Immune Cell Induced Stimulation Assays

Immune Cell Stimulation Assays

Numerous techniques have been developed for stimulating B and T lymphocytes ex vivo so as to alter their state of activation, differentiation, and/or proliferation. Freshly isolated blood lymphocytes from normal individuals are typically quiescent, unless recently stimulated in vivo for some reason. Thus, stimulation is usually required to induce cell-type specific functions or to induce the synthesis of detectable levels of specific molecules - e.g., DNA, mRNA, and/or proteins, modified proteins, etc. - for subsequent analysis ex vivo. Although lymphocytes can be activated through their unique antigen-binding receptors, only an exceedingly small fraction of the B and T lymphocytes in any sample will respond to direct antigenic stimulation because the antigen receptors on these lymphocytes are highly diverse and highly specific for different antigens. Thus, antigen-nonspecific polyclonal activators are frequently used to stimulate lymphocytes ex vivo. Combinations of different activators are typically used for many of the diagnostic test results presented here in order to trigger multiple intracellular signaling pathways as required for optimal lymphocyte activation.

B Cell-Specific Stimulation

Anti-immunoglobulin (Ig) antibodies crosslink the heavy (H) and/or light (L) chains of B cell receptors (BCR) and thereby mimic antigen binding to these receptors and activate B cells polyclonally.
Anti-CD40 plus anti-BCR antibodies together trigger a two-signal induction of B cell differentiation and/or activation ex vivo that is more potent than activation by either stimulus alone. Anti-CD40 antibodies crosslink CD40 co-stimulatory molecules expressed on the membranes of activated B cells, thereby mimicking the co-stimulatory crosslinking effects induced by CD40L (CD154) expressed on the membranes of activated T helper cells.
IL-4 plus anti-CD40 antibodies induced C cells to undergo Ig class switching and eventually begin synthesizing IgE antibodies. IL-4 also induces activated B cell surface expression of two differentiation markers - the low affinity IgE receptor, FcεRII (CD23), and the intercellular adhesion molecule-1, ICAM-1 (CD54). (See Cytokine Production)
Mitogenic plant lectins have multivalent carbohydrate-binding properties that can also polyclonally activate B cells by crosslinking membrane glycosylated proteins, including the BCRs on B cells, thus mimicking antigen binding to these receptors. Certain lectins, such as pokeweed mitogen (PWM), are particularly effective activators of human B cells.
Bacterial lipopolysaccharide (LPS) is a potent activator of B cells and monocytes. Cell activation is initiated by LPS complexes formed with an acute phase LPS-binding protein (LBP) found in serum. LPS/LBP complexes activate B cells and monocytes by similar but distinct mechanisms. With B cells, LPS/LBP complexes bind to or engage CD19 (a B cell surface differentiation marker) and also bind to innate immune toll-like receptors - TRL4 and RP105 - and possibly other molecules - in order to polyclonally activate B cells. With monocytes, LPS/LBP complexes bind to or engage CD14 (a monocyte differentiation marker), TLR4, and possibly other monocyte activating molecules thereby inducing monocytes to release cytokines, some of which amplify B cell responses. Thus, B cell activation by LPS is both antigen and T helper cell independent.

T Cell-Specific Stimulation

Anti-CD3 or anti-T cell receptor (TCR) antibodies crosslink the TCR/CD3 receptor complexes on T cells and thereby mimic peptide antigen/MHC class I or class II complex binding to these receptors as normally presented by antigen presenting cells (APC) - i.e., dendritic cells, macrophages, monocytes, and/or B cells. Thus, T cells in the sample are polyclonally activated.
Anti-CD28 plus anti-TCR or anti-CD3 antibodies together trigger a two-signal induction of T cell differentiation and/or activation ex vivo that is more potent than the stimulus produced by either alone. Anti-CD28 antibodies crosslink CD28 co-stimulatory molecules expressed on activated T cells, thereby mimicking the co-stimulatory effects induced by B7-1 (CD80) and B7-2 (CD86) co-receptors expressed on the membrane surfaces of conventional APC.
IL-2 plus anti-CD3 antibodies induce T cells to proliferative vigorously and up-regulate T cell surface expression of the IL-2Rα alpha subunit, gp55 (CD25), and IL-2Rβ beta subunit. gp75 (CD122) of the multimeric IL-2-receptor complex. (See Cytokine Production)
Phytohemagglutinin (PHA) and Concanavalin A (Con A): These carbohydrate-binding plant lectins are primarily mitogenic for T-cells because they polyclonally activate T-cells by crosslinking the membrane glycosylated proteins on these cells, including TCR/CD3 complexes, which mimicks and thereby bypasses direct antigen binding to the receptors.
Antigen-pulsed APC can clonally activate T cells with receptors that specifically recognize the peptide antigen/MHC class I or class II complexes presented by APC. For example, when white blood cells (WBC) isolated from an individual are incubated (i.e., "pulsed") with tetanus toxoid (TT) antigen ex vivo, any TT-specific T cells present usually proliferate (See Proliferation Assays) if that individual has had tetanus shots and retains some residual level of TT-specific immunological memory.
Alloantigens stimulate T cell functions and proliferation when WBC from different individuals are incubated together for a few days in culture. T cell activation arises from structural differences between MHC class I and/or class II molecules expressed on the surfaces of different cell populations. Specifically, T cytotoxic (T_C) lymphocytes are activated by recognizing (binding) foreign allo-MHC class I molecules expressed on "non-self" cells in the cell mixture. Similarly, T helper (T_H) lymphocytes are activated by recognizing (binding) foreign allo-MHC class II molecules expressed "non-self" APC in the cell mixture.
Bacterial lipopolysaccharide (LPS), while a potent activator of B cells and monocytes, only induces limited T cell activation through bystander effects where cytokines released from activated monoctyes amplify certain T cell responses, such as induced IFN-γ production.

