Regulating T-cells and Their Response to Cancer最新文献

{"title":"Abstract A187: RIG-I agonists reinforce antitumor adaptive immunity and decrease Treg activity in breast cancer","authors":"David L. Elion, Max E. Jacobson, D. Hicks, Bushra Rahman, V. Sanchez, Paula I Gonzales-Ericsson, O. Fedorova, A. Pyle, John T. Wilson, R. Cook","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A187","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A187","url":null,"abstract":"RIG-I like receptors, RNA helicases that sense viral oligonucleotide motifs and activate innate immunity, are gaining interest in cancer therapy, given their ability to redirect immune responses within the tumor microenvironment (TME), and increase efficacy of experimental cancer vaccines. RIG-I agonists are not well studied in breast cancers, a type of cancer that is often considered immunologically “silent.” We recently reported that therapeutic delivery of RIG-I agonists increase tumor-infiltrating leukocytes (TILs) and expression of proinflammatory Th1 cytokines in the 4T1 mouse model of aggressive, metastatic breast cancer through tumor cell-intrinsic mechanisms. However, these studies do not rule out the importance of myeloid immune responders (e.g., macrophages and dendritic cells) in propagating the effects of RIG-I agonists against tumor cells in vivo, nor do they rule out the impact of RIG-I agonists on adaptive antitumor immunity, a subject that is relatively understudied. We assessed the effects of the RIG-I agonist SLR20 on the the activity of effector T-lymphocytes (TEff) and regulatory T-lymphocytes (TReg) in the TME. Interestingly, SLR20 treatment of mouse and human breast tumor cells increased expression of FAS and MHC-I on tumor cells, and caused tumor cells to express T-cell chemoattractants (e.g., CXCL10, RANTES), potentially increasing T-cells recruitment to tumors, and increasing tumor cell susceptibility to TEff recognition and killing. Using an ex vivo co-culture assay in which 4T1 mouse mammary tumor cells were co-cultured with CD8+ T-cells harvested from mice pre-inoculated with SLR20-treated 4T1 tumor cells, we measured the rate of CD8+-mediated tumor cell killing. This approach revealed that T-cells harvested from mice inoculated with SLR20-treated cells caused greater tumor cell killing than what was seen by CD8+ T-cells harvested from untreated mice. We also found that conditioned media harvested from 4T1 cells treated with SLR20 increased clonal expansion of CD3/CD28-activated T-cells above what was seen with conditioned media harvested from 4T1 cells treated with a control oligonucleotide or from untreated 4T1 cells. TGFβ-mediated differentiation of CD4+ T-cells into tolerogenic and immunosuppressive TRegs was measured in cultures of CD4+ T-cells treated with cultured media derived from SLR20-treated 4T1 cells. These studies showed that cultured media harvested from 4T1 cells treated with SLR20, but not from untreated 4T1 cells or 4T1 cells treated with a control ligand, diminished TReg differentiation, and decreased CD4+ T-cells surface expression of PD-1, CTLA4, and CCR8. Importantly, in vivo experiments assessing therapeutic treatment of 4T1 tumors with SLR20 revealed greater tumor growth inhibition when SLR20 was combined with PD-L1 targeting antibodies. Taken together, these findings indicate that therapeutic activation of RIG-I signaling operates at the interface of innate and adaptive immunity within breast ","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"50 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":"127003103","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 A165: Depletion of macrophages in the tumor-draining lymph node enhances dendritic cell-induced antitumor immunity and survival","authors":"Floris Dammeijer, Mandy van Gulijk, Melanie Lukkes, M. Nimwegen, R. Hendriks, T. V. Hall, H. Vroman, J. Aerts","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A165","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A165","url":null,"abstract":"Effective antitumor immunity involves successful priming of tumor-specific T-cells by dendritic cells (DC) in the lymph node (LN), followed by trafficking, infiltration and sustained elimination of tumor cells by T-cells. Immunotherapies aim to facilitate or further invigorate these processes, but at which anatomic sites these therapies act and in which specific patients remains unknown. We and others have identified macrophages as key mediators of immune suppression in the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are capable of negating the effectiveness of multiple conventional- and