In-depth characterization of T cell responses with a combined Activation-Induced Marker (AIM) and Intracellular Cytokine Staining (ICS) assay.

Since T cells are key mediators in the adaptive immune system, evaluating antigen-specific T cell immune responses is pivotal to understanding immune function. Commonly used methods for measuring T cell responses include Activation-Induced Marker (AIM) assays and Intracellular Cytokine Staining (ICS). However, combining these approaches has rarely been reported. This study describes a combined AIM + ICS assay and the effect of collecting the supernatant. Peripheral blood mononuclear cells (PBMCs) from seven healthy donors were stimulated with DMSO (negative control), Epstein-Barr virus (EBV) peptide pools, and PHA (positive control). The AIM markers OX40 + CD137+ were used for CD4+ T cells and CD69 + CD137+ and CD107a + CD137+ for CD8+ T cells. Cytokine-secreting cells were identified as CD40L+ cytokine+ for CD4+ and CD69+ or CD107 + cytokine+ for CD8+ T cells. Half of the supernatant was collected before adding the BFA/Monensin/CD137 antibody solution to assess the impact on T cell responses. The CD107a + CD137+ AIM markers combination had a lower background than CD69 + CD137+, making CD107a+ a more sensitive marker for CD8+ AIM markers. Collecting half of the supernatant did not significantly affect the immune responses. Our AIM + ICS combined protocol enables the simultaneous assessment of activation and cytokine release reducing the sample volume for testing T cell responses. We also show that collecting half of the supernatant does not significantly interfere with immune responses detection.