1 Introduction
2 CD4⁺ T cells in spleen and lymphoid tissue
3 T cells from non-lymphoid tissues
- 3.1 Cell subsets, frequencies, and marker expression
- 3.2 Sample preparation of different tissues

Table of Content

Mouse CD4⁺ T helper cells

1 Introduction

T helper cells (T_Hcells) play a pivotal role in adaptive immunity. Following antigen recognition, naive T_H cells are activated, undergo further differentiation, and secrete cytokines to promote ('help') and steer the immune response. All T_H cells are positive for CD4, a coreceptor that complements the T cell receptor (TCR) to communicate with an antigen-presenting cell (APC).

MACS Handbook:

Pan T cells (mouse)

CD8+ T cells (mouse)

Regulatory T cells (mouse)

2 CD4⁺ T cells in spleen and lymphoid tissue

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% of the T cells, most of them in a double-positive stage, expressing both CD4 and CD8.

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Spleen (mouse)

Lymph node (mouse)

2.1 Cell subsets, frequencies, and marker expression

At a glance: TH cell subsets derived from naive CD4⁺ T cells

TH cell subset	Cytokines secreted	Function
T_H1	IFN‑γ, IL‑2, TNF-α	Activate macrophagesPromote cytotoxic T cell proliferation
T_H2	IL‑4, IL‑5, IL‑6, IL‑13	Promote humoral immune responseActivate B cells for expansion and antibody production
T_H17	IL‑17A, IL‑17F, IL‑21, IL‑22, IL-26	Maintain mucosal barrierClear pathogens at mucosal surfaces
T_H9	IL‑9, IL-10	Important role in several inflammatory scenarios: asthma, ulcerative colitis, and helminth infections
T_H22	IL-6, IL-10, IL-12, IL-21	Affect mainly epithelial and stromal cellsRecruited mainly to the skin, contributing to host defense against microbial pathogensContribute to tissue repair and remodeling
T_fH	IL-6, IL-10, IL-12, IL-21	Essential for the germinal center formation, affinity maturation, and the development of most high-affinity antibodies and memory B cells

Depending on the cytokine environment, naive T_H cells can differentiate into several subsets, including T_H1, T_H2, T_H17, T_H9, T_H22 cells, and T follicular helper cells (T_fH). They can also differentiate into induced regulatory T cells (iTreg; PMID: 26688349). See chapter Regulatory 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.

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Overview of mouse T cell differentiation from naive to memory T cells (PMID: 24258910, 26999211).

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 keeping 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.

2.2 Miltenyi Biotec application protocols for CD4⁺ T cells from spleen and lymphoid tissues

Miltenyi Biotec has created dedicated application protocols to expand and polarize CD4⁺ T cells.

2.3 Sample preparation of spleen and lymphoid tissue

The main source for mouse CD4⁺ T_H 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.

MACS Handbook:

Spleen (mouse)

Lymph node (mouse)

Sample preparation

2.4 Magnetic cell separation of CD4⁺ TH cells from spleen and lymph nodes

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

2.4.1 Isolation of CD4⁺ T cells

At a glance: Kits and reagents for the separation of CD4⁺ 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 CD4 antigen is expressed on most thymocytes and mature T helper cells, on a subset of NKT cells, and at lower levels on a subpopulation of dendritic cells.	Yes*	CD4 (L3T4) MicroBeads, mouse
Single-cell suspensions from spleen and lymph nodes	Depletion of non-target cells	Isolation of CD4⁺ T cells by depletion of all non-target cells using antibodies against CD8a, CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, Anti-MHC-class II, Ter-119, and TCRγ/δ.	Yes*	CD4⁺ 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.

CD4 (L3T4) MicroBeads, mouse, were developed for positive selection or depletion of mouse CD4⁺ 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, CD4 (L3T4) MciroBeads offer the fastest way to isolate CD4⁺ 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.

CD4 (L3T4) MicroBeads, mouse

Before separation

After separation

Before separation

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After separation

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Fast isolation of CD4⁺ T cells. A single-cell suspension from mouse spleen was prepared using the gentleMACS Dissociator. CD4⁺ T cells were isolated from this single-cell suspension using the CD4 (L3T4) 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.

As an alternative to positive selection by CD4 MicroBeads, the CD4⁺ T Cell Isolation Kit, mouse enables isolation of untouched CD4⁺ T cells from single-cell suspensions of mouse spleen and/or lymph nodes by depletion of non-CD4⁺ T cells. The untouched target cell fraction can then be used for a further separation round to isolate other subsets.

