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T-cell tolerance is a major obstacle to successful cancer immunotherapy; thus, developing strategies to break immune tolerance is a high priority. Here we show that expression of the E3 ubiquitin ligase Grail is upregulated in CD8+ T cells that have infiltrated into transplanted lymphoma tumours, and Grail deficiency confers long-term tumour control. Importantly, therapeutic transfer of Grail-deficient CD8+ T cells is sufficient to repress established tumours. Mechanistically, loss of Grail enhances anti-tumour reactivity and functionality of CD8+ T cells. In addition, Grail-deficient CD8+ T cells have increased IL-21 receptor (IL-21R) expression and hyperresponsiveness to IL-21 signalling as Grail promotes IL-21R ubiquitination and degradation. Moreover, CD8+ T cells isolated from lymphoma patients express higher levels of Grail and lower levels of IL-21R, compared with CD8+ T cells from normal donors. Our data demonstrate that Grail is a crucial factor controlling CD8+ T-cell function and is a potential target to improve cytotoxic T-cell activity.
The adaptive immune system, especially CD8+ cytotoxic T lymphocytes (CTL), have a crucial function in controlling the development of neoplastic lesions1. Effector CD8+ T cells can efficiently destroy target cells with death cell ligands such as tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) or execution of the perforin/granzyme and interferon (IFN)-γ-dependent machinery2. Although CD4+ T cells are important in anti-cancer immunity, their predominant function at tumour sites is to maintain function of tumour-specific CTLs by producing cytokines3, 4.
Cancer immunotherapy aims to reactivate a patient’s immune system to fight against tumours; however, T-cell tolerance induced by the inhibitory tumour microenvironment is an obstacle5. Therefore, understanding the cellular and molecular mechanisms that underlie T-cell tolerance in cancer would guide the development of effective therapies. E3 ubiquitin ligases, including Cbl-b, Itch and Grail are important regulators of T-cell tolerance6. Cbl-b and Itch have been reported to be involved in tumour development7,8,9,10 and expression of Grail, a type I transmembrane protein localised to the endosomal compartment, is associated with T-cell anergy11. We previously showed that Grail-deficient mice are resistant to immune tolerance induction in vitro and in vivo12, 13. We showed that Grail is required for downregulating TCR signalling in recently activated CD4+ T cells, and that lack of Grail leads to hyperproliferation, excessive cytokine production and abrogation of the suppressive function of regulatory T (Treg) cells12. However, the role of Grail in CD8+ T cells is unclear.
In the current study, we find high expression of Grail in mouse CD8+ T cells that have infiltrated into lymphoma tumours and we examine the role of Grail in EL-4 and EG-7 lymphoma models. Grail deficiency provides the host with spontaneous protection against tumours, which is mediated mainly by CD8+ T cells in Grail-deficient mice. In tumours, loss of Grail enhances anti-tumour reactivity of CD8+ T cells. Moreover, in mouse CD8+ T cells, Grail regulates the expression of IL-21 receptor (IL-21R) and naive Grail −/− CD8+ T cells are hyperresponsiveness to IL-21 in vitro due to augmented IL-21R expression, which is regulated by Grail-dependent ubiquitination and degradation. Importantly, similar to the mouse model, we show high expression of Grail and diminished expression of IL-21R in CD8+ T cells from lymphoma patients compared with healthy donors. Altogether, our results indicate a suppressive function of Grail in CD8+ T cells and suggest that Grail-ablated CD8+ T cells could be an efficient tool for eliciting immune responses against tumours.
Grail loss correlates with enhanced CD8+ TILs infiltration
To investigate the potential role of Grail in CD8+ T cells, we utilised subcutaneous EL-4 and EG-7 murine lymphoma models. EL-4 cells are derived from a T-lineage lymphoma developed in C57BL/6 mouse treated with 9,10-dimethyl-1,2-benzanthracene and represents a tumour model with weak immunogenicity. EG-7 cells are EL-4 transfectants that express chicken ovalbumin (OVA), which serves as tumour antigen and are therefore considered highly immunogenic tumours. EG-7 lymphoma cells were injected subcutaneously into the flanks of wild-type (WT) mice and 17 days later CD4+ and CD8+ tumour-infiltrating lymphocytes (TIL) or T cells residing in the spleen were sorted and analysed for Grail expression by real-time (RT)-PCR. Remarkably, Grail messenger RNA (mRNA) levels were significantly upregulated in CD8+ T cells from tumours compared to those in spleens, suggesting a role of Grail in controlling the function of tumour-specific CTLs (Supplementary Fig. 1a). In contrast, we did not detect any significant upregulation of Grail in tumour-infiltrated CD4+ T cells. Interestingly, Cbl-b expression was not increased in CD4+ and CD8+ TILs (Supplementary Fig. 1b), suggesting a distinct regulation and function of Grail and Cbl-b in tumours. Similarly, when EL-4 cells were injected in WT mice, Grail but not Cbl-b expression was selectively upregulated in CD8+ T cells infiltrated in tumours (Supplementary Fig. 1c and d), suggesting that both strong or weak immunogenic tumours selectively induced Grail expression in tumour-infiltrating CD8+ T cells in vivo.
To assess whether Grail contributes to anti-tumour immunity in vivo, we inoculated EG-7 cells into sex- and age-matched WT and Grail −/− mice and monitored the kinetics of tumour growth (Fig. 1a). Tumour growth was comparable between WT and Grail −/− mice in the first 10 days after tumour inoculation. Intriguingly, while between day 10 and 17 tumours grew rapidly in WT mice; Grail-deficient mice were able to control tumour growth until day 55 demonstrating a delay in tumour growth in the absence of Grail (Fig. 1a; Supplementary Fig. 2a). To explore the underlying immune mechanisms of reduced tumour growth in Grail −/− mice, we analysed TILs. In comparison to TILs from WT mice, we observed that the percentage of total CD8+ TILs is comparable at day 7 but increased by day 12 in Grail −/− mice (Fig. 1b). This increased frequency of tumour antigen-specific CD8+ TILs from Grail −/− mice was maintained at day 17 (Fig. 1c, d); however, we did not detect changes in the percentage of Grail −/− CD4+ TILs compared to WT (Fig. 1b, c). By day 55, the ratio of CD4+:CD8+ TIL was 1:1 but CD8+ TIL were still found to have cytotoxic capability as demonstrated by granzyme B and IFNγ expression (Supplementary Fig. 2b). Similarly to the EG-7 model, EL-4 tumours rapidly grew in WT mice, while being well-controlled in Grail −/− mice (Supplementary Fig. 3). Moreover, the total number of CD8+ T cells but not CD4+ T cells was increased in the tumours of Grail −/− mice inoculated with EL-4 tumour cells compared to WT mice. Thus, these data show that loss of Grail expression results in increased infiltration of CD8+ T cells into the tumour tissues and importantly in attenuation of tumour growth with either strong or weak immunogenicity.
For further studies, we evaluated the accumulation, activation and effector function of tumour antigen-specific CD8+ T cells. First, we assessed whether accumulation of Grail −/− CD8+ TILs could be due to either a decreased suppressive capability of Grail −/− Tregs or a gained resistance to suppression by the CD8+ T cells. To address this possibility, we examined the suppressive function of Treg cells from Foxp3-GFP and Grail −/− Foxp3-GFP mice towards WT and Grail −/− CD8+ T cells (Supplementary Fig. 4a). Similar to CD4+ T cell, CD4+ Foxp3-GFP+ T cells from Grail −/− mice exhibited reduced suppression activity to both WT and Grail −/− CD8+ T cells12. However, Grail −/− CD8+ T cells appeared to have gained a resistance to WT Treg-mediated suppression both in terms of proliferation and production of granzyme B and IFNγ (Supplementary Fig. 4a). This suggests that the delayed tumour growth observed in vivo may be due to a combination of reduced suppressive activity of Tregs and an enhanced resistance to suppression by CD8+ T cells in the absence of Grail.
To further explore the possible mechanism(s) for accumulation of Grail −/− CD8+ T cells in the EG-7 model of lymphoma, next we analysed the susceptibility of WT and Grail −/− CD8+ TILs to apoptosis; however, we did not detect any difference in the percentage of apoptotic markers Annexin V and 7AAD in CD8+ TIL between WT and Grail −/− groups on days 7 and 12 after tumour inoculation. In addition, we analysed the expression of pro-survival costimulatory receptors OX-40 and 4-1BB and anti-apoptotic factor Bcl-2 in CD8+ TIL from WT and Grail −/− mice. Interestingly, CD8+ TIL from Grail −/− mice had increased expression of OX-40, 4-1BB and Bcl-2 at both time points (Supplementary Fig. 4b). Moreover, mRNA levels of pro-survival costimulatory molecules OX-40 and 4-1BB, and anti-apoptotic gene Bcl-xL (B-cell lymphoma-extra large) were enhanced on day 17 as well, suggesting that Grail could control the survival of CTLs infiltrated into the tumours and thus result in the accumulation of this subset within the tumour (Supplementary Fig. 4c).
To assess whether the absence of Grail has an effect on activation and effector function of CD8+ T cells, we analysed OVA-specific responses ex vivo upon OT-I peptide (chicken OVA peptide 257–264 SIINFEKL) restimulation. In fact, we observed a significantly higher percentage of IFN-γ and granzyme B double-positive cells in Grail −/− CD8+ T cells compared to WT CD8+ T cells (Fig. 1e), suggesting that Grail ablation elicits effector function of CTLs. Since WT CD8+ T cells also highly expressed granzyme B, the impact of Grail loss may be specific to IFNγ responses. As we did not detect any IFNγ single producing cells, we focused on the granzyme B+IFNγ+ subset.
To confirm the functional state of Grail −/− CD8+ T cells in the tumours, we sorted tumour-infiltrating CD8+ T cells from WT and Grail −/− mice and analysed the expression of cytolytic factors by RT-PCR. We detected a statistically significant increase in mRNA levels of IFN-γ, granzyme B, TNF-α and perforin 1 in CD8+ TIL from Grail −/− mice (Supplementary Fig. 4c), further supporting that Grail controls the effector function of CTLs infiltrated into the tumours.
Grail −/− CD8+ T cells are essential for EG-7 tumour control
To test whether CD8+ T cells were necessary to control tumour growth in Grail −/− mice, we depleted CD8+ T cells using a specific antibody before and during EG-7 tumour growth. For WT mice, both CD8+ T cell-depleted and rIgG-treated groups showed similar tumour growth (Fig. 2a), suggesting the nonresponsive state of WT CD8+ T cells. Interestingly, tumour growth was dramatically increased when CD8+ T cells were depleted in Grail −/− mice (Fig. 2a), supporting the critical role of CD8+ T cells in tumour control in Grail −/− mice.
Next we examined whether absence of Grail in CD8+ T cells would be sufficient to confer a protective role against established tumours in normal host. To answer this, we used an adoptive cell transfer therapeutic model where Grail −/− mice were crossed with OT-I TCR transgenic mice to obtain Grail −/− OT-I mice. CD8+ T cells (CD45.2+) from OT-I and Grail −/− OT-I mice were transferred intravenously into congenic CD45.1 mice 5 days after EG-7 inoculation when tumour was palpable. The kinetics and extent of tumour growth were similar between WT control mice (no cell transfer) and mice that received WT OT-I cells (Fig. 2b). In contrast, tumour growth was markedly reduced in mice which received Grail −/− OT-I cells (Fig. 2b). These data indicated that therapeutic transfer of OVA-specific Grail −/− CD8+ T cells was sufficient to mediate regression of established EG-7 tumours.
To investigate the underlying mechanisms, we analysed the transferred CD8+ T cells in TILs. We found that there was a marked increase in percentage of total, donor and host CD8+ T cells in TILs from the Grail −/− OT-I-transferred group (Fig. 2c, d), while there was no difference between control group and OT-I-transferred group. The increase in host CD8+ T cells could be due to the increased level of apoptosis and/or inflammation at the tumour site mediated by Grail −/− CD8+ T cells. Analysis of transferred OVA-specific CD8+ T cells showed significantly higher percentage for IFN-γ and granzyme B double-positive or total IFN-γ+ cells in transferred Grail −/− OT-I cells than WT OT-I cells (Fig. 2e, f). These data suggested that in the therapeutic model, transferred Grail −/− OT-I cells retained their cytolytic function in the suppressive tumour microenvironment and are sufficient to suppress established tumours.
Enhanced effector function of Grail-deficient CD8+ T cells
In order to understand the mechanism for enhanced function of Grail-deficient CD8+ T cells, first we analysed the role of Grail during CD8+ T-cell activation in vivo. Purified CD45.2+ OT-I and Grail −/− OT-I cells were labelled with 5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester (CFDA SE, CFSE) and adoptively transferred into CD45.1+ congenic mice that were inoculated with EG-7 tumour cells 5 days earlier. Three days later, proliferation of transferred cells and their cytokine production were examined. We found that proliferation and IL-2 production by transferred Grail −/− OT-I cells were comparable to WT OT-I cells (Supplementary Fig. 5a, b). However, in contrast to WT OT-I cells, Grail −/− OT-I cells in TILs showed a higher frequency of IFN-γ+ granzyme B+ expression upon in vitro restimulation with OT-I peptide (Fig. 3a), which suggested that Grail negatively regulates CD8+ T-cell effector function in vivo.
To check whether the increased CD8+ T-cell function in Grail-deficient mice is T-cell intrinsic, we generated mixed bone marrow chimeras by transferring a mixture of congenic CD45.1+ WT and CD45.2+ Grail-deficient bone marrow cells into sub-lethally irradiated Rag1 −/− mice. Eight weeks after reconstitution, we inoculated mice with EG-7 tumour cells. Grail-deficient CD8+ T cells infiltrated into the tumours exhibited greatly enhanced production of IFN-γ when compared to WT cells in response to OT-I restimulation (Fig. 3b). The above results confirm that Grail expression in T cells is required to control CD8+ T-cell effector function in vivo.
To further analyse the function of Grail in CD8+ T cells, naive CD8+ T cells from WT and Grail −/− mice were activated with plate-bound anti-CD3 alone or together with anti-CD28. WT and Grail −/− CD8+ T cells exhibited similar proliferation and IL-2 production (Supplementary Fig. 5c). Consistent with the above results, Grail −/− OT-I cells showed comparable proliferation and IL-2 production with WT OT-I cells when activated with OVA peptide and WT antigen-presenting cells (APCs) (Supplementary Fig. 5d). These data indicate that in contrast to CD4+ T cells, Grail does not control proliferation and IL-2 production in CD8+ T cells12. We also activated OT-I or Grail −/− OT-I splenocytes using anti-CD3 or OVA peptide and checked the upregulation of activation markers CD69, CD44 and CD25 on CD8+ T cells. There was no difference in upregulation of these markers between WT and Grail −/− CD8+ T cells (Supplementary Fig. 6). However, we found that Grail −/− CD8+ T cells exhibited greatly increased production of IFN-γ in response to anti-CD3 stimulation alone, which was further enhanced by addition of anti-CD28 (Fig. 3c). Similarly, significant differences in IFN-γ production between WT and Grail −/− OT-I cells were observed in the presence of OVA peptide (Fig. 3d). Most importantly, significant improvement in cytokine expression in Grail −/− CD8+ T cells led to their enhanced killing function both in vitro and in vivo compared to WT CD8+ T-cell effectors (Fig. 3e, f). An extended in vivo killing assay demonstrates that WT CD8+ T cells required additional time to perform their efficient antigen-specific cytolytic function as compared to Grail −/− CD8+ T cells (Fig. 3f; Supplementary Fig. 7). These results indicate that Grail controls the cytotoxic function of CD8+ T cells.
Previously we reported that Grail-deficient CD4+ T cells are less efficient in downregulation of TCR-CD3 expression12. To assess whether enhanced cytotoxic function of Grail −/− CD8+ T cells is associated with their inefficiency in TCR downmodulation, WT and Grail −/− OT-I T cells were activated with OVA peptide and changes in TCRβ expression analysed by flow cytometry over a 6 h time course. OVA peptide stimulation led to TCR downmodulation on WT cells, whereas TCR modulation was markedly attenuated in Grail −/− CD8+ T cells, suggesting that Grail-deficient CD8+ T cells are more resistant to activation-induced TCR downmodulation than WT cells (Supplementary Fig. 8a). To investigate further, we analysed the ubiquitination of CD3ζ in CD8+ T cells from WT and Grail −/− mice. Six hours after activation in the presence of MG132 to prevent degradation, T-cell lysates were subject to immunoprecipitation with CD3ζ antibodies and immunoblotting with anti-Ub antibodies. In comparison to WT cells, ubiquitination of CD3ζ in Grail −/− CD8+ T cells was substantially reduced demonstrating that Grail targets CD3 through its ubiquitination and subsequent degradation (Supplementary Fig. 8b). Thus, sustained TCR-CD3 signalling in Grail −/− CD8+ T cells could be one of the possible mechanisms for their elevated effector function.
Grail loss causes hyperresponsiveness to IL-21R signalling
Members of the common γ-chain (γc) cytokine family, including IL-2, IL-7 and IL-15 have important roles in regulating CD8+ T-cell response. IL-21, a member of the γc family, has also been reported to improve CD8+ T-cell function through regulation of IFN-γ, granzyme B and perforin production, and survival in infection and tumour models14,15,16. Interestingly, we found that Grail −/− CD8+ TILs expressed significantly higher level of IL-21R compared to WT TILs (Fig. 4a) suggesting a potential role of Grail in the regulation of IL-21 signalling.
To assess whether IL-21R signalling could contribute to enhanced effector function of Grail −/− CD8+ T cells, FACS-sorted naive WT and Grail −/− CD8+ T cells were activated with plate-bound anti-CD3 in the absence or presence of IL-21. Interestingly, though WT and Grail −/− CD8+ T cells proliferated similarly with anti-CD3 treatment alone, proliferation of Grail −/− CD8+ T cells significantly increased after IL-21 treatment compared to WT CD8+ T cells (Fig. 4b). In addition, whereas anti-CD3-induced IFN-γ, granzyme B, perforin1 and IL-21Rα mRNA, and protein expression in WT CD8+ T cells was increased, there was significantly higher expression of these molecules (both mRNA and protein levels) in Grail −/− CD8+ T cells upon IL-21 treatment (Fig. 4c–e; Supplementary Fig. 9). The enhanced production of cytolytic factors and IL-21R by Grail −/− CD8+ T cells was not simply due to the increased proliferation of these cells in response to IL-21 as this was also observed at the per cell level by flow cytometry staining. These data suggested that as Grail −/− CD8+ T cells express higher levels of IL-21R, they are more sensitive to IL-21 treatment than WT cells and that the expression of IL-21R may be driven in part by IL-21.
To further assess the role of IL-21 signalling in effector function of Grail −/− CD8+ T cells in vivo, WT, Grail −/− and Grail −/− Il21 −/− mice were inoculated with EG-7 tumour cells. Interestingly, absence of IL-21 caused progressive tumour growth in Grail −/− mice comparable to WT mice (Fig. 4f). Consistent with increased tumour growth, percentage of CD8+ T cells infiltrated to the tumours of Grail −/− Il21 −/− mice was decreased compared to Grail −/− mice (Fig. 4g). However, tumour growth was comparable between WT and Il21 −/− mice demonstrating that the effect seen in the Grail −/− Il21 −/− and Grail −/− mice was due to the enhanced IL-21R signalling in the absence of Grail (Supplementary Fig. 10). Moreover, there were fewer IFN-γ+ granzyme B+ antigen-specific CD8+ T cells from the tumours of Grail −/− Il21 −/− mice compared to Grail −/− mice (Fig. 4h), supporting the role of IL-21R signalling in Grail −/− CD8+ T-cell expansion and function. However, CD8+ TIL from WT and Il21 −/− mice had similar levels of functionality (Supplementary Fig. 10). Thus, the above data demonstrate that Grail deficiency results in increased IL-21R signalling in CD8+ T cells and consequently enhanced cytolytic function against tumours in vivo.
Grail regulates IL-21R expression
Next we dissected the molecular mechanism for hyper-responsiveness of Grail −/− CD8+ T cells to IL-21R signalling. IL-21 functions through IL-21R, which is a heterodimer that consists of γc (CD132) and IL-21Rα chain17. After binding of IL-21 to the IL-21Rα/γc complex, Janus-activated kinase 1 (JAK1) and JAK3 undergo autophosphorylation and subsequent phosphorylation of signal transducer and activator of transcription (STAT)-1 and STAT3 and to a lesser extent, STAT5. To determine which of the IL-21R signalling components is controlled by Grail; first, we checked the phosphorylation levels along with total protein levels of STAT1, STAT3 and STAT5 in naive WT and Grail −/− CD8+ T cells after they were activated with anti-CD3 in the presence or absence of IL-21. TCR signalling alone did not induce phosphorylation of STAT1, STAT3 and STAT5 in either WT or Grail −/− CD8+ T cells. Consistent with previous reports18, IL-21 stimulation induced the phosphorylation of STAT1 and STAT3 in WT CD8+ T cells (Fig. 5a). Compared to WT cells, Grail −/− CD8+ T cells showed enhanced phosphorylation of these STATs as well as STAT5 (Fig. 5a). However, the total levels of these STATs were comparable between WT and Grail −/− CD8+ T cells (Fig. 5a), suggesting that molecules upstream of STATs account for the phenotype of Grail-deficient CD8+ T cells. Next, we examined difference between IL-21-activated WT and Grail −/− CD8+ T cells in JAK1 expression and activation, and observed augmented JAK1 phosphorylation in Grail −/− CD8+ T cells upon IL-21 treatment, while total JAK1 level was comparable between WT and Grail −/− CD8+ T cells (Fig. 5b).
Since STATs and JAK1 protein levels in CD8+ T cells were not affected by Grail deficiency, we examined whether Grail controls the surface IL-21R expression. Interestingly, analysis of surface expression of γc/CD132 and IL-21Rα in naive CD8+ T cells revealed increased protein levels of γC/CD132 and IL-21Rα level upon Grail deficiency (Fig. 5c). Since γc also constitutes the receptors for IL-2, IL-7 and IL-15, we checked expression of IL-7Rα, IL-2Rβ/IL-15Rb and IL-2Rα on naive CD8+ T cells, and found that their expressions were comparable between WT and Grail −/− CD8+ T cells (Supplementary Fig. 11a), suggesting that Grail specifically controls IL-21R levels on CD8+ T cells. Consistent with receptor levels, Grail −/− CD8+ T cells did not show enhanced proliferation and mRNA expression of IFN-γ and granzyme B in response to IL-2, IL-7 and IL-15 treatments (Supplementary Fig. 11b, c).
Next, we hypothesised Grail may regulate IL-21R protein level post-translationally through ubiquitination and degradation. To check whether Grail directly targets IL-21R subunits, we either overexpressed γc/CD132 or flag-tagged IL-21Rα together with WT or Grail mutant in 293T cells along with HA-tagged ubiquitin. After immunoprecipitation of γc/CD132 with CD132 antibody or IL-21Rα–Flag with Flag antibody and reprobing with HA-HRP antibodies, we observed the formation of high molecular weight ubiquitin conjugates in the presence of WT Grail, indicating polyubiquitin chain formation which was not observed on IL-15Ra (Fig. 5d; Supplementary Fig. 12). This ubiquitination was not present upon overexpression of the Grail mutant, which lacks a functional RING-finger domain (Fig. 5d, right lanes). Thus, IL-21R is a specific substrate for Grail-mediated ubiquitination in CD8+ T cells. To determine whether Grail regulates the expression of IL-21R through endosome-mediated degradation, naive OT-I T cells from WT mice were activated with OVA peptide and WT APCs in the presence of the endosome inhibitor Latrunculin B. Addition of Latrunculin B led to upregulation of IL-21Rα and CD132 but not CD25, CD122 or IL-7R expression, suggesting that Grail may specifically target the endocytosed IL-21R complex via endosome-mediated degradation (Fig. 5e).
Regulation of Grail expression by TGF-β
We found that Grail expression levels were significantly upregulated in CD8+ T cells infiltrated into the tumours compared to CD8+ T cells that had homed to the spleens, indicating that tumour-specific factors contribute to Grail expression in CD8+ TILs. It has been reported that EL-4 cells suppress immune responses by secreting large amount of TGF-β19. Interestingly, we found that Grail is significantly upregulated in TGF-β-treated CD8+ T cells (Fig. 6a). Thus, we further examined whether TGF-β signalling is responsible for Grail expression in tumour CD8+ TILs by administering TGF-β blocking antibodies in WT mice during EG-7 tumour growth. In fact, blockade of TGF-β resulted in a reduced rate of tumour growth and decreased tumour size at day 17 (Fig. 6b). Importantly, analysis of sorted CD8+ TILs from both groups revealed a correlation between anti-TGF-β-mediated tumour blockade and diminished Grail (but not Cbl-b) expression in CD8+ TILs (Fig. 6c). Furthermore, CD8+ TIL from mice that received TGF-β blockade showed increased IL-21R expression by RT-PCR (Fig. 6d). Overall this data suggest that TGF-β signalling is one of the mechanisms responsible for Grail expression in CD8+ T cells infiltrated into tumours and consequently to their unresponsiveness.
Grail expression by CD8+ T cells from patients with lymphoma
The above data presented demonstrate a suppressive function of Grail in CD8+ T cells in both an antigen-restricted and non-antigen-restricted mouse model of lymphoma. This led us to explore Grail and IL-21R expression patterns in CD8+ T cells from the peripheral blood of patients with either diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma (FL). To this end, peripheral blood mononuclear cells (PBMCs) were isolated from normal donors (ND PBMCs), DLBCL and FL, and stained for Grail expression by flow cytometry (Fig. 7a). Interestingly, Grail expression by CD8+ T cells was significantly higher in patients with both subtypes of lymphoma as compared to ND PBMCs (p = 0.024, Fig. 7b). Similarly, to the murine models, the enhanced expression of Grail was specific to this E3 ubiquitin ligase as expression of Cbl-b was not different between ND and patient PBMCs (Fig. 7c). Furthermore, the level of IL-21R was significantly lost in the patient PBMC samples as compared to the ND (Fig. 7d). As levels of TGF-β present in the serum of patients with both FL and DLBCL have shown to be significantly higher than that found from ND, this could provide an explanation for the high level of Grail expression observed in the CD8+ T cells present in the blood20, 21. Indeed, analysis of lymphoma patient serum samples showed significantly higher levels of TGF-β as compared to ND controls (Fig. 7e). Overall, this data suggest that the model described in the murine system where Grail expression is induced by TGF-β signalling and is involved in ubiquitination and degradation of the IL-21R may also occur in human patients with lymphoma. Further study of Grail in patients with lymphoma and solid tumours is warranted.
To date, cancer immunotherapy is a promising approach to reactivate a patient’s immune system to fight against tumours; however, it is a difficult process due to various tumour evasion mechanisms, particularly by T-cell tolerance induction. Thus, today it is of high priority to develop strategies to break T-cell tolerance and thereafter enhance T-cell cytotoxicity towards tumour cells. Previously, we have acknowledged the E3 ubiquitin ligase, Grail, as an essential component of CD4+ T-cell tolerance12, 13. In the current study, we have determined the intrinsic role of Grail in controlling CD8+ T-cell CTL functions. In fact, in CD8+ T cells, Grail controls TCR and IL-21R signalling and restricts their effector cytokine expression and cytolytic function. As a result, Grail-deficient CD8+ T cells are sufficient to control established tumours.
