- 1 Introduction
- 2 CD8⁺ cytotoxic T cells in spleen and lymphoid tissue
- 3 T cells from non-lymphoid tissues
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 then leads to 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 cells 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 capacity of this T cell subset is of great interest to scientists in the context of immunotherapy.
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A typical mouse spleen weighs between 80 and 120 mg and contains up to 1×108 splenocytes. T cells constitute roughly 25% of all splenocytes, 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. Cell numbers in lymph nodes vary, but up to 80% of the total lymph node cell population are T cells, again with a CD4+:CD8+ T cell ratio of 2:1. Cells in the thymus of a young mouse, however, may account for more than 90% T cells, most of them in a double-positive stage, expressing both CD4 and CD8.
Most T cell subtypes can undergo several differentiation steps after activation by their respective antigen. Apart from differentiating into effector T cells, some naive T cells (TNAIVE) may differentiate into various memory T cell subsets, including stem cell–like memory T cells (TSCM), central memory T cells (TCM), and effector memory T cells (TEM). Each subset is defined by distinct surface markers. Antigen-inexperienced T cells express homing receptors CD62L and CCR7, but lack expression of activation markers CD44 and CD95. With ongoing differentiation towards the memory phenotypes, CD62L and CCR7 are down-regulated, while CD44 and CD95 are gradually up-regulated. With progressive differentiation towards the memory phenotype, antigen-dependence, tissue tropism, effector function, and senescence increase (PMID: 24258910, 26999211)
Shared features of all memory T cell subtypes are 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 naive 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 naive 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 naive 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 naive 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 naive and memory T cells greatly depends on age, living conditions, and individual history of immune responses.
The main source for mouse CD8+ T cells are single-cell suspensions from spleen and lymph nodes. The tissue must be dissociated into a single-cell suspension for many downstream applications, including isolation of cell subpopulations, cell culture, or flow cytometry analysis.
Dissociation can be accomplished fully automatically using the gentleMACS™ Dissociator and specific Tissue Dissociation Kits (e.g. Spleen Dissociation Kit, mouse). A special protocol for the preparation of single-cell suspensions from mouse spleen without enzymatic treatment can be downloaded from the Related resources panel to the right. Alternatively, tissues can be dissociated using a manual procedure. For details, see chapter Mouse cell sources.
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Miltenyi Biotec has developed numerous products for the efficient magnetic separation of T cells from single-cell suspensions generated from mouse spleen and lymph nodes. For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic cell separation .
MACS Handbook:
Magnetic cell separation
At a glance: Kits and reagents for the separation of CD8⁺ T cells from spleen and lymph nodes
| Starting material | Isolation strategy | Comments | Automation | Product |
|---|---|---|---|---|
| Single-cell suspensions from spleen and lymph nodes | Positive selection of target cells | The marker CD8 is predominantly expressed on cytotoxic T cells. Suitable for the depletion or enrichment of CD8+ cells. | Yes* | CD8a (Ly-2) MicroBeads, mouse |
| Single-cell suspensions from spleen and lymph nodes | Depletion of non-target cells | Isolation of CD8+ T cells by depletion of all non-target cells using antibodies against CD4, CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, Anti-MHC-class II, Ter-119, and TCRγ/δ. | Yes* | CD8a+ T Cell Isolation Kit, mouse |
| *Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X. | ||||
CD8a (Ly-2) MicroBeads, mouse, were developed for positive selection or depletion of mouse CD8+ T cells from spleen and lymphoid tissue–derived single-cell suspensions. With only 13.5 minutes processing time from single-cell suspension to target cells, CD8a (Ly-2) MicroBeads offer the fastest way to isolate CD8+ T cells without activation or any other effect on the cells' function. The non-target cell fraction can be used for a further separation round to isolate other subsets.
Fast isolation of CD8+ T cells. A single-cell suspension from mouse spleen was prepared using the gentleMACS™ Dissociator. CD8a+ T cells were isolated from this single-cell suspension using CD8a (Ly-2) MicroBeads, an LS Column, and a QuadroMACS™ Separator. Cells were fluorescently stained with CD45-VioGreen™, CD4-VioBlue®, and CD8a-PE-Vio®770, and analyzed by flow cytometry using a MACSQuant® Analyzer. Viable leukocytes were gated for analysis based on scatter signals, 7-AAD fluorescence, and CD45 expression.
Effective isolation of untouched CD8+ T cells. A single-cell suspension from mouse spleen was prepared using the program m_spleen_01.01 on the gentleMACS Dissociator. CD8a+ T cells were isolated from this single-cell suspension using the CD8a+ T Cell Isolation Kit, an LS Column, and a MidiMACS™ Separator. Cells were fluorescently stained with the MC CD8a T Cell Cocktail and analyzed by flow cytometry using a MACSQuant Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
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At a glance: Kits and reagents for the separation of naive/memory CD8⁺ T cell subsets from spleen and lymph nodes
| Starting material | Isolation strategy | Comments | Automation | Product |
|---|---|---|---|---|
| Single-cell suspensions from spleen and lymph nodes | Depletion of non-target cells | Isolation of naive CD8+ T cells via depletion of all memory T cells, CD4+ T cells, and non-T cell types | Yes* | Naive CD8a+ T Cell Isolation Kit, mouse |
| Single-cell suspensions from spleen and lymph nodes | Positive selection of target cells | CD62L (L-Selectin) is a naive/early memory T cell marker for central memory T cells. In combination with pre-selection via the CD8a+ T Cell Isolation Kit, CD8+ central memory T cells can be separated based on CD62L expression. | Yes* | CD62L MicroBeads, mouse(after pre-selection with CD8a+ T Cell Isolation Kit, mouse) |
| *Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X. | ||||
Specialized kits are available for the isolation of further CD8+ T cell subsets. Mouse naive CD8+ T cells can be isolated by depletion of memory T cells, CD4+ T cells, and non-T cell types using the Naive CD8a+ T Cell Isolation Kit, mouse.
