1. Developmental Biology
  2. Immunology and Inflammation
Download icon

Differential requirement of kindlin-3 for T cell progenitor homing to the non-vascularized and vascularized thymus

  1. Federico Andrea Moretti
  2. Sarah Klapproth
  3. Raphael Ruppert
  4. Andreas Margraf
  5. Jasmin Weber
  6. Robert Pick
  7. Christoph Scheiermann
  8. Markus Sperandio
  9. Reinhard Fässler
  10. Markus Moser  Is a corresponding author
  1. Max-Planck-Institute of Biochemistry, Germany
  2. Ludwig-Maximilians-Universität, Germany
Research Article
Cite this article as: eLife 2018;7:e35816 doi: 10.7554/eLife.35816
9 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Kindlin-3 deficiency causes thymus atrophy.

(A and B) Images from Fermt3+/+ (upper panel) and Fermt3-/- (lower panel) thymi isolated at the indicated embryonic and postnatal stages. Scale bars 1 mm. (C) Total thymocyte numbers at indicated time points (E14.5 to P8). N(E14.5)=28/28; N(E16.5)=12/12; N(E18.5)=30/21; N(P3)=6/4; N(P8)=3/2. (D) Haematoxylin and Eosin staining of paraffin-embedded thymus sections. Scale bar 1 mm. (E and F) Frequencies of CD4 and CD8 double negative (DN), double positive (DP) and single positive CD4 and CD8 thymocytes isolated from P3 and P6 thymi. N(P3)=16/18; N(P6)=8/4. Bars indicate means ± standard errors. **p<0.01; ***p<0.001. See also Figure 1—figure supplement 1.

https://doi.org/10.7554/eLife.35816.002
Figure 1—figure supplement 1
Kindlin-3 deficiency causes thymus atrophy.

(A) Kindlin-3 expression in protein lysates from FACS sorted CD4/CD8 double negative (DN), CD4/CD8 double positive (DP), CD4 and CD8 single positive thymocytes. GAPDH served as loading control. (B) Expression of kindlin-1,–2 and −3 in protein lysates of Fermt3+/+ and Fermt3-/- thymi. Keratinocyte and embryonic stem cell (ESC) lysates were used as positive controls for kindlin-1 and −2 expression, respectively. GAPDH was used as loading control. (C) Cryosections of P3 Fermt3+/+ and Fermt3-/- thymi were stained with anti-pan-laminin (red), anti-CD4 (green) and anti-CD8 (blue) antibodies (left panel), or with anti-pan-cytokeratin (green), anti-PECAM-1 (red) antibodies and DAPI (blue) (right panel). Cells in cyan indicate CD4/CD8 double positive cells. M, medulla; C, cortex; V, vessel; CMJ, cortico-medullary junction. Scale bar 100 µm. Absolute numbers of DN, DP, CD4+ and CD8+ thymocytes present in P3 (D) and P6 (E) thymi. (F) Expression of TCRβ, CD3, CD5 and CD24 on DP, CD4+ and CD8+ thymocytes from P3 Fermt3+/+ and Fermt3-/- thymi analysed by flow cytometry. Isotype matched controls (blue); Fermt3+/+ cells (green); Fermt3-/- cells (red). Bars indicate means ± standard errors. **p<0.01; ***p<0.001.

https://doi.org/10.7554/eLife.35816.003
Figure 2 with 1 supplement
Reduced thymocyte proliferation contributes to thymus atrophy of kindlin-3-deficient mice.

(A) Staining for cleaved caspase-3 counterstained with Mayer´s haematoxylin on paraffin sections of Fermt3+/+ and Fermt3-/- P3 thymi. Scale bar 100 µm. (B) Single-cell suspensions of thymocytes from control and Fermt3-/- P3 thymi were stained with the apoptotic (Annexin V) and dead cell (7-AAD) markers. Numbers within the FACS blots represent percent of cells within each quadrant. (C) Number of apoptotic cells per field of view (fov) observed in (A). N = 10. Percentage of early apoptotic cells (Annexin V+, 7-AAD-) in control and Fermt3-/- thymi (D) and in distinct T cell subpopulations (CD4/CD8 DN, CD4/CD8 DP, CD4 single positive and CD8 single positive) (E). N = 7/10. (F) Sections of Fermt3+/+ and Fermt3-/- P3 thymi stained for BrdU incorporation and counterstained with Mayer´s haematoxylin. Scale bar 100 µm. (G) BrdU incorporation in thymocytes analysed by flow cytometry. (H) Numbers of BrdU positive cells per field of view (fov) observed in (F). N = 8/6. (I) Percentage of proliferating, BrdU positive thymocytes measured by flow cytometry. N = 14. (J) Percentage of BrdU positive cells in distinct T cell subpopulations. N = 14. (K) CD4 T cells isolated from spleens of control Fermt3fl/fl/2D2 and Fermt3fl/fl/2D2/CD4Cre mice were stained with CFSE and stimulated either with DCs loaded with different concentrations of MOG35-55 peptide or primed with anti-CD3e/CD28 antibodies and PMA. Representative histograms show CSFE dilution. Red-lined histograms represent cells incubated with not-loaded DCs or no antibodies. Bars indicate means ± standard errors. **p<0.01; ***p<0.001. See also Figure 2—figure supplement 1.

https://doi.org/10.7554/eLife.35816.004
Figure 2—figure supplement 1
Reduced thymocyte proliferation contributes to thymus atrophy of kindlin-3-deficient mice.

Kindlin-3-deficient CD4+ T cells are able to form immune synapses with dendritic cells. Mature wild-type dendritic cells loaded with MOG35-55 peptide were incubated with CD4+ T cells from Fermt3fl/+ (K3fl/+) and Fermt3fl/fl (K3fl/fl) mice expressing Cre-recombinase under the control of the CD4 promoter, a MOG35-35 specific TCR (2D2) and a double fluorescent reporter transgene to verify Cre activity (mT/mG). Cells were stained for intergin LFA-1, tyrosin-phosphorylated proteins and actin and imaged by confocal microscopy. Size bar 10 µm.

https://doi.org/10.7554/eLife.35816.005
Figure 3 with 1 supplement
Reduced frequencies of T cell progenitors in Fermt3-/- thymi.

(A) Thymocytes from Fermt3+/+ and Fermt3-/- mice were stained for lineage markers (B220, CD19, TER119, NK1.1, CD11b, Gr-1, CD8α, CD3e, TCRβ, TCRγδ and CD11c), CD44 and c-kit to identify DN1-2 (Linneg, c-kithi, CD44hi) and the DN3-4 (Linneg, c-kitlow, CD44low) populations. (B and C) PB from Fermt3+/+ and Fermt3-/- animals (P3) were stained for lineage markers (TER119, B220, CD11b, Gr-1, CD11c, NK1.1, CD4 and CD8α), c-kit and Sca-1 to identify hematopoietic progenitor cells and analysed by flow cytometry. N = 4. Numbers within the representative FACS plots indicate cell percentages. Bars indicate means ± standard errors. **p<0.01; ***p<0.001. See also Figure 3—figure supplement 1.

https://doi.org/10.7554/eLife.35816.006
Figure 3—figure supplement 1
Reduced frequencies of T cell prognitors in Fermt3-/- thymi.

Thymocytes from Fermt3+/+ and Fermt3-/- P3 mice were stained for lineage markers (B220, CD19, TER119, NK1.1, CD11b, Gr-1, CD8α, CD3ε, TCRβ, TCRγδ and CD11c), CD44 and CD25 to identify and quantify DN1 (Linneg, CD44hi, CD25-), DN2 (Linneg, CD44hi, CD25+), DN3 (Linneg, CD44low, CD25+) and DN4 (Linneg, CD44low, CD25-) populations by flow cytometry. Numbers within FACS plots indicate cell percentages. Total number of DN1, DN2, DN3 and DN4 cells were measured by flow cytometry. N = 20. Bars indicate means ± standard errors. *p<0.05; **p<0.01; ***p<0.001.

https://doi.org/10.7554/eLife.35816.007
Figure 4 with 2 supplements
Kindlin-3-deficient T cell progenitors are severely impaired in seeding the adult thymus.

(A–C) Images (A), weights (B) and cell numbers (C) of thymi from adult WT and Rag2-/- mice and Rag2-/- mice 10 weeks after reconstitution with Fermt3+/+ and Fermt3-/- fetal liver cells. Scale bar 5 mm. N = 10. (D) Single-cell suspensions were stained for lineage markers (B220, CD19, TER119, NK1.1, CD11b, Gr-1, CD11c, CD8α, CD3e, TCRβ, and TCRγδ), c-kit, CD44 and CD25 to identify the DN1-2 (Linneg, c-kithi, CD44hi) and the DN3-4 (Linneg, c-kitlow, CD44low) populations (upper panel), and DN1 (Linneg, CD44hi, CD25-), DN2 (Linneg, CD44hi, CD25+), DN3 (Linneg, CD44low, CD25+) and DN4 (Linneg, CD44low, CD25-) populations (lower panel) by flow cytometry. Frequencies of DN1-2 (E) and DN4 (F) thymocytes. N = 8–10. (G) Analysis of peripheral blood T-lymphocytes. (H and I) Frequencies of circulating CD4+ and CD8+ T cells calculated from flow cytometric analyses shown in (G). N = 10. Numbers within the representative FACS plots indicate cell percentages. (J) Expression of talin-1 and kindlin-3 in CD4T cells isolated from the spleen of Rag2-/- mice after reconstitution with Fermt3+/+ and Fermt3-/- fetal liver cells. GAPDH serves as loading control. (K) genomic PCR of CD8+ T cells sorted from the spleen of Rag2-/-/Fermt3+/+ and Rag2-/-/Fermt3-/- chimeras. Numbers within the representative FACS plots indicate cell percentages. Bars indicate means ± standard errors. **p<0.01; ***p<0.001. See also Figure 4—figure supplements 1 and 2.

https://doi.org/10.7554/eLife.35816.008
Figure 4—figure supplement 1
Kindlin-3-deficient T cell progenitors are severely impaired in seeding the adult thymus.

(A) Thymocytes from polyIC-induced Fermt3fl/fl/Mx1-Cre-negative and Fermt3fl/fl/Mx1-Cre-positive mice were stained for lineage markers (B220, CD19, TER119, NK1.1, CD11b, Gr-1, CD8α, CD3ε, TCRβ, TCRγδ and CD11c), CD44 and c-kit to identify the DN1-2 population (Linneg, c-kithi, CD44hi). (B) Cell numbers, weight and flow cytometric analyses of thymi from mixed FL cell chimeras, which received a 9:1 ratio of FL cells from Fermt3+/+ or Fermt3-/- embryos (CD45.2) and WT B6.SJL FL cells (CD45.1). Cells were stained for CD45.2, CD4 and CD8. Numbers within FACS plots indicate cell percentages. Frequencies of DP, DN and SP CD4 and CD8 T cells were calculated from flow cytometric analyses. N = 8/10. Bars indicate means ± standard errors.

https://doi.org/10.7554/eLife.35816.009
Figure 4—figure supplement 2
Kindlin-3-deficient T cell progenitors are severely impaired in seeding the adult thymus.

Kindlin-3 is exclusively expressed in hematopoietic cells of the thymus. (A) Single cell suspensions of thymi from E15.5 embryos of WT (+/+) and Kindlin-3-EGFP knockin (Fermt3-EGFP ki/ki) mice were stained for CD45.2 and the thymic epithelial marker EpCAM. Both populations were analysed for EGFP expression. (B) Thymic cells from adult WT and Fermt3-EGFP knockin mice were stained for CD45.2, CD3, CD31 and EpCAM to distinguish between hematopoietic, endothelial and thymic epithelial cells. All subpopulations were analysed for EGFP expression.

https://doi.org/10.7554/eLife.35816.010
Figure 5 with 1 supplement
Kindlin-3-independent colonization of the fetal thymus.

