Cell Surface Protein Expression Research Articles

Targeting cancer stem cells in refractory cancer.

Although common cancer therapies, such as chemotherapy and radiation therapy, have recently improved and yielded good results, evaluated as tumor shrinkage, disease recurrence is still a common event for most cancer patients. This is termed refractory cancer. This tumor regrowth following therapy is generally thought to be caused by a small, specific population of tumor cells called cancer stem cells (CSCs). Similar to other stem cells, CSCs have the capacity for self-renewal and multipotent differentiation, and they have been identified in many tumor types based on cell surface protein expression. This specific cell population has stemness characteristics as examined by serial transplantation in animal models. Previous studies have developed a specific signature of cell surface markers and biological functions that can identify CSCs in many solid tumors. In this review, we summarize the characterization of CSCs using new techniques for identifying and quantifying them in situ. These techniques and concepts could be valuable for evaluating the effects of therapies on this cell population. Finally, we conclude by discussing several unique preclinical treatment strategies to targets CSCs, such as reprogramming CSCs or inducing attack by immune cells. Therapeutic and diagnostic methodologies that can target and quantify CSCs will be valuable tools for eradicating refractory cancer.

Collagen Binding Proteins of Gram-Positive Pathogens.

Collagens are the primary structural components of mammalian extracellular matrices. In addition, collagens regulate tissue development, regeneration and host defense through interaction with specific cellular receptors. Their unique triple helix structure, which requires a glycine residue every third amino acid, is the defining structural feature of collagens. There are 28 genetically distinct collagens in humans. In addition, several other unrelated human proteins contain a collagen domain. Gram-positive bacteria of the genera Staphylococcus, Streptococcus, Enterococcus, and Bacillus express cell surface proteins that bind to collagen. These proteins of Gram-positive pathogens are modular proteins that can be classified into different structural families. This review will focus on the different structural families of collagen binding proteins of Gram-positive pathogen. We will describe how these proteins interact with the triple helix in collagens and other host proteins containing a collagenous domain and discuss how these interactions can contribute to the pathogenic processes.

A rice bran phytochemical, cyanidin 3-glucoside, inhibits the progression of PC3 prostate cancer cell.

Prostate cancer is one of the high incidences and the most invasive cancer that is also highly resistant to chemotherapy. Currently, several natural products have been considering using as the supplements for anti-cancer therapy. This study aims to identify the potential active anti-cancer ingredients in the bran extracts of the native Thai rice (Luempua cultivar). Rice bran fraction enriched in anthocyanins was successively isolated and processed until the major purified compound obtained. The sub-fractions and the purified, rice bran, cyanidin 3-glucoside (RBC3G), were studied for biological effects (cell viability, migration, and invasion assays) on human prostatic cancer (PC3) cells using immunohistochemical-staining and immuno-blotting approaches. The sub-fractions and the purified RBC3G inhibited epithelial mesenchymal transition (EMT) characteristics of PC3 cells by blocking the expression of several cytoskeletal associate proteins in a concentration dependent manner, leading to decreasing of the cancer cell motility. RBC3G reduced the expression of Smad/Snail signaling molecules but enhanced the expression of cell surface protein, E-cadherin, leading to a delay tumor transformation. The RBC3G also inhibited matrix metalloproteinase-9 and nuclear factor-kappa B expression levels and the enzymes activity in PC3 cells, leading to a slow cell migration/invasion process. The results suggested that RBC3G blunt and/or delay the progressive cancer cell behaviors by inhibit EMT through Smad signaling pathway(s) mediating Snail/E-cadherin expression.

Mesenchymal Stem Cells Successfully Deliver Oncolytic Virotherapy to Diffuse Intrinsic Pontine Glioma

Diffuse intrinsic pontine glioma (DIPG) is among the deadliest of pediatric brain tumors. Radiotherapy is the standard-of-care treatment for DIPG, but offers only transient relief of symptoms for patients with DIPG without providing significant survival benefit. Oncolytic virotherapy is an anticancer treatment that has been investigated for treating various types of brain tumors. Here, we have explored the use of mesenchymal stem cells (MSC) for oncolytic virus (OV) delivery and evaluated treatment efficacy using preclinical models of DIPG. The survivin promoter drives the conditional replication of OV used in our studies. The efficiency of OV entry into the cells is mediated by fiber modification with seven lysine residues (CRAd.S.pK7). Patients' samples and cell lines were analyzed for the expression of viral entry proteins and survivin. The ability of MSCs to deliver OV to DIPG was studied in the context of a low dose of irradiation. Our results show that DIPG cells and tumors exhibit robust expression of cell surface proteins and survivin that enable efficient OV entry and replication in DIPG cells. MSCs loaded with OV disseminate within a tumor and release OV throughout the DIPG brainstem xenografts in mice. Administration of OV-loaded MSCs with radiotherapy to mice bearing brainstem DIPG xenografts results in more prolonged survival relative to that conferred by either therapy alone (P < 0.01). Our study supports OV, CRAd.S.pK7, encapsulated within MSCs as a therapeutic strategy that merits further investigation and potential translation for DIPG treatment.

IMMU-30. DISSECTING THE DYNAMIC RESPONSES OF GLIOBLASTOMA TO CAR T CELL THERAPY USING PATIENT-DERIVED TUMOR ORGANOIDS AND SINGLE-CELL MULTI-OMICS ANALYSES

Abstract Immunotherapy, including chimeric antigen receptor (CAR) T cell therapy, has yielded major advancements for a number of hard-to-treat cancers; however, its transformative potential has yet to be realized in glioblastoma (GBM). Clinical studies of EGFRvIII targeted CAR T cell therapy have indicated that tumor heterogeneity, immune microenvironment, and adaptive responses to treatment may play important roles in limiting overall efficacy. We sought to examine comprehensively the dynamic molecular landscape of CAR T cell therapy in GBM using patient-derived GBM organoids (GBOs), a newly established laboratory model of inter- and intra- tumoral heterogeneity. Especially advantageous in these studies, GBOs preserve the intrinsic composition of somatic mutations, transcriptomic states, and non-neoplastic cells that contribute to the tumor microenvironment. Complementing the complexity of this biological system, we constructed an integrated single-cell multi-omics platform to interrogate gene expression, cell surface protein expression, somatic variants, and TCR sequences all from the same cell. Co-culture of GBOs and EGFRvIII targeted CAR T cells led to rapid T cell activation with concomitant upregulation of cytokine response gene programs in both antigen-positive and antigen-negative tumor cells. The adaptive tumor response also included expression of immune checkpoint receptor (ICR) ligands, such as PD-L1. At later time points, T cells transitioned into a dysfunctional or exhausted state, as characterized by increased cell surface expression of inhibitory receptors, such as PD-1, and decreased markers of effector activity despite the presence of antigen. Interestingly, CAR T cell therapy not only led to changes in immune-related pathways and tumor microenvironment, but it also induced shifts in tumor cell states with the depletion of an oligodendrocyte precursor cell-like state and corresponding enrichment in an astrocyte-like state. This finding suggests that EGFRvIII targeted CAR T cell therapy may leverage intrinsic cellular plasticity to induce differentiation-like effects in the surviving tumor cells.

Cite-Seq Profiling of T Cells in Multiple Myeloma Patients Undergoing BCMA Targeting CAR-T or Bites Immunotherapy

