RhoH GTPase recruits and activates Zap70 required for T cell receptor signaling and thymocyte development

Yi Gu^1, 3, Hee-Don Chae^1, 3, Jamie E Siefring¹, Aparna C Jasti¹, David A Hildeman² & David A Williams¹

¹ Division of Experimental Hematology, Cincinnati Children's Research Foundation and Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229, USA.

² Division of Immunobiology, Cincinnati Children's Research Foundation and Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229, USA.

³ These authors contributed equally to this work.

RhoH is a hematopoietic-specific, GTPase-deficient member of the Rho GTPase family with unknown physiological function. Here we demonstrate that Rhoh^-/- mice have impaired T cell receptor (TCR)–mediated thymocyte selection and maturation, resulting in T cell deficiency. RhoH deficiency resulted in defective CD3 phosphorylation, impaired translocation of the signaling molecule Zap70 to the immunological synapse and reduced activation of Zap70-mediated signaling in thymic and peripheral T cells. Proteomic analyses demonstrated that RhoH is a component of TCR signaling and is required for recruitment of Zap70 to the TCR through interaction with RhoH noncanonical immunoreceptor tyrosine-based activation motifs (ITAMs). In vivo reconstitution studies also demonstrated that RhoH function depends on phosphorylation of the RhoH ITAMs. These findings suggest that RhoH is a critical regulator of thymocyte development and TCR signaling by mediating recruitment and activation of Zap70.

As demonstrated by studies of human immunophenotypes and a variety of gene-targeted mouse lines, functional T cell development is critical for the adaptive immune system. Maturation of T lymphocytes with the appropriate immune repertoire occurs in the thymus and involves a tightly controlled, sequential process of T cell receptor (TCR) gene rearrangement, positive and negative selection and proliferative expansion¹. There are three critical checkpoints in the TCR lineage maturation, comprising -selection regulated by a pre-TCR signaling complex^{2, 3} and positive and negative selection controlled by signals derived from a fully rearranged TCR antigen complex⁴.

The Rho family GTPases are Ras-like molecules increasingly recognized as important signaling proteins involved in the pre-TCR and TCR pathways during T cell differentiation and proliferation⁵. In general, Rho GTPases cycle between GTP-bound, active and GDP-bound, inactive states and are regulated by the activity of guanine nucleotide–exchange factors and GTPase-activating proteins. Rhoh, first identified as a hypermutable gene in non-Hodgkin lymphoma^{6, 7} and a member of the RhoE subfamily, lacks intrinsic and/or agonist-induced GTPase activity and remains in the GTP-bound, active conformation⁸. Thus, regulation of the cellular activity of this GTPase remains unclear but it may be depend in part on cell-specific expression that determines the amount of intracellular protein. Indeed, RhoH is hematopoietic specific and has high expression in mouse thymus and human T cells^{8, 9}. Experimental alteration of RhoH expression affects the proliferation and engraftment of hematopoietic progenitor cells⁹ and integrin-mediated adhesion in Jurkat cells¹⁰. Studies have reported that transcriptional regulation of the Rhoh gene and alternative splicing of 5' exons of Rhoh mRNA occur in hematopoietic and lymphoid cells^{8, 11}. However, key unanswered biological issues remain, including the mechanism of physiological regulation and the function of RhoH in the hematopoietic lineages.

Intrathymic selection events are dependent on TCR signal transduction mediated by protein tyrosine kinases¹². Engagement of antigen receptors by self antigens expressed on the thymic stromal cells initiates activation of the Src family kinases Lck and Fyn. Phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of CD3 subunits by Src family kinases induces interaction of CD3 with Zap70 in the plasma membrane and cytoskeleton-enriched fractions and activation of Zap70 (refs. 13,14). Zap70 subsequently stimulates 'downstream' signaling cascades, including the activation (phosphorylation) of mitogen-activated protein kinases¹⁵. Zap70-deficient mice have developmental arrest of thymocytes at the CD4⁺CD8⁺ double-positive (DP) stage¹⁶. The importance of Zap70 in human T cell differentiation and/or function has also been demonstrated in patients with loss-of-function mutations in the gene encoding Zap70 (refs. 17,18) or defective recruitment of Zap70 to the signaling competent TCR complex¹⁹. So far, the molecular mechanism involved in regulating the recruitment of Zap70 to the TCR subunits for signaling activation is incompletely understood, and as-yet-unidentified molecules have been linked to the process. Our studies here of gene-targeted Rhoh-knockout mice demonstrate a critical function for RhoH in thymocyte development and peripheral T cell function by regulating the recruitment and activation of Zap70 in TCR signal transduction and also suggest a previously unknown regulatory mechanism for RhoH activity by tyrosine phosphorylation of ITAMs, which may have broad implications for GTPase biology.

Figure 1. Substantial blockade of intrathymic T cell development in Rhoh-/- mice. (a) Immunoblot of lysates from wild-type (WT) and Rhoh-/- thymocytes. -, antibody to. (b) Total thymocytes in wild-type and Rhoh-/- mice. Data represent mean s.d.; n = 8 mice per genotype. *, P < 0.01, Rhoh-/- versus wild-type mice. (c) Flow cytometry showing the distribution of CD4+CD8+ thymocyte subsets in thymi from 2-week-old wild-type and Rhoh-/- mice. Numbers in dot plots indicate the percent of each CD4 and CD8 subset in the corresponding quadrant. Cell number (right) is calculated as (frequency) (thymic cellularity). (d) Flow cytometry of thymocytes from 6-week-old wild-type and Rhoh-/- mice stained for Thy-1.2, CD4, CD8, CD25 and CD44. Numbers in dot plots indicate the percent of each CD25 and CD44 subpopulation in the corresponding quadrant, after gating on the DN cells. Cell number (right) is calculated as (frequency) (total DN cells). Data in c,d represent mean s.d.; n = 10 mice per genotype. Data are representative of three experiments. Full Figure and legend (47K)

Intrathymic T cell maturation involves sequential differentiation stages that can be distinguished on the basis of CD4 and CD8 coreceptor expression²⁰. Very few CD4⁺ or CD8⁺ single-positive (SP) cells were present in Rhoh^-/- thymus (Fig. 1c). Rhoh^-/- mice had fewer DP thymocytes and slightly more CD4^-CD8^- double-negative (DN) thymocytes than did their wild-type littermates (Fig. 1c). The smaller DP, CD4 SP and CD8 SP subpopulations in Rhoh^-/- thymocytes were associated with more apoptosis than that of wild-type cells (Supplementary Fig. 2 online). Immature DN thymocytes can be further categorized into four distinct pre–T cell developmental stages depending on the differential expression of CD25 (interleukin 2 receptor -chain) and CD44 (Pgp-1)²¹. Rhoh^-/- thymuses had significantly more CD44⁺CD25⁺ (DN2) and CD44^-CD25⁺ (DN3) cells than did wild-type thymuses but had fewer Rhoh^-/- CD44^-CD25^- (DN4) cells (Fig. 1d). The greater number of Rhoh^-/- DN2 and DN3 cells was associated with moderately more proliferation, whereas the smaller number of Rhoh^-/- DN4 cells was associated with more apoptosis (Supplementary Fig. 2). These data collectively suggested that the deletion of Rhoh results in substantial blocks in thymocyte development at the DN3 and DP stages, leading to many fewer mature T cells in the Rhoh^-/- thymus as well as peripheral lymphoid tissues.

TCR is essential in thymocyte development through interaction with self major histocompatibility complex class I and class II molecules expressed on thymic stromal cells. TCR -chain gene rearrangement and surface expression are critical for the transition of DN thymocytes to DP thymocytes². Flow cytometry showed that Rhoh^-/- DP thymocytes included fewer TCR^hi cells than did wild-type thymocytes (Fig. 2a). In contrast, the proportion of TCR^lo cells in Rhoh^-/- thymuses was normal. Furthermore, semiquantitative RT-PCR analysis showed that Rhoh^-/- and wild-type thymocytes had similar amounts of rearranged Tcrb variable (V), diversity (D) and joining (J) segments and Cd3e transcripts (Fig. 2b). These data suggested that rearrangement of the Tcrb locus and expression of Cd3e are normal in Rhoh^-/- thymocytes.

Figure 2. Defective TCR-mediated thymic positive selection in Rhoh-/- mice. (a) Flow cytometry of thymocytes from wild-type and Rhoh-/- mice, stained with fluorescein isothiocyanate–anti-CD4, allophycocyanin–anti-CD8 and phycoerythrin–anti-TCR. Numbers above bracketed lines indicate percent TCRhi cells. n = 8 mice per genotype. (b) RT-PCR of Tcrb VDJ and Cd3e transcripts in wild-type and Rhoh-/- thymocytes. VC and DC, products of RT-PCR with primers V8 LV-5' and C2A-3' and primers D2-5' and C2B-3' (Supplementary Methods online), respectively. Hprt1 (hypoxanthine guanine phosphoribosyl transferase), loading control. (c,d) Flow cytometry of the expression of CD5 (c) and CD69 (d) by wild-type and Rhoh-/- DP thymocytes. Gray (c), isotype antibody control. Numbers above bracketed lines (d) indicate percent CD69hi cells. n = 6 mice per genotype. (e) Flow cytometry of the distribution of the CD4+ and CD8+ subsets of thymocytes from wild-type mice (Non-tg Rhoh+/+), p14tg/+Rhoh+/+ mice (Rhoh+/+) and p14tg/+Rhoh-/- mice (Rhoh-/-). Numbers in dot plots (top) indicate percent cells in each corresponding quadrant; numbers above bracketed lines (bottom) indicate percent TCR V8hi cells among DP thymocytes. n = 10 mice per genotype. (f) Proliferation of CD3+ cells isolated from splenocyte samples from wild-type and Rhoh-/- mice and cultured for 48 h in the presence of plate-coated anti-CD3 (dose, below graph) or medium alone. Data represent mean s.d. (n = 6 mice). *, P < 0.001, Rhoh-/- versus wild-type cells. Data are representative of more than three experiments. Full Figure and legend (57K)

TCR engagement initiates intracellular signals that induce the expression of thymocyte positive-selection and maturation markers, such as CD5 and CD69 (refs. 22, 23, 24, 25). Surface expression of CD5 and CD69 was much lower on Rhoh^-/- DP thymocytes than on wild-type DP thymocytes (Fig. 2c,d), suggesting that TCR-mediated positive selection might be defective in Rhoh^-/- thymocytes. Furthermore, wild-type thymocytes expressing transgenic p14 TCR (V2V8), a TCR specific for the major histocompatibility complex class I–restricted epitope of the lymphocytic choriomeningitis virus glycoprotein²⁶ (called 'p14^tg/+' here), were positively selected to the CD8 SP subset. In contrast, that selection, accompanied by skewing toward CD8 SP thymocytes (p14^tg/+Rhoh^+/+; Fig. 2e, middle), was mostly absent from p14^tg/+Rhoh^-/- mice (Fig. 2e, right), and the thymocyte phenotype of the p14^tg/+Rhoh^-/- mice resembled that of Rhoh^-/- mice (Fig. 1c and Supplementary Fig. 3 online). Also, p14^tg/+Rhoh^-/- DP thymocytes had lower expression of the transgenic V8 TCR than did p14^tg/+Rhoh^+/+ cells (Fig. 2e). These results suggested that RhoH is required for the TCR-mediated signaling that regulates thymocyte positive selection and maturation.

To further determine if the few peripheral T cells present in Rhoh^-/- mice were competent for TCR signal transduction, we isolated CD3⁺ cells from wild-type and Rhoh^-/- splenocytes, stimulated the cells with antibody to CD3 (anti-CD3) and assessed their proliferative response. Surface expression of TCR was similar in wild-type and Rhoh^-/- spleen T cells (mean fluorescent intensity of TCR, wild-type versus Rhoh^-/-: CD4⁺ cells, 175 8 versus 187 10; CD8⁺ cells, 102 6 versus 95 5). Despite having normal amounts of TCR, Rhoh^-/- T cells proliferated much less after stimulation with anti-CD3 than did wild-type cells (Fig. 2f), suggesting that RhoH is required for TCR signaling in peripheral T cells, probably 'downstream' of the TCR.

To determine whether the thymocyte developmental defects in Rhoh^-/- mice were directly related to the loss of RhoH, we transduced Rhoh^-/- bone marrow cells with a bicistronic retroviral vector coexpressing enhanced green fluorescent protein (EGFP) and hemagglutinin-tagged RhoH (HA–RhoH), which has been shown to produce RhoH expression similar to endogenous expression in those cells⁹. We transplanted EGFP⁺ sorted bone marrow cells by intravenous injection into sublethally irradiated recipient mice deficient in recombination-activating gene 2 (Rag2^-/-), which have less thymic cellularity and lack mature T cells²⁷. At 9 weeks after transplantation, transgenic expression of RhoH was associated with more thymic cellularity (Fig. 3a). The number of CD4 SP or CD8 SP thymocytes in Rag2^-/- mice that received RhoH-transduced Rhoh^-/- bone marrow cells was similar to the number in mice infused with wild-type cells (Fig. 3b,c); in contrast, mice that received Rhoh^-/- cells lacked thymocyte development. Transgenic expression of RhoH in Rhoh^-/- cells also led to many more mature CD4⁺ or CD8⁺ T cells in the spleen and lymph nodes (Supplementary Table 2 online). All recipient mice had similar degrees of engraftment, as assessed by the EGFP⁺ chimerism in the thymus and peripheral blood, suggesting that those phenotypic changes were related to enhanced thymic T cell development (Supplementary Table 2). We also isolated Rhoh^-/- and RhoH-reconstituted Rhoh^-/- CD3⁺ splenocytes from the Rag2^-/- recipient mice, stimulated the cells with anti-CD3 and assessed their proliferative responses. Transgenic expression of RhoH in the reconstituted cells resulted in significant correction of the proliferation defect of Rhoh^-/- T cells (Fig. 3d). These results demonstrated that the thymocyte development defect associated with RhoH deficiency is intrinsic to hematopoietic cells and can be completely 'rescued' by transgenic expression of RhoH.

Figure 3. Transgenic expression of RhoH restores Rhoh-/- intrathymic development and T cell function. EGFP+ wild-type and Rhoh-/- bone marrow cells transduced with a retroviral vector coexpressing EGFP and RhoH or empty vector (control) were injected intravenously into sublethally irradiated Rag2-/- recipient mice; 9 weeks later, thymuses and spleens from the reconstituted recipient mice were analyzed. None, no bone marrow transplanted. (a) Thymic cellularity. (b) Flow cytometry of the distribution of CD4+ and CD8+ subsets. Numbers in dot plots indicate percent cells in each corresponding quadrant. (c) Number of cells in the CD4+ or CD8+ SP subset, calculated as (frequency) (total thymocytes). (d) In vitro proliferation of equal numbers of CD3+ cells isolated from splenocytes of the reconstituted Rag2-/- recipient mice cultured for 48 h in plates coated with anti-CD3 (10 g/ml) or medium alone. Data represent mean s.d. (n = 5–10 mice per transplant group (a–c) or n = 3 (d)) and are representative of more than two experiments. Full Figure and legend (39K)

Figure 4. RhoH is tyrosine-phosphorylated and interacts with Zap70. (a) GST affinity assay of Jurkat cell lysates, detected by SDS-PAGE and Coomassie blue staining. Far right, nonspecific bacterial proteins. *, identified by mass spectrometry as Zap70. (b) Immunoprecipitation (IP) and immunoblot (IB) of lysates of HEK293 cells transfected with various combinations (above lanes) of DNA constructs expressing HA–RhoH, Zap70 and/or constitutively active Lck (ca-Lck). (c) Immunoprecipitation and immunoblot of Jurkat cells left untransduced or transduced with a retroviral vector expressing HA–RhoH and left unstimulated or stimulated anti-CD3. pTyr, phosphorylated tyrosine; pZap70, phosphorylated Zap70; pp23 and pp21, phosphorylated p23 and p21; pCD3, phosphorylated CD3. Data are representative of more than three experiments. Full Figure and legend (33K)

Zap70 is a key signaling molecule in the TCR complex through its interaction with the tyrosine-phosphorylated CD3 isoforms p21 and p23^{14, 28, 29}. To determine whether RhoH is regulated by tyrosine phosphorylation after TCR engagement and is involved in the CD3-Zap70 complex in T cells, we expressed HA–RhoH in Jurkat cells. Stimulation with anti-CD3 increased tyrosine phosphorylation of HA–RhoH (Fig. 4c, bottom) and HA–RhoH immunopreciptated together with Zap70 (Fig. 4c, top). These results suggested that TCR engagement can induce the tyrosine phosphorylation of RhoH and association of RhoH with Zap70. In contrast to the interaction with p21 and p23 only after stimulation with anti-CD3 (Fig. 4c, top), we detected the Zap70-RhoH association in the resting state, suggesting that RhoH probably interacts with Zap70 before Zap70 is recruited to the phosphorylated CD3 induced by TCR engagement.

Zap70 is known to recognize tyrosine-phosphorylated ITAMs containing the consensus sequence YXX(L/I)X_6–8YXX(L/I) (where 'X' is any amino acid and 'L/I' means 'leucine or isoleucine')³⁰. Both human and mouse RhoH have ITAM-like motifs between residue 73 and residue 86, which contain alanine as an aliphatic amino acid at the '+3' position^{31, 32} (Fig. 5a), differing from the leucine or isoleucine in the conventional ITAMs. Alignment of the amino acid sequences of RhoH and other known Rho GTPases, including Rac1, Cdc42, RhoA, and RhoE, suggested that the motifs are unique to RhoH. To test whether the two tyrosine residues in the motifs were required for the interaction of RhoH with Zap70, we substituted the tyrosine residues at positions 73 and 83 with phenylalanine. The resultant 'RhoHF73F83' mutant had much less interaction with Zap70 than did wild-type RhoH in Jurkat cells after stimulation with anti-CD3 (Fig. 5b, top), suggesting that the ITAM motifs in RhoH are probably involved in the interaction with Zap70. The weak binding between the RhoHF73F83 mutant or nonphosphorylated GST–RhoH and Zap70 (Fig. 4a) indicated that additional, unidentified sequences in RhoH may also be involved in modifying this interaction. Zap70 binds to phosphorylated ITAMs via the two cooperative Src homology 2 (SH2) domains¹³. Zap70 mutants lacking either the N-terminal or C-terminal SH2 domain showed defective binding to RhoH when expressed together with constitutively active Lck in HEK293 cells (Fig. 5c). These results collectively suggested that the interaction between RhoH and Zap70 depends mainly on the tyrosine-phosphorylated ITAMs in RhoH and the SH2 domains in Zap70.

Figure 5. Conserved tyrosine-phosphorylated sequence motifs in RhoH mediate the interaction with Zap70 and RhoH function in thymocyte development. (a) Alignment of the amino acid sequences of RhoH, Rac1, Cdc42, RhoA and RhoE. Bold (RhoH), ITAM-like motifs; 'a' (ITAM consensus sequence), aliphatic amino acid. (b) Immunoprecipitation and immunoblot analysis of lysates of Jurkat cells left untransduced (None) or transduced with retroviral vector expressing HA–RhoH or mutant HA–RhoHF73F83 and left unstimulated (0) or stimulated with anti-CD3 (10) before immunoprecipitation. (c) Immunoprecipitation of total lysates of HEK293 cells expressing HA–RhoH and constitutively active Lck and transfected with Zap70, or Zap70 mutants lacking the N-terminal SH2 domain (Zap70N–SH2) or the C-terminal SH2 domain (Zap70C–SH2). Bottom, binding affinity, determined by the ratio of bound to total HA–RhoH. (d) Flow cytometry of thymocytes from Rag2-/- recipient mice 9 weeks after transplantation with wild-type bone marrow cells (open bars) or with Rhoh-/- bone marrow cells transduced with a retroviral vector expressing EGFP (light gray bars) or coexpressing EGFP and either RhoH (filled bars) or RhoHF73F83 (dark gray bars). None, no bone marrow transplanted. Number of cells in the CD4+ or CD8+ SP subset is calculated as (frequency) (total thymocytes). Data represent mean s.d. (n = 4–6 mice per transplant group). (e) Immunoblot analysis of HA–RhoH and HA–RhoHF73F83 or -actin (loading control) in EGFP+ thymocytes isolated from the Rag2-/- recipients in d. Bottom, EGFP chimerism and mean fluorescence intensity (MFI) of thymocytes from individual recipient mice. Data are representative of more than three experiments. Full Figure and legend (47K)

TCR engagement triggers the recruitment of Zap70 to the TCR complex at the cytoskeleton-enriched plasma membrane, leading to activation of Zap70 and the Zap70-induced 'downstream' kinase cascades^{13, 14}. Given the functional interaction of RhoH with Zap70 in thymocyte development and the known involvement of Rho GTPases in cytoskeleton rearrangements in many mammalian cell types, we hypothesized that RhoH may be involved in recruitment of Zap70 to the plasma membrane. To test that, we examined the subcellular localization of Zap70 in wild-type and Rhoh^-/- thymocytes. Without stimulation with anti-CD3, there was substantial Zap70 in wild-type thymocytes in the soluble cytoplasm-containing (S100) and detergent-soluble membrane-containing (P100) fractions and little in the detergent-insoluble cytoskeleton-containing fraction (Fig. 6a). In the resting Rhoh^-/- thymocytes, there was much less Zap70 in the P100 fraction and it was barely detectable in the cytoskeleton fraction. Stimulation with anti-CD3 further increased the translocation of Zap70 to the cytoskeleton fraction in wild-type thymocytes. That recruitment was almost completely blocked in Rhoh^-/- thymocytes. In contrast to loss of Zap70, loss of RhoH had little affect on the subcellular distribution of CD3, which we detected in the P100 and cytoskeleton fractions. These results suggested that RhoH is required for the proper localization of Zap70 to the plasma membrane and cytoskeleton fractions in thymocytes.

Figure 6. Rhoh-/- thymocytes have impaired translocation of Zap70 to the plasma membrane and the immunological synapse and reduced phosphorylation of Zap70, CD3 and p42-p44. (a) Immunoblot of lysates of wild-type and Rhoh-/- thymocytes left unstimulated (0) or stimulated anti-CD3 (10). Lysates were separated by centrifugation into the soluble S100, detergent-soluble P100 and detergent-insoluble cytoskeleton fractions. (b) Fluorescence microscopy of p14 TCR–transgenic CD8+ splenocytes transduced with EGFP–RhoH and conjugated for 5 min with CH.B2 cells preloaded with gp33 peptide, then fixed and stained with anti-Zap70 (red). Differential interference contrast (DIC) images show antigen-specific T cell–APC conjugates. EGFP–RhoH and Zap70 are localized together in the immunological synapse (yellow; arrowheads). (c) Fluorescence microscopy of thymocytes from p14tg/+Rhoh+/+ (WT) or p14tg/+Rhoh-/- transgenic mice, conjugated and stained as described in b. Zap70 is recruited to the immunological synapse in wild-type cells (arrowheads). (d,e) Immunoblot of phosphorylated and unphosphorylated Zap70 and CD3 (d) or phosphorylated and unphosphorylated p42-p44 (e) in lysates of freshly isolated wild-type and Rhoh-/- thymocytes left unstimulated (0) or stimulated anti-CD3 (10). Data are representative of three experiments. Full Figure and legend (95K)

Consistent with the defective plasma membrane translocation, there was less phosphorylation of Zap70 at Y319, which is important for Zap70 activation³⁵, in Rhoh^-/- thymocytes and spleen T cells than in wild-type cells, after stimulation with anti-CD3 (Fig. 6d and Supplementary Fig. 4). The lower phosphorylation of Zap70 in Rhoh^-/- T cells was associated with defective phosphorylation of the membrane adaptor Lat and p42-p44 mitogen-activated protein kinases (Fig. 6e and Supplementary Fig. 4), critical 'downstream' signaling molecules activated by Zap70 after TCR engagement^{15, 36}. However, phosphorylation of p38 mitogen-activated protein kinase and the kinase Akt was not affected in Rhoh^-/- cells (data not shown). Also, phosphorylation of CD3 was impaired in Rhoh^-/- thymocytes and spleen T cells (Fig. 6d and Supplementary Fig. 4). We detected neither p21 nor p23 in Rhoh^-/- T cells, in contrast to wild-type cells. Those data suggested that RhoH is an important intracellular regulator for activation of the CD3-Zap70 signaling pathways.

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That developmental defect was also associated with less expression of CD5, CD69 and TCR in Rhoh^-/- thymocytes, which is regulated by TCR signaling⁴⁰. Impaired TCR signaling in Rhoh^-/- T cells was further demonstrated by less proliferation and less activation of kinase signaling cascades in response to TCR engagement. The moderately lower TCR surface expression on Rhoh^-/- thymocytes may have contributed in part to the defective TCR signaling. However, Rhoh^-/- spleen T cells with normal expression of TCR also demonstrated similar defects. Those results suggested that RhoH is required for intracellular TCR signal transduction. Thus, the lower TCR expression on Rhoh^-/- thymocytes is probably a result rather than a cause of the developmental arrest and the defective TCR signaling.

One important issue is how RhoH regulates Zap70 membrane translocation and recruitment to the TCR. Studies have suggested that the binding of peptide–major histocompatibility complex to the TCR stimulates sequential phosphorylation of the ITAMs of CD3, resulting in the p21 and p23 isoforms²⁸. Only the fully phosphorylated p23 is responsible for the SH2-dependent Zap70 interaction, whereas p21 binds weakly to Zap70 and is not able to activate Zap70 (ref. 43). The p23 isoform is not detectable in Rhoh^-/- T cells after TCR engagement. That may explain the defective activation of Zap70 in Rhoh^-/- cells, due to the lack of high-affinity binding to p23. Lck is the main kinase responsible for phosphorylation of the ITAMs of CD3⁴⁴, and its activity is required for the transition of pre–T cells from the DN to the DP stage⁴¹, apparantly similar to RhoH protein. Further studies of the activity and subcellular localization of Lck in Rhoh^-/- T cells will provide better understanding of the functions of RhoH in TCR-mediated Zap70 activation.

Our biochemical studies have also provided evidence of a second mechanism by which RhoH may regulate Zap70 membrane translocation and activation in T cells. We have shown that RhoH directly bound to the SH2 domains of Zap70. That interaction was greatly enhanced by tyrosine phosphorylation of the ITAMs of RhoH after TCR engagement. Notably, the ITAMs of RhoH were critical for its interaction with Zap70 and its function in thymocyte development. Those findings, along with the finding of a defective Zap70 membrane-recruitment phenotype associated with Rhoh^-/- T cells, led us to propose that RhoH may be involved in the translocation of Zap70 to the TCR in the cytoskeleton-enriched and plasma membrane fractions through that interaction. In support of that hypothesis, we have shown that the EGFP–RhoH fusion protein was present in the cytoplasm and was also targeted to the plasma membrane in normal T cells, which is probably regulated by the C-terminal prenylation site CKIF⁸. Furthermore, immunofluorescence staining showed that RhoH was localized together with Zap70 in the immunological synapse, where the TCR subunits were present. Comparison of the ITAM consensus sequences in RhoH and CD3 has indicated that unlike CD3, RhoH does not have a typical leucine or isoleucine residue at the '+3' position for high-affinity SH2 domain–dependent binding^{30, 32}. Thus, it can be predicted that when both are fully phosphorylated and localized together with Zap70, CD3 would have higher affinity binding for Zap70 than would RhoH. Therefore, one possible function of RhoH is to shuttle intracellular Zap70 to the membrane-associated CD3, leading to the effective, high-affinity interaction of Zap70 with p23 after TCR engagement. Indeed, we have shown that Zap70 immunoprecipitated together with RhoH in resting T cells, in contrast to the Zap70-p23 interaction, which we detected only in T cells stimulated with anti-CD3.

Notably, impaired recruitment of Zap70 to the p23 isoform has also been reported to be associated with defective TCR signaling in patients with common variable immunodeficiency with T cell defects¹⁹. T cells from those patients have normal expression of known TCR signaling components, including Lck, CD3 and Zap70, and the proteins expressed from alleles of those patients are competent in signaling assays in vitro. That would suggest that as-yet-undefined molecules are needed to mediate recruitment of Zap70 to the TCR. Given the phenotype of Rhoh^-/- T cells, it remains a likely working hypothesis that RhoH serves as an adaptor molecule for the effective recruitment and activation of Zap70 in TCR signaling. RhoH may represent a new molecular target for the study of common variable immunodeficiency with T cell defects.

In summary, we have shown that RhoH is critical in normal T cell development and signaling. RhoH was required for full phosphorylation of CD3, membrane translocation of Zap70 and subsequent activation of the Zap70-mediated pathways. In addition to its biological function in T cells, our biochemical evidence has shown that RhoH function is probably regulated by tyrosine phosphorylation of ITAMs, a mechanism not linked before to the physiological regulation of Rho GTPases. Given that RhoH belongs to the RhoE subfamily of Rho GTPases that are GTPase deficient and therefore lack regulation by the guanine nucleotide–exchange factors and GTPase-activating proteins⁸, this regulation may be critical for the biological activity of RhoH. Phosphorylation may be an important additional mechanism by which Rho GTPases are regulated⁴⁵.

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The 129/Sv Rhoh^-/- mice were generated by Targeting Laboratory. The entire coding region of mouse Rhoh is in its third exon; the targeting vector was designed to replace the third exon of Rhoh with a neomycin-resistance cassette. The genotypes of Rhoh gene-targeted embryonic stem cells and transgenic mice were determined by Southern blot analysis of DNA digested with SpeI using a 5' Rhoh genomic DNA probe or by PCR analysis with primers. The 129/Sv Rhoh^-/- mice were crossed with wild-type or p14 TCR (V2V8) transgenic mice on a C57BL/6J background to generate Rhoh^-/- or p14^tg/+Rhoh^-/- compound mice. Mice used were littermates derived from backcross generations with an N of more than 2. The 129S6/SvEvTac-Rag2^-/- mice were purchased from Taconic Animal Models. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Cincinnati Children's Hospital Research Foundation (Cincinnati, Ohio).

For the transduction of bone marrow, primary T cells and Jurkat cells, the following mouse stem cell virus–based retroviral vectors were used: MIEG3 (empty vector), HA–RhoH⁹, HA–RhoHF73F83, generated by with QuiChange Site-Directed Mutagenesis Kit (Stratagene), and EGFP–RhoH, constructed as described⁴⁶. For the production of GST–RhoH fusion proteins, a BamHI–EcoRI fragment of full-length Rhoh cDNA was subcloned into plasmid pGEX-2T (GE Healthcare Life Science). Full-length Zap70 cDNA and Zap70 cDNA with deletion of the SH2 domain were subcloned into pCDNA3 (Invitrogen) at the EcoRI and SalI sites from pGEM3Z-Zap70, pGEM3Z-ZapP70N–SH2 and pGEM3Z-Zap70C-SH2 constructs¹³. For the transfection of HEK293 cells, the following DNA constructs were used: HA–RhoH, pCDNA3-Zap70, pCDNA3-Zap70N–SH2 and pCDNA3-Zap70C–SH2, and pRc-CMV-ca-Lck.

Fluorescence-conjugated monoclonal antibodies to the following mouse antigens were used for flow cytometry: CD4 (RM4-5), CD8 (53-6.7), CD25 (7D4), CD44 (IM7), TCR -chain (H57-597), TCR (GL3), TCR V8, TCR V5 (MR9-4), CD69 (H1.2F3), CD5 (53-7.3), Gr-1 (RB6-8C5), Mac-1 (M1-70), NK1.1 (PK136), Thy1.2 (53-2.1), CD45R–B220 (RA3-6B2), IgM (R6-60.2), BrdU (3D4) and Ter119 (Ly-76; all from Pharmingen). For immunoblot analyses, antibodies to the following were used: RhoH⁹ (B4998), Zap70 phosphorylated at Y319 (17a), phosphorylated tyrosine (4G10) and Lat (45; Pharmingen); hemagglutinin (3F10; Roche); -actin (AC-15; Sigma); CD3 (6B10.2; Santa Cruz Biotechnology); and Lat phosphorylated at Y191 (3584), Zap70 (99F2), phosphorylated p42-p44 (Thr202-Tyr204; 197G2) and p42-p44 (9102; Cell Signaling Technology). Primary antibodies were detected with the secondary antibodies horseradish peroxidase–conjugated goat anti-mouse (7076) or goat anti-rabbit (7074; both Cell Signaling Technology), or donkey anti-rat (sc-2956; Santa Cruz Biotechnology) using enhanced chemiluminescence detection (Cell Signaling Technology). GST fusion proteins were expressed in Escherichia coli BL21 (DE3) cells and were purified according to the manufacturer's recommendations (GE Healthcare Life Science). Purified GST fusion protein lysates were incubated for 1 h at 4 °C with glutathione–Sepharose 4B beads. Bead-bound GST fusion proteins were separated by SDS-PAGE and were quantified by Coomassie blue staining.

Single-cell suspensions were generated from thymus and spleen. Total cells were counted with a Z1 Coulter counter (Beckman Coulter) or a hemocytometer. Cells were stained with fluorescence-conjugated antibodies for flow cytometry as described⁴⁷. For isolation of CD3⁺ T cells without stimulation of the cells with anti-CD3 during purification, splenocytes were labeled with fluorescein isothiocyanate–conjugated antibodies to mouse antigens, including CD45R–B220, Gr-1 and Ter119, for negative selection with anti–fluorescein isothiocyanate magnetic beads, according to the manufacturer's recommendations (Miltenyi Biotec). For TCR activation, thymocyte samples containing more than 90% DP cells or CD3⁺ T cells (more than 90% purity) were incubated on plates precoated with purified anti–mouse CD3 (2c11; 25 g/ml; Pharmingen) as described⁴⁸.

