MACS Handbook

Human CD8+ cytotoxic T cells

1 Introduction

CD8+ cytotoxic T cells are a subtype of T cells and the main effectors of cell-mediated adaptive immune responses. They kill aberrant cells, such as cancer cells, infected cells (particularly with viruses), or cells that are damaged in other ways.

Through binding to their T cell receptor (TCR), cytotoxic T cells recognize their cognate antigen presented on the surface of a target cell by a class I MHC molecule. For efficient binding of the TCR to the class I MHC molecule, the former must be accompanied by a glycoprotein called CD8. Therefore, these T cells are called CD8+ T cells. Successful recognition of an antigen leads then to the killing of the target cell.

Cytotoxic T cells have two main mechanisms of killing a target cell. First, release of perforin, granzymes, and granulysin permeabilizes the cell membrane, triggers the caspase cascade, and thereby ultimately leads to apoptosis (programmed cell death) of the target cell. A second strategy induces apoptosis through Fas-mediated, direct cell-cell interaction of the cytotoxic T cell and the target cell. Activated cytotoxic T cell express Fas ligand (FasL) on their surface that can bind to the Fas receptor (Fas) on the target cell. This interaction again leads to caspase-induced apoptosis of the target cell.

The cytotoxic ability of this T cell subset is of great interest to scientists in the context of immune therapy.

2 CD8+ cytotoxic T cells in peripheral blood

In human peripheral blood, 15–30% of all CD45+ leukocytes are T cells, with the CD4+ T cells accounting for approximately two thirds of the total T cell population, and CD8+ T cells making up the remaining one third. In isolated PBMCs, T cells are with 45–70% of total cells by far the most abundant cell type. Up to 60% of total PBMCs are CD4+ T cell and up to 30% are CD8+ T cells.

2.1 Cell subsets, frequencies, marker expression

Most T cell subtypes can undergo memory differentiation steps after activation by their respective antigen. Apart from differentiating into effector T cells, some naïve T cells (TNAIVE) may differentiate into various memory T cells subsets, such as stem cell-like memory T cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM) and effector memory RA+ T cells (TEMRA). Each differentiated subset is defined by distinct surface markers. Antigen-inexperienced T cells express naïve marker CD45RA, as well as homing receptors CD62L and CCR7, but lack CD45RO and CD95 expression. With ongoing differentiation towards memory phenotypes, CD45RA, CD62L, and CCR7 are downregulated, while memory marker CD45RO and activation marker CD95 are gradually upregulated. With progressive differentiation towards the memory phenotype, antigen-dependency, tissue tropism, effector function, and senescence increase (PMID: 24258910, 26999211).

Shared features of all memory T cell subtypes is that they are long-lived and can quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thereby mounting a faster and more potent immune response than the first immune response to a given pathogen. The different subtypes exert different functions and exhibit different properties, such as tissue tropism or capacity for self-renewal, reflecting the specific immune-related circumstances that led to their differentiation into a given memory subtype.
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Overview of human T cell differentiation from naïve to memory T cells (PMID: 24258910, 26999211).

Typically, the frequency of naïve T cells specific for a given antigen is very low, ranging between 0.01 and 0.001% of the total T cell count, depending on the respective specificity. When a naïve T cell encounters its cognate antigen and is consequently activated, clonal expansion begins, boosting the frequency of those antigen-specific T cells by several orders of magnitude. This way, they can efficiently fulfill their role as effectors in the immune response (PMID: 22517866, 17707129).

Most clonally expanded antigen-specific T cells die after the termination of the immune response, but a small percentage survive as memory T cells. Memory T cells have a long lifespan and can quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen. At birth, the T cell repertoire is almost exclusively composed of naïve T cells. With progressing age and antigen-experience, memory T cells may become the most abundant T cell population, constituting up to 35% of all circulating T cells (PMID: 24336101).

Notably, laboratory mice carry almost exclusively naïve T cells due to their specific holding conditions and relatively young average age. This, of course, changes dramatically in certain disease-related experimental settings. In humans, the frequency of naïve and memory T cells greatly depends on age, living conditions, and individual history of immune responses.

2.2 Miltenyi Biotec application protocols for CD8+ cytotoxic T cells from peripheral blood

Miltenyi Biotec has created dedicated application protocols for working with and analyzing CD8+ cytotoxic T cells.

2.3 Sample Preparation of peripheral blood

Miltenyi Biotec offers various kits for the direct isolation of pan T cells from peripheral blood and blood products. No specific sample preparation is needed when using those kits. T cells can also be isolated from peripheral blood mononuclear cells (PBMC), which can be generated either by density gradient centrifugation or using the MACSprep™ PBMC Isolation Kit, human. For details, see the MACS Handbook chapter Human  blood.