immune-targeted anticancer therapies. Several current therapeutic strategies aim to deplete or reprogram macrophages; however, as these drugs act systemically, their precise mechanism and site of action remains unclear. Besides TAMs, macrophages in the LN have been identified as potent immune modulatory cells in diverse settings. Using a novel method that allows for the specific interrogation of LN-macrophages, we aimed to investigate the role of LN macrophages in regulating DC-induced anti-tumor immunity. To determine the immune modulatory functions of the tumor on the LN, we extensively characterized the immune contexture and phenotype of the tumor-draining lymph node (TDLN) compared to a distant non-tumor draining lymph node (non-TDLN) using multicolor flow- and histo-cytometry, in an orthotopic mouse model of peritoneal mesothelioma. In addition, the effects of systemic macrophage depletion using an CSF1R-kinase inhibitor or clodronate encapsulated liposomes (CEL) were evaluated. We developed a method to specifically deplete TDLN-macrophages while leaving TAMs and other tissue macrophages intact, by intrapleural (i.pl) injection of low-dose CEL. Using this model, we investigated the immune-regulatory properties of LN-macrophages following adoptive transfer of activated, tumor-loaded DCs in the TDLN by both flow- and histo-cytometry. Comparison of the TDLN and non-TDLN immune contexture revealed prominent shifts in immune cell frequencies and phenotypes, including a decrease in T-cell frequencies and a marked increase in CD169+ LN-macrophages in the TDLN. Systemic macrophage targeting using CSF1R-kinase inhibition or CEL effectively minimized LN-macrophage subsets as well as TAMs, therefore preventing the specific interrogation of LN-macrophage biology during tumor growth and in the context of immune activation. Conversely, titrating CEL doses down to 5% of the total dosing volume injected i.pl. allowed for the specific depletion of LN-macrophages while leaving systemic macrophages undisturbed. LN-macrophages limited the presence of migratory ex vivo activated and tumor-loaded DCs, as indicated by increased counts of CFSE+ DCs in the in the TDLN following adoptive transfer in CEL-pretreated mice. Depletion of TDLN-macrophages increased LN- and blood frequencies and activation status of DC-induced CD8+ T-cells and CD4+ T-helper cells, indi","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"106 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":"132558127","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 A211: Requirement of Treg-intrinsic CTLA4-PKCeta signaling pathway for suppressing tumor immunity","authors":"Christophe Pedros, Hsin-Yu Liu, Ann J. Canonigo-Balancio, K. Kong, A. Altman","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A211","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A211","url":null,"abstract":"The ability of Tregs to control the development of immune responses is essential for maintaining immune system homeostasis. However, Tregs also inhibit the development of efficient antitumor immune responses. Here we explored the characteristics and mechanistic basis of the Treg-intrinsic CTLA4-PKCη signaling pathway that we recently found to be required for contact-dependent Treg-mediated suppression. We show that PKCη is required for the Treg-mediated suppression of tumor immunity in vivo. The presence of PKCη-deficient (Prkch-/-) Tregs in the tumor microenvironment was associated with a significantly increased expression of the costimulatory molecule CD86 on intratumoral CD103+ DCs, enhanced priming of antigen-specific CD8+ T-cells, and greater levels of effector cytokines produced by these cells. Tumor development was reduced similarly in Treg-specific (Prkch-flox x Foxp3-Cre) and globally deficient recipients (Prkch-/-) as compared to wt mice, indicating that PKCη expression by effector cells is dispensable for the development of efficient anti-tumor responses. Similar to mouse Tregs, the GIT-PAK-PIX complex also operated downstream of CTLA4 and PKCη in human Tregs, and GIT2 knockdown in Tregs promoted antitumor immunity. We are now exploring the therapeutic impact of PKCη deletion in tumor-bearing mice using tamoxifen treatment of Prkch-flox x Foxp3-ERT2Cre and Prkch-flox x UBC-ERT2Cre mice as a surrogate for treatment with a highly selective and efficient PKCη inhibitor. Collectively, our data suggest that targeting the CTLA4-PKCη-GIT-PAK-PIX signaling pathway in Tregs could represent a novel immunotherapeutic strategy to alleviate the negative impact of Tregs on antitumor immune responses. Citation Format: Christophe