CD4⁺ T Cell Isolation Kit, mouse

Before separation

CD4⁺T cells after depletion of non-CD4⁺ T cells

Before separation

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CD4⁺T cells after depletion of non-CD4⁺ T cells

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Effective isolation of untouched CD4⁺ T cells. A single-cell suspension from mouse spleen was prepared using the program m_spleen_01.01 on the gentleMACS Dissociator. CD4⁺ T cells were isolated from this single-cell suspension using the CD4⁺ T Cell Isolation Kit, an LS Column, and a MidiMACS™ Separator. Cells were fluorescently stained with the MC CD4 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.

2.4.2 Isolation of naive/memory CD4⁺ T cells

At a glance: Kits and reagents for the separation of naive/memory CD4⁺ 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 CD4⁺ T cells by depletion of all memory T cells, CD8⁺ T cells, and non-T cell types	Yes*	Naive CD4⁺ T Cell Isolation Kit, mouse
Single-cell suspensions from spleen and lymph nodes	Sequential separation	Separation of CD4⁺ CD62L⁺ T cells in combination with depletion of CD8a⁺ and non-T cells. CD62L is expressed on naive T cells and early stages of memory T cells (T_SCM, T_CM). Samples from younger mice tend to have less of those early memory T cells, so that the kit mainly separates naive CD4⁺ T cells. Older mice have a higher frequency of memory T cells, so that separation with this kit results in a mixed population of naive and early memory T cells.	Yes*	CD4⁺CD62L⁺ 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 by the CD4⁺ T Cell Isolation Kit, CD4⁺ central memory T cells can be separated based on CD62L expression.	Yes*	CD62L MicroBeads, mouse(after pre-selection with CD4⁺ 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 CD4⁺ T cell subsets. Mouse naive CD4⁺ T cells can be isolated by depletion of all memory T cells, CD8⁺ T cells, and non-T cell types using the Naive CD4⁺ T Cell Isolation Kit, mouse.

Naive CD4⁺ T Cell Isolation Kit, mouse.

Before separation,
unseparated fraction

Before separation,
gated on CD45⁺CD3ε⁺CD4⁺ T cells

Isolated naive CD4⁺ cells

Isolated naive CD4⁺ cells, gated on CD45⁺CD3ε⁺ CD4⁺ T cells

Before separation,
unseparated fraction

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Before separation,
gated on CD45⁺CD3ε⁺CD4⁺ T cells

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Isolated naive CD4⁺ cells

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Isolated naive CD4⁺ cells, gated on CD45⁺CD3ε⁺ CD4⁺ T cells

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Isolation of naive CD4⁺ T cells from mouse spleen. Naive CD4⁺ T cells were isolated from a single-cell suspension from mouse spleen using the Naive CD4⁺ T Cell Isolation Kit, an LS Column, and a MidiMACS™ Separator. Cells were fluorescently stained with CD45-VioGreen™, CD4-VioBlue®, CD62L-PE, CD3ε-APC-Vio®770, and CD44-FITC 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 and a gate was set on CD45⁺ cells.

2.5 Characterization of CD4⁺ T cells by flow cytometry

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Mouse T_H cell subsets and corresponding polarizing cytokines, surface markers, transcription factors, and secreted cytokines.

CD4⁺ T cell subsets and their differentiation status can be determined by flow cytometry based on their expression of cell surface markers, transcription factors, and the cytokines they produce. Miltenyi Biotec offers a vast portfolio of conventional and recombinant REAfinity™ Antibodies and flow analysis kits for comprehensive analysis.

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MACS Flow Cytometry - antibodies

2.5.1 Flow cytometry panels

The following surface markers, cytokines, and transcription factors can be used to identify CD4⁺ T cells and subsets according to their development state or subtype.

At a glance: Markers for the detection of CD4⁺ T cell subsets by flow cytometry

T cell development – naive vs. memory (T_SCM/T_CM/T_EM/T_RM)	CD4⁺ T cell subsets (T_H1/T_H2/T_H9/T_H17/T_H22/T_fH)	Activated T cells	Exhausted T cells
CD3	CD3	CD3	CD3
CD4	CD4	CD4	CD4
CD8	CD44	CD8a	CD8
CD27	CD161 (NK1.1)	CD8b	CD96 (TACTILE)
CD28	CD183 (CXCR3)	CD25 (IL2A)	CD152 (CTLA-4)
CD44	CD185 (CXCR5)	CD27	CD160 (NK1)
CD62L	CD194 (CCR4)	CD28	CD223 (LAG-3)
CD69	CD196 (CCR6)	CD44	CD244
CD103	CD197 (CCR7)	CD69	CD272 (BTLA)
CD127	CCR10	CD95 (FAS-R)	CD278 (ICOS)
CD197 (CCR7)	IL-2	CD134 (OX40)	CD279 (PD1)
	IL-4	CD137 (4-1BB)	CD366 (TIM-3)
	IL-5	CD154 (CD40L)	TIGIT
	IL-9	Ki-67	VISTA
	IL-10	KLRG1	EOMES
	IL-13
	IL-17
	IL-22
	IL-26
	IFN-γ
	T-bet
	RORγT
	GATA3

