Fig 2: Tissue-Specific Oscillations in Endothelial Cell Adhesion Molecules(A) Map of rhythmic protein expression of endothelial cell-adhesion molecules of various organs (n = 3–6 mice with 6 time points measured each; one-way ANOVA).(B) Integration of all expressed molecules over all organs across the day (n = 3–6 mice with 6 time points measured each; one-way ANOVA).(C) Integration of ICAM-1 and VCAM-1 expression over all organs across the day (n = 3–6 mice with 6 time points measured each; one-way ANOVA).(D) Adoptive transfer of donor cells to recipients treated with functional blocking antibodies directed against the indicated molecules at ZT1 and ZT13. Results are presented as percentages of injected cells (n = 4–12 mice; one-way ANOVA followed by Dunnett comparison to control groups and unpaired Student’s t test for comparisons between ZT1 and ZT13 groups).(E) Fold change of donor cells remaining in recipient blood at ZT1 and ZT13 after anti-VCAM-1 or anti-ICAM-1 antibody treatment, respectively, in comparison with numbers of isotype antibody controls (n = 7–11 mice; unpaired Student’s t test).(F) Adoptive transfer of donor cells to Icam1-/- recipients at ZT1 and ZT13 (n = 6–8 mice; unpaired Student’s t test).(G) Endogenous blood leukocyte numbers in Icam1-/- mice at ZT1 and ZT13 (n = 6–8 mice; unpaired Student’s t test).*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; #, ##, ###, #### indicate significance levels analogous to those of control groups. All data are represented as mean ± SEM. ns, not significant; MFI, mean fluorescence intensity. See also Figure S2 and Table S1.

Fig 3: Kindlin-3 is important to stabilize the adhesion of T cell progenitors and mature T cells to vascular integrin ligands when blood flow velocities and shear rate levels increase during development and in vessel segments of higher blood flow within lymph nodes.(A) Talin and kindlin-3 expression in FACS-sorted Pir+ T cell progenitor cells from WT FL of E13.5 embryos compared to DN1 cells sorted from the thymus of P6 mice. GAPDH served as loading control. (B) Talin and kindlin-3 expression in control and kindlin-3-/- Pir+ T cell progenitor cells isolated from E13.5 FL. GAPDH served as loading control. Mean blood flow velocities (C) and shear rates (D) within the yolk sac vasculature of E12.5 to E16.5 embryos were determined by intravital microscopy. N = 14/3/8/21/18. (E and F) Relative adhesion of Pir+ T cell progenitor cells FACS-sorted from the FLs of control and Fermt3-/- E13.5 embryos on ICAM-1, P-selectin, CCL21 and CCL25 or on VCAM-1, P-selectin, CCL21 and CCL25 coated ibidi flow chambers with stepwise increasing shear rates. N = 5. Bars indicate means ± standard deviation. (G) Purity of CD4+ T cells from WT and Fermt3 hypomorphic (n/-) mice that have been labelled with CFSE and Far Red and mixed in a 1:1 ratio. Grey line represents isotype control. (H,I) Adhesion of CD4+ T cells in vivo. (H) Representative microscopic images of adherent (+/+, red) and (n/-, green) cells in the lymph node vasculature after adoptive transfer. Sum intensity Z projections of confocal stacks are shown. Segmented lines indicate vessel outlines. Scale bar = 50 µm. (I) Quantification of adherent CD4+ T cells (N = 18–19 vessels from three mice). (J, K) Microvascular blood flow in the lymph node vasculature. (J) Centerline blood flow velocity and (K) vascular shear rate in LN microvessel segments (N = 25–27 field of views from three mice). Bars indicate means ± standard deviation. **p<0.01; ***p<0.001. See also Figure 8—figure supplements 1 and 2.