|TH1||IFN-γ, IL-2, TNF-α|
|TH2||IL-4, IL-5, IL-6, IL-13|
|TH17||IL-17A, IL-17F, IL-21, IL-22, IL-26|
|TH9||IL-9 in high amounts, IL-10|
|TH22||IL-13, IL-22, TNF-α|
|Tfh||IL-6, IL-10, IL-12, IL-21|
Depending on the cytokine environment, naive CD4+ TH cells might differentiate into several subsets, including TH1, TH2, TH17, TH9, TH22 cells, and T follicular helper cells (Tfh). Naive CD4+ T cells can furthermore differentiate into induced regulatory T cells (iTreg; PMID: 26688349). See chapter Regulatory T cells.
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.
Miltenyi Biotec has created dedicated application protocols for the isolation of different CD4+ T cell subsets.
Miltenyi Biotec has developed numerous products for the straightforward magnetic separation of CD4+ T cells and corresponding subsets. T cells can be isolated either straight from whole blood, buffy coat, leukoreduction system chambers (LRSC), or Leukopak without density gradient centrifugation and erythrocyte lysis, or from PBMCs.
|Starting material||Isolation strategy||Comments||Automation||Product|
|Pan CD4+ T cells|
|Whole Blood||Positive selection of target cells||CD4 is predominantly expressed on TH cells. Suitable for smaller sample volumes (total capacity: 40 mL whole blood).||Yes||StraightFrom™ Whole Blood CD4 MicroBeads, human|
|Buffy coat||Positive selection of target cells||CD4 is predominantly expressed on TH 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 CD4 MicroBead Kit, human|
|LRSC||Positive selection of target cells||CD4 is predominantly expressed on TH cells. Allows direct processing of an entire LRSC (up to 40 mL) without density centrifugation and includes the needed columns.||Yes||StraightFrom LRSC CD4 MicroBead Kit, human|
|Leukopak||Positive selection of target cells||CD4 is predominantly expressed on TH cells. Allows direct processing of ½ a LeukoPak without density centrifugation and includes the needed columns.||Yes||StraightFrom Leukopak CD4 MicroBead Kit, human|
|Whole blood||Depletion of non-target cells||Column-free isolation of CD4+ T cells (total capacity: 3x30 mL whole blood).||No||MACSxpress CD4 T Cell Isolation Kit, human|
|Whole blood||Depletion of non-target cells||Column-free isolation of CD4+CD45RO+ cells via depletion of CD45RA+ cells (total capacity: 3x30 mL whole blood).||No||MACSxpress CD4 Memory T Cell Isolation 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 CD4 MicroBead Kit, human, CD4+ cells are separated from an entire buffy coat in less than 30 minutes. The kit can be used in combination with a QuadroMACS Separator for manual separation, but is best combined with the MultiMACS™ Cell24 Separator Plus .
Isolation of CD4+ T cells directly from buffy coat. Separation of a buffy coat sample using the StraightFrom™ Buffy Coat CD4 MicroBead Kit and the MultiMACS™ Cell24 Separator Plus with the Single-Column Adapter and Whole Blood Columns. Cells were fluorescently stained with CD4-PE, CD14-APC, as well as CD45-VioBlue® and analyzed by flow cytometry using 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 3x30 mL)
The MACSxpress CD4 T Cell Isolation Kit was developed for the fast and easy isolation of highly pure CD4+ 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.
|Starting material||Isolation strategy||Comments||Automation||Product|
|PBMCs||Positive selection of target cells||CD4 is predominantly expressed on TH cells. Suitable for the depletion or enrichment of CD4+ cells form a PBMC sample.||Yes||CD4 MicroBeads, human|
|PBMCs||Depletion of non-target cells||Isolation of CD4+ TH cells via depletion of CD8+ T cells, monocytes, neutrophils, eosinophils, B cells, dendritic cells, NK cells, granulocytes, γ/δ T cells, and erythroid cells||Yes||CD4+ T Cell Isolation Kit, human|
|PBMCs||Positive selection of target cells||CD4 is predominantly expressed on TH cells. MicroBead and antibody labeling fully removable.||Yes||REAlease CD4 MicroBead Kit|
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 TH cell isolation.
