This research workflow for antigen-specific T cells enables potent stimulation using specific PepTivator® Peptide Pools. Besides that, it enables the efficient enrichment of antigen-activated T cells and a reliable multiparameter flow analysis of antigen-specific T cells.
Antigen-specific T cells play a central role in the adaptive immune response in various pathologies. In cancer, tumor-specific T cells are involved in effective anti-tumor immunity, whereas in infectious diseases, pathogen-specific T cells coordinate specific defense mechanisms. Apart from pathological contexts, antigen-specific T cells are also crucial for the formation of immunological memory (e.g. after vaccination) or for the maintenance of tolerance to self-antigens. However, due to low target cell frequencies, efficient enrichment and sensitive detection methods are of great interest. Miltenyi Biotec has developed a complete workflow to ensure successful analysis of rare antigen-specific T cells.
Discover our different workflows for antigen-specific T cells and find the one that fits your experimental needs.
Covered in this webinar are Miltenyi Biotec’s unique solutions for the stimulation, pre-enrichment, isolation, and analysis of these rare cells.
To analyze antigen-specific T cells, human peripheral blood mononuclear cells (PBMCs) are traditionally used as a source of effector cells due to their cellular composition and their convenient accessibility. The MACSprep™ PBMC Isolation Kit, human has been developed for the fast and untouched isolation of human PBMCs without density centrifugation
Using StraightFrom® Technology, T cells and antigen-presenting cells (e.g. monocytes) can be quickly enriched directly from whole blood and blood products (buffy coat, LRSC, and Leukopak®). Isolated T cells and antigen-presenting cells can be used for antigen-presentation assays (e.g. mixed lymphocyte reaction, MLR) to assess antigen-specific restimulation T cells.
Maike Schlemminger, Silke Weber-Lohmann, Martin Kolnik, Zhong Yu, Susanne Höher-Peters, and Gregor Winkels
Antigen-specific in vitro stimulation of T cells is commonly performed to characterize antigen-specific T cell responses. Our PepTivator® Peptide Pools enable efficient antigen-specific stimulation of both CD4+ and CD8+ T cells.
Every stimulation experiment requires a robust positive control. For the stimulation of antigen-specific T cells we recommend CytoStim™, human for polyclonal activation of human T cells. CytoStim Reagent is an antibody-based reagent that causes T cell activation by cross-linking the TCR with an MHC molecule of an antigen-presenting cell.
Reliable analysis of viable antigen-specific T cells is challenging and often hampered by critically low cell frequencies. With our MicroBeads and cell enrichment kits, you can isolate and analyze viable antigen-specific T cells based on specific activation markers or cytokine expression.
CD154, also known as CD40L, gp39, T-BAM, TRAP, or Ly-62 is transiently up-regulated on activated CD4+ T cells and plays an important role as a co-stimulatory molecule in T cell/antigen-presenting cell (APC) interactions through ligation of CD40. Due to its transient expression within hours after activation, CD154 is an ideal a marker for the isolation of activated antigen-specific CD4+ T cells. Optionally, pre-enrichment can be performed using the CD4+ T Cell Isolation Kit, human or the Pan T Cell Isolation Kit, human.
CD137 is mainly expressed on activated CD4+ and CD8+ T cells, activated B cells, and on natural killer cells, but can also be found on resting monocytes and dendritic cells. CD137 has been described to be a suitable marker for antigen-specific activation of human CD8+ T cells, as CD137 is not expressed on resting CD8+ T cells and its expression is reliably induced after 24 hours of stimulation. For the isolation of activated antigen-specific CD8+ T cells, the CD137 MicroBead Kit can be combined with the CD8+ T Cell Isolation Kit, human.
By combining magnetic cell enrichment and multiparameter flow cytometry analysis of CD154+CD4+ T cells, Rapid ARTE allows the processing of larger cell numbers and the direct ex vivo detection and characterization of rare antigen-specific T cells. The complete Rapid ARTE protocol can be found in our collection of step-by-step application protocols.
Michaela Niemöller, Mario Assenmacher, and Anne Richter
Following in vitro stimulation, T cells start to secrete specific cytokines. MACS Cytokine Secretion Assays are unique tools for the enrichment and detection of a wide range of cytokines secreted by viable cells, even directly from whole blood or PBMCs.
The MACSQuant Tyto Cell Sorter revolutionizes classical cell sorting. With its small footprint, the gentle sorting approach, and disposable cartridges, the MACSQuant Tyto is ideally suited for isolating even the rarest antigen-specific T cells in research and clinical settings. Additionally, pre-enrichment of rare cell populations can be achieved by performing two consecutive sorts on the same population.
Find out more about the easy cell sorting procedure on the MACSQuant Tyto: It's simple, it's gentle, it's sterile. This video will give you an overview of the straightforward operator steps from sample loading to retrieval.
Find out more about the MACSQuant Tyto’s ability to isolate rare antigen-specific cytotoxic T cells to a purity above 90%.
As the final step in many workflows, flow analysis plays an important part in various T cell applications. We provide reagents, optimized and validated flow panels, and detailed application notes that help you make sure your flow analysis and phenotypic characterization of rare antigen-specific T cells are reliable and consistent.
Phenotypic characterization of antigen-specific T cells by flow cytometry can be performed using optimized panels of fluorochrome-conjugated REAfinity Recombinant Antibodies. Our Miltenyi Biotec tested panels (MBTPs) for flow cytometry applications are developed and validated by Miltenyi Biotec’s R&D experts, as well as by customers who use our antibodies.
Relevant flow panels for antigen-specific T cells:
Petra Bacher and Alexander Scheffold
Department of Cellular Immunology, Clinic for Rheumatology and Clinical Immunology, Charité – University Medicine Berlin, Berlin, Germany
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