Visualizing DC morphology and T cell motility to characterize DC-T cell encounters in mouse lymph nodes under mTOR inhibition

Mammalian target of rapamycin (mTOR), a serine/threonine kinase orchestrating cellular metabolism, is a crucial immune system regulator. However, it remains unclear how mTOR regulates dendritic cell (DC) function in vivo , especially DC-T cell encounters, a critical step for initiating adaptive immune responses. We dynamically visualized DC-T contacts in mouse lymph node using confocal microscopy and established an encounter model to characterize the effect of mTOR inhibition on DC-T cell encounters using DC morphology. Quantitative data showed mTOR inhibition via rapamycin altered DC shape, with an increased form factor (30.17%) and decreased cellular surface area (20.36%) and perimeter (22.43%), which were associated with Cdc42 protein downregulation (52.71%). Additionally, DCs adopted a similar morphological change with Cdc42 inhibition via ZCL278 as that observed with mTOR inhibition. These morphologically altered DCs displayed low encounter rates with T cells. Time-lapse imaging data of T cell motility supported the simulated result of the encounter model, where antigen-specific T cells appeared to reduce arrest in the lymph nodes of rapamycin-pretreated mice relative to the untreated group. Therefore, mTOR inhibition altered DC morphology in vivo and decreased the DC-T cell encounter rate, as well as Cdc42 inhibition. By establishing an encounter model, our study provides an intuitive approach for the early prediction of DC function through morphological quantification of form factor and area.