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

{"title":"Abstract A174: The anti-LAG-3 antibody REGN3767 promotes immune activation in the tumor microenvironment and enhances antitumor activity of anti-PD-1 antibody REGN2810 in PD-1/LAG-3 humanized mice","authors":"E. Burova, Gabor Halls, Omaira Allbritton, Wen Zhang, W. Olson, M. Mohrs, G. Thurston, E. Ioffe","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A174","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A174","url":null,"abstract":"The immune checkpoint receptor lymphocyte activation gene 3 (LAG-3) inhibits proliferation and cytokine production by activated CD4 and CD8 T-cells upon binding to major histocompatibility complex class II (MHC II). Another inhibitory receptor, programmed death 1 (PD-1), and its ligands PD-L1 and PD-L2, are critical for the establishment and maintenance of peripheral T-cell tolerance. Because PD-1 and LAG-3 signaling in the tumor microenvironment enables tumors to escape immune surveillance, combined targeting of PD-1 and LAG-3 has emerged as a promising therapeutic strategy. REGN3767 is a human monoclonal antibody that binds LAG-3 with high affinity and rescues T-cell function by blocking its interaction with MHC II. Similarly, the human monoclonal antibody REGN2810 blocks PD-1 interaction with PD-L1 and PD-L2. To test the in vivo activity of REGN3767 alone or in combination with REGN2810, we generated humanized PD-1/LAG-3 knock-in mice, in which the extracellular domains of mouse Pdcd1 and Lag3 genes were replaced with the corresponding regions of human PD-1 and human LAG-3, respