High levels of human antigen-specific CD4+ T cells in peripheral blood revealed by stimulated coexpression of CD25 and CD134 (OX40).

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.