(A–D) Sagittal sections of whole embryos at gestational ages E11.5 (A), E12 (B), E12.5 (C) and E13.5 (D) were stained for fibronectin (perithymic mesenchyme, blue), cytokeratin (thymic primordium, green) and CD45 (potential T cell progenitor cells, red). Scale bars 100 µm. (E) Numbers of CD45+ cells within the thymic primordium and in the perithymic mesenchyme at the indicated time points. N = 3–4. (F) Representative flow cytometric profiles of fetal thymocytes from control and Fermt3-/- thymi at the indicated time points stained for lineage markers (B220, CD19, TER119, NK1.1, CD11b, Gr-1, CD11c, CD8α, CD3e, TCRβ, and TCRγδ), c-kit and CD44 to identify the DN1-2 (Linneg, c-kithi, CD44hi) and the DN3-4 (Linneg, c-kitlow, CD44low) populations. Numbers within the representative FACS plots indicate cell percentages. Frequencies (G) and total number (H) of DN1-2 cells measured in the flow cytometric analyses shown in (F). N = 7–10 Bars indicate means ± standard errors. *p<0.05; **p<0.01; ***p<0.001. See also Figure 5—figure supplement 1.

https://doi.org/10.7554/eLife.35816.011
Figure 5—figure supplement 1
Kindlin-3-independent colonization of the fetal thymus.

Kindlin-3-deficient T cell progenitors colonize the fetal thymus but decrease with age. Thymocytes from Fermt3+/+ and Fermt3-/- E15.5 (A) and E18.5 (B) embryos were stained for lineage markers (B220, CD19, TER119, NK1.1, CD11b, Gr-1, CD8α, CD3ε, TCRβ, TCRγδ and CD11c), CD44 and CD25 to identify and quantify DN1 (Linneg, CD44hi, CD25-), DN2 (Linneg, CD44hi, CD25+), DN3 (Linneg, CD44low, CD25+) and DN4 (Linneg, CD44low, CD25-) populations by flow cytometry. Numbers within FACS plots indicate cell percentages. Total number of DN1, DN2, DN3 and DN4 cells were measured by flow cytometry. N = 6–8. Bars indicate means ± standard errors. *p<0.05; **p<0.01; ***p<0.001.

https://doi.org/10.7554/eLife.35816.012
Fetal liver-derived T cell progenitors accumulate in the blood.

(A) Fetal liver cells from control and Fermt3-/- embryos from indicated gestational stages were stained for lineage markers (B220, CD19, TER119, NK1.1, Gr-1 and Thy1.2), c-kit, CD45 and PIR and analysed by flow cytometry. Frequencies (B) and total numbers (C) of PIR+ T cell progenitors per fetal liver. N(E12.5)=7/6; N(E13.5)=8/5; N(E14.5)=10/11; N(E15.5)=4/7. (D) FB from Fermt3+/+ and Fermt3-/- embryos were stained for lineage markers (B220, CD19, TER119, NK1.1, Gr-1 and Thy1.2), c-kit and PIR and analysed by flow cytometry. (E) Frequencies of circulating PIR+ T cell progenitors determined in (D). N(E11.5)=5; N(E12)=8/3; N(E12.5)=4/5; N(E13.5)=7/6. Numbers within the representative FACS plots indicate cell percentages. Bars indicate means ± standard errors. *p<0.05; **p<0.01; ***p<0.001.

https://doi.org/10.7554/eLife.35816.013
Kindlin-3-deficient T cell progenitors home to the fetal thymus and further develop into SP T cells ex vivo.

(A) Schematic description of the thymus attraction experiment. (B) Representative images of the thymus attraction experiment at the beginning and 40 hr after starting the experiment. Linneg fetal liver cells from control or Fermt3-/- embryos (both CD45.2+) are seen in the upper right corner of the images, a dGuo-treated wild-type fetal thymus lobe (FT, CD45.1+) is located at the lower left corner. Scale bar 250 µm. (C) Schematic description of the fetal thymus organ culture. (D) dGuo-treated fetal thymus lobes seeded with fetal liver cells as performed in (B) were further cultured for 18 days. Cells were stained for CD45.1, CD45.2, CD4, and CD8. Numbers within the representative FACS plots indicate cell percentages.

https://doi.org/10.7554/eLife.35816.014
Figure 8 with 2 supplements
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.

https://doi.org/10.7554/eLife.35816.015
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.

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.

https://doi.org/10.7554/eLife.35816.016
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.

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.

https://doi.org/10.7554/eLife.35816.017
Author response image 1
Adhesion efficiencies of PMNs in cremaster muscle venules of wild-type (WT), Kindlin-3 hypomorph (neo) mice and kindlin-3 knockout (KO) chimeras at indicated shear rates.
https://doi.org/10.7554/eLife.35816.020

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent
(M.musculus)
Fermt3-/-PMID: 18278053RRID: MGI:2147790Dr. Reinhard Fässler
(Max Planck Institute
of Biochemistry)
Genetic reagent
(M.musculus)
Fermt3floxPMID: 24089451RRID: MGI:5551978Dr. Reinhard Fässler
(Max Planck Institute
of Biochemistry)
Genetic reagent
(M.musculus)
Fermt3n/-PMID: 26438512RRID: MGI:3785479Dr. Reinhard Fässler
(Max Planck Institute
of Biochemistry)
Genetic reagent
(M.musculus)
Rag2-/-PMID: 1547487RRID: MGI:1858556Dr. Frederick W Alt
(Howard Hughes
Medical Institute)
Genetic reagent
(M.musculus)
Mx1-CrePMID: 7660125RRID: MGI:2176073Dr. Klaus Rajewsky
(Max Delbrück Center
for Molecular Medicine)
Genetic reagent
(M.musculus)
CD4-CrePMID: 11728338RRID: MGI: 2386448Dr. Christopher B.
Wilson, (University
of Washington)
Genetic reagent
(M.musculus)
2D2PMID: 12732654RRID: MGI:3700794Dr. Vijay K Kuchroo
(Center for Neurologic
Diseases)
Genetic reagent
(M.musculus)
mTmGPMID: 17868096RRID: MGI:3716464Dr. Liqun Luo
(Howard Hughes
Medical Institute)
Antibodyanti-B220-biotinBD PharmingenCat. #: 553085;
RRID: AB_394615
FACS (1:200)
AntibodyPOD-coupled
anti-BrdU
RocheCat. #: 11 585 860 001;
RRID: AB_514485
IHC (1:30)
Antibodyrabbit anti-mouse
cleaved caspase-3
Cell SignallingCat. #: 9661;
RRID: AB_2341188
IHC (1:100)
Antibodyanti-CD3eeBioscienceCat. #: 14-0031-82;
RRID: AB_467049
10 µg/ml
Antibodyanti-CD3e-biotinBD PharmingenCat. #: 553060;
RRID: AB_394593
FACS (1:200)
Antibodyanti-CD3-PEBD PharmingenCat. #: 555275;
RRID: AB_395699
FACS (1:200)
Antibodyanti-CD4-biotinBD PharmingenCat. #: 553649;
RRID: AB_394969
FACS (1:200)
Antibodyanti-CD4-FITCBD PharmingenCat. #: 553651;
RRID: AB_394971
FACS (1:200)
Antibodyanti-CD4-PerCPBD PharmingenCat. #: 550954;
RRID: AB_393977
FACS (1:200)
Antibodyanti-CD5-PEeBioscienceCat. #: 12-0051-82;
RRID: AB_465523
FACS (1:200)
Antibodyanti-CD8α-biotinBD PharmingenCat. #: 553028;
RRID: AB_394566
FACS (1:200)
Antibodyanti-CD8-APCeBioscienceCat. #: 17-0081-81;
RRID: AB_469334
FACS (1:200)
Antibodyanti-CD8-PEeBioscienceCat. #: 12-0081-81;
RRID: AB_465529
FACS (1:200)
Antibodyanti-CD11b-biotinBD PharmingenCat. #: 557395;
RRID: AB_2296385
FACS (1:200)
Antibodyanti-CD11c-biotinBD PharmingenCat. #: 553800;
RRID: AB_395059
FACS (1:200)
Antibodyanti-CD16/CD32BD PharmingenCat. #: 553142;
RRID:AB_394657
FACS (1:400)
Antibodyanti-CD19-biotineBioscienceCat. #: 13-0193-82;
RRID: AB_657656
FACS (1:200)
Antibodyanti-CD24-PEBD PharmingenCat. #: 553262;
RRID: AB_394741
FACS (1:200)
Antibodyanti-CD25-FITCBD PharmingenCat. #: 553071;
RRID: AB_394603
FACS (1:200)
Antibodyanti-CD28eBioscienceCat. #: 14-0281-82;
RRID: AB_467190
2 µg/ml
Antibodyrat anti-CD31 (PECAM-1)BD PharmingenCat. #: 557355;
RRID: AB_396660
IHC (1:300)
Antibodyanti-CD31-
Alexa Fluor 647
BiolegendCat. #: 102415;
RRID: AB_493411
IHC (1:200)
Antibodyanti-CD31-eFLour450eBioscienceCat. #: 48-0311-82;
RRID: AB_10598807
FACS (1:200)
Antibodyanti-CD44-PEBD PharmingenCat. #: 553134;
RRID: AB_394649
FACS (1:200)
Antibodyanti-CD45-FITCeBioscienceCat. #: 14-0451-82;
RRID: AB_467251
IHC (1:100)
Antibodyanti-CD45.2-biotinBD PharmingenCat. #: 553771;
RRID: AB_395040
FACS (1:200)
Antibodyanti-CD45.2-APCeBioscienceCat. #: 17-0454-82;
RRID: AB_469400
FACS (1:200)
Antibodyhamster anti-
CD54 (ICAM)
BD PharmingenCat. #: 553250;
RRID: AB_394732
IHC (1:100)
Antibodygoat anti-
CD106 (VCAM)
R and D SystemsCat. #: AF643;
RRID: AB_355499
IHC (1:80)
Antibodyanti-c-kit-APCeBioscienceCat. #: 17-1171-81;
RRID: AB_469429
FACS (1:200)
Antibodyanti-c-kit-PerCPBiolegendCat. #: 105821;
RRID: AB_893230
FACS (1:200)
Antibodymouse anti-pan
-cytokeratin
Sigma-AldrichCat. #: C2562;
RRID: AB_476839
IHC (1:400)
Antibodyanti-EpCAM-APC
-eFlour780
Thermo Fisher ScientificCat. #: 47-5791-82;
RRID: AB_2573986
FACS (1:200)
Antibodyrat anti-ER-TR4Eric Vroegindeweij
(Princess Maxima
center of pediatric
oncology)
IHC undiluted
Antibodyrat anti-ER-TR5Eric Vroegindeweij
(Princess Maxima
center of pediatric
oncology)
IHC undiluted
Antibodyanti-F4/80-biotinBio-RadCat. #: MCA497GA;
RRID: AB_323806
FACS (1:200)
Antibodyalexa-fluor-488-
conjugated anti-
Fluorescin IgG
Thermo Fisher
Scientific
Cat. #: A-11090;
RRID: AB_221562
IHC (1:500)
Antibodyrat anti-ICAM-1 PEBiolegendCat. #:116107;
