1. ECs form a damage-resistant regenerative niche in the thymus.
ECs form a damage-resistant regenerative niche in the thymus. (A) Cell subsets in the thymus were assessed at day 7 after TBI, and the depletion was calculated compared with an untreated age-matched control cohort (n = 10 to 25 per subset). Subsets analyzed include DN1 (CD44+CD25−), early T-lineage progenitor (ETP; CD44+CD25−c-kit+), DN2 (CD44+CD25−), DN3 (CD44+CD25−), DN4 (CD44+CD25−), DP, SP4, SP8, CD8+ or CD8− DCs, MHCIIhi or MHCIIlo cTEChi/lo (CD45−EpCAM+MHCII+Ly51+UEA1lo), MHCIIhi or MHCIIlo mTEChi/lo (CD45−EpCAM+MHCII+Ly51loUEA1+), fibroblast, ECs, and ILCs (CD45+CD3−CD8−IL7Rα+CD4+RORγt+CCR6+). (B and C) Six-week-old female C57BL/6 mice were treated with PBS (n = 10), Dex (50 mg/kg, ip, on day 0; n = 10), Cyclo (100 mg/kg per day, ip, on days −1 and 0; n = 10), or TBI (550 cGy on day 0; n = 10). On day 4, mice were perfused with 25 μg of anti–VE-cadherin antibody (BV13) conjugated to Alexa Fluor 647 and sacrificed, and total thymic cellularity and EC number were assessed. (B) Total thymic cellularity and absolute number of ECs. (C) Concatenated flow cytometry plots detailing the proportion of VE-cadherin+CD45− cells in the thymus. SL-TBI, sublethal TBI. (D and E) C57BL/6 mice were given TBI (n = 10 to 15 per group) and assessed on days 4, 7, and 14. On the day of harvest, mice were perfused with 25 μg of anti–VE-cadherin antibody (BV13) conjugated to Alexa Fluor 647. (D) Total cellularity (open circles) and absolute number of ECs (closed circles) in the thymus were calculated using flow cytometry. (E) Proportion of VE-cadherin+ ECs as a function of CD45− stromal cells. Flow cytometry plots represent concatenated data from one experiment. (F) Proportion of ECs as a function of total thymic cellularity (n = 14 to 20 from four independent experiments). (G to M) Three-dimensional reconstruction of thymus vasculature at days 0, 4, 7, and 14 after TBI using LSFM (n = 3 per time point). (G) Visualization of VE-cadherin staining in the thymus. (H) Calculation of the volume of whole thymus and vasculature. (I) Total number of vessel segments in the thymus vasculature after damage where segments were defined as the length of the vessel between two branching points. (J) Vessel segments were binned according to their length. Total vasculature length was calculated. (K) Vascular segments were color-coded on the basis of branch level. (L) Number of segments per branch level and the total number of vessel branches. (M) Vascular density was calculated as a ratio of vascular network volume and as a function of total thymus volume [from (H)]. Bar graphs represent means ± SEM. *P < 0.05, **P < 0.01, ***P <
Tobias Wertheimer et al. Sci. Immunol. 2018;3:eaal2736
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