Journal: Frontiers in Immunology

Article Title: Intact lymph node homing and CD8 + T-cell priming abilities of Sprouty2-deficient dendritic cells

doi: 10.3389/fimmu.2026.1761675

Figure Lengend Snippet: Spry2-deficient DCs show efficient CCR7-driven migration and in vivo LN homing. (A, B) Spry2 +/+ and Spry2 -/- mBMDCs were embedded into a 3D collagen matrix using µ-slide chemotaxis chambers and allowed to migrate along CCL19 gradients. Cell migration was recorded by time-lapse video microscopy and cells were manually tracked. (A) Experimental setup using the µ-slide chemotaxis chamber (left panel) and migration tracks of 30 mBMDCs from one representative experiment centered on the same starting point (right panels). (B) Quantification of velocity, directionality and forward migration index (xFMI). Individual data points represent means of independent experiments, bars show mean ± SEM of 79 cells pooled from 3 independent experiments. (C-F) Spry2 +/+ and Spry2 -/- mBMDCs were labeled with CellTracker Green (CTG) or CellTracker Deep Red (CTDR) and a 1:1 mixture was injected into the footpad of C57BL/6J recipient mice. Immigration of mBMDCs in draining and contralateral popliteal LNs (dPLNs and clPLNs) was determined after 24 h by flow cytometry. (C) Scheme of the LN homing assay. (D) Ratio of injected CTG or CTDR labeled mBMDCs. (E) Representative contour plots showing immigrated CTG- and CTDR-labeled mBMDC populations gated on live/CD11c + MHC-II high migratory DCs in PLNs. (F) Frequencies of Spry2 +/+ and Spry2 -/- mBMDCs in dPLNs, pre-gated on live/CD11c + MHC-II high cells in dPLNs. Data points of 12 mice pooled from 3 independent experiments. (G-I) Emigration of skin DCs from Spry2 +/+ and Spry2 -/- split ear explants floating on media containing CCL19 and CCL21 (20 nM each) for 72 h was quantified by flow cytometry. (G) Scheme of the ear crawl-out assay. (H) Representative contour plots depicting frequencies of CD11c + MHC-II + DCs emigrated from skin explants. (I) Frequencies (% of live cells; left panel) and absolute numbers (right panel) of emigrated CD11c + MHC-II + DCs. Bars show mean ± SEM from explants of 5 mice from at least 4 independent experiments. (J) Ear skin of Spry2 +/+ and Spry2 -/- mice was digested using liberase TL and DC populations were analyzed by flow cytometry. Frequencies of CD11c + MHC-II + DCs in the skin, pre-gated on live cells in the skin. Bars show mean ± SEM of 4 individual mice from 3–4 independent experiments. (K-M) Abdomen of Spry2 +/+ and Spry2 -/- mice were shaved and a dibutylphtalate (DBP) containing FITC solution was applied. Frequencies of FITC + skin DCs in dLNs (axial and brachial) were determined after 24 h by flow cytometry. (K) Scheme of the FITC skin painting assay. (L) Representative contour plots depicting FITC + populations gated on CD11c + MHC-II high migratory DCs in dLNs of unpainted control (w/o FITC) and FITC-painted (w/FITC) Spry2 +/+ and Spry2 -/- mice. (M) Frequencies of FITC + CD11c + MHC-II high DCs that migrated to dLNs (% within CD11c + MHC-II high cells in the LN). Bars show mean ± SEM of 11 mice from 4 independent experiments. Statistical differences were determined using Student’s unpaired (B, I, J, M) and paired (F) t-test; ns = not significant (p > 0.05).

Article Snippet: C57BL/6J (H-2 b ) mice were originally purchased from Charles River, Germany.

Techniques: Migration, In Vivo, Chemotaxis Assay, Microscopy, Labeling, Injection, Flow Cytometry, Control