Simultaneous, Single-Cell Measurement of Messenger RNA, Cell Surface Proteins, and Intracellular Proteins.

Nucleic acid content can be quantified by flow cytometry through the use of intercalating compounds; however, measuring the presence of specific sequences has hitherto been difficult to achieve by this methodology. The primary obstacle to detecting discrete nucleic acid sequences by flow cytometry is their low quantity and the presence of high background signals, rendering the detection of hybridized fluorescent probes challenging. Amplification of nucleic acid sequences by molecular techniques such as in situ PCR have been applied to single-cell suspensions, but these approaches have not been easily adapted to conventional flow cytometry. An alternative strategy implements a Branched DNA technique, comprising target-specific probes and sequentially hybridized amplification reagents, resulting in a theoretical 8,000- to 16,000-fold increase in fluorescence signal amplification. The Branched DNA technique allows for the quantification of native and unmanipulated mRNA content with increased signal detection and reduced background. This procedure utilizes gentle fixation steps with low hybridization temperatures, leaving the assayed cells intact to permit their concomitant immunophenotyping. This technology has the potential to advance scientific discovery by correlating potentially small quantities of mRNA with many biological measurements at the single-cell level.

Dextramer reagents are effective tools for quantifying CMV antigen-specific T cells from peripheral blood samples.

The enumeration of antigen-specific T cells is increasingly relevant in clinical and research settings. This information is useful for evaluating immune responses to treatment, monitoring the efficacy of anticancer vaccines, and for detecting self-reactive T cells in autoimmune disorders. Quantifying antigen-specific T cells can be accomplished via IFNγ ELISpot assay, the measurement of intracellular cytokine production by flow cytometry, or by lymphocyte proliferation assays in response to antigen. While robust, these technologies are labor-intensive and can take several days to obtain results. New technology has led to more powerful tools for quickly and accurately measuring antigen-specific T cells by flow cytometry via fluorescently-labeled TCR-specific multimers. In this study, we evaluated the use of an assay based on Dextramer reagents for enumerating cytomegalovirus (CMV) antigen-specific T cells (CASTs). Assay performance characteristics were assessed by establishing Dextramers' sensitivity (median=0.4; range=0.1-1.4 CASTs µl(-1) ), determining their specificity (100%), evaluating assay robustness with different leukocyte sources and assay reproducibility via interlaboratory and interinstrument investigations. Furthermore, the levels of CASTs in 95 peripheral blood samples from 62 unique blood and marrow transplants recipients correlated well between Dextramers and Tetramers (R(2) =0.9042).

Tracking immune cell proliferation and cytotoxic potential using flow cytometry.

In the second edition of this series, we described the use of cell tracking dyes in combination with tetramer reagents and traditional phenotyping protocols to monitor levels of proliferation and cytokine production in antigen-specific CD8(+) T cells. In particular, we illustrated how tracking dye fluorescence profiles could be used to ascertain the precursor frequencies of different subsets in the T-cell pool that are able to bind tetramer, synthesize cytokines, undergo antigen-driven proliferation, and/or carry out various combinations of these functional responses.Analysis of antigen-specific proliferative responses represents just one of many functions that can be monitored using cell tracking dyes and flow cytometry. In this third edition, we address issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T-cell functions. We summarize key characteristics of and differences between general protein- and membrane-labeling dyes, discuss determination of optimal staining concentrations, and provide detailed labeling protocols for both dye types. Examples of the advantages of two-color cell tracking are provided in the form of protocols for (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay and (b) simultaneous monitoring of proliferative responses in effector and regulatory T cells.

Multiparameter flow cytometry for the diagnosis and monitoring of small GPI-deficient cellular populations.

BACKGROUND: Glycosyl-phosphatidylinositol (GPI)-negative blood cells are diagnostic for Paroxysmal Nocturnal Hemoglobinuria (PNH). Marrow failure states are often associated with GPI-negative cell populations. Quantification of small clonal populations of GPI-negative cells influences clinical decisions to administer immunosuppressive therapy in marrow failure states (aplastic anemia or myelodysplastic syndrome) and to monitor minimal residual disease after allogeneic blood or marrow transplantation (BMT). We studied the reliability of high-resolution flow cytometry markers operating at the limits of detection. METHODS: We performed serial quantification of the PNH clone size in 38 samples using multiparameter flow cytometry. Granulocytes, monocytes, and RBCs were gated using forward and side scatter as well as lineage-specific markers. The GPI-linked markers fluorescent aerolysin (FLAER), CD55, and CD59 were comparatively evaluated. We also evaluated CD16 on granulocytes and CD14 on monocytes. The sensitivity of detection by each marker was further defined by serial dilution experiments on a flow-sorted sample. Two patients had quantification of their GPI-negative clones before and after allogeneic BMT. RESULTS: FLAER was the most discriminant marker and allowed identification of 0.1% of GPI-negative cells despite other markers having superior signal-to-noise characteristics. CD14 and CD16 were inferior to CD55 at lower concentrations and in clinical application. CONCLUSIONS: Multiparameter flow cytometry permits quantification of small GPI-negative clones with a sensitivity limit of about 0.1%. The single most reliable marker to monitor small granulocyte or monocyte PNH clones is FLAER, especially in conditions such as myelodysplastic syndromes or BMT, when traditional GPI-linked surface marker expression can be significantly altered. © 2010 International Clinical Cytometry Society.