MACS Handbook:

Blood (human)

2.4 Magnetic separation of CD8+ cytotoxic T cells from peripheral blood and blood products

Miltenyi Biotec has developed numerous products for the straightforward magnetic separation of CD8+ T cells and corresponding subsets. CD8+ T cells can be isolated either straight from whole blood or blood products without density gradient centrifugation and erythrocyte lysis, or from PBMCs.

For details on MACS® Cell Separation Technology, see the MACS handbook chapter Magnetic cell separation

2.4.1 Isolation of CD8+ T cells from whole blood, buffy coat, LRSC, and LeukoPak
At a glance: Kits and reagents for the direct separation of CD8+ T cells from peripheral blood and blood products
Starting materialIsolation strategyCommentsAutomationProduct
Whole BloodDepletion of non-target cellsColumn-free isolation of CD8+ T cells (total capacity: 3´30 mL whole blood).NoMACSxpress CD8 T Cell Isolation Kit, human
Whole BloodPositive selection of target cellsCD8 is predominantly expressed on cytotoxic T cells. Suitable for smaller sample volumes (total capacity: 40 mL whole blood).YesStraightFrom Whole Blood CD8 MicroBeads, human
LRSCPositive selection of target cellsCD8 is predominantly expressed on cytotoxic T cells. Allows direct processing of an entire LRSC (up to 40 mL) without density centrifugation and includes the needed columns. YesStraightFrom LRSC CD8 MicroBead Kit, human
Buffy CoatPositive selection of target cellsCD8 is predominantly expressed on cytotoxic T cells. Allows direct processing of an entire buffy coat (up to 80 mL) without density centrifugation and includes the needed columns.YesStraightFrom Buffy Coat CD8 MicroBead Kit, human
LeukoPakPositive selection of target cellsCD8 is predominantly expressed on cytotoxic T cells. Allows direct processing of ½ a LeukoPak without density centrifugation and includes the needed columns.YesStraightFrom Leukopak CD8 MicroBead Kit, human

The StraightFrom™ MicroBead Kits were developed for the rapid positive selection of target cells directly from whole blood, buffy coat, LRSC, or Leukopak. None of the kits require any sample preparation, like density centrifugation, erythrocyte lysis. or cell count. The appropriate kit is chosen based on the starting material.

With the StraightFrom Buffy Coat CD8 MicroBead Kit, human, CD8+ cells are separated from an entire buffy coat in less than 30 minutes. The Kit can be in combination with a QuadroMACS™ Separator for manual separation, but is best combined with the MultiMACS™ Cell24 Separator Plus for fast and convenient semi-automated separation.

StraightFrom Buffy Coat CD8 MicroBead Kit, human
Analysis gate
CD8+ cells

CD8+ T cells isolated from buffy coat without any sample preparation. A human buffy coat sample was processed using the StraightFrom Buffy Coat CD8 MicroBead Kit and the MultiMACS Cell24 Separator Plus with the Single-Column Adapter and Whole Blood Columns. Cells were fluorescently stained with CD3-PE, CD8‑APC, as well as CD45‑VioBlue® and analyzed by flow cytometry on the MACSQuant® Analyzer. Cells were triggered via CD45-VioBlue, cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

Alternatively, the MACSxpress® Isolation Kits allow the column-free isolation of target cells from freshly drawn anti-coagulated whole blood via the depletion of non-target cells. MACSxpress Kits are ideal for the processing of larger sample volumes (total capacity 3´30 mL).

The MACSxpress CD8 T Cell Isolation Kit, human,was developed for the fast and easy isolation of highly pure CD8+ T cells directly from whole blood.Non-target cells are removed by immunomagnetic depletion using MACSxpress Beads. Simultaneously, erythrocytes are sedimented, yielding target cells of high purity.
MACSxpress CD8 T Cell Isolation Kit, human
Unseparated fraction
Unseparated fraction
After separation
After separation

Untouched CD8+ T cells isolated from whole blood. EDTA-anticoagulated blood samples were processed using the MACSxpress CD8 T Cell Isolation Kit, a MACSmix™ Tube Rotator, and a MACSxpress Separator. The isolated cells were fluorescently stained with CD45- VioBlue, CD3-APC, CD8-FITC, and CD56-PE, and then analyzed by flow cytometry on the MACSQuant Analyzer. Cell debris, non-leukocytes, and dead cells were excluded from the analysis based on CD45 expression, scatter signals, and propidium iodide fluorescence.

