High Levels of Human Antigen-Specific CD4+ T Cells in Peripheral Blood Revealed by Stimulated Coexpression of CD25 and CD134 (OX40)

Abstract

Ag-specific human CD4+ memory T lymphocytes have mostly been studied using assays of proliferation in vitro. Intracellular cytokine and ELISPOT assays quantify effector cell populations but barely detect responses to certain recall Ags that elicit strong proliferative responses, e.g., tetanus toxoid, that comprise non-Th1 CD4+ cells. We have found that culturing whole blood with Ag for 40–48 h induces specific CD4+ T cells to simultaneously express CD25 and CD134. This new technique readily detects responses to well-described CD4+ T cell recall Ags, including preparations of mycobacteria, CMV, HSV-1, influenza, tetanus toxoid, Candida albicans, and streptokinase, as well as HIV-1 peptides, with high specificity. The assay detects much higher levels of Ag-specific cells than intracellular cytokine assays, plus the cells retain viability and can be sorted for in vitro expansion. Furthermore, current in vitro assays for human CD4+ memory T lymphocytes are too labor-intensive and difficult to standardize for routine diagnostic laboratories, whereas the whole-blood CD25+CD134+ assay combines simplicity of setup with a straightforward cell surface flow cytometry readout. In addition to revealing the true extent of Ag-specific human CD4+ memory T lymphocytes, its greatest use will be as a simple in vitro monitor of CD4+ T cell responses to Ags such as tuberculosis infection or vaccines.

Human antigen‐specific CD4+CD25+CD134+CD39+ T cells are enriched for regulatory T cells and comprise a substantial proportion of recall responses

“…1). This result, plus our previously published data [19], confirmed that CD4 + CD25 + CD134 + T cells identified after a 44 hours Ag stimulation, and the CD39 + and CD39 − populations therein, up-regulate the activation markers CD25 and CD134 due to TCR stimulation, not through bystander activation.Regardless of Ag stimulus, we consistently observed that a substantial proportion of Ag-specific CD4 + CD25 + CD134 + T cells responding to stimulation expressed CD39 (30-60%), with the magnitude varying by Ag and individual (Fig. 1A).…”

“…In order to test whether CD39 staining would reveal a FOXP3 + sub-population of memory CD4 + T cells, we utilised our previously described 44 hours activation assay that identifies CD4 + T-cell Ag recall responses through the induced co-expression of CD25 and CD134 [19]. We incubated healthy donor whole blood with staphylococcus enterotoxin B (SEB), PHA, tetanus toxoid (Tet Tox), CMV lysate and HSV for 44 hours and then stained with anti-CD3, -CD4, -CD25, -CD134, -CD39 and -FOXP3 mAbs.…”

“…Importantly, we have previously found that using an activation time period of 40-44 hours means that the quantification of the identified response is not affected by either cell proliferation, bystander activation or substantial cell death via apoptosis [19]. CD134, a member of the TNF/TNFR family, functions as a co-stimulatory molecule and is important for the generation of the later stages of a recall response [28,29], but the role of CD134 signalling in Treg-cell function is currently debated.…”

“…This assay was performed as described previously [19]. Briefly, either fresh whole blood diluted 1:1 with RPMI-1640 media (Invitrogen) or isolated PBMCs in media at 1 × 10 6 cells/mL, was placed in 24-well plates, Ag was added and plates were incubated for 44 hours at 37°C with 5% CO 2 (humidified atmosphere).…”

“…CMV lysates were prepared and used at a final concentration of 1/250, as previously described [19]. Tet Tox was obtained from Commonwealth Serum Laboratories (CSL, VIC, Australia) and used at a final concentration of 2 Lf U/mL.…”