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Research ArticleHEALTH AND MEDICINE
Antibody-mediated neutralization of soluble MIC significantly enhances CTLA4 blockade therapy
Jingyu Zhang1,2,
Dai Liu3,
Guangfu Li3,
Kevin F. Staveley-O'Carroll3,
Julie N. Graff4,
Zihai Li1,5 and
Jennifer D. Wu1,5,*
1Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29425, USA.
2CanCure LLC, Everett, WA 98208, USA.
3School of Medicine, University of Missouri, Columbia, MO 65211, USA.
4Knight Cancer Institute, Oregon Health and Science University, Portland, OR 97239, USA.
5Cancer Immunology Program, Hollings Cancer Center, Charleston, SC 29425, USA.
↵*Corresponding author. Email: wujjd{at}musc.edu
Science Advances 17 May 2017:
Vol. 3, no. 5, e1602133
Jingyu Zhang
Dai Liu
Guangfu Li
Kevin F. Staveley-O'Carroll
Julie N. Graff
Zihai Li
Jennifer D. Wu
For correspondence: wujjd@musc.edu
Antibody therapy targeting cytotoxic T lymphocyte–associated antigen 4 (CTLA4) elicited survival benefits in cancer patients; however, the overall response rate is limited. In addition, anti-CTLA4 antibody therapy induces a high rate of immune-related adverse events. The underlying factors that may influence anti-CTLA4 antibody therapy are not well defined. We report the impact of a cancer-derived immune modulator, the human-soluble natural killer group 2D (NKG2D) ligand sMIC (soluble major histocompatibility complex I chain–related molecule), on the therapeutic outcome of anti-CTLA4 antibody using an MIC transgenic spontaneous TRAMP (transgenic adenocarcinoma of the mouse prostate)/MIC tumor model. Unexpectedly, animals with elevated serum sMIC (sMIChi) responded poorly to anti-CTLA4 antibody therapy, with significantly shortened survival due to increased lung metastasis. These sMIChi animals also developed colitis in response to anti-CTLA4 antibody therapy. Coadministration of an sMIC-neutralizing monoclonal antibody with the anti-CTLA4 antibody alleviated treatment-induced colitis in sMIChi animals and generated a cooperative antitumor therapeutic effect by synergistically augmenting innate and adoptive antitumor immune responses. Our findings imply that a new combination therapy could improve the clinical response to anti-CTLA4 antibody therapy. Our findings also suggest that prescreening cancer patients for serum sMIC may help in selecting candidates who will elicit a better response to anti-CTLA4 antibody therapy.
NKG2D ligand
soluble MIC (sMIC)
anti-sMIC antibody
anti-CTLA4 antibody therapy
Cytotoxic T lymphocyte–associated antigen 4 (CTLA4) is a co-inhibitory receptor that controls cytotoxic effector T cell activation during initiation and maintenance of healthy adaptive immune responses (1–3). The expression of CTLA4 is often up-regulated during T cell activation upon interacting with antigen-presenting cells (APCs) (1–3). CTLA4 competes with the costimulatory T cell receptor (TCR) CD28 in binding to the B7 molecules (B7.1 and B7.2) expressed on APCs and has a higher binding affinity than CD28 (1–4). In contrast to CD28/B7 binding, which acts as a costimulatory signal to promote T cell activation and proliferation, the binding of CTLA4 to B7 transmits an inhibitory signal. CTLA4 is considered the "off switch" or "immune checkpoint" for effector T cell function. CTLA4 can also control effector T cell function through the expansion of CD4+FoxP3+ regulatory T cells, in which CTLA4 is constitutively expressed and critical for maintaining the suppressive function (5, 6). Therapy with anti-CTLA4 antibody elicited remarkable effectiveness in controlling tumor growth in mice (7–12).
Preclinical investigations have led to the development of anti-CTLA4 antibodies for cancer immunotherapy. Ipilimumab, a full human monoclonal anti-CTLA4 antibody that blocks the binding of CTLA4 to the B7 molecule, was approved by the U.S. Food and Drug Administration (FDA) for the treatment of advanced melanoma (4, 13–15). Currently, ipilimumab is also undergoing clinical trials for the treatment of non–small cell lung carcinoma, small cell lung cancer, and bladder cancer (16–18). Despite the remarkable tumor control ability of anti-CTLA4 antibody demonstrated in preclinical animal models, the overall clinical response rate of anti-CTLA4 antibody as a stand-alone agent is limited to 15% in melanoma (4). Notably, therapy with anti-CTLA4 antibody has been associated with a significant risk of immune-related adverse events (IRAEs) in patients, with most of the severe cases restricted to gastrointestinal inflammation (13, 19–21). Combination therapy of anti–programmed cell death protein 1 antibody and anti-CTLA4 antibody has achieved greater overall response than monotherapy in patients with melanoma, the most immunogenic cancer; however, the response remains limited to a subgroup of patients and is accompanied by increased toxicity (22–24). It is thus imperative to understand the circumstances that may limit the clinical efficacy of anti-CTLA4 antibody therapy and the risk of toxicity to develop more effective yet safe regimens.
The differential expression of immune modulators by human and rodent tumors may contribute to the response disparity between clinical and preclinical anti-CTLA4 antibody therapy. The major histocompatibility complex (MHC) I chain–related family molecules, including the molecules MICA and MICB (collectively termed MIC), are an example of such immune modulators. MIC is frequently induced in human tumors upon genotoxic insults but is absent in rodents (25–31). The induction of MIC on the cancer cell surface can activate natural killer (NK) cells and costimulate cytotoxic CD8 T cells, NK-like T cells, and subsets of γδ T cells through engaging the activating immune receptor natural killer group 2D (NKG2D) (32, 33). The activation of NKG2D has been shown to provide unique costimulation to antigen-specific CD8 T cells that is nonredundant to CD28 costimulation (32, 34–37). In cancer patients, MIC is often released as the highly immunosuppressive soluble form sMIC through shedding (25, 28, 29). Tumor-derived sMIC at large suppresses host immune response using multiple strategies including (but not limited to) down-regulating NKG2D expression and destabilizing CD3ζ in the TCR/CD3 complex on CD8 T cells (25, 38), perturbing NK cell function and peripheral maintenance (39, 40), and cultivating an immunosuppressive tumor microenvironment by driving the expansion of myeloid suppressor cells and skewing macrophage into an alternatively activated phenotype (41). Using a clinically relevant MIC-transgenic spontaneous mouse tumor model that closely recapitulates the onco-immune dynamic of human cancer (39), we have demonstrated that therapy with the nonblocking anti-sMIC monoclonal antibody (mAb) B10G5 globally abrogates the immunosuppressive effect of sMIC, immobilizes NK and CD8 T cells to the tumor microenvironment, and overcomes immune tolerance of antigen-specific CD8 T cells (42, 43). In line with these understandings, we herein investigated the impact of tumor-derived human sMIC on the therapeutic efficacy of anti-CTLA4 antibody. We show that high levels of serum sMIC not only negatively influenced the antitumor efficacy of anti-CTLA4 antibody but also evoked colitis development during anti-CTLA4 antibody therapy. Coadministration of the sMIC-neutralizing antibody B10G5 with anti-CTLA4 antibody generated a cooperative antitumor effect and alleviated therapy-induced colitis. These results suggest a new avenue for combination immunotherapy of cancer.
Animals with high serum levels of sMIC respond poorly to anti-CTLA4 antibody therapy
Tumor-derived sMIC negatively affects tumor immunity through multiple mechanisms (25, 39–41). To address the impact of sMIC on the efficacy of anti-CTLA4 antibody therapy, we treated cohorts of 27- to 28-week-old TRAMP (transgenic adenocarcinoma of the mouse prostate) and TRAMP/MIC littermates with anti-CTLA4 antibody (3.0 mg/kg) or control immunoglobulin G (cIgG), respectively (Fig. 1A). We previously showed that more than 40% of TRAMP/MICB mice at the indicated age had elevated serum sMIC as a result of tumor shedding (39). Consistent with other studies (44, 45), TRAMP mice presented no or marginal response to anti-CTLA4 antibody monotherapy (Fig. 1, B and D). TRAMP/MIC mice that received anti-CTLA4 antibody therapy presented reduced overall survival compared to littermates that received cIgG therapy (Fig. 1B). Animals that succumbed to diseases during therapy, whether receiving cIgG or anti-CTLA4 antibody, had significantly higher levels of serum sMIC before therapy than those that survived to the study end point (Fig. 1C). However, in animals that had high levels of sMIC before therapy, tumors progressed more aggressively in response to anti-CTLA4 antibody therapy. The disease progression was manifested through large primary tumor burdens and massive lung metastasis (Fig. 1, D to F). Animals that had high serum sMIC and had received anti-CTLA4 antibody therapy also developed colitis (fig. S1). These data indicate that high levels of circulating sMIC adversely affect tumor response to anti-CTLA4 antibody therapy and also contribute, at least in part, to anti-CTLA4 antibody–induced autoimmune colitis.
Fig. 1 Mice with high circulating tumor-shed soluble MIC (sMIChi) had poor response to anti-CTLA4 antibody therapy.
(A) Schematic depiction of therapy. (B) Kaplan-Meier survival curve. αCTLA4, anti-CTLA4. *P < 0.05. (C) Prostate (spontaneous tumor site) weight at necropsy. Boxed animals succumbed to disease before the study end point. (D and E) Representative micrographs (D) and overall number (E) of lung metastasis in TRAMP/MIC mice in response to anti-CTLA4 antibody therapy. Arrows, lung nodules. (F) Serum levels of sMIC in TRAMP/MICB mice before receiving therapy. Sera from TRAMP were used as negative controls. ns, not significant.
We substantiated our observations in syngeneic TRAMP-C2 and sMICB-expressing TRAMP-C2-sMICB transplantable prostate tumor models (fig. S2), in which both tumor cell lines were subcutaneously inoculated into MICB/B6 male transgenic mice, as we have previously described (39, 42, 46). In the absence of sMIC, TRAMP-C2 tumors presented no response to anti-CTLA4 antibody therapy. Consistent with our observations in transgenic TRAMP/MICB mice, TRAMP-C2-sMICB tumors grew more aggressively in response to anti-CTLA4 antibody therapy (fig. S2B). In animals that received anti-CTLA4 antibody therapy, TRAMP-C2-sMICB tumor–bearing mice developed more severe colon inflammation than TRAMP-C2 tumor–bearing mice (fig. S2C).
Antibody neutralizing sMIC markedly enhances therapeutic responses to anti-CTLA4 antibody
To confirm that sMIC contributes to subjects' poor response to anti-CTLA4 antibody therapy and to determine whether neutralizing sMIC can enhance the therapeutic efficacy of anti-CTLA4 antibody or MIC+ tumors, we coadministered the anti-sMIC mAb B10G5 (3.0 mg/kg) with anti-CTLA4 antibody to a cohort of TRAMP/MICB littermates at 26 to 27 weeks old, as depicted in Fig. 1A. In our recent publications, we described that B10G5 can effectively neutralize the immunosuppressive effect of sMIC to induce the debulking of primary tumors and eliminate metastasis (42). In agreement with our previous findings, all animals responded to mAb B10G5 monotherapy (Fig. 2, A to E). Remarkably, regardless of serum levels of sMIC before therapy, all animals survived to the study end point with clearance of lung metastasis in response to anti-CTLA4 antibody therapy with the coadministration of B10G5 (Fig. 2, A and E). The coadministration of B10G5 significantly reduced the circulating level of sMIC (Fig. 2B) and generated a cooperative therapeutic effect with anti-CTLA4 antibody, eliciting a significant reduction in primary tumors at the study end point compared to monotherapies (Fig. 2C). The coadministration of B10G5 also alleviated anti-CTLA4 antibody therapy–induced colitis (fig. S3). These data demonstrate the benefit of the sMIC-neutralizing antibody B10G5 in enhancing anti-CTLA4 antibody therapy.
Fig. 2 Antibody B10G5 cotargeting sMIC remarkably enhances response to anti-CTLA4 antibody therapy.
(A) Kaplan-Meier survival curve. (B) Serum levels of sMIC at necropsy. (C) Prostate weight at necropsy. (D and E) Number (D) and representative micrographs (E) of lung micrometastasis in animals of each treatment cohort.
A melanoma patient who developed anti-MIC autoantibody during anti-CTLA4 antibody therapy was found to elicit a superior response (47). We thus assayed the presence of anti-sMIC autoantibody during early response to anti-CTLA4 antibody (ipilimumab) in a phase 1/2 clinical trial (NCT01498978) on a small cohort (n = 10) of metastatic prostate cancer patients who were also receiving androgen suppression therapy. A high titer of anti-MIC autoantibody was detected in one patient (ID: OHSU 5254-8) after one cycle of ipilimumab (fig. S4). Patient OHSU 5254-8 elicited a durable response with a prostate-specific antigen decrease from 191 to 4.6 ng/ml after eight cycles of ipilimumab. No autoimmune colitis has been noted in patient OHSU 5254-8 thus far. This case study reinforces the utility of sMIC-neutralizing antibody in enhancing anti-CTLA4 antibody therapy.
B10G5 neutralizing sMIC heightens CD8 T cell response to anti-CTLA4 antibody therapy
The therapeutic efficacy of anti-CTLA4 antibody is commonly determined by the activation status of effector CD8 T cells. The coadministration of B10G5 with anti-CTLA4 antibody to TRAMP/MICB mice resulted in a significant increase of CD8 T cells in tumor-draining lymph nodes (dLNs) and tumor infiltrates compared to the monotherapy with anti-CTLA4 antibody or B10G5 (Fig. 3A and fig. S5A). Consistent with our previous observation, B10G5 therapy alone increased the expression of NKG2D on CD8 T cells (42). Notably, NKG2D as a nonconventional effector T cell costimulatory molecule is expressed by all human CD8 T cells but is only expressed by activated mouse CD8 T cells (48). A cocktail therapy of anti-CTLA4 antibody and B10G5 further increased the number of NKG2D+ CD8 T cells, signifying that more CD8 T cells were activated (Fig. 3B and fig. S5B). NKG2D and CD28 can deliver nonredundant costimulatory signals to CD8 T cells (35, 49). Thus, the further increased NKG2D expression in response to cocktail therapy would presumably provide a nonconventional costimulatory signal to augment antigen-specific CD8 T cell responses to MIC+ tumors, in addition to the diminution of CTLA4 co-inhibitory signals.
Fig. 3 Combination therapy of anti-sMIC and anti-CTLA4 antibody remarkably increases CD8 T cell population, activation, and functional potential in tumor-dLNs and tumor beds.
(A to C) Percentage of CD8 T cells (A), NKG2D+ CD8 T cells (B), and activated or memory-like CD8 T cells (C) in the spleen (spln), tumor-dLNs, and tumor infiltrates (TILs). (D) CD8 T cell response to ex vivo restimulation with PMA and ionomycin, as measured by IFN-γ production. All representative graphs of flow cytometry analyses are shown in fig. S5.
The coadministration of B10G5 with anti-CTLA4 antibody therapy significantly increased the effector memory-like CD44hi CD8 T cell compartments in the spleen, dLNs, and tumor infiltrates (Fig. 3C and fig. S5C). The combination therapy also increased the intrinsic ability of CD8 T cells to produce interferon-γ (IFN-γ) in response to ex vivo phorbol 12-myristate 13-acetate (PMA) and ionomycin restimulation (Fig. 3D and fig. S5D).
We retrospectively compared the functional potential of CD8 T cells in TRAMP and TRAMP/MICB mice that had received anti-CTLA4 antibody monotherapy. CD8 T cells in all TRAMP mice uniformly exhibited a nominal response to anti-CTLA4 therapy (fig. S6). In TRAMP/MIC mice that had received anti-CTLA4 antibody therapy, the CD8 T cell response to ex vivo PMA/ionomycin restimulation varied in accordance to serum levels of sMIC; however, the percentage of CD8 T cells was not profoundly affected (fig. S7A). A severely impaired response was observed in mice with high levels of sMIC, whereas the responses in TRAMP/MICB mice with low levels of sMIC and in TRAMP mice remain comparable. One of the 11 TRAMP/MICB mice that developed anti-sMIC autoantibody during anti-CTLA4 antibody monotherapy demonstrated remarkable responsiveness to ex vivo restimulation (fig. S7B). Notably, the polarization of CD8 T cell functional potential was correlative with the presence and function of NK cells in the dLNs (fig. S6B). Given that sMIC perturbs NK cell homeostatic maintenance and function and that NK cell–dendritic cell (DC) cross-talk is critical in regulating T cell priming (39, 50–53), these observations further highlight a potential important role of NK cells in effectuating CTLA4 blockade therapy.
Combination therapy of antibody neutralizing sMIC and anti-CTLA4 antibody markedly overturns antigen-specific CD8 T cell tolerance
We have previously shown that B10G5 monotherapy overcomes antigen-specific CD8 T cell tolerance (42). To determine whether combination therapy with B10G5 and anti-CTLA4 antibody can further enhance CD8 T cell antitumor response in an antigen-specific manner, we adoptively transferred carboxyfluorescein succinimidyl ester (CFSE)–labeled SV40TAg-specific TCR-I CD8 T cells into cohorts of mice that had received therapies for 4 weeks, as indicated and schematically depicted in Fig. 1A. Notably, these TCR-I CD8 T cells bear TCRs specific for the TRAMP/MIC oncogene SV40TAg and can be detected by the SV40TAg peptide I–specific Db/I-tetramer.
Adoptively transferred antigen-specific TCR-I CD8 T cells would normally fail to sustain after initial expansion in TRAMP or TRAMP/MICB mice because of clonal deletion (42, 54). Whereas anti-CTLA4 antibody monotherapy only had a marginal effect on sustaining Db/I-tetramer+ TCR-I CD8 T cells in dLNs, tumor infiltrates, or spleen in TRAMP/MIC mice, B10G5 therapy achieved consistent sustainability of the adoptively transferred TCR-I CD8 T cells with a great percentage in the tumor infiltrates, as we have previously presented (Fig. 4A and fig. S8A) (42). Remarkably, the combination antibody therapy with B10G5 and anti-CTLA4 further significantly enhanced the sustainability of TCR-I CD8 T cells compared to B10G5 monotherapy (Fig. 4A and fig. S8A).
Fig. 4 Combination therapy of anti-sMIC antibody and anti-CTLA4 antibody cooperatively enhances antigen-specific CD8 T cell antitumor responses.
CFSE-labeled tumor antigen SV40TAg–specific TCR-I CD8 T cells were transferred into TRAMP/MICB mice that have received variable antibody regimens for 4 weeks, which were continued after transfer of TCR-I CD8 T cells. Shown are data obtained 14 days after the transfer of CFSE-labeled TCR-I CD8 T cells. (A) Percentage of Db/I-tetramer+ SV40TAg-specific CD8 T cells in tumor infiltrates, dLNs, and spleen. (B) Proliferation of SV40Tag-specific CD8 T cells represented by CFSElo population. (C) Activation of Db/I-tetramer+ SV40TAg-specific CD8 T cells in response to therapy shown by the CD44hi population. (D) Response of Db/I-tetramer+ SV40TAg-specific CD8 T cells to ex vivo SV40TAg peptide restimulation, as measured by IFN-γ production. Representative graphs of flow cytometry analyses are shown in fig. S8.
Adoptively transferred naïve Db/I-tetramer+ TCR-1 CD8 T cells experienced initial expansion, as shown by the dilution of CFSEhi Db/I-tetramer+ CD8 T cells in all cohorts of animals (Fig. 4B and fig. S8B). However, therapy with B10G5 or B10G5 combined with anti-CTLA4 antibody provoked the continuous expansion of Db/I-tetramer+ CD8 T cells, shown as a significantly increased percentage of CFSElo Db/I-tetramer+ CD8 T cells compared to cIgG therapy (Fig. 4B and fig. S8B). The combination therapy resulted in a substantially higher percentage of CFSElo Db/I-tetramer+ CD8 T cells compared to B10G5 monotherapy (Fig. 4B and fig. S8B).
The continuous expansion of antigen-specific CD8 T cells in the tumor microenvironment and tumor-dLNs is an indication of the activation of antigen-specific CD8 T cells. Anti-CTLA4 antibody monotherapy generally did not elicit a benefit in activating Db/I-tetramer+ CD8 T cells (Fig. 4, C and D). Consistent with our previous studies (42), B10G5 monotherapy evoked a significantly higher percentage of CD44hi Db/I-tetramer+ CD8 T cells (Fig. 4C and fig. S8C) and greater intrinsic cellular response to restimulation (Fig. 4D and fig. S8D). Combination therapy of B10G5 and anti-CTLA4 antibody induced a more pronounced increase in the number of CD44hi Db/I-tetramer+ CD8 T cells (Fig. 4C and fig. S8C) and in IFN-γ production by Db/I-tetramer+ CD8 T cells in response to PMA/ionomycin restimulation (Fig. 4D and fig. S8D). Together, these data further reveal the effector mechanism whereby combination therapy of B10G5 and anti-CTLA4 antibody heightens antigen-specific CD8 T cell antitumor responses.
B10G5 neutralizing sMIC and anti-CTLA4 antibody cooperatively heighten DC costimulatory potential
We investigated the mechanisms whereby the cocktail therapy with B10G5 and anti-CTLA4 antibody would synergistically bolster antigen-specific CD8 T cell responses. We previously showed that B10G5 therapy alone can enhance DC activation in tumor-dLNs and increase the expression of the DC costimulatory molecules CD80 and CD86 (42). With the antibody cocktail therapy, a further significant increase occurs in the DC surface costimulatory molecules CD80 and CD86, along with the DC activation molecule CD40 (Fig. 5, A and B). Such an effect was not observed in non–tumor-dLNs (fig. S9). Given that CD80 and CD86 can engage both the costimulatory molecule CD28 and the co-inhibitory molecule CTLA4, these data indicate that B10G5 neutralizing sMIC in combination with CTLA4 blockade would allow more robust CD28-mediated costimulatory signal delivered to antigen-specific CD8 T cells that may in part confer the synergistic effect on sustaining antigen-specific CD8 T cell activity.
Fig. 5 B10G5 and anti-CTLA4 antibody therapy cooperatively increases DC activation (CD40) and the expression of costimulatory molecules CD80 and CD86 in tumor sites.
(A) Representative histograms from flow cytometry analyses of CD40, CD80, and CD86 expression on DCs from tumor-dLNs and tumor beds. Gray-filled profiles, control isotype staining; open dark profiles, antibody to specific DC surface molecules. (B) Summary data of mean fluorescence intensity (MFI) of CD40, CD80, and CD86 on DCs.
Cotargeting sMIC increases TCR clonality and repertoire complexity
In general, better clinical outcome in response to immunotherapy is associated with higher clonal expansion of CD8 T cells in patients (55–58). In metastatic melanoma and castration-resistant prostate cancer patients, improved survival in response to anti-CTLA4 antibody therapy has been shown to be associated with TCR clonal-type stability as well as repertoire complexity (55, 57). Surveying TCR3β sequences within the TRAMP/MIC prostate tumor tissues using in situ immunoSEQ technology revealed that B10G5 cotargeting sMIC remarkably increased not only TCR content in tumor tissues but also TCR clonality (Fig. 6, A and B). Immunohistochemistry (IHC) assessing CD8 T cell infiltration in tumors further confirmed the relevance of an increase in tumor TCR content to B10G5 cotargeting sMIC (Fig. 6C). The data indicate that cotargeting sMIC facilitates the recruitment of T cells into the tumors and the subsequent expansion, which presumably leads to the enhanced therapeutic effect of CTLA4 blockade.
Fig. 6 B10G5 cotargeting sMIC during CTLA4 blockade therapy increases T cell content and TCR clonality in tumors.
DNA were isolated from paraffin-embedded tumor sections and subjected to immunoSEQ assay, as previously described (Adaptive Biotechnologies). Tumor in situ TCR abundance (A) and clonality (B) were assayed by surveying TCR3β expression and analyzed with the Shannon diversity index using immunoSEQ Analyzer software. (C) Representative IHC confirming increased CD8 T cell infiltration in tumors with B10G5 cotargeting sMIC.
B10G5 therapy stabilizes CD3ζ in CD8 T cells
We sought to further understand the mechanisms whereby anti-sMIC therapy increases antigen-specific CD8 T cell maintenance during anti-CTLA4 antibody therapy. In vitro studies have shown that sMIC not only down-modulates NKG2D expression but also impairs the TCR/CD3 complex signaling of CD8 T cells by destabilizing its key downstream signaling molecule CD3ζ (38). We thus analyzed the intracellular levels of CD3ζ in splenic CD8 T cells from the cohorts of experimental animals by flow cytometry. Unstimulated splenic CD8 T cells from TRAMP/MIC mice with high levels of circulating sMIC (sMIChi) had diminished intracellular CD3ζ with anti-CTLA4 antibody therapy compared to those with low levels of circulating sMIC (sMIClo) (Fig. 7A). All TRAMP/MICB mice that received B10G5 and anti-CTLA4 antibody therapy had an increased intracellular level of CD3ζ in CD8 T cells (Fig. 7A). A pronounced increase in the level of CD3ζ was also observed in prostate tissues with B10G5 cotargeting sMIC (Fig. 7B). These data suggest that neutralizing sMIC to stabilize CD3ζ and thus sustain TCR activation would allow clonal expansion of antigen-specific T cells and increases in TCR clonality in tumor infiltrates.
Fig. 7 Neutralizing sMIC stabilizes the TCR signaling molecule TCR3ζ.
(A and B) Splenocytes from TRAMP/MIC mice that received anti-CTLA4 monotherapy and a cocktail therapy of B10G5 and anti-CTLA4 were intracellularly stained for CD3ζ. (A) Representative fluorescence-activated cell sorting graphs demonstrating CD3ζ expression in CD8 T cells with different regimens. (B) Summary data of the MFI of CD3ζ expression in CD8 T cells. (C) Representative IHC staining demonstrating the level of CD3ζ in tumor parenchyma.
Using a clinically relevant TRAMP/MICB double transgenic mouse model that closely recapitulates the onco-immunology dynamics of human cancer (39), we demonstrate that the antibody cocktail cotargeting the soluble NKG2D ligand sMIC not only remarkably augmented the therapeutic efficacy of immune checkpoint blockade anti-CTLA4 antibody but also generated a cooperative therapeutic effect with anti-CTLA4 therapy. We show that a cocktail therapy of antibody neutralizing the immunosuppressive effect of sMIC and CTLA4 blockade cooperatively primed DC activation and up-regulated the expression of CD80/CD86 costimulatory molecule on DCs, overcame CD8 T cell tolerance, enhanced TCR/CD3 signaling capacity in CD8 T cells, and increased T cell clonality or repertoire complexity in tumor infiltrates. Given the clinical observations that melanoma patients who developed anti-sMIC autoantibody during anti-CTLA4 therapy displayed better prognosis (47), this is the first proof-of-concept preclinical study to demonstrate that antibody cotargeting soluble NKG2D sMIC and anti-CTLA4 antibody therapy can generate a cooperative therapeutic effect. Our study launched a new viable avenue of combination immune therapy of cancer.
The anti-CTLA4 antibody ipilimumab was approved by the FDA for the treatment of unresectable or metastatic melanoma; however, ipilimumab was discontinued for adverse reactions in 10% of patients in a clinical trial for melanoma (www.cancer.gov/about-cancer/treatment/drugs/fda-ipilimumab). In clinical trials with castration-resistant prostate cancer patients, there were ipilimumab toxicity–related deaths, although some subgroups of patients benefited from ipilimumab (18, 59–62). Of all the treatment toxicity–related clinical manifestations, the IRAE of colitis was the most frequent and most severe event that had even led to death (13, 20). Currently, there is no biomarker to predict the patient population that will elicit colitis-related adverse reactions to ipilimumab therapy. Our unexpected findings in TRAMP/MIC mice suggest that high levels of tumor-derived sMIC may be one of the co-founders and also indicators for anti-CTLA4 antibody therapy–induced autoimmune colitis in MIC+ cancer patients. We acknowledge that our current findings cannot be generalized to MIC− cancer patients. However, because MIC is broadly expressed by nearly all human tumors, prescreening cancer patients for serum sMIC may be necessary to eliminate the potential intestinal inflammation–related IRAEs of anti-CTLA4 antibody therapy. Although not addressed here, large-scale retrospective clinical investigations on anti-CTLA4 antibody therapy are warranted to support this concept.
Our data demonstrate that multiple pathways may be involved in the cooperative therapeutic effects of antibody targeting sMIC and anti-CTLA4 antibody. We previously showed that monotherapy with the sMIC-neutralizing antibody B10G5 up-regulates CD80 and CD86 expression on DCs in tumor-dLNs (42). Therefore, neutralizing sMIC in combination with anti-CTLA4 antibody would provide superior availability of CD80 and CD86 to CD28 compared to respective monotherapy. This mechanism was supported by our data demonstrating that combination therapy resulted in a marked increase in the expression of CD80 and CD86 on DCs from tumor-dLNs and tumor parenchyma. Given that NKG2D and CD28 are known to provide nonredundant costimulatory signals to CD8 T cells (35) and that neutralizing sMIC has been shown to restore surface NKG2D expression on CD8 T cells (42), increased dual costimulation by NKG2D and CD28 would also contribute to the heightened effects of combination therapy. Last, colleagues and our current study consistently showed that sMIC destabilizes the critical TCR signaling molecule CD3ζ on CD8 T cells during prolonged stimulation (38), which would impair antigen-specific CD8 T cell function and, in turn, restrict its clonal expansion. Thus, stabilizing the TCR/CD3 complex and restoring antigen-specific TCR signaling in CD8 T cells by B10G5 also contribute to the enhanced response to anti-CTLA4 antibody therapy. Our data of increased TCR repertoire clonality and CD3ζ expression in the tumor tissue in response to B10G5 therapy corroborated this underlying mechanism.
Notably, animals with high levels of circulating sMIC developed severe colon inflammation or colitis in response to anti-CTLA4 antibody therapy; an antibody neutralizing sMIC alleviated this adverse autoimmune effect. This biology was never observed or reported in other preclinical mouse models with anti-CTLA4 antibody therapy. The NKG2D signaling pathway has been associated with inflammatory bowel disease in humans (63–65); however, this finding is not conclusive in mice because of the differences in the models studied, some of which do not naturally express NKG2D ligands (66, 67).
We also observed progressive lung metastasis in sMIChi TRAMP/MICB mice in response to anti-CTLA4 antibody monotherapy. One limitation to the current study is that we were not be able to determine whether increased lung metastasis and development of colitis were related as a cause and an effect or whether both were the collateral or concurrent events of a common cause. Studies have shown that anti-CTLA4 antibody therapy selectively propagates specific species of gut microbiota (68). Studies have also shown that gut microbiota could regulate NKG2D expression in the gut and that NKG2D signaling in IFN-γ–producing intestinal CD4 T cells plays a role in the pathogenesis of colitis (66, 69, 70). Together, it is conceivable to propose that anti-CTLA4 antibody therapy–induced colitis in sMIChi mice may result from the interplay of microbiota composition and NKG2D signaling in pathogenic CD4 T cells in the gut. Moreover, gut microbiota has also been shown to regulate the therapeutic efficacy of CTLA4 blockade (68). Thus, whether the development of colitis and increased lung metastasis are related and how they interplay could be complicated biological events. These questions are under active investigation.
MIC is a human molecule that is not expressed by rodents. Because of this limitation, preclinical studies on CTLA4 blockade therapy to date have not had the opportunity to consider the immune modulatory impact of sMIC. Using an "MIC humanized" double transgenic model, we demonstrated that antibody neutralizing sMIC not only enhances the therapeutic efficacy of anti-CTLA4 antibody but also generates a cooperative therapeutic effect with anti-CTLA4 antibody. Given that sMIC was prevalent in a broad range of malignancies and that anti-CTLA4 antibody is currently in active clinical trial for cancers other than melanoma, our study revealed a new avenue to enhance the therapeutic effect of anti-CTLA4 antibody and concurrently eliminate therapy-induced autoimmune colitis. This concept was supported by a published case report in a melanoma patient (47) and our current case study in a metastatic prostate cancer patient that reveals the association of better clinical prognosis with the development of anti-sMIC autoantibody during early response to ipilimumab. The impact of sMIC on colon inflammation during anti-CTLA4 antibody therapy is an unexpected aspect of our findings. Although the underlying mechanisms may be complex and are not currently understood, our findings nevertheless also suggest a potential clinical biomarker to predict responsiveness or toxicity of anti-CTLA4 antibody therapy in cancer patients. Large-scale clinical investigations are warranted.
Antibodies and flow cytometry
Single-cell suspensions from spleens, dLNs, non-dLNs, or tumor tissues were prepared as previously described (42). Combinations of the following fluorochrome-conjugated antibody were used for cell surface or intracellular staining to define populations of NK, CD8, and subsets of CD4 T cells: CD3e (clone 145-2c11), CD8a (clone 53-6.7), CD4 (clone GK1.5), NK1.1 (clone PK136), NKG2D (clone CX5), CD44 (clone eBio4B10), CD11c (clone N418), MHCII (clone M5/114.15.2), CD80 (clone 16-10A1), CD86 (clone PO3), CD40 (clone 1C10), and CD3ζ (clone 6B10.2, BioLegend). For ex vivo restimulation, freshly isolated single-cell suspension was cultured in complete RPMI 1640 medium containing PMA (50 ng/ml) and ionomycin (500 ng/ml) for 6 hours before it was analyzed for IFN-γ production by intracellular staining with an antibody specific to IFN-γ (XMG1.2). Multicolored flow cytometry analyses were performed on LSR II (BD). Data were analyzed with FlowJo software (Tree Star).
Animals and antibody therapy
All animal experimental procedures were approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina (MUSC). Mice were maintained at the MUSC Hollings Cancer Center animal facility under specific pathogen–free conditions. Male TRAMP mice at the age of 27 to 28 weeks were randomized into two treatment cohorts: (i) cIgG or (ii) anti-CTLA4 antibody (clone 9H10, BioXCell). In parallel, age-matched TRAMP/MICB double transgenic mice were randomized into four treatment cohorts: (i) anti-sMIC mAb B10G5 [which was previously described (39)], (ii) anti-CTLA4 antibody (clone 9H10), (iii) a cocktail of B10G5 and anti-CTLA4 antibody, and (iv) a cocktail of cIgGs. All antibodies were administered at a dose of 3 mg/kg body weight through intraperitoneal injection twice per week. All animals received the treatment for 8 weeks before they were euthanized at the study end point, unless they succumbed to diseases or adverse effects at an earlier time point.
Antigen-specific T cell tolerance experiment
The experimental procedure was described previously (42). Briefly, CD8 T cells from TCR-I transgenic mice were labeled with CFSE and injected intravenously into animals (2 × 106 cells per mouse) in the respective experimental groups. Animals were sacrificed at the indicated time points to assess TCR-I T cell in vivo frequency with TCR-I–specific H-2Db/TAg epitope I–tetramer (Db/I-tetramer) (71). To assay antigen-specific CD8 T cell responses, a bulked single-cell suspension prepared from the spleen, lymph nodes, and tumor digests was stimulated overnight with 0.5 μM TAg epitope I peptide (SAINNYAQKL) and assayed for intracellular IFN-γ expression of CD8+ or Db/I-tetramer+ T cells by flow cytometry.
Tissue collection
Blood was collected via tail bleeding during therapy and via cardiac puncture after euthanasia. Splenocytes, tumor-dLNs, and non–tumor-dLNs were collected for immunological analyses. Prostates, lungs, livers, kidneys, pancreata, and colons were collected and fixed in 10% neutral fixation buffer, followed by paraffin or Tissue-Tek OCT (optimum cutting temperature) compound embedding for pathological and histological examinations. In some experiments, portions of prostate tumors were used for parathion in single-cell suspensions.
Histology and IHC staining
All collected tissues were sectioned and stained with hematoxylin and eosin for histological evaluation. For IHC staining to detect specific antigens, the following antibodies were used: anti-Ki67 (Neomarkers), anti–cleaved caspase-3 (clone 5A1E, Cell Signaling), anti-CD8 (BD Biosciences), anti-NK1.1 (PK136, eBioscience), anti-SV40Tag (Santa Cruz), anti–arginase I (Santa Cruz), and anti-p63 (Neomarkers). The IHC staining protocol has been previously described (39, 42). All sections were counterstained with hematoxylin for nucleus visualization.
Serum sMIC and cytokine detection
Serum levels of sMICB were assessed using respective sandwich enzyme-linked immunosorbent assay kits (R&D Systems). Serum levels of cytokines were assayed by the Eve Technologies Corporation using the Luminex technology.
High-throughput TCR sequencing in tumor tissues
A high-throughput TCR sequence survey from paraffin-embedded tissue was performed according to protocol described elsewhere (56). Briefly, sections from paraffin blocks of the prostate were collected for DNA extraction using the QIAamp DNA FFPE Tissue Kit (QIAGEN Inc.), per the manufacturer's instructions. The TCRβ CD3 (CDR3β) region was amplified, sequenced, and quantified from a standardized 1200 ng of DNA using the immunoSEQ assay as previously described (Adaptive Biotechnologies) (56, 72). TCR sequence and clonality were determined by the Shannon diversity index using immunoSEQ Analyzer software (Adaptive Biotechnologies) (56).
All results are means ± SEM, unless specified otherwise. Mouse and sample group sizes were n > 5, unless indicated otherwise. Data were analyzed using the analysis of variance unpaired t test. Differences between means were considered significant at P < 0.05. Kaplan-Meier survival curves were generated and analyzed using GraphPad Prism software.
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/5/e1602133/DC1
fig. S1. Induction of subclinical colitis in sMIChi TRAMP/MIC mice in response to anti-CTLA4 therapy.
fig. S2. Recapitulation of the negative effect of sMIC on anti-CTLA4 therapy in a transplantable tumor model.
fig. S3. Antibody neutralizing sMIC eliminates colitis in TRAMP/MICB mice that received anti-CTLA4 therapy.
fig. S4. Detection of anti-sMIC autoantibody in the sera of a small cohort of prostate cancer patients who have metastatic disease and enrolled in a clinical trial (NCT01498978) of ipilimumab in combination with hormone suppression at the Knight Cancer Institute.
fig. S5. Representative graphs of flow cytometry analyses demonstrate that combination therapy of anti-sMIC and anti-CTLA4 antibody remarkably increases CD8 T cell population, activation, and functional potential in tumor-draining lymph nodes and tumor beds.
fig. S6. Marginal response of CD8 T cells to anti-CTLA4 therapy in TRAMP mice.
fig. S7. Circulating sMIC or anti-sMIC autoantibody affects response to anti-CTLA4 therapy in TRAMP/MICB mice compared to MIC-negative TRAMP mice.
fig. S8. Representative graphs of flow cytometry analyses demonstrate that combination therapy of anti-sMIC antibody and anti-CTLA4 antibody cooperatively enhances antigen-specific CD8 T cell anti-tumor responses.
fig. S9. Therapy has no effect on the activation or costimulatory molecule on DCs in the spleen or non–tumor-dLNs.
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S. Cording,
G. Eberl,
A. Schlitzer,
F. Ginhoux,
S. Mani,
T. Yamazaki,
N. Jacquelot,
D. P. Enot,
M. Bérard,
J. Nigou,
P. Opolon,
A. Eggermont,
P.-L. Woerther,
E. Chachaty,
N. Chaput,
G. Kroemer,
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, Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. Science 350, 1079–1084 (2015).
C. H. F. Hansen,
T. L. Holm,
Ł. Krych,
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, Gut microbiota regulates NKG2D ligand expression on intestinal epithelial cells. Eur. J. Immunol. 43, 447–457 (2013).
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Acknowledgments: Funding: This work was supported by the NIH–National Cancer Institute (NCI) grants 1R01CA149405, 1R01CA208246, and 1R01CA204021; by the Department of Defense–Prostate Cancer Research Program award W81XWH-15-1-0406 (to J.D.W.); partly by the Flow Cytometry Core Facility Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313); and by CanCure LLC (1R41CA206688). Z.L. was funded by NIH–NCI (P01CA186866) (funding period: 01 September 2015 to 31 August 2020). D.L., G.L., and K.F.S.-O. were supported by NIH–NCI grant 1R01CA164335-01A1. Author contributions: J.D.W. conceived the concept, directed all the studies, and prepared the manuscript. J.Z. performed all animal experiments and data analyses. D.L., G.L., and K.F.S.-O. prepared the TCR-I CD8 T cells and assisted with the experiments. J.N.G. provided patient serum samples and associated clinical information. J.N.G. also contributed to manuscript preparation. Z.L. participated in the manuscript preparation. Competing interests: J.D.W. is on the scientific advisory board of CanCure LLC. All other authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.
Vol 3, No. 5
You are going to email the following Antibody-mediated neutralization of soluble MIC significantly enhances CTLA4 blockade therapy
By Jingyu Zhang, Dai Liu, Guangfu Li, Kevin F. Staveley-O'Carroll, Julie N. Graff, Zihai Li, Jennifer D. Wu
Science Advances 17 May 2017 : e1602133
Cooperative therapeutic effect of anti-CTLA4 and anti-sMIC antibodies.

redpajama_set_name: RedPajamaCommonCrawl


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text:
Find us Search
Archive (2011-2018)
Hive Spring
Witte de With & Spring present
Moderation(s)
Do you believe in productive misunderstandings? Does moderation always entail translation? Are you more, or less, defined by geography? When I say "satellite geography," are you annoyed or amused? A project is a fragment of the future: true or false? If asked, could you explain the difference between moderation and mediation? Without being cynical: aren't you tired of cynicism? Would you say accommodation is of great or only moderate importance to you? How often, if ever, do you feel manipulated? When you hear "a cross-continental stage for play and production," does it confound you? Would you say yes if I asked you to moderate me? Do you believe streams of energy can persist beyond a year's time? Are you taking pleasure in any of this? Should you?
January 7, 2013 – August 17, 2014
Moderation(s) brings together an international group of artists, curators, and writers including A Constructed World, Nadim Abbas, Lee Ambrozy, Oscar van den Boogaard, Michael Lee, Gabriel Lester, Christina Li, Bik van der Pol, Eszter Salamon, and Koki Tanaka, who will participate in a program of contemporary discourse and production lasting for more than year and taking place between initiators Spring Workshop and Witte de With Center for Contemporary Art in Rotterdam. Within this framework, Spring and Witte de With invited Heman Chong, a Singaporean artist, curator, and writer, to steer the program which will involve more than fifty artists and engender a conference, three exhibitions, three residencies, and a book of short stories.
The first project part of Moderation(s) is Bibliotheek (Library), a list of books that functions both as a bibliography for Moderation(s), as well as a physical library installed at Spring and Witte de With. All participants in Moderation(s) are invited to suggest books, creating an ever-expanding common library that can be replicated easily by any individual, community, or institution. You are welcome to download the latest version of Bibliotheek (Library) here.
An overview of Moderation(s) projects:
Curatorial duo Latitudes – no strangers to Rotterdam, having curated Portscapes in collaboration with the Port of Rotterdam and SKOR in 2009 – will be in residence at Spring and produce Incidents of Travel (January 2013), for which the duo has invited artists including Ho Sin Tung, Yuk King Tan, Nadim Abbas, and Samson Young to develop day-long tours that articulate the city of Hong Kong and their artistic practice through routes and waypoints.
Artist Michael Lee will join the project to develop a series of maps and charts that trace the larger constellation of Moderation(s) that will punctuate the year's program with multiple cartographic interventions.
Performances by artists including Mette Edvardsen, Anthony Marcellini, Eszter Salamon, Benjamin Seror, and Koki Tanaka will take place in and around Witte de With in A Thing At A Time during the weekend of 19-20 April 2013. Alongside the programmed performances RoseLee Goldberg, Director of Performa, gives a talk and writer Guy Mannes-Abbott is invited as a witness to reflect upon the two-day program.
For A Fictional Residency, Dutch novelist Oscar van den Boogaard has been invited to be writer-in-residence at Spring. During his stay in Hong Kong he will meet and work with Hong Kong based Nadim Abbas (visual artist), Enoch Cheng (director and writer), Doretta Lau (journalist), and Laurent Gutierrez and Valérie Portefaix, the architectural duo MAP Office.These encounters will inspire a volume of short stories.
Stories And Situations: The Moderation(s) Conference is a day-long conference taking place at Witte de With on 5 October 2013, where discourse and distance are the quintessential subject matters. For this, invited curators Lee Ambrozy, Amira Gad, Christina Li and Xiaoyu Weng work in collaboration with moderator Heman Chong.
A Constructed World will produce The Social Contract at Spring in October 2013, a work in which audiences are asked to sign a legal contract restraining them from speaking about what they saw inside the exhibition.
Finally, from May 22 to August 17, 2014, The Part In The Story Where A Part Becomes A Part Of Something Else, a large-scale show is mounted at Witte de With. This exhibition sublimates many of the conversations and gestures found within Moderation(s) with works from artists including Nadim Abbas, Bik Van der Pol, Michael Lee, Gabriel Lester, Ang Song-Ming, Praneet Soi, Ho Sin Tung, Warren Leung and Sara Wong, Johan Zetterquist.
In speaking about this project, moderator Heman Chong proposes "to make 'soft' the practices of both artist and curator, so that one becomes easily soluble in the other, while retaining their unique forms and patterns of working. The participants will be encouraged to indulge in the pleasures of exchanging knowledge and tools without any pressure to collaborate."
The program kicked off in August 2012 with a performative teaser. Presented at Spring, in Hong Kong, Guilty Pleasures by artist Ang Song Ming engaged the assembled audience as participants in the setting of a listening party. Having been asked to bring along a song they considered to be their "guilty pleasure," the audience members then took turns "confessing" as to why they found their song both enjoyable and incriminating, before playing it for the crowd.
Moderation(s) is initiated by Witte de With's Director Defne Ayas in collaboration with Spring Workshop Founder Mimi Brown.
Date2013 - 2014
TypeEvent
Related Events and Artists
Witte de With
Ang Song Ming
Artist-in-Residence
Moderator of Moderation(s); co-editor of Stationary
Curator-in-Residence
Nadim Abbas
Ho Sin Tung
Yuk King Tan
Latitudes:
Incidents of Travel
*Nadim Abbas Tour
*Open Day
Writer / Publications
Writer-in-Residence
A Fictional Residency
November 1 - December 15, 2013
Betti-Sue Hertz
Writer/Curator
Alana Kushnir
Nov 1 - Dec 15, 2013
Asia Art Archive 亞洲藝術文獻庫
Heman Chong 張奕滿
Hong Kong New Music Ensemble
Claire Hsu 徐文玠
M+ Museum of Visual Culture
Para Site Art Space
Yana Peel
Alexandra A. Seno
Tai Kwun Contemporary 大館當代美術館
Witte de With Center for Contemporary Art
The Hive Spring
info@thehive.com.hk
Spring Workshop
us@springworkshop.org
3/F Remex Centre
42 Wong Chuk Hang Road
Aberdeen, Hong Kong
Click here for map of Wong Chuk Hang neighbourhood
BOOK | A Fictional Residency
A Witness' Blog by Christina Li
Bibliotheek (Library)
PRESS RELEASE: Moderation(s) launch
3/F Remex Centre, 42 Wong Chuk Hang Road, Aberdeen, Hong Kong
us@springworkshop.org / Newsletters / Mailing List / Hive Spring


text:
SIU in the News
SIU in the News - December, 2019
Saturated Buffer Design Research Improves Water Quality
The Capital Journal -- Dec. 31, 2019
Researchers Jon Schoonover, Karl Williard and Gurbir Singh from the College of Agricultural Sciences, Southern Illinois University, Carbondale, Illinois, are building upon past research.
Naked mole-rat has bite force of a 'Tasmanian devil' and may advance treatments for human tooth loss, SIU researcher finds
Southern Illinoisan – Dec. 26, 2019
Diana Sarko, Ph.D., an assistant professor in the anatomy department with the Southern Illinois University School of Medicine, recently published a fifth paper about the strange little hairless, buck-toothed creatures.
New report casts more doubt that museum's stovepipe hat belonged to Lincoln
Chicago Sun-Times – Dec. 24, 2019
pages of writings by Waller's son, Elbert, the former state legislator, held by archivists at Southern Illinois University in Carbondale.
Connector task force finishes report
Republic-Times – Dec. 24, 2019
… like public safety and economic development. Southern Illinois University Carbondale Director of Small Business Incubator Programs Deborah Barnett, Task Force …
The Grandma Test: Are You Explaining What You Do In A Way That Makes Sense?
Forbes.com – Dec. 23. 2019
… emerging industries, Open Assembly engaged the help of linguist Jeffrey Punske, Ph.D., director of undergraduate studies in Linguistics at Southern Illinois University Carbondale. We wanted to explore …
Debate coach: Women won the night
CNN.com – Dec. 20, 2019
Todd Graham is the director of debate at Southern Illinois University. His debate teams have won five national championships and he has been recognized three times as the national debate coach of the year.
40 years later, Southern Illinois archaeologist's finds return to SIU, shedding light on early cultures
… writing books, publishing in academic journals and earning a job as Southern Illinois University Carbondale's curator of archaeology …
SIU Carbondale will no longer require SAT, ACT for admission
Associated Press-KMOV-TV – Dec. 15, 2019
Southern Illinois University Carbondale will no longer require students to take the ACT or SAT to be admitted to the university.
SIU gives honorary degree to actor Bob Odenkirk
"Better Call Saul" star Bob Odenkirk has been designated with a honorary degree from his alma mater in southern Illinois. Southern Illinois University 's Board of Trustees has confirmed the honorary Doctor of Performing Arts degree …
SIU celebrates more than a thousand new graduates, Saturday
WSIL-TV – Dec. 14, 2019
More students can now call themselves graduates of Southern Illinois University Carbondale.
NBC5Chicago – Dec. 14, 2019
SIU Carbondale announces end to SAT/ACT admissions requirements
KFVS-TV – Dec. 13, 2019
Applications for students wanting to attend Southern Illinois University Carbondale may start to look a little different.
SIU Carbondale drops SAT, ACT admission requirement to 'level the playing field'
Joining a national trend, Southern Illinois University Carbondale will no longer require students to take the SAT or ACT standardized tests to gain admission …
ACT and SAT scores no longer required for SIU Carbondale applications
WPSD-TV – Dec. 13, 2019
Your student will not have to stress over standardized testing anymore to get into Southern Illinois University at Carbondale.
Beer 101: As breweries multiply, St. Louis area universities tap into the demand
St. Louis Post-Dispatch – Dec. 12, 2019
… as a way for non-science majors to learn the principles of chemistry through beer. Southern Illinois University Carbondale invested in creating a Fermentation Science Institute in 2013 to host brewing classes and research.
SIU sees increase in student applications
SIU is seeing a significant increase in student applications.
Symbolic Egyptian Head Cones Were A Reality, Archaeologists Find
Forbes.com – Dec. 11, 2019
Writing in the journal Antiquity, an international group of scholars that includes Anna Stevens (Monash University), Corina Rogge (Museum of Fine Arts Houston), Jolanda Bos (Archaeology & Heritage Consultancy), and Gretchen Dabbs (Southern Illinois University) have published the first ever physical evidence of people wearing these head cones.
Scientists Are Contemplating a 1,000-Year Space Mission to Save Humanity
Know All Options – Dec. 11, 2019
… Jeffrey Punske, linguists from the University of Kansas and the University of Southern Illinois, write that "[i]f a trip takes several generations to complete, the language may differ …
Bob Odenkirk says he would 'love to come back' to Carbondale to receive honorary SIU degree
… actor Bob Odenkirk would "love to come back" to one of his "favorite parts of this whole country," he said, to receive the honorary Doctor of Performing Arts degree recently designated to him by SIU Carbondale, his alma mater.
SIU Carbondale applications up significantly as recruitment efforts pay off
Southern Illinoisan – Dec. 4, 2019
"I'm glad to say I can stand here and share some good news with you for a change," said Interim Provost Meera Komarraju on Wednesday as she updated the Southern Illinois University Board of Trustees on SIU Carbondale's efforts to stem two decades of enrollment decline.
SIU School of Law teams with medical students to help veterans
KFVS-TV – Dec. 3, 2019
A unique partnership at Southern Illinois University aims to help Heartland veterans in need.
Poshards' gift to Simon Institute to take SIU student summit to new heights
Former SIU President, Illinois State Senator and U.S. Congressman Glenn Poshard has donated $100,000 to the SIU Carbondale Paul Simon Public Policy Institute …
Southern Illinois University selects next president, pending board confirmation
After a national search, Southern Illinois University has chosen its next leader.
SIU in the News highlights selected media coverage featuring the work and contributions of the university and members of the SIU community. Contact Andrea Steen, University Communications and Marketing, asteen@siu.edu, 618-453-2518, to add or remove faculty and staff from the internal distribution list.


As I type this, it's still Ada Lovelace Day. I intended to write more, but alas have time only to say this: computing and IT companies want to hire more women. IBM UK (where I work) want to see substantially more women applicants for their hundreds of graduate trainee vacancies.
While I don't represent the company on this blog, I'm happy to point people to a recruitment site that talks about what it's like to work in the IBM UK Ideas Lab. The hope is that many more modern-day Ada Lovelaces will apply — not favouritism, just encouragement.
It's also worth noting that these vacancies are for a training scheme. So while applicants, men or women, may have a computing or IT degree, they do not have to — a good degree in any discipline is acceptable.
I have blogged a bit more about this on eightbar.
Ideally, I would have blogged about the achievements of a woman in technology and science. So I'm going to cheat and post a link to a blog by Laura Cowen, who is herself a splendid example of a woman in The Ideas Lab, but who has also helpfully linked to blogs by women who inpired her: Laura Czajkowski and Ana Nelson.
Edited to add: I pressed "save" at one-minute-past-Lovelace! Tsk!
Last year, IBM published one of my inventions and another where I was a co-inventor. I was quite pleased, even though they were published, not patented. Then I saw a churlish article in The Register about IBM's patents — prompting this grumbly personal blog response.
Mind you, some of The Register's other calculations were inaccurate too. And I think they missed a more interesting analysis of the figures that they were handed on a plate by the various source documents.
For example, The Register claims that a company called Hon Hai Precision grew fastest on USPTO patent awards, but then refers to a table of data that shows Hon Hai was up 39% year-to-year whereas Microsoft was up 43%.
On trends, the article says: "if IBM and Microsoft continue at the same pace, Microsoft should slide into the number-two spot behind IBM. Then it's just a matter of time and filings before Microsoft deposes IBM at the top." It fails to make a connection with another observation in the article that "the size of portfolio is the currency that you use to trade to another company".
So how many years on current trends will it be before Microsoft amasses the same amount of total patent "currency" as IBM, Samsung, Canon, Sony or others? Especially as some new patents derive from that existing intellectual property, and those other companies have been amassing thousands every year for many years… in IBM's case, for decades. Maybe that's why in 2003 Microsoft hired Marshall Phelps, the former IBMer who Newsweek said turned IBM's patent portfolio into a $2 billion business.
But why can't companies — IBM or otherwise — do different things for different kinds of patents? And if the article's questioning IBM's accumulation of intellectual "currency", perhaps it could have admitted something else made plain in the press release: IBM also had nearly 4,000 additional technical inventions in 2009, but published them directly instead of seeking patent protection, thereby making the inventions freely available to others in a public database of prior art. Including mine. Fly, my pretties, fly!
Companies like IBM, Samsung, Canon, Sony and (increasingly) Microsoft have a big portfolio of existing patents on which they can develop new intellectual property; and IBM also freely publishes thousands of new technical inventions that others can build on. There you have it, fanbois.
I suppose journalists prefer to write a story about "the Beast of Redmond breathing down everyone's necks" or "Big Blue's mask finally slips after nearly a decade". (Or is it 17 years? Let me check that press release again.) And that's easier to do if you get the basic maths wrong, selectively quote the data, and if you don't bother with much real analysis of the underlying trends. That much is patently obvious.
With my new job I have a new office. The view has improved. Now when I look out of my window, I can see a rather splendid view across the grounds of Hursley Park from my vantage point in the House. Previously, I had a less-than-splendid view across an open-tread stairwell in A Block. The old office was newer and better-appointed than my new home, but opposite me was the lift and the loos.
When I was working at my desk, I might spot some movement in the corner of my eye. I had to resist the instinct to look up and see what it was. Because that inevitably would give the unfortunate impression that I was keeping track of everyone who was using the lift. Or the toilets. It could have been worse, I suppose. I could have been holding a stopwatch.
Now I could use binoculars every morning to track activity from the House as far as the site boundary, and no-one would notice. But I promise not to. Besides, no-one in my new department looks out of the window in the morning. Because that would give us nothing to do in the afternoon.
You can look around the site for yourself with this handy Explore Hursley web page.


text:
Josh Taylor signs with Top Rank
After becoming the winner of the World Boxing Super Series, unified super lightweight champion Josh Taylor was looking forward to big fights in 2020. Now that wish may be one step closer to being true.
Earlier today, Taylor signed a multi-year agreement with Top Rank Boxing and will have his first fight under the promotional banner be televised on ESPN soon. The "Tartan Tornado" is coming off his win over Regis Prograis which was one of the best fights in 2019 to unify the IBF and WBA "super" titles at 140 pounds. That win places him at the top of the division, and he is ready to get in gear under his new contract.
"A new year, a new decade with lots of new beginnings, and I'm starting this new decade with a big bang," Taylor said. "2019 was a huge year for me, but 2020 looks set to be even bigger and I'm delighted to have signed a deal with Top Rank and ESPN and an advisory contract with MTK Global. I believe I am with the best team to take my career to the next level."
Josh Taylor looks to pick up the momentum of his 2019 run by securing big fights in 2020.
This signing was a big addition to Top Rank, as they also have the WBC and WBO champion Jose Ramirez under contract. This sets the stage for a potential fight between Taylor and Ramirez for the undisputed super lightweight championship. That may have been one of the major factors as to why Top Rank wanted to add Taylor to their roster. Even so, Top Rank chairman Bob Arum feels that having Taylor is a great addition.
"Josh Taylor is one of the world's best fighters, and he is a fight fan's fighter, a tough guy willing to fight anyone we put in front of him," said Arum. "Whether it's Jose Ramirez in a fight for the undisputed junior welterweight title or any of the welterweights out there, he's ready for the biggest challenges. I want to thank Josh's advisors at MTK Global, who have the same goal as us, which is to make him an international star."
With Taylor's style being fan-friendly and having two of the major world titles at the weight, he is going to attract good crowds at his fights. MTK Global, who advises Taylor, now have the promotional backing of Top Rank to try to make him one of the most popular fighters in the sport. Taylor is very happy that Top Rank and MTK are working together for him to place his career to bigger heights.
"I couldn't ask for a better partnership, and I know the future looks bright with this team lighting the way."
Cover photo by Stephen Pond/Getty Images
The Boxing Source January 9, 2020 April 29, 2020 boxing, boxing news, Josh Taylor, Top Rank
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Named after its founder, Seymour Mann was a import company that originally specialized in giftware and home accessories. It was founded in l946. Later he, with his wife, Eda began offering dolls, many of which his wife designed.
Not sure if Seymour Mann named all their pattern pieces but Asparagus would seem appropriate for this piece.


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Password will be the root password for your server. If you don't know the root password or haven't set one up, please see https://my.FastDomain.com/hosting/help/whm-login#root-password.


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611502 – SELinux is preventing /bin/bash "getattr" access on /etc/rc.d/init.d/openvpn.
Bug 611502 - SELinux is preventing /bin/bash "getattr" access on /etc/rc.d/init.d/openvpn.
SELinux is preventing /bin/bash "getattr" access on /etc/rc.d/init.d/openvpn.


Aidan (Zach Braff) is a struggling Los Angeles actor who hasn't had a decent role since appearing in a dandruff shampoo commercial ages ago. His beautiful wife Sarah (Kate Hudson) has a well-paying job at the Water Department but her cubemate Jerry (Michael Weston) is a sexist foul-mouthed jerk who doesn't seem to understand just how obnoxious he actually is. The pair have two clever children in the form of Grace (Joey King) and Tucker (Pierce Gagnon), their tuition at the local Hebrew private school paid for by Aidan's judgmental and domineering father Gabe (Mandy Patinkin).
Admittedly, it isn't the life Aidan expected he'd be living, he and his younger brother - something of a minor genius, in actuality a rather lazy slacker - Noah (Josh Gad) dreaming as kids they'd be something akin to superheroes saving the world on a nightly basis. But when Gabe gets sick all of them are forced to put their lives in perspective, coming to grips with the fact that reality and fantasy don't always mix and that the dreams of youth often give way to the practicalities of adulthood - which, as Wish I Was Here takes great pains to point out, isn't necessarily a bad thing.
In fact, discovering the adult life one is living for what it really is can lead to astonishing revelations regarding family, love and commitment that are far more powerful than any dream or fantasy could ever live up to. Reveling in how we mature, understanding the relationships we take the time to establish and explore, learning to relate to one's children as affable generals leading them into a world of unlimited discovery, all of that and more can be there for the taking; making the decision to embrace the challenge and go for that particular brass ring the only choice that truly matters.
Written alongside his brother Adam, the film is Zach Braff's first trip back behind the camera since he released cult existential coming-of-age comedy favorite Garden State back in 2004. While the overall tone and emotional tenor has certainly matured, the motion picture itself offers up many of the same positives and minuses as its predecessor. Showing a knack for human insight, having an idiosyncratic, almost whimsical eye for creative detail, the filmmaker still can't resist slipping into sitcom pabulum from time to time, the narrative thrust sadly stalling out every single time he does.
All the same, when things work they do so marvelously. More than that, Braff has a gift for staging sublime moments of intimate, insightful maturation that are difficult to resist, showcasing Aidan's evolution with a knowing candor that hits close to home. The moments he spends with his two children are by far some of the strongest the film has to offer - one out in the secluded rock-strewn corners of the California desert outstandingly so.
The bits chronicling Noah's ineptitude and overall lackadaisical approach to life and what he wants from it don't resonate near as well as the other elements of the narrative do; and while Gad isn't bad, I can't say I found him particularly pleasing a presence, either. A supposed relationship with a ComicCon costume enthusiast (a massively underutilized Ashley Greene) goes nowhere other than to put an exclamation mark on a rather tired joke, while gags involving the brother's geek street cred are too obvious to be even slightly funny.
On the flipside, Braff's handling of Hudson is magical, the filmmaker reminding us all just how talented the former Almost Famous Oscar nominee truly is. After over a decade in romantic comedy hell, a confinement mostly of her own making, it must be said, the actress comes alive in ways I can't recall seeing in quite some time, making the most of every moment and every scene with magnetic enthusiasm. While what she says isn't always as insightful or as knowing as the Braff brothers hope it is, Hudson makes her lines connect with sparkling authenticity, a seminal moment in a hospital room between her and Patinkin downright stunning and deserving of every tear.
The movie made something of a minor name for itself for being the first high-profile project to be mostly funded via Kickstarter in the wake of the success of Veronica Mars, some trying to make the case a star and filmmaker of Braff's caliber shouldn't have to go this route in order to get his features off the ground. I'm more neutral as far as that debate is concerned, and if there are people willing to give money in order to see films like this come to life I can't begrudge them if they feel the need to donate.
More than that, I'm extremely happy to live in a world in which Wish I Was Here exists. Little missteps aside, moments of melodramatic excess notwithstanding, there is a pleasing sincerity to Braff's story I responded to body and soul. I was moved by Aidan's journey, found that it spoke to me far more often than it did not, his revelations having a profound simplicity to them I was thoroughly captivated by. I cherished my time spent watching this movie. More importantly, I can't wait until I get the opportunity to watch it again.


As one of Chicago's most beloved parks, Maggie Daley is home to plenty of options to spend a gorgeous fall day outside, including tennis courts, a skating ribbon, a climbing wall and running trails. Named after Chicago's longtime first lady, Maggie C. Daley, the park is dedicated to providing blissful outdoor space for Chicago's residents. A weekend or two at Maggie Daley Park before the cold weather sets in is the perfect way to get outdoors.
A healthy lifestyle isn't complete without focusing on the mind. Chill offers meditation sessions, massage appointments and yoga classes, all designed to help you relax and find peace within your hectic schedule. A gentle yoga session is a great antidote for a long workday (and a massage afterwards is always a good idea).
What better way to welcome autumn than with an afternoon of strolling through the Zoo? Embrace the start of the season with a short trip up to Lincoln Park for the Zoo's fourth annual Fall Fest. Pick a few pumpkins, ride a Ferris wheel and wave hello to the animals, all within the heart of the city. Fall Fest starts on September 28th, running until the end of October.
If you prefer to workout in the comfort of your own home, 1000M Level 11 Fitness Center is equipped with a Strength Training Room, Cardio Room, and a Multi-purpose Studio for yoga, barre or HIIT classes. If the weather cooperates, our Level 11 Sun Deck has an outdoor yoga area and meditation space for al fresco fitness. Or let your mind take the day off with our Virtual Meditation Room. Whatever way you prefer to stay active, 1000M has everything you need to curate a healthy, holistic life.


I would like to have a chart of my planets and their statistics. I prefer to see the efficiency, the production, type, population, dissatisfaction, and etc. This way, I can manage my planets much better. For example, I won't have to look at every planet to see which is inefficient and etc.


Take note of this name: Roya Sadat, born in 1981 in Herat, Afghanistan. Her first name translates as "Dream." Becoming a filmmaker was her innermost dream, one that she has now been able to realize despite immense barriers. Her debut film as director, "Three Dots," was screened at the first Afghanistan Film Festival at the Cologne Filmhaus that took place April 14-24.
Roya acquired her cinematic skills through "learning by doing." Since her puberty she has been grappling with the theoretical imperatives of the seventh art.
During those years she sat alone in a small room in Herat and read handbooks on filmmaking, by American author Syd Field, among others – the books were translated into Persian by Iranian translators – while the Taliban tried with all their might to make her country into a zone free of film and art.
Just after the barbarian Taliban regime was ousted, Roya took up contact with a handful of Afghan cineastes in Kabul who had begun with the cultural reconstruction of the capital. The director Siddik Barmak – who was at the time busy shooting what became his internationally renowned film "Osama" – thought Sadat's film projects were worth supporting, so he took her on as a script writer for two short films and got a Japanese production company to fund her feature project "Three Dots – Se Noughta."
"Se Noughta" tells the sad story of a single woman named Gul Afrooz living in a village in northwestern Afghanistan along the border to Iran and struggling to raise her three helpless children. Although she is engaged to her lover Firooz, she is forced to marry an old warlord, a khan. Caught up in traditional structures, the Khan sends her to Iran as a drug courier for an opium smuggling ring. The ring is busted and she is arrested.
Sadat uses realistic images to narrate the bitter fate of this woman who flees from misery, poverty, drought and hunger only to land in a prison in Iran.
Is the life of Gul Afrooz typical of Afghani women who live in rural areas?
Roya Sadat: During the period of drought in Afghanistan, which lasted for over six years, many women in this area of the country were left without means and had to fight to survive. They were exposed not only to the forces of nature, but also to the arbitrary rule and the coercion of the khans who ruled in the region. They were forced by the khans to serve as drug couriers. While we were shooting the film, their followers gave us trouble. On the sixth day of shooting some armed clan lords drove us out of the area. They said we were strangers and would have a negative influence on their women. In this region they don't even have television sets. The clans had never seen a film team on which men and women work together.
What does the collaboration between men and women look like in an Afghan film team?
Sadat: Just like a badly equipped, understaffed film team suffering from a lack of professionalism like anywhere else in the world. Only we had guts and faith. We were lacking just about everything: money, lighting, professional support. Sometimes I even had to serve as a cook! We had a lot of problems, right from the beginning.
Sadat: I spent two years looking for a young woman to play the lead role. Under the condition that Gul Afrooz would not carry on any long dialogs with men in the film, her husband said she could play the role. After ten days of shooting he changed his mind, Gul Afrooz had to go home. But then she continued to play the part. Without her strong will and her dedication our project would have been doomed. She was even able to get through the crisis in her marriage afterwards.
Except for the technical problems with your film, "Three Dots" has an aura of authenticity. Is this becoming a trend in Afghan cinema? Is there a general language for Afghan cinema?
Sadat: It is too early yet to speak of "Afghan cinema." It will take a while until such a thing can be discovered and defined. But if you are talking about the objective content of a film, then perhaps you can speak of Afghan cinema. That is, films that grapple with the realities in our society and offer an unsparing description of it, for example the film "Osama." The fact is, however, that "Afghan cinema" at the moment is trying to show reality rather than create it on film. Because at the moment we are in a phase of working through things. We are busy dealing with reality, which is unfortunately composed of misery, poverty and injustice.
Is this development being influenced by outside elements – for example by Iranian film?
Sadat: Iranian cinema certainly has a longer tradition than ours and is more progressive. The Afghani filmmakers imitate but not their Iranian colleagues. In addition, Iran and Afghanistan have – historically speaking – a similar culture and a common language. The aesthetic similarities can perhaps be traced to these communalities. I am sure that Afghan cinema will soon develop its own film language and its own unmistakable identity.


The Defense Ministry today signed a memorandum of cooperation with the Chung-Shan Institute of Science and Technology and CSBC, one of the nation's largest shipbuilders, to domestically develop and manufacture submarines in Taiwan. President Tsai Ing-wen hailed the agreement, which authorities hope could lead to Taiwan deploying its first sub around 2027.
President Tsai Ing-wen was at Zuoying Military Harbor in Kaohsiung, where she boarded one of the nation's new locally made Panshi fast combat support ships before it took to sea as part of the Navy's Dunmu Fleet. It was the first time that a sitting president had personally helped send off a departing naval fleet.
The fleet will carry out a series of training exercises intended to demonstrate Taiwan's naval readiness. The occasion also marked a step forward for Taiwan's efforts to build its own submarines.
Building submarines is set to be the most challenging part of our policy for domestically producing defense systems. I also know that there are some out there who are skeptical of this program. But I want to tell everyone that the people of Taiwan have always been ready to take on challenges.
Tsai also boarded the ROCS Hai Hu, one of Taiwan's two Dutch-built Chien Lung-class submarines, where she was taken on a tour of the vessel's facilities. CSBC Chairman Cheng Wen-lon was confident that Taiwan could commission its own submarine within 10 years.
I think that 10 years is definitely doable, because 8 years is when we're aiming to do sea tests. The first four years of our current contract is for design work, and it's not until the second stage when we'll start building the vessels. The building stage should take three to four years.
Previous reports suggest that after European countries rebuffed Taiwanese requests for technological assistance in its submarine program, authorities may now be seeking US help to retrofit decommissioned subs from Japan. CSBC, though, denied such speculation.


Today, as the blogger once again, I didn't see any caterpillars. I saw at least 3 pods that were opened but that was about it. But there could be some in other sections. Usually, there would and should be caterpillars out and about. There were more last year than this year. Is there something wrong this year?


text:
Diekholzen – gmina samodzielna () w Niemczech, w kraju związkowym Dolna Saksonia, w powiecie Hildesheim.

Współpraca
Miejscowości partnerskie:
  Combloux, Francja
  Karow, Meklemburgia-Pomorze Przednie

Przypisy

Bibliografia
 Landesbetrieb für Statistik und Kommunikationstechnologie Niedersachsen, Hanower, 2009

Powiat Hildesheim
Gminy w Dolnej Saksonii

redpajama_set_name: RedPajamaWikipedia


Hanukkah begins at sunset today, so I thought I would write a little about this holiday. Hanukkah is the Jewish Festival of Lights. It is an eight day celebration which lasts from the twenty-fifth day on Kislev to the second day of Tevet. Since the Hebrew calendar is a lunisolar calendar, the days float around from November to December in the Gregorian calendar. This year the days of Hanukkah are celebrated December 24-Jamuary 1.