Lymphocyte Proliferation to Antigens, Blood
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
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) The widely used method for assessing lymphocyte proliferation to antigens has hitherto been the measurement of 3H-thymidine incorporated into the DNA of proliferating cells. The disadvantages with the 3H-thymidine method of lymphocyte proliferation are:
1. The technique is cumbersome due to the use of radioactivity
2. It does not allow discrimination of responding cell populations in response 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 decrease in proliferation of only a subset of T cells, or an apparent decrease in total lymphocyte proliferation due to T-cell lymphopenia and underrepresentation of T cells in the PBMC pool. None of these can be discriminated 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 sample.
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 (TCR) 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).
For this assay, we use 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-AAD and Annexin V. The Click-iT-EdU assay has already been shown to be an acceptable alternative to the 3H-thymidine assay for measuring lymphocyte/T-cell proliferation.(3)
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.(4) 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.(5)
Assessing T-cell function in patients on immunosuppressive therapy, including solid-organ transplant patients
Evaluating patients suspected of having impairment in cellular immunity
Evaluation of T-cell function in patients with primary immunodeficiencies, either cellular (DiGeorge syndrome, T-negative severe combined immunodeficiency: SCID, etc) or combined T- and B-cell immunodeficiencies (T- and B-negative SCID, Wiskott Aldrich syndrome, ataxia telangiectasia, common variable immunodeficiency, among others) where T-cell function may be impaired
Evaluation of T-cell function in patients with secondary immunodeficiency, either disease related or iatrogenic
Evaluation of recovery of T-cell function and competence following bone marrow transplantation or hematopoietic stem cell transplantation
Abnormal test results to antigen stimulation are indicative of impaired T-cell function, if T-cell counts are normal or only modestly decreased. If there is profound T-cell lymphopenia, it must be kept in mind that there could be a "dilution" effect with underrepresentation of T cells within the peripheral blood mononuclear cell (PBMC) population that could result in lower T-cell proliferative responses. However, this is not a significant concern in the flow cytometry assay, since acquisition of additional cellular events during analysis can compensate for artificial reduction in proliferation due to lower T-cell counts. In the case of antigen-specific T-cell responses to tetanus toxoid (TT), there can be absent responses due to natural waning of cellular immunity, if the interval between vaccinations has exceeded the recommended period, especially in adults. In such circumstances, it would be appropriate to measure TT-specific T-cell responses 4 to 6 weeks after a booster vaccination.
There is no absolute correlation between T-cell proliferation in vitro and a clinically significant immunodeficiency, whether primary or secondary, since T-cell proliferation in response to activation is necessary, but not sufficient, for an effective immune response. Therefore, the proliferative response to antigens can be regarded as a more sensitive, but less specific, test for the diagnosis of infection susceptibility.
It should also be kept in mind that there is no single laboratory test that can identify or define impaired cellular immunity, with the exception of an opportunistic infection.
Controls in this laboratory and most clinical laboratories are healthy adults. Since this test is used for screening and evaluating cellular immune dysfunction in infants and children, it is reasonable to question the comparability of proliferative responses between healthy infants, children, and adults. It is reasonable to expect robust T-cell-specific responses to TT in children without cellular immune compromise, as a result of repeated childhood vaccinations. The response to Candida albicans can be more variable depending on the extent of exposure and age of exposure. A comment will be provided in the report documenting the comparison of pediatric results with an adult reference range and correlation with clinical context for appropriate interpretation.
It should be noted that without obtaining formal pediatric reference values, it remains a possibility that the response in infants and children can be underestimated. However, the practical challenges of generating a pediatric range for this assay necessitate comparison of pediatric data with adult reference values or controls.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
When interpreting results it should be kept in mind that the range of lymphocyte proliferative responses observed in healthy, immunologically competent individuals is large. The reference ranges provided will be helpful in ascertaining the magnitude of the normal response.
Lymphocyte proliferation to mitogens is known to be affected by concomitant use of steroids, immunosuppressive agents, including cyclosporine, tacrolimus (FK506), Cellcept (mycophenolate mofetil), immunomodulatory agents, alcohol, and physiological and social stress.
Lymphocyte proliferation responses to antigens (and mitogens) are significantly affected by time elapsed since blood collection. Results have been shown to be variable for specimens assessed >24 and <48 hours post-blood collection. Therefore, lymphocyte proliferation results must be interpreted with due caution and results should be correlated with clinical context. Specimens >24-hours old may give spurious results.
Diminished results may be obtained in cultures that contain excess neutrophils or nonviable cells.(8)
Timing, and consistency in timing, of blood collection is critical when serially monitoring patients lymphocyte subsets (specifically T cells in this context) and their diurnal variation can potentially affect the magnitude of the proliferative response, especially in patients who already have severe T-cell lymphopenia. 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 (NK)-cell counts, on the other hand, are constant throughout the day. Circadian variations in circulating T-cell counts have been shown to be negatively correlated 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, and during summer compared to winter.
Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
Viability of lymphocytes at day 0: > or =75.0%
Maximum proliferation of Candida albicans as % CD45: > or =5.7%
Maximum proliferation of Candida albicans as % CD3: > or =3.0%
Maximum proliferation of tetanus toxoid as % CD45: > or =5.2%
Maximum proliferation of tetanus toxoid as % CD3: > or =3.3%
Clinical References Provides recommendations for further in-depth reading of a clinical nature
1. Dupont B, Good RA: Lymphocyte transformation in vitro in patients with immunodeficiency diseases: use in diagnosis, histocompatibility testing and monitoring treatment. Birth Defects Orig Artic Ser 1975;11:477-485
2. Stone KD, Feldman HA, Huisman C, et al: Analysis of in vitro lymphocyte proliferation as a screening tool for cellular immunodeficiency. Clin Immunol 2009;131:41-49
3. Yu Y, Arora A, Min W, et al: EdU-Click iT flow cytometry assay as an alternative to 3H-thymidine for measuring proliferation of human and mice lymphocytes. J Allergy Clin Immunol 2009;123(2):S87
4. Davis CM, Kancheria VS, Reddy A, et al: Development of specific T cell responses to Candida and tetanus antigens in partial DiGeorge syndrome. J Allergy Clin Immunol 2008,122:1194-1199
5. Semba RD, Muhilal, Scott AL, et al: Depressed immune response to tetanus in children with vitamin A deficiency. J Nutr 1992;122:101-107
6. Lis H, Sharon N: Lectins: carbohydrate-specific proteins that mediate cellular recognition. Chem Rev 1998;98:637-674
7. Salic A, Mitchison TJ: A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci USA 2008;105:2415-2420
8. Fletcher MA, Urban RG, Asthana D, et al: Lymphocyte proliferation. In Manual of Clinical Laboratory Immunology. Fifth edition. Edited by NR Rose, EC de Macario, JD Folds, et al. Washington, DC. ASM Press, 1997, pp 313-319