Establishment of the Reference Intervals of Lymphocyte Function in Healthy Adults Based on IFN-γ Secretion Assay upon Phorbol-12-Myristate-13-Acetate/Ionomycin Stimulation

Abstract

The function of lymphocytes is the key to reflect the immune status of hosts. Evaluation of lymphocyte function is a useful tool to monitor the effect of immunosuppressive treatment and predict the prognosis of immune-mediated diseases (e.g., cancer, autoimmune diseases, and infectious diseases). As the lymphocytes have various activities, such as activation, cytotoxicity, and cytokine secretion, it is a challenge to evaluate the function of lymphocytes in clinical practice and the reference intervals (RIs) of lymphocyte function are rarely reported. The present study showed that the secretion of IFN-γ was well correlated with the activation, chemotaxis, and cytotoxicity of CD4+, CD8+ T cells, and NK cells, which suggests that IFN-γ production can be used as a symbol of lymphocyte function. We therefore created a simple method to detect the function of CD4+, CD8+ T cells, and NK cells simultaneously according to IFN-γ secretion by using whole blood instead of peripheral blood mononuclear cells. We further established the RIs of lymphocyte function (CD4+ T cells: 15.31–34.98%; CD8+ T cells: 26.11–66.59%; NK cells: 39.43–70.79%) in healthy adults. This method showed good reproducibility for the evaluation of lymphocyte function. The established RIs were suitable for use in other centers based on the validation data. We also validated the RIs in individuals with different immune status, and the results showed that kidney transplant recipients and infants (0–1 year) had a decreased lymphocyte function, whereas T cells in systemic lupus erythematosus patients exhibited an opposite trend. Overall, we have successfully established the RIs of lymphocyte function in healthy adults in a simple way, which might be of important clinical value in the diagnosis, monitoring, and prognosis of immune-related diseases.

Variability of ex-vivo stimulated T-cells secretory profile in healthy subjects

“…Although macrophages are the main source of TNF-α (49), the production of this cytokine was also described for T-cells (46). A recent study, also aiming to establish a reference interval, reported the following proportions of activated cytokine-producing cells, compared with us: CD4+ 30% IL-2+ (vs. 61%), 25% TNF-α+ (vs. 27%), 23% IFN-γ+ (vs. 7%); together with CD8+ cells 3% IL-2+ (vs. 25%), 25% TNF-α+ (vs. 7%) and 43% IFN-γ+ (vs. 8%) (35). Such significant differences are very common between ICS protocols and, to a certain extent, between other flow cytometry experiments.…”

“…Nevertheless, our subsequent experience with PBMC density gradient isolation (this study included) showed us that processing blood volumes higher than 3mL will in fact significantly lower the cell yield. Several concentrations of PMA/ION are reported in the literature for PBMC activation, ranging from 1 to 100ng/mL PMA (32-39) and from 0.4 to 1.4µM for ION (that is roughly 300-1000ng/ mL) (32)(33)(34)(35)(36)38), depending on protocol requirements and personal preferences. While the exact PMA concentration values seem to be chosen arbitrarily (e.g.…”

The laboratory tests and host immunity of COVID-19 patients with different severity of illness

“…Lymphocyte function. PMA/ionomycin-stimulated lymphocyte function assay was performed as described in our previous studies (22,24). Briefly, 100 μL of whole blood was diluted with 400 μL of IMDM medium and stimulated with Leukocyte Activation Cocktail (containing PMA, ionomycin and brefeldin A, BD GolgiPlug) for 4 hours.…”

Dysregulation of Immune Response in Patients With Coronavirus 2019 (COVID-19) in Wuhan, China

“…PMA/ionomycin-stimulated lymphocyte function assay was performed as described previously. [11] The percentages of IFN-γ positive cells in different cell subsets were defined as the function of them. The data were reviewed by a trained team of physicians in Tongji Hospital.…”