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The finding that many chemotherapeutic agents have immunostimulatory effects has provided the impetus to combine chemotherapy and immunotherapy for synergistic antitumor effects. However, the critical determinants of effective antitumor immunity after chemotherapy have not been defined. Here we report that adoptive transfer of tumor-specific CD4+ T cells after chemotherapy with cyclophosphamide gave rise to polyfunctional CD4+ effector cells, which in turn intensified the inflammatory milieu and enhanced the activation of CD8+ T cells in the tumor microenvironment. Although this combined chemoimmunotherapy initially resulted in progressive regression of advanced B-cell lymphoma, its therapeutic efficacy was not durable and most mice succumbed to late relapse. Notably, relapse was associated with acquisition of a tolerized phenotype in tumor-specific CD4+ T cells, characterized by overexpression of program death-1 (PD-1). Remarkably, effective antitumor immunity was maintained and cure became prevalent when polyfunctional CD4+ effector cells were prevented from undergoing PD-1–mediated tolerization, either by antibody blockade of the PD-1–PD-L1 pathway, or targeted ablation of PD-1 in tumor-specific CD4+ T cells. Our study suggests that tumor-reactive CD4+ T cells act as the "gatekeepers" of the host antitumor immunity in the postchemotherapy setting, thereby their functional status governs the choice between eradication versus regrowth of residual tumors.
Chemotherapy is a major treatment modality for many types of cancers. Chemotherapeutic drugs are chosen for their cytotoxicity toward cancerous cells. Nevertheless, increasing numbers of anticancer drugs have been found to exert immune enhancing effects through inducing various forms of immunogenic tumor cell death, which leads to enhanced antigen processing and presentation.1 In addition, some anticancer drugs can mitigate tumor-induced immunoregulatory mechanisms by depleting T regulatory cells (Tregs) or myeloid derived suppressor cells (MDSCs).2,3 Moreover, certain agents can cause transient lymphopenia,4 along with induction of a wealth of growth factors, resulting in robust expansion and survival of tumor-reactive immune cells.5 These findings have led to a growing recognition that elicitation of the endogenous host immunity may intrinsically contribute to the efficacy of some widely used antineoplastic agents.1,6 However, although standard chemotherapy can reduce the symptoms of cancer, in adult malignancies it is rarely curative. Tumor relapses often occur in patients after the initial treatment, suggesting that the endogenous antitumor immunity elicited by chemotherapy is not sufficient to provide durable therapeutic benefit.
It is generally believed that by somehow "resetting" the tumor microenvironment, chemotherapy creates a window of opportunity for therapeutic immune interventions. There is accumulating evidence that incorporation of chemotherapy with immunologic maneuvers, such as adoptive immunotherapy and therapeutic vaccines, can effectively treat established tumors.6 In this regard, cyclophosphamide (Cy), a prototypical alkylating agent widely used in cancer treatment, has been shown to be particularly effective in augmenting the effectiveness of adoptive T-cell therapy (ACT) in various animal models and some clinical studies.7-10 A great deal has been learned about the acute effects of Cy on potentiating CD8+ T cells, enhancing antigen presentation, and reducing/inactivating Tregs.11,12 However, to date the impact of Cy on tumor-reactive CD4+ effector T cells has received little attention. Understanding the role of CD4+ T cells in the postchemotherapy period has direct clinical relevance, because one consistent observation from human ACT trials is that beneficial clinical responses are often associated with the presence of tumor-reactive CD4+ T cells.10,13 Cy is a common component of the preconditioning chemotherapy regimens used in many ACT protocols, including recent clinical studies on treating B-cell malignancies with autologous T cells engineered to express a chimeric antigen receptor (CAR).14-16 Particularly, a phase 1 study conducted by Sadelain and colleagues in patients with chronic lymphocytic leukemia (CLL) demonstrated the therapeutic potential of the combination of Cy-treatment and CD4-dominant T-cell therapy.16 These studies achieved encouraging patient outcomes, although the long-term efficacy of this treatment strategy remains to be evaluated in a larger patient population.
In this study, we aimed to gain mechanistic insight into the role of CD4+ T cells in the postchemotherapy setting. Using a biologically relevant preclinical model in which mice with advanced B-cell lymphoma were treated with Cy followed by adoptive transfer of tumor-specific CD4+ T cells, we demonstrated that CD4+ T cells with a polyfunctional effector phenotype were not only critical for mounting robust antitumor immune responses that led to continuous tumor regression after chemotherapy, but also essential for maintaining a protective immunity against tumor recurrence.
Female BALB/c mice of 4 to 6 weeks old were purchased from the National Cancer Institute. TCR transgenic mice on a BALB/c background expressing an αβTCR-specific for amino acids 110 to 120 from influenza hemagglutinin (HA) presented by major histocompatibility complex (MHC) class II molecule IAd were originally generated in the laboratory of Dr H. von Boehmer (Harvard Medical School). Thy1.1+/+ HA-TCR Tg mice were generous gifts from Dr Hyam I. Levitsky (The Johns Hopkins University School of Medicine). The PD-1KO mice on a BALB/c background (PD-1-KO-N12[BALB/c]), generated in the laboratory of Dr Tasuku Honjo,17 were purchased from RIKEN BioResource Center. Thy1.1+/+ PD-1KOHA-TCR Tg mice were obtained by crossing the 2 transgenic strains. All mice were housed under specific pathogen-free conditions by Laboratory Animal Services (LAS) of the Georgia Health Sciences University (GHSU). Rag2−/− mice on BALB/c background were purchased from Tarconic. All animal experiments were approved by the Institutional Animal Care and Use Committee of GHSU.
Antibodies and reagents
Detailed descriptions of reagents are given in supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article).
Tumor cells and animal tumor models
The generation and maintenance of the wild-type (WT) murine B-cell lymphoma cell line A20 (A20WT) and its derivative cell lines A20HA were previously described.18 A20 cell line expressing granulocyte macrophage colony-stimulating factor (GM-CSF; A20GMCSF) was a gift from Dr Hyam I. Levitsky. Colorectal tumor cell line CT26, purchased from ATCC, was engineered to express HA (CT26HA). For T-cell adoptive transfer, spleens, and lymph nodes from specified Tg mice were harvested, homogenized, lysed by ACK lysing buffer (Invitrogen) to remove red blood cells. Cells were enriched for CD4+ T cells by magnetic-activated cell sorting (Miltenyi Biotec), and labeled with 2uM carboxyfluorescein diacetate succinimidyl ester (CFSE). The percentage of lymphocytes positive for CD4 and the clonotypic TCR (monoclonal antibody 6.5 for HA-specific CD4+ T cells) was determined by flow cytometry. A total of 2.5 ∼ 3 × 106 CD4+ TCR+ T cells were injected intravenously into each recipient.
A20HA tumor cells were inoculated to mice via 2 different routes as specified in study. When injected via tail vein (1 × 106 per mouse), A20HA tumors systemically disseminated to secondary lymphoid tissues, and infiltrated the liver and bone marrow at late stages, closely mimicking human B-cell malignancy.19 By contrast, subcutaneous injection of A20HA (5 × 106) on the hind flank led to localized tumor growth. CT26HA tumors (0.5 × 106) injected to mice via tail vein exclusively localized to the lungs. For subcutaneous A20HA tumors, tumor growth was monitored by caliper measurement of the tumor area every 3 days, and expressed as the product of 2 perpendicular diameters in square millimeters. Mice were euthanized when tumor size reached 400 mm2 or when tumor sites ulcerated. For systemic A20HA tumors, the experimental endpoints for survival studies were limb paralysis, appearance of ascites, and death. Long-term survival was defined as no sign of tumor presence and no symptom of sickness by day 40 after treatments. Cures were defined as no abnormalities on anatomical inspection of mice that have met the criteria for long-term survival.
Combinatorial chemoimmunotherapy
Details of the treatment procedures are given in supplemental Methods.
Tumors were flash-frozen in optimal cutting temperature solution (Sakura). Sections (8 μm) were cut with a microcryotome (Leica), fixed for 10 minutes in cold acetone, and stained with rat anti-CD8 antibody (clone 53.67). After wash, sections were stained with Dylight 488–conjugated donkey anti–rat IgG (Jackson ImmunoResearch Laboratories) and DAPI, and analyzed on an Axio Observer inverted microscope (Zeiss) under a 20× objective.
Cell preparation and flow cytometry analysis
Detailed methods are given in supplemental Methods.
Quantitative real-time PCR
Detailed methods are given in supplemental Methods.
Statistical analysis
Data were analyzed using Prism 4.0 software (GraphPad). The statistical significance of the results was determined using the Student t test. Data for tumor survival were analyzed using a log-rank test. P values less than .05 were considered statistically significant.
Chemotherapy with Cy drives a productive CD4+ T-cell response among adoptively transferred tumor-specific CD4+ T cells that otherwise would be tolerized
We recently reported that chemotherapy with Cy can potentiate antitumor CD4+ T-cell responses by driving the effector differentiation of adoptively transferred tumor-specific CD4+ T cells.9 In this study, we sought to identify the immunologic parameters that can reflect the functional status of tumor-specific CD4+ T cells in the postchemotherapy setting. Figure 1A shows that Cy-treatment of mice bearing systemic A20HA tumors led to a sharp increase in the frequency of the transferred HA-specific CD4+ T cells, accompanied by enhanced cell division. Correspondingly, there was substantial accumulation of highly divided tumor-specific CD4+ T cells in Cy-treated mice (Figure 1B). In addition, the divided CD4+ T cells in Cy-treated versus untreated mice showed marked differences in their phenotype and functionality as measured by a panel of selected phenotypic and functional markers. Among these markers, sustained PD-1 expression is associated with clonal exhaustion in CD8+ T cells,20 and Foxp3 is the hallmark of CD4+ regulatory T cells21 ; thus prominent expression of these molecules would be suggestive of a defective immune response. On the other hand, CD40L and Th1-type cytokines (such as IFNγ, TNFα, and IL2) are associated with CD4+ effector/helper activities,22,23 and CD127 (IL7R) expression correlates with effector to memory transition;24 thus expression of these molecules would imply a productive immune response. After adoptive transfer, tumor-specific CD4+ T cells in mice without chemotherapy exhibited a defective response signature, characterized by high levels of PD-1 and Foxp3, low levels of CD127 and CD40L, and a paucity of Th1-type cytokines [Figure 1C (−)Cy]. By contrast, pretreatment of the hosts with a single dose of Cy had profound beneficial effects on the transferred tumor-specific CD4+ T cells, resulting in reduced levels of PD-1 and Foxp3, but pronounced expression of CD127 and CD40L [Figure 1C (+)Cy]; and importantly, these cells acquired polyfunctionality—the ability to simultaneously produce multiple proinflammatory cytokines including IFNγ, TNFα, and IL2.
Of note, we have previously shown in this model that polyfunctional CD4+ effector cells do not arise if tumor cells do not express the cognate antigen,9 suggesting the importance of chemotherapy-induced antigen release. In addition to releasing tumor antigens by directly killing tumor cells, Cy used at certain doses can also cause lymphopenia, which can drive homeostatic proliferation of T cells.5,11 To dissect the relevance of antigen provision and lymphopenia, we conducted a series of experiments in which A20HA-bearing Rag2−/− mice that had received HA-specific CD4+ T cells were or were not subsequently immunized with irradiated A20HA tumor cells (supplemental Figure 1). The rationales for the experimental design were that Rag2−/− mice would provide a lymphopenic environment, and irradiated tumor cells would die in vivo and thus release antigens. The conclusion of this set of experiments was that the mere combination of lymphopenia and tumor cell death did not recapitulate the robust CD4-potentiating effects of Cy. Our results support the notion that the potent stimulatory effects of Cy on adoptive T-cell therapy is the combined result of its multiplex immunomodulating properties, including mitigation of immunosuppression and creation of an immunogenic milieu, in addition to inducing tumor cell death and lymphopenia.
The immune-potentiating effects of Cy on tumor-specific CD4+ T cells are applicable to MHC-II–negative tumors
Of B-cell origin, A20 tumor cells can potentially act as APCs for CD4+ T cells,25 raising the question whether the observed CD4-potentiating effects of Cy were unique to the lymphoma model. To address this, the same experimental procedures used in the A20HA lymphoma model were applied to a colorectal tumor model in which HA-expressing CT26 tumors (CT26HA) were inoculated to mice via tail vein. Analogous to the results seen in the A20HA tumor model, Cy significantly enhanced the expansion of the transferred CD4+ T cells in the draining lymph node and spleen (Figure 1D), and promoted the generation of polyfunctional CD4+ effector cells, marked by elevated CD40L expression and the ability to produce multiple proinflammatory cytokines (Figure 1E). Similar results were obtained in mice bearing HA-expressing renal cell or mammary carcinoma, RencaHA, or 4T1HA, respectively (data not shown). It is important to note that CT26 tumor cells, unlike A20 tumor cells, do not constitutively express MHC II molecules, and cannot be induced to express MHC II by IFNγ (Figure 1F). Collectively, the data indicate that the immune-potentiating effects of Cy on CD4+ T cells are not restricted to hematologic malignancy, but also applicable to solid tumors that cannot directly present antigens to CD4+ T cells.
CD4+ effector T cells significantly contribute to reprogramming of the immune milieu in the tumor microenvironment after chemotherapy
There is accumulating evidence that CD4+ T cells are critically involved in modulating the immune milieu in the tumor microenvironment.26-28 Given the robust CD4+ T-cell response after the combination of Cy-treatment and CD4+ T-cell transfer, we hypothesized that activated CD4+ effector cells might play a significant role in conditioning an immunogenic tumor milieu after chemotherapy. To facilitate the study of intratumoral milieu, A20HA tumors were subcutaneously inoculated to the flank of a mouse where they grew into a localized tumor mass. Mice with large (∼ 170 mm2) established A20HA tumors were treated or not treated with Cy, followed by adoptive transfer of HA-specific CD4+ T cells the next day (Figure 2 schema). Replicating the results seen in the systemic tumor model shown in Figure 1, Cy treatment led to vigorous expansion of the transferred tumor-specific CD4+ T cells in the tumor mass, and these infiltrating CD4+ T cells exhibited a polyfunctional effector phenotype with the signature expression of CD40L and proinflammatory cytokines including IFNγ and TNFα (Figure 2A). We reasoned that the prominent expression of CD40L and IFNγ by tumor-specific CD4+ T cells might lead to augmented activation and maturation of APCs. Supporting this notion, tumor-infiltrating CD11c+ dendritic cells (DC)s in mice receiving Cy plus CD4+ T-cell transfer had increased expression of CD40 and B7.1 compared with their counterparts in mice receiving Cy alone (Figure 2B). In mice not receiving chemotherapy (untreated and CD4+ T-cell transfer only), CD11c+ DCs were barely detectable in tumors (data not shown), suggesting that chemotherapy promotes tumor infiltration by DCs.
To assess the tumor milieu, tumor masses were resected from mice and assayed for the expression profiles of inflammatory cytokines and chemokines using quantitative real-time PCR. The transcripts of a panel of genes tested, including IL6, IL1β, IL17, CXCL10/IP10, and GMCSF, were low or undetectable in tumors recovered from untreated mice or mice that received CD4+ T-cell transfer only (Figure 2C). In line with published studies,29,30 Cy-treatment increased the transcripts of these inflammation-related genes (Figure 2C Cy only). Addition of tumor-specific CD4+ T cells after Cy further boosted the transcripts of these genes (Figure 2C Cy/CD4). Of note, IL17 protein was detected in a subset of donor CD4+ T cells on antigenic restimulation (supplemental Figure 2A), suggesting that the transferred tumor-specific CD4+ T cells contribute to elevated IL17 production in vivo. Taken together, these data suggest that Cy-treatment fosters a proinflammatory tumor milieu, and tumor-specific CD4+ effector cells can significantly reinforce this effect.
CD4+ effector T cells provoke functional maturation of intratumoral CD8+ T cells with which they collaborate to markedly enhance the efficacy of chemotherapy
We next asked whether the presence of CD4+ effector cells would augment the recruitment and function of CD8+ T cells in tumor. In mice receiving no treatment or CD4+ T-cell transfer only, immunofluorescence (IF) staining of the tumors revealed that there was only scarce presence of endogenous CD8+ T cells; however, the tumors became heavily infiltrated by CD8+ T cells after Cy treatment, regardless of CD4+ T-cell transfer (Figure 3A). FACS analyses confirmed the observations of IF, and further demonstrated that the frequencies of tumor-infiltrating CD8+ T cells were comparable in mice that either received Cy only or Cy plus CD4+ T-cell transfer (Figure 3B). However, notably the intratumoral CD8+ T cells in mice that received Cy plus CD4+ T-cell transfer exhibited enhanced functional maturation, as reflected by the heightened levels of granzyme B and IFNγ, compared with their counterparts in mice that received Cy only (Figure 3C).
The copresence of the donor CD4+ T cells and the endogenous CD8+ T cells in the tumor lesion prompted us to determine their relative contribution to antitumor efficacy. Using tumor growth rate as a functional readout, Figure 3D shows that CD4+ T-cell transfer alone did not alter the tumor growth rate compared with untreated mice (CD4 only versus no treatment). When chemotherapy was given alone, tumor growth was briefly arrested but resumed shortly (Cy only). By contrast, the combination of Cy and CD4+ T-cell transfer (Cy/CD4) led to progressive regression of large established tumors over a period of 3 weeks. To evaluate the contribution of CD8+ T cells to tumor rejection, mice were injected with CD8-depleting antibody before receiving Cy plus CD4+ T-cell transfer. In the absence of the endogenous CD8+ T cells, the combination of Cy and CD4+ T-cell transfer was less effective in mediating tumor regression (Figure 3D CD8 depleted, Cy/CD4), suggesting the involvement of the host endogenous CD8+ T cells in tumor rejection. The results indicate that adoptive transfer of tumor-specific CD4+ T cells augments the efficacy of chemotherapy, and that the optimal antitumor effect of this chemoimmunotherapy requires the contribution of the endogenous CD8+ T cells.
Polyfunctional CD4+ T cells can be converted into a PD-1–dominant dysfunctional state in a tumor environment
Chemotherapy can often reduce the symptoms of cancer, but it is rarely curative. Given the pivotal role of tumor-specific CD4+ effector cells in postchemotherapy hosts, it was important to know whether these cells could maintain their phenotype in the face of persistent residual tumors. To address this, we asked whether polyfunctional CD4+ effector cells were susceptible to tolerization after being re-exposed to tumors. We used CD40L as a marker for polyfunctional CD4+ effector cells. This was based on our observations that intracellular CD40L (intraCD40L) was abundant in highly divided tumor-specific CD4+ T cells recovered from tumor-bearing mice that received Cy plus CD4+ T-cell transfer (Figure 4A); and CD40L was rapidly mobilized to the surface (sCD40L) and primarily associated with IFNγ expression after a brief antigenic restimulation in vitro (Figure 4B). The highly activated CD4+ effector cells (CFSElow sCD40Lhigh) were isolated by FACS-sorting, and reinfused into mice bearing 10-day-old tumors (Figure 4C schema). Two weeks after T-cell transfer, the input cells lost expression of CD40L and CD127, became predominantly PD-1+ and acquired Foxp3 expression, manifesting a complete conversion from the effector cell signature to the defective cell signature (Figure 4C). The results demonstrate that highly activated CD4+ effector cells are at risk of being tolerized by residual tumors that persist after chemotherapy.
Tumor relapses after chemoimmunotherapy correlate with loss of the polyfunctional effector phenotype in tumor-specific CD4+ T cells
We next asked whether late relapse would occur in mice after receiving the combined chemoimmunotherapy, and if so, whether it would be associated with tolerization of the activated antitumor CD4+ T cells. We knew from Figure 1 that virtually all transferred CD4+ T cells initially became activated in Cy-treated tumor-bearing mice. When these mice were followed long-term after treatment, however, most mice eventually relapsed and died, and only a small cohort of mice (∼ 20%) achieved long-term survival or cure (Figure 5A). Serial analysis of peripheral blood revealed that tumor relapses correlated with a steep decrease in the frequencies of the transferred CD4+ T cells in circulation (Figure 5B). Furthermore, tumor-specific CD4+ T cells in relapsed mice lost the effector-cell signature and acquired the defective-cell signature (Figure 5C). Particularly, acquisition of PD-1 expression in these CD4+ T cells was accompanied by loss of cytokine-producing capability (Figure 5D). These changes in donor CD4+ T cells were also accompanied by loss of function in host CD8+ T cells in relapsed mice (Figure 5E). By contrast, mice that remained in remission (or cure) had a stable population of the transferred CD4+ T cells that continued to exhibit the effector cell signature (Figure 5B-D), and maintained a pool of responsive endogenous CD8+ T cells with memory attribute (more responsive than naive CD8+ T cells on challenge as shown in Figure 5E). Collectively, these data demonstrate that tumor relapses after chemoimmunotherapy correlate with acquisition of a PD-1–dominant dysfunctional phenotype in tumor-specific CD4+ T cells.
PD-1 blockade after chemoimmunotherapy markedly reduces the occurrence of relapse
Given the prominent expression of PD-1 in tolerized CD4+ effector cells, we hypothesized that disruption of the PD-1 pathway after chemoimmunotherapy can invigorate antitumor CD4+ T cells and consequently prevent tumor recurrence. To test this, mice with localized large A20HA tumors were treated with Cy plus CD4+ T-cell transfer, and then were injected or not injected with a mixture of blocking antibodies targeting PD-1 and PD-L1 (Figure 6A schema). Despite a period of progressive tumor regression, relapses were common (11 of 13) in mice receiving chemoimmunotherapy without subsequent PD-1/PD-L1 blockade (Figure 6A Cy/CD4). The relapsed tumors still expressed the target antigen HA (supplemental Figure 3), suggesting that relapses were not because of selective outgrowth of antigen-loss variants. By contrast, addition of PD-1/PD-L1 blockade after chemoimmunotherapy dramatically reduced relapse occurrence (1 of 9; Figure 6A Cy/CD4/αPD1+αPDL1 antibodies). As controls, mice treated with the combination of Cy and PD-1/PD-L1 blockade all had rapid tumor regrowth (Figure 6B Cy/αPD1+αPDL1 antibodies), indicating that PD-1/PD-L1 blockade by itself had minimal effects on residual tumors.
We next sought to confirm the findings of the preceding experiments, which employed surrogate tumor antigen and naive TCR Tg CD4+ T cells, in a more stringent experimental setting in which tumor-reactive, polyclonal CD4+ T cells derived from the endogenous repertoire were used to target physiologic tumor antigens. As shown in the Figure 6C schema, CD4+ T cells reactive to A20 tumor–derived endogenous antigens (not transgene HA) were obtained from the draining lymph nodes of tumor-free mice that had been vaccinated with irradiated GMCSF-producing A20 tumors (GVAX); these polyclonal CD4+ T cells were expanded to sufficient quantities in vitro, and then adoptively transferred to Cy-treated tumor-bearing mice, with or without subsequent PD-1/PD-L1 blockade. The tumor-reactivity of the vaccine-primed CD4+ T cells (GVAX-CD4) was documented by their capability to produce IFNγ on incubation with A20 tumors in vitro (Figure 6C dot plots). It is noteworthy that A20HA cells were still used as tumor inoculants in this set of experiment, thus the results can be compared with the relevant controls shown in preceding experiments. However, under this experimental setting HA was irrelevant to the antitumor immunity because it was not the target of vaccine-primed CD4+ T cells. Replicating the results seen in experiments using TCR Tg CD4+ T cells, chemoimmunotherapy using polyclonal CD4+ T cells followed by PD-1/PD-L1 blockade led to complete tumor regression and mice remained symptom-free by the time of analysis, whereas chemoimmunotherapy without PD-1/PD-L1 blockade resulted in only a transient remission (Figure 6D). Taken together, our data provide strong evidence that the PD-1/PD-L1 axis critically contributes to immune tolerance and tumor relapse after chemoimmunotherapy.
Targeted ablation of PD-1 in tumor-specific CD4+ T cells results in durable antitumor immunity after chemoimmunotherapy
Blocking antibodies specific for PD-1 or PD-L1 may act on multiple targets, including both CD4+ and CD8+ T cells. To definitively address the role of PD-1 specifically in the CD4 population, we bred our HA-specific TCR Tg mice onto a PD-1–deficient background. Figure 7A shows that adoptive transfer of HA-specific PD-1KOCD4+ T cells after chemotherapy led to dramatically improved therapeutic benefits, achieving a 90% cure rate (Figure 7A) that was comparable with the outcome of PD-1/PD-L1 blockade shown in Figure 6A. Moreover, the cured mice were all protected from subsequent tumor re-challenge (Figure 7B), suggesting the development of memory. In those protected mice, the transferred tumor-specific CD4+ T cells were readily detectable, and retained the polyfunctional effector phenotype (Figure 7C).We further showed that the survival benefit was lost in 4 of 5 mice when endogenous CD8+ T cells were depleted (Figure 7D), suggesting that the curative therapeutic effect conferred by PD1-deficient CD4+ T cells in postchemotherapy hosts still requires the involvement of endogenous CD8+ T cells. Collectively, these results indicate that tumor-specific CD4+ effector cells are key determinants of durable antitumor immunity in the postchemotherapy setting, and their functional status is dictated by cell-intrinsic PD-1 signaling.
Although the concept of combined chemoimmunotherapy for cancer can be dated back to at least 3 decades ago,31 it was only recently that the mechanistic basis for the synergy between these 2 treatment modalities began to be elucidated at the cellular and molecular level.1,6 A successful chemoimmunotherapy should not only result in effective tumor destruction, but also prevent posttherapy relapse. Using a mouse model of posttherapy relapse, the current study provides novel insight into the dynamic interplay between tumors and antitumor CD4+ T cells in the context of chemoimmunotherapy. We showed that the mutually reinforcing effects between chemotherapy and tumor-reactive CD4+ T cells can forge an enhanced antitumor response. Importantly, we demonstrated that an effective and durable antitumor immunity can be achieved after chemotherapy if tumor-specific CD4+ T cells are maintained in the polyfunctional effector state by avoidance of PD-1–mediated tolerization.
It has been shown that activated CD4+ T cells, through CD40L-CD40 interaction, can mediate DC licensing, which is essential for priming and maintaining CD8+ effector cells in tumor microenvironment.32,33 Activated CD4+ T cells also enhance CD8+ T cell–mediated antitumor immunity by exerting important "postlicensing" roles, such as recruiting activated CD8+ T cells to tumor via the action of IFNγ, and promoting CD8+ T cell cytolytic function and proliferation through IL2.27,34 Moreover, CD4+ T cells may act on stromal cells to inhibit tumor angiogenesis through the effects of IFNγ and TNFα.35,36 Activated CD4+ T cells that can concurrently produce multiple proinflammatory cytokines (including IFNγ, TNFα, and IL2) are often referred as polyfunctional CD4+ T cells.23 The polyfunctional CD4+ T cells described in our study coexpressed CD40L and proinflammatory cytokines, endowing these cells with the potential to carry out diverse functions in a highly coordinated and synergistic fashion. Our demonstration of induction of polyfunctionality in transferred tumor-specific CD4+ T cells is potentially clinically relevant, because adoptive T-cell therapy using tumor antigen-specific CD4+ T cell clones has now become a viable treatment option for certain types of cancer.37 Our finding that the curative therapeutic effect conferred by CD4+ T cells in postchemotherapy hosts still required endogenous CD8+ T cells supports the concept that polyfunctional antitumor CD4+ T cells act as gatekeepers to maintain an effective host antitumor immunity, leading to durable remission or even cure.38
The present study confirms previous reports that Cy induces the release of an array of growth factors, inflammatory cytokines, and chemokines, including IL6, IL1β, IL17, CXCL10, and GMCSF, and so on.29,39 Here we showed that IL17 transcripts induced by Cy were further elevated by CD4+ T-cell transfer (Figure 2C), and the transferred tumor-specific CD4+ T cells may contribute to IL17 production in tumor (supplemental Figure 2A). Several recent studies demonstrate that Th17 cells can also be polyfunctional (coexpressing IL17 and IFNγ), exhibit certain memory cell attributes, and collaborate with CD8+ T cells to mediate long-term antitumor immunity.40-43 However, similar to other published studies using Cy treatment,29,39 we did not find significant population of CD4+ T cells that coexpressed IL17 and IFNγ (supplemental Figure 2B), suggesting that Cy may preferentially drive the development of Th1-type polyfunctional effector cells. In addition to IL17, we showed that the presence of activated CD4+ T cells also augmented the levels of IL1β, IL6, and GM-CSF. Intriguingly, these inflammatory molecules can have both beneficial and detrimental effects on antitumor immunity.38,44 Our results, in line with the report by Haabeth et al,28 suggest that tumor-specific CD4+ effector cells may play an essential role in driving therapy-induced inflammation toward tumor inhibition.
Given the importance of tumor-specific polyfunctional CD4+ effector cells, it is alarming that these cells are susceptible to tolerization by persistent residual tumors. Importantly, we identified the PD-1 pathway as a major mechanism mediating tolerization of CD4+ effector cells in the postchemotherapy setting. The therapeutic potential of PD-1/PD-L1 blockade has long been established in preclinical models,45,46 and has been validated in recent clinical trials.47,48 However, the therapeutic significance of PD-1 blockade on CD4+ T cells has been obscure. We showed that PD-1 blockade markedly improved the long-term efficacy of chemoimmunotherapy in the form of Cy-treatment plus adoptive transfer of monoclonal CD4+ T cells specific for a single tumor antigen (HA; Figure 6A). Moreover, the therapeutic benefit of PD-1 blockade also extended to chemoimmunotherapy using vaccine-primed polyclonal CD4+ T cells reactive to physiologic tumor antigens (epitopes undefined; Figure 6B-C). Our data suggest that PD-1 blockade immunotherapy, when used in combination with chemotherapy and adoptive immunotherapy, may sustain tumor-reactive CD4+ effector cells in addition to invigorating antitumor CD8+ T cells, and hence may greatly improve long-term patient outcomes. From an application standpoint, whereas the vaccine-primed CD4+ T cells used in our study were derived from immunized tumor-free donors, we believe that these cells can be obtained from vaccinated tumor-bearing hosts. This notion is supported by some recent clinical studies showing that polyfunctional CD4+ T cells can be induced in cancer patients after vaccination.49,50
We showed that targeted ablation of PD-1 in donor CD4+ T cells dramatically improved long-term survival after chemoimmunotherapy (Figure 7A), providing unequivocal evidence that the key relevant PD-1 expression was on tumor-specific CD4+ T cells. To our best knowledge, our study provides the first evidence that targeted disruption of PD-1 in tumor-specific CD4+ T cells is sufficient to result in sustained antitumor immunity and durable therapeutic outcome in postchemotherapy hosts. Interestingly, we previously showed in the same model that transfer of PD-1–deficient naive CD4+ T cells did not reject tumor in the absence of Cy-treatment, and the transferred cells still became tolerized.9 The results of this study suggest that PD-1 is not involved in the initial tolerance induction in naive CD4+ T cells, but is critical in tolerizing activated CD4+ T cells at the effector phase, thereby profoundly affecting the magnitude and durability of the antitumor immune responses elicited by chemoimmunotherapy.
In conclusion, our study unveils a previously unappreciated pivotal role of CD4+ T cells in controlling host antitumor immunity after chemotherapy, underscoring the importance of tumor-reactive CD4+ T cells in both initiating and maintaining an effective antitumor immunity in postchemotherapy hosts. The findings may have important implications for the design and implementation of more effective anticancer chemoimmunotherapy.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
The authors thank Dr Hyam I. Levitsky and Dr Tasuku Honjo for transgenic mice; and Dr Yong Teng and Dr Lingqian Li for technical assistance.
This work was supported by a Research Scholar Grant (RSG-12-169-01-LIB) from the American Cancer Society and GHSU Special Funding Initiative to G.Z.; R01 CA72669 and P01 AI056299 to B.R.B.; AI065175, the JDRF and the Mason Trust to A.L.M.; and R01 CA096651 to D.H.M.
National Institutes of Health
Contribution: Z.-C.D. performed research and analyzed results; L.H. performed research; H.Y. provided reagents; B.R.B., A.L.M., and D.H.M. contributed reagents, discussed results, and edited the paper; and G.Z. designed and performed research, analyzed results, and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Gang Zhou, Cancer Immunology, Inflammation and Tolerance Program, Cancer Center, Georgia Health Sciences University, 1120 15th St, CN-4140, Augusta, GA 30912; e-mail: email@example.com.


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Location: Chile, Latin America
Operator: Gold Fields Limited
Royalty: NSR: 2%
In early 2019, Franco-Nevada acquired an existing 2% NSR on the Salares Norte project being advanced by Gold Fields Limited ("Gold Fields") in the Atacama region of Northern Chile. The royalty is subject to a 1% buyback by Gold Fields within 24 months of announcing commercial production for $6 million. Salares Norte is a blind epithermal gold and silver deposit and represents one of Gold Fields key development projects. The royalty covers 9 km2 of the Salares Norte mining concession and an additional 12 km2 of concessions at the nearby Piedra concessions.
Gold Fields recently completed a feasibility study for the project which included a maiden Mineral Reserve containing 3.5 million ounces of gold and 39.3 million ounces of silver (21.1 million tonnes @ 5.1 g/t gold and 57.9 g/t silver). The feasibility study envisages an open pit mining operation and an initial mine life of 11.5 years producing 450,000 ounces of gold equivalent per year for the first 7 years. The environmental impact assessment was accepted for review in July 2018 with an 18-24 month review period anticipated. Detailed engineering is underway and Gold Fields estimates that construction could commence in late 2020.


text:
Minesweeper is a nice little puzzle game packed with every version of Microsoft Windows up to Windows 7. When you start the game, first you must select the difficulty: beginner, intermediate or expert. The levels will affect the size of the board (9×9, 16×16, 16×30) and the number of mines (10, 40, 99) respectively. The level is randomly generated. By left-clicking any square on the grid, you will either open a new area, detonate a mine or find a number. That number tells you the number of adjacent mines. Right-clicking places flags where you think there's a mine. Middle-clicking a number with a matching number of flags adjacent to it uncovers all squares adjacent to it that are not flagged, which will set off a mine if even one flag is in the wrong square.
Minesweeper is known to have quite a few little strategies:
- If you have a 1 on a corner, it's a mine. Why? Because there's only one available tile adjacent. Be very careful, because while this technique is useful (and integral), if you fail a spot check and don't see that there's already a mine diagonal to your 1, you will probably die.
- A 2 at the very edge of a wall adjacent to two hidden tiles means they're both mines.
- 3 on a wall: they're all mines.
- If you see the numbers "2 1 2" on a wall, the spaces adjacent to the 2s are both safe and the space adjacent to the 1 is a mine, and similarly if you see "1 2 1", the space in the middle is safe and both spaces adjacent to the 1s are mines. You may intuitively expect "2 1 2", averaging to 5/3, would imply more mines tend to be found adjacent to those three squares and "1 2 1", averaging to 4/3, would imply less, but you would be wrong.
- A generalization of 3 along a wall: If you see two numbers that are adjacent (not diagonal, but sharing a side) to each other and they differ by 3 (such as 4 and 1, or 5 and 2), then all three squares on the other side of the larger number are mines and all three on the other side of the smaller are safe.
The world record for Expert difficulty is currently 31 seconds (previous record).
Examples of tropes used in Minesweeper:
- Anti-Frustration Features:
- You cannot die on the first click of a new game, no matter what. The grid is randomized before your first click, and if that first click happens to be on a mine, it will either randomize the board again (non-Windows versions) or move the mine to the top right corner (Windows versions). Vista and Windows 7 tweak this some more so not only is the first square empty, so are all the adjacent squares. Naturally, this is turned off for when you replay the same layout.
- Super Minesweeper introduces a "Damage" mechanic. Uncovering a mine no longer leads to an instant Game Over but instead adds a penalty to the in-game clock when the game ends after all non-mine tiles are uncovered, the exact amount of time being shown on the sidebar. This was a deliberate design choice as according to the main developer, some board layouts and game variants featured within can became so Nintendo Hard that even the average experienced player would not be able to do a No-Damage Run ala traditional Minesweeper.
- Bomb Disposal: The goal is to isolate mines without setting them off.
- Bowdlerise: As of Windows Vista, you can play Flowersweeper instead, with the mines replaced with flowers. This was done in case countries like Laos, which have lots of unexploded bombs left over from past wars that kill innocent people every year, threw a fit about Minesweeper hitting too close to home; Microsoft could simply make Flowersweeper the default in exports of Windows.
- Classic Cheat Code: XYZZY
- Cool Shades: Your sole reward for winning.
- Easter Egg:
- In the pre-Vista versions, typing in "XYZZY" on the keyboard and then pressing Shift causes the uppermost pixel on the top left corner of the screen to change color depending on where the cursor is. The pixel will be black if it's on a mine, and white otherwise.
- The Vista and 7 versions are fully compatible with Xbox 360 controllers, a feature that isn't described anywhere in help files. The controller even rumbles if you hit a mine.
- Failed a Spot Check: KABOOM!
- Game Mod: There's a lot of clones of this game, including several odd variants including hexagonal Minesweeper (imagine playing it in a beehive) and spherical Minesweeper. It seems in general to be a fairly popular programming exercise, and some version or another is standard on nearly every Linux distribution.
- "Have a Nice Day" Smile: The player's "avatar", in a way.
- Level Goal: One take on the rules is to require the player to guide a character across the board one step at a time. The Power Pro-kun Pocket games had examples of such gameplay since 2 in 2000. Microsoft themselves eventually added it for the Windows 8 version.
- Luck-Based Mission: Happens fairly frequently. See the article's image for an example. Basically, it amounts to knowing exactly how many tiles in a set have mines but not being able to confirm (with perfect accuracy) which ones in that set have mines, despite having cleared out the rest of the board. It's also the reason a perfect (non-cheating) AI doesn't exist for Minesweeper, too. There's also a couple of other reasons a perfect AI doesn't exist, not least the NP-completeness of the problem.
- Nintendo Hard:
- Larger boards with more hidden mines require a larger amount of skill and luck to detect the location of mines.
- Crazy Minesweeper and Super Minesweeper contain modes with medium and large-sized bombs that count as two and three bombs respectively, making it harder to determine which tiles contain mines in a given area.
- One-Hit-Point Wonder: Click a tile with a mine under it and the game is over. Averted in Super Minesweeper thanks to bombs being reconfigured to not immediately end the game.
- Race Against the Clock: That clock will keep ticking until it reaches 999."What happens then?"
"Nothing. You just suck."
— Minesweeper The Movie Trailer
- Random Number God: At some point, you're probably going to have to guess. Better hope the god is on your side, or you can lose ten minutes of cautious probing.
- Randomly Generated Levels: Each level is different every time.
- Real Trailer, Fake Movie: Minesweeper: The Movie
- Schrödinger's Gun: See Anti-Frustration Features above.
- Too Dumb to Live: "Hmm... There must be three mines around that 2, let's click he- Crap."
- What Happened to the Mouse?: The smiley is strangely missing from the Vista/Windows 7 version.
- Wingding Eyes: When the smiley dies, it sports X eyes.
- Wiki Rule: Right here


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Who knows what happens at the end of The Dark Knight?
i had to leave before the end because little cousin got scared of harvey dents gross face. i understand we left way before it was over but if anyone wants to tell me the ending it would be cool.
4 answers 4
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text: They are coming back! After an amazing 3 days in 2017 The WATOTO CHILDREN'S CHOIR are coming back again for 1 night only. They will be at Kingsway Hall in Dovercourt on Tuesday 28 May 2019. Admission is free and there is no need to book tickets, just come. The doors open at 6:30pm and the concert begins at 7:00pm.


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I'm a mess when it comes to anything material. I'm just a creature of habit, and I hate replacing things. Not because of the cost, necessarily, but rather because I'm someone who rarely knows what I want, but rather I typically only know what I don't want.
My entire travel setup is so bad right now. So bad. Both of my carry-ons are basically broken. My camera is basically broken. My MacBook Air is falling apart. My Bose headphones are basically broken… and that's only the beginning.
Both Ford and Tiffany have been telling me that I need to start replacing this stuff, and at this point I've basically been given an ultimatum to make a decision by this weekend, or else.
After doing quite a bit of research I'm not sure there's a perfect option out there for me. So I think I just need to be talked into something. Let me share my current setup, and then what I think I'm looking for.
I recently asked you guys for recommendations on a camera as well, and I really appreciate all the feedback, which has given me so much to think about. Tiffany pointed out that Sony is coming out with the RXII soon, which is supposed to be a significant upgrade from the camera I have right now. I've decided to wait until that comes out to make a final decision (given my familiarity with that camera), so hopefully that's not a mistake.
But at this point it's in awful shape. Several of the zippers have fallen off, including the ones to open the main part of the bag. Furthermore, when I roll the bag it makes horrible squeaking noises, and the wheel has quite a bit of resistance. It's just embarrassing at this point.
It would appear that Tumi has some sort of a five year warranty, though I guess I'm just outside of that, so won't get it fixed.
Then I also have a cool weekender bag I like. It has served its purpose, as I've had it for many years. One of the zippers is broken, and it also doesn't really meet my needs anymore.
Why? Because when I bought the bag I lived in hotels full time, and my top priority was maximizing my carry-on space. The weekender bag is huge, and basically the size of my 20″ carry-on. Amazingly they consistently let me take both on a plane.
But nowadays I'm looking for a smaller bag, and also one with more compartments, so I can better stay organized (this one has virtually no compartments).
Why do I have a strong preference for two wheels over four wheels? Mainly because I have wrist pain (which I'm being treated for), and the act of dragging sideways hurts my wrists.
My current setup is perfect in that regard. I have my carry-on, and then I strap the other bag on the back. The weight balances out, and I could literally drag the whole thing with one finger and feel nothing. It's perfect. So I'd really like a setup that allows me to strap my personal item to the back of my carry-on.
My recent trip to Japan reinforced my love of two wheel bags. Ford has a four wheeler and struggled because in Japan they seem to have those little yellow things that stick up from the floor everywhere, and that's a pain if you have four wheels, while it wasn't an issue at all for me.
I recognize you can just elect to use two wheels of a four wheel carry-on, but in my experience it's just not as practical as a two wheeler.
I'm looking for a much smaller personal item that allows me to keep my electronics well organized. It only needs to have room for a laptop, headphones, chargers, a passport wallet, converter, etc.
I'd be happy having a much smaller bag than I currently have. Ideally it would also strap to the back of my bag, and wouldn't look horrible either.
I'm flexible as to whether that comes in the form of a backpack, a smaller duffel/weekender, or whatever…
I feel like I'm one of the few people who still prefers a bag with two wheels, which is a real challenge, because most new bags nowadays seem to have four wheels. It really does minimize the impact on my wrists to have two wheels, but it could also be that I'm just thinking of four wheel luggage wrong.
Then I try to complement that with a backpack or duffel or something, and then I get overwhelmed and get stuck with my current setup.
So, brilliant readers, please enlighten me! What do I do? For me this isn't a decision I take lightly, given just how much I use my luggage.
Well here comes an A321XLR order that shouldn't be a surprise to anyone. American Airlines has just placed an order for 50 Airbus A321XLR aircraft. This deal includes the conversion of 30 A321neo orders (they have a total of 100 A321neos on order), as…
It goes without saying that Boeing is in quite a pickle with the 737 MAX. The plane is grounded globally following two accidents. Boeing and regulators are now working on getting the plane back in the sky. Boeing claims the necessary software updates…
I'm sure everyone is tired of hearing about the A321XLR by now, but in this post I wanted to address a big picture concern that many readers have had with the plane. The A321XLR is no doubt incredibly innovative in terms of the route possibilities it…
I have a bone to pick with people. Let me start by acknowledging that I'm a selective germaphobe and I'm also really bad at science (great combo, I know!), so I might end up being totally off base here. That's a risk I'm willing to take. Heck, I'd much…
Airlines don't always do an amazing job keeping passengers updated when they anticipate that a flight will be delayed. I can't even count the number of times I've been at a gate 10 minutes before the scheduled departure time (20 minutes after boarding…
Over the past few years we've seen Delta SkyMiles, United MileagePlus, and American AAdvantage, all adopt revenue based frequent flyer programs. With these changes, you generally accrue miles based on how much you spend on your ticket rather than how…
Several months ago I wrote a guide about Priority Pass restaurants in the US. In light of the news we learned earlier in the week regarding Priority Pass memberships issued through Amex cards no longer getting access to Priority Pass restaurants, I figured…
There are places you travel to with a certain vision of what the experience will be like -- sitting streetside in a Parisian cafe while an accordion plays; inhaling scents of saffron and sumac in a crowded Turkish spice market; being enveloped in the…
About Ben (Lucky)Ben Schlappig (aka Lucky) is a travel consultant, blogger, and avid points collector. He travels about 400,000 miles a year, primarily using miles and points to enhance his first class experiences. He chronicles his adventures, along with industry news, here at One Mile at a Time.
It's less capacity than you currently have, but may suit your needs. Both are highly adaptable luggage, albeit a bit less stylish than what you're probably used to.
Tumi zippers are designed to break easily so they don't mess with actual zipping system. Bring it in to any store they will attach a new zippers in few seconds. You can definitely stuff more in the two wheeler over four wheeler. I went for a travelpro crew 5 for a very reason, but newer TUMI Alpha3 series is much more classy.
Stick to Tumi, I have an Alpha 3 carry-on, a Sheppard bagpack and a weekender and couldn't be happier.
Soft-side, expandable, two-wheels, pockets galore, lifetime warranty, looks very nice. You just described this….
I think they look almost exactly like Tumi but better quality and more features. Note lifetime warranty is for repair. If the bag is unrepairable, they give you a credit for a new bag but it wouldn't cover the complete price.
I'm in the Briggs and Riley camp and love their stuff. For a cabin unwheeled bag, it's hard to beat the Framed Weekender. My wife says it looks like a large doctors bag – but it is awesome, with a computer pouch and the best passport small items side pocket ever. You should look at this bag.
Couple this with a 2 wheel domestic or international carry on bag (again Briggs and Riley) and you can pack from 4 days to three weeks (with a little laundry on the road). There is a navy blue collection that is very snazzy.
You don't need to do it stat if it is just zipper pulls and wheels. Get a pack of replacement zipper pulls and google luggage rollerblade wheel replacement.
Wirecutter just ran a review of the TravelPro Crew Expert. Looks like it is really well made, but that adds weight that may not be worth the trade-off.
"those little yellow things that stick up from the floor everywhere…" Tactile paving?


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A splendid and vivacious perlage comes face-to-face with a unique and complex bouquet in which flowery notes, accents of honey and white chocolate as well as dried fruits come together and simultaneously reveal a component of exotic fruits such as pineapple, grapefruit and mango which is rounded off by a spicy, fresh-roasted trace, originating the aging process of the cuvee in wooden barrels.
In the mouth, this sparkling wine, which thanks to its heady structure and a component must still be "tamed" and find its balance or a more leisurely and familiar expression, immediately reveals its penchant to full-bodiedness and intensity. Nonetheless, even in the interim phases, it appears precious, due its freshness and tastefulness as well as due to its robustness, which is so charcteristic of great sparkling wine sorts.
To be enjoyed together with fish-based meals or along with white meat dishes, which can also be rather lavish.
Bright yellow with topaz reflexes. The aroma is complex, reminiscent of acacia and lime blossom. On the palate the wine is full-flavoured, expressive and mellow, elegantly structured, tangy with excellent length. The wine will continue to develop and age for several years.
>> dettaglio
A lovely, lively perlage is matched by an extravagant and complex bouquet in which flowery notes and accents of honey, white chocolate, and dried fruits all come together. At the same time, components of exotic fruits such as pineapple, grapefruit, and mango become noticeable. The bouquet of the Cuvée Marianna is rounded out by a spicy, freshly roasted note that originates the aging and refining of the still wines in small oak casks. In the mouth, this sparkling wine immediately shows its tendency toward fullness and substance, thanks to its dense structure and a component that still needs to be "tamed" and find its balance. As a result of its freshness, its luxuriant juiciness, and its tastiness, it is an extraordinary product at every phase up to and including its long-lasting and opulent finish.
Table Parmesan - an original Italian Stravecchio. The cheese is compact in his structure with little punching, like the Grana (Parmesan). With increasing maturity this cheese tends to become emery and fragile.
Spicy, piquant mountain cheese of the Casearia Trentina, 7-9 months matured


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text:
16th March
Concert will begin at 12:15 hours GMT
Thorvaldsdottir, Sibelius with Khachatryan and Rachmaninov from Amsterdam
Live stream
Live stream will be available right here at the beginning of the concert
Sergey Khachatryan, violin, and the Netherlands Radio Philharmonic Orchestra conducted by
Stanislav Kochanovsky play:
THORVALDSDOTTIR: Aeriality.
SIBLIUS: Violin Concerto.
RACHMANINOV: Symphony No. 1.
NPO Radio 4


text:
Notice: On April 23, 2014, Statalist moved from an email list to a forum, based at statalist.org.
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st: explanation of stdf
"Ariel Linden, DrPH" <firstname.lastname@example.com>
st: explanation of stdf
Wed, 7 Apr 2010 17:37:13 -0700
I am not sure if it is a matter of nomenclature, but I am a little confused
on the meaning of the stdf (found in predict). More specifically, I am not
sure what I get if I build 95% CIs using this value. It is not really a
Confidence Interval, it is not a Prediction Interval, and it is definitely
not a Tolerance Interval.
Here is the stata definition:
"stdf calculates the standard error of the forecast, which is the standard
error of the point prediction for one observation. It is commonly referred
to as the standard error of the future or forecast value. By construction,
the standard errors produced by stdf are always
larger than those produced by stdp; see [R] regress postestimation Methods
and formulas."
* For searches and help try:
* http://www.stata.com/help.cgi?search
* http://www.stata.com/support/statalist/faq
* http://www.ats.ucla.edu/stat/stata/


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Bon Jovi's Music- Issue 4
By Shelia
Bon Jovi's music sounds like other iconic '80s figures. Guitar riffs, heavy drumming, and epic guitar solos were all the rage. But, like any other great music artist, Bon Jovi has his own style. I listen to his "Greatest Hits" album all the time, and though his songs sound different, some songs have a very distinct country vibe along with the rock music. Jon Bon Jovi almost sounds like someone off of a country-music radio station. But the lyrics themselves definitely have a similar style. His songs are often about acting for change and standing up for yourself. Take "Livin' on a Prayer": a couple is struggling, but they're working to get out of that struggle. "We Weren't Born to Follow" is exactly what it sounds like; we aren't born to be followers. "... You gotta stand up for what you believe." "It's My Life": "I ain't gonna be just a face in the crowd; you're gonna hear my voice when I shout it out loud... I just wanna live while I'm alive." Bon Jovi's music definitely has that heavy rock that was all the rage in the '80s, but he was able to make his music stand out as his own with his lyrics and blend of the country and rock genres. If you are definitely not a fan of '80s rock music or country, I don't recommend listening to Bon Jovi. That said, I think the band does have good music. I recommend listening to one or two of his songs to at least give you a feel for him and his style.
"Napal Baji" by Psy- Issue 4
By Shelia F.
"Napal Baji" by Psy is just like his other songs, oddly good if that makes any sense. Like all of his music, a lot of words aren't English so you can't understand it but the beat just has you listening on. The music video had its ups and downs though, as usual. I'll start with downs because there actually isn't many of them. First, in parts of the video, mainly the beginning, it just looked like Psy was lip syncing. The words came out before his mouth moved. It got better as the video played though. And the second thing is both an up and a down, the fashion. It was so colorful, with pinks, and purples, and blues, and white. I thought I was gonna start seeing rainbows. The positive twist to this though, is that the outfits worked. Everyone pulled their outfit off, no matter how odd. And that's a good transition into the positive category. The editing, great. Not too much, but what was done seemed real and natural. Also, the dancing seemed better, despite using some of the same dance moves as he always does. Overall, good song, good video, I wouldn't suggest it if the person doesn't like Psy or not understanding lyrics. Other than that though, it's great and should be recommended.
Adele "Hello" - Issue 3
By Sheila F.
Currently number one on the boards is Adele's "Hello." There's a couple things about the music video that could be changed. First thing is lip-syncing. I don't know if she is, I don't know if she isn't, but there are at least two scenes in the beginning of the video where her mouth closed before the singing ends. Also, at the end, what's that move she makes? It looks like she's about to shoot a basketball. Odd moments aside though, it's a good music video. The scenes are dramatic and the effects seem real. The song itself is also great. Personally I don't like Adele. She's usually depressing and her lyrics don't go with her voice very well if that makes sense. But although this is still depressing, it's more intense and her voice matches better. She also sings a bit louder in this which adds to the intensity. All in all, good song, and if you have the same opinion about Adele, I would still suggest listening to this at least once.
Justin Bieber "Sorry" - Issue 3
By Sheila F.
If you're into completely edited, pop music, than you should listen to "Sorry" by Justin Bieber. Compared to his original music, and the albums that came afterwards, it isn't too bad. But compared to other artists on the radio it isn't too good either. No matter how much anyone dislikes Justin Bieber, this isn't personal. He doesn't know how to sing without computers fixing his music. Although if you take out the "oh's" that were repeated so many times, the lyrics are good. It kind of makes you feel like he's apologizing to the audience for all he's done to people since he became famous. However, that feeling stops once he mentions "body." This is just one person's opinion, you may like the song, you also may like the person. But that's up to you to decide. I personally wouldn't recommend this unless you know the person is either into pop, or Justin.
By Natalia Littau
"Daddy" by Psy - Issue 3
By Shelia F.
Ok, "Daddy" by Psy, where do I begin? Well we all originally know Psy because of his song "Gangman Style" but he also has new songs. His song "Daddy," aside from the chorus which consists of only that word, the lyrics are pretty good. Especially for a song where half the lyrics aren't in English. The music video on the other hand, was quite disturbing. Not only did it start out in a hospital with a woman giving birth, Psy's face was on the baby. Then in the school scenes, his face is also on what I think is meant to be a little boy's body but it looks like a girl's. Either way, the face doesn't fit the body. Then later, an old man with facial hair is wearing pink women's clothing. Which in general is innocent, but it just made him look really creepy. Same with the "swaggy" guy (at least I think that was their intention). Also, despite good ones, there were also some horrible effects throughout the whole video. His dancing however, has gotten better. All in all, it's a good song but I suggest watching a lyric video.
Calvert Voices
A publication created by Calvert High School students for the Calvert School community.
May 2016


text:
Tees Maar Khan (2010) Hindi Movie Songs Download
Tees Maar Khan (2010) Songs Download And Information :
- Film Released Year: 2010.
- Director: Farah Khan
- Artist: Vishal-Shekhar, Shirish Kunder
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- Music Category: Hindi Songs
- Files Format: High-Quality Mp3 and 128Kbps & 320Kbps Zip File (Full Album).
- Audio Bit Rate: 128Kbps and 320Kbps.
- Country: India.
- Movie's Language: Hindi.
Download Tees Maar Khan Hindi Song is a 2010 Hindi movie which directed by Farah Khan. The movie features Akshay Kumar, Akshaye Khanna, Katrina Kaif in the main roles. And the music was composed by Vishal-Shekhar, Shirish Kunder Song. Bollywood New Hindi Songs Tees Maar Khan movie officially release on 2010.
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Esther McVey took two months to respond to a challenge to her hated "rape clause".
And the answer wasn't worth waiting for.
She is sticking to the callous policy that forces women to reveal details of how their child was conceived in an act of horrifying sexual violence.
It's the sickening by-product of Theresa May's two-child cap on tax credits.
The only way to get more benefit for a subsequent child is if you're covered by exemptions like the rape clause.
McVey – known as McVile for her support for welfare cuts – was put on the spot on her first day as Work and Pensions Secretary.
Former Scottish Labour leader Kezia Dudgale and the SNP's Alison Thewliss, among others, have been consistent in their calls for the policy to be ditched.
Both will have to keep fighting, along with every other politicians with a conscience.
McVey says the way the clause is handled strikes the right balance for women who've been raped and need more welfare.
Re-read that sentence and pause to imagine how that policy can ever be worth the cash it saves the bean counters at the Treasury.
The UK Government should do the decent thing and scrap the clause and rethink the welfare cap.
Maybe then the Government can pursue the rich tax evaders who really could boost our national bank balance.
Work for change
The tiny number of disabled people in paid work shows how far we have to go as a nation.
Just five per cent of adults with learning disabilities have a wage.
Down's Syndrome Awareness Week is another opportunity to put that right.
That's why we're highlighting Fiona Dawson's experience.
She's on the British Paralympics team – and she's a swimming coach in Prestwick.
Fiona's not paid for that role, instead getting money from a shop job and administrative work, but she is determined to live her life to the full.
Too many people think disability must always be a block.
It took 10 years of trying until Fiona landed a paid position.
With more effort, and better awareness, that five per cent figure can be transformed.
Mews property
A warring household can make you feel like you're crawling up the walls.
Which is literally what Simba the cat does to keep away from her canine housemate.
Splitting your home with a creative outdoor ladder might seem excessive – but it certainly stops them fighting like cat and dog.


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This is being billed as the most expensive flat to rent in Edinburgh - take a look inside
Edinburgh has no shortage of amazingly expensive flats to rent, but this one takes the cake for being the most expensive.
Monday, 9th September 2019, 11:17 am
This £1.7m penthouse comes from Tay Letting. It is a three bedroom apartment which overlooks Donaldson's School and will be available to rent for upwards of £5k per month - take a look inside here.


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New Delhi, 3 September
With a record highest number of bookings, Indian Railway Catering and Tourism Corporation (IRCTC) Ltd, through its e-ticketing website www.irctc.co.in, had on 2 September, booked 5.72 lakh e-tickets which was the highest ever e-tickets booked on a single day. This has surpassed the earlier highest of 5.04 lakhs recorded last month on 12 August. This is the third time it booked more than 5 lakh tickets on a day this year, the first time it was on 1 March.
"IRCTC has booked a total of 135 lakh e-tickets with an average of 4.34 lakhs daily in the just concluded month of August 2013 against a total of 123 lakh e-tickets with an average of 3.99 lakhs daily in the corresponding month of August 2012 registering an increase of nearly 10 percent in the total number of tickets booked in the comparing months of August in 2012 and 2013," said Mr Pradip Kundu, spokesperson, IRCTC. The overall daily average booking was around 3.85 lakhs last year (2012-13).
According to Mr Kundu, the total number of passengers booked by IRCTC has also gone up to 233 lakhs in August 2013 against 220 lakhs achieved in August 2012.
Further, the IRCTC has also recently launched the non-internet SMS-based ticketing through which one can book a railway ticket with a basic feature mobile phone, by sending simple SMS, which does not require any internet connection.


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text: Mark Zuckerberg and Priscilla Chan, who just had their first child earlier this year are pledging to cure all diseases in their child's life time. With a $3bn investment over 10 years they hope to put the money to research instead of treatment. "We spend 50x more on treatment than we do on research of why we are sick." Mark stated when addressing the tools they hope for in the future.


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2 BR / 2 BA / 6 PPL - St Pete Beach, FL
Caprice Resort is a direct Gulf Front property located on St. Pete Beach. Enjoy the stunning beach views. The property features a heated pool, private beach, bbq grills, shuffle board court and a sun deck. The Caprice is walking distance to many shops and restaurants. Unit #106 is a two bedroom, two bathroom condo with a fully equipped kitchen. Private balcony with a direct Gulf Front view. The master bedroom has a King size bed. The second bedroom has two Twin beds. The living room has a sleeper sofa. The kitchen and bathrooms have been updated. Wood laminate flooring throughout. Free Wi-Fi and cable.


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NEW NAME, NEW LOOK FOR MID-HUDSON CIVIC CENTER Read Full Story Here NEW SKATING DIRECTOR We are so excited to share with everyone that starting March 19, 2018, Gilberto Viadana will become a part of the Mid-Hudson Civic Center Inc. family as the new Skating Director.Check price
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More on GUE (Global Underwater Explorers)
As I mentioned in my last post, I recently took an introductory course, GUE Fundamentals. I mentioned that I learned a lot and I did. I learned several new methods of propulsion, that is variations on the standard fin kicks that we typically use. I'll go into a little more of that next time.


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Taking Down The Giants - Sunday Pm
August 30, 2015
Awakening Youth Pastor Tom Crandall talks about taking down giants. We've all had to face things in our lives that appear bigger than they really are. But, giants shrink to real sizes in the presence of the Lord. You're a giant killer! Scripture Reference: Psalm 27, 1 Samuel 5, 1 Samuel 17


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- Price$367,500
- SQ. Feet4,380
- Built2019
2 Fairview Ln., Clarksville
Essential Information
- Price$367,500
- Bathrooms0.00
- Square Footage4,380
- Acres0.00
- Year Built2019
- TypeResidential Income
- StatusUnder Contract - Not Showing
- Parking Spaces8
- GaragesParking Lot
- ConstructionBrick,Vinyl Siding
Additional Information
- Date ListedMay 30th, 2019
- ZoningR4
Listing Details
- Listing OfficeCory Real Estate Services
Community Information
- Address2 Fairview Ln.
- Area76 - Montgomery County (St. Bethlehem, Dunbar Cave)
- SubdivisionFairview
- CityClarksville
- CountyMontgomery County
- StateTN
- Zip Code37042
- HeatingCentral
- CoolingCentral
- # of Stories2
School Information
- ElementaryMinglewood Elementary
- MiddleNew Providence Middle School
- HighNorthwest High School
HOA Information
- HOANone
The data relating to real estate for sale on this web site comes in part from the Internet Data Exchange Program of RealTracs Solutions. Real estate listings held by brokerage firms other than RE/MAX Elite are marked with the Internet Data Exchange Program logo or thumbnail logo and detailed information about them includes the name of the listing brokers.
Disclaimer: All information is believed to be accurate but not guaranteed and should be independently verified. All properties are subject to prior sale, change or withdrawal.
Copyright 2019 RealTracs Solutions.
Listing information last updated on October 23rd, 2019 at 4:59pm CDT.


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Things are rockin! We are closin deals left and right! Visit us at www.Twitter.com/TheMcDAgency . We are in the process of launching two new divisions: The Urban Retail Division (specializing in the leasing and sales of Uptown area street level retail and restaurant space) and The Sports and Entertainment Division (handling the special needs of Charlotte's sports and entertainment professionals). We are also about to bring on 2 more of the Uptown area's top brokers to join our team! Follow me on Twitter at http://www.shanemcdevitt.com/ and visit http://www.askuptown.com/ for quick and easy answers to any of your real estate questions. http://www.touruptowncharlotte.com/ is coming soon. We are the sponsor of the Uptown Cool Down party series, the next two will be Aug 14 and 28. August 14th till be at NIX at 5pm, be there! We will also be the premier sponsor of an Uptown Restaurant Week that will soon be announced. Lots happenin! Tryin to keep up!
Labels: ask uptown, askuptown.com, downtown charlotte retail, shane mcdevitt, tour uptown charlotte, twitter uptown charlotte, uptown charlotte retail


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Repair Guides | Wiring Diagrams | Wiring Diagrams | AutoZone.com 12: 1993-95 MX6/626 (2.0L A/T) Engine SchematicsWiring Harness Mazda Mx 6 - > Mazda 6 Parts and Mazda 6 Accessories > Mazda Miata MX-5 Parts and Mazda Miata MX-5 Accessories 2017 2018 Mazda CX-7 Trailer Hitch with Wiring Harness.Pull your toys to your favorite destination with this Class I-rated receiver designed especially for your Mazda CX-5. See your Mazda Dealer for details.. Mazda Stereo Wiring Harness Diagram (Nov 09, 2018) - Welcome to Wiring Diagram Pictures. Most of us have used on line for finding info, suggestions, articles or other resource for their purposes. Like everyone else are.. Turboing a second gen mx-6; An MX-6 For Racing? Stuck in 2nd gear; Shaking at 30-40 mph; I need help on my auto trans. i need a mx-6 engine!! Looking 4 a moonroof panel; Needed a working power antenna; Is there a idle adjustment on these mx-6's? fuse cover diagram; problem starting; How to repair chipping clear coat?.
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As the way we work changes, mobile and cloud technology adoption have seen the dissolution of the traditional enterprise security perimeter. As a result, new threat vectors have been introduced which legacy security frameworks are often incapable of sufficiently defending against.
With a "never trust, always verify" approach, MobileIron goes the extra step.
Mobile use is at an all-time high:
Recent years have seen users benefit hugely from the flow of data across a wide variety of devices, apps, networks and cloud services. But the expectation of having anytime, anywhere access has eliminated the need for a traditional security perimeter, whilst creating a massive-attack surface which is open to a whole host of new risks and threats.
Desktops are being replaced by mobile endpoints, and data centres are moving more and more information to the cloud. As a result, data sits on devices and clouds that an enterprise business owns, as well at those they don't, in addition to crossing multiple networks. Security on these unknown networks can often have far less robust security than in the workplace.
Hackers follow opportunity
Hackers follow the data, as this is what is valuable to them. It's far more efficient and less time-consuming for malicious actors to find and expose new doors rather than break through old ones which have layers of PC protection. Enterprises have a multitude of devices so they're an obvious target for hackers.
Mobile attacks are growing in sophistication, therefore organisations need to focus on a mobile-centric security approach rather than relying on security models built for the PC and data-centre world that poorly translate to the mobile-cloud world.
So, what is zero-trust?
The zero-trust approach works by recommending that a security strategy is built based on the assumption that your network is already compromised. As such, secure access is determined with a "never trust, always verify" approach, requiring consistent verification enforcement of the likes of the device, user, apps, networks and presence of threats.
There are multiple different approaches to the zero-trust model, but all key methods focus on identity, gateway and the device. But a mobile-centric approach is essential to address the security challenges which are raised by a perimeter-less modern enterprise, whilst still enabling the agility and anytime access required by businesses.
The MobileIron Solution
MobileIron operates under the belief that the mobile is the very centre of the enterprise, leading them to create a security platform that starts from the device. A mobile-centric, zero trust framework goes well beyond any traditional identity management and gateway point solutions. Access is granted based on a comprehensive set of attributes, involving validating the devices, establishing user context, checking app authorisation, verifying the network, whilst detecting and remediating any threats – all before granting access to any user or device. Significantly, this all happens instantaneously without interrupting the user experience, whilst ensuring the business is in complete control of data as it flows across apps, devices, networks and cloud services.
Your device is your ID
With a user's device becoming their secure ID, the need for passwords is eliminated whilst ensuring access to business information is more secure and simple. As a result, data is protected whilst mobile users have the freedom and flexibility to be productive for mobile working.
The four-step process
What powers MobileIron's zero-trust, mobile-centric solution?
- MobileIron UEM – Provides visibility and IT control required to secure, manage and monitor all corporate and BYO devices, or desktop which accesses any business-critical data. A vast range of employee devices can be secure and managed throughout their entire lifecycle.
- Mobile Threat Defence – Built-in threat detection and remediation protects devices without the need for Internet connectivity or concerns about user adoption – essential as hackers develop ever-increasing attacks on mobile devices and apps.
- MobileIron Access – Seamless, conditional access is achieved through a combination of passwordless single sign-on (SSO) and multi-factor authentication (MFA). Ensuring only those authorised can access and share corporate data from any device, OS or location to any services supports a zero-trust framework.
An enhanced experience
- Simple device on-boarding and automatic configuration means no lengthy employee set up guides for users to struggle through.
- Passwordless access – eliminates the need to remember password.
- Continuous on-device threat detection – users are not required to take any action
- Intuitive remediation workflows – non-compliant devices are easily fixed without the need for helpdesk involvement
The MobileIron solution is embraced by both users for the seamless experience it allows, and by IT due to its easy implementation and the dramatic reduction in help-ticket requests.
The MobileIron solution is embraced by both users for the seamless experience it allows, and by IT due to its easy implementation and the dramatic reduction in help-ticket requests.
MobileIron Access - Blocking Safari
How Can We Help?
Mobliciti can provide you with this zero-trust, mobile-centric framework that allows you to confidently adopt mobile and cloud technologies whilst driving productivity and reducing your risk of data breaches. We can offer full Cloud Security solutions that protect your data beyond the perimeter.
To find out more, please get in touch.


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OverviewThis pack contains 1 copy of Communication Sciences and Disorders: A Clinical Evidence-Based Approach and 1 printed access card to the Video eText.
For courses in Communication Sciences and Disorders.
A clinical, case-based approach to communication sciences and disorders.
Communication Sciences and Disorders, A Contemporary Evidence-Based Perspective introduces students to the field through clinicians, patients and families. Rich with examples, the first part covers foundational concepts of development, anatomy, physiology, augmentative and complex communication. Following these foundations, the second part highlights inherited and acquired disorders affecting children and older adults. A new chapter features multicultural topics, while an expanded chapter delves into advancements in assessment and intervention. Strong themes of literacy weave throughout.