Our initial observation that Grail is preferentially upregulated in CD8+ TIL compared to CD8+ T cells localised in lymphoid tissues, suggested the potential role of Grail in CD8+ T-cell anti-tumour activity. In fact, we observed a correlation between Grail expression in CD8+ T cells and their unresponsiveness to tumours that was abolished upon Grail deletion. Importantly, we detected high levels of Grail expression in CD8+ T cells from lymphoma patients compared to CD8+ T cells from healthy donors, which further suggested that Grail could serve as a functional marker for nonresponsive CTLs. This hypothesis requires testing in a larger patient cohort and comparisons between liquid and solid tumours would be informative, especially as of TGF-β expression patterns could vary within the local tumour microenvironment. Surprisingly, we did not detect the upregulation of another E3 ubiquitin ligase, Cbl-b, in either CTLs infiltrated in mouse lymphoma or in CD8+ T cells from lymphoma patients, indicating the distinct regulation and function of Grail and Cbl-b in CD8+ T cells.
The ability of adoptively transferred Grail −/− CD8+ T cells to control antigenic tumours provides evidence on the intrinsic role of Grail in controlling CD8+ T-cell activation and function. Surprisingly, unlike Grail −/− CD4+ T cells12, 13, our in vitro and in vivo analysis showed that Grail deficiency did not change CD8+ T proliferation and IL-2 production upon antigen encounter but did enhance resistance to Treg-mediated suppression. As Grail −/− Tregs also showed a weakened ability to suppress CD8+ T cells, this deficiency could also play a role in the observed delay in tumour growth in the Grail −/− mice. However, production of the effector cytokine IFN-γ by Grail-deficient CD8+ T cells and their cytolytic activity towards tumour cells were significantly enhanced, suggesting that Grail controls effector function of CD8+ T cells. Since Grail selectively targets TCR-CD3 signalling for degradation in CD4+ T cells12, enhanced effector function of Grail −/− CD8+ T cells could be attributed to sustained TCR signalling. We determined that Grail −/− CD8+ T cells are defective in TCR downmodulation and furthermore Grail targets CD3ζ for ubiquitination and degradation, suggesting that sustained TCR-CD3 signalling is one of the mechanisms for excessive function of Grail −/− CTLs.
In addition to TCR engagement and positive costimulation, CD8+ T cells require a third signal, cytokines for supporting their effector functions, survival and memory formation22. IL-21 is a cytokine produced by activated CD4+ T cells and natural killer T cells 17 whose activity has been shown to stimulate CD8+ T-cell cytotoxic function and survival17, 23. We found that Grail expression in patients with lymphoma CD8+ T cells and mouse CD8+ TILs negatively correlates with IL-21R expression while expression of other receptors involved in common gamma chain cytokine signalling (IL-2R, IL-7R and IL-15R) did not depend on the level of Grail. Our data also suggest the essential role of IL-21R signalling in Grail −/− CD8+ T-cell function, since Grail −/− CD8+ T cells in the absence of IL-21 signalling expressed significantly low level of cytolytic cytokines and failed to suppress tumour growth. Analysis of IL-21R downstream signalling components including Jak1, STAT1, STAT3 and STAT5 did not show any difference in their protein level upon Grail deficiency, while their phosphorylation levels were significantly enhanced. Interestingly, we found that protein levels of IL-21R subunits, γc/CD132 and IL-21Rα were already increased in naive Grail −/− CD8+ T cells. Moreover, Grail specifically controls the expression of IL-21R subunits via ubiquitination and endosome-mediated degradation. Altogether, our study for the first time indicates that Grail controls the effector function of CD8+ T cells by directly targeting and controlling IL-21R expression.
Overall, our results elucidate pivotal functions of Grail in the regulation of CD8+ T-cell function. Sustained TCR-CD3 and IL-21R signalling helps Grail-deficient CD8+ TILs to maintain their CTL activity. Thus modulation of Grail expression in CD8+ T cells might be a potent new strategy to augment the effectiveness of tumour-specific CD8+ T cells for anti-cancer immunotherapy.
C57BL/6J, SJL, RAG1 −/− and OT-I transgenic mice were purchased from Jackson Laboratories. Il21 −/− mice, Grail −/− mice have been previously described12, 24. Grail −/− mice were crossed with OT-I mice or Il21 −/− mice to get Grail −/− OT-I mice or Grail −/− Il21 −/− mice, respectively. All mice are on a C57BL/6 background. About 8–10 week-old age- and sex-matched mice (either male or female) were used for experiments. Mice were housed in the SPF animal facility at M.D. Anderson Cancer Center and all animal experiments were approved by Institutional Animal Care and Use Committee.
EL-4 (murine lymphoma) and EG-7 were purchased from ATCC and maintained in DMEM medium supplemented with 10% fetal bovine serum and antibiotics. Cells lines were tested for mycoplasma contamination prior to any experiments.
All blood and tissue samples were obtained after written informed consent from patients through a University of Texas MD Anderson Cancer Center IRB-approved protocol (2005-0656).
To analyse the expression of Grail in human CD8+ T cells, human PBMCs were first stained for surface antigens (CD3, CD8) and a live/dead dye followed by fixation and permeabilized for staining with Grail. The following antibodies were obtained from BD Biosciences: PerCP-Cy5.5-conjugated anti-CD3 (clone SK7, 1:20 dilution), Pacific blue-conjugated anti-CD8 (clone RPA-T8, 1:50 dilution). Alexa488-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, 1:300 dilution). Rabbit monoclonal Grail antibody (anti-RNF 128, 1:100 dilution) was obtained from Abcam. Live/dead Aqua dye (1:200 dilution) was purchased from Invitrogen. For animal experiments, the following antibodies were purchased from Biolegend: CD8 PerCP-Cy5.5 (clone 53-6.7, 1:1000 dilution), CD4 FITC (clone GK1.5, 1:100 dilution), CD25 PE (clone PC61, 1:100 dilution), CD62L FITC (clone MEL-14, 1:800 dilution) CD44 APC (clone IM7, 1:500 dilution), CD69 PE (clone H1.2F3, 1:100 dilution), CD132 PE (clone TUGM2, 1:100 dilution), CD127 PE (IL-7R, clone CB1199, 1:100 dilution), Rag IgG2ak biotin (clone RTK2758, 1:100 dilution), Annexin V FITC (1:20 dilution), TCRβ FITC (clone H57-597, 1:100 dilution), OX-40 APC (clone OX86, 1:100 dilution) and Bcl-2 Alexa Fluor 647 (clone Bcl/10C4, 1:100 dilution). Antibodies were also purchased from BD Biosciences: granzyme B Alexa Fluor 647 (clone GB-11, 1:100 dilution), IL-2 APC (clone JES6-5H4, 1:100 dilution), 7AAD (1:20 dilution) and caspase-3 PE (C92-605, 1:5 dilution). Antibodies from eBiosciences were also purchased: IL-21R biotin (clone eBio4Ag, 1:100 dilution), IFNγ PE (clone XMG1.2, 1:200 dilution), CD122 PE (clone IM-b1, 1:100 dilution) and 4-1BB PE (clone 17B5, 1:100 dilution). Streptavidin APC (1:500 dilution) was purchased from Jackson ImmunoResearch. Gating was determined using the Fluorescence minus one approach. Samples were acquired using a BD FACs Canto and analysed using FlowJo v 10.0.7. The gating strategy for CD8+ T-cell phenotyping is described in Supplementary Fig. 13.
Detection of serum TGF-β
TGF-β protein levels in serum of healthy individuals and lymphoma patients were detected by enzyme-linked immunosorbent assay (ELISA) using the manufacturer’s instructions (eBioscience).
T-cell function analysis in vitro
Naive WT or Grail −/− CD8+ T cells (CD8+CD25−CD62LhiCD44lo) were flow cytometry-sorted and activated with plate-bound indicated doses of anti-CD3 and/or anti-CD28 (eBioscience). Naive OT-I or Grail −/− OT-I cells (CD8+CD25−CD62LhiCD44lo) were sorted and activated with indicated doses of OT-I peptide (SIINFEKL) (RS Synthesis) in the presence of irradiated WT APCs. IL-2 production was determined 24 h after T-cell activation by ELISA using manufacturer’s instructions (BD Biosciences). Proliferation was assayed after 2 days (for 0.001 µg/ml OVA stimulation) or 3 days (for anti-CD3 (2 µg/ml, clone 2C11, eBioscience) and/or anti-CD28 (2 µg/ml, clone 37.51, eBioscience) stimulation) of activation by adding [3H] thymidine (Perkin Elmer) to the culture for the last 8 h. IFN-γ and granzyme B production was analysed by flow cytometry 2 days (OVA stimulation) or 3 days (anti-CD3 and/or anti-CD28 stimulation) later. In Fig. 4b, c, FACS-sorted naive CD8+ T cells from WT and Grail −/− mice were activated with plate-bound anti-CD3 (2 µg/ml, eBioscience) alone or together with IL-21 (50 ng/ml, Peprotech). Cells were harvested 24 h later for gene expression analysis. About 72 h after activation, proliferation was assayed by adding [3H] thymidine (Perkin Elmer) and IFNγ and granzyme B were detected by ELISA using manufacturer’s instructions (BD Bioscience) and flow cytometry staining. For Treg suppression assay, naive CD8+ T cells were cultured with plate-bound anti-CD3 and irradiated WT APCs or together with WT or Grail −/− Foxp3-GFP+CD4+ nTreg cells in triplicate wells. Proliferation was analysed 72 h after treatment by adding [3H] thymidine (Perkin Elmer) to the culture for the last 8 h. In Supplementary Fig. 10c, FACS-sorted naive CD8+ T cells from WT and Grail −/− mice were activated with plate-bound anti-CD3 (2 µg/ml, eBioscience) alone or together with hIL-2 (30 U/ml, Peprotech) or mIL-7 (5 ng/ml, Peprotech) or mIL-15 (5 ng/ml, Peprotech) and 24 h later cells were harvested for gene expression analysis.
The in vitro SIINFEKL-specific CTL response was measured using a caspase-3 cleavage assay25. In brief, naive CD8+ T cells from WT and Grail −/− OT-I mice were activated with OT-I peptide (0.001 µg/ml; RS Synthesis) and irradiated WT APCs for 2 days. EL-4 cells were used as target cells after labelling with DDAO-SE and 1 µg/ml peptide. Activated CD8+ T cells were co-cultured with the target EL-4 cells at a 1:1 ratio for 2 h at 37 °C. The cells were permeabilized and stained with PE-conjugated anti-cleaved caspase-3 antibody. The percentage of cleaved caspase-3-positive cells among DDAO-SE-labelled target cells were analysed by flow cytometry.
The in vivo SIINFEKL-specific cytolytic activity of CD8+ T-cell responses was measured as follows. Briefly, spleen cells from OT-I or Grail −/− OT-I mice were adoptively transferred (iv) into C57BL/6J mice (6–8 week old, 106 cells per mouse) followed by s.c. vaccination with OVA peptide (100 µg per mouse, RS Synthesis) and anti-CD40 (50 µg per mouse, clone FGK4.5, BioXcell). Imiquimod cream 5% (50 µg/mouse, Aldara, Fougera) was applied on the vaccination site. In addition, mice intraperitoneally (i.p.) received 100,000 IU rhIL-2 (TECIN, Hoffman LaRoche Inc.) as previously described26. Three days after vaccination, mice were injected with a 1:1 mix of target cells (5 × 106 of each). Target cells were prepared using splenocytes from C57BL/6 mice either loaded with 1 µg/ml peptide and labelled with 5 µmol/l CFSE or unloaded and labelled with 0.5 µmol/l CFSE. Four and 8 h later, splenocytes from the recipients were analysed by flow cytometry to assess peptide-specific killing of the CFSEhi labelled cells.
Quantitative real-time PCR
Total RNA was prepared using TriZol reagent (Life Technology). Complimentary DNA (cDNA) was made using Superscript reverse transcriptase and oligo(dT) primers (Life Technology), and gene expression was detected with a Bio-Rad iCycler Optical System using iQ SYBR green RT PCR kit (Bio-Rad Laboratories, Inc.). The data were normalised to the reference gene β-actin. The primers for Grail, IFN-γ, TNF, IL-21, IL-21R, granzyme B, Cbl-b, OX-40, 4-1BB, Bcl-xL and β-actin were previously described12, 16, 24, 27,28,29,30,31,32,33,34,35,36. Briefly, the pairs used for mouse are: Grail forward: 5′-TAGCTGTGCTGTGTGCATTG-3′, reverse: 5′-CTTCATGGGGAGAGGCAGTA-3′; IFN-γ forward: 5′-GATGCATTCATGAGTATTGCCAAGT-3′, reverse: 5′-GTGGACCACTCGGATGAGCTC-3′; TNF-α forward: 5′-AATGGCCTCCCTCTCATCAGT-3′, reverse: 5′-GCTACAGGCTTGTCACTCGAATT-3′; IL-21R forward: 5′-TCCCACGCCCAGGAGACCACC-3′, reverse: 5′-ATGCTGGGGTTGGGGCTCCGT-3′; granzyme B forward: 5′-ATCAAGGATCAGCAGCCTGA-3′, reverse: 5′-TGATGTCATTGGAGAATGTCT-3′; Cbl-b forward: 5′-TTCCAGATGGCAAACTCAATG-3′, reverse: 5′-TACATTCTCTCTCCTTGCCTTCTTTA-3′; OX-40 forward: 5′-AACCTCGGCAGGACAGCGGC-3′, reverse: 5′-CACTGGCTGGGTGGCGGGTC-3′; 4-1BB forward: 5′-CCCCCACATATTCAAGCAAC-3′, reverse: 5′-TAGCCTCCTCCTCCTCCTTC-3′; Bcl-xL forward: 5′-TGGTGGTCGACTTTCTCTCC-3′, reverse: 5′-CTCCATCCCGAAAGAGTTCA-3′; perforin 1 forward: 5′-AGGCAAACATGCGCGCCTCCGT-3′, reverse: 5′-GGACCGAGATGCGGCCACCGA-3′; b-actin forward: 5′-TGGAATCCTGTGGCATCCATGAAAC-3′, reverse: 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′. For human samples the following pairs were utilised: Grail forward: 5′-CTGCTCGAAGGCTACGGAAT-3′, reverse: 5′-GGGCCAATTTCCTTGTCTCCT-3′; IL-21R forward: 5′-GGGCTCTGTGATGTAGGCAG-3′, reverse: 5′-CTGGTCTTGCCAGGTAAGGG-3′; Cbl-b forward: 5′-TGTCTCTGGACAGCTACGGC-3′, reverse: 5′-GTCTTACCACTTTGTCCATGAGC-3′; actin forward: 5′-TCCCTGGAGAAGAGCTACGA-3′, reverse: 5′-AGCACTGTGTTGGCGTACAG-3′.
Immunoblot analysis and ubiquitination assay
Cells were lysed in kinase assay lysis buffer supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitors (10 mM NaF and 1 mM Na3VO4). Protein concentration was determined by Bio-Rad Bradford protein assay and equal amounts of protein were loaded for immunoblot analysis with antibodies against phospho-STAT1 (Tyr701), phospho-STAT3 (Ser727), phospho-STAT5 (Tyr694), phosphor-JAK1 (Tyr1022/1023), STAT1, STAT3, JAK1 (Cell Signaling), STAT5 (Santa Cruz Biotechnology), Grail (Abcam) (1:100 dilution). Anti-β-actin (1:1000 dilution), anti-mouse IgG-HRP (1:5000 dilution) and anti-rabbit IgG-HRP (1:5000 dilution) secondary antibodies were from Thermo scientific. β-actin was used as the loading control for all experiments.
For the ubiquitination assays, 293 T cells were transfected in six-well plates with bicistronic retroviral expression vector pGFP-RV containing either IL-21Rα, CD132 or IL-15Rα, and Grail or Grail mutant and pcDNA-HA-ubiquitin. Cells were lysed in kinase lysis buffer supplemented with N-ethylmaleimide (NEM). Lysates were incubated with anti-FLAG M2 mAb (Sigma Aldrich), anti-CD132 (Santa Cruz) or anti-IL-15Rα (Santa Cruz) (5 µg per sample) and then Protein A-agarose (Sigma Aldrich), followed by immunoblotting using anti-HA-HRP, anti-IL-15Ra, anti-CD132 (Santa Cruz Biotechnology) and anti-Flag antibodies (Sigma) (1:1000 dilution). Uncropped gel images are shown in Supplementary Fig. 14.
EL-4 and EG-7 tumour models
1 × 106 EG-7 or EL-4 cells were inoculated s.c. to the right flanks of experimental animals (8–10 week old) on day 0 and tumours were measured every other day using a digital caliper from day 7. Tumour sizes were calculated by ½(ab 2), where a is the length and b is the width. The maximum tumour size allowed and adhered to is 1.5 cm tumour diameter. For the adoptive transfer therapeutic model, congenic SJL (8–10 week old) recipient mice were inoculated with EG-7 cells and 5 days later when tumours were palpable transferred with 3 × 106 OT-I or Grail −/− OT-I cells by i.v. In some experiments, purified OT-I and Grail −/− OT-I cells were labelled with 5-(and 6)-CFDA SE, CFSE before being transferred. For CD8+ T-cell depletion experiments, 150 μg anti-CD8 (clone 2.43, BioXcell) was given by i.p. 3 days before tumour inoculation and weekly after that until the end of experiments. Rat IgG (BioXcell) was administered to control mice. The depletion efficiency was checked by blood staining with corresponding antibodies for each experiment. For TGF-β blockade, 100 μg anti-TGF-β (clone 1D11, BioXcell) was given 5 days after EG-7 inoculation three times with 2 day intervals. Rat IgG (BioXcell) was administered to control mice.
Graphical presentation and statistical analysis of the data were performed using GraphPad Prism (Version 5.0d, GraphPad software, San Diego, CA, USA) and Excel. Data are displayed as mean and SEM. Results between experimental groups were compared using a one-way analysis of variance or Student’s t-test. p < 0.05 was considered statistically significant. Statistical significance is displayed as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.
The authors declare that the data supporting the findings of this study are available within the article and its supplementary information files, or are available from the corresponding author upon request.