Adaptive T cell therapy using chimeric antigen receptor (CAR) T cells and bispecific T cell engagers (BiTEs) have demonstrated encouraging responses in heavily pre-treated multiple myeloma (MM) patients. However, the cellular and molecular predictors of clinical response are not fully understood as well as the mediators of acquired resistance remain elusive. Local immune suppression and T cell exhaustion are important mediators of responses therefore, it is plausible to speculate that a tolerant tumor microenvironment and the expansion of specific T cell populations may dictate clinical responses. In this study, we performed at the single cell level a broad immunophenotypic and transcriptomic characterization of the blood and bone marrow (BM) T cells of sensitive and resistant MM patients treated with adaptive T cell therapies. Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) we measured the expansion of variable T cell subsets, T cell specific activation and inhibitor markers and their functional states in order to identify cellular mediators of resistance to these adoptive immune therapies. Serial blood samples and BM aspirates (n=12) were collected from patients treated with anti-BCMA CAR-T or BCMA-CD3 BiTEs at variable time points, prior and post initiation of therapy and at relapse. Bone marrow mononuclear fractions were isolated through ficoll density gradients coupled with magnetic sorting of CD3pos T cells. Unbiased mRNA profiling coupled with feature barcoding technology for cell surface protein (TotalSeq-B) of BM CD3pos T cells was then performed by using the chromium single cell (10x Genomics). Paired-end sequencing was performed on Illumina platform. Cell Ranger and Seurat pipeline were used for sample de-multiplexing, barcode processing, single-cell 3′ gene counting, cell surface protein expression and data analysis. CAR-T cells were identified by the expression of the chimeric CAR-T cell transcript. The parallel measurement of transcripts and cell surface protein phenotypes of CD3pos T cells using a panel of 19 immune surface markers underlined the T cell repertoire diversity and identified different T cell subsets among the CD8pos and CD4pos T cells. Notably, the cell surface protein information overlaid on the transcript-generated UMA allowed accurate identification of all main immune clusters, in particular for the CD45RA and CD45RO positive cells. Comparison of CITE-Seq features revealed that the T cells composition of the blood and BM niches differed significantly between sensitive and resistant patients. As such an enrichment of CD4pos T cells with a higher CD4:CD8 ratio was noted in responding patients. Phenotypic (CD45RA, CD45RO, CD95, CCR7, CD62L, CD28, CD27) and transcriptional signatures (TCF7, LEF1, GATA3, EOMES, TBX21, PRDM1) also identified a higher proportion of memory like T cells (Tscm, Tcm) in responding patients. In contrast, T cells of resistant patients were enriched with terminally exhausted (Tex) and senescent cells with loss of CD28, high GMZHand GMZB, CD57pos, CD69pos and CD160pos as well as upregulation of TBX21. Expression of T cell checkpoint inhibitors such as LAG3, TIGIT and PD1 was high in these Tex cells as well as in some Tem. Of note, ex vivo T cell activation studies with TIGIT blockade demonstrated T cell activation in an autologous MM and T cell co-culture system with enhanced MM cells death. An expanded cluster of regulatory T cells (Treg) FOXP3pos,CD25pos was also observed in two resistant patients. Of note, no loss of BCMA transcript or surface expression was noted in MM cells at the time of acquired resistance. Single cell transcriptome of primary MM cells and chromatin accessibility (ATAC-seq) analyses of T cells of these patients are ongoing to investigate the transcriptional programs and epigenetic factors underlying the immune escape. Combined single cell features profiling of the transcriptome and surface protein expression of T cells from MM patients receiving BCMA targeted CAR-T or BiTEs therapies revealed potential mediators of resistance. In particular, T cells composition (low CD4:CD8 ratio and reduced population of Tscm, Tcm) along with an enrichment of terminally exhausted T cells are the main features observed in resistant patients. Delineating these mechanisms will guide future T cells engineering studies to enhance the efficacy and response durability of adoptive immunotherapy in MM. Disclosures McCulloch: Amgen: Honoraria; Sanofi: Honoraria; Celgene: Honoraria; Janssen: Honoraria. Duggan:Jannsen: Consultancy; Amgen: Consultancy; Novartis: Honoraria; Celgene: Consultancy; Astra Zeneca: Consultancy. Jimenez-Zepeda:Janssen, Celgene, Amgen, Takeda: Honoraria. Bahlis:AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; BMS/Celgene and Janssen: Consultancy, Honoraria, Other: Travel, Accomodations, Research Funding; Karyopharm Therapeutics: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria. Neri:Celgene/BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

Immune-Related Adverse Events Associated With Immune Checkpoint Inhibitor Therapy

As part of immune surveillance, killer T lymphocytes search for cancer cells and destroy them. Some cancer cells, however, develop escape mechanisms to evade detection and destruction. One of these mechanisms is the expression of cell surface proteins which allow the cancer cell to bind to proteins on T cells called checkpoints to switch off and effectively evade T-cell-mediated destruction. Immune checkpoint inhibitors (ICIs) are antibodies that block the binding of cancer cell proteins to T-cell checkpoints, preventing the T-cell response from being turned off by cancer cells and enabling killer T cells to attack. In other words, ICIs restore innate antitumor immunity, as opposed to traditional chemotherapies that directly kill cancer cells. Given their relatively excellent risk-benefit ratio when compared to other forms of cancer treatment modalities, ICIs are now becoming ubiquitous and have revolutionized the treatment of many types of cancer. Indeed, the prognosis of some patients is so much improved that the threshold for admission for intensive care should be adjusted accordingly. Nevertheless, by modulating immune checkpoint activity, ICIs can disrupt the intricate homeostasis between inhibition and stimulation of immune response, leading to decreased immune self-tolerance and, ultimately, autoimmune complications. These immune-related adverse events (IRAEs) may virtually affect all body systems. Multiple IRAEs are common and may range from mild to life-threatening. Management requires a multidisciplinary approach and consists mainly of immunosuppression, cessation or postponement of ICI treatment, and supportive therapy, which may require surgical intervention and/or intensive care. We herein review the current literature surrounding IRAEs of interest to anesthesiologists and intensivists. With proper care, fatality (0.3%-1.3%) is rare.

