Clinical Information

Determining impaired T-cell function by culturing human peripheral blood mononuclear cells (PBMC) in vitro with recall antigens, including Candida albicans (CA) and tetanus toxoid (TT), has been part of the diagnostic immunology repertoire for many years.(1,2) A widely used method for assessing lymphocyte proliferation to antigens 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:

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.

Antigens, like CA and TT, have been widely used to measure antigen-specific recall (anamnestic) T-cell responses when assessing cellular immunity. In fact, it may be more revealing about cellular immune compromise than assessing the response of lymphocytes to mitogens because the latter can induce T-cell proliferative responses even if those T cells are incapable of responding adequately to antigenic (physiologic) stimuli. Therefore, abnormal T-cell responses to antigens are considered a diagnostically more sensitive, but less specific, test of aberrant T-cell function.(2)

Antigens used in recall assays measure the ability of T cells bearing specific T-cell receptors to respond to such antigens when processed and presented by antigen-presenting cells. The antigens used for assessment of the cellular immune response are selected to represent antigens, seen by a majority of the population, either through natural exposure (CA) or as a result of vaccination (TT).

This assay uses a method that directly measures the S-phase proliferation of lymphocytes through the use of Click chemistry. 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.(3,4)

The degree of impairment of antigen-specific T-cell responses can vary depending on the nature of the cellular immune compromise. For example, some, but not all, patients with partial DiGeorge syndrome, a primary cellular immunodeficiency, have been reported to have either decreased or absent T-cell responses to CA and TT.(5) Similarly, relative immune compromise, especially to TT, has been reported in children with vitamin A deficiency, but the measurements have been largely of the humoral immune response. Since this requires participation of the cellular immune compartment, it can be postulated that there could be a potential impairment of antigen-specific T-cell responses as well.(6)