ABSTRACT
Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.
Subject(s)
DNA/analysis , Fluorescent Dyes/chemistry , Multiplex Polymerase Chain Reaction/methods , DNA/metabolism , Fluorescent Dyes/metabolism , Humans , Protein Binding/physiology , Real-Time Polymerase Chain Reaction/methodsABSTRACT
CD45 is dynamically repositioned within lipid rafts and the immune synapse during T cell activation, although the molecular consequences of CD45 repositioning remain unclear. In this study we examine the role of CD45 membrane compartmentalization in regulating murine T cell activation. We find that raft-localized CD45 antagonizes IL-2 production by opposing processive TCR signals, whereas raft-excluded CD45 promotes ERK-dependent polarized synaptic lipid raft clustering and IL-2 production. We propose that these dual CD45 activities ensure that only robust TCR signals proceed, whereas signals meeting threshold requirements are potentiated. Our findings highlight membrane compartmentalization as a key regulator of CD45 function and elucidate a novel signal transduction pathway by which raft-excluded CD45 positively regulates T cell activation.
Subject(s)
Extracellular Signal-Regulated MAP Kinases/metabolism , Interleukin-2/biosynthesis , Leukocyte Common Antigens/physiology , Membrane Microdomains/enzymology , Membrane Microdomains/immunology , Receptors, Antigen, T-Cell/physiology , Signal Transduction/immunology , T-Lymphocytes/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Line , Cell Line, Tumor , Down-Regulation/immunology , Enzyme Activation/immunology , Humans , Hybridomas , Interleukin-2/antagonists & inhibitors , Leukocyte Common Antigens/biosynthesis , Leukocyte Common Antigens/genetics , Lymphocyte Activation/genetics , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Membrane Microdomains/metabolism , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction/genetics , Son of Sevenless Proteins/genetics , Son of Sevenless Proteins/metabolism , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , Tyrosine/antagonists & inhibitors , Tyrosine/metabolism , Up-Regulation/immunology , src Homology Domains/geneticsABSTRACT
It was previously shown that costimulation of CD8(+) lymphocytes results in de novo expression of CD4. This study expanded on this observation to investigate the function of CD4 on CD8 cells. The ability of costimulated CD8 cells to respond to interleukin 16 (IL-16), a ligand that binds CD4 and induces cellular chemotaxis, was examined. IL-16-mediated ligation of CD4 expressed on CD8 T cells was found to induce an intracellular signal that directs migration of these cells in vitro. Thus, expression of CD4 on a CD8 lymphocyte has functional importance and may serve to control distribution of newly activated CD8 T cells in vivo.