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We thank the members of Dr. Chen Dong laboratory, as well as Dr. S.-C.S. laboratory for discussions; and the FACS core facility at MD Anderson Cancer Center for assistance. This work is supported by NIH research (AI083761 (R.N.), A1R03AI120027 (R.N.), 1R21AI20012 (R.N.), AI064639 (S.-C.S.), CA184845 (C.B.), CA178580 (C.B.), CA155143 (S.S.N.) and P30CA16672 (flow core)), Institutional Research Grant (R.N.), start-up grant (R.N.), MD Anderson CIC seed grant (R.N.). L.W.K. is recipient of Leukemia & Lymphoma Society Quest for Cures grant (0855-14). K.R. is recipient of CPRIT Fellowship (RP101503). The primary blood and tumour samples were provided by The University of Texas MD Anderson Cancer Center Lymphoma Tissue Bank, which is supported by the Lymphoma SPORE P50CA136411 (NIH), Cancer Center Support Grant CA16672 (NIH) and Fredrick B Hagemeister Research Fund.
The authors declare no competing financial interests.
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Haymaker, C., Yang, Y., Wang, J. et al. Absence of Grail promotes CD8+ T cell anti-tumour activity. Nat Commun 8, 239 (2017). https://doi.org/10.1038/s41467-017-00252-w
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January 8 – Give Pets A Chance: Cornflake and PorkChop
Cornflake ID# A048318 is a white and tan 7 year old chihuahua and Porkchop ID# A048897 is a 5 year old Cairn Terrier mix.
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Napa’s Araujo Estate Bought by Owners of Bordeaux’s Château Latour
Araujo Estate, one of Napa Valley’s elite Cabernet Sauvignon producers, has been purchased by the owners of French first-growth Château Latour. The Artemis Group, the financial company founded by French businessman François Pinault, announced it has added Araujo to an impressive roster of wineries in Bordeaux, Burgundy and the Rhône. The purchase price was not disclosed, but the deal includes the historic Eisele Vineyard and its 38 acres of vines, the winery and cave complex, the brand and existing inventory.
“Araujo Estate and its jewel, the unique Eisele Vineyard, have been producing consistently one of the very best wines of Napa Valley,” said Frédéric Engerer, CEO of Château Latour, in a statement.
Araujo is the latest purchase by the Pinault family, headed by François and son François-Henri, which through Artémis Group owns Latour, Domaine d’Eugénie in Burgundy and Château-Grillet in the Rhône Valley.
Bart and Daphne Araujo were not available for comment. The couple purchased Eisele Vineyard in 1990 from Milt and Barbara Eisele. Situated at the foot of the Palisades area southeast of Calistoga, Eisele Vineyard had been planted to vines since 1884 and is one of the most notable vineyards in Napa Valley. Its track record of stellar Cabernets dates back to the 1970s, including bottlings by Joseph Phelps Winery and Conn Creek.
The Araujos undertook a major replanting after the purchase and added new varieties, including Syrah and Sauvignon Blanc. In 2000, Bart told Wine Spectator why he quit his previous career in home construction to focus on wine: “Daphne and I believe that this property is too important not to devote full attention to it.”
The Araujos continued to make improvements on the property, first by hiring Bordeaux enologist Michel Rolland as a consultant, and then converting to the intensive farming practices of biodynamics in 2001. Engerer has been experimenting with biodynamics at Latour.
The current Araujo staff—including the winemaking team led by Françoise Peschon—will remain with the estate under its new ownership. The Araujos have said they will stay in residence at the estate until January to assist in the transition.

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ATTORNEYS AT LAW
“I do general business, bankruptcy and real estate litigation. As part of that, I handle contract disputes, represent lenders/creditors in bankruptcy court and do health care-related litigation. I’m also proud that I’m able to be an important resource to Sacramento’s Arab American community. Because of my fluency in Arabic and being from the Middle East, I’ve been able to help clients who are looking for an Arab-speaking attorney.”
Bashar Ahmad handles all types of civil litigation matters, with a particular emphasis on business healthcare and real estate disputes. He has been the lead attorney on a number of large litigation matters, including cases dealing with complex real estate and healthcare issues.
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Outside of work, people know me as a huge 'car guy.' I learned how to rebuild cars as a teenager, and barely a day goes by when I'm not buying tools and tinkering with cars, including my favorites, high performance cars and raised 4X4s.
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- Obtained prejudgment orders of attachment, injunctions and writs of possession in complex commercial cases.
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- Represented a medical group in a number of disputes with a capitated health plan, including a dispute over the method of computing the capitated payments owed to the group under the parties’ agreement.
- Represented an employer in a dispute with a hospital over amounts for certain health care services covered under the employer’s federal Employee Retirement Income Security Act (ERISA) plan.
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Design is everywhere in our lives, perhaps most importantly in the places where we’ve just stopped noticing. 99% Invisible is a weekly exploration of the process and power of design and architecture. From award winning producer Roman Mars. Learn more at 99percentinvisible.org.
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February 4th, 2011
Episode 16 of 275 episodes
Everyone knows it when they see it. The classic “castle with turrets” periodic table is a beautiful and concise icon that contains a great deal of amazing information, if you only know how to read it. And even if you don’t know anything about the table, it’s still easy to admire and get lost in. Author of The Disappearing Spoon, Sam Kean, talks us through the design of the table that hung in the front of your science class for years, but you probably never really understood.
In a time where we're all threatened by a rhetoric of hate from the people in power; A Gay And A NonGay challenges many of our differences head on and promises that no matter who you are, or what you're into (Bruce Springsteen or Britney), love is love and gay and nongays can be friends. Contact us on Twitter @gaynongay

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Published July 23, 2002
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A Second Course in Statistics: Regression Analysis, 7th Edition with / MINITAB Release 14 for Windows CD [MENDENHALL] on *FREE* shipping on qualifying offers. A Second Course in Statistics: Regression Analysis, 7th Edition with / MINITAB Release 14 for Windows CDAuthor: MENDENHALL. Description Elementary Statistics, Seventh Edition, features a thorough presentation of the reasoning behind statistics, balanced with analysis and exploration of real data. This text emphasizes the development of statistical thinking rather than rote drill and practice. This new edition continues the author’s tradition of cutting-edge statistical pedagogy and seamless integration of. Introduction to the Practice of Statistics, 7th Edition J.K. out of 5 stars 5. Paperback. $ Usually ships within 1 to 3 weeks. Introduction to the Practice of Statistics w/CD 6th (sixth) edition byMoore David S. Moore. out of 5 stars and am fairly familiar with statistics. The number of associated Minitab files are nice /5(90). A Second Course in Statistics: Regression Analysis, Seventh and actually a fairly easy read. For our class we are using R, and the cd in the back of the book allows us to read the data off of the cd, and use it with R. My last statistics class used Design and Analysis of Experiments 8th edition, and to me, this book was a lot more helpful Reviews:
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Calculus Applied Approach Seventh Edition Plus Student Solutions Guide Plus Mathspace CD Plus Smarthinking. BoD Books on Demand , Edition 1 Category: Engineering Level: Intermediate. Diseño y análisis de experimentos. Elementary Statistics with CD-ROM and InfoTrac. Johnson, Robert R. and Kuby, Patricia Six Sigma Statistics using Mini Green Belt Edition. About. Khan, Rehman Amazon , Edition 1. Understanding Statistics with Statistics CD-ROM and Student Solutions Manual, Seventh Edition and DV by Brase Hardcover | Not Avail | Pub. Date: ISBN: | ISBN More Details Similar Books»Compare Prices» Add to Wish List» Tag this book» Add book review. Second Course in Statistics, A: Regression Analysis (2-downloads) - Kindle edition by Mendenhall, William, Terry Sincich. Download it once and read it on your Kindle device, PC, phones or tablets. Use features like bookmarks, note taking and highlighting while reading Second Course in Statistics, A: Regression Analysis (2-downloads).Reviews:

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text:
Louthiana: or, an Introduction to the Antiquities of Ireland. First Edition (1758)
Book ID: 60964
Louthiana: or, an Introduction to the Antiquities of Ireland. In upwards of Ninety Views and Plans: Representing, with Proper Explanations, the Principal Ruins, Curiosities, and Ancient Dwellings, in the County of Louth. Divided into Three Books. Engraved by Paul Fourdrinier. London: Thomas Payne, 1758. First Edition. Folio. Rebound in recent half crushed morocco & marbled boards. Two small library ink stamps to front free endpaper & upper right margin of title-page; small worm holes to outer margins of leaves, text not affected, otherwise a fine bright copy. The work divided as follows: (Book 1) Views and plans of the Most remarkable Bodes, Forts, and Mounts in the County of Louth, with an Account of their Origin as attributed to the Natives or Foreign Colonies. In Twenty Plates. (Book 2) Plans and View of the Principal Castles, Keeps, and Towers in the County of Louth, Formerly The Habitations of Nobility and Gentry. In Twenty-Four Plates. (Book 3) A Collection of the Most Remarkable Remains of the Works of the Danes and Druids in the County of Louth. In Twenty-Two Plates. One of the earliest examples of archaeological publishing in Ireland or Britain, a second edition, revised and corrected, with a few additions by the author was issued later the same year.
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The Queen: Portraits of a Monarch exhibition at Windsor Castle features work by many different artists, who all have Queen Elizabeth II as their subject.
For more on this story visit the ITV News Meridian website.
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The plaque at number 16 Cowley Street in Westminster marks the brown brick Georgian townhouse where Sir John lived for 31 years.
A man reportedly armed with several knives has been arrested in Whitehall on suspicion of planning a terror attack, police have said.
Metropolitan Police officer, Tom Harrison, has been crawling the course since the race began on Sunday morning.


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Kelly Kay » Toy Time
It's a no brainer that a naked Kelly Kay gives us a boner. But we were surprised to learn that being nude is also a turn-on for our favorite busty Brit. In fact, wearing her birthday suit excites Kelly so much, she takes matters-and a dildo- into her own hands. And lets us watch!
April 26th, 2018

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Series A-end of the city’s iconic, San Siro/Giuseppe Meazza stadium is on the horizon. Thursday morning will present the city of Milan, and the plans for a new stadium that can be re-shared by both Inter and AC Milan. Among other things, a skeleton in a glass of the new temple, which is good for up to 60,000 seats), referring to the famous Duomo in Milan, italy.
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CANBERRA (Australia) -- Violence has receded in the Solomon Islands capital on Friday, but the government did not show any signs of addressing the...

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The choices available from the main menu will vary depending on the state of the current tune in memory.
Obviously one cannot play/save/alter/view a non-existent tune nor is there any point in playing a tune when one of the channels is empty, nor saving such a tune to tape as machine code.
When the program is first run, or when a machine code copy of a tune has been saved to tape, there will be no tune in memory. At this point a saved tune file may be loaded, or a new tune entered. (The tempo and play shifts may be altered at any time.)
All the main menu options are explained below.
1. Enter a New Tape
On pressing 'I', you will first be asked if you are sure you wish to do this, as any tune already in memory will automatically be deleted by the new tune.
If you press 'N' you will be returned to the main menu without any adverse effects to the current tune; pressing 'Y' will allow you to continue 'entering a new tune'.
After pressing 'Y' you must first select the correct key signature* as instructed. Using the space-bar will allow you to view the current key signature on the staves, so you can select the correct key signature when copying from sheet music even if you don't know C# from F♭ by checking that any #'s or ♭'s are on the same lines and spaces as on the sheet music.
(* See later: Explanation of Musical Terms)
When you have the correct key signature press 'Enter' and again you will be asked if you are sure and may press' N' to re-select a key signature or 'Y' to continue.
When you have chosen a key signature you must select the correct time signature*.
As far as the program is concerned the time signature selected simply alters the bar length*. Hence, for 2/3 us 4/6, for 1/2 use 2/4, for 2/2 use 4/4, etc. Simply press '2', for 2/4, '7' for 7/8, etc. as required. Again you may change your mind in response to the 'Are You Sure' question.
The next thing to do is select the required number of channels or voices by simply pressing the number required. As before, you may change your mind by pressing 'Y' or 'N'.
Now the graphics screen will appear.
On the screen are two full staves, with a small figure in between. This small figure denotes the current channel for note entry and will initially be '1'. Underneath the bottom stave are the note lengths which may be selected. On the quaver is a cursor: this may be moved onto the required note/rest length using the left and right arrow keys. (The #/♭ etc. in the bottom right hand corner should be ignored.)
To enter a note or rest, first of all select the required length by moving the cursor onto the correct length at the bottom of the screen using the arrow keys and then:
a. To Enter a Note
Press 'N' and a cursor will appear on the lower stave. At this point if you think you have selected the wrong length - or if you should have entered a rest - you may press 'Clear' to change your mind.
The cursor on the stave may be moved using the 'up' and 'down' arrow keys until it is on the correct line/space for the current note. (Pressing 'Shift' and cursor up, or 'Shift' and cursor down will move the cursor to the top and bottom of the stave respectively.) Once the cursor is in the correct place you may press '#' for sharp*, '!' for flat*, 'N' for natural*, or 'Enter' if 'none of these' is required.
The note will now appear on the stave with a sharp/flat/natural/nothing before it according to your choice, and you may proceed as described in 'c', altering Note/Rest length'.
b. To Enter a Rest
Press 'R' and a rest of the required length will immediately appear on the stave. N.B. Press 'F' for a full bar rest*. Once the rest has appeared, you may proceed as below.
c. Altering Note/Rest length
If a note or rest is to be dotted* you may now press '.' once to do so, and '.' again if 'double dotting' is required. Once the note has been dotted or not as required, if it is possible to link* it to the previous note on the current channel, press 'L' to do so, or press 'Enter' if it is not to be linked. N.B. Notes/Rests can only be linked until they reach a certain length.
The cursor will now re-appear on the note lengths at the bottom of the screen and the number indicating the current channel will change to that of the next logical channel for input, and you may proceed to enter the next note/rest in the same way. Always refer to the channel number to ensure you are entering the correct note/rest.
When the program is waiting for the next note/rest length, you can:
Enter the next note/rest length as already described.
Press the space-bar to delete the last note or rest entered.
Press 'P' to switch note back back on and off. When there is a 'P' displayed on the right of the note lengths the notes you enter will be played immediately and also when copied to the upper stave.
Press 'Q' to return to main menu.
Press 'B' to force the first Bar position. This is necessary when the first bar of a tune is not a full bar.
2. Load a File from Tape
This allows you to load a tune that has been saved previously using 7. Save the File to Tape. There are three tunes following the main program on the Music Maker tape which may be loaded using this option. As soon as you press '2' the motor and audio will be switched on to allow you to position the source tape at the start of the tune file to be loaded. Once the tape is in position press STOP on the cassette deck.
Type in the required filename (max. 8 characters). If you just want to load the next tune file on the tape just press 'Enter' without typing a file name. You may type 'QUIT' as a file name if you decide not to load a file.
After entering a file name you may change it by answering 'N'.
After pressing 'Y', ensure the tape is in the correct place and that the remote if you have one is plugged into the cassette deck and then press a key and press PLAY on the cassette deck.
If you do not have a remote, be ready to press STOP on the cassette deck as soon as your Dragon stops loading.
The question regarding the key signature is explained later, and normally you should answer 'Y'.
Once all the file has loaded you will be asked from which bar you wish to view the loaded tune: the more bars you choose to view, the longer you will have to wait before you can continue.
Whenever the top stave is full the program will wait for a key-press before carrying on.
Once all the tune has, been displayed, again the program will wait for a key-press and 'then. you will be able to add to the tune if you wish or press 'Q' to return to the menu.
3. Change the Tempo
This allows you to change the speed at which the tune is played: the greater the value, the slower the tune.
N.B. When a tune file is loaded the tempo is set to that at which the tune file was saved.
4. Change the Play Shift
The notes on a channel will be changed by the value of the Play Shift for that channel before they are played (using Option 8) or saved to machine code (using Option 9).
This allows notes to be played which cannot be typed on the display staves.
The value of the Play Shift indicates the change - in semitones* - from the note that is actually displayed.
up one octave
down one octave
down two octaves
5. Alter the Tune
On pressing '5', the second menu will appear; this is explained below:
i. Add to the Current Tune
This allows, you to add more notes to the current tune in the manner already described under Main Menu section 1.
ii. Delete all Notes to the Start of the Current Bar
This deletes the notes on all chahnels to that start of the current bar. If the tune is at the start of a bar, this option will delete all the previous bar.
iii. Alter a Particular Note
This allows you to change the pitch* of an individual note, to change a note to a rest, or to change a test to a note.
You must first select which channel you require and then proceed as instructed.
iv. Transpose the Tune
This allows you to shift the current tune into a new key and automatically changes the key signature as necessary.
Since any change in key may be achieved by moving the tune up or down in pitch you will usually be given a choice of up or down for the transposition. You may move each channel separately.
In order to ensure that all the notes in the tune can still be displayed on the staves it will sometimes not be possible to move a channel up or down in which case the transposition cannot be achieved. In some cases one of the channels can only be moved in one direction in which case this will be done automatically. As the channels are transposed you will be informed as to the direction in which each is altered.
v. Repeat a Section of the Tune
This allows sections of the current tune already in memory to be 'added to the end of the tune. Any number of whole bars from anywhere in the current tune may be repeated in this way, including the whole tune.
N.B. Using Option iii (Alter a Particular Note), is better than using the space-bar to delete an incorrect note/rest as long as the length of the required note/rest is the same length as the incorrect note/rest.
6. View the Tune
This allows you to view the tune from the start of a selected bar, pausing to wait for a keypress whenever the top stave is full and when all the tune has been output to the stave.
7. Save the File to Tape
The current tune may be saved to tape using this option so that it can be reloaded using Option 2, 'Load a File from Tape', and subsequently altered.
Positioning of the save tape and giving a file name is done as described for Option 2.
After pressing 'Y' to indicate that the file name is correct it is preferable to remove the remote from the cassette deck and then press 'Play + Record' on the cassette deck before pressing a key to save the tune file to tape.
8. Play the Tune
After pressing 8, the notes will be altered by the relevant play shifts and then the tune will be played.
When you press' N' to return to the menu, any alterations made using the play shifts will be removed before returning.
9. Save the Tune to Tape
This option allows you to save the tune to tape as a standalone machine code routine which may be reloaded using CLOADM, and EXEC'ed from the normal basic.
First of all you will be asked if you are sure you wish to do this since it will result in the tune in memory being deleted after it has been saved. For this reason it is a good idea to save the tune file to tape using Option 7 before using this Option.
Since before the tune is saved the notes are altered by the relevant play shifts you should also check that the play shifts are correct by playing the tune using Option 8 before saving the tune using this Option.
Position the tape, enter the filename, and save the tune as in Option 7.
While the machine code is being saved, the start, end and exec addresses will be given. It is useful to make a note of the start address as this is different for different tunes.
The end and exec addresses are always the same, being 32,767 and 32,159 respectively.
Pressing '0' on the main menu will result in a 'keybeep' being toggled on and off in turn. This is useful if you like a more positive reaction to pressing keys, or if you find the auto-repeat a bit too fast when entering a tune, etc.
The three tunes following the main program require a change in the play shifts in order to be played correctly.
The first two -'IMNTSMGD' and 'SLMBRSNG' require a play shift of -12 on channel 4 and the other 'HGRNMRCH' requires a play shift of +12 on channel l and -12 on channel 4.
Explanation of Musical Terms re: Music Maker
a. Key Signature
The key signature is the arrangement of sharps or flats in front of the time signature.
A sharp or a flat on a line or space alters the pitch of all notes in that position by a semitone. A sharp causes the pitch to be made higher; a flat causes the pitch to be made lower.
When there are no sharps or flats as the key signature, the key represented is that of C. This can be said to be the fundamental key and in this-case the lines and spaces represent C~ D, E, F, G, A and B in turn. There are other notes, which are:
C# or D♭
D# or E♭
F# or G♭
G# or A♭
A# or B♭
These may be obtained in the key of C by putting a sharp or flat in front of the note concerned.
In keys other than C there will be one or more sharps or one or m ore flats as the key signature. In these cases the arrangement of notes represented by the lines and spaces will be different. e.g. A sharp on the line/ space representing C means that all notes on all lines/spaces representing C are C♭.
In the cases where there is no sharp or flat between two fundamental notes, e.g. between B and C, the addition of a sharp on the lower note or a flat on the higher note results in a change in the fundamental note, e.g. B# is C and C♭ is B.
The sharps or flats in a key signature may be ignored for a particular note by putting a natural in front of that note. This results in the note concerned being the fundamental note of that line or space (as if the key were C).
b. The Time Signature
The time signature in Music Maker is as already stated solely concerned with bar length.
Time signatures appear as two figures on the stave. The lower figure indicates the note length that is concerned. The different values of this figure and their meanings are given below:
'1' indicates the length of a semibreve
'2' indicates the length of a minim
'4' indicates the length of a crotchet
'8' indicates the length of a quaver
'16' indicates the length of a semi-quaver
'6' indicates a length equal to 2/3rds of a crotchet
The upper figure indicates the number of times the length represented by the lower figure occurs in a whole bar. e.g. 3/8 indicates three quavers in a whole bar; 5/6 indicates a bar length of three and one third crotchets, etc.
c. Bar Length
The bar length as just mentioned is a direct result of the time signature and is the length of the section of tune displayed on the stave between two bar lines. The bar lines are the solid vertical lines dividing the tune into sections.
d. Sharps, Flats and Naturals
These are the symbols used to alter the 'usual' pitch of notes. (See (a) above). As well as in the key signature, however, they can be used to alter particular notes: in this way a note may be made sharp or flat if there is no sharp or flat for that note in the key signature.
A natural is used to cancel any sharp or flat in the key signature for a particular note.
e. Full Bar Rest
A Full bar rest is - as might be expected - a rest lasting for the length of a full bar. Thus for tunes with different bar lengths, full bar rests will also vary in length. This is why they are entered in a different manner from the fixed length rests.
f. Dotting Notes
A dot following a note or rest indicates that the note/rest is to last half as long again as normal. e.g. a dotted minim will last for 1 1/2 times the length of a minim, i.e. the length of a minim plus the length of a crotchet.
A double dotted note/rest is to last three quarters as long again as normal in a similar manner to that above.
g. Linked Notes
Linked notes and rests are to last, without break, for the total length of the linked notes/rests.
N.B. It follows that to link two notes, they must be of equal pitch and notes or rests to be linked must be consecutive.
If a note/rest is to last part the end of a bar without break, the section of that note/rest in the second bar must be linked to the length at the end of the first bar.
In Music Maker, the maximum length which can be reached by linking notes/rests is equal of a semibreve + a crotchet + a semi-quaver + a semi-demi-semi-quaver.
h. An Octave
An octave is an interval of pitch equal to 12 semitones.
As stated earlier, there are 12 different notes:
The next note in the sequence is C, and this C is said to be an octave higher than the C at the start of the sequence, The notes with sharps/flats correspond to the black notes on a piano.
The pitch of a note is the height or depth of the note. The letters used and sharps/flats indicate the pitch of notes.
Entering a Tune from Sheet Music
There are a few differences between sheet music and Music Maker. Essentially these are:
Repeated sections of a tune are always repeated in the display on Music Maker as opposed to the indications sometimes used in sheet music.
There is a limit to the highest/lowest notes which can be typed in and displayed on Music Maker. If any notes for a voice/channel in the sheet music are too high to be reached on the Music Maker display all notes for this voice/channel should be typed in an octave lower than on the sheet music. To play this voice/channel correctly it is necessary to alter the play shift for the voice/channel to '+12'.
Similarly, if any notes for a voice/channel in the sheet music are too low for the Music Maker display, all the notes for that voice/channel must be entered an octave higher. This voice/channel can then be played correctly by altering its play shift to '-12'.
Sharps, flats and naturals, other than sharps or flats in the key signature, last only for the note they precede in Music Maker as opposed to lasting to the end of the bar or until changed as in sheet music. Thus sharps/flats/naturalised notes must always have the relevant sharp/flat or natural added in Music Maker.
Certain time signatures are found in sheet music that differ from those available in Music Maker.
A time signature from Music Maker must be used wherever possible which results in the same bar length as in the sheet music.
In sheet music notes/rests played 'together are displayed' vertically together. In Music Maker notes played together are offset slightly to avoid confusion as to the voice/ channel and separated from the previous and following notes by vertical dotted lines.
There is a maximum of four voices/channels for Music Maker. In cases where more voices/channels appear on the sheet music it is better to avoid voices/channels which appear to duplicate any already being used and to choose those with shorter notes (more active ones). Always choose the voice/channel with the highest pitch and usually it is better to also use the one with the lowest pitch.
There is often extra notation, above, to the side of, in the middle of, the staves in sheet music; since none of this can be used in Music Maker, it must be ignored. (Except in cases of pitch.) Apart from the differences in notation between sheet music and Music Maker, the following advice might be useful.
Never have exactly the same pitch of note being played on two channels at the same time. If possible put one in an octave below the other; if not put in a rest for the required length of note in place of one of the notes.
When using one channel only in Music Maker always type in the melody from the sheet music (the highest notes). Shown below are the lengths of rests as they generally appear in sheet music alongside the note length to choose to get the correct length of rest:
vii. Full bar rest
The lengths shown in Music Maker with a small figure '3' nearby are those which must be used to make up triplets. In sheet music triplets are normally shown as below:
and double triplets (made up of six notes) may appear similarly but with a figure six rather than a figure 3 nearby.
In the third triplet above the double bar indicates each note is 1/3 of a quaver in length rather than a third of a crotchet as in the
first two triplets.
Enter each note of a triplet using the relevant third of a crotchet or third of a quaver lengths as normal notes on the relevant voice/channel.
N.B. Occasionally a rest is omitted on a channel in sheet music. When this occurs you must put in a rest of the correct length in Music Maker.
Explanation of the question regarding the original key signature (When a tune file is loading)
A tune file may be loaded without its original key signature if you wish, by answering 'N' in response to this question. The key signature used on the display will be that used for the previous tune in memory.
You may select the key signature using which you wish to display a tune before loading the tune file. To do this, first select Option 1 from the main menu and choose the key signature you require. You may then select any time signature and any number of channels as these are irrelevant. When the staves appear, immediately press 'Q' to return to the main menu and then load the tune file. If you answer the question regarding the key signature with 'N' the key signature you selected before loading the file will be used to display the tune.
Quick Reference Guide
1. Entering a Tune
i. Cursor on note lengths
Full bar test
Toggle playback on/off
Quit (back to main menu)
Delete last entry
forces the first bar line position
ii. Cursor on the stave
Cursor up one
Cursor down one
Shift + ↑
Cursor to top
Shift + ↓
Cursor to bottom
iii. Note or rest on the stave
To dot note/rest
To link the note/rest
to continue to the next note'rest
2. Use 'QUIT' as a file name to return immediately to the menu when loading or saving a file.
3. The value of lines/spaces in the key of C are below:
4. The following table gives the major chords.
5. Using '/' and '/' when altering a value on Music Maker instead of '-' and '-' will alter the value in tens instead of units.
6. The note/rest lengths displayed on Music Maker are related as below:
7. Use a playshift of -12 on channel 4 for "I'm into something good" and "Slumber Song" and playshifts of -12 on channel 1 and -12 on channel 4 for "Hungarian March".
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