotopography of CTCs. Bovine serum albumin (BSA) as a linker and DNA aptamer against the epithelial cell adhesion molecule (EpCAM) as a specific affinity molecule are then introduced onto the cell membrane interfaces to achieve the highly efficient and specific capture of CTCs. Finally, the captured target cells can be intactlyreleased from the substrate using the complementary DNA sequence with controlling the incubation time. This study provides a smart strategy in the development of a natural biological interface for the isolation and release of CTCs with high purity.

Blood Leukocyte Indices in Male Rats of Different Ages

Blood leukocyte indices were studied in outbred male of rats of five age groups - 1,5; 6, 12, 18 and 24 months. Six month's old rats were served as the control. The increase in age in rats was accompanied by an increase of total leukocyte count (starting from the 12-month-old), in the percentage of neutrophils (both banded and segmented), eosinophils and monocytes as well as by a decrease in the number of lymphocytes, the appearance (starting from the 18-month-old) of polychromatophilic and immature cells, and cytoplasm plasmatization in some leukocytes. In immature 1,5-month-old rats, only the percentage of eosinophils (as in 12-month-old rats) and the total number of leukocytes changed (decreased). The process of restructuring of the immune system altered links in body reflected the dynamics of integral leukocyte indices (ILI). Changes in ILI at increase in age (starting from 18 months of age) indicated an increase in infectious intoxication and inflammation, activation of cellular immunity and effector link, the prevalence of non-specific protection cells (neutrophils) and their immature forms, as well as a delayed type of hypersensitivity, decrease in immunoreactivity and level of adaptation of the body. The lowest Garkavi adaptation index was in 24-month-old rats. Only in 1,5-month-old rats, the microphagocytes increased in the blood, as well as the body level of allergization was the lowest and the level of intoxication level was the highest compared to the other studied age groups.

Abstract A36: Single-cell characterization of tumor-infiltrating T cells from renal cell carcinoma

Abstract Purpose: T-cell infiltration of renal cell carcinoma (RCC) is commonly viewed as evidence for antitumor immunity that may be harnessed by T cell-directed immunotherapies. We hypothesize that bona fide tumor-reactive T cells constitute only a fraction of tumor-infiltrating lymphocytes (TIL) that may be composed largely of nonspecific passenger cells. This study deployed TRB-CDR3 repertoire characterization and targeted single-cell RNAseq of RCC versus normal tissue and peripheral blood mononuclear cells (PBMC)-derived T cells to analyze phenotypes that may associate with tumor-antigen specificity. Experimental Procedures: RCC tumor, normal adjacent kidney tissue (NAT), skin, and PBMC were collected from 9 RCC patients at the time of nephrectomy surgery. Tissue samples were digested to single-cell suspensions and CD45+CD8+CD3+ T cells were sorted. Sequencing of TCR-β CDR3 regions (TRB-CDR3) was carried out on polyclonal CD8+ T-cell populations. Targeted single-cell RNAseq was then applied to sorted CD8+ T cells from the same samples focusing on TRB-CDR3 and TRA-CDR3 regions and 25 T-cell phenotype genes. Full-length T-cell receptor (TCR) genes from selected, clonally expanded, tumor-associated CD8+ T cells were cloned into lentiviral expression vectors for re-expression in CD8+ T cells from healthy donors to facilitate analysis of TCR specificity. Results: TCR-β CDR3 sequencing demonstrated the frequency of clonally expanded TRB was higher in tumors (mean 45%; range 27%-65%) compared with PBMCs (mean 14%; range 4%-22%) or NATs (mean 15%; range 2%-26%) for all 9 cases. Expanded T-cell clonotypes present in the tumor and not detected in NAT or PBMC samples were present in every case. Dimensionality reduction of targeted single-cell RNAseq analysis identified phenotypic differences between clonally expanded and nonexpanded T cells in 3 of 9 tumors. Overall, IFN-γ, perforin, and granzymeB were upregulated in clonally expanded T cells, while IL-2, IL-13, and CTLA-4 were upregulated in nonexpanded cells. A total of 116 TRB-CDR3 and TRA-CDR3 sequence pairs were identified from clonally expanded RCC TIL, among which 49 were only found in tumor tissue. Based on effector gene expression, as well as the availability of an autologous RCC tumor line, a total of 12 expanded TRAB pairs were cloned into lentiviral expression vectors. Analysis of TCR specificity for autologous tumor as well as HLA-matched RCC cell lines will be updated. Conclusions: T-cell clonal expansions associated with the microenvironment of RCC tumors and may represent antigen engagement. Phenotypic differences in effector gene expression between clonally expanded and nonexpanded CD8+ T cells from RCC TIL are heterogeneous between tumors. The association of such phenotypic differences with clinical outcomes is a priority for further investigation. Evaluation of alternate 10x platform and analysis of tumor antigen recognition by TCRs from clonally expanded T cells with cytotoxic phenotype unique to the RCC microenvironment is ongoing. Citation Format: Yuexin Xu, Alicia J. Morales, Andrea M.H. Towlerton, Edus H. Warren, Scott S. Tykodi. Single-cell characterization of tumor-infiltrating T cells from renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A36.

Exploring and analysing single cell multi-omics data with VDJView

BackgroundSingle cell RNA sequencing provides unprecedented opportunity to simultaneously explore the transcriptomic and immune receptor diversity of T and B cells. However, there are limited tools available that simultaneously analyse large multi-omics datasets integrated with metadata such as patient and clinical information.ResultsWe developed VDJView, which permits the simultaneous or independent analysis and visualisation of gene expression, immune receptors, and clinical metadata of both T and B cells. This tool is implemented as an easy-to-use R shiny web-application, which integrates numerous gene expression and TCR analysis tools, and accepts data from plate-based sorted or high-throughput single cell platforms. We utilised VDJView to analyse several 10X scRNA-seq datasets, including a recent dataset of 150,000 CD8+ T cells with available gene expression, TCR sequences, quantification of 15 surface proteins, and 44 antigen specificities (across viruses, cancer, and self-antigens). We performed quality control, filtering of tetramer non-specific cells, clustering, random sampling and hypothesis testing to discover antigen specific gene signatures which were associated with immune cell differentiation states and clonal expansion across the pathogen specific T cells. We also analysed 563 single cells (plate-based sorted) obtained from 11 subjects, revealing clonally expanded T and B cells across primary cancer tissues and metastatic lymph-node. These immune cells clustered with distinct gene signatures according to the breast cancer molecular subtype. VDJView has been tested in lab meetings and peer-to-peer discussions, showing effective data generation and discussion without the need to consult bioinformaticians.ConclusionsVDJView enables researchers without profound bioinformatics skills to analyse immune scRNA-seq data, integrating and visualising this with clonality and metadata profiles, thus accelerating the process of hypothesis testing, data interpretation and discovery of cellular heterogeneity. VDJView is freely available at https://bitbucket.org/kirbyvisp/vdjview.

Heart non-specific effector CD4+ T cells protect from postinflammatory fibrosis and cardiac dysfunction in experimental autoimmune myocarditis

