Skip to main content
. 2018 Sep 6;7:e35816. doi: 10.7554/eLife.35816

Figure 8. 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.

Figure 8.

Figure 8—figure supplement 1. 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.

Figure 8—figure supplement 1.

Surface expression of integrins on HPCs (Linneg, c-kit+) and T cell progenitors (Linneg, c-kit+, PIR+). FL cells from E13.5 control and Fermt3-/- embryos were stained for lineage markers (B220, CD19, TER119, NK1.1, Gr-1 and Thy1.2), c-kit, PIR and one of the indicated integrins.
Figure 8—figure supplement 2. 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.

Figure 8—figure supplement 2.

ICAM-1 (A) and VCAM-1 (B) expression was quantified by measuring the fluorescence intensities of immunofluorescence stainings of CD31+ pharyngeal vessels at E12.5 and E13.5 and intrathymic vessels at E18.5. N = 3. (C–E) Flow cytometric analysis of ICAM-1 (C), ICAM-2 (D) and VCAM-1 (E) expression of CD31+ endothelial cells from the thymus/neck region of E12.5, E13.5 and E18.5 embryos. N = 3. Bars indicate means ± standard deviation. Bars indicate means ±standard deviation.