ABSTRACT
The peptidase inhibitor PI16 was shown previously by microarray analysis to be over-expressed by CD4-positive/CD25-positive Treg compared with CD4-positive/CD25-negative Th cells. Using a monoclonal antibody to the human PI16 protein, we found that PI16-positive Treg have a memory (CD45RO-positive) phenotype and express higher levels of FOXP3 than PI16-negative Treg. PI16-positive Treg are functional in suppressor assays in vitro with potency similar to PI16-negative Treg. Further phenotyping of the PI16-positive Treg revealed that the chemokine receptors CCR4 and CCR6 are expressed by more of the PI16-positive/CD45RO-positive Treg compared with PI16-negative/CD45RO-positive Treg or Th cells. PI16-positive Treg showed enhanced in vitro migration towards the inflammatory chemokines CCL17 and CCL20, suggesting they can migrate to sites of inflammation. We conclude that PI16 identifies a novel distinct subset of functional memory Treg which can migrate to sites of inflammation and regulate the pro-inflammatory response at those sites.
Subject(s)
Carrier Proteins/immunology , Cell Movement , Chemokine CCL17/immunology , Chemokine CCL20/immunology , Glycoproteins/immunology , Immunologic Memory , T-Lymphocytes, Regulatory/immunology , Cell Proliferation , Cytokines/immunology , Forkhead Transcription Factors/immunology , Humans , Leukocyte Common Antigens/immunology , Phenotype , T-Lymphocytes, Regulatory/cytologyABSTRACT
The analysis of membrane molecules using antibodies detected by immunofluorescence staining and flow cytometry is used widely in research and diagnostic immunology. Conventional staining techniques readily detect molecules present at concentrations of around 2000 molecules per cell, but some molecules are expressed and function at much lower abundance. We described previously a method for the detection of molecules present at 100 molecules per cell or less based on the use of phycoerythrin as the fluorophore, a three-layer amplification process, and careful selection of available reagents. In recent years, a number of new reagents, fluorophores and kits, have become available, some of them intended for high-sensitivity applications. In this paper, a number of these reagents have been compared with the published method. While some of the reagents gave variable results or high nonspecific staining in our hands, several reagents were comparable with the published method. Furthermore, the new fluorophores allow improved simultaneous detection of two low-abundance markers.
Subject(s)
Antigens, CD/analysis , Flow Cytometry/methods , Fluorescent Dyes , Membrane Proteins/analysis , Antibodies/chemistry , Antibodies/immunology , Antigens, CD/immunology , Fluorescent Antibody Technique/methods , Fluorescent Dyes/chemistry , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Indicators and Reagents , Membrane Proteins/immunologyABSTRACT
We describe an economical 20 litre bench-top fermenter suitable for production of recombinant antibody fragments in bacterial expression systems. The bacterial culture contained within a polycarbonate carboy is mixed (400-600 rpm) and aerated (1 vessel vol./min) by a high-shear radial flow impeller mounted on a hollow stainless steel shaft, through which pressurised air is pumped. Air is dispersed as fine bubbles into the culture medium by the turbine impeller, without the need for a porous sparger. A stainless steel baffle stabilised by a gliding counterweight increases mixing. The components can easily be disassembled for cleaning and sterilisation. Temperature (range 20-37 degrees C) and pH (range 7.0-7.5) are controlled manually. Using the apparatus, it proved possible to achieve Escherichia coli cell culture densities equivalent to an optical density at 600 nm (OD(600)) of 30-32, compared with OD(600) 4-6 in shake flasks. A yield of 40 mg/litre/day of a recombinant antibody fragment was obtained with the fermenter, which was 15-fold more than the yield of 2.5mg/litre/day achieved in shake flasks. The fermenter may be particularly suited for research purposes.
