Clinical Information

T-cell stimulation in vitro is used extensively in the diagnostic immunology arena for facilitating T-cell proliferation and evaluation of T-cell function in a variety of clinical contexts.(1,2) A widely used method for assessing lymphocyte proliferation has hitherto been the measurement of (3)H-thymidine incorporated into the DNA of proliferating cells. The disadvantages of the (3)H-thymidine method of lymphocyte proliferation are as follows:

1. The technique is cumbersome due to the use of radioactivity.

2. It does not distinguish between different cell populations responding to stimulation.

3. It does not provide any information on the contribution of apoptosis or cell death to the interpretation of the final result.

Further, decreased lymphocyte proliferation could be due to several factors, including overall diminution of T-cell proliferation, or an apparent decrease in total lymphocyte proliferation due to T-cell lymphopenia and under-representation of T cells in the peripheral blood mononuclear cell pool. None of these can be distinguished by the thymidine uptake assay but can be assessed by flow cytometry, which uses antibodies to identify specific responder cell populations. Cell viability can also be measured within the same assay without requiring additional cell manipulation or specimen.

While mitogens such as phytohemagglutinin activate T cells by binding to cell membrane glycoproteins, including the T-cell receptor (TCR)-CD3 complex, there are mitogenic or co-mitogenic antibodies, including those directed against the CD3 coreceptor that can stimulate T-cell proliferation. Typically, anti-CD3 antibodies provide an initial activation signal but do not induce significant proliferation, and the addition of a costimulatory antibody (anti-CD28) provides the stimulus for robust proliferation.(3) An exogenous T-cell growth factor, such as interleukin-2 (IL-2), may also be used as an alternate to anti-CD28 costimulation, and in patients with suspected IL-2 receptor-associated signaling defects, it may be more helpful than the use of anti-CD28. IL-2, an autocrine cytokine, has been demonstrated to be critical in T-cell proliferation.(4,5) The interaction of IL-2 with the IL-2 receptor (IL-2R) plays a central role in regulation of T-cell proliferation.(4) Triggering of the TCR leads to synthesis of IL-2 in certain T-cell subsets and induction of high-affinity IL-2Rs in antigen- or mitogen-activated T cells, and the binding of IL-2 to IL-2R ultimately leads to T-cell proliferation. The use of exogenous IL-2 in association with anti-CD3 allows discrimination of whether T cells, which cannot proliferate to other mitogenic signals, can respond to a potent growth factor such as IL-2. Stimulation of T cells with soluble antibodies to anti-CD3 (and the associated TCR complex) causes mobilization of cytoplasmic calcium and translocation of protein kinase C from the cytoplasm to the cell membrane. This stimulation also causes induction of phosphatidylinositol metabolism and subsequent IL-2 production for proliferation.(6) T-cell activation induced by anti-CD3 antibody requires prolonged stimulation of protein kinase C, which apparently can be achieved by the concomitant use of the anti-CD28 antibody for costimulation without addition of other mitogenic stimuli, such as phorbol myristate acetate.(7)

This assay uses a method that directly measures the S-phase proliferation of lymphocytes through the use of click chemistry. In the Invitrogen Click-iT-EdU assay, the click chemistry has been adapted to measure cell proliferation through direct detection of nucleotide incorporation. The Click-iT-EdU assay has been shown to be an acceptable alternative to the (3)H-thymidine assay for measuring lymphocyte/T-cell proliferation.(8,9)

In the assay, an alkyne-modified nucleoside is supplied in cell-growth media for a defined period and is incorporated within cells. The cells are subsequently fixed, permeabilized, and reacted with a dye-labeled azide, catalyzed by copper. A covalent bond is formed between the dye and the incorporated nucleotide, and the fluorescent signal is then measured by flow cytometry.(10) Specific proliferating cell populations can be visualized by the addition of cell-specific antibodies. Cell viability, apoptosis, and death can also be measured by flow cytometry using 7-aminoactinomycin D and annexin V.

The absolute counts of lymphocyte subsets are known to be influenced by a variety of biological factors, including hormones, the environment, and temperature. The studies on diurnal (circadian) variation in lymphocyte counts have demonstrated progressive increase in CD4 T-cell count throughout the day, while CD8 T cells and CD19+ B cells increase between 8:30 a.m. and noon, with no change between noon and afternoon. Natural killer-cell counts, on the other hand, is constant throughout the day. Circadian variations in circulating T-cell counts negatively correlate with plasma cortisol concentration. In fact, cortisol and catecholamine concentrations control distribution and, therefore, the numbers of naive versus effector CD4 and CD8 T cells. It is generally accepted that lower CD4 T-cell counts are seen in the morning compared with the evening(11) and during summer compared to winter.(12) These data therefore indicate that timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets.