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{"title":"Abstract B190: WASP-dependent actin protrusions mechanically potentiate killing by cytotoxic T-cells","authors":"Fella Tamzalit, Mitchell S. Wang, Weiyang Jin, V. Boyko, John M. Heddleston, C. Black, L. Kam, M. Huse","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B190","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B190","url":null,"abstract":"Cytotoxic T Lymphocytes (CTLs) play a central role in immune responses against intracellular pathogens and cancer. Cytotoxic responses are induced by the formation of the immunologic synapse after the recognition of the peptide-major histocompatibility complex by the T-cell receptor. Synapse formation is associated dramatic reorganization of both microtubules and filamentous actin (F-actin) within the T-cell. This promotes the directional secretion of toxic perforin and granzymes into the intercellular space, enhancing both the potency and the specificity of targeT-cell killing. We have previously demonstrated that force exertion by CTLs at the immunologic synapse was strongly correlated with the cytotoxic potential. Indeed, force exertion enhances cytotoxicity by increasing membrane tension on the target cell, which in turn promotes the pore-forming activity of secreted perforin. This correlation between applied force and biochemical responses, which we referr to as mechanopotentiation, raised the prospect that CTLs might use three-dimensional structures at the immunologic synapse to coordinate force exertion and lytic granules secretion. In the present study, we investigated the mechanisms underlying mechanopotentiation in CTLs using a combination of three-dimensional micropatterned stimulatory substrates and high-resolution imaging. Our results revealed that CTLs couple lytic granule release with the formation of highly dynamic F-actin rich protrusions. These protrusions, which are generated by the Wiskott-Aldrich Syndrome protein (WASP) and the Arp2/3 actin nucleation complex, are required for coordinating force exertion with cytolytic secretion allowing efficient killing. Our results provide insight into how cells organize mechanical output and emphasize the importance of studying complex, communicative interfaces in three dimensions. Citation Format: Fella Tamzalit, Mitchell S. Wang, Weiyang Jin, Vitaly Boyko, John M. Heddleston, Charles T. Black, Lance C. Kam, Morgan Huse. WASP-dependent actin protrusions mechanically potentiate killing by cytotoxic T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B190.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"2 3 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"124336065","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B197: Translational control in macrophages during inflammatory response","authors":"K. Xiang, D. Bartel","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B197","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B197","url":null,"abstract":"Translational control has been increasingly appreciated as a critical gene-regulatory mechanism during immune responses. Previous studies suggested that translation of select cytokines could be regulated by mRNA poly(A)-tail lengths in memory T-cells and macrophages. We carried out transcriptome-wide analysis with mRNA sequencing, ribosome-footprinting profiling as well as poly(A)-tail length profiling in both RAW264.7 cells and mouse thioglycollate-elicited peritoneal macrophages. No coupling between poly(A)-tail length and translational efficiency (TE) was observed in either cell type, arguing against a global translational-regulatory regime mediated by poly(A)-tail lengths. Immune activation with the treatment of lipopolysaccharides (LPS) to these cells did not change the uncoupled regime. Moreover, gene expression landscape in LPS-treated macrophages was dominated by mRNA level changes, with little contribution from TE changes. The up- or down-regulation of TE was not accompanied by concerted lengthening or shortening of poly(A) tails, implying existence of yet unclear translational control mechanisms in these immune cells. Finally, our studies in other biologic systems, including HeLa cells, Xenopus oocytes and yeast suggest that saturating levels of poly(A)-binding proteins might suppress poly(A) tail length-mediated translational control in various uncoupled systems, including immune cells. Citation Format: Kehui Xiang, David P. Bartel. Translational control in macrophages during inflammatory response [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B197.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"125840664","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B152: Generation of monoclonal antibodies against a newly identified target for invasive prostate cancer and their use in the development of an antibody-based targeting therapy","authors":"A. D. Biase, T. Regad, J. Vadakekolathu, D. Powe, H. Jäck, E. Roth","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B152","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B152","url":null,"abstract":"In 2013, cancer immunotherapy was selected as the breakthrough of the year by Science’s editors and monoclonal antibody therapy is considered one of its most successful tools. When castration-resistant prostate cancer (PCa) disease progresses and despite androgen deprivation treatment, it typically metastasizes, leading to poor patients’ prognosis and survival. In this case, immunotherapy represents the only chance of treatment. In a previous study carried out by our team on the role of cytoplasmic PML1 in the context of epithelial to mesenchymal transition (EMT) in PCa2, we identified a new promising PCa target for antibody-based therapy. Our target, of which we cannot disclose the name for patenting reasons and that we will further refer to as Protein-X, is a type 1 transmembrane protein that is specifically expressed by PCa cell. Transcriptome profiling obtained using Nanostring technology with a cancer progression panel showed that the knockdown of Protein-X leads to an upregulation of the epithelial EMT marker E-cadherin in PCa cells, suggesting its role in the process. Furthermore, the clinical value of Protein-X as a diagnostic and/or prognostic biomarker of invasive and aggressive PCa, is also being investigated by a histopathologist using immunohistochemistry analysis of Protein-X’s expression on tissue microarrays (TMAs). To develop a monoclonal antibody-based therapy against Protein-X, mouse monoclonal antibodies (mAbs) were generated using hybridoma technology. MAbs-producing hybridomas were sequenced and purified mAbs were tested for their specificity by flow cytometry, immunofluorescence, ELISA and immunoblotting assays. Using in vitro antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay, we show that the antibodies mediate cell cytotoxicity. This result will be further investigated in vivo using a xenograft mouse model. Future experiments will focus on designing and generating chimeric antigen receptor (CARs) modified adoptive T-cell targeting of Protein-X-expressing tumor cells and bispecific antibodies. In summary, we aim to develop an anti-Protein-X antibody-based therapy against invasive prostate cancer, which will potentially represent a new tool to target aggressive PCa responsible for cancer metastasis and poor prognosis in PCs patients. Citation Format: Anna Di Biase, Tarik Regad, Jayakumar Vadakekolathu, Desmond Powe, Hans-Martin Jack, Edith Roth. Generation of monoclonal antibodies against a newly identified target for invasive prostate cancer and their use in the development of an antibody-based targeting therapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B152.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"122 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134598529","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B183: LFA-1 signals via Crk adapter proteins to induce actin-dependent T-cell migration and mechanosensing","authors":"Nathan H. Roy, Joanna L Mackay, Alexander Buffone, K. Newell, T. F. Robertson, S. Agarwal, M. Karimi, D. Hammer, J. Burkhardt","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B183","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B183","url":null,"abstract":"Allogeneic hematopoietic stem cell transplants (HSCT) are used to treat many malignancies, but the prevalence of graft-versus-host disease (GvHD) limits their overall success. Manipulating T-cell trafficking has emerged as an effective countermeasure, yet downstream integrin signaling pathways have yet to be targeted. We previously found that T-cells lacking the adapter proteins Crk and CrkL exhibit a robust antitumor response while causing little GvHD. We now find that T-cells from mice lacking Crk adapter proteins exhibit defects in LFA-1/ICAM-1 induced actin polymerization, leading edge formation, 2D migration, and integrin-mediated mechanosensing. Under shear flow, Crk/CrkL deficient T-cells fail to migrate upstream on ICAM-1 but migrate normally on VCAM-1, suggesting these integrin ligands relay different outside-in signals. Analysis of LFA-1 signaling reveals that Crk protein expression is required for phosphorylation of c-Cbl and its subsequent interaction with the PI3K subunit p85. Through this mechanism, Crk proteins promote PI3K activity and cytoskeletal remodeling downstream of LFA-1 engagement. Interestingly, this signaling pathway was largely specific to the LFA-1/ICAM-1 interaction, as WT-cells plated on VCAM-1 (which binds and signals through VLA-4) failed to induce high levels of phospho-Cbl, showed diminished PI3K activity, and did not migrate with a defined actin-rich leading edge. Finally, we show that knocking out CrkL alone is sufficient to alleviate GvHD, pointing toward unique roles of the Crk isoforms in T-cell biology. Together, these studies identify key signaling differences downstream of β2 vs β1 integrins that drive T-cell migratory behavior, and identify CrkL as an important factor during GvHD. Importantly, these data provide insight into integrin signaling that could be used to manipulate T-cell trafficking. Citation Format: Nathan H. Roy, Joanna L. MacKay, Alexander Buffone Jr., Krista Newell, Tanner F. Robertson, Sangya Agarwal, Mobin Karimi, Daniel A Hammer, Janis K. Burkhardt. LFA-1 signals via Crk adapter proteins to induce actin-dependent T-cell migration and mechanosensing [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B183.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"20 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"132859792","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B153: Human NK cell distribution memory and residence in tissue sites","authors":"P. Dogra, T. Senda, P. Szabo, D. Carpenter, M. Tóth, Puspa Thapa, M. Snyder, M. Miron, Brahma V. Kumar, D. Farber","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B153","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B153","url":null,"abstract":"Natural killer (NK) cells are innate immune cells with the ability to kill tumor cells without prior exposure. NK cells express multiple activating and inhibitory receptors in addition to the low-affinity immunoglobulin G binding receptor CD16. Accumulating evidence implicates a role of NK cells in not only direct killing of tumor cells, but also in cancer immunosurveillance and preventing metastasis to tissues sites. However, at present the distribution, diversity and tissue driven differences in NK cell function are not well characterized which may have an implication on the anti-cancer potential of tissue NK cells. Through collaboration with LiveOnNY our local organ procurement organization, we receive blood, bone marrow (BM), lung, intestines, tonsil and associated lymph nodes (LN) from research consented organ donors. Here we used this unique tissue resource to investigate the distribution, phenotypic, functional and transcriptional diversity of NK cell subsets in human tissues. We found that NK cells are ubiquitously distributed in human tissues comprising up to 40% of the CD45+ CD14/19- cells in blood, BM, spleen and lung, while only a small fraction (up to 2%) in the intestine and the LN. While blood, BM, spleen and lung are dominated by mature NK cells (CD56dim CD16+), majority of the NK cells in intestine and LN are immature (CD56hi CD16-). Age, sex and CMV sero-status do not show any correlation with NK cell distribution or subset frequency in tissues. Notably, NK cell subset distribution seems to drive functional differences between tissue NK cells, with lymphoid site NK cells expressing lower levels of effector molecules granzyme B, TNFα, Prf, Ifnγ and displaying reduced degranulation compared with their counterparts from blood, BM and spleen. For an in-depth analysis of tissue-mediated effects on NK cell subset functionality, we performed whole-transcriptome profiling on immature and mature NK cells isolated from blood, BM, spleen, lung and LN. Our analysis identified several effector molecules and NK cell surface receptors being differentially expressed between immature and mature NK cells. Furthermore, while mature NK cells of blood and tissues have similar transcriptional profiles, the transcriptional profiles of immature blood and tissue NK cells show tissue driven heterogeneity with differential expression of transcription factors, metabolic enzymes and NK cell surface receptors. Interestingly, the transcriptional signature of immature NK cells is reminiscent of the transcriptional signature of tissue resident memory T-cells showing increased expression of CD103, CD49a, CXCR6. We validated the expression of these markers using multiparameter flow cytometry and found that subset of immature NK cells in mucosal sites (lung and intestine) do indeed express markers of tissue residence. Additionally, by applying trajectory projection algorithm on NK cells from tissue sites, we show that resident NK cells comprise a distinct populati","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"127394339","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B146: The tumor necrosis factor superfamily member RANKL suppresses effector cytokine production in group 3 innate lymphoid cells","authors":"Jennifer K. Bando, S. Gilfillan, Christina Song, Keely G. McDonald, S. C. Huang, R. Newberry, Yasuhiro Kobayashi, D. Allan, J. Carlyle, M. Cella, M. Colonna","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B146","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B146","url":null,"abstract":"While signals that activate group 3 innate lymphoid cells (ILC3s) have been described, the factors that negatively regulate these cells are less well understood. Here we found that the tumor necrosis factor (TNF) superfamily member receptor activator of nuclear factor κB ligand (RANKL) suppressed ILC3 activity in the intestine. Deletion of RANKL in ILC3s and T-cells increased C-C motif chemokine receptor 6 (CCR6)+ ILC3 abundance and enhanced production of interleukin-17A (IL-17A) and IL-22 in response to IL-23 and during infection with the enteric murine pathogen Citrobacter rodentium. Additionally, CCR6+ ILC3s produced higher amounts of the master transcriptional regulator RORγt at steady state in the absence of RANKL. RANKL-mediated suppression was independent of T-cells, and instead occurred via interactions between CCR6+ ILC3s that expressed both RANKL and its receptor, RANK. Thus, RANK-RANKL interactions between ILC3s regulate ILC3 abundance and activation, suggesting that cell clustering may control ILC3 activity. Citation Format: Jennifer Kaoru Bando, Susan Gilfillan, Christina Song, Keely McDonald, Stanley C-C. Huang, Rodney D. Newberry, Yasuhiro Kobayashi, David S.J. Allan, James R. Carlyle, Marina Cella, Marco Colonna. The tumor necrosis factor superfamily member RANKL suppresses effector cytokine production in group 3 innate lymphoid cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B146.