Mouse CD8⁺ cytotoxic T cells

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 (T_NAIVE) may differentiate into various memory T cell subsets, including stem cell–like memory T cells (T_SCM), central memory T cells (T_CM), and effector memory T cells (T_EM). 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.

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 .

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.

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.

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.