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2.4.2 Isolation of CD8+ T cells from PBMCs
At a glance: Kits and reagents for the separation of CD8+ T cells and subsets from PBMCs
Starting materialIsolation strategyCommentsAutomation possible Product
Pan CD8+ T cells
PBMCPositive selection of target cellsCD8 is predominantly expressed on cytotoxic T cells. Suitable for the depletion or enrichment of CD8+ cells form a PBMC sample.YesCD8 MicroBeads, human
PBMCDepletion of non-target cellsIsolation of CD8+ T cells via depletion of CD4+ T cells, monocytes, neutrophils, eosinophils, B cells, dendritic cells, NK cells, granulocytes, γ/δT cells, and erythroid cellsYesCD8+ T Cell Isolation Kit, human
PBMCDirect isolation of target cells and label removalCD8 is predominantly expressed on cytotoxic T cells. MicroBead and antibody labeling fully removable.NoREAlease CD8 MicroBead Kit, human
CD8+ T cell subsets
PBMCDepletion of non-target cells followed by positive selection of target cellsDepletion of CD45RO+, CD56+, and CCR7+ cells, followed by isolation of all CD8+ T cells. Isolated cell are CD8+ CD45RA+.YesCD8+ CD45RA+ Effector T Cell Isolation Kit, human
PBMCDepletion of non-target cells followed by positive selection of target cellsDepletion of CD45RO+, CD56+, CD57+, and CD244+ cells followed by isolation of all CD8+ T cells.YesNaive CD8+ T Cell Isolation Kit, human
PBMCTwo-step positive selection of target cellsIsolation of CD8+ cells followed by isolation of CD57+ cells. CD57 is expressed by late-stage CD8+ effector cells.YesCD8+ CD57+ T Cell Isolation Kit, human
PBMCDepletion of non-target cellsSerial depletion of non-target cells.YesCD8+ Memory T Cell Isolation Kit, human

Instead of working directly with whole blood or blood products, samples can be processed over a density gradient centrifugation to pre-enrich peripheral blood mononuclear cells (PBMCs) as starting material for subsequent cell isolation. 

CD8 MicroBeads, human, enable positive selection or depletion of CD8+ cells from PBMC samples by direct magnetic labeling.
CD8 MicroBeads, human
PBMCs before separation
CD8- cells
CD8+ cells

Example of a separation using CD8 MicroBeads. CD8+cells were isolated from human PBMCs using CD8 MicroBeads, a LS Column, and a MidiMACS™ Separator.

The CD8+ T Cell Isolation Kit, human, enables fast isolation of untouched CD8+ cytotoxic T cells from PBMCs in only 18 minutes. This updated kit offers even better performance and a significantly shorter protocol, replacing its well-known predecessor.
CD8+ T Cell Isolation Kit, human
Unseparated fraction
Isolated CD8+ T cells

Untouched CD8+ T cells isolated from human PBMCs. Samples were processed using the CD8+ T Cell Isolation Kit, an LS Column, and a MidiMACS Separator. Cells were fluorescently stained with CD8-FITC, CD56-PE, and CD3-APC to visualize the target cell fraction. Isolated CD8+ T cells were CD3+ and CD56. Cells were analyzed by flow cytometry using the MACSQuant Analyzer.

The REAlease® CD8 MicroBead Kit, human, offers a rapid solution for the positive selection of CD8+ T cells from PBMCs with the option to fully remove the MicroBead and antibody labeling. Cells are thus completely label-free and ready for any downstream application, including a second round of magnetic labeling and separation for further subset isolation.
REAlease® CD8 MicroBead Kit, human

A) Cell purity

Before separation
Label-free CD8+ cells

B) Label-free cells: REAlease Biotin Complex release

Before separation
MicroBead-free CD8+ cells
Label-free CD8+ cells

Label-free, highly pure CD8+ T cells. (A) CD8+ T cells were isolated from human PBMCs using the REAlease CD8 MicroBead Kit, MS Columns, and a MiniMACS™ Separator. Cells were fluorescently stained with CD8-PE and analyzed by flow cytometry on the MACSQuant Analyzer X. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence. (B) The efficient removal of all labels is shown using Anti-Biotin-APC to detect the presence of REAlease Biotin Complex by flow cytometry. Directly after isolation, the cells showed staining of biotin ("MicroBead-free CD8+ cells"), whereas the label-free CD8+ cells after the REAlease Biotin Complex release were negative for biotin, similar to the non-labeled cells before separation.

2.5 Characterization of CD8+ cytotoxic T cells by flow cytometry

CD8+ T cell subsets and their differentiation status can be determined by flow cytometry based on the expression of cell surface markers, transcription factors, and their secretion of cytokines. Miltenyi Biotec offers a vast portfolio of conventional and recombinant REAfinity™ antibodies for comprehensive analysis.
2.5.1 Flow cytometry panels

The following antibody combinations for surface marker, intracellular cytokine, and transcription factor staining can be used to identify CD8+ T cell subsets by flow cytometry.


At a glance: Markers for the analysis of CD8+ T cell subsets using MACS antibodies
CD8+ cytotoxic T cellsT cell development – naïve vs. memory (TSCM/TCM/TTM/TEM/TEMRA)Activated T cellsExhausted T cells
CD3CD3CD3CD3
CD8CD4CD4CD4
CD107a (LAMP-1)CD8CD8CD8
CD178 (FasL)CD27CD25 (IL2RA)CD96 (TACTILE)
Granzyme BCD28CD27CD152 (CTLA-4)
IFN-γCD45RACD28CD160 (NK1)
PerforinCD45ROCD69CD223 (LAG-3)
CD253 (TRAIL)CD57CD95 (FasR)CD244 (2B4)
TNF-αCD62L (L-Selectin)CD134 (OX40)CD272 (BTLA)
CD69CD137 (4-1BB)CD278 (ICOS)
CD95 (FasR)CD154 (CD40L)CD279 (PD1)
CD127CD154 (CD40L)CD366 (TIM-3)
CD197 (CCR7)Ki-67TIGIT
KLRG1VISTA
EOMES
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2.5.2 Analysis of surface markers and cytokines

Miltenyi Biotec offers a range of solutions for the analysis of T cell-associated surface markers and cytokines: 

  • MACS Antibodies are the ideal solution for the staining of T cell surface markers or the intracellular staining of cytokines.
  • MACS Cytokine Secretion Assays allow detection and enrichment of viable cytokine-secreting cells. The human kits are available for a large number of cytokines, including TNF-α, GMCSF, IFN-α, IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17 and IL-22, and can be used for further characterization of T cell subsets.
  • The human MACSPlex 12 Cytokine Kits are used for multiplex analysis of secreted cytokines in serum and cell culture supernatants using a standard flow cytometer. Cytokines that can be analyzed in a single sample include: GM-CSF, IFN-α, IFN-γ, lL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-17A, and TNF-α.
  • The Rapid Cytokine Inspectors allow for rapid analysis of activated T cells by combining surface marker analysis (e.g., CD4, CD8, CD154) with intracellular cytokine staining (e.g. IFN-γ, TNF-α, IL-2 and IL-17).
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Principle of the Cytokine Secretion Assay – Cell Enrichment and Detection Kits.

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Principle of MACSPlex Assays. Samples containing unknown levels of analytes are incubated with the antibody-coated MACSPlex (MPx) Capture Beads, and analytes bind to the specific antibody. A Detection Reagent, composed of a cocktail of APC-conjugated antibodies specific for the analytes, is added. Consequently, sandwich complexes are formed between the MPx Capture Bead, the analyte and the Detection Reagent. These complexes can be analyzed based on the fluorescence characteristics of both the MPx Capture Bead and the Detection Reagent. Standards of known quantities of given analytes are provided with the kit and are used for the quantification of the analytes within the unknown samples.

2.6 Cell culture of CD8+ cytotoxic T cells

The Miltenyi Biotec cell culture and stimulation portfolio offers a specialized and versatile range of culture media and reagents for the stimulation, activation/expansion, and differentiation of T cells.
MACS Handbook:

Cell culture

2.6.1 Cultivation, activation, and expansion of T cells
At a glance: Kits and reagents for the cultivation, activation, and expansion of T cells
UseCommentsProduct
Culture mediumOptimized T cell media without serum or animal-derived components. Also available in MACS GMP grade and with and without phenol red.TexMACS Medium
SupplementConsistent, high-quality recombinant cytokines for successful cell culture. Available in premium, research and MACS GMP grades.MACS Cytokines
StimulationNanomatrix-based activation of T cells via CD3/CD28 engagement.Available in research and MACS GMP gradesT Cell TransAct
StimulationCell-sized activation beads (‘artificial APCs’) loaded with activating CD2, CD3 and CD28 antibodies.T Cell Activation/Expansion Kit, human
StimulationNon-toxic alternative to Staphylococcal enterotoxin B (SEB). Functions as a superantigen.CytoStim
StimulationExtensive panels of tumor-, virus-, fungi- and microbiota-specific antigens for the stimulation of antigen-specific CD4+ and CD8+ T cells. Available in premium, research and MACS GMP grade as well as in 96-well cell culture plate format.PepTivator Peptide Pools
StimulationIn vitro T cell activation and expansion.

