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.
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.
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.
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.
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.
|Starting material||Isolation strategy||Comments||Automation||Product|
|Whole Blood||Depletion of non-target cells||Column-free isolation of CD8+ T cells (total capacity: 3´30 mL whole blood).||No||MACSxpress CD8 T Cell Isolation Kit, human|
|Whole Blood||Positive selection of target cells||CD8 is predominantly expressed on cytotoxic T cells. Suitable for smaller sample volumes (total capacity: 40 mL whole blood).||Yes||StraightFrom Whole Blood CD8 MicroBeads, human|
|LRSC||Positive selection of target cells||CD8 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.||Yes||StraightFrom LRSC CD8 MicroBead Kit, human|
|Buffy Coat||Positive selection of target cells||CD8 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.||Yes||StraightFrom Buffy Coat CD8 MicroBead Kit, human|
|LeukoPak||Positive selection of target cells||CD8 is predominantly expressed on cytotoxic T cells. Allows direct processing of ½ a LeukoPak without density centrifugation and includes the needed columns.||Yes||StraightFrom 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.
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.
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.
|Starting material||Isolation strategy||Comments||Automation possible||Product|
|Pan CD8+ T cells|
|PBMC||Positive selection of target cells||CD8 is predominantly expressed on cytotoxic T cells. Suitable for the depletion or enrichment of CD8+ cells form a PBMC sample.||Yes||CD8 MicroBeads, human|
|PBMC||Depletion of non-target cells||Isolation 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 cells||Yes||CD8+ T Cell Isolation Kit, human|
|PBMC||Direct isolation of target cells and label removal||CD8 is predominantly expressed on cytotoxic T cells. MicroBead and antibody labeling fully removable.||No||REAlease CD8 MicroBead Kit, human|
|CD8+ T cell subsets|
|PBMC||Depletion of non-target cells followed by positive selection of target cells||Depletion of CD45RO+, CD56+, and CCR7+ cells, followed by isolation of all CD8+ T cells. Isolated cell are CD8+ CD45RA+.||Yes||CD8+ CD45RA+ Effector T Cell Isolation Kit, human|
|PBMC||Depletion of non-target cells followed by positive selection of target cells||Depletion of CD45RO+, CD56+, CD57+, and CD244+ cells followed by isolation of all CD8+ T cells.||Yes||Naive CD8+ T Cell Isolation Kit, human|
|PBMC||Two-step positive selection of target cells||Isolation of CD8+ cells followed by isolation of CD57+ cells. CD57 is expressed by late-stage CD8+ effector cells.||Yes||CD8+ CD57+ T Cell Isolation Kit, human|
|PBMC||Depletion of non-target cells||Serial depletion of non-target cells.||Yes||CD8+ 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.
Example of a separation using CD8 MicroBeads. CD8+cells were isolated from human PBMCs using CD8 MicroBeads, a LS Column, and a MidiMACS™ Separator.
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.
A) Cell purity
B) Label-free cells: REAlease Biotin Complex release
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.
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.
|CD8+ cytotoxic T cells||T cell development – naïve vs. memory (TSCM/TCM/TTM/TEM/TEMRA)||Activated T cells||Exhausted T cells|
|CD178 (FasL)||CD27||CD25 (IL2RA)||CD96 (TACTILE)|
|Granzyme B||CD28||CD27||CD152 (CTLA-4)|
|CD253 (TRAIL)||CD57||CD95 (FasR)||CD244 (2B4)|
|TNF-α||CD62L (L-Selectin)||CD134 (OX40)||CD272 (BTLA)|
|CD69||CD137 (4-1BB)||CD278 (ICOS)|
|CD95 (FasR)||CD154 (CD40L)||CD279 (PD1)|
|CD127||CD154 (CD40L)||CD366 (TIM-3)|
Miltenyi Biotec offers a range of solutions for the analysis of T cell-associated surface markers and cytokines:
|Culture medium||Optimized T cell media without serum or animal-derived components. Also available in MACS GMP grade and with and without phenol red.||TexMACS Medium|
|Supplement||Consistent, high-quality recombinant cytokines for successful cell culture. Available in premium, research and MACS GMP grades.||MACS Cytokines|
|Stimulation||Nanomatrix-based activation of T cells via CD3/CD28 engagement.Available in research and MACS GMP grades||T Cell TransAct|
|Stimulation||Cell-sized activation beads (‘artificial APCs’) loaded with activating CD2, CD3 and CD28 antibodies.||T Cell Activation/Expansion Kit, human|
|Stimulation||Non-toxic alternative to Staphylococcal enterotoxin B (SEB). Functions as a superantigen.||CytoStim|
|Stimulation||Extensive 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|
|Stimulation||In vitro T cell activation and expansion.||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.
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.
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.
Miltenyi Biotec has created dedicated application protocols to enrich T cells from various human tissues: