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.

The CXCR4-CXCL12 axis promotes T cell reconstitution via efficient hematopoietic immigration

T cells play a critical role in immunity to protect against pathogens and malignant cells.T cell immunodeficiency is detrimental,especially when T cell perturbation occurs during severe infection,irradiation,chemotherapy,and age-related thymic atrophy.Therefore,strategies that enhance T cell reconstitution provide considerable benefit and warrant intensive investigation.Here,we report the construction of a T cell ablation model in Tg(coro1a:Den NTR)zebrafish via metronidazole administration.The nascent T cells are mainly derived from the hematopoietic cells migrated from the kidney,the functional homolog of bone marrow and the complete recovery time is 6.5 days post-treatment.The cxcr4b gene is upregulated in the responsive hematopoietic cells.Functional interference of CXCR4 via both genetic and chemical manipulations does not greatly affect T lymphopoiesis,but delays T cell regeneration by disrupting hematopoietic migration.In contrast,cxcr4b accelerates the replenishment of hematopoietic cells in the thymus.Consistently,Cxcl12b,a ligand of Cxcr4,is increased in the thymic epithelial cells of the injured animals.Decreased or increased expression of Cxcl12b results in compromised or accelerated T cell recovery,respectively,similar to those observed with Cxcr4b.Taken together,our study reveals a role of CXCR4-CXCL12 signaling in promoting T cell recovery and provides a promising target for the treatment of immunodeficiency due to T cell injury.