Isolation of naive CD8a+ T cells from mouse spleen. Naive CD8a+ T cells were isolated from a single-cell suspension from mouse spleen using the Naive CD8a+ T Cell Isolation Kit, an LS Column, and a MidiMACS Separator. Cells were fluorescently stained with CD3ε-APC, CD8a-VioBlue, CD44-FITC,and CD62L-PE, and analyzed by flow cytometry using a MACSQuant Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
CD8+ T cell subsets and their differentiation status can be determined by flow cytometry based on their expression of cell surface markers, transcription factors, and cytokines. Miltenyi Biotec offers a vast portfolio of conventional and recombinant REAfinity™ Antibodies and flow analysis kits for comprehensive analysis.
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The following surface markers, cytokines, and transcription factors can be used to identify CD8+ T cells and subsets according to their development state or subtype.
At a glance: Markers for the detection of CD8⁺ T cell subsets by flow cytometry
| CD8+ cytotoxic T cells | T cell development – naive vs. memory (TSCM/TCM/TEM/TRM) | Activated T cells | Exhausted T cells |
|---|---|---|---|
| CD3 | CD3 | CD3 | CD3 |
| CD8 | CD4 | CD4 | CD4 |
| CD107a (LAMP-1) | CD8 | CD8a | CD8 |
| CD178 (FasL) | CD27 | CD8b | CD96 (TACTILE) |
| CD253 (TRAIL) | CD28 | CD25 (IL2A) | CD152 (CTLA-4) |
| IFN-γ | CD44 | CD27 | CD160 (NK1) |
| Perforin | CD62L | CD28 | CD223 (LAG-3) |
| Granzyme B | CD69 | CD44 | CD244 |
| TNF-α | CD103 | CD69 | CD272 (BTLA) |
| CD127 | CD95 (FAS-R) | CD278 (ICOS) | |
| CD197 (CCR7) | CD134 (OX40) | CD279 (PD1) | |
| CD137 (4-1BB) | CD366 (TIM-3) | ||
| CD154 (CD40L) | TIGIT | ||
| Ki-67 | VISTA | ||
| KLRG1 | EOMES |
Miltenyi Biotec offers a range of solutions for the analysis of T cell–associated surface markers and cytokines:
MACS Handbook:
Cell culture
At a glance: Kits and reagents for the cultivation, activation, and expansion of T cells
| Use | Comments | Product |
| Culture medium | Optimized T cell media without serum or animal-derived components. Also available in MACS GMP grade and with or 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 | Cell-sized activation beads (‘artificial APCs’) loaded with activating CD3 and CD28 antibodies. | T Cell Activation/Expansion Kit, mouse |
| Stimulation | In vitro T cell activation and expansion | CD3ε pure – functional grade, mouse CD28 pure – functional grade, mouse |
Miltenyi Biotec offers high-quality recombinant cytokines for cell culture, as well as reagents for polyclonal stimulation, such as the T Cell Activation/Expansion Kit, mouse. This kit employs large, cell-sized particles loaded with biotinylated antibodies to activate and expand primary cells. The particles mimic antigen-presenting cells and, when loaded with CD3 and CD28 antibodies and applied in a specific bead-to-cell ratio, lead to efficient T cell activation. Different T cell Activation/Expansion Kits are available for the stimulation of T cells as well as Treg cells of mouse, human, and non-human primate origin.
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 antibodies CD3ε and CD28 pure – functional grade, mouse are suitable for in vitro T cell activation and expansion. The CD3ε (145-2C11) and CD28 (37.51) antibodies recognize the respective mouse receptors. Upon binding of the antibodies to the receptors, a stimulatory signal is transferred that, in combination with additional cytokines (e.g., IL-2), leads to the activation and expansion of T cells.
Depending on the respective organ and the inflammatory context, T cell frequencies are highly variable in non-lymphoid tissue. Furthermore, T cells vary in the expression of certain surface markers, such as homing receptors, adhesion molecules, and chemokine receptors, with regard to their tissue tropism. Although the expression of those markers somewhat depends on the specific T cell subtype and differentiation status, certain receptors, especially in memory T cells, can be defined as hallmarks for tissue tropism.
For the flow cytometry analysis of tissue-derived T cells, Miltenyi Biotec offers comprehensive panels of antibodies corresponding to the markers indicated in the table above.
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Tissues must be dissociated into a single-cell suspension for many downstream applications, including isolation of cell subpopulations, cell culture, or flow cytometry analysis. The combination of mechanical dissociation and enzymatic treatment based on the gentleMACS Octo 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 lamina propria, lung, liver, and tumor. For details, see chapter Mouse cell sources.
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