Clinical Information

The method of determining impaired T-cell function by culturing human peripheral blood mononuclear cells (PBMC) in vitro with mitogenic plant lectins (mitogens), such as phytohemagglutinin (PHA) and pokeweed mitogen (PWM) has been part of the diagnostic immunology repertoire for many years.(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 with 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 contribution of activation-induced 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 PBMC 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.

Mitogens are very potent stimulators of T-cell activation and proliferation independent of their antigenic specificity.(3) It has been suggested that mitogens can induce T-cell proliferative responses even if they are incapable of responding adequately to antigenic (physiologic) stimuli. Therefore, abnormal T-cell responses to mitogens are considered a diagnostically less sensitive, but more specific, test of aberrant T-cell function. Lectin mitogens have been shown to bind the T-cell receptor, which is glycosylated through its carbohydrate moiety, thereby activating quiescent T cells. Mitogenic stimulation has been shown to increase intracellular calcium (Ca[2+]) in T cells, which is essential for T-cell proliferation. While PHA is a strong T-cell mitogen, PWM is a weak T-cell mitogen but induces B-cell activation and proliferation as well.

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. 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.(4) 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 (7-AAD) and annexin V.

The Click-iT-EdU assay has shown to be an acceptable alternative to the (3)H-thymidine assay for measuring lymphocyte/T-cell proliferation.(5,6)

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, are 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, 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(7) and during summer compared to winter.(8) These data therefore indicate that timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets.