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.

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) (11). 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 (12). WASp-deficient mice (13)(14)(15) 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 (2). In the first, the DNFA1 allele of the DRF1/DIAPH1 (5q31) gene for human mDia1 is affected in nonsyndromic deafness (16). The DFNA1 mutation results in a frameshift mutation predicted to cause a truncation near the C-terminal Dia autoregulatory domain (17) of mDia1, potentially affecting mDia1 autoinhibition and regulation by small GTPases (9). 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 (18). 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.
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. Beadbound 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 ϫ 10 5 ) in serum-free RPMI were added to the upper well of a 3-m pore-size transwell insert (Corning) previously coated overnight with 20 g/ml fibronectin or BSA. Where indicated, 10% serumcontaining 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 Na 3 VO 4 ) and 1 g/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 l 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 ϫ 10 5 ) in serum-free (or serumcontaining, where indicated) RPMI were added to a 96-well plate previously coated overnight with 20 g/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 ϫ 10 5 mouse T cells were incubated on ice for 30 min with 5 g/ml each anti-CD3 and -CD28, followed by cross-linking with 5 g of antihamster IgG for 5 min at 37°C. Cells were immediately used in intracellular flow cytometric analysis for determining F-actin accumulation. Intracellular Flow Cytometry-T cells (2 ϫ 10 5 ) were washed in phosphate-buffered saline and fixed using Fix and Perm Cell Permeabilization kit (Caltag Laboratories) according to the manufacturer's protocol. Briefly, after fixation and washing, cells were simultaneously permeabilized (when indicated) and stained with fluorescent-conjugated primary antibodies (1 g, 2 ϫ 10 5 cells) and/or Texas Red-conjugated phalloidin (for F-actin accumulation) for 20 min at room temperature in the dark. Cells were washed in phosphate-buffered saline, and fluorescence was detected using a FACSCalibur flow cytometer (BD Biosciences). Gene Targeting and Genotyping-Gene targeting and genotyping were performed exactly as described (7). Embryonic stem cells used in the initial targeting were 129Sv and chimeric mice were B6; mice used in this study were of a mixed 129/B6 background.

RESULTS
T Cell Lymphopenia in Drf1-targeted Mice-To investigate the physiological role of mDia1, the gene encoding p140mDia1 protein was targeted as described (7). Three-month-old Drf1targeted mice were used for these studies. p140mDia1 protein expression loss was demonstrated in splenic T cells by Western blotting and intracellular flow cytometry (Fig. 1, A and B) as well as in tissues by quantitative reverse transcription PCR (data not shown). Expression of the related mDia2 and mDia3 proteins was unchanged in T cells from 3-month-old Drf1 Ϫ/Ϫ relative to Drf1 ϩ/ϩ mice (data not shown).
Hematoxylin and eosin staining of lymph node and splenic sections from Drf1 Ϫ/Ϫ mice demonstrated diminished T cell infiltration in these organs ( Fig. 1C and data not shown, respectively), suggesting T cell lymphopenia within peripheral lymphoid tissues. Quantifying absolute numbers of splenic T cells isolated by negative selection (see "Experimental Procedures") from Drf1 ϩ/ϩ , Drf1 ϩ/Ϫ , and Drf1 Ϫ/Ϫ mice showed a dramatic reduction in null animals (Fig. 1D). However, flow cytometric analysis of blood, bone marrow, and spleen revealed little change in the overall percentages of CD45 ϩ lymphocytes or CD45ϩCD19ϩ B cells between Drf1 ϩ/ϩ and Drf1 Ϫ/Ϫ animals (supplemental Table S1 and gating strategy in supplemental Fig. S1). Despite equal expression of T cell receptor across genotypes and in their respective tissues (data not shown), mature CD8ϩ T cell percentages were diminished in Drf1 Ϫ/Ϫ bone marrow, spleen, thymus, and, to a lesser extent, blood (Fig. 2, A  and B), suggesting a potential role for mDia1 in T cell development and/or proliferation. This is consistent with a role for actin nucleation-promoting factors in T cell development, as WASp Ϫ/Ϫ mice showed defective CD4ϩ T regulatory cells (20,21). Furthermore, mice lacking coronin, a protein regulating Arp2/3, showed reduction in the CD8ϩ T cell compartment (22). Collectively, these data suggest an important role for regulation of the actin cytoskeleton in maintaining T cell homeostasis.
Adhesion, Migration, and Ruffling Defects Observed in Drf1null T cells-Rho GTPase-controlled cytoskeletal remodeling directs adhesion and migration during development and immune responses (23). In addition to impaired T cell maturation, loss of mDia1 protein expression may dually affect T cell emigration into peripheral lymphoid organs, thereby leading to the observed T cell lymphopenia in these tissues. Therefore, we assessed whether T cell adhesion and/or migration were impaired in Drf1 Ϫ/Ϫ cells.
Drf1 ϩ/ϩ and Drf1 ϩ/Ϫ T cells adhered to fibronectin (FN), yet adhesion was reduced about 50% in Drf1 Ϫ/Ϫ T cells (Fig. 3A). Because the N terminus of mDia1 binds to and regulates integrin in vitro (24), we analyzed whether impaired integrin expression accounted for adhesion defects to FN. ␤1 integrin (CD29) expression was unchanged upon CD3/CD28 stimulation over time, and the overall percentages of CD29 ϩ lympho-cytes within the bone marrow and spleen were unchanged across Drf1 genotypes (data not shown and supplemental Fig.  S2, respectively). Migration toward serum was completely impaired in Drf1 Ϫ/Ϫ cells, while Drf1 ϩ/Ϫ cell migration was comparable with that of Drf1 ϩ/ϩ T cells (Fig. 3B). Chemotactic cell migration toward the chemokine CXCL12 revealed a complete or severe block in Drf1 Ϫ/Ϫ and Drf1 ϩ/Ϫ T cell migration, respectively (Fig. 3C). To assess whether the migratory defects . Impaired Drf1 ؊/؊ splenic T cell adhesion and migration. A, calcein AM-labeled splenic T cells were allowed to adhere to 20 g/ml BSA or fibronectin (FN) in the presence of 10% FBS (fetal bovine serum) or in serumfree medium (SFM), and adhesion was quantified after 60 min. B and C, transwell migration assays using BSA or FN-coated (20 g/ml) transwell inserts and either SFM, 10% FBS (B) or 30 ng/ml CXCL12 (C) in serum-free medium placed in the lower well. Migration into the lower well was quantified after 5 h in triplicate. Error bars represent standard deviations. p values were determined from n Ͼ 3 using Student's t-test assuming equal variances. Shown is a representative experiment of three.
in Drf1 Ϫ/Ϫ T cells could simply be attributed to severe adhesion defects, we allowed Drf1 Ϫ/Ϫ T cells to adhere to FN through 5 h, the duration of our migration assays. Upon extending the adhesion assay through 5 h, adhesion was 70% recovered in Drf1 Ϫ/Ϫ T cells relative to Drf1 ϩ/ϩ cells (supplemental Fig. S3A). Thus, although possible that this reduction in adhesion at 5 h could adversely affect migration, it is likely that the migratory defects observed toward both serum and CXCL12 were not solely attributable to lack of adhesion. Furthermore, expression of total or surface CXCR4, the receptor for CXCL12, was unaffected in Drf1 Ϫ/Ϫ T cells (supplemental Fig. S3B). Therefore, while Drf1 knock out affects the cytoskeletal remodeling machinery necessary for migration, it does so in the absence of down-regulation of cell surface expression of either ␤1 integrin or CXCR4.
Diminished F-actin Accumulation in Drf1-null T Cells-Activated Drf1-targeted T cells may fail to reorganize their cytoskeleton, a process required for normal T cell responses such as adhesion and migration (25). Isolated splenic T cells were plated on CD3/CD28-coated coverslips to engage the TCR, and cell morphology was examined by differential interference contrast time-lapse microscopy. Drf1 ϩ/ϩ T cells ruffled extensively 30 min postplating, whereas Drf1 Ϫ/Ϫ cells were weakly adherent and failed to ruffle despite extended ligation (through 240 min) (Fig. 4A and supplemental  movie). Comparable results were observed in T cells adhered to FN and ICAM-2 (intercellular adhesion molecule 2) (data not shown), suggesting a role for p140mDia1 in dynamic membrane ruffling in activated T cells. Indeed, Jurkat cells adhered to anti-CD3 antibodycoated coverslips and immunostained for p140mDia1 localized mDia1 to ruffles (Fig. 4B).
As an actin nucleation-promoting factor, the mDia1 formin homology-2 domain potently assembles F-actin filaments (2). F-actin accumulation within the immunological synapse (formed at the site of T cell interaction with antigen-presenting cells) is a hallmark of T cell activation (23,26). To assess whether mDia1 plays a role in F-actin accumulation in activated T cells, splenic T cells adherent to anti-CD3/CD28 antibody-coated coverslips were imaged by confocal three-dimensional reconstruction (Fig. 4C). Drf1 ϩ/ϩ T cells generated an F-actin-rich immune synapse adjacent to the coverslip, whereas F-actin failed to accumulate at the immune synapse in Drf1 Ϫ/Ϫ T cells. Furthermore, splenic T cells stimulated with anti-CD3/ CD28 antibodies were stained with fluorescent-labeled phalloidin and analyzed by flow cytometry (Fig. 4D) to assess overall F-actin polymerization. F-actin accumulation was marginally defective (64% of wild type; inset) in T cells from Drf1 ϩ/Ϫ animals, yet Drf1 Ϫ/Ϫ cells were dramatically impaired (25% of wild type). Collectively, these data suggest that F-actin assembly necessary for immune synapse formation requires p140mDia1.
Impaired Proliferation upon TCR Activation in Drf1 Ϫ/Ϫ T Cells-Formation of the actin-rich immune synapse is not only required for sustained T cell receptor signaling, interleukin 2 production and proliferation, but it also functions in regulating the strength of the signals emanating from the T cell receptor (27). Therefore, in addition to adhesion and migratory defects, proliferation defects upon T cell activation in Drf1 Ϫ/Ϫ animals could account for the diminished splenic T cell compartment. Indeed, a role for mDia1 in cytokinesis was previously described, and mDia1 has been localized to the midbodies

cells.
A, isolated splenic T cells from Drf1 ϩ/ϩ or Drf1 Ϫ/Ϫ mice adhered to anti-CD3/CD28-coated glass coverslips for 30 min and were imaged by time-lapse differential interference contrast microscopy for 10 min (supplemental movie). B, Jurkat E6 cells were allowed to adhere to anti-CD3-or poly-L-lysine-coated coverslips for 30 min. Cells were fixed, permeabilized, and stained with anti-mDia1 antibodies along with Texas Red phalloidin to visualize actin architecture. C, splenic T cells were adhered to CD3/CD28 antibody-coated coverslips for 30 min. Cells were fixed and stained with Hoechst 33258 (blue) to visualize nuclei and Texas Red phalloidin (red) to visualize F-actin architecture by serial Z sectioning and three-dimensional reconstruction by confocal microscopy. Overlays of individual images rotated 90°are depicted, with the coverslip position noted. D, F-actin accumulation was assessed by intracellular flow cytometry using isolated splenic T cells stimulated with control IgG or anti-CD3/CD28 antibodies. Fixed, permeabilized cells were stained with fluorescent phalloidin. Inset depicts data as the percentage of mean fluorescence of the wild type.

DISCUSSION
The results presented here indicate a crucial role for p140mDia1 in cytoskeletal remodeling during T cell activation. Our data reveal the involvement of p140mDia1 in T cell proliferation and migration, consistent with considerable genetic evidence that Rho GTPases, regulatory exchange factors (i.e. Vav), and their effectors (i.e., WASp) participate in normal T cell function. For example, Vav Ϫ/Ϫ or WASp Ϫ/Ϫ animals are defective in dynamic remodeling of actin following TCR ligation (29,30). Furthermore, the GTPase RhoA regulates integrinmediated T cell adhesion and chemotactic migration (31,32). Although p140mDia1 is a known RhoA effector, it is plausible that the disruption in adhesion and chemotactic migration is due to disruption of RhoA-mediated p140mDia1 activation and dysregulation of forminmediated actin dynamics. Indeed, a role for mDia1 and the related budding yeast formin Bni1 have been extensively characterized as promoting cell polarity and mediating both directional cell migration (33)(34)(35) and spontaneous cell migration (36). Furthermore, expression of constructs interfering with mDia1 function or small hairpin RNA constructs directed against mDia1 also disrupt microtubule organizing center reorientation in activated T cells, a hallmark of T cell polarization (36,37).
Although our results indicate that loss of mDia1 protein expression does not affect integrin expression, they do not preclude the possibility that the defects observed in either adhesion or chemotactic migration were attributed to changes in integrin affinity. Indeed, neutrophils derived from WASp patients and knock-out animals are defective in integrin clustering and signaling and fail to polarize upon activation (38), so it is plausible that disruption of actin nucleation-promoting factors affects integrin function without altering protein expression. Studies have been initiated to assess this possibility in activated Drf1 Ϫ/Ϫ T cells.
Our data also implicate p140mDia1 in membrane ruffling, possibly through RhoA interaction, consistent with recent fluorescence resonance energy transfer (FRET)-based evidence (39,40). The contribution of p140mDia1 to ruffling potentially involves cooperation between cytoskeletal remodeling activities mediating branched (WASp/Arp2/3) and non-branched actin nucleators (formins) (41). In this regard, it is compelling that defects in WASp, the canonical activator of Arp2/3 (42), lead to a impaired T cell activation and F-actin assembly nearly identical to defects observed here. Future studies investigating mechanisms whereby formins and activated Arp2/3 cooperate to elaborate dendritic networks within cells are currently underway.
In contrast to our results, a recent study showed that depletion of human mDia1 (i.e. DIA1) by short hairpin RNA did not affect actin dynamics at the immune synapse upon TCR stimulation (37). However, p140mDia1 protein depletion was incomplete in those studies. Our data show that F-actin accumulation/polymerization (Fig. 4) and FN adhesion (Fig. 3) in T cells from Drf1 ϩ/Ϫ mice were modestly, if at all, affected, sug- T cells were treated with Concanavalin A or plate-bound TCR-activating antibodies: (5 g/ml anti-CD3 Ϯ 2 or 5 g/ml anti-CD28 as described) (28). Error bars represent standard deviations (n ϭ 3). B, isolated thymocytes were labeled with CFDA-S.E. and stimulated with interleukin 2 for 96 h, after which CFDA-S.E. dye dilution was assessed by flow cytometry. C, isolated splenic T cells were stimulated for 30 min with anti-CD3/CD28 antibodies and lysates immunoblotted for phospho-ERK1/2, total ERK1/2, or tubulin.
gesting that lower levels of p140mDia1 are sufficient to mediate actin dynamics in stimulated T cells.
Disrupting the dynamic actin cytoskeleton has been demonstrated to be detrimental to normal T cell function. Indeed, T cells lacking WASp fail to proliferate in response to proliferation stimuli (30). Moreover, our results suggest that upon loss of mDia1 F-actin content is dramatically decreased and chemotactic migration is ablated. However, defective T lymphocyte trafficking and homeostasis were observed in coronin-1 knockout mice (22); coronin-1 is a protein demonstrated to inhibit Arp2/3-directed actin polymerization (43).
In that study, coronin-1 was required for normal actin dynamics, as loss of coronin-1 expression induced a 2-fold increase in F-actin content, subsequently inhibiting chemotactic migration. Moreover, constitutively activated mDia1 (lacking the GTPase binding and a portion of the Dia inhibitory domain) enhanced actin accumulation while decreasing chemotactic migration upon TCR engagement (36). Indeed, activating mDia1 can also function to arrest the normally dynamic actin and microtubule cytoskeletons (34, 44 -46) and inhibit normal T cell function (36), consistent with our results. Collectively, these studies in addition to data presented here suggest that exquisite control of the dynamic actin cytoskeleton through actin nucleation-promoting factors is crucial for maintaining normal T cell function. Control of mDia1 expression and/or function may therefore be a component in regulating the cytoskeleton of activated T cells. Studies currently underway are investigating whether genetic defects in human Drf1 gene are observed in instances of human hematological diseases.