Activation-induced marker (AIM) assay to detect Chlamydia trachomatis-specific T cells in highly exposed women 2261007

Abstract Introduction Natural immunity to Chlamydia trachomatis (CT) develops in some women but is weak and requires sensitive assays. Activation-induced marker (AIM) assays detect antigen-specific T cells via surface markers and offer higher sensitivity, reduced cytokine bias, and intact viability compared with intracellular cytokine staining, important for sorting cells for downstream single-cell analyses. Methods PBMCs from 115 women highly exposed to CT (TRAC2) and 12 seronegative controls were stimulated for 24h with chlamydial CPAF or MOMP peptide pools. Cells were stained for viability, CD4, CD8, and six AIMs (CD69, PD-L1, CD25, OX40, CD40L, 4-1BB). AIM+ cells (expressing ≥2 markers) were normalized to a Leukopak control in each batch and background-subtracted using paired media controls. Results TRAC2 CT+ participants had significantly higher AIM+ CPAF- (p = 0.001) and MOMP-specific (p = 0.007) CD4 responses than controls, while CD8 responses were not significantly elevated (p = 0.18 and p = 0.27, respectively). Of CT+ participants, 41% (39/95) and 40% (37/93) had AIM+ CD4 responses to CPAF and MOMP (>2X control mean), respectively, while 20% (19/95) and 15% (14/93) showed corresponding CD8 responses. CD69 and PD-L1 were most commonly co-expressed, but with variable patterns across individuals. Conclusion AIM assays markedly improve detection of CT-specific T cells and support the central role of CD4 T cells in responses to CT infection. This platform will enable sorting and multi-omic profiling of CT-specific T cells across blood, cervical, and endometrial TRAC2 tissues by Ab-, mRNA-, and TCR-seq. AIM+ PBMCs will define individualized CT-specific TCR repertoires to match clonotypes in fragile mucosal samples without ex vivo stimulation. We will assess whether CT-specific cells are skewed toward distinct T-helper phenotypes or transcriptional profiles, whether they differ by antigen (CPAF vs MOMP), and which are linked to reduced recurrent or ascending infection. Funding Source T32 AI007001, R01 AI170959 Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)