RRID: AB_313698
FACS (1:200)
Antibodyrabbit anti-FibronectinMerck MilliporeCat. #: AB2033;
RRID: AB_2105702
IHC (1:300)
Antibodymouse anti-GAPDHMerck MilliporeCat. #: CB1001;
RRID: AB_2107426
WB (1:20.000)
Antibodyanti-Gr-1-biotinBD PharmingenCat. #: 553125;
RRID: AB_394641
FACS (1:200)
Antibodyalexa-fluor-546
-conjugated
anti-goat IgG
Thermo Fisher
Scientific
Cat: #: A-11056;
RRID: AB_2534103
IHC (1:300)
Antibodyalexa-fluor-546-
conjugated
anti-hamster IgG
Thermo Fisher
Scientific
Cat: #: A-21111;
RRID: AB_2535760
IHC (1:300)
Antibodyalexa-fluor-546-
conjugated
anti-mouse IgG
Thermo Fisher
Scientific
Cat: #: A-11003;
RRID: AB_2534071
IHC (1:300)
Antibodyalexa-fluor-647
-conjugated
anti-mouse IgG
Thermo Fisher
Scientific
Cat: #: A-31571;
RRID: AB_162542
IHC (1:300)
Antibodyalexa-fluor-647
-conjugated
anti-rabbit IgG
Thermo Fisher
Scientific
Cat: #: A-21244;
RRID: AB_2535812
IHC (1:300)
Antibodyalexa-fluor-488-
conjugated
anti-rat IgG
Thermo Fisher
Scientific
Cat: #: A-11006;
RRID: AB_2534074
IHC (1:300)
Antibodyalexa-fluor-647-
conjugated
anti-rat IgG
Thermo Fisher
Scientific
Cat: #: A-21247;
RRID: AB_141778
IHC (1:300)
Antibodyanti-Integrin α4-PEBD PharmingenCat. #: 553157;
RRID: AB_394670
FACS (1:200)
Antibodyanti-Integrin αL-PEBD PharmingenCat. #: 553121;
RRID: AB_394637
FACS (1:200)
Antibodyanti-Integrin β1-PEBiolegendCat. #: 102207;
RRID: AB_312884
FACS (1:200)
Antibodyanti-Integrin β2-PEBD PharmingenCat. #: 553293;
RRID: AB_394762
FACS (1:200)
Antibodyanti-Integrin β3-PEeBioscienceCat. #: 12-0611-81;
RRID: AB_465717
FACS (1:200)
Antibodyanti-Integrin β7-PEBD PharmingenCat. #: 557498;
RRID: AB_396735
FACS (1:200)
Antibodyrabbit anti-Kindlin-1Markus Moser
(MPI of Biochemistry)
WB (1:1000)
Antibodymouse anti-Kindlin-2Sigma-AldrichCat. # SAB4200525
-200UL
WB (1:1000)
Antibodyrabbit anti-Kindlin-3Markus Moser
(MPI of Biochemistry)
WB (1:3000)
Antibodyrabbit
anti-pan-laminin
Rupert TimplIHC (1:700)
Antibodyanti-LFA-1-PEBD PharmingenCat. #: 553121;
RRID: AB_394637
IF (1:100)
Antibodyanti-NK1.1-biotinBD PharmingenCat. #: 553163;
RRID: AB_394675
FACS (1:200)
Antibodyanti-PIR-APCeBioscienceCat. #: 17-3101-82;
RRID: AB_1944406
FACS (1:200)
Antibodyanti-PIR-PEBD PharmingenCat. #: 550349;
RRID: AB_393628
FACS (1:200)
Antibodyanti-p-TyrosineSanta Cruz
Biotechnology
Cat. #: sc-7020;
RRID: AB_628123
IF (1:50)
Antibodyanti-Sca-1-PEeBioscienceCat. #: 12-5981-82;
RRID: AB_466086
FACS (1:200)
Antibodymouse anti-talinSigma-AldrichCat. #: T3287;
RRID: AB_477572
WB (1:20.000)
Antibodyanti-TCRβ-PEeBioscienceCat. #: 12-5961-82;
RRID: AB_466066
FACS (1:200)
Antibodyanti-TCRγδ-biotinBD PharmingenCat. #: 553176;
RRID: AB_394687
FACS (1:200)
Antibodyanti-Ter119-biotinBD PharmingenCat. #: 553672;
RRID: AB_394985
FACS (1:200)
Antibodyanti-Thy1.2-biotinBD PharmingenCat. #: 553001;
RRID: AB_394540
FACS (1:200)