An in vivo BrdU (5-bromo-2'-deoxyuridine) incorporation assay was done as described⁹. Mice were fed 1 mg/ml of BrdU (Sigma) for 36 h and then were killed for thymus collection. Thymocytes from each mouse were stained with anti-CD4, anti-CD8, anti-CD25, anti-CD44 and anti-BrdU for proliferation analysis or with annexin V (550474; Pharmingen) for apoptosis analysis (all from Pharmingen) and then were analyzed by flow cytometry. For in vitro proliferation assays, equal amounts of CD3⁺ cells isolated from wild-type and Rhoh^-/- splenocytes were plated in 96-well culture plates without precoating or precoated with the 2c11 mAb to mouse CD3 (Pharmingen) in medium containing 10% FCS (HyClone). After 48 h, cells were incubated for 18 h with 1 Ci/well of [³H]thymidine (GE Healthcare Life Science). Incorporation of [³H]thymidine was measured with a scintillation counter (Beckman).

Jurkat cells were transduced with the high-titer retrovirus supernatant as described above for bone marrow cells and then were sorted to obtain EGFP⁺ cells. HEK293 cells were transfected by the calcium phosphate method according to the manufacturer's instructions (Invitrogen). The immunoprecipitation assay was done as described⁴⁸. Cells were lysed in Mg²⁺ lysis wash buffer (MLB) (Upstate) containing Complete Protease Inhibitors (Roche) and phosphatase inhibitors (1 mM Na₃VO₄ and 10 mM NaF). Then, 500 g of cell lysate was immunoprecipitated with anti-Zap70 or anti-hemagglutinin and protein A–protein G agarose (Santa Cruz). The immunoprecipitates or 20 g of cell lysates were separated by 12% SDS-PAGE and then were analyzed by immunoblot.

Splenocytes were isolated from p14 TCR–transgenic mice. Isolated splenocytes were labeled with fluorescein isothiocyanate–labeled anti–mouse CD8 for positive selection with anti–fluorescein isothiocyanate microbeads according to the manufacturer's recommendations (Miltenyi Biotech) to obtain CD8⁺ T cells. CD8⁺ T cells were cultured in Iscove's modified Dulbecco's medium plus 10% FCS supplemented with recombinant mouse interleukin 2 (10 ng/ml; Peprotech) and 50 M -mercaptoethanol on tissue culture plates precoated with mAb to CD3 (Pharmingen). For transduction, a published centrifugation method was used⁵². Cells were resuspended in retroviral supernatant containing interleukin 2 and protamine sulfate (4 g/ml) and were centrifuged for 2 h at 30 °C at 2,400g on plates coated with mAb to CD3. The transduction process was repeated five times. EGFP⁺ T cells were isolated with a FACSVantage (BD Biotechnology).

CHB.2B cells⁵³, a B cell lymphoma line used as APCs, were preloaded for 12 h with 1 g/ml of gp33 peptide. Preloaded CHB.2B cells were plated on coverslips coated with poly-L-lysine. Equal amount of freshly isolated thymocyte samples containing more than 80% DP cells or the transduced and sorted EGFP⁺CD8⁺ T cells from wild-type and Rhoh^-/- p14 transgenic mice were then added onto the coverslips. After 5 min, cells were fixed with cytofix buffer (BD Biosciences) and were stained with anti-Zap70 (Upstate) followed by Alexa Fluor 555–conjugated mouse immunoglobulin G antibody. Stained cells were mounted for fluorescence imaging analysis with a fluorescence microscope equipped with a 40 objective lens and a deconvolution system (Leica) 'driven' by Openlab software (Improvision).

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We thank D. Schumann and Y. Yuan (Genome Research Institute, University of Cincinnati, Cincinnati, Ohio) for assistance with mass spectrometry analysis; A.S. Shaw (Washington University School of Medicine, St. Louis. Missouri) for the pGEM3Z-Zap70, pGEM3Z-Zap70N-SH2 and pGEM3Z-Zap70C-SH2 constructs; C.T. Baldari (University of Siena, Siena, Italy) for the constitutively active Lck construct; P. Marrack (National Jewish Medical and Research Center, Denver, Colorado) for breeding pairs of p14 lymphocytic choriomeningitis virus TCR–transgenic mice; S. Li, J. Bailey and V. Summer (Cincinnati Children's Hospital, Cincinnati, Ohio) for technical assistance; and Y. Zheng for discussions and critical comments on the manuscript. Supported by the National Cancer Institute (KO1 CA107110 to Y.G.) and the US National Institutes of Health (RO1 DK62757 to D.A.W. and RO1 CA113969).

Competing interests statement:  The authors declare that they have no competing financial interests.