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"53 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"123574576","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B171: Elucidating the mechanism of RAG tracking and its impacts on V(D)J recombination","authors":"Cheng-Sheng Lee, Jiazhi Hu, F. Alt","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B171","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B171","url":null,"abstract":"RAG endonuclease initiates V(D)J recombination by binding to the recombination signal sequences (RSSs), introducing DNA breaks between two V(D)J gene segments, and mediating their joining. This process is tightly regulated to generate diverse antigen receptor repertoires and prevent aberrant events that could cause genomic translocations/deletions and lymphoid cancer. RAG can track directionally over chromosomal loop domain in which they reside. Tracking is evidenced by cleavage/joining of short cryptic RSSs lying exclusively in convergent orientation to a bona fide RSS. However, the mechanism of RAG tracking and its impacts remain unknown. Here I established an unbiased assay system by utilizing allele-specific barcode and employing high throughput genome-wide translocation sequencing (HTGTS) to quantitatively evaluate RAG-mediated recombination frequency. I found that insertion of one single bona fide RSS is sufficient to initiate RAG tracking in the c-Myc domain and the recombination between the RSS and its cryptic RSSs seems to follow the 12/23 rule to some degree. When ectopically inserted bona fide RSS pair is 0.5kb apart, convergent configuration of the RSS pair is 4.8-fold more frequent than the tandem configuration. When the distance increases to 267kb, the difference increases to 14-fold, suggesting the contribution of RAG tracking over diffusion-based mechanism increases when the distance between RSS pair increases. In addition, the presence of CTCF-binding element promotes recombination by more than 10-fold. Depletion of Wapl, cohesin remover, to disrupt cohesin turnover reduces V(D)J recombination. The distal RSSs are much preferentially affected. The data suggest that RAG tracking may be driven by cohesin sliding along chromatin. Citation Format: Cheng-Sheng Lee, Jiazhi Hu, Frederick W. Alt. Elucidating the mechanism of RAG tracking and its impacts on V(D)J recombination [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B171.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"5 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"124004481","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B155: Impact of transcription initation on translation regulation by the mTOR pathway","authors":"S. Duttke, M. Chang, C. Glass, A. Berman, C. Benner","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B155","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B155","url":null,"abstract":"The two major processes of eukaryotic gene expression, transcription and translation, are spatially and temporally separated and commonly perceived to be independently regulated. Unexpectedly, the analysis of nascent transcription start sites (TSSs) genome-wide, proposes that alternative TSS can target mRNAs of the same gene to distinct modes of translational regulation.Our preliminary data found the TCT core promoter motif to function as a transcriptionally suboptimal Initiator (Inr) motif. Despite the rapid turnover of most regulatory elements, the TCT motif is remarkably conserved among the promoters of orthologous bilaterians genes. However, the translation of mRNAs starting from TCT but not Inr promoters is preferentially regulated by the Mammalian target of rapamycin complex 1 (mTORC1), likely via the La-related protein 1 (Larp1) that binds mRNAs starting on pyrimidine but not purines. These findings reveal a functional dependency between transcription initiation and translation regulation, and highlight the importance of TSS selection in promoters. Inhibitors of mTORC1, and more recently Larp1, are rapidly moving into clinical cancer therapy trials but did not live up to the expectations in patients. Markers for patient stratification and predicting mTOR inhibitor efficacy are needed. As TSSs reflect the integrated signals of gene regulatory pathways and also appear to impact translational regulation, our future research will address how far alternative TSSs facilitate acquired resistance to mTOR inhibitors in tumors and 2) TSSs can serve as a predictive marker for mTOR inhibitors efficacy. Citation Format: Sascha H. Duttke, Max Chang, Christopher K. Glass, Andrea Berman, Christopher Benner. Impact of transcription initation on translation regulation by the mTOR pathway [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B155.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"17 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"123342190","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B192: CD56 participation in immune effector cell activation and tumor cell eradication: A role for interleukin-15","authors":"H. V. Acker, Maarten Versteven, H. D. Reu, P. Ponsaerts, Z. Berneman, V. Tendeloo, E. Smits","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B192","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B192","url":null,"abstract":"Oncoimmunologists are in a constant search for more effective immunotherapeutic treatments, helping to cure cancer. In this respect, immune cell participation is a key issue. A particularly interesting marker to identify antitumor immune cells is the neural cell adhesion molecule (NCAM), known as CD56. Namely, hematopoietic expression of CD56 seems to be confined to activated immune cells exhibiting some level of cytotoxic properties (Van Acker et al., Frontiers in Immunology 2017). Unfortunately, the current knowledge on the expression and functional role of CD56 is very fragmented. Therefore, we sought to elucidate the role of CD56 expression on various killer immune cells. First, we identified the high motility NCAM-120 isoform to be the main subset on immune cells. Next, through neutralization of surface CD56, we were able to demonstrate for the first time a direct involvement of CD56 in tumor cell lysis exerted by CD56-expressing killer cells such as natural killer cells, gamma delta (γδ) T-cells and interleukin (IL)-15-cultured dendritic cells (DCs). We also detected a putative crosstalk mechanism, suggesting CD56 as a co-stimulatory molecule in the interaction between IL-15 DCs and CD8 T-cells. Finally, by blocking the mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase (PI3K)–AKT pathway, with respectively trametinib and afuresertib, we confirmed our hypothesis that IL-15 stimulation directly leads to CD56 upregulation via the recruitment of shc, binding a phosphotyrosine residue on the IL-2/15Rβ chain. In conclusion, these results underscore the previously neglected importance of CD56 expression on immune cells, benefiting current and future immune therapeutic options. Citation Format: Heleen H. Van Acker, Maarten Versteven, Hans De Reu, Peter Ponsaerts, Zwi N Berneman, Viggo F. Van Tendeloo, Evelien L. Smits. CD56 participation in immune effector cell activation and tumor cell eradication: A role for interleukin-15 [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B192.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"47 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"127829201","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B169: Engineering phase separation of multivalent signaling proteins through the solubility of folded protein domains","authors":"Jonggul Kim, J. Ditlev, M. Rosen","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B169","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B169","url":null,"abstract":"Similar to three-dimensional membraneless organelles, it has been demonstrated that T-cell LAT signaling clusters form through multi-valency driven phase separation. This phase separated cluster was found to promote Arp2/3-complex driven actin assembly in vitro and correlated with increased ERK phosphorylation in cells. However, in this initial study, perturbations used to study phase separation also affect interactions known to be important for T-cell signaling, convoluting the impact of phase separation on signaling with effects due to changes in canonical biochemical interactions. To understand more precisely the role of phase separation in T-cell signaling, I will alter the solubility of folded domains through mutations of surface charged residues to modulate their phase separation propensities, independent of their high-affinity ligand binding interactions. To demonstrate the validity of this approach, I have first applied it to a synthetic system, based on multivalent interactions between the small ubiquitin-like modifier (SUMO) and the SUMO interaction motif (SIM), which will enable us to provide design principles that can be used on T-cell receptor signaling proteins. I demonstrate that it is possible to specifically alter the solubility of folded domains without altering their canonical molecular interactions. The solubility of individual folded domains serves as a predictor of the concentration threshold of phase separation, and phase separation can either be enhanced or abolished with a single mutation. Principles gained from this work on SUMO/SIM will be applied to multivalent proteins involved T-cell receptor signaling, namely Grb2 and Nck1, and the relationship between phase separation and signaling will be firmly established with future implications for rational engineering of T-cells. Citation Format: Jonggul Kim, Jonathon Ditlev, Michael K. Rosen. Engineering phase separation of multivalent signaling proteins through the solubility of folded protein domains [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B169.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"1021 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"132002632","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}