CD4 MicroBeads, human enable positive selection or depletion of CD4+ cells by direct magnetic labeling.The CD4+ T Cell Isolation Kit, human enables fast isolation of untouched CD8+ cytotoxic CD4+ 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.
a) Cell purity
Label-free, highly pure CD4+ T cells.
(A) CD4+ T cells were isolated from human PBMCs using the REAlease CD4 MicroBead Kit, MS Columns, and a MiniMACS Separator. Cells were fluorescently stained with CD4-FITC and analyzed by flow cytometry using the MACSQuant Analyzer X. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
b) Label-free cells: REAlease Biotin Complex release
(B) The efficient removal of all labels was shown by using Anti-Biotin-APC to analyze the cells by flow cytometry for the presence of REAlease Biotin Complex. Directly after isolation, the cells showed staining of biotin ("MicroBead-free CD4+ cells"), whereas the label-free CD4+ cells after the REAlease Biotin Complex release were negative for biotin similar to the non-labeled cells before separation. Release efficiency was higher than 99% for the REAlease Anti-Biotin MicroBeads (CD4).
|Starting material||Isolation strategy||Comments||Automation||Product|
|PBMCs||Depletion of non-target cells||Isolation of all naïve CD4+ T cells via depletion of CD45RO+ and non-T cells.||Yes||Naive CD4+ T cell Isolation Kit II, human|
|PBMCs||Depletion of non-target cells||Isolation of all memory CD4+ cells via depletion of CD45RA+ and non-T helper cells.||Yes||Memory CD4+ T cell Isolation Kit, human|
|PBMCs||Depletion of non-target cells||Isolation of all CD4+ effector memory T cells via depletion of non-T helper, CD45RA+, and CCR7+ cells.||Yes||CD4+ Effector Memory T cell Isolation Kit, human|
|PBMCs||Sequential separation||Depletion of non-CD4+ cells and naïve CD4+ T cells, followed by separation of TCM via CD197.||Yes||CD4+ Central Memory T cell Isolation Kit, human|
|PBMCs||Positive selection of target cells||CD62L (L-Selectin) is a naive/early memory T cell marker.||Yes||CD62L MicroBeads, human (after pre-selection with CD4 T cell Isolation Kit, human)|
|PBMCs||Positive selection of target cells||CD54RA is expressed on naive T cells, but not on memory T cells||Yes||CD45RA MicroBeads, human (after pre-selection with CD4 T cell Isolation Kit, human)|
|PBMCs||Positive selection of target cells||CD54RO is expressed on memory T cells, but not on naive T cells||Yes||CD45RO MicroBeads, human (after pre-selection with CD4 T cell Isolation Kit, human)|
The Naive CD4+ T Cell Isolation Kit II, human was developed for the isolation of untouched naive CD4+ T cells from PBMCs. Memory CD4+ T cells and non-CD4+ T cells are labeled with a cocktail of biotinylated CD45RO, CD8, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD56, CD123, Anti-TCRγ/δ, Anti-HLA-DR, and CD235a (Glycophorin A) antibodies, and then magnetically labeled with Anti-Biotin MicroBeads for subsequent depletion.
The Memory CD4+ T Cell Isolation Kit, human is used to isolate untouched memory T helper cells from PBMCs via depletion of naive T cells, CD8+ T cells, B cells, NK cells, γ/δ T cells, monocytes, DCs, granulocytes, platelets, and erythroid cells. PBMCs are incubated with a cocktail of biotinylated CD45RA, CD8, CD14, CD16, CD19, CD56, CD36, CD123, Anti-TCRγ/δ, and CD235a (Glycophorin A) antibodies. Non-target cells are then magnetically labeled with Anti-Biotin MicroBeads for subsequent depletion.
