Cell Separation using Pro5® Pentamers and Magnetic Beads
In addition to detecting antigen-specific T cells, Pro5® MHC Class I Pentamers can be used in conjunction with magnetic beads to enrich for the T cell population of interest. Magnetic bead sorting is a simple solution for applications requiring enrichment of antigen-specific T cells; for example, therapeutic applications, generation of T cell lines, PCR cloning, gene profiling, and cell culture experiments. Alternatively, if magnetic bead sorting is used as a pre-enrichment step before Fluorescence-Activated Cell Sorting (FACS) it will reduce both the number of cells that will have to be sorted and the required sorting time.
There are currently two main methods for magnetic bead separation. Both technologies employ mixing cells with paramagnetic beads (these only exhibit magnetic properties when placed within a magnetic field), which may be purchased with a specific affinity, or can be coated with an antibody of your choice. In the first, tube-based method, target-bead complexes are removed from the cell suspension using an external magnet that draws the complexes to the inner edge of the tube, allowing supernatant to be removed. Removing the tube from the magnetic field enables resuspension of the target-bead complexes. Separation is gentle and does not require centrifugation or columns. In the alternative, column-based method, target-bead complexes pass through a separation column, which is placed in a strong, permanent magnet. The column matrix serves to create a high-gradient magnetic field that retains bead-bound complexes while non-labeled cells flow through. Following removal of the column from the magnetic field, the retained cells may be eluted.
A Comparison of Methods
The figure below shows an experiment in which the column-based method of magnetic separation was used to enrich antigen-specific T cells from peripheral blood cells (PBMCs). 1x107 PBMCs were incubated with 5 tests (50μl) R-PE labeled A*02:01/EBV Pentamer (GLCTLVAML) for 10 minutes at room temperature (22ºC), followed by 20μl anti-R-PE microbeads (Miltenyi Biotec) for 15 minutes at 4°C. Cells were passed over an ‘MS’ column using the MidiMACS system and the flow-through fraction discarded. The column was then removed from the magnetic field and the retained target cells flushed out as positively selected cells (positive fraction 1). These cells were then passed over a second ‘MS’ column in order to enrich the antigen-specific T cells of interest further (positive fraction 2). Resultant cells were then incubated with anti-CD8 antibody for 20 minutes on ice prior to fixation and analysis by flow cytometry. Note, due to the small size of the Miltenyi microbeads, cells may be analyzed by flow cytometry with beads still attached.
The figure below shows the results of an experiment in which antigen-specific cells were depleted from a peripheral blood suspension using a biotin-labeled Pro5® Pentamer and streptavidin microbeads (LodeStars™ 2.7 Streptavidin; Polymer Laboratories) exactly as detailed in the protocol linked above. A sample of the original cell population (pre-depletion) and supernatant following isolation (post-depletion) were incubated with anti-CD8-FITC antibody plus streptavidin-PE to visualize antigen-specific cells. The antigen-specific population was reduced from 1.53% to 0.04%, confirming that bead isolation was successful (97.4% of antigen-specific cells removed).
Case Study: Pro5® MHC Class I Pentamers used to separate HBV-specific T cells for subsequent phenotypic analysis
Using virus-specific CD8+ T cell clones and primary human hepatocytes, Gehring et al. analyzed the modulation of CD8+ T cell function following recognition of peptide pulsed or virally infected hepatocytes. The T cell clones were generated from PBMC from HLA-A2+ HBV infected donors by labeling the cells with R-PE-conjugated HBc18-27-specific Pro5® Pentamer and purifying via magnetic cell sorting using anti-R-PE microbeads. Separated cells were cloned by limiting dilution assay and clones were expanded using allogeneic irradiated PBMC feeder cells.
Figure: The production of CD107a and TNF-alpha from the HBc18-27-specific T cells (generated through magnetic cell sorting with an HBc18-27-specific Pentamer) was measured after 5 hours of incubation with EBV B cells or HEPG2 cells pulsed with the peptide concentrations shown.
It was observed that limiting the amount of viral antigen in infected human hepatocytes preferentially stimulates CD8+ T cell degranulation, which may lead to hepatocyte damage that is directly caused by virus specific T cells in chronic hepatitis patients.
Magnetic bead sorting with R-PE- or biotin-labeled Pro5® Pentamers is a simple solution for applications requiring enrichment or depletion of antigen-specific T cells. If used as a pre-enrichment step before flow cytometry sorting, it will reduce both the number of cells that will have to be sorted and the required sorting time. Biotin-labeled Pentamers can be used in conjunction with streptavidin coated paramagnetic beads and are compatible with any bead system. Isolating antigen-specific T cells in this manner is useful if viable cells need to be obtained for further culture or analysis, such as expression profiling.
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