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• W3136571528 abstract "Article15 March 2021Open Access Source DataTransparent process ARHGAP45 controls naïve T- and B-cell entry into lymph nodes and T-cell progenitor thymus seeding Le He Le He orcid.org/0000-0001-5639-377X Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Henan Key Laboratory for Immunology and Targeted Therapy, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China Search for more papers by this author Marie-Pierre Valignat Marie-Pierre Valignat orcid.org/0000-0002-6031-3813 LAI, CNRS, INSERM, Aix Marseille Univ, Marseille, France Search for more papers by this author Lichen Zhang Lichen Zhang orcid.org/0000-0002-7810-9941 Henan Key Laboratory for Immunology and Targeted Therapy, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China Search for more papers by this author Lena Gelard Lena Gelard orcid.org/0000-0003-1969-0032 Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Centre d’Immunophénomique, INSERM, CNRS UMR, Aix Marseille Université, Marseille, France Search for more papers by this author Fanghui Zhang Fanghui Zhang orcid.org/0000-0003-3833-7066 Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Henan Key Laboratory for Immunology and Targeted Therapy, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China Search for more papers by this author Valentin Le Guen Valentin Le Guen Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Search for more papers by this author Stéphane Audebert Stéphane Audebert orcid.org/0000-0002-9409-2588 CNRS, INSERM, Institut Paoli-Calmettes, CRCM, Marseille Protéomique, Aix Marseille Univ, Marseille, France Search for more papers by this author Luc Camoin Luc Camoin orcid.org/0000-0002-1230-4787 CNRS, INSERM, Institut Paoli-Calmettes, CRCM, Marseille Protéomique, Aix Marseille Univ, Marseille, France Search for more papers by this author Even Fossum Even Fossum orcid.org/0000-0001-7064-0327 Institute of Clinical Medicine, University of Oslo and Oslo University Hospital, Oslo, Norway Search for more papers by this author Bjarne Bogen Bjarne Bogen Institute of Clinical Medicine, University of Oslo and Oslo University Hospital, Oslo, Norway Search for more papers by this author Hui Wang Hui Wang Henan Key Laboratory for Immunology and Targeted Therapy, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China Search for more papers by this author Sandrine Henri Sandrine Henri Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Search for more papers by this author Romain Roncagalli Romain Roncagalli orcid.org/0000-0001-7554-0552 Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Search for more papers by this author Olivier Theodoly Olivier Theodoly orcid.org/0000-0001-8787-3709 LAI, CNRS, INSERM, Aix Marseille Univ, Marseille, France Search for more papers by this author Yinming Liang Corresponding Author Yinming Liang [email protected] orcid.org/0000-0001-9174-4037 Henan Key Laboratory for Immunology and Targeted Therapy, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China Search for more papers by this author Marie Malissen Corresponding Author Marie Malissen [email protected] orcid.org/0000-0003-2331-1445 Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Centre d’Immunophénomique, INSERM, CNRS UMR, Aix Marseille Université, Marseille, France Laboratory of Immunophenomics, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China Search for more papers by this author Bernard Malissen Corresponding Author Bernard Malissen [email protected] orcid.org/0000-0003-1340-9342 Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Centre d’Immunophénomique, INSERM, CNRS UMR, Aix Marseille Université, Marseille, France Laboratory of Immunophenomics, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China Search for more papers by this author Le He Le He orcid.org/0000-0001-5639-377X Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Henan Key Laboratory for Immunology and Targeted Therapy, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China Search for more papers by this author Marie-Pierre Valignat Marie-Pierre Valignat orcid.org/0000-0002-6031-3813 LAI, CNRS, INSERM, Aix Marseille Univ, Marseille, France Search for more papers by this author Lichen Zhang Lichen Zhang orcid.org/0000-0002-7810-9941 Henan Key Laboratory for Immunology and Targeted Therapy, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China Search for more papers by this author Lena Gelard Lena Gelard orcid.org/0000-0003-1969-0032 Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Centre d’Immunophénomique, INSERM, CNRS UMR, Aix Marseille Université, Marseille, France Search for more papers by this author Fanghui Zhang Fanghui Zhang orcid.org/0000-0003-3833-7066 Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Henan Key Laboratory for Immunology and Targeted Therapy, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China Search for more papers by this author Valentin Le Guen Valentin Le Guen Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Search for more papers by this author Stéphane Audebert Stéphane Audebert orcid.org/0000-0002-9409-2588 CNRS, INSERM, Institut Paoli-Calmettes, CRCM, Marseille Protéomique, Aix Marseille Univ, Marseille, France Search for more papers by this author Luc Camoin Luc Camoin orcid.org/0000-0002-1230-4787 CNRS, INSERM, Institut Paoli-Calmettes, CRCM, Marseille Protéomique, Aix Marseille Univ, Marseille, France Search for more papers by this author Even Fossum Even Fossum orcid.org/0000-0001-7064-0327 Institute of Clinical Medicine, University of Oslo and Oslo University Hospital, Oslo, Norway Search for more papers by this author Bjarne Bogen Bjarne Bogen Institute of Clinical Medicine, University of Oslo and Oslo University Hospital, Oslo, Norway Search for more papers by this author Hui Wang Hui Wang Henan Key Laboratory for Immunology and Targeted Therapy, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China Search for more papers by this author Sandrine Henri Sandrine Henri Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Search for more papers by this author Romain Roncagalli Romain Roncagalli orcid.org/0000-0001-7554-0552 Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Search for more papers by this author Olivier Theodoly Olivier Theodoly orcid.org/0000-0001-8787-3709 LAI, CNRS, INSERM, Aix Marseille Univ, Marseille, France Search for more papers by this author Yinming Liang Corresponding Author Yinming Liang [email protected] orcid.org/0000-0001-9174-4037 Henan Key Laboratory for Immunology and Targeted Therapy, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China Search for more papers by this author Marie Malissen Corresponding Author Marie Malissen [email protected] orcid.org/0000-0003-2331-1445 Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Centre d’Immunophénomique, INSERM, CNRS UMR, Aix Marseille Université, Marseille, France Laboratory of Immunophenomics, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China Search for more papers by this author Bernard Malissen Corresponding Author Bernard Malissen [email protected] orcid.org/0000-0003-1340-9342 Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France Centre d’Immunophénomique, INSERM, CNRS UMR, Aix Marseille Université, Marseille, France Laboratory of Immunophenomics, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China Search for more papers by this author Author Information Le He1,2, Marie-Pierre Valignat3, Lichen Zhang2, Lena Gelard1,4, Fanghui Zhang1,2, Valentin Le Guen1, Stéphane Audebert5, Luc Camoin5, Even Fossum6, Bjarne Bogen6, Hui Wang2, Sandrine Henri1, Romain Roncagalli1, Olivier Theodoly3, Yinming Liang *,2, Marie Malissen *,1,4,7 and Bernard Malissen *,1,4,7 1Centre d’Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France 2Henan Key Laboratory for Immunology and Targeted Therapy, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China 3LAI, CNRS, INSERM, Aix Marseille Univ, Marseille, France 4Centre d’Immunophénomique, INSERM, CNRS UMR, Aix Marseille Université, Marseille, France 5CNRS, INSERM, Institut Paoli-Calmettes, CRCM, Marseille Protéomique, Aix Marseille Univ, Marseille, France 6Institute of Clinical Medicine, University of Oslo and Oslo University Hospital, Oslo, Norway 7Laboratory of Immunophenomics, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang City, China *Corresponding author. Tel: +86 373 383 1237; E-mail: [email protected] *Corresponding author. Tel: +33 6 33 24 54 04; E-mail: [email protected] *Corresponding author. Tel: +33 7 86 28 29 83; E-mail: [email protected] EMBO Reports (2021)22:e52196https://doi.org/10.15252/embr.202052196 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Synopsis T and B cells continually recirculate between blood and secondary lymphoid organs. The RHO GTPase activating protein ARHGAP45 regulates naïve T cell deformability, motility, and chemotaxis, and thereby regulates key trafficking steps