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Primary antibodies

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 staining of T cell surface markers or intracellular staining of cytokines.
MACS Cytokine Secretion Assays allow detection and enrichment of viable cytokine-secreting cells. The mouse kits are available for a large number of cytokines, including IFN-γ, IL-2, IL-4, IL-5, IL-10, and IL-17, and can be used for further characterization of T cell subsets.
The mouse MACSPlex Cytokine 10 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-γ, IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-17A, IL-23, and TNF-α.

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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. The MPx Detection Reagent, composed of a cocktail of APC-conjugated antibodies specific for the analytes, is added. Consequently, sandwich complexes are formed between MPx Capture Bead, analyte, and MPx Detection Reagent. These complexes can be analyzed based on the fluorescence characteristics of both MPx Capture Bead and 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 CD4⁺ 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 helper 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 and research grades as well as MACS GMP Grade.	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.

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Efficient expansion of T cells. Expansion rates of human and mouse CD4⁺ and CD8⁺ T cells over the course of several days after activation and expansion with the respective T Cell Activation/Expansion Kit.

MACS Cytokines are available in three different grades – research and premium grades as well as 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 pretesting.

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.

2.6.2 Differentiation of CD4⁺ T cells

At a glance: Kits and reagents for the differentiation of CD4⁺ T cells

Use	Comments	Product
Differentiation	Consistent, high-quality recombinant cytokines for successful cell culture. Available in premium and research grades as well as MACS GMP Grade.	MACS Cytokines
Differentiation	Functional-grade antibodies effectively mimic or inhibit ligand-receptor interactions.	Functional-grade antibodies
Differentiation	Complete starter kit for polarization of T_H1 cells, including all needed cytokines and functional-grade antibodies.	CytoBox Th1, mouse
Differentiation	Complete starter kit for polarization of T_H2 cells, including all needed cytokines and functional-grade antibodies.	CytoBox Th2, mouse
Differentiation	Complete starter kit for polarization of T_H17 cells, including all needed cytokines and functional-grade antibodies.	CytoBox Th17, mouse

By combining polarizing cytokines and blocking or activating antibodies (‘functional-grade’), naive CD4⁺ cells can be differentiated into different T_H cell subtypes. With the optimized TexMACS™ Medium, premium-grade cytokines, and a vast array of functional-grade antibodies, Miltenyi Biotec offers all the reagents required for efficient polarization of naive CD4⁺ T cells.

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MACS Cytokines and functional-grade antibodies available for mouse T_H cell polarization.

CytoBoxes T_H1, T_H2, and T_H17 are the ideal tools for the polarization of mouse CD4⁺ T cells into the respective T_H cell subset. The CytoBoxes comprise all cytokines and functional-grade antibodies needed for an efficient polarization – ready-to-use at an optimal ratio. Differentiation efficiency is boosted with the use of TexMACS Medium.

For detailed information about Miltenyi Biotech media optimized for T cells, see chapter Cell culture media.

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Comparison of cytokine expression levels of mouse T_H cells differentiated with CytoBoxes T_H1, T_H2, and T_H17. Isolated mouse naive CD4⁺ T cells were cultivated with reagents of each CytoBox in TexMACS Medium (+10% FBS) or basal medium (RPMI + 10% FBS). Lineage-specific effector cytokine expression was analyzed after differentiation.

3 T cells from non-lymphoid tissues

Mouse T cells can also be obtained from non-lymphoid tissue. Miltenyi Biotec offers a variety of solutions for the preparation of non-lymphoid mouse tissue as well as the subsequent separation, cultivation, and analysis of the respective T cell populations.

3.1 Cell subsets, frequencies, and marker expression

Overview of surface markers to analyze mouse T cell tissue tropism. (PMID: 27038487)

Overview of surface markers to analyze mouse T cell tissue tropism.

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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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Primary antibodies

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. 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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Sample preparation

Tumor tissue (mouse)

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Mouse CD4+ T helper cells

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Mouse CD4⁺ T helper cells