Human Pan T cells

The StraightFrom® Whole Blood CD3 MicroBeads, human, were developed for the rapid positive selection of CD3⁺ T cells directly from whole blood. The kit requires no sample preparation, like density gradient centrifugation, erythrocyte lysis, or cell count.

Instead of working directly with whole blood, samples can be processed by density gradient centrifugation to pre-enrich peripheral blood mononuclear cells (PBMCs) as starting material for subsequent T cell isolation.

The CD62L MicroBeads, CD45RA MicroBeads, or CD45RO MicroBeads are used to positively select the respective cells according to the given marker. Isolation of CD62L⁺, CD45RA⁺, or CD45RO⁺ T cells can be accomplished by using these MicroBeads in combination with kits enabling the isolation of untouched T cells, like the Pan T Cell Isolation Kit.

T cell activation can be i) antigen specific, by recognition of the cognate antigen, or ii) polyclonal via, e.g., antibody-mediated CD3/CD28 ligation, binding of chemical agents like PMA/ionomycin, or exposure to a super-antigen like staphylococcal enterotoxin B (SEB) or the non-toxic alternative CytoStim™. T cell activation is characterized by up-regulation of surface markers and the expression of proinflammatory cytokines. Depending on the T cell subtype and the activation mode, activation markers and cytokines include CD25 (IL2RA), CD30, CD38, CD69, CD95 (FASR), CD137 (4-1BB), CD154 (CD40L) and IL-2, IL-4, IL-5, IL-6, IL-9, IL-13, IL17, TNF-α, IFN-γ, respectively. For details on using these markers to identify activated T cells, see Characterization of T cells by flow cytometry below.

Antigen-activated T cells are especially interesting for research because of their specificity and potential for cell-based therapies. However, the frequency of antigen-specific T cells in peripheral blood can be as low as 0.01% of all T cells. This low frequency makes processing of high cell numbers and the use of specialized analysis methods essential for reliable detection and phenotypic characterization. Miltenyi Biotec has developed dedicated application protocols for working with antigen-specific T cells.

CD137 and CD154 were described as surrogate markers for antigen-activated T cells and can be used for analysis and enrichment of these rare cells. CD137 is mainly expressed on activated CD4⁺ and CD8⁺ T cells, activated B cells, and on natural killer cells, but can also be found on resting monocytes and dendritic cells. CD137 has been described as a suitable marker for antigen-specific activation of human CD8⁺ T cells, as CD137 is not expressed on resting CD8⁺ T cells and its expression is reliably induced after 24 hours of stimulation.  Thus, activated antigen-specific CD8⁺ T cells can be isolated by combining the CD137 MicroBead Kit, human with the CD8⁺ T Cell Isolation Kit, human.

The CD154 antibody specifically recognizes the human CD154 antigen, also known as CD40L, gp39, T-BAM, TRAP, or Ly-62. CD154 is transiently up-regulated on activated CD4⁺ T cells and plays an important role as a costimulatory molecule in T cell/antigen-presenting cell interactions through ligation of CD40. Due to the transient expression of CD154 within hours after activation, the CD154 MicroBead Kit, human can be used to isolate activated antigen-specific CD4⁺ T cells. 

Secreted cytokines can be used either as markers for antigen-activated T cells, or as a means to isolate and analyze these cells. For details, see Analysis of surface markers and cytokines below. Miltenyi Biotec has also created dedicated application protocols for working with cytokine-secreting cells.

• Expansion of antigen-specific cytokine-secreting T cells
   
• Detection and enrichment of cytokine-secreting cells  
  
   

Gamma/delta T cells (γ/δ T cells) have a distinctive T cell receptor (TCR) on their surface that consists of one γ chain and one δ chain, as opposed to the 'conventional' α/β T cells that express a TCR composed of α and β TCR chains. Since most T cells are α/β T cells, γ/δ T cells are relatively rare (<10% of all T cells in peripheral blood), showing highest abundance in a population of lymphocytes of the gut mucosa known as intraepithelial lymphocytes. 

