Quantification of Human T Cell Responses to Chlamydia trachomatis: Intracellular Cytokine Staining (ICS) and Activation-Induced Marker (AIM) Assays.

High-resolution characterization of antigen-specific T cell responses is essential for understanding cellular immunity to Chlamydia trachomatis infection in humans. This chapter describes two complementary flow cytometry-based approaches for measuring human antigen-specific T cell responses: intracellular cytokine staining (ICS) and activation-induced marker (AIM) staining. Both assays enable single-cell-level analysis following antigen stimulation but differ in their readouts. ICS quantifies functional cytokine production in response to short- or long-term stimulation, whereas the AIM assay identifies antigen-specific T cells based on transient surface expression of activation markers without fixation or permeabilization, preserving cells for downstream applications. Detailed protocols are provided for peripheral blood mononuclear cell (PBMC) thawing and preparation, short-term or long-term antigen stimulation, and flow cytometry staining, as well as practical considerations for assay selection, experimental controls, optimization, and data acquisition.