Epithelial Cell Adhesion Molecule: An Anchor to Isolate Clinically Relevant Circulating Tumor Cells.

In the last few decades, the epithelial cell adhesion molecule (EpCAM) has received increased attention as the main membrane marker used in many enrichment technologies to isolate circulating tumor cells (CTCs). Although there has been a great deal of progress in the implementation of EpCAM-based CTC detection technologies in medical settings, several issues continue to limit their clinical utility. The biology of EpCAM and its role are not completely understood but evidence suggests that the expression of this epithelial cell-surface protein is crucial for metastasis-competent CTCs and may not be lost completely during the epithelial-to-mesenchymal transition. In this review, we summarize the most significant advantages and disadvantages of using EpCAM as a marker for CTC enrichment and its potential biological role in the metastatic cascade.

Alterations in the chondrocyte surfaceome in response to pro-inflammatory cytokines

BackgroundChondrocytes are exposed to an inflammatory micro-environment in the extracellular matrix (ECM) of articular cartilage in joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). In OA, degenerative changes and low-grade inflammation within the joint transform the behaviour and metabolism of chondrocytes, disturb the balance between ECM synthesis and degradation, and alter the osmolality and ionic composition of the micro-environment. We hypothesize that chondrocytes adjust their physiology to the inflammatory microenvironment by modulating the expression of cell surface proteins, collectively referred to as the ‘surfaceome’. Therefore, the aim of this study was to characterize the surfaceome of primary equine chondrocytes isolated from healthy joints following exposure to the pro-inflammatory cytokines interleukin-1-beta (IL-1β) and tumour necrosis factor-alpha (TNF-α). We employed combined methodology that we recently developed for investigating the surfaceome in stem cells. Membrane proteins were isolated using an aminooxy-biotinylation technique and analysed by mass spectrometry using high throughput shotgun proteomics. Selected proteins were validated by western blotting.ResultsAmongst the 431 unique cell surface proteins identified, a high percentage of low-abundance proteins, such as ion channels, receptors and transporter molecules were detected. Data are available via ProteomeXchange with identifier PXD014773. A high number of proteins exhibited different expression patterns following chondrocyte stimulation with pro-inflammatory cytokines. Low density lipoprotein related protein 1 (LPR-1), thrombospondin-1 (TSP-1), voltage dependent anion channel (VDAC) 1–2 and annexin A1 were considered to be of special interest and were analysed further by western blotting.ConclusionsOur results provide, for the first time, a repository for proteomic data on differentially expressed low-abundance membrane proteins on the surface of chondrocytes in response to pro-inflammatory stimuli.

Structure-guided engineering of the affinity and specificity of CARs against Tn-glycopeptides

The potency of adoptive T cell therapies targeting the cell surface antigen CD19 has been demonstrated in hematopoietic cancers. It has been difficult to identify appropriate targets in nonhematopoietic tumors, but one class of antigens that have shown promise is aberrant O-glycoprotein epitopes. It has long been known that dysregulated synthesis of O-linked (threonine or serine) sugars occurs in many cancers, and that this can lead to the expression of cell surface proteins containing O-glycans comprised of a single N-acetylgalactosamine (GalNAc, known as Tn antigen) rather than the normally extended carbohydrate. Previously, we used the scFv fragment of antibody 237 as a chimeric antigen receptor (CAR) to mediate recognition of mouse tumor cells that bear its cognate Tn-glycopeptide epitope in podoplanin, also called OTS8. Guided by the structure of the 237 Fab:Tn-OTS8-glycopeptide complex, here we conducted a deep mutational scan showing that residues flanking the Tn-glycan contributed significant binding energy to the interaction. Design of 237-scFv libraries in the yeast display system allowed us to isolate scFv variants with higher affinity for Tn-OTS8. Selection with a noncognate human antigen, Tn-MUC1, yielded scFv variants that were broadly reactive with multiple Tn-glycoproteins. When configured as CARs, engineered T cells expressing these scFv variants showed improved activity against mouse and human cancer cell lines defective in O-linked glycosylation. This strategy provides CARs with Tn-peptide specificities, all based on a single scFv scaffold, that allows the same CAR to be tested for toxicity in mice and efficacy against mouse and human tumors.

