Optimization and Use of ELISpot and Proliferation Assays: Characterizing a CD4+ T-Cell Immune Response to C. trachomatis in Adolescent Women

Chlamydia trachomatis is the most prevalent STI in the United States, with the highest rates of infection reported among adolescent females6. The development of treatment strategies to prevent infection and the associated reproductive pathology
requires a better understanding of the CD4+ T-cell immune responses to Chlamydia infection. PBMCs isolated from a seropositive donor and a seronegative donor were stimulated with various concentrations of chlamydial antigens (Serovar D EB and
CHSP60-l) and underwent 24- and 48-hour incubations. T-cell proliferation and cytokine secretion was quantified by 3H proliferation and dual-color ELISpot assays, respectively. Experimental assays revealed that PBMCs stimulated with 1 uglmL of chlamydial antigens and incubated for 48 hours was optimal for stimulating T -cells from a seropositive donor to proliferate more robustly and to display more specific secretion of IFN-y than T-cells from a seronegative donor. In a follow-up study with a small cohort of adolescent women (13 :'S Mage:'S 25) at high risk for Chlamydia infection, statistical analyses of experimental assays suggested that alamar blue T-cell proliferation and ELISpot assays were less accurate at diagnosing Chlamydia than current PCR diagnostic methods. Furthermore, analyses revealed that experimental assays were unable to predict immune status differences and to detect pathology in adolescent women. Although, exploratory ROC analyses suggested that experimental assays may have the potential to detect pathology among patients, such that AUC values for experimental assays did better than chance (AUC > 0.5) at predicting pathology when CD4+ T-cells were stimulated
with chlamydial antigens. "