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* Centre for Immunology, St. Vincent’s Hospital, Sydney, New South Wales, Australia;
National Centre in HIV Epidemiology and Clinical Research, University of New South Wales, Sydney, New South Wales, Australia;
Australian Red Cross Blood Service and Transfusion Medicine and Immunogenetics Research Unit, Central Clinical School, Faculty of Medicine, University of Sydney, Sydney, New South Wales, Australia;
Westmead Millennium Institute, Sydney University, Sydney, New South Wales, Australia; and
¶ Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia
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.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 J.J.Z., A.D.K., D.A.C., M.K., and A.L.C. are partly supported by program grants from the Australian National Health and Medical Research Council. M.L.M. and A.D.K. are recipients of a Dora Lush postgraduate scholarship and a Practitioner Fellowship, respectively, from the National Health and Medical Research Council. The National Centre in HIV Epidemiology and Clinical Research is supported by the Commonwealth Department of Health and Ageing.
2 Address correspondence and reprint requests to Dr. John J. Zaunders, Centre for Immunology, St. Vincent’s Hospital, Victoria Street, Darlinghurst, New South Wales 2010, Australia. E-mail address: j.zaunders{at}cfi.unsw.edu.au
3 Abbreviations used in this paper: ICC, intracellular cytokine; Flu, influenza A; LPA, lymphoproliferation assay; MAI, Mycobacterium avium; MTB, Mycobacterium tuberculosis; PET, polyester; SEB, staphylococcal enteroantigen B; SKSD, streptokinase; TT, tetanus toxoid.
4 The online version of this article contains supplemental material.
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