Notch3 signaling promotes tumor cell adhesion and progression in a murine epithelial ovarian cancer model.

High grade serous ovarian cancer (HGSC) is the most common and deadly type of ovarian cancer, largely due to difficulties in early diagnosis and rapid metastasis throughout the peritoneal cavity. Previous studies have shown that expression of Notch3 correlates with worse prognosis and increased tumorigenic cell behaviors in HGSC. We investigated the mechanistic role of Notch3 in a model of metastatic ovarian cancer using the murine ovarian surface epithelial cell line, ID8 IP2. Notch3 was activated in ID8 IP2 cells via expression of the Notch3 intracellular domain (Notch3IC). Notch3IC ID8 IP2 cells injected intraperitoneally caused accelerated ascites and reduced survival compared to control ID8 IP2, particularly in early stages of disease. We interrogated downstream targets of Notch3IC in ID8 IP2 cells by RNA sequencing and found significant induction of genes that encode adhesion and extracellular matrix proteins. Notch3IC ID8 IP2 showed increased expression of ITGA1 mRNA and cell-surface protein. Notch3IC-mediated increase of ITGA1 was also seen in two human ovarian cancer cells. Notch3IC ID8 IP2 cells showed increased adhesion to collagens I and IV in vitro. We propose that Notch3 activation in ovarian cancer cells causes increased adherence to collagen-rich peritoneal surfaces. Thus, the correlation between increased Notch3 signaling and poor prognosis may be influenced by increased metastasis of HGSC via increased adherence of disseminating cells to new metastatic sites in the peritoneum.

SAT-598 Shared Signaling Profile Between Human MRAPα-Induced Human MC4R Constitutive Activity and Obesity-Associated Human MC4R Constitutive Activity

The human melanocortin 4 receptor (hMC4R) plays a critical role in the regulation of energy balance with more than 150 distinct human obesity-associated mutations. Most exhibit defective MC4R functionality but six have been reported to associate with constitutive activity. This represents a conundrum since a lean phenotype is expected for enhanced MC4R signaling. Human melanocortin 2 receptor accessory protein alpha (hMRAPα) induces hMC4R constitutive activity in transfected HEK293 cells (1,2). We do not know whether the hMRAPα-induced gain-in-function for hMC4R would cause, or prevent, obesity because of this conundrum. Here, we hypothesize that wild-type hMC4R, obesity-associated constitutively active hMC4R and hMRAPα-induced constitutive active hMC4R can exist in distinct conformational states and elicit distinct signaling profiles. To test this, we compared transiently expressed HA-hMC4R in HEK293 cells for basal and agonist activation for adenylyl cyclase, Cre driven β-galactosidase reporter transcription, and receptor protein expression. Six previously reported obesity-associated hMC4R constitutively active variants were compared with two hMC4R constitutively active mutations not associated with obesity, two hMC4R variants associated with protection from development of obesity, five non-constitutively active hMC4R mutations associated with obesity, hMRAPα co-expressed with hMC4R, and wild-type hMC4R. Our data confirm hMC4R constitutive activity coupling to both adenylyl cyclase and Cre β-galactosidase reporter for only two hMC4R variants associated with obesity (H76R & L250Q), one hMC4R mutation (H158R) not associated with obesity, and hMRAPα co-expressed with hMC4R. We show α-MSH stimulated concentration curves for wild-type hMC4R, H76R, L250Q & H158R hMC4R variants and hMRAPα co-expressed with hMC4R coupling to adenylyl cyclase. Surprisingly, out of these, only wild-type hMC4R and H158R hMC4R variant exhibited α-MSH-stimulated Cre β-galactosidase reporter concentration curves. Western blotting and ELISA showed ~70% reduced cell surface and total receptor protein expression for hMC4R co-expressed with hMRAPα and obesity-associated constitutively active hMC4R variants, compared to wild-type hMC4R. To summarize, two constitutively active hMC4R variants (H76R and L250Q) associated with obesity, and hMC4R co-expressed with hMRAPα, share a signaling profile comprising protein expression and α-MSH stimulated functional coupling to adenylyl cyclase and Cre-reporter gene expression. We conclude (1) if hMC4R is co-expressed with hMRAPα in vivo it would likely contribute to human obesity, and (2) obesity-associated constitutively active hMC4R variants exhibit a signaling anomaly that may underpin development of anti-obesity therapeutics.1. Kay EI, et al. J Mol Endocrinol. 2013;50:203-215.2. Kay EI, et al. PLoS ONE. 2015;10(10):e0140320.