Pedros, Hsin-Yu Liu, Ann J. Canonigo-Balancio, Kok-Fai Kong, Amnon Altman. Requirement of Treg-intrinsic CTLA4-PKCeta signaling pathway for suppressing tumor immunity [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 A211.","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"25 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":"131516192","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 A178: Nr4a transcription factors limit CAR T-cell function in solid tumors","authors":"Joyce Chen, J. Scott-Browne, Isaac F. López-Moyado, Laura J. Hempleman, Hyungseok Seo, T. Sekiya, A. Yoshimura, A. Rao","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A178","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A178","url":null,"abstract":"In cancer immunotherapy, CD19-targeted CAR T-cells have exhibited impressive clinical efficacy against B cell leukemias and lymphomas; however, they have been less effective against solid tumors. This is in part because CAR T-cells enter a hyporesponsive (“exhausted” or “dysfunctional”) state that is triggered by chronic antigen stimulation and characterized by upregulation of several inhibitory receptors and loss of effector function. To identify transcriptional regulators and other candidates contributing to the diminished function of CAR T-cells in solid tumors, we developed a CAR T-cell model in which recipient mice bearing murine melanoma tumors expressing the human CD19 antigen were adoptively transferred with CD19-targeted CAR T-cells. Genome-wide analyses of these mouse tumor-infiltrating lymphocytes (TILs) showed that endogenous CD8+ TILs and CAR T TILs selected for expression of high levels of PD-1 and TIM3 exhibited similar profiles of gene expression and chromatin accessibility, associated with secondary activation of the Nr4a nuclear receptor family of transcription factors by the transcription factor NFAT. We demonstrate that in both CAR T TILs and endogenous CD8+ TILs, the Nr4a proteins Nr4a1 (Nur77), Nr4a2 (Nurr1), and Nr4a3 (Nor1) are prominent effectors of the transcriptional program downstream of NFAT: they promote the expression of inhibitory receptors and genes associated with early stages of the exhausted/ dysfunctional state, and limit effector function. Most importantly, treatment of tumor-bearing mice with CAR T-cells lacking all three Nr4a transcription factors (Nr4aTKO) resulted in tumor regression and prolonged survival. Nr4aTKO tumor-infiltrating CAR T-cells displayed a gene expression profile characteristic of effector function, including increased expression of granzymes, cytokines and the transcription factor T-bet. Many of these gene expression changes were associated with altered regulatory element accessibility near effector genes. Our data identify Nr4a transcription factors as major players in the cell-intrinsic program of T-cell hyporesponsiveness and point to Nr4a inhibition as a promising strategy for cancer immunotherapy. Citation Format: Joyce Chen, James P. Scott-Browne, Isaac F. Lopez-Moyado, Laura J. Hempleman, Hyungseok Seo, Takashi Sekiya, Akihiko Yoshimura, Anjana Rao. Nr4a transcription factors limit CAR T-cell function in solid tumors [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 A178.","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"118 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":"132972909","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 A198: The inhibitory checkpoint molecule NKG2A is upregulated on tumor-infiltrating NK cells and CD8 T-cells in human head and neck tumors","authors":"Michael Korrer, Young J Kim","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A198","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A198","url":null,"abstract":"Background: Immunotherapy has revolutionized cancer therapy by targeting checkpoint molecules found on endogenous immune cells. Presently, the most commonly targeted checkpoint molecules are CTLA-4 and PD-1, which results in response rates of approximately 25% in head and neck squamous cell carcinomas (HNSCC). To improve treatment, additional immune checkpoint molecules expressed in the tumor microenvironment must be identified. Natural Killer Group 2 A (NKG2A) is an inhibitory receptor found on NK cells and CD8 T-cells, the receptor for which is HLA-E, a non-classical MHC molecule often overexpressed in solid tumors. This study aimed to identify if NKG2A is expressed on tumor infiltrating NK cells and CD8 T-cells from human HNSCC tumors as it is a potential therapeutic target. Methods: Fresh human HNSCC tumors were digested with Miltenyi Human Tumor Dissociation Kit and Gentle Macs machine following manufacturers’ instructions. Cells were then stained for surface phenotyping or frozen for functional assays. Results: We analyzed and compared tumor-infiltrating NK cells and CD8 T-cells from HNSCC patients with matched PBMC by flow cytometry for the expression of activating and inhibitory receptors. We found a unique population of effector memory PD-1+ NKG2A+ CD8 T-cells which was absent from the blood. NKG2A+ PD-1+ CD8 T-cells expressed higher levels of CTLA-4 and LAG3 as well as produced lower IFN-γ than NKG2A- PD-1+ CD8 T-cells. Interestingly, NKG2A+ PD-1+ CD8 T-cells expressed higher levels of both Perforin and Granzyme B, suggesting that these cells are cytotoxically potent. We found a similar upregulation of NKG2A in the NK cell population. NK cells from the primary tumor, but not the blood of HNSCC patients, significantly increased expression of inhibitory KIR2DL4, PD-1 and NKG2A. In addition, we determined that the ligand for NKG2A, HLA-E, was abundantly expressed on CD45+ monocytes and T-cells, but was absent on CD45- cells in the tumor. Finally, using the murine melanoma tumor model B16, we found that blocking NKG2A with monoclonal antibodies in conjunction with intratumoral STING injections resulted in significant immune mediated tumor rejection. Conclusions: We believe that this study provides the first characterization of human tumor-infiltrating NKG2A+ PD-1+ CD8 T-cells. We have shown that NKG2A+ CD8 T-cells express the highest levels of cytotoxic markers, suggesting an enhanced capacity to kill tumor cells, yet also the highest levels of checkpoint molecule expression. We have also shown that NKG2A is upregulated in tumor-infiltrating NK cells. Furthermore, we have demonstrated that blocking NKG2A in in vivo mouse models results in tumor rejection. Taken together, we believe this makes NKG2A an ideal therapeutic target to improve antitumor cytotoxic responses. Citation Format: Michael J. Korrer, Young Kim. The inhibitory checkpoint molecule NKG2A is upregulated on tumor-infiltrating NK cells and CD8 T-cells in human head and neck tum","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","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":"125884458","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 A221: Epigenetic therapy restores polyfunctionality of malignant pleural effusion T-cells in patients with non-small cell lung cancer without downregulation of PD-1","authors":"Hsing-Chen Tsai, Yi-Chieh Wu, Shu-Yung Lin, I-Yu Chen, Jih-hsiang Lee, Kuang-Hua Cheng, Ping Wang, Huan-Jang Ko, Wen-Chien Huang, Yi-Jhen Huang, Kai Wei, Chong-Jen Yu, Y. Chiu","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A221","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A221","url":null,"abstract":"Cytotoxic T-cells in patients with advanced tumors are often dysfunctional due to persistent cancer antigen exposure. These dysfunctional T-cells, known as exhausted T-cells, are characterized by an increased expression of inhibitory receptors and the gradual loss of effector functions, which contribute to immune evasion of cancer cells. Immune checkpoint blockade therapy works by removing the inhibitory signals, reversing the T-cell exhausted state, thereby leading to immune-mediated tumor regression. While immunotherapy has become a front-line treatment for many lung cancer patients for its promising clinical outcome, the durability of this reversal of T-cell exhaustion still remains unsatisfactory, which contributes to eventual treatment failure. Emerging evidence in animal models suggests that exhausted T-cells have a distinct epigenetic landscape that remains unaltered following immune checkpoint blockade therapy. Thus, we hypothesized that epigenetic therapy may reprogram tumor-reactive T-cells to enhance their effector functions. We isolated CD3+ T-cells from malignant pleural effusion or primary tumor tissues of 20 lung cancer patients and examined their immunophenotypes, including surface markers (CD4 and CD8), proliferative index (Ki67), as well as inhibitory receptors (PD-1, TIM3, and CTLA4). T-cells were subject to in vitro daily treatment of decitabine, a DNA methyltransferase inhibitor, for 72 hours. Polyfunctional T-cells that co-express effector functions (i.e., TNF-α, IFN-γ, IL-2 and CD107a) were analyzed by multichromatic flow cytometry. We discovered that T-cells in the malignant pleural effusion or primary lung cancer tissues have higher CD4/CD8 ratios as compared with peripheral blood mononuclear cells from healthy volunteers. The majority of T-cells from lung cancer patients exhibited the exhausted phenotype and expressed at least one inhibitory receptor at higher levels. This indicates that T-cells from malignant pleural effusion could potentially reflect dysfunctional immune states in lung cancer patients when tumor tissues are not available. Moreover, treatment of decitabine at clinically relevant concentrations significantly increased the polyfunctionality of PD-1 positive CD4+ and CD8+ T-cells from selected patients. Interestingly, the reinvigoration of polyfunctional T-cell responses did not associate with the downregulation of PD-1. On the other hand, the effects of decitabine on PD-1 negative CD4+ and CD8+ T-cells were only modest. This suggests that malignant pleural effusion T-cells, which are often PD-1 positive, may respond to epigenetic reprogramming via PD-1-independent mechanisms. Our data shed light on the potential uses of epigenetic therapy in lung cancer treatment by immunomodulation. Citation Format: Hsing-Chen Tsai, Yi-Chieh Wu, Shu-Yung Lin, I-Yu Chen, Jih-Hsiang Lee, Kuang-Hua Cheng, Ping-Huai Wang, Huan-Jang Ko, Wen-Chien Huang, Yi-Jhen Huang, Kai-Lin Wei, Chong-Jen Yu, Yen-Ling Chiu. Epigenetic thera","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"4 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":"129856385","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 B202: Defining the role of IL-27 signaling on responses to metastatic melanoma","authors":"Anthony T. Phan, C. Hunter","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B202","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B202","url":null,"abstract":"Checkpoint blockade and cellular therapies for cancer treatment have yielded impressive clinical results. These approaches harness the ability of T-cells, critical mediators of the body’s immune response to infection, to eliminate tumors. Entry of T-cells into the tumor microenvironment exposes them to an array of regulatory signals that impact T-cell differentiation and function. The immunomodulatory cytokine, interleukin 27 (IL-27), produced by tumor cells and immune cells, is a critical regulator of the immune system. A complex literature exists regarding the role of IL-27 in tumor responses and the conflicting conclusions from these studies can be attributed to differences in tumor models and a lack of lineage-specific genetic tools to dissect the impact of IL-27 signaling on particular cell types. We find that IL-27 receptor alpha (WSX1)-deficient mice exhibit reduced tumor burdens following intravenous administration of B16 melanoma in comparison to wildtype mice. Further, the reduced number of macroscopic nodules corresponds with reduced tumor area within the lungs. Analysis of IL-27 reporter mice reveals a population of nonalveolar macrophage/monocytes to be the primary source of IL-27 in the lung as early as day 10 following B16 melanoma challenge. The production of IL-27 in the lung following tumor challenge and the improved resistance to intravenous dissemination of B16 melanoma by WSX1-deficient mice suggests production of IL-27 suppresses immune responses to melanoma within the lung. In line with these data, we find that tumor nodules in the lungs of WSX1-deficient mice have increased infiltration of CD3+ cells observed by histologic analysis in comparison to tumor nodules in wild-type mice. These data support a model where IL-27 suppresses the infiltration or activity of T-cells responding to metastatic melanoma and suggest that novel strategies targeting IL-27 signaling in the tumor microenvironment may synergize with current immunotherapy approaches to improve T-cell responses to tumors. Citation Format: Anthony T. Phan, Christopher A. Hunter. Defining the role of IL-27 signaling on responses to metastatic melanoma [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 B202.","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"9 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":"128497671","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 A173: The extracellular ATP receptor P2RX7 is required for CD8+ T-cells to maintain and respond to chronic virus and melanoma tumors","authors":"Henrique B Silva, S. Jameson","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A173","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A173","url":null,"abstract":"T-cell exhaustion is an acquired state of T-cell dysfunction induced by sustained antigen stimulation (such as in chronic viral infections and cancer). Recently, it was described that exhausted CD8+ T-cells are heterogeneous, with some being preferentially rescued by checkpoint blockade therapy. These cells typically express the chemokine receptor CXCR5 