Antibodyrat anti-VCAM-1 PEBiolegendCat. #: 105713;
RRID: AB_1134166
FACS (1:200)
Peptide,
recombinant protein
recombinant
mouse CCL21
R and D SystemsCat. #: 457–6C;
Accession #:
P84444.1
Peptide,
recombinant protein
recombinant
mouse CCL25
R and D SystemsCat. #: 481-TK;
Accession #:
O35903.1
Peptide,
recombinant protein
recombinant
mouse ICAM-1-FC
R and D SystemsCat. #: 796-IC;
Accession #: Q3U8M7
Peptide,
recombinant protein
MOG35-55MPI of
Biochemistry,
Core facility
Peptide,
recombinant protein
recombinant
mouse P-Selectin-FC
R and D SystemsCat. #: 737-PS;
Accession #:
Q01102
Peptide,
recombinant protein
recombinant
mouse VCAM-1-FC
R and D SystemsCat. #: 643-VM;
Accession #:
CAA47989
Commercial
assay or kit
PE Annexin V
Apoptosis
Detection Kit
BD BiosciencesCat. #: 559763
Commercial
assay or kit
BrdU Flow KitBD PharmingenCat. #: 559619;
RRID: AB_2617060
Commercial
assay or kit
anti-biotin
MicroBeads
MiltenyiCat. #: 130-090-485;
RRID: AB_244365
Commercial
assay or kit
CD4 T cell isolation
kit
MiltenyiCat. #: 130-104-454
Commercial
assay or kit
Vectastain Elite ABC
HRP Kit, rabbit IgG
VECTOR
laboratories
Cat. #: PK-6101;
RRID: AB_2336820
Chemical
compound, drug
CellTrace CFSE
Cell Proliferation Kit
Thermo Fisher
Scientific
Cat. #: C34554
Chemical
compound, drug
Collagenase DSigma-AldrichCat. #: C01301 mg/ml
Chemical
compound, drug
FITC-DextranSigma-AldrichCat. #: FD150S
Chemical
compound, drug
DNaseISigma-AldrichCat. #: D5025100 U/ml
Chemical
compound, drug
dGuoSigma-AldrichCat. #: D09011.35 mM
Chemical
compound, drug
CellTrace Far Red
Cell Proliferation Kit
Thermo Fisher
Scientific
Cat. #: C345641 µM
Chemical
compound, drug
FluoSpheres 580/606Thermo Fisher
Scientific
Cat. #: F88211 µM
Chemical
compound, drug
LPSSigma-AldrichCat. #: L26541 mg/ml
Chemical
compound, drug
poly-L-LysineSigma-AldrichCat. #: P8920pure
Chemical
compound, drug
Phalloidin Alexa350Thermo Fisher
Scientific
Cat. #: A22281IF (1:40)
Chemical
compound, drug
PureColAdvanced BioMatrixCat. #: 5005–100 ML
Chemical
compound, drug
Streptavidin Cy3Jackson Immuno
Research
Cat. #: 016-160-084;
RRID: AB_2337244
FACS (1:400)
Chemical
compound, drug
Streptavidin FITCThermo Fisher
Scientific
Cat. #: SA10002FACS (1:200)
Chemical
compound, drug
Streptavidin PerCP-Cy5.5BD PharmingenCat. #: 551419FACS (1:200)
Software, algorithmImageJNational Institute
of Health
RRID:SCR_003070
Software, algorithmImSpector ProLaVision
BioTec GmbH
Software, algorithmPhotoshopAdobeRRID:SCR_014199
Software, algorithmSlidebook 6.0.8 SoftwareIntelligant
Imaging
Innovations
RRID:SCR_014300
Software, algorithmVirtualDubGNU General
Public License (GPL)
OtherGlass Bottom
Microwell Dishes
MatTek CorporationCat. #: P35G-1.5–20 C
OtherIbidi flow chambers
µ-Slide I 0.8 Luer IbiTreat
IbidiCat. #: 80196

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)