Heart-specific CD4+ T cells have been implicated in development and progression of myocarditis in mice and in humans. Here, using mouse models of experimental autoimmune myocarditis (EAM) we investigated the role of heart non-specific CD4+ T cells in the progression of the disease. Heart non-specific CD4+ T cells were obtained from DO11.10 mice expressing transgenic T cell receptor recognizing chicken ovalbumin. We found that heart infiltrating CD4+ T cells expressed exclusively effector (Teff) phenotype in the EAM model and in hearts of patients with lymphocytic myocarditis. Adoptive transfer experiments showed that while heart-specific Teff infiltrated the heart shortly after injection, heart non-specific Teff effectively accumulated during myocarditis and became the major heart-infiltrating CD4+ T cell subset at later stage. Restimulation of co-cultured heart-specific and heart non-specific CD4+ T cells with alpha-myosin heavy chain antigen showed mainly Th1/Th17 response for heart-specific Teff and up-regulation of a distinct set of extracellular signalling molecules in heart non-specific Teff. Adoptive transfer of heart non-specific Teff in mice with myocarditis did not affect inflammation severity at the peak of disease, but protected the heart from adverse post-inflammatory fibrotic remodelling and cardiac dysfunction at later stages of disease. Furthermore, mouse and human Teff stimulated in vitro with common gamma cytokines suppressed expression of profibrotic genes, reduced amount of α-smooth muscle actin filaments and decreased contraction of cardiac fibroblasts. In this study, we provided a proof-of-concept that heart non-specific Teff cells could effectively contribute to myocarditis and protect the heart from the dilated cardiomyopathy outcome.

The Role of Neutrophils in the Immune System: An Overview.

Neutrophils, also known as polymorphonuclear neutrophils (PMNs), have long been considered as the short-lived, nonspecific white cells that form pus-and also happen to kill invading microbes. Indeed, neutrophils were often neglected (and largely not considered) as immune cells. This historic view of neutrophils has changed considerably over the past several decades, and we now know that in addition to playing the predominant role in the clearance of bacteria and fungi, they have a major role in shaping the host response to infection and immune system homeostasis. The change in our view of the role of neutrophils in the immune system has been due in large part to the study of these cells in vitro. Such work has been made possible by new and/or improved methods and approaches used to investigate neutrophils. These methods are the focus of this volume.

Chimeric Antigen Receptor Modified Memory-like (CAR-ML) NK Cells Exhibit Potent Responses to NK-Resistant Tumors