of T and B cells in vivo. ARHGAP45 is a GTPase activating protein predominantly expressed in hematopoietic cells. T and B cells lacking ARHGAP45 show markedly reduced deformability, motility, and chemotaxis. ARHGAP45 controls naïve T and B cell entry into lymph nodes. ARHGAP45 further controls hematopoietic progenitor cell engraftment, and T-cell progenitor thymus seeding. Introduction Secondary lymphoid organs are anatomical sites in which adaptive immune responses are initiated. They include lymph nodes (LNs), and the spleen and naïve T cells continually recirculate between them and the blood. Entry into LNs involves a multistep cascade in which the lymphocyte-homing receptor L-selectin (CD62L) supports initial rolling of blood-borne, naive T cells along high endothelial venules (HEVs). The chemokines CCL19 and CCL21 on the luminal surface of HEVs bind to the CCR7 chemokine receptor expressed on rolling naïve T cells, leading successively to LFA-1-ICAM-1-mediated firm adhesion, T-cell polarization, and subsequent crawling over and diapedesis through HEVs. Blood-borne, naive B cells also use CCR7 together with the CXCR4 and CXCR5 chemokine receptors to enter LNs (Schulz et al, 2016). Among the eight sub-families that constitute the RHO family of small GTPases, the RAC, RHO and CDC42 sub-families control cell polarity, shape, and migration by regulating actin cytoskeletal dynamics (Lawson & Ridley, 2018). The activity of these small GTPases is controlled by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that allow them to cycle between a GTP-bound form which activates downstream effectors and an inactive GDP-bound form. To promote trans-endothelial migration (TEM), CCR7 activates various phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K) isoforms. PLCs generate inositol trisphosphate and diacylglycerol (DAG), whereas PI3Ks generate phosphatidylinositol 3,4,5 trisphosphate (PIP3) (Schulz et al, 2016). Such intracellular second messengers control the intracellular distribution and function of RHO GEFs and GAPs. T cells express more than 20 GAPs, and the function and mechanism of action of most of them remain to be elucidated (Stein & Ruef, 2019). We recently identified the ARHGAP45 GAP among the signaling protein complexes that assemble in mouse primary T cells (Voisinne et al, 2019; Mori et al, 2021). ARHGAP45, also known as HMHA1 (human minor histocompatibility antigen 1), has been first described in human and comprises an N-terminal BAR domain followed by a C1 and a RHO GAP domain (de Kreuk et al, 2013). BAR domains consist of a helical bundle of 200–280 amino acids that associates in antiparallel fashion to form dimers that bind to membranes according to their curvature (Carman & Dominguez, 2018), whereas C1 domains bind membrane-bound DAG. Heterologous expression of human ARHGAP45 in HeLa cells showed that it regulates the actin cytoskeleton and cell spreading through undefined mechanism (de Kreuk et al, 2013). ARHGAP45 GAP activity is autoinhibited by an intramolecular interaction involving the BAR domain (de Kreuk et al, 2013), suggesting that ARHGAP45 adopts an active conformation when its C1 and BAR domains simultaneously bind to DAG-containing membranes with an appropriate curvature. To analyze the role of ARHGAP45 in vivo, we developed two mouse models where ARHGAP45 is ablated either systemically or selectively in T cells. We also determined the constellation of proteins that specifically interact with ARHGAP45 in T cells and assessed the role of ARGHAP45 in T-cell deformability, polarization and motility. Altogether, our data demonstrate that ARHGAP45 plays a critical role in the entry of naïve T and B cells into LNs, in the engraftment of hematopoietic progenitor cells in the bone marrow (BM), and in T-cell progenitor thymus seeding. Results Characterization of the ARGHAP45 interactome in Jurkat T cells Although ARHGAP45 is predominantly expressed in hematopoietic cells (www.immgen.org, http://biogps.org, https://genevisible.com/search), previous studies on ARHGAP45 relied on overexpression in heterologous cells (de Kreuk et al, 2013). Therefore, before analyzing the functional consequences resulting from the lack of ARHGAP45 in vivo, we used quantitative interactomics (Voisinne et al, 2019) to determine which proteins associate with endogenous ARHGAP45 molecules in T cells. Using CRISPR/Cas9 editing, the ARHGAP45 molecules present in the human leukemic T-cell line Jurkat were tagged at their amino terminus with an affinity Twin-Strep-tag (OST; Fig 1A). The proteins (“the preys”) associating to the ARHGAP45OST “bait” were affinity purified before or after stimulation with anti-CD3 for 120 s and analyzed by MS. To distinguish proteins truly associating with ARHGAP45 from nonspecific contaminants, we compared our data with control AP-MS experiments involving wild-type (WT) Jurkat T cells. ARHGAP45 interactors were identified (Fig 1B and Dataset EV1), and the ARHGAP45-prey stoichiometry was also measured using intensity-based absolute quantification (Voisinne et al, 2019). By determining the number of copies per cell of each protein expressed in Jurkat cells (Dataset EV2) and combining them with interaction stoichiometries, we organized the proteins interacting with ARHGAP45 (the “ARHGAP45 interactome”) into a stoichiometry plot (Fig 1C). Figure 1. The ARHGAP45 interactome of Jurkat T cells Jurkat T cells (WT), ARHGAP45-deficient Jurkat T cells (ARHGAP45−/−), and five independent clones (C1–C5) of Jurkat T cells expressing ARHGAP45OST molecules were analyzed for ARHGAP45 protein expression. Immunoblot analysis of equal amounts of proteins from cell lysates that were either directly analyzed (Total lysates), or subjected to affinity purification on Strep-Tactin Sepharose beads followed by elution of proteins with D-biotin (Affinity purification), and probed with antibody to ARGHAP45 or anti-HSP60 (loading control). Left margin, molecular size in kilodaltons (kDa). Volcano plot showing proteins enrichment (fold change in log10 scale) after affinity purification in Jurkat T cells expressing ARHGAP45OST molecules compared to affinity purification in control Jurkat T cells expressing similar levels of WT (untagged) ARHGAP45 proteins prior to (unstimulated) and at 120 s after (stimulated) TCR stimulation. ARHGAP45 interacting proteins with a > 2-fold enrichment and a P-value < 0.01 were selected as specific ARHGAP45 interactors (Dataset EV1) and some of them are specified in red. Red lines represent the thresholds set on P-value and enrichment to identify specific ARHGAP45 interactors. ARHGAP45-specific interactors highlighted in the text are displayed in a stoichiometry plot (Voisinne et al, 2019) where the ratios of the prey to bait cellular abundances (“abundance stoichiometry”) are plotted as a function of their maximal interaction stoichiometries (“interaction stoichiometry”), both using log10 scale (see Datasets EV1 and EV2). The ARHGAP45 bait is shown as a yellow dot. The size of the dots is commensurate to the maximal protein enrichment in ARHGAP45OST samples as compared to WT control samples. The TNKS prey for which it was not possible to determine the cellular abundance is shown at the bottom of the stoichiometry plot. For each time point, three independent biological replicates were performed and each biological replicate was analyzed in triplicate by MS. Source data are available online for this figure. Source Data for Figure 1 [embr202052196-sup-0008-SDataFig1.pdf] Download figure Download PowerPoint Analysis of the GAP activity of ARHGAP45 in a cell-free system showed that it can catalyze RHOA, RAC1 and CDCD42 GTP hydrolysis (de Kreuk et al, 2013). In contrast, our stoichiometry plot showed that RHOA was the major RHO GTPase capable of associating with ARHGAP45 in cellulo and constituted one of the most abundant ARHGAP45 interactors (Fig 1C), suggesting that in T cells ARHGAP45 primarily regulates the RHOA GTPase. RHOC, a second member of the RHO family of small GTPases, was also found among ARHGAP45 interactors, and its interaction stoichiometry was 31-fold lower than that of the ARHGAP45-RHOA interaction (Fig 1C). This might result from the fact that ARHGAP45 is a better GAP for RHOC than RHOA, leading to its rapid dissociation from ARHGAP45 after GTP hydrolysis. Some BAR domain-containing proteins bind to members of the 14-3-3 protein family in a phosphoserine-dependent manner (Carman & Dominguez, 2018). Along that line, YWHAQ, also known as 14-3-3 protein θ, associated with ARHGAP45 in a TCR-inducible manner (Fig 1C), a finding consistent with the presence of several TCR-inducible phosphorylated serine residues in ARGHAP45 (Locard-Paulet et al, 2020). Proteins involved in regulation of cortical actin tension (MYO1G), cell migration (CORO1C), vesicular transport (RAB11B), or colocalizing with the chemokine receptor CXCR4 and F-actin in T cells (DBN1) also associated with