Validated protocol for FoxP3 reveals increased expression in type 1 diabetes patients.

BACKGROUND: FoxP3 has become a key identifier of regulatory T cells. Investigators have used a variety of antibodies and methods for detecting FoxP3 by flow cytometry. To standardize FoxP3 antibody staining for use in clinical trial samples, we tested various antibodies from different vendors, cell preparation protocols and fix/perm reagents, and cell isolation procedures. Using this optimized staining protocol, we evaluated clinical specimens from patients with multiple sclerosis (MS) or type 1 diabetes. METHODS: FoxP3 antibodies from eBioscience (236A/E7 and PCH101) and BioLegend (206D) were evaluated along with their respective methods and fix/perm reagents for preparation and staining of FoxP3 for flow cytometry. Fresh washed blood and frozen or fresh PBMC were evaluated. Upon optimization of the protocol, clinical samples (frozen PBMC) from patients with MS or type 1 diabetes and healthy control donors were evaluated with the BioLegend antibody. RESULTS: Clone 206D from BioLegend yielded optimal staining and the fix/perm reagents from both eBioscience and BioLegend were comparable. Data were also comparable between cells separated by Ficoll (fresh or frozen) and washed blood samples, allowing this protocol to be applicable to different types of samples. We validated this protocol using clinical samples and saw a significant increase in FoxP3 expression in the patients with type 1 diabetes but not in the MS. CONCLUSIONS: The results from this study will allow the assessment of FoxP3 by flow cytometry on samples from clinical sites that are analyzed in real time on fresh blood or frozen PBMC.

Prostate-derived Ets transcription factor (PDEF) downregulates survivin expression and inhibits breast cancer cell growth in vitro and xenograft tumor formation in vivo.

Previous studies using immunohistochemistry suggest that loss of the expression of the prostate-derived Ets transcription factor (PDEF) is a strong indicator for cancer cell malignancy. However, the underlying mechanism for this has not been well elucidated. We determined the role of PDEF in breast cancer cell growth and tumor formation using a series of experiments including Western blotting, promoter-luciferase reporter assay, RNA interference technology and a mouse xenograft model. We also determined the relationship between PDEF expression in human breast tumor specimen and cancer patient survivability. These studies revealed that PDEF expression is inversely associated with survivin expression and breast cancer cell xenograft tumor formation. PDEF-specific shRNA-mediated silencing of PDEF expression resulted in the upregulation of survivin expression in MCF-7 cells, which was associated with increased cell growth and resistance to drug-induced DNA fragmentation (apoptosis). In contrast, survivin-specific siRNA-mediated silencing of survivin expression decreased MCF-7 cell growth. Ectopic expression of PDEF inhibited both survivin promoter activity and endogenous survivin expression. Importantly, shRNA-mediated silencing of PDEF expression in MCF-7 breast cancer cells enhanced survivin expression and xenograft tumor formation in vivo. Furthermore, loss of PDEF expression in breast cancer tissues tends to be associated with unfavorable prognosis. These studies provide new information for the role of PDEF and survivin in breast cancer cell growth and tumor formation.

Molecular mechanism of inhibition of survivin transcription by the GC-rich sequence-selective DNA binding antitumor agent, hedamycin: evidence of survivin down-regulation associated with drug sensitivity.

Expression of the antiapoptotic protein survivin is associated with cancer cell viability and drug resistance. Thus, control of its expression in cancer cells has significant consequences for cancer therapeutics. Here we have shown that hedamycin, a GC-rich DNA binding drug, down-regulated survivin expression. Using a series of survivin promoter-luciferase constructs, we have identified an 86-bp GC-rich DNA element (-124 to -39) that mediates the ability of hedamycin to down-regulate survivin expression. Furthermore, both in vivo foot-printing and in vitro gel mobility shift experiments revealed that hedamycin bound to a 21-bp GC-rich DNA element (-115 to -95) in the survivin promoter. This drug-DNA interaction abrogated the binding of Sp-1 or Sp1-like proteins to the 21-bp cis-acting DNA element, and mutagenesis of this region consistently diminished survivin promoter activity. Finally, down-regulation of survivin transcription by hedamycin modulated the viability of cancer cells. These data suggest that abrogation of Sp-1 or Sp1-like protein binding to the 21-bp DNA element in the survivin promoter contributes at least in part to the inhibitory effect of hedamycin on survivin gene transcription. Drug-induced modulation of survivin gene expression may provide novel approaches for cancer therapeutics.