Experimental approaches to lymphocyte proliferation

Several experimental in vitro models exist to study antigen-specific and nonspecific (polyclonal) T cell proliferation (Table 3). The use of antigen-specific proliferation is limited by the fact that cell proliferation is limited to the frequency of cells having a clonally rearranged antigen receptor that is specific for the test antigen. For example, the frequency of circulating human T cells responding to alloantigen is on the order of 10~2, the frequency of cells responding to soluble 'memory' antigens such as tetanus toxin is 10~5, and the frequency of cells responding to a primary antigen is <10 6. For this reason, T cell clones of antigen-specific cells are used to study antigen-specific responses, and have proved quite useful for analysis of the molecular basis of cellular interactions. A limitation of this approach, however, is that the triggering properties of T cell clones are much less stringent than primary T cells. Antibodies to the T cell receptor (TCR) are a con venient and effective way to cause polyclonal T cell proliferation. Antibodies to either the heterodimeric T cell receptor or the associated CD3 complex are effective mitogens. Antibodies may be added in solution or immobilized onto the surface of culture vessels or beads. The manner of addition of the antibody to the culture system is a critical experimental variable. For example, soluble antibodies are much more dependent upon the presence of accessory cells for their mitogenic effect than are immobilized antibodies. Furthermore, when using soluble antibody as a stimulus for human T cells, the isotype of the anti-TCR antibody is important due to the varying affinity of Fc receptors on accessory cells for murine monoclonal antibodies. Antibodies to the CD2 and CD28 coreceptors also cause polyclonal T cell proliferation.

Chemical reagents can also be used to mimic the biochemical signals provided by TCR-induced T cell activation, as was previously discussed with respect to B cell activation. This concept has been termed 'membrane bypass activation'. As in B cells, calcium ionophorcs together with phorbol esters cause T cell proliferation, however the proliferation is accompanied by high rates of apoptosis unless appropriate costimulatory signals are provided. A general principle of lymphocyte proliferation is that multiple stimuli are required in order to elicit a full proliferative response and to avoid induction of apoptosis. For example, lectin-stimulated proliferation requires the presence of accessory cells such as monocytes. Pure populations of T cells will not divide unless the primary stimulus as well as the biochemical signals provided by the accessory cells are supplied. In practice, small and variable quantities of monocytes are required for in vitro proliferation (0.1% monocytes for phytohemagglutinin (PHA)-induced proliferation; 1% monocytes for anti-CD3 and calcium ionophore-induced proliferation).

Both B and T cell proliferation can be assayed in a variety of ways. pHlThymidine added to stimulated cells becomes incorporated into newly synthesized DNA, and thus is a measure of stimulation of the initial cell population. Bromodeoxyuridine measures proliferation using the same principle, but is detected by flow cytometry with a fluorescent antibody. Proliferation measured using these agents must take into account ongoing cell death in the population as a whole. Hoechst 33342, a DNA-binding dye and Pyronin Y, a RNA-binding dye can also be detected by flow cytometry and measure cell cycle phase. PKH-26 and other vital membrane dyes can discern divided and nondivided cells, but become less accurate the more a population of cells has divided. Tetrazolium salts are converted by metabolically active cells to formazan, which can be quantified by measuring absorbance at 490 nm.

See also: Apoptosis; B lymphocyte activation; B lymphocyte differentiation; CD3; CD28; CD40 and its ligand; Endotoxin (lipopolysaccharide (LPS)); Lifespan of immune cells and molecules; Memory, immunological; MHC restriction; Polyclonal activators; Superantigens; T lymphocyte activation; T lymphocyte clonal expansion; T lymphocyte differentiation.

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