ARHGAP45, however, with a lower stoichiometry than RHOA and YWHAQ (Fig 1C). Interestingly, ARHGAP45 associated with another BAR-containing GAP protein called GMIP (also known as ARHGAP46; Fig 1C), a result consistent with the possibility for distinct RHO GAP to heterodimerize via their BAR domain (Carman & Dominguez, 2018). Therefore, the composition of the ARHGAP45 interactome of Jurkat T cells suggests that ARHGAP45 acts as a GAP specific for RHOA and presumably RHOC. Effect of ARHGAP45 deficiency on T- and B-cell development To analyze the role of ARHGAP45 in vivo, we established mice homozygous for an Arhgap45 allele lacking a critical exon (Arhgap45 exon 4; Fig EV1A). The resulting Arhgap45−/− mice were born at expected Mendelian frequencies and lacked detectable ARHGAP45 protein as exemplified using developing T cells and mature T and B cells (Fig EV1B). Arghap45–/– thymi were of normal size (Fig 2A) and contained normal numbers of DN2, DN3, DN4, and DP cells (Fig 2B; see legend for a definition of T-cell developmental stages). The presence of reduced numbers of DN1 cells (1.5-fold), and of both CD4+ (1.3-fold) and CD8+ (1.4-fold) SP thymocytes suggested that thymus seeding and transit from the medulla to the cortex, two steps depending on CCR7 signals (Kurobe et al, 2006; Calderon & Boehm, 2011), were slightly impeded by the lack of ARHGAP45. The presence of normal DN2-3 cell numbers further suggested that some compensatory cell divisions occurred at the DN1 to DN2-3 transition. Click here to expand this figure. Figure EV1. Generation of mutant mouse lacking ARHGAP45, or expressing a loxP-flanked Arhgap45 allele Schematic representation of the WT Arghap45 and Arghap45− alleles. Exons 1-23 are shown and numbered and the 5’ and 3’UTR shown as gray box. The deletion engineered in the Arghap45− allele encompasses exon 4 (http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000035697;r=10:80016653-80031472;t=ENSMUST00000099501). Immunoblot analysis of equal amounts of total lysates of thymocytes (left panel) and of B and T cells (right panel) purified from WT, Arhgap45−/−, and Arhgap45∆T/∆T mice probed with anti-ARHGAP45 and anti-β-Actin (loading control). Molecular weights are shown on the left. Results are representative of two experiments. Schematic representation of the Arhgap45tm1a, Arhgap45fl, and Arhgap45∆T 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. Download figure Download PowerPoint Figure 2. Development of T and B cells in Arhgap45−/− mice Total cellularity of thymus spleen and of mesenteric and peripheral LNs of WT and Arhgap45−/− mice (see key in upper right corner). Upon thymus colonization, ETP develop into CD4−CD8−double negative (DN) cells that mature into CD4+CD8+ double positive (DP) cells, some of which proceed further into CD4+ and CD8+ single positive (SP) cells that egress from the thymus. Based on the expression of CD25 and CD44, DN cells can be further organized according to the following developmental series: DN1 (CD44+CD25−) → DN2 (CD44+CD25+) → DN3 (CD44−CD251+) → DN4 (CD44−CD25−). After excluding cells positive for CD11b, CD11c, CD45R, or CD161c, WT and Arhgap45−/− thymocytes were analyzed by flow cytometry for expression of CD4, CD8, CD25, and CD44 and the numbers of cells present in each of the specified T-cell developmental stages determined. Numbers of T and B cells found in the blood of WT and Arhgap45−/− mice. Numbers of T and B cells found in pooled mesenteric and peripheral LNs of WT and Arhgap45−/− mice. Numbers of T and B cells found in the Peyer’s patches of WT and Arhgap45−/− mice. In the spleen, IgMhiIgDlo transitional 1 (T1-Bc) B cells constitute recent immigrant from the BM that develop into IgMhiIgDhi transitional (T2-Bc) B cells, which differentiate into mature IgMloIgDhi, or follicular recirculating B cells Fol-Bc (Carsetti, 2004)). WT and Arhgap45−/− splenocytes were analyzed by flow cytometry for expression of CD19, CD45R, IgM and IgD and the numbers of cells present in each of the specified B-cell developmental stages determined. Numbers of total CD4+ and CD8+ T cells, and of naïve and effectors CD4+ and CD8+ T cells found in peripheral LNs of WT and Arhgap45−/− mice. Also shown are CD62L levels (MFI) on naïve CD4+ and CD8+ T cells from WT and Arhgap45−/−. CD4+ and CD8+ T cells from peripheral LNs analyzed for expression of CD44 and CD62L. Numbers in quadrants indicate percent naïve (CD44loCD62Lhi) and central memory CD8+ T cells (CD44hiCD62Lhi). Data information: In (A)–(H) the results for each mouse are shown as a dot and correspond to three to four experiments involving a total of 12–25 mice. *P ≤ 0.033, **P ≤ 0.002, ***P ≤ 0.001, ****P ≤ 0.0001; unpaired Student’s t-test. Mean and SD (A, B, G) or SEM (C, D, E, F) are also shown. Download figure Download PowerPoint The numbers of T and B cells found in the blood of Arhgap45−/− mice were 3.7- and 3.2-fold reduced, respectively (Fig 2C). Arhgap45−/− LNs had a reduced cellularity (Fig 2A) due to diminished numbers of naïve T (2.2-fold) and B (2.5-fold) cells (Fig 2D), whereas Arhgap45−/− Peyer’s patches showed an even stronger reduction of naïve T (4-fold) and B (17-fold) cell numbers (Fig 2E). In contrast, Arhgap45−/− spleens showed a normal cellularity with normal T- and B-cell numbers and an almost normal representation of T1, T2, and follicular B cells (Fig 2A and F, see legend for a definition of B-cell developmental stages). Comparable number of effector memory CD4+ and CD8+ T cells were found in Arhgap45−/− and WT LNs (Fig 2G). Due to the reduced numbers of naïve T cells found in Arhgap45−/− LNs, increased percentages of effector memory cells were observed in Arhgap45−/− CD44-CD62L dot plots (Fig 2H). Therefore, considering that the spleen differs from LNs and Peyer’s patches by its lacks of HEV (Schulz et al, 2016), and that effector memory T cells primarily reach LNs via afferent lymphatic vessels (Jackson, 2019), our data suggest that the absence of ARHGAP45 specifically impedes the TEM of naïve T and B cells through the HEV of LNs and Peyer’s patches. ARHGAP45 deficiency impedes LN entry rate of naïve T and B cells To determine whether the reduced numbers of naïve T and B cells found in the LNs of Arhgap45−/− mice were due to defective LN entry rate, we measured the short-term LN homing efficiency of adoptively transferred ARHGAP45-deficient T and B cells and compared it to that of co-transferred WT T and B cells. Accordingly, total spleen cells from WT and Arhgap45−/− mice were labeled with CTV and CMTPX dyes, respectively, mixed at a 1 to 1 ratio, and injected intravenously into WT recipient mouse. For both T and B cells, the ratio of Arhgap45−/− to WT cells that entered LNs and spleen was measured 4 h after transfer (Fig 3A). Significantly decreased Arhgap45−/−/WT ratios were observed in LNs for both T (1.8-fold) and B (2.2-fold) cells as compared to pre-injection ratios (Fig 3B). In contrast, ARHGAP45 deficiency was without measurable effects on the entry of B and T cells into the spleen (Fig 3B). In control experiments involving 1 to 1 mixture of WT spleen cells labeled with CTV or CMTPX, no decrease was observed in the T- and B-cell CTV-CMTPX ratio found in LNs (Fig 3A and B). The naïve T cells found in the blood and LNs of WT and Arhgap45−/− mice expressed levels of CD62L (Fig 2H), CCR7 and LFA-1 (Fig. 3C and D) comparable or even slightly increased (CD62L) as compared to their WT counterparts. Therefore, naïve T and B cells deficient in ARHGAP45 showed an impeded entry into LNs despite their expression of normal levels of CD62L, CCR7 and LFA-1 molecules. Figure 3. Arhgap45−/− naïve T and B cells showed impeded LN entry CTV-labeled splenocytes (20 × 106 cells) from either Arhgap45−/−or WT mice were mixed with CMTPX-labeled WT splenocytes (20 × 106) and injected intravenously in WT recipient mice. After 4 h, single-cell suspensions were prepared from the spleen and mesenteric LNs (mLN) and the percentages of CTV- and CMTPX-labeled B and T cells determined by flow cytometry. The ratio of CTV/CMTPX-labeled cells present in each organ (Ro) was determined and normalized by dividing it with Ri, the ratio of CTV/CMTPX-labeled cells present in the cell mixtures prior to injection. Ro/Ri ratio corresponding to each of the specified mice are shown. Data shown for Arhgap45−/− mice correspond to 4 independent experiments (E1–E4) involving a total of 15 individual mice whereas data shown for WT mice correspond to two independent experiments (E1 and E2) involving a total of five individual mice. The Arhgap45−/− mice analyzed on the same day as the control mice corresponded to the E1 and E2 experiments. Mean and SD are shown. ns, non-significant, ****P ≤ 0.0001; multiple unpaired T-tests. Expression of CCR7, and LFA-1 on naïve CD4+ and CD8+ T cells found in LNs of WT and Arhgap45−/− mice, analyzed by flow cytometry. Gray shaded curves, isot" @default.
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