Activation phenotypes defined by the coordinated expression of activation markers discriminate TCR-mediated and...

Studying activation of antigen-specific T cells is essential for understanding adaptive immune response to pathogens, tumors, and vaccines. Flow cytometry is commonly used to identify antigen-specific T cells based on the induction of activation-induced markers (AIMs). However, these markers are also expressed on bystander T cells activated by cytokines produced by antigen-activated T cells. This complicates the distinction between true T cell receptor (TCR)-activated- and bystander T cells. We developed an approach to differentiate between these types of cells. We stimulated human PBMCs with anti-CD3 antibody (TCR-mediated) or supernatant of activated T cells, IL-2, or IL-15 (bystander activation). We analyzed AIM co-expression patterns on CD4⁺ and CD8⁺ T cells, and defined activation phenotypes that distinguish TCR-activated from bystander-activated T cells. In anti-viral responses, peptide stimulation mainly induced bystander activation, yet bystander T cells contributed minimally to cytokine production. Our findings provide a framework for identifying T cell response types in diverse clinical contexts.