Complete workflow for the stimulation, enrichment, and analysis of antigen-specific T cells

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.

Workflow for antigen-specific T cell research

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.

Webinar
Working with antigen-specific T cells

Covered in this webinar are Miltenyi Biotec’s unique solutions for the stimulation, pre-enrichment, isolation, and analysis of these rare cells.

Comparison of PBMC populations isolated with the MACSprep PBMC Isolation Kit or by density gradient centrifugation.

Untouched PBMC isolation

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

  • Untouched isolation of PBMCs from 1–8 mL of human whole blood.
  • No density centrifugation, no erythrocyte lysis.
  • Fast processing in only 25 minutes.
  • Superior reproducibility, recovery, and cell viability.
  • Parallelization of multiple samples (MultiMACS™ Cell24 Separator Plus).
  • Isolated PBMCs are suitable for various downstream applications.
Immune cell composition before and after enrichment using StraightFrom Technology.

Isolation of immune cells directly from whole blood and blood products 

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.

  • Fast isolation of immune cells directly from whole blood and blood products.
  • No density gradient centrifugation, no erythrocyte lysis.
  • Preservation of cell functionality for full downstream compatibility.
  • Automated  isolation of multiple samples with MultiMACS Cell24 Separator Plus.

Scientific poster
Isolation of functional cells straight from buffy coat, LRSC, and Leukopak®

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.

Production of IFN-γ by human CD4+ or CD8+ T cells after stimulation with different antigens using PepTivator Peptide Pools.

Antigen-specific stimulation with PepTivator Peptide Pools 

  • Efficient antigen-specific stimulation of both CD4+ and CD8+ T cells with our extensive panel of antigens.
  • Pool of 15-mer peptides with 11-amino-acid overlap that cover the complete sequence of the respective antigen.
  • Available in 96-well format for high-throughput T cell activation.
  • MACS® GMP-Grade PepTivator Peptide Pools for clinical applications.
  • Constantly growing portfolio.

Broschure
PepTivator Peptide Pools – Standardize your T cell assays

Comparison of T cell activation using CytoStim, human, SEB, and PMA/lonomycin.

Polyclonal stimulation as positive control 

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.

  • Rapid and efficient restimulation of human effector/memory T cells.
  • Non-toxic alternative to the superantigen Staphylococcal enterotoxin B (SEB).

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.

Magnetic enrichment of CD154+ cells allows for a sensitive characterization of rare BKV-reactive CD4+ T cells. 

Isolation of antigen-activated CD4+ T cells with the CD154 MicroBead Kit, human 

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.

Isolation of antigen-activated CD8+ T cells with the CD137 MicroBead 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.

Rapid ARTE – Enrichment of rare antigen-specific CD154+CD4+ T cells for sensitive flow analysis

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.

  • Fast protocol with only 1-hour hands-on time.
  • Unique combination of cell enrichment via CD154 marker and multiparameter flow analysis.
  •  Direct ex vivo detection and high-resolution characterization of rare antigen-specific T cells.

Scientific poster
The rapid antigen-reactive T cell enrichment procedure enables direct ex vivo BKV-specific CD4+ T cell characterization

Michaela Niemöller, Mario Assenmacher, and Anne Richter

Principle of the MACS Cytokine Secretion Assays

Isolation of viable cytokine-secreting T cells with Cytokine Secretion Assay – Cell Enrichment and Detection Kits

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.

  • Unique technology for the isolation of viable cytokine-secreting cells.
  • Accurate analysis of cytokine secretion at single-cell level.
  • Sensitive detection of one T cell per million cells.
  • Available for human and mouse cells secreting TNF-α, GM-CSF, IFN-α, IFN-γ, or IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, IL-22.
  • Isolated cells are suitable for various downstream applications (e.g. cell expansion, TCR epitope mapping, genomic analysis).
     
Sterile Sorting of antigen-specific T cells to high purity using the MACSQuant Tyto Cell Sorter. 

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.

  • Gentle and fast sorting process that does not affect cell viability.
  • Fully closed and sterile cartridge system providing maximal safety to cells and operator.
  • Sort and even re-sort cells for the enrichment of ultra-rare cell populations.
  • Enrich antigen-specific T cells from <0.05% to >90% purity in a single run.

Video
MACSQuant Tyto: Cell sorting as easy as it can be 

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.

Application note
Isolation of CD8+ antigen-specific T cells

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.

  • REAfinityTM Recombinant Antibodies are recombinantly generated antibodies that provide superior lot-to-lot consistency and eliminate background signal.
  • Cytokine Secretion Assay – Detection Kits allow for flow analysis of cytokine secretion at a detection level of one T cell in a million.
  • MACSplex Cytokine Kits enable multiplexed analysis of secreted cytokines in cell culture supernatant.
  • Rapid Cytokine Inspector combines surface marker and intra-cellular cytokine staining for multiparameter analysis of activated human T cells.
  • MACSQuant® Analyzers are fully automated benchtop flow cytometers for highly sensitive, multiparameter flow analyses.
  • Build your own T cell flow panel with our Flow Panel Builder.

Optimized multicolor flow cytometry panels

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:

Application note
Detection, enrichment, and direct phenotypic and functional characterization of antigen-specific naive and memory T cell subsets

Petra Bacher and Alexander Scheffold

Department of Cellular Immunology,  Clinic for Rheumatology and Clinical Immunology,  Charité – University Medicine Berlin, Berlin, Germany

Get acquainted with our protocols and antibody panels for your research in antigen-specific T cells covering workflow steps from cell preparation to flow cytometric analysis. 

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