T Cell Responses in Mammalian Diaphanous-related Formin mDia1 Knock-out Mice

Activated T cells rapidly assemble filamentous (F-) actin networks in response to ligation of the T cell receptor or upon interaction with adhesive stimuli in order to facilitate cell migration and the formation of the immune synapse. Branched filament assembly is crucial for this process and is dependent upon activation of the Arp2/3 complex by the actin nucleation-promoting factor Wiskott-Aldrich Syndrome protein (WASp). Genetic disruption of the WAS gene has been linked to hematopoietic malignancies and various cytopenias. Although the contributions of WASp and Arp2/3 to T cell responses are fairly well characterized, the role of the mammalian Diaphanous (mDia)-related formins, which both nucleate and processively elongate non-branched F-actin, has not been demonstrated. Here, we report the effects on T cell development and function following the knock out of the murine Drf1 gene encoding the canonical formin p140mDia1. Drf1-/- mice develop lymphopenia characterized by diminished T cell populations in lymphoid tissues. Consistent with a role for p140mDia1 in the regulation of the actin cytoskeleton, isolated Drf1-/- splenic T cells adhered poorly to extracellular matrix proteins and migration in response to chemotactic stimuli was completely abrogated. Both integrin and chemokine receptor expression was unaffected by Drf1-/- targeting. In response to proliferative stimuli, both thymic and splenic Drf1-/- T cells failed to proliferate; ERK1/2 activation was also diminished in activated Drf1-/- T cells. These data suggest a central role for p140mDia1 in vivo in dynamic cytoskeletal remodeling events driving normal T cell responses. Activated T cells rapidly assemble filamentous (F-) actin networks in response to ligation of the T cell receptor or upon interaction with adhesive stimuli in order to facilitate cell migration and the formation of the immune synapse. Branched filament assembly is crucial for this process and is dependent upon activation of the Arp2/3 complex by the actin nucleation-promoting factor Wiskott-Aldrich Syndrome protein (WASp). Genetic disruption of the WAS gene has been linked to hematopoietic malignancies and various cytopenias. Although the contributions of WASp and Arp2/3 to T cell responses are fairly well characterized, the role of the mammalian Diaphanous (mDia)-related formins, which both nucleate and processively elongate non-branched F-actin, has not been demonstrated. Here, we report the effects on T cell development and function following the knock out of the murine Drf1 gene encoding the canonical formin p140mDia1. Drf1-/- mice develop lymphopenia characterized by diminished T cell populations in lymphoid tissues. Consistent with a role for p140mDia1 in the regulation of the actin cytoskeleton, isolated Drf1-/- splenic T cells adhered poorly to extracellular matrix proteins and migration in response to chemotactic stimuli was completely abrogated. Both integrin and chemokine receptor expression was unaffected by Drf1-/- targeting. In response to proliferative stimuli, both thymic and splenic Drf1-/- T cells failed to proliferate; ERK1/2 activation was also diminished in activated Drf1-/- T cells. These data suggest a central role for p140mDia1 in vivo in dynamic cytoskeletal remodeling events driving normal T cell responses. Rho family GTPases regulate diverse cellular activities, including actin and microtubule dynamics, gene transcription, the cell cycle, and membrane trafficking (1Jaffe A.B. Hall A. Annu. Rev. Cell Dev. Biol. 2005; 21: 247-269Crossref PubMed Scopus (2298) Google Scholar). Rho GTPase signaling is propagated through interactions with downstream effectors such as autoregulated mammalian Diaphanous-related formins (mDia1, 2The abbreviations used are: mDiamammalian DiaphanousF-actinfilamentous actinWASpWiskott-Aldrich Syndrome proteinTCRT cell receptorBSAbovine serum albuminFNfibronectinERKextracellular signal-regulated kinase 2The abbreviations used are: mDiamammalian DiaphanousF-actinfilamentous actinWASpWiskott-Aldrich Syndrome proteinTCRT cell receptorBSAbovine serum albuminFNfibronectinERKextracellular signal-regulated kinase mDia2, and mDia3) (2Wallar B.J. Alberts A.S. Trends Cell Biol. 2003; 13: 435-446Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). mDia1 and mDia2, for example, act as effectors for both RhoA and RhoB (3Wallar B.J. Deward A.D. Resau J.H. Alberts A.S. Exp. Cell Res. 2007; 313: 560-571Crossref PubMed Scopus (85) Google Scholar, 4Fernandez-Borja M. Janssen L. Verwoerd D. Hordijk P. Neefjes J. J. Cell Sci. 2005; 118: 2661-2670Crossref PubMed Scopus (126) Google Scholar, 5Watanabe N. Madaule P. Reid T. Ishizaki T. Watanabe G. Kakizuka A. Saito Y. Nakao K. Jockusch B.M. Narumiya S. EMBO J. 1997; 16: 3044-3056Crossref PubMed Scopus (677) Google Scholar, 6Alberts A.S. Bouquin N. Johnston L.H. Treisman R. J. Biol. Chem. 1998; 273: 8616-8622Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Cdc42 is known to bind and regulate mDia2 (7Peng J. Wallar B.J. Flanders A. Swiatek P.J. Alberts A.S. Curr. Biol. 2003; 13: 534-545Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). mDia proteins, like other formins, nucleate, processively elongate, and (in some cases) bundle non-branched F-actin (8Goode B.L. Eck M.J. Annu. Rev. Biochem. 2007; 76: 593-627Crossref PubMed Scopus (582) Google Scholar). Actin assembly by formins is mediated by the conserved formin homology-2 (FH2) domain (9Higgs H.N. Trends Biochem. Sci. 2005; 30: 342-353Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar); FH2 domains dimerize and associate with elongating barbed ends of growing actin filaments (10Kovar D.R. Curr. Opin. Cell Biol. 2006; 18: 11-17Crossref PubMed Scopus (190) Google Scholar). mammalian Diaphanous filamentous actin Wiskott-Aldrich Syndrome protein T cell receptor bovine serum albumin fibronectin extracellular signal-regulated kinase mammalian Diaphanous filamentous actin Wiskott-Aldrich Syndrome protein T cell receptor bovine serum albumin fibronectin extracellular signal-regulated kinase Both Wiskott-Aldrich syndrome protein (WASp) and the related N-WASp proteins function as GTPase-regulated actin nucleation-promoting factors by binding to and inducing the Arp2/3 complex to generate branched filamentous (F-) actin) (11Nicholson-Dykstra S. Higgs H.N. Harris E.S. Curr. Biol. 2005; 15: R346-R357Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). WASp is the product of the Wiskott-Aldrich syndrome (WAS) gene and is mutated in that X-linked hereditary disease that is characterized by thrombocytopenia, neutropenia, eczema, increased susceptibility to infection, lymphoma, and leukemia (12Snapper S.B. Rosen F.S. N. Engl. J. Med. 2003; 348: 350-351Crossref PubMed Scopus (26) Google Scholar). WASp-deficient mice (13Snapper S.B. Rosen F.S. Mizoguchi E. Cohen P. Khan W. Liu C.H. Hagemann T.L. Kwan S.P. Ferrini R. Davidson L. Bhan A.K. Alt F.W. Immunity. 1998; 9: 81-91Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 14Snapper S.B. Rosen F.S. Annu. Rev. Immunol. 1999; 17: 905-929Crossref PubMed Scopus (193) Google Scholar, 15Lacout C. Haddad E. Sabri S. Svinarchouk F. Garcon L. Capron C. Foudi A. Mzali R. Snapper S.B. Louache F. Vainchenker W. Dumenil D. Blood. 2003; 102: 1282-1289Crossref PubMed Scopus (67) Google Scholar) have T cell deficiencies similar to those in WAS patients, specifically, poor T cell proliferation and the secretion of cytokines in response to T cell receptor (TCR) stimulation. Mutations in WASp serve as a paradigm for how defects in cytoskeletal remodeling can lead to disease. Although defects in WASp lead to various malignancies, nothing is known about the role of mDia proteins in vivo. To date, only two genetic disorders are associated with mutations in genes encoding mDia proteins (2Wallar B.J. Alberts A.S. Trends Cell Biol. 2003; 13: 435-446Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). In the first, the DNFA1 allele of the DRF1/DIAPH1 (5q31) gene for human mDia1 is affected in nonsyndromic deafness (16Lynch E.D. Lee M.K. Morrow J.E. Welcsh P.L. Leon P.E. King M.C. Science. 1997; 278: 1315-1318Crossref PubMed Scopus (328) Google Scholar). The DFNA1 mutation results in a frameshift mutation predicted to cause a truncation near the C-terminal Dia autoregulatory domain (17Alberts A.S. J. Biol. Chem. 2001; 276: 2824-2830Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar) of mDia1, potentially affecting mDia1 autoinhibition and regulation by small GTPases (9Higgs H.N. Trends Biochem. Sci. 2005; 30: 342-353Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). In the second genetic defect, a breakpoint translocation in the last exon of the DRF2/DIAPH2 (Xq22) gene for human mDia3 protein was identified and was associated with premature ovarian failure in one patient (18Bione S. Sala C. Manzini C. Arrigo G. Zuffardi O. Banfi S. Borsani G. Jonveaux P. Philippe C. Zuccotti M. Ballabio A. Toniolo D. Am. J. Hum. Genet. 1998; 62: 533-541Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). There has been no demonstration that expression or function of mDia3 protein was affected by this mutation. In the present study, the first analysis of knocking out expression of an mDia protein, we demonstrate a role for mDia1 in normal T cell function. Disruption of mDia1 leads to fewer T cells in secondary lymphoid organs in Drf1 null animals. T cell adhesion, migration, and proliferation upon activation were all impaired in T cells derived from Drf1-targeted mice. These results suggest a crucial role for mDia1 in the dynamic regulation of the actin cytoskeleton in activated T cells. Cells, Antibodies, and Reagents—Jurkat E6-1 human leukemic T cells were obtained from ATTC and cultured in RPMI supplemented with 10% fetal bovine serum and penicillin. The following antibodies were used in these studies: anti-mouse CD3 (145-2C11) and anti-human OKT3 hybridomas were from ATCC; anti-mouse CD28 and anti-mouse CD19 from Pharmingen; anti-mouse CD28 from Chemicon; anti-mouse CD29 from Biolegend; anti-mouse CD4, CD3, CD8, and CD45 antibodies from Caltag Laboratories; anti-mouse CXCR4 from Abcam; and anti-hamster secondary antibody from Jackson Laboratory. Anti-p140mDia1 from BD Transduction and anti-mDia2 (pAb 158) antibodies were previously described (19Tominaga T. Sahai E. Chardin P. McCormick F. Courtneidge S.A. Alberts A.S. Mol. Cell. 2000; 5: 13-25Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). CXCL12 was from R&D Systems. Texas Red-conjugated phalloidin and carboxy-fluorescein diacetate, succinyl ester (CFDA-S.E.) were from Molecular Probes. Splenic T Cell Isolation—Three-month-old mice were sacrificed and spleens were removed immediately. Splenic T cells were isolated using the T cell Negative Isolation kit (Dynal) according to the manufacturer's specifications. Briefly, spleens were dissociated with a scalpel and the cell suspension passed through a mesh strainer (BD Biosciences). Red blood cells were lysed with red blood cell (RBC) lysing buffer (Sigma), and the suspension was treated with DNase I (Invitrogen). The remaining strained cells were treated with negative selection antibody mix, followed by incubation with magnetic depletion beads. Bead-bound cells were removed with a magnet, and the remaining purified T cells were washed in RPMI medium with 0.1% fetal bovine serum and resuspended in a minimal volume in serum-free medium. T Cell Migration Assay—Isolated splenic T cells were loaded with Calcein AM (Molecular Probes) for 15 min at 37 °C prior to migration assay. T cells (1 × 105) in serum-free RPMI were added to the upper well of a 3-μm pore-size transwell insert (Corning) previously coated overnight with 20 μg/ml fibronectin or BSA. Where indicated, 10% serum-containing RPMI or 30 ng/ml CXCL12 was added to the lower well; otherwise, serum-free conditions were maintained. Cells were allowed to migrate for 5 h at 37 °C. The inserts were aspirated and upper membranes were wiped with a cotton swab. Inserts were then moved into wells containing ice-cold lysis buffer (20 mm Tris-HCl, pH 7.5, 1% Triton X-100, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4) and 1 μg/ml each aprotinin, leupeptin, and pepstatin. Concurrently, cells in the lower well were transferred to an Eppendorf tube, pelleted, and lysed. Cells adherent to the bottom well were also washed and lysed. All three lysates were pooled and 200 μl was loaded into a 96-well plate; fluorescent units were read in a fluorescent plate reader (485 nm excitation, 520 nm emission). Total cells migrated was extrapolated from a standard curve derived from readings from a known number of labeled cells. Standard deviations are expressed from triplicate wells. T Cell Adhesion Assay—Isolated splenic T cells were loaded with Calcein AM (Molecular Probes) for 15 min at 37 °C prior to adhesion assay. T cells (1 × 105) in serum-free (or serum-containing, where indicated) RPMI were added to a 96-well plate previously coated overnight with 20 μg/ml of fibronectin or BSA. Cells were incubated for 60 min or longer (where indicated) at 37 °C, after which wells were aspirated and washed three times with phosphate-buffered saline/0.5% BSA, rotating the plate 90° with each wash. Adherent cells were read in a fluorescent plate reader (485 nm excitation, 520 nm emission), with total adherent cells extrapolated from a standard curve derived from a known number of labeled cells. Standard deviations are expressed from triplicate wells. T Cell Stimulation—For T cell activation, 2 × 105 mouse T cells were incubated on ice for 30 min with 5 μg/ml each anti-CD3 and -CD28, followed by cross-linking with 5 μg of anti-hamster IgG for 5 min at 37 °C. Cells were immediately used in intracellular flow cytometric analysis for determining F-actin accumulation. cells × 105) were washed in phosphate-buffered and using and Cell kit according to the manufacturer's Briefly, after and cells were indicated) and with antibodies (1 2 × 105 Texas Red-conjugated phalloidin F-actin for 20 min at in the Cells were washed in phosphate-buffered and was using a flow (BD Biosciences). and and were as described (7Peng J. Wallar B.J. Flanders A. Swiatek P.J. Alberts A.S. Curr. Biol. 2003; 13: 534-545Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). cells used in the were and mice were mice used in this were of a T Cell in Drf1-targeted the role of mDia1, the gene encoding p140mDia1 protein was as described (7Peng J. Wallar B.J. Flanders A. Swiatek P.J. Alberts A.S. Curr. Biol. 2003; 13: 534-545Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Drf1-targeted mice were used for these p140mDia1 protein expression was in splenic T cells by and intracellular flow and as well as in by not of the related mDia2 and mDia3 proteins was in T cells from to mice not and of and splenic from Drf1-/- mice diminished T cell in these organs and data not T cell lymphopenia lymphoid tissues. of splenic T cells isolated by negative selection from and Drf1-/- mice a in null flow cytometric analysis of and in the of or cells and Drf1-/- and in expression of T cell receptor and in not T cell were diminished in Drf1-/- to a blood and a role for mDia1 in T cell development is with a role for actin nucleation-promoting factors in T cell as mice T cells S. S. S. S. T. K. T. J. 2007; PubMed Scopus Google Scholar, F. P. C. Bhan A.K. Snapper S.B. J. Exp. 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Biol. 2006; 16: Full Text Full Text PDF PubMed Scopus Google we impaired integrin expression for adhesion defects to integrin expression was upon and the of the and were Drf1 not and Migration serum was completely impaired in Drf1-/- cell migration was with that of T cells cell migration the chemokine CXCL12 a or in Drf1-/- and T cell migration, To the defects in Drf1-/- T cells to adhesion we allowed Drf1-/- T cells to to through 5 the of migration the adhesion through 5 adhesion was in Drf1-/- T cells to cells that this in adhesion at 5 h migration, is that the defects both serum and CXCL12 were not to of expression of total or the receptor for was unaffected in Drf1-/- T cells Drf1 knock out the cytoskeletal remodeling for migration, in the of of cell expression of integrin or F-actin in T Drf1-targeted T cells to cytoskeleton, a process for normal T cell responses such as adhesion and migration K. J. F. M.K. D. 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Immunol. 2003; PubMed Scopus Google with these in to data suggest that of the dynamic actin cytoskeleton through actin nucleation-promoting factors is crucial for normal T cell function. of mDia1 expression function a in the cytoskeleton of activated T cells. are genetic defects in human Drf1 gene are in of human and for of the and Resau and for also Alberts for with

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