CD3 pure – functional grade, human

CD28 pure – functional grade, human

TexMACS™ Medium is a serum-free cell culture medium developed specifically for T cells. It has been used in a variety of applications and, in combination with MACS cytokines, is an ideal starting point for reliable cultivation conditions. The medium is also available in MACS GMP grade, and with or without phenol red.

For detailed information about Miltenyi Biotech media optimized for T cells, see chapter Cell culture media.
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Comparison of expansion rates of human T cells in different media. Cells were expanded in TexMACS Medium, a competitor product, and serum containing basal medium (RPMI + 10% FBS) using the T cell Activation/Expansion Kit, human.

T cell activation is essential for a variety of downstream application. Miltenyi Biotec offers polyclonal stimulation reagents that have been carefully designed to ensure optimal stimulation conditions.

T Cell TransAct™ is a ready-to-use reagent that is applied volumetrically, eliminating the need for bead-to-cell ratio calculations. Excess reagent is simply removed via culture wash. T Cell TransAct is available in both research and MACS GMP grades for a seamless transfer of workflows into clinical settings.
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Activation of human purified T cells. T cells isolated using the Pan T Cell Isolation Kit were activated for 48 hours using  T Cell TransAct™  in TexMACS Medium supplemented with Human IL-2.  Cells were fluorescently stained and analyzed by flow cytometry using the MACSQuant Analyzer.

The T Cell Activation/Expansion Kit, human, employs large cell-sized particles loaded with biotinylated antibodies of choice to activate and expand primary cells. The large cell-sized particles mimic antigen-presenting cells and, when loaded with CD2, CD3 and CD28 and applied in a specific bead-to-cell ratio, lead to efficient T cell activation. Use the decision tree to determine the right T cell activation product for your project.
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Decision tree: T Cell Activation and Expansion Kit vs. T Cell TransAct.

CytoStim® is an antibody-based reagent that rapidly stimulates T cells. It can be used as a non-toxic alternative to SEB, e.g., for the positive control of antigen-specific T cell stimulation assays or intracellular cytokine staining experiments to detect cytokine or activation marker expression.
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A comparison of different T cell stimulation reagents. Freshly isolated human PBMCs were stimulated for 4 h using either CytoStim, SEB, or PMA/Ionomycin. Cells were subsequently stained with CD4, CD154, and Anti-IFN-γ antibodies and analyzed for CD154 and IFN-γ expression after pre-gating for CD4.

PepTivator® Peptide Pools enable the antigen-specific stimulation of both CD4+ and CD8+ T cells with an extensive panel of tumor-, virus-, fungi- and microbiota-specific antigens. Consisting of 15-mer peptides with 11-amino-acid overlaps, PepTivator Peptide Pools cover the complete sequence of the respective antigen. Available in research-, premium- and MACS GMP-grade. The most popular PepTivator Peptide Pools are also available in a 96-well cell culture plate format for high-throughput cell activation.

MACS Cytokines  are available in three different grades – research, premium, and MACS GMP grade – to provide best flexibility in any assay setup. Notably, premium-grade MACS Cytokines exhibit well-defined biological activities, normalized to international reference standards (IU/mg), that allow exact unit dosing for reproducible results without laborious pre-testing.

Finally, the CD3 and CD28 pure – functional grade antibodies, human are suitable for in vitro T cell activation and expansion. The CD3 (OKT3) andCD28 (15E8) antibodies recognize the respective human receptors. Upon receptor binding, a stimulatory signal is transferred that, in combination with additional cytokines (e.g., IL-2 or IL-7/IL-15), leads to the activation and expansion of T cells.
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3 CD8+ cytotoxic T cells from other tissues

T cells can also be obtained from tissues other than peripheral blood. Miltenyi Biotec offers a variety of solutions for the preparation of human tissues, as well as the subsequent separation, cultivation, and analysis of the respective T cell populations.

3.1 Miltenyi Biotec application protocols for T cells from other tissues

3.2 Sample preparation of different tissues

Tissues must be dissociated into a single-cell suspension for many downstream applications, including isolation of cell subpopulations, cell culture, or flow cytometry analysis. Combining mechanical dissociation and enzymatic treatment by using the gentleMACS™ Dissociator with Heaters and specific Tissue Dissociation Kits enables reproducible T cell isolation. T cells can be obtained with excellent yield, high viability rate, and preserved cell epitopes even from hard-to-process tissues, including tumor, skin, and brain tissue, among others. See Human cells and organs to learn more about the sample preparation of different tissue types.