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PLoS One. 2017 Oct 24;12(10):e0186998. doi: 10.1371/journal.pone.0186998. eCollection 2017.

Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells.

Author information

Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California, United States of America.
CR-CHUM, Université de Montréal, Montreal, Québec, Canada.
Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID), La Jolla, California, United States of America.
UCSD School of Medicine, Division of Infectious Diseases, La Jolla, California, United States of America.
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Yerkes National Primate Research Center and Emory Vaccine Center, Atlanta, Georgia, United States of America.
Chronic Viral Illnesses Service and Division of Hematology, McGill University Health Centre, Montreal, Québec, Canada.


The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells.

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