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The activation\u2010induced marker (AIM) assay is a robust technique to identify and characterize antigen\u2010specific CD4 and CD8 T cells. However, there is variability in the AIM assay, particularly in the type and number of activation markers used. In this study, we set out to define which marker combinations are most suited to optimally detect antigen\u2010specific CD4 and CD8 T cells and if certain marker combinations preferentially detect specific CD4 T helper subsets. A multiparameter flow cytometry panel, including six common activation markers: CD40L, CD137, CD69, OX40, CD25, and PD\u2010L1, was used for detecting antigen\u2010specific T cells following infection (SARS\u2010CoV\u20102 and CMV) or vaccination (mRNA\u20101273 SARS\u2010CoV\u20102). We demonstrate that combining multiple activation markers increases the detection frequency of antigen\u2010specific CD4 T cells compared to commonly used dual marker combinations. In addition, marker combinations including PD\u2010L1 detected a higher frequency of antigen\u2010specific CD4 T cells in SARS\u2010CoV\u20102 and CMV infected but not in SARS\u2010CoV\u20102\u2013vaccinated individuals. Certain dual marker combinations preferentially detected specific CD4 T helper subsets. The majority of antigen\u2010specific CD8 T cells were captured by the dual combination of CD69 plus CD25. In conclusion, combining CD137, CD69, OX40, CD25, and PD\u2010L1 in an AIM assay results in robust and optimal detection of both specific CD4 T helper subsets and CD8 T cells in different antigenic contexts.<\/jats:p>","DOI":"10.1155\/jimr\/1284907","type":"journal-article","created":{"date-parts":[[2026,2,5]],"date-time":"2026-02-05T07:55:33Z","timestamp":1770278133000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Combining Activation\u2010Induced Markers With PD\u2010L1 Selectively Enhances Detection of Antigen\u2010Specific T Cells in Virus\u2010Infected Individuals"],"prefix":"10.1155","volume":"2026","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8524-0622","authenticated-orcid":false,"given":"Christine","family":"Kreher","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-1575-2973","authenticated-orcid":false,"given":"Laura Y. 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