and depend on the transcription factor TCF1 for their development. However, little is known about the extracellular signals required for the generation and function of this subset. Our group has been working with the “danger signal” extracellular ATP (eATP), which sense cellular damage and is recognized by purinergic receptors in mammals. Notably, we recently discovered that the eATP purinergic receptor P2RX7 is crucial for the generation and maintenance of long-lived memory CD8+ T-cells in a cell-intrinsic way, by controlling the mitochondrial function and viability of these cells during acute viral infections. Here, we show that P2RX7 is also required, in a cell-intrinsic way, for CD8+ T-cells to survive and maintain in the presence of chronic antigen. In response to chronic lymphocytic choriomeningitis virus (LCMV Clone 13) infection, we tracked WT vs P2RX7KO LCMV-specific CD8+ T-cells, using specific tetramer staining in germline knock-out vs WT mice, or co-adoptive transfer of antigen-specific (P14) cells into CD4-depleted recipient mice. In response to LCMV-Clone 13, P2RX7KO CD8+ T-cells initially expand but fail to maintain after the first week of infection. This is accompanied by almost no expansion of the CXCR5+ subset – which express higher surface P2RX7 levels. The few CXCR5+ cells that form in the absence of P2RX7 are aberrant in their phenotype, with higher expression of PD1 and Tim3 and lower expression of TCF1. During chronic LCMV, P2RX7KO CD8+ T-cells have increased cell death as early as one week after infection, but no proliferation defects. Correlating with this, both mitochondrial mass and membrane potential (a readout of mitochondrial functionality) of P2RX7KO CD8+ T-cells are severely impaired. Consequently, germline P2RX7KO mice have defective control of viral titers in comparison to WT counterparts. Despite having higher PD1 expression, anti-PD1 therapy with do not induce expansion of adoptively transferred P2RX7KO P14 cells in comparison to WT P14 cells, in pair with the absence of the CXCR5+ subset. Importantly, pharmacologic P2RX7 blockade after establishment of chronic infection leads to a decrease in WT LCMV-specific CD8+ T-cell numbers, indicating P2RX7-mediated eATP sensing is constantly required for CD8+ T-cells to survive during chronic infection. Finally, we used adoptive transfer of in vitro activated WT vs P2RX7KO OT-I cells (in which we observed similar initial activation) and observed that CD8+ T-cells require cell-intrinsic P2RX7 to infiltrate B16-OVA melanoma tumors. Additionally, P2RX7KO mice have impaired B16 melanoma growth control. These results show tha","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"9 11 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":"123803943","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 A196: Systematic identification of markers and drug targets on exhausted CD8+ T-cells","authors":"W. Hudson, Julia Gensheimer, H. Kissick, R. Ahmed","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A196","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A196","url":null,"abstract":"CD8+ T-cells are responsible for detection and killing of hosT-cells expressing non-self proteins. Upon antigen persistence, as in cancer or chronic viral infection, antigen-specific CD8+ T-cells lose their effector function, a phenomenon known as T-cell exhaustion. This process is concomitant with increased surface expression of negative regulatory proteins such as PD-1 and CTLA-4. Blockade of these molecules has become a widespread and successful clinical strategy for the treatment of malignancies. Unfortunately, not all tumor types are responsive to checkpoint molecule blockade, nor do all patients with susceptible tumor types exhibit clinical responses. These failures highlight the need for identification of additional negative regulators on exhausted, tumor-specific CD8+ T-cells. Here, we perform a systematic approach to identify surface proteins expressed on exhausted murine and human CD8+ T-cells. Using RNA-sequencing data from antigen-specific CD8+ T-cells from the mouse lymphocytic choriomeningitis virus (LCMV) infection model as well as data from human tumor-infiltrating CD8+ T-cells, we show high cross-species concordance of genes encoding membrane proteins in T-cell exhaustion. These expression profiles not only include many known checkpoint molecules such as PD-1, but also several proteins that negatively regulate T-cell receptor signaling but are largely unexplored in the process of CD8+ T-cell exhaustion. We validate these results performing by flow cytometry on both mouse and human CD8+ T-cells, including on T-cells obtained from resected human tumor tissue. To substantiate the importance of these markers, we further explore the function of one identified surface protein, CD101, in the murine LCMV infection model. We demonstrate that CD101 is induced on antigen-specific of CD8+ T-cells weeks after infection, in contrast with the early expression of canonical exhaustion markers such as PD-1. We show that antigen-specific CD101+ cells arise from CD101- precursors and that this differentiation process results in a loss of effector function, cytokine production, and proliferative capacity with simultaneous large transcriptomic changes. Together, these results demonstrate that exhaustion-associated surface protein expression patterns are largely conserved between humans and mice and provide a comprehensive catalog of phenotypic markers and potential drug targets for CD8+ T-cell-directed immunotherapy. Citation Format: Will H. Hudson, Julia L. Gensheimer, Haydn T. Kissick, Rafi Ahmed. Systematic identification of markers and drug targets on exhausted CD8+ 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 A196.","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"12 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":"123837555","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 A183: ATOR-1017: A 4-1BB antibody designed for superior safety/efficacy profile in cancer immunotherapy","authors":"E. Dahlén, A. Rosén, K. Barchan, A. Dahlman, P. Ellmark, Tina Furebring, K. Smith","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A183","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A183","url":null,"abstract":"4-1BB (CD137) is an inducible co-stimulatory receptor expressed on activated T-cells and natural killer (NK) cells. Activation of 4-1BB induces proliferation, cytokine production, cytotoxic function and survival of T and NK cells. Most importantly, it has been shown to result in tumor eradication and long-term tumor immunity in numerous experimental tumor models. Thus, 4-1BB agonists may have great potential in cancer immunotherapy. However, the 4-1BB antibodies in clinical development are associated with toxic side effects and/or poor efficacy, potentially limiting their clinical use. ATOR-1017 is an anti-4-1BB IgG4 antibody designed to overcome the limitations observed with other 4-1BB antibodies. It binds to a distinct 4-1BB epitope located in domain 2 and blocks 4-1BB ligand binding. Furthermore, its agonistic effect is dependent on crosslinking by Fcγ Receptor (FcγR) I and IIb. A potentially advantageous safety profile was supported by the lack of observed immunotoxicity in a cytokine release assay based on human PBMC using a panel of proinflammatory cytokines as readout. Furthermore, expression profiling of human tumor and normal tissue demonstrates that 4-1BB and FcγRs are highly expressed in the tumor environment. In contrast, co-expression of 4-1BB and FcγRs in non-tumor tissues, such as the liver, is low. Therefore, ATOR-1017 is expected to result in tumor-localized immune activation, thereby avoiding systemic adverse events, including hepatotoxicity, which has been observed by another, FcγR-independent 4-1BB antibody. Finally, to further investigate the safety profile of ATOR-1017, a pilot toxicology study was conducted. ATOR-1017 binds with similar affinity of approximately 0.2 nM to human and cynomolgus monkey 4-1BB, whereas no detectable binding to mouse 4-1BB is observed. Thus, cynomolgus monkeys were administered four weekly doses of 5, 15 or 50 mg/kg ATOR-1017. No clinical signs were observed at any dose, indicating a favorable safety profile. In conclusion, ATOR-1017 was designed to be highly active in the tumor environment, and to have minimal systemic effects, thereby enabling a superior safety/efficacy profile. This is supported by a preclinical data package, demonstrating dependency on FcγR-mediated crosslinking, a clean profile in cytokine release assay, 4-1BB and FcγRs co-expression in the tumor and no clinical adverse events observed in a repeat dosing pilot toxicology study. ATOR-1017 is in preclinical development with initiation of phase I anticipated in 2019. Citation Format: Eva Dahlen, Anna Rosen, Karin Barchan, Anna Dahlman, Peter Ellmark, Tina Furebring, Karin Enell Smith. ATOR-1017: A 4-1BB antibody designed for superior safety/efficacy profile in cancer immunotherapy [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 ","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"21 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":"132926762","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}