Isolation of untouched memory CD4+ T helper cells from PBMCs. Samples were processed using the Memory CD4+ T Cell Isolation Kit, an LS Column, and a MidiMACS Separator. Cells were stained with CD4 and CD45RO antibodies.
Example separation with CD62L MicroBeads, human. CD62L+ cells were separated from PBMCs using CD62L MicroBeads. For positive selection, CD62L+ cells were isolated using an MS Column and a MiniMACS™ Separator. For depletion of CD62L+ cells, the sample was separated over an LD Column in a MidiMACS Separator.
|T cell subset||Isolation strategy||Comments||Automation||Product|
|TH2||Direct isolation of target cells||CD294 (CRTH2) is a marker for TH2 cells.||Yes||CD294 (CRTH2) MicroBead Kit, human|
|Skin-homing T cells||Direct isolation of target cells||Cutaneous lymphocyte-associated antigen (CLA) is a marker for skin-homing T cells||Yes||Anti-CLA MicroBead Kit, human|
|Skin-homing CD4+ T cells||Sequential separation||First isolate CD4+ T cells and then select cells expressing the skin-homing receptor CLA||Yes||Combine:|
CD4+ T Cell Isolation Kit, human
Anti-CLA-PE, human (clone HECA-452)
|Recent thymic emigrant T cells||Sequential separation||First non-CD4+ cells and memory CD4+ T cells are depleted. Then RTEs are separated via CD31.||Yes|
|T cell development – naive vs. memory (TSCM/TCM/TTM/TEM/TEMRA)|
CD4+ TH subsets (TH1/TH2/TH9/TH17/TH22/Tfh)
|Activated TH cells||Exhausted TH cells|
|CD27||CD183 (CXCR3)||CD25 (IL2RA)||CD96 (TACTILE)|
|CD28||CD184 (CXCR4)||CD27||CD152 (CTLA-4)|
|CD45RA||CD185 (CXCR5)||CD28||CD160 (NK1)|
|CD45RO||CD194 (CCD4)||CD69||CD223 (LAG-3)|
|CD57||CD196 (CCR6)||CD95 (FasR)||CD244 (2B4)|
|CD62L (L-Selectin)||CD197 (CCR7)||CD134 (OX40)||CD278 (ICOS)|
|CD95 (FasR)||CCR10||CD137 (4-1BB)||CD279 (PD1)|
|CD127||IL-2||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, research grade|
|Supplement||Consistent, high-quality recombinant cytokines for successful cell culture. Available in premium, research and GMP grades.||MACS Cytokines|
|Stimulation||Nanomatrix-based activation of T cells via CD3/CD28 engagement. Available in research and MACS GMP grades||T Cell TansAct™|
|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||CD3 pure – functional grade, human |
CD28 pure – functional grade, human
TexMACS Medium, research grade 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 Biotec media optimized for T cells, see the 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 GMP grades, for a seamless transfer of workflows into clinical settings.
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.
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) and CD28 (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.
|Differentiation||Consistent, high-quality recombinant cytokines for successful cell culture. Available in premium, research, and MACS GMP grades.||MACS Cytokines|
|Differentiation||Functional-grade antibodies effectively mimic or inhibit ligand-receptor interactions.||Functional-grade antibodies, human|
By combining polarizing cytokines and blocking or activating antibodies (‘functional-grade’), naive CD4+ cells can be differentiated into a variety of different TH cell subtypes. With the optimized TexMACS Medium, premium-grade cytokines, and a vast array of functional-grade antibodies, Miltenyi Biotec offers multiple tools for the efficient polarization of naive CD4+ T cells.
Miltenyi Biotec has created dedicated application protocols to enrich T cells from various human tissues: