Essential role for Vav1 in activation, but not development, of 
T cells
Wojciech Swat1,3,6,
Ramnik Xavier4,
Atsushi Mizoguchi4,
Emiko Mizoguchi4,
Jessica Fredericks1,
Keiko Fujikawa1,
Atul K. Bhan4 and
Frederick W. Alt1,3,5
1 The Center for Blood Research, 2 Howard Hughes Medical Institute, 3 Department of Pediatrics, The Childrens Hospital, 4 Department of Pathology, Medicine and Molecular Biology, Massachusetts General Hospital, and 5 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA 6 Present address: Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA
Correspondence to: F. W. Alt, The Childrens Hospital, Howard Hughes Medical Institute, 320 Longwood Avenue, Boston, MA 02115, USA; E-mail: alt{at}rascal.med.harvard.edu
Transmitting editor: K. M. Murphy
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Abstract
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Vav1 is a guanine nucleotide exchange factor essential in the development and function of
ß lineage T cells. Here we report that in contrast to profound effects on pre-TCR- or
ß TCR-dependent events in thymocyte development, Vav1 deficiency has no detectable effect on the development of 
T cells. Strikingly, however, these 
T cells are markedly deficient in signaling through the 
TCR, as evidenced by a lack of proliferation and cytokine production in response to stimulation with anti-
TCR antibodies. We propose that Vav1 has a unique and non-redundant role in the initiation of signaling downstream of the 
TCR in lymphocytes.
Keywords: antigen receptor, lymphocyte activation, lymphocyte development, intestinal epithelial lymphocyte, Vav1
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Introduction
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T cells can be divided into two lineages based on the expression of
ß or 
TCR (1). While
ß T cells have a well-defined role in adaptive immune responses, the role of the 
T cells is less clear (2). Both types of T cells develop in the thymus; however, mature
ß T cells migrate to the lymph nodes and spleen, whereas 
T cells favor residence in mucosal environments, especially the intraepithelial compartment of skin and gut (2). During development,
ß and 
T cells derive from a pool of common progenitors contained among CD4CD8 [double-negative (DN)] thymocytes (3,4). Of these, the most immature CD44+CD25 (or DN1) cells can give rise to several lymphoid lineages including T, B, NK and thymic dendritic cells (4). The subsequent CD44+CD25+ (or DN2) population comprises pro-T cells for both
ß and 
lineages (3). These DN2 cells are dependent on cytokine signaling for survival and initiation of rearrangements in TCR
and
loci (5), while TCRß genes rearrange during transition to the CD44CD25+ (or DN3) stage (68). At the DN3 stage, thymocytes encounter a checkpoint (referred to as ß selection), which allows only cells with functional TCRß to differentiate into CD4+CD8+ [double-positive (DP)] cells (9). DP cells can further develop into mature CD4+ or CD8+ T lymphocytes following TCR
gene assembly and expression.
Unlike
ß T cells, 
T cells in general do not progress through the DP stage during their development; moreover, only
ß, but not 
, lineage cells are thought to express pre-TCR and undergo proliferative expansion (9). In this context, 
and pre-TCR expression can direct common progenitor cells to differentiate respectively into either the 
or
ß lineage. Thus, cells that successfully rearrange and express TCR
and
genes develop along the 
lineage, whereas TCR
and
genes in
ß T cells are mostly out-of-frame (1012). In contrast, successful rearrangement and expression of TCRß genes, in a complex with pre-TCR (pT
) chains, commits progenitor cells to the
ß lineage (13).
Various lines of evidence suggest that
ß and 
receptors may employ common signaling pathways. Development of both
ß and 
T cells was impaired in mice with disrupted expression of any of several common components of the TCR signaling apparatus, including CD3
(14), CD3
(15,16), TCR
(17,18), TCR
(19,20), Lck and Fyn (21, 22), CD45 (23), and SLP76 (24). Thus, both types of TCR seem to share at least several signaling components. However, differential usage of signaling cascades by 
and
ß T cells was suggested by transcriptional profiling approaches (25,26). In addition, recent observations suggested that the pre-TCR may employ a distinct mechanism of receptor clustering to exert its function, as only the pre-TCR, but not 
TCR, was capable of spontaneously localizing to glycolipid-enriched membrane rafts in DN3 thymocytes (27), most probably independently of ligand binding (28). Such differences in receptor clustering mechanism could, conceivably, underlie distinct signaling properties of these receptors even if downstream components were shared. Indeed, biochemical evidence suggests that despite differences in their behavior on cell surface, pre-TCR and
ß TCR use similar downstream effectors to transduce signals, including phospholipase C (PLC)
1-mediated activation of Ca2+, protein kinase C, Ras, NF-
B and NFAT (29).
In this context, mice deficient in Vav1, a Rho guanine nucleotide exchange factor and a key regulator of PLC
1 activity, demonstrated a strict requirement for Vav1 in
ß T cell development and activation (3033). However, it is not known if Vav1 is also required for signaling downstream of 
TCR. To determine whether Vav1 has a role in development and activation of 
lineage cells, we used Vav1-deficient mice. Here we report the results of our experiments, which establish an essential role for Vav1 in signaling downstream of 
TCR, but not development of 
lineage T cells.
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Methods
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Antibodies and flow cytometry
Cell suspensions were counted and stained with antibodies following standard procedures. The following antibody conjugates were used (PharMingen): phycoerythrin (PE)H129.19 (anti-CD4), FITC, PE, cytochrome c or biotin53-6.7 (anti-CD8a), PE3C7 or FITC7D4 (anti-IL-2R
), FITC-14-2C11 (anti-CD3
) and cytochrome cRA3-6B2 (anti-B220). Biotinylated antibodies were detected with streptavidinPE or streptavidincytochrome c (PharMingen). Subsets of DN thymocytes were analyzed based on expression of CD44 and CD25, after gating out all cells staining with a cocktail of biotinylated antibodies to CD4, CD8, B220, Mac-1 and Gr-1 followed by streptavidinCyChrome. For intracellular staining of the TCRß cells were first stained with PECD4 and CyChromeCD8
on the surface. Subsequently, cells were fixed, permeabilized in 1% saponin and stained with FITC labeled anti-Cß specific antibodies (H57; PharMingen) and analyzed on a FACSCalibur flow cytometer (Becton Dickinson) with CellQuest software. Data are displayed as histograms or dot-plots with a logarithmic scale. Each plot represents analysis of
2 x 105 events collected as listmode files.
Isolation and stimulation of intraepithelial lymphocytes (IEL)
The IEL were extracted as previously described (34). Briefly, after removal of Peyers patches, small intestine was inverted with the aid of polyethylene tubing and incubated in RPMI containing 1% FBS at 37°C for 30 min with shaking. After vortexing for 30 s, the cells were passed through a glass wool column. The IEL were further purified using 40/72% Percoll gradient centrifugation. Flow cytometric analysis showed that >95% of the extracted cells were CD3+.
ß T cells were purified from lymph node cell suspensions by magnetic sorting and removal of B cells with anti-Ig coated Dynabeads (Dynal, Oslo, Norway) to avoid receptor cross-linking during the purification process, using standard procedures. The purity of resulting T cells was >90%, as confirmed by FACS analysis. Lymphocytes were cultured in RPMI 1640 medium supplemented with 10% FCS, penicillin/streptomycin, L-glutamine, sodium pyruvate, non-essential amino acids and 2-mercaptoethanol. For cytokine analysis, the IEL (1 x 105 / well) were stimulated with anti-TCR
mAb (UC7, 10 mg/ml)- or hamster Ig-coated plates for 72 h (34). In some experiments expression of 
TCR on cell blasts was confirmed by staining with a different anti-
TCR mAb (clone GL4; PharMingen). The concentration of cytokines in supernatants was analyzed by ELISA according to the manufacturers instruction (PharMingen). For proliferation assay, IEL (1 x 105/well) were cultured in anti-TCR
mAb (UC7, 10 mg/ml)- or hamster Ig-coated plates.
ß T cells were cultured at the concentration of 5 x 104 cells/well plate-bound anti-CD3
. After 48 h culture, the cells were pulsed with 1 µCi [3H]thymidine for 16 h. Thymidine incorporation was determined by scintillation counting. The data are displayed as raw c.p.m. values. All assays were conducted in triplicates.
Mice
Vav1/ germline mice were previously described (32) and kindly provided by Dr Victor Tybulewicz. Mice were housed in a barrier-type facility of the Childrens Hospital (Boston, MA). In all experiments, littermate mice were analyzed at the age between 4 and 8 weeks. Since the results of all experiments with either Vav1+/+ or Vav1+/ cells were the same, we did not further distinguish between the two.
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Results
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Impaired ß selection in Vav1-deficient mice
A role for Vav1 in signaling by the
ß TCR was previously established (3033). Vav1-deficient mice had a reduction in single-positive (SP) and DP thymocytes, while DN cells were increased [(3033) and Fig. 1], suggesting a potential role for Vav1 in signaling ß selection by the pre-TCR. To determine if pre-TCR signaling and ß selection was specifically impaired in Vav1-deficient mice, we first analyzed DN3 thymocytes for expression of TCRß chains. To that end, we stained wild-type, Vav1/ or RAG2/ CD25+ thymocytes for intracellular TCRß (Fig. 1B, left panels). Vav1-deficient mice had a significantly increased proportion of DN3 cells that expressed cytoplasmic TCRß chains (15%), as compared to wild-type (5%) (Fig. 1B). As expected, RAG2/ thymocytes did not stain with anti-TCRß antibodies (Fig. 1B). Strikingly, analyses of light scatter properties of DN3 thymocytes revealed that Vav1/ cells were significantly smaller in size than those from wild-type mice (Fig. 1B). In addition, both percentages and total numbers of large (blasting) cells within the DN and DP populations were
4- to 5-fold less in Vav1/ mice than in wild-type mice (Fig. 1B, right panels). In this regard, increases in cell size were previously correlated with ß selection by others (10). Together, these results are consistent with the notion that pre-TCR signaling is impaired in Vav1-deficient mice leading to the accumulation of TCRß+ DN3 cells due to inefficient ß selection.
Vav1-deficient 
T cells develop normally
While Vav1 was shown to be required for proliferative expansion of
ß lineage thymocytes (3033), it is thought that 
lineage cells do not undergo a 
TCR-driven stage of proliferative expansion. In fact, in contrast to the reduction in
ß-bearing cells, Vav1/ mice showed slightly increased numbers of thymic 
T cells (Fig. 2A and Table 1). Consistent with previous reports, our examination of peripheral lymphoid organs showed reduced numbers of
ß T cells in Vav1-deficient mice (3033) (Fig. 2B, Table 1 and data not shown). However, both Vav1-deficient and wild-type mice showed comparable numbers and percentages of 
T cells in spleens (Fig. 2B and Table 1). In addition, both types of mice yielded similar numbers of IEL extracted from lamina propria of small intestines (
0.51 x 106 cells per mouse, data not shown), which contained 5060% of 
T cells (Fig. 2B, middle panel). We conclude from these experiments that unlike
ß T cells, 
lineage cells do not require Vav1 for their development.
Vav1 is required for activation of 
T cells via the TCR
Our data clearly showed that 
T cells do not need Vav1 for their development; however, Vav1 may still be required for activation via the 
TCR. To determine if such requirement existed in this lineage, we first analyzed the capability of wild-type and Vav1/ 
T cells to proliferate in response to anti-
TCR antibodies. In these studies we used populations of IEL which were isolated without the use of anti-
TCR antibodies (see Methods). IEL from either wild-type or Vav1/ mice, which typically contained 5060% 
T cells, showed comparable levels of surface expression of 
TCR as measured by flow cytometry (Fig. 2B and data not shown). IEL were subsequently cultured in the presence or absence of various concentrations of stimulatory anti-
TCR antibodies for 2 days, after which proliferation was measured by [3H]thymidine incorporation (Fig. 3A and see Methods). Strikingly, while wild-type cells responded to anti-
TCR cross-linking with vigorous proliferation, Vav1/ cells completely failed to respond in this assay (Fig. 3A). Staining with TCR
antibodies and flow cytometry analyses confirmed the majority of cells undergoing blastic transformation to be 
TCR+ (see Methods and data not shown). The failure of Vav1-deficient 
T cells to proliferate was also demonstrated by flow cytometry, which clearly showed a lack of blast formation in Vav1/ cultures at 36 h following stimulation (Fig. 3B, right panel). As expected based on previous studies, Vav1/
ß T lymphocytes also failed to proliferate in response to triggering with anti-
ß TCR antibodies in these assays and Fig. 3B, left panel). Thus, similar to
ß T cells, 
T cells also require Vav1 for TCR-induced proliferative responses. Furthermore, Vav1/ IEL cells were completely unable to produce IFN-
in response to stimulation with anti-
TCR antibodies, which was in clear contrast to wild-type cells (Fig. 3C). We conclude from these experiments that Vav1 is absolutely required for activation of 
T cells by antigen receptor.
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Discussion
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Previous studies demonstrated a role for Vav1 in signaling by
ß TCR and T cell activation (3033,35). In this report we show that Vav1 is also required for signaling by 
TCR. Despite its essential role in 
T cell activation, Vav1 is dispensable for 
T cell development. We also extend previous observations on the role of Vav1 in the development of
ß T cells (3033). Specifically, we show that Vav1 deficiency results in a partial block at the DN3 stage, suggesting that Vav1 is required for efficient signaling by the pre-TCR. This developmental block is not due to a defect in assembly or expression of TCRß chain genes, since a large fraction of Vav1/ DN3 cells have cytoplasmic TCRß chains. Notably, Vav1-deficient mice also have reduced numbers of DP thymocytes and fewer large blasting DN3 TCRß+ thymocytes, suggesting that Vav1 functions, at least in part, to promote the proliferative expansion and developmental stage progression directed by the pre-TCR.
Strikingly, 
T cell development is unperturbed in Vav1-deficient mice. Unlike development along the
ß lineage pathway, commitment to the 
lineage is thought not to require TCRß-driven proliferative expansion. In this regard, earlier reports suggested a possibility that 
T cell precursors undergo TCRß-driven proliferation (36,37). However, more recent data showed that only precursors of
ß, but not 
, lineage proliferate in a pre-TCR-dependent manner (13). Notably, pT
-deficiency specifically impaired the development of
ß, but not 
, lineage cells (9). These observations lend further support to a view that unlike
ß, 
T cells do not undergo extensive TCR-dependent proliferation during development. Therefore, our finding that 
T cells can be generated in normal numbers in the absence of Vav1 is in accord with the notion that the TCR signaling apparatus requires Vav1 to trigger proliferation.
Development of 
T cells is crucially dependent on signaling by cytokine receptors sharing the common
chain (38,39). In this regard, our findings clearly demonstrate that Vav1 is not required for cytokine receptor signaling essential for development of 
T cells. However, our results are also consistent with the possibility that Vav1 is involved in such signaling, as suggested by previous reports, e.g. that Vav1 can bind Jak2 kinase in myeloid cell lines (40). If this is the case, functional redundancy between Vav family members could explain the lack of apparent developmental defects of 
T cells in Vav1/ mice, while Vav1 function is clearly not redundant for
ß T cells (41). Notably, recent studies also demonstrated redundancy between Vav1 and Vav2 in B cells (42,43). In this context, while Vav1 homologous proteins, Vav2 and Vav3, are expressed in DN3 thymocytes in RAG2/ mice (Swat et al., unpublished data), their specific functions in these cells remain to be elucidated.
Although Vav1 is dispensable in 
T cell development, we show in this report that it is strictly required for activation of mature 
T cells. In this regard, requirements for Vav1 for activation of 
T cells appear to be similar to those of
ß lineage T cells. Vav1 deficiency severely impairs the ability of
ß T cells to proliferate and produce IL2 in response to stimulation with antigen or anti-TCR antibodies (30,31,33). Likewise, Vav1-deficient 
T cells also show a complete block in TCR-induced proliferation and cytokine production. Several TCR-proximal signaling proteins were previously implicated in 
TCR function (1424). However, in contrast to Vav1 deficiency, all of those mutations also disrupted 
T cell development. Therefore, it is plausible that Vav1 is specifically involved in the regulation of only a subset of signals downstream of TCRCD3 modules that are required for efficient proliferative expansion. On the other hand, Vav1 may not be involved in regulating
ß or 
T cells by other signaling pathways implicated in lineage commitment, including Notch family receptors (44), their effectors, basic helix-loop-helix (bHLH) transcription factors (45) or a repressor of bHLH, the Id3 (46), or, alternatively, a role for Vav1 in regulating these pathways may be redundant with Vav2 or Vav3. As Vav1-deficient mice develop normal numbers of 
T cells phenotypically indistinguishable from wild-type, but severely impaired in 
TCR signaling, these mice offer a new model to study the function of 
T cells.
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Acknowledgements
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We thank Drs Fred Rosen and Barry Sleckman for helpful advice and critical review of the manuscript. This work was supported by the National Institutes of Health grants HL59561 (F. W. A.), DK47677 (A. K. B.) and AI01472 (R. J. X.). F. W. A. is an investigator of the Howard Hughes Medical Institute. W. S. was a recipient of the Arthritis Foundation Hulda Irene Duggan Investigator Award.
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Abbreviations
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bHLHbasic helix-loop-helix
DNdouble negative
DPdouble positive
IELintraepithelial lymphocyte
PEphycoerythrin
PLCphospholipase C
SPsingle positive
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