Regulation of human macrophage phenotype and GPBAR1 expression by cell seeding density and glucocorticosteroids

Abstract Macrophages are key mediators of tissue homeostasis and inflammation and do so by virtue of differential expression of secreted and cell surface proteins. Cytokines such as IFNγ or IL-4 and IL-13 are known to generate classical proinflammatory M1 and alternatively activated M2 macrophages, respectively, but it is less clear how cell density, cell-cell contact and corticosteroids work together to affect macrophage function. Corticosteroids such as cortisol are known to downregulate the inflammatory signaling but they can also alter the expression of cell surface proteins, such as CD163, on steady-state macrophages. Here we examined the effect of monocyte seeding density and cortisol on the expression of cell surface proteins on human macrophages. The expression of the macrophage markers CD16, CD163, CD204 and VSIG4 were found to correlate with monocyte seeding density such that at low seeding densities, monocyte-derived macrophages were found to express minimal amounts of CD16, CD163, CD204 and VSIG4 while at high seeding densities, expression of these markers was restored. When cultured in the presence of cortisol, macrophages seeded at low densities were found to upregulate expression of these markers, but not CD204, to levels comparable to macrophages seeded at high densities grown in the presence of MCSF. Additionally, in the presence of cortisol, macrophages expressed higher levels of GPBAR1 which has also been shown to reduce classical proinflammatory macrophage activation in colitis models. This data suggests that corticosteroids may regulate macrophage function by modulating the expression of cell surface proteins such as GPBAR1 and not just interfering with morer conventional inflammatory cell signaling pathways..

Evaluation of oligonucleotide conjugated antibodies as reporter molecules in single-cell assays

Abstract Multiplexing in conventional flow and mass cytometric analysis has been constrained by both the number of available reporter molecules as well as signal spillover between these molecules. Monoclonal antibodies(mAbs) conjugated to oligonucleotides have been used in the design of ultrasensitive immunoassays but the multiplexing potential of these reagents have only recently been realized. The Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) method is established and uses oligo-conjugated mAbs and single-cell RNA sequencing technology to simultaneously enable the quantitation of both mRNA and protein molecules. Using the BD Rhapsody™ platform and BD® AbSeq, we examined the expression of 399 genes and 40 proteins in human PBMCs. The ability of single cell RNA-seq (scRNA-seq) to differentiate major and minor subsets of immune cells was evaluated. As in conventional cytometric analysis, immune cell populations could be identified based on the expression of canonical cell surface proteins and we compared the frequency of immune cell populations identified via scRNA-seq to CyTOF technology. Additionally, genes expected to be up- or down-regulated between established major populations such as T- and B-cells were observed thereby encouraging further analysis of gene expression in minor sub-populations. Overall, immune cell frequencies were found to be highly concordant between technologies. Single-cell “multi-omics” in which mRNA, proteins, and immune cell clonality are simultaneously measured on single cells thus stands poised to innovate drug development by providing unprecedented depth in the analysis of cells expressing therapeutic targets and driving disease pathogenesis.

Aging T cell Immunity to Conserved Viral Epitopes

Abstract Although seasonal vaccination is widely accessible, its efficacy is inconsistent. Older individuals, in particular, tend to have difficulty mounting adequate immunity to infection. A possible reason for this struggle may be due to high mutagenesis in influenza hemagglutinin (HA), the major component of vaccines. Since older individuals possess diminished immune repertoire diversity compared to younger individuals, large sweeps in the viral proteome can be devastating. Unlike the antibody response, which primarily targets mutagenic surface proteins like HA; however, the T cell response can target more conserved regions of the viral proteome. Vaccines designed to stimulate T cells may thus provide greater protection in older individuals. To better understand the T cell repertoire in relation to the conserved regions of the viral proteome, we have predicted highly conserved viral T cell epitopes from various influenza strains and used our next-generation sequencing based method, TetTCR-seq HD, to pair T cell receptor (TCR) sequence, antigen specificity, transcriptome, and surface protein expression at the single cell level. In addition to novel antigen discovery, our high dimensional data have revealed the presence of immunity toward highly conserved influenza epitopes in older and younger individuals. Additionally, although age appears to decrease TCR repertoire and antigenic diversity, other factors such as immunodominance toward other viral epitopes, such as human cytomegalovirus, interestingly have a greater impact on the influenza-specific repertoire and phenotype. Our data ultimately indicate that healthy individuals harbor a reservoir of influenza-specific T cells that may be targetable by vaccines.

Cell-Surface Proteomics Identifies Differences in Signaling and Adhesion Protein Expression between Naive and Primed Human Pluripotent Stem Cells.

SummaryNaive and primed human pluripotent stem cells (hPSC) provide valuable models to study cellular and molecular developmental processes. The lack of detailed information about cell-surface protein expression in these two pluripotent cell types prevents an understanding of how the cells communicate and interact with their microenvironments. Here, we used plasma membrane profiling to directly measure cell-surface protein expression in naive and primed hPSC. This unbiased approach quantified over 1,700 plasma membrane proteins, including those involved in cell adhesion, signaling, and cell interactions. Notably, multiple cytokine receptors upstream of JAK-STAT signaling were more abundant in naive hPSC. In addition, functional experiments showed that FOLR1 and SUSD2 proteins are highly expressed at the cell surface in naive hPSC but are not required to establish human naive pluripotency. This study provides a comprehensive stem cell proteomic resource that uncovers differences in signaling pathway activity and has identified new markers to define human pluripotent states.

Antibacterial and Antifungal Agent, Triclosan (TCS) Alters Secretion of Interleukin beta and Interleukin 6 from Human Immune Cells

Triclosan (TCS) is an antimicrobial compound that is widely used in personal hygiene products such as mouthwash and toothpaste. TCS can be ingested or absorbed through the skin and has been found in human blood, breast milk, and urine. Interleukin (IL)‐6 and IL‐1 beta (IL‐1β) are important pro‐inflammatory cytokines produced by lymphocytes, monocytes, and other cells. Both regulate cell growth, tissue repair, and immune function, increased levels of each have been associated with a number of diseases including rheumatoid arthritis and certain cancers. TCS has been shown to inhibit the lytic function of human natural killer (NK) lymphocytes and to decrease expression of key cell surface proteins on NK cells. Here we examine whether TCS alters the secretion of IL‐1β and IL‐6 from human immune cell preparations. Human peripheral blood mononuclear cells (PBMCs) and monocyte‐depleted (MD)‐PBMCs were exposed to TCS at concentrations of 0–5 μM. Cytokine secretion was measured at 24 h, 48 h, and 6 days using enzyme‐linked immunosorbent assay (ELISA). In this study, both IL‐1β and IL‐6 secretion were altered at one or more concentration of TCS at one or more length of exposure. IL‐1β secretion was increased at 1, 2.5 or 5 μM TCS. Effects of 24 h exposure to 1, 2.5 and 5 μM TCS on IL‐1β secretion from PBMCs treated with selective MAPK pathway component inhibitors were also measured. These results indicate that TCS has the capacity to disrupt secretion of these two important pro‐inflammatory cytokines. Additionally the data show that TCS‐induced increases in IL‐β secretion were dependent on p38 MAPK.Support or Funding InformationSupported by NIH grant U54CA163066.

High CD206 levels in Hodgkin lymphoma-educated macrophages are linked to matrix-remodeling and lymphoma dissemination.

Macrophages (Mφ) are abundantly present in the tumor microenvironment and may predict outcome in solid tumors and defined lymphoma subtypes. Mφ heterogeneity, the mechanisms of their recruitment, and their differentiation into lymphoma‐promoting, alternatively activated M2‐like phenotypes are still not fully understood. Therefore, further functional studies are required to understand biological mechanisms associated with human tumor‐associated Mφ (TAM). Here, we show that the global mRNA expression and protein abundance of human Mφ differentiated in Hodgkin lymphoma (HL)‐conditioned medium (CM) differ from those of Mφ educated by conditioned media from diffuse large B‐cell lymphoma (DLBCL) cells or, classically, by macrophage colony‐stimulating factor (M‐CSF). Conditioned media from HL cells support TAM differentiation through upregulation of surface antigens such as CD40, CD163, CD206, and PD‐L1. In particular, RNA and cell surface protein expression of mannose receptor 1 (MRC1)/CD206 significantly exceed the levels induced by classical M‐CSF stimulation in M2‐like Mφ; this is regulated by interleukin 13 to a large extent. Functionally, high CD206 enhances mannose‐dependent endocytosis and uptake of type I collagen. Together with high matrix metalloprotease9 secretion, HL‐TAMs appear to be active modulators of the tumor matrix. Preclinical in ovo models show that co‐cultures of HL cells with monocytes or Mφ support dissemination of lymphoma cells via lymphatic vessels, while tumor size and vessel destruction are decreased in comparison with lymphoma‐only tumors. Immunohistology of human HL tissues reveals a fraction of cases feature large numbers of CD206‐positive cells, with high MRC1 expression being characteristic of HL‐stage IV. In summary, the lymphoma‐TAM interaction contributes to matrix‐remodeling and lymphoma cell dissemination.

Interaction between SDF1 and CXCR4 Promotes Photoreceptor Differentiation via Upregulation of NFκB Pathway Signaling Activity in Pax6 Gene-Transfected Photoreceptor Precursors

Background: CCL2 (also known as monocyte chemoattractant protein 1) and CX3CR1 (also known as Fractalkine receptor)-deficient mice have damaged photoreceptors. Objectives: We examined the interaction of SDF1 and CXCR4 on the differentiation of retinal progenitors into rhodopsin-positive photoreceptors. Methods: Cloned retinal progenitors were obtained by Pax6 gene transfection of mouse iPS cells followed by serial dilution. Clones were selected by expression of nestin, Musashi1, Six3, and Chx10 mRNA. Cell surface protein expression was analyzed by flow cytometry. The levels of mRNA and intracellular protein were examined by real-time PCR and immunochemistry, respectively. Transient transfection experiments of retinal progenitors were conducted using a human rhodopsin promoter luciferase plasmid. Results: We selected 10 clones that expressed Six3, Chx10, Crx, Rx1, Nrl, CD73, and rhodopsin mRNA, which, except for rhodopsin, are photoreceptor precursor markers. Clones expressed both CD73 and CXCR4 on the cell surface and differentiated into rhodopsin-positive photoreceptors, which was reinforced by the addition of exogenous SDF1. A CXCR4 inhibitor AMD3100 blocked SDF1-mediated differentiation of progenitors into photoreceptors. SDF1 enhanced human rhodopsin promoter transcription activity, possibly via the NFκB pathway. Addition of SDF1 to the cell culture induced nuclear translocation of NFκB on retinal progenitor cell clones. Neonatal and newborn mouse retinas expressed SDF1 and CXCR4. Cells in the outer nuclear layer where photoreceptors are located expressed CXCR4 at P14 and P56. Cells in the inner nuclear layer expressed SDF1. Conclusions: These findings suggest that retinal progenitor cell differentiation was at least partly regulated by SDF1 and CXCR4 via upregulation of NFκB activity.

Flow Cytometry Analysis of dog Bone Marrow Stem Cell Surface Protein Expression

The present study aimed to study the Flow cytometry analysis of dog bone marrow stem cell surface protein expression. Five healthy mongrel dogs (3 male dogs and 2 female dogs). The dogs weight was (24 - 50 kg b.wt) and average age was ( 8 - 24 Months), the dogs examined against diseases. The dogs were completely sterilized against any external parasites or any external diseases. Flow cytometry analysis of the surface markers against the selected phenotypes following cell surface protein expression (CD44, CD29, CD73, CD90, CD105, CD166, CD271, CD45, CD34, CD13, and c-kit (all from BD Biosciences). BM-MSC at third passage were cultured in DMEM (Dulbecco’s Modified Eagle Medium) deprived of FCS (fetal calf serum), and supernatants were collected after 6 or 24 hours microvesicles. MVs purification was performed using the ExoQuick-TC exosome precipitation solution (ExoQuick; System Biosiences) and storage at −80°C until further use. It could be concluded that, the canine BM-MSCs play an important role in veterinary medicine and can help in treating many diseases and injury that the animals facing it, flow cytometery can help for analysis and identification of this cells, so it can be used easily for treating certain diseases and injuries. Our results provide fundamental information to enable for more reproducible and reliable quality control in the identification of canine BM-MSCs.