NK cells are cytotoxic innate lymphoid cells that mediate anti-tumor responses and traffic to most tissues. NK cells activated briefly with IL-12, IL-15, and IL-18 differentiate into memory-like (ML) NK cells with enhanced anti-tumor effects, which has been translated into clinical trials for patients with leukemia. Adoptive cellular therapy with donor ML NK cells were safe and induced CR/CRi in >50% of rel/ref AML patients in a first-in-human phase 1 clinical trial at Washington University (PMID27655849). In preliminary results, donor ML NK cells expand and can persist for months in an immune-compatible recipients during or after allogeneic hematopoietic cell transplantation (HCT), and maintain potent effector function. However, NK cell recognition of many cancer types is limited, since they depend on expression of stress-induced activating receptor ligands. We hypothesized that ML NK cells engineered with chimeric antigen receptors (CAR) would demonstrate improved anti-tumor responses against classically NK-resistant targets. To test this idea, ML NK cells were engineered to express an anti-CD19-CAR, and responses against NK-resistant B-cell malignancies evaluated in vitro and in vivo. CAR-modified primary human ML NK cells (CAR-ML) were transduced with an anti-CD19-CD8a-41BB-CD3z-GFP (CD19-CAR-ML) lentivirus. This differentiation and transfection approach resulted in approximately 15-25% of ML NK cells transduced with the CAR construct, as determined flow cytometry staining for soluble CD19 and GFP. As an additional control, αCD33-CD8a-41BB-CD3z-GFP CAR-transduced ML NK cells and GFP- internal control ML NK cells were used. Here, in vitro functional assays were used to determine if CD19-CAR enhances ML NK cells in an antigen (CD19)-specific manner. CD19-CAR-ML (GFP+) and control ML NK (GFP-) cells were evaluated for functional responses to CD19-positive or CD19-negative tumor targets in vitro. CD19-CAR-ML NK cells demonstrated significantly increased IFN-γ production (44±4% vs. 15±3%, p<0.001); mean ± SEM) and degranulation (31±4% vs. 5±1%, p<0.001) against NK-resistant CD19+ Raji targets, compared to control GFP- and CD33-CAR ML NK cells. To understand the contributions of ML differentiation on the enhanced functionality of CD19-CAR modified NK cells, we compared CD19-CAR-ML NK cells to control CD19-CAR NK cells that were treated with IL-15 only. CD19-CAR-ML NK cells also exhibited significantly increased effector responses compared to control CAR NK cells against CD19+ targets (p<0.01). CD19-CAR-ML NK cells responded similarly to GFP- ML NK cells against CD19-negative Kasumi leukemia targets. Finally, CD19-CAR-ML NK cells also exhibited significantly enhanced killing, degranulation, and IFN-γ production against primary CD19+ follicular lymphoma targets from patient lymph nodes (p<0.01). To establish the translational utility of this approach, autologous CD19-CAR-ML NK cells generated from lymphoma patients demonstrated significantly increased IFN-γ production (p<0.05) and degranulation (p<0.01) against their own CD19+ lymphoma targets, compared to control ML NK cells (GFP-). These data confirm contributions of both ML differentiation and CAR expression in the enhanced antigen-specific, anti-tumor responses observed in CAR-ML NK cells. To test the expansion and persistence of ML NK cells transduced with CD19 or CD33-CAR were transferred into CD19+ Raji-bearing NSG mice and supported in vivo with IL-15. After three weeks, mice were sacrificed and NK (CD56) cell persistence and tumor (CD19) burden assessed by flow cytometry. CD19-CAR ML recipient mice had reduced tumor burden in the BM, spleen, and blood compared to CD33-CAR ML treated mice. Notably, CD19-CAR-ML (GFP+) were increased to >70% of human NK cells from 20% in the CD33-CAR-ML recipient mice, suggesting antigen-specific CAR-expressing NK cells expand or survive better in vivo than non-transduced ML or non-specific CAR-ML NK cells in vivo. Thus, combining CAR with ML differentiation results in NK cells with enhanced responses to NK resistant tumors. These studies warrant continued CAR-ML development and provide the pre-clinical rationale for translating this combination NK cell therapy approach to the clinic. Figure Disclosures Fehniger: Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse).

Modulation of Shape and Size-Dependent Characteristics of Nanoparticles

Background: Nanoparticles have considerably been studied for biomedical applications which include biosensing, bioimaging diagnostics, etc. but the effect of shape and size on the nanoparticles has received little attention. The execution of nanoparticles is affected by various factors such as size, shape, surface charge, etc. Objective: In this manuscript, the effects of nanoparticles size and shape on cellular uptake, biodistribution, and half-life of nanoparticles are discussed. The shape of nanoparticles affects the biodistribution, cellular uptake, adhesion strength, half-life circulation, and drug release kinetics and drug clearance. Methods and Results: The shape of the particles enhances the distribution ratio. Particles with a different shape such as spherical shape particles, improve oral drug bioavailability. Rod-like structure of particles showed more specific uptake and less nonspecific uptake in cells related to sphere particles. The size of the particles also affects the binding properties to the receptor and the enthalpic and entropic properties to control the adhesive strength in nanoparticles. Maximum in vitro cellular uptake showed a 10-60nm size range. Nanoparticles that are more than 100nm in size have the ability to penetrate through leaky vasculature into tumors. The size of the nanoparticles affects the biological fate and the large size particles are accumulated in the liver and spleen. Conclusion: The present manuscript specifically deals with the nanocarrier formulation such as cubosomes and hexasomes. The nanoparticles shape has an impact on hydrodynamics significantly and interfaces of vascular targeting. Due to the small particle size of the cubosomes nanoparticles, the cubic phase is more appropriate for the controlled release drug delivery system. It can be concluded from the findings of the literature survey that the size and shape of nanocarriers have a significant effect on pharmaceutical and biomedical adaptation.