ARHGAP45 controls naïve T‐ and B‐cell entry into lymph nodes and T‐cell progenitor thymus seeding

Abstract T and B cells continually recirculate between blood and secondary lymphoid organs. To promote their trans‐endothelial migration (TEM), chemokine receptors control the activity of RHO family small GTPases in part via GTPase‐activating proteins (GAPs). T and B cells express several RHO‐GAPs, the function of most of which remains unknown. The ARHGAP45 GAP is predominantly expressed in hematopoietic cells. To define its in vivo function, we describe two mouse models where ARHGAP45 is ablated systemically or selectively in T cells. We combine their analysis with affinity purification coupled to mass spectrometry to determine the ARHGAP45 interactome in T cells and with time‐lapse and reflection interference contrast microscopy to assess the role of ARGHAP45 in T‐cell polarization and motility. We demonstrate that ARHGAP45 regulates naïve T‐cell deformability and motility. Under physiological conditions, ARHGAP45 controls the entry of naïve T and B cells into lymph nodes whereas under competitive repopulation it further regulates hematopoietic progenitor cell engraftment in the bone marrow, and T‐cell progenitor thymus seeding. Therefore, the ARGHAP45 GAP controls multiple key steps in the life of T and B cells.

Ex23-3'UT Ex10  A Schematic representation of the WT Arghap45 and Arghap45 À alleles. Exons 1-23 are shown and numbered and the 5' and 3'UTR shown as gray box. The deletion engineered in the Arghap45 À allele encompasses exon 4 (http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000035697;r=10: 80016653-80031472;t=ENSMUST00000099501). B Immunoblot analysis of equal amounts of total lysates of thymocytes (left panel) and of B and T cells (right panel) purified from WT, Arhgap45 À/À , and Arhgap45 ΔT/ΔT mice probed with anti-ARHGAP45 and anti-b-Actin (loading control). Molecular weights are shown on the left. Results are representative of two experiments. C Schematic representation of the Arhgap45 tm1a , Arhgap45 fl , and Arhgap45 ΔT alleles. See "Generation of mice with a loxP-flanked Arhgap45 allele and of mice conditionally deprived of ARHGAP45 in T cells" in Materials and Methods. Exons 1-23 are shown and numbered and the 5' and 3'UTR shown as gray boxes.
Source data are available online for this figure.  Figure EV2. Numbers of B and T cells in the specified organs of WT mice and of mice deficient in ARHGAP45 (Arhgap45 À/À ), or expressing a loxP-flanked Arhgap45 allele prior to (Arhgap45 fl/fl ) or after (Arhgap45 ΔT/ΔT ) crossing with CD4-Cre transgenic mice.
A Cellularity of thymus from the specified mice. B Numbers of T cells found in the blood of the specified mice. C Numbers of B cells found in the blood of the specified mice. D Numbers of T cells found in the LNs of the specified mice. E Numbers of B cells found in the LNs of the specified mice. F Numbers of T cells found in the spleen of the specified mice. G Numbers of B cells found in the spleen of the specified mice.
Data information: Each dot corresponds to a mouse and the mean and SD are indicated. Data are representative of three independent experiments involving each a total of 8-16 mice. A one-way Anova test was used to compare each mouse model against WT mouse controls. The resulting probability is indicated above each model. ns, non-significant, **P ≤ 0.002, ****P ≤ 0.0001. A Analysis of migration patterns of WT and Arghap45 À/À naive T cells on 2D surface coated with ICAM-1. Each track represents the migratory path of individual WT and Arghap45 À/À naive T cells recorded over 466 s in a single field of view.
Trajectories were plotted to a common starting point, and > 200 T cells were recorded per plot using time-lapse microscopy at 10× magnification.

B Average cell speed of WT cell (black) and
Arghap45 À/À naive T cells (red). Three independent experiments were performed involving more than 200 cells and each dot corresponds to the mean of the speed in one given experiment. The speed of 10 µm/min correspond to Brownian motion, which means that cells are not motile (two-tailed Student's ttest). Figure EV4. Analysis of the projected adhesion area of WT and Arghap45 À/À naive T cells on 2D surface coated with ICAM-1 CCL21.

A B C D G H E F
A-F Processing of images from RICM microscopy to infer projected adhesion area. The projected area of cells is extracted from bright field images (A), which are binarized (B) to extract the contour in red (C). The area of adhesion fingerprint is assessed from RICM images (D) that are inverted and binarized (E) to extract the area of the contact zone in green (F). To illustrate image processing, the final image of the migration sequence shown in Fig 6C has Figure EV5. Analysis of hematopoietic progenitors in the BM of WT and Arhgap45 À/À mice.
A Gating strategy for the specified BM hematopoietic cell progenitors as described in Cordeiro Gomes et al (2016). HSC: hematopoietic stem cells, MPP: multipotent progenitors, CLP: common lymphoid progenitors, CMP: common myeloid progenitors, GMP: granulocyte and monocyte progenitors, and MEP: megakaryocyte and erythroid progenitors. B Numbers of HSC, MPP and CLP per femur of WT and Arhgap45 À/À mice. C Numbers of CMP, GMP and MEP per femur of WT and Arhgap45 À/À mice.
Data information: Each dot corresponds to a mouse and the mean and SD are indicated. Data are representative of three independent experiments. *P ≤ 0.01, ***P ≤ 0.001; unpaired Student's t-test.