T Cell Proliferation in Mixed Leukocyte Reactions

"Two-way" MLRs (mixed leukocyte reactions): WBCs from two individuals, even related individuals, will typically begin proliferating spontaneously because the two cell populations are likely to express nonmatching MHC class I and/or class II alloantigens on their surfaces. The strongest proliferative responses arise when T helper cells from one individual engage allo-MHC class II molecules selectively expressed on the APC from the other individual. A similar but weaker proliferative response also arises when T cytotoxic cells from one individual engage allo-MHC class I molecules ubiquitously expressed on almost every cell type of the other individual. In this way, alloantigen activated T cytotoxic cells gain the capacity to kill cells expressing allo-MHC class I molecules. Without further intervention (see below), the lymphocytes from both individuals act as responders against the allo-APC of the opposite individual, thereby resulting in a "two-way" MLR.
"One-way" MLRs: Such reactions can be set up so that only one WBC population proliferatves in response to the MHC class I and/or II alloantigens expressed on the cells from another individual. If radioactive deoxynucleotides are used to monitor proliferation (by measuring the incorporation of radioactivity into newly synthesized DNA), it is necessary to inactivate the stimulating cell population so that it can no longer proliferate in response to the other cell population in the culture. Cell-division by one cell population can be prevented by pre-treating the "stimulator" cell population with a sublethal dose of an anti-mitotic compound, such as mytomycin C, or by irradiating these cells with a sublethal dose of X-rays. However, if one investigates MLR-induced cell proliferation with the CFSE proliferation assay, it is not necessary to inactivate the stimulator cell population. In this case, the responding cell population is pre-labeled with 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) before being mixed with the stimulator cell population. As the fluorescently labeled responder cells proliferate, their average CFSE fluorescence intensity diminishes by half with each round of cell division. By FACS analysis, the responder population will show up a series of distinct bands, each with a stepwise loss in fluorescence intensity depending on the number of daughter cell divisions that have occurred in the original cell population.

B and T Cell Co-stimulation Co-stimulatory events are critical for B and T cell activation because at least two, if not more, independent stimulation signals are typically required for optimal lymphocyte activation and function. One signal is normally generated when antigen binds to and cross-links a lymphocyte antigen receptor, i.e., the surface BCR of a B cell or the TCR of a T cell. Another signal is normally generated through the activation of other cell surface co-stimulatory receptors including cytokine receptors and cell-adhesion receptors. However, certain compounds can bypass some or all of these early lymphocyte activating signals. For example, LPS is a strong polyclonal activator of B cells (See above) because it effectively delivers multiple signals to B cells by more than on biochemical mechanism. However, most other drugs used to artificially stimulate lymphocytes deliver only one signal and alone they generally induce only weak responses. Thus, many other stimulatory compounds, such as those listed below, are used in combinations to promote strong B or T lymphocyte activation.

Phorbol myristic acid (PMA) directly binds to and activates protein kinase C (PKC), an intracellular signaling molecule that normally triggers one of several downstream signaling pathways that are activated after B and T lymphocytes have been stimulated through their antigen receptors. This pathway is normally triggered intracellularly by diacylglycerol (DAG), which is generated by activated phosphylase C-gamma (PLC-γ) when it splits plasma membrane-associated phosphoinositol 4,5-bisphosphate (PIP₂) into DAG plus inositol triphosphate (IP₃). Thus, PMA bypasses some of the early receptor-induced cell signaling events and thereby facilitates the polyclonal activation of B and T cells.
Ionomycin is a Ca²⁺ ionophore that facilitates Ca²⁺ entry into the cell. A rise in intracellular Ca²⁺ is normally one of several downstream signaling events that accompany the activation of B and T lymphocytes through their antigen receptors. This pathway is normally triggered by IP₃, which is generated by activated PLC-γ when it splits PIP₂, as discussed above. Thus, ionomycin bypasses some of the early receptor-induced cell signaling events and thereby facilitates the polyclonal activation of B and T cells.