The antigens that activate γ/δ T cells and their specific function are still largely unknown. They recognize antigens without the presence of major histocompatibility complex molecules, are able to function as antigen-presenting cells, and they exhibit cytotoxicity. This makes them ideal candidates as therapeutic targets to induce durable immunity in the context of different pathologies. Their functional responses are induced upon recognition of stress antigens, such as heat-shock proteins, which promotes cytokine production and regulates various stages of the immune reaction in response to stress. Thereby, γ/δ T cells can attack target cells directly through their cytotoxic activity or indirectly through the activation of other immune cells.

Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties with both T cells and natural killer cells. Many of these cells recognize the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids. As such, NKT cells are important in recognizing glycolipids from organisms, such as Mycobacterium tuberculosis. 

NKT cells express an α/β T cell receptor, but also a variety of molecular markers that are typically associated with NK cells, such as NK1.1. The best-known NKT cells differ from conventional α/β T cells in that their T cell receptors are far more limited in diversity ('invariant' or 'type 1' NKT cells, also termed iNKT cells). However, also other CD1d-restricted T cells ('type 2' NKT cells) recognize lipids and glycolipids presented by CD1d molecules, rather than peptide-MHC complexes. NKT cells include both NK1.1⁺ and NK1.1⁻, as well as CD4⁺, CD4⁻, CD8⁺ and CD8⁻ cells. NKT cells may share other features with NK cells, such as CD16 and CD56 expression and granzyme production.

In humans, iNKT cells express a highly conserved TCR, consisting of Va24-Ja18 paired with Vb11, which is specific for glycolipid antigens. They are notable for their ability to respond rapidly to danger signals and pro-inflammatory cytokines. Once activated, they engage in effector functions, like NK transactivation, T cell activation and differentiation, B cell activation, dendritic cell activation and cross-presentation activity, and macrophage activation.

The CD3⁺CD56⁺ NKT Cell Isolation Kit, human, was developed for the sequential separation of CD3⁺CD56⁺ NKT cells from human PBMCs and other single-cell suspensions.

 

The down-regulation of immune responses by regulatory T cells plays a key role in the induction of tolerance. It has been reported that TCRα/β⁺CD4^–CD8^– double-negative T cells also have the ability to specifically down-regulate immune responses towards allo-antigens, both in humans and mice. Double-negative T cells are found in lymphoid as well as in non-lymphoid tissues. They constitute about 1–3% of peripheral CD3⁺ cells.

The Double-negative T Cell Isolation Kit enables the isolation of double-negative T cells (CD4^–CD8^–CD56^–CD3⁺TCRα/β⁺ cells) from PBMCs by sequential sorting. The isolation is performed in a two-step procedure.

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

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 the staining of T cell surface markers or the intracellular staining of cytokines.
• MACS Cytokine Secretion Assays allow detection and enrichment of viable cytokine-secreting cells. The human kits are available for a large number of cytokines, including TNF-α, GM-CSF, IFN-α, IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, and IL-22, and can be used for further characterization of T cell subsets.
• The MACSPlex Cytokine 12 Kit, human is 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-α, IFN-γ, lL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-17A, and TNF-α.
• The Rapid Cytokine Inspectors allow for rapid analysis of activated T cells by combining surface marker analysis (e.g., CD4, CD8, CD154) with intracellular cytokine staining (e.g. IFN-γ, TNF-α, IL-2, and IL-17).

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 Biotec media optimized for T cells, see the MACS Handbook chapter Cell culture. 

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.

The T Cell Activation/Expansion Kit, human, employs large cell-sized particles loaded with biotinylated antibodies of choice to activate and expand primary cells. The large cell-sized particles mimic antigen-presenting cells and, when loaded with CD2, CD3, and CD28 antibodies and applied in a specific bead-to-cell ratio, lead to efficient T cell activation. The decision tree below helps to find the right T cell activation product for a particular experiment.

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 grades. 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 grades – 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.