High sensitivity 8-color flow cytometry assay for paroxysmal nocturnal hemoglobinuria granulocyte and monocyte detections.

Flow cytometry is the gold standard in diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) by detecting the absence of glycol-phosphatidyl inositol (GPI)-linked protein expression on granulocyte and monocyte surfaces. However, the current assays are not optimized and require improvement, particularly in reducing background fluorescence and optimizing sensitivity and specificity. With more fluorochromes available and with advances in instrument engineering, rare populations may be identified with high sensitivity. The present study assessed an 8-color combination of comprehensive GPI-linked markers, namely fluorescein-labeled proaerolysin (FLAER), cluster of differentiation 157 (CD157), CD24 and CD14, and the lineage markers for granulocyte (CD15) and monocyte (CD64) cells to detect PNH clones. Additionally, to optimize the PNH flow assay, a 'dump' channel was used, comprised of CD5 and CD19, to exclude non-specific binding in order to reduce background. This method aimed to improve sensitivity and reduce the background to create an optimized PNH flow cocktail. The results demonstrated that the current 4-color PNH combination identifies a CD55- and FLAER+ population that is not PNH clones. By contrast, the 8-color panel delineated PNH clones from both monocyte and granulocytes by using granulocyte antigen (CD15) and monocyte antigen (CD64) as a gating strategy. The sensitivity was 0.01% for granulocytes and 0.05% for monocytes with an acquisition of 100,000 monocyte and granulocyte events. The background on a normal whole blood sample was 0.00076% on monocytes and 0.00277% on granulocytes. Thus, overall, the 8-color PNH assay exhibited high levels of specificity and sensitivity. The 8-color combination facilitated the improvement and enhancement of sensitivity in PNH clone identification, and may provide a useful tool for pathologists in PNH diagnosis and for monitoring patients at risk of developing classical/hemolytic PNH, to enable treatment to be delivered promptly.

Stabilization of pre-optimized multicolor antibody cocktails for flow cytometry applications.

BACKGROUND: Flow cytometry has a multitude of applications in nearly all fields of biology. Newly described biological markers enable the creation of novel reagents which then aid in the elucidation of unique subsets of cells and their potential role in health and disease. In order to enable the simultaneous detection of an even greater number of parameters, the future progress of flow cytometry relies on advances in instrument engineering and the parallel development of new fluorophores. METHODS: In order to address the issues of reagent reliability, reproducibility, and work-flow optimization, we have used the freeze-dry technique to stabilize pre-mixed, pre-optimized, multicolor 'cocktails' of antibodies within 12 × 75 mm flow cytometry tubes (Lyotube). In this study we describe several lyophilized stabilized reagent combinations that are functional for extended periods of time (18 months and beyond), and can be stored at ambient temperature, eliminating cold-chain requirements during transportation and storage. This improves precision and reduces the redundant labor and error-potential associated with mixing antibodies to create "home-brew" cocktails. RESULTS: We have stained different types of samples including normal and leukemic whole blood, bone marrow, and PBMCs, as well as cell lines, directly with BD Lyotube reagents: The data show comparable and consistent performance of multiple batches of dehydrated, stabilized mixtures of antibodies and their liquid counterparts. CONCLUSIONS: The approach we describe here, the Lyotube, facilitates the improvement and implementation of standardization measures in clinical settings and in multi-site studies, a useful tool which can also be applied to determining the efficacy and safety of candidate therapeutics and vaccines. © 2016 International Clinical Cytometry Society.

Toward development of a comprehensive external quality assurance program for polyfunctional intracellular cytokine staining assays.

The External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program assesses the proficiency of NIH/NIAID/DAIDS-supported and potentially other interested research laboratories in performing Intracellular Cytokine Staining (ICS) assays. The goal of the EQAPOL Flow Cytometry External Quality Assurance Program (EQAP) is to provide proficiency testing and remediation for participating sites. The program is not punitive; rather, EQAPOL aims to help sites identify areas for improvement. EQAPOL utilizes a highly standardized ICS assay to minimize variability and readily identify those sites experiencing technical difficulties with their assays. Here, we report the results of External Proficiency 3 (EP3) where participating sites performed a 7-color ICS assay. On average, sites perform well in the Flow Cytometry EQAP (median score is "Good"). The most common technical issues identified by the program involve protocol adherence and data analysis; these areas have been the focus of site remediation. The EQAPOL Flow Cytometry team is now in the process of expanding the program to 8-color ICS assays. Evaluating polyfunctional ICS responses would align the program with assays currently being performed in support of HIV immune monitoring assays.

Iron administration before stem cell harvest enables MR imaging tracking after transplantation.

PURPOSE: To determine whether intravenous ferumoxytol can be used to effectively label mesenchymal stem cells (MSCs) in vivo and can be used for tracking of stem cell transplants. MATERIALS AND METHODS: This study was approved by the institutional animal care and use committee. Sprague-Dawley rats (6-8 weeks old) were injected with ferumoxytol 48 hours prior to extraction of MSCs from bone marrow. Ferumoxytol uptake by these MSCs was evaluated with fluorescence, confocal, and electron microscopy and compared with results of traditional ex vivo-labeling procedures. The in vivo-labeled cells were subsequently transplanted in osteochondral defects of 14 knees of seven athymic rats and were evaluated with magnetic resonance (MR) imaging up to 4 weeks after transplantation. T2 relaxation times of in vivo-labeled MSC transplants and unlabeled control transplants were compared by using t tests. MR data were correlated with histopathologic results. RESULTS: In vivo-labeled MSCs demonstrated significantly higher ferumoxytol uptake compared with ex vivo-labeled cells. With electron microscopy, iron oxide nanoparticles were localized in secondary lysosomes. In vivo-labeled cells demonstrated significant T2 shortening effects in vitro and in vivo when they were compared with unlabeled control cells (T2 in vivo, 15.4 vs 24.4 msec; P < .05) and could be tracked in osteochondral defects for 4 weeks. Histologic examination confirmed the presence of iron in labeled transplants and defect remodeling. CONCLUSION: Intravenous ferumoxytol can be used to effectively label MSCs in vivo and can be used for tracking of stem cell transplants with MR imaging. This method eliminates risks of contamination and biologic alteration of MSCs associated with ex vivo-labeling procedures.

Tumor vaccines in 2010: need for integration.

Induction of tumor-specific immunity is an attractive approach to cancer therapy, however to date every major pivotal trial has resulted in failure. While the phenomena of tumor-mediated immune suppression has been known for decades, only recently have specific molecular pathways been elucidated, and for the first time, rationale means of intervening and observing results of intervention have been developed. In this review we describe major advances in our understanding of tumor escape from immunological pressure and provide some possible therapeutic scenarios for enhancement of efficacy in future cancer vaccine trials.

Enhancement of DNA cancer vaccine efficacy by combination with anti-angiogenesis in regression of established subcutaneous B16 melanoma.

Immunotherapy of cancer offers great promise, however translation into human studies has yielded relatively poor results to date. The concept of combining cancer vaccination with angiogenesis inhibition is appealing, due to favorable safety profile of both approaches, as well as possible biological synergies. Here we studied the anti-tumor effects of combining plasmid DNA (pDNA) vaccination and anti-angiogenesis in B16F10 murine model. By using electroporation-mediated gene/pDNA delivery, the anti-tumor efficacy of vaccination with pDNAs encoding gp100, TRP2 and Ii-PADRE was facilitated by administration of soluble form of EphB4 fused with human serum albumin (sEphB4-HSA), or by co-delivery of pDNAs encoding Angiostatin and/or Endostatin. In an optimized administration protocol, melanoma vaccination together with intratumoral delivery of pDNAs encoding Angiostatin and Endostatin resulted in 57% tumor-free survival over 90 days after challenge. These data support the general concept that suppression of angiogenesis may allow for enhanced efficacy of anti-tumor immunity, suggesting the synergetic effects of therapeutic pDNA vaccination and angiogenesis inhibition in cancer therapy.

Induction of cytotoxic T cell response against HCA661 positive cancer cells through activation with novel HLA-A *0201 restricted epitopes.

HCA661 is a cancer-testis (CT) antigen frequently expressed in human hepatocellular carcinoma (HCC). To search for immunogenic peptides of HCA661, bioinformatics analysis and CD8(+) T cell IFN-gamma ELISPOT assay were employed, and two HLA-A *0201 restricted peptides, H110 and H246, were identified. These two HCA661 peptides are naturally processed in dendritic cells (DCs) and when used for DCs loading, they are sufficient to prime autologous CD8(+) T cells to elicit cytotoxic response against HCA661(+) human cancer cells. The HCA661 peptides, H110 and H246, are hence attractive candidates for human cancer immunotherapy.

Glutathione depletion inhibits dendritic cell maturation and delayed-type hypersensitivity: implications for systemic disease and immunosenescence.

BACKGROUND: Dendritic cells (DCs) play a key role as antigen-presenting cells in the immune system. There is growing evidence that the redox equilibrium of these cells influences their ability to induce T-cell activation and to regulate the polarity of the immune response. This could affect the outcome of the immune response during systemic diseases and aging. OBJECTIVE: Our aim was to elucidate the mechanism by which the redox equilibrium of antigen-presenting DCs affects the delayed-type hypersensitivity (DTH) response during experimental modification of glutathione levels, as well as during aging. METHODS: We looked at the effect of glutathione depletion by diethyl maleate in DCs as well as during systemic administration on the DTH response to the contact-sensitizing antigens, oxazolone, and 2,4-dinitro-1-fluorobenzene. We also determined whether glutathione repletion with N-acetyl cysteine could influence the decline of the DTH response in aged mice. RESULTS: Glutathione depletion in bone marrow-derived DCs interfered in their ability to mount a DTH response on adoptive transfer into recipient mice. Glutathione depletion interfered in IL-12 production and costimulatory receptor expression in DCs, leading to decreased IFN-gamma production in the skin of recipient mice. Systemic diethyl maleate treatment exerted similar effects on the DTH response and IFN-gamma production, whereas N-acetyl cysteine administration reversed the decline of the DTH response in aged animals. CONCLUSION: Glutathione depletion downregulates T(H)1 immunity through a perturbation of DC maturation and IL-12 production. CLINICAL IMPLICATIONS: These data show that the induction of oxidative stress in the immune system, under disease conditions and aging, interferes in T(H)1 immunity.

Pro-oxidative diesel exhaust particle chemicals inhibit LPS-induced dendritic cell responses involved in T-helper differentiation.

BACKGROUND: Epidemiologic studies show that exposure to ambient particulate matter leads to asthma exacerbation. Diesel exhaust particles (DEPs), a model pollutant, act as an adjuvant for allergic sensitization. Increasing evidence shows that this effect could be mediated by an effect on dendritic cells (DCs). OBJECTIVE: Our aim was to elucidate the mechanism by which pro-oxidative DEP chemicals change DC function so that these antigen-presenting cells strengthen the immune response to an experimental allergen. METHODS: We exposed murine bone marrow-derived DCs and a homogeneous myeloid DC line, BC1, to DEPs and organic extracts made from these particles to determine how the induction of oxidative stress affects cellular maturation, cytokine production, and activation of antigen-specific T cells. RESULTS: DEP extracts induced oxidative stress in DCs. This change in redox equilibrium interfered in the ability of Toll-like receptor agonists to induce the expression of maturation receptors (eg, CD86, CD54, and I-A(d)) and IL-12 production. This perturbation of DC function was accompanied by decreased IFN-gamma and increased IL-10 induction in antigen-specific T cells. The molecular basis for the perturbation of DC function is the activation of a nuclear factor-erythroid 2 (NF-E2)-related factor 2-mediated signaling pathway that suppresses IL-12 production. NF-E2-related factor 2 deficiency abrogates the perturbation of DC function by DEPs. CONCLUSION: These data provide the first report that pro-oxidative DEP chemicals can interfere in T(H)1-promoting response pathways in a homogeneous DC population and provide a novel explanation for the adjuvant effect of DEPs on allergic inflammation. CLINICAL IMPLICATIONS: These data clarify the adjuvant effect of particulate air pollutants in allergic inflammatory disease.

CD80 transfected human hepatocellular carcinoma cells activate cytotoxic T lymphocytes to target HCC cells with shared tumor antigens.

Human HCC cell lines (BEL-7402, SMMC-7721 and QGY-7703) do not express CD80 molecules, although they express MHC class I molecules and ICAM-1. HCC's poor immunogenicity may therefore be due to lack of CD80 molecules. This study first investigated whether CD80 molecules could provide minimal co-stimulatory signal for establishing an efficient anti-tumor immunity in HCC and second, whether the transfection of CD80 into the BEL-7402 cell line could induce T cell activation for targeting other HCC cell lines expressing shared common antigens. The transfection of cDNA encoding CD80 into ICAM-1+ HCC BEL-7402 cells was confirmed by flow cytometrical analysis. The CD80-transfected cells could enhance the immunogenicity of BEL-7402 cells as detected by T cell proliferation assay, and also activated the T cells at a higher proliferation rate comparing with the BEL-7402 cells transfected with vector only. The CD80-transfected cell line was also found able to activate T cells which subsequently induced cell lysis of SMMC-7721, QGY-7703 and parent BEL-7402 cell lines as detected by cytotoxicity assay. It can be concluded that the cytotoxicity was due to MHC class I restricted CD8+ cytotoxic T lymphocytes, but not natural killer (NK) cells, since this cytotoxic effect could be blocked by anti-MHC class I antibody and the cytotoxicity was shown very low in NK-cell-sensitive K562 cell line. Electroporation of CD80 cDNA into human HCC cells could increase the expression of the functional CD80 molecules and enhance the immunogenicity of the genetically-modified HCC cells to activate T cells for targeting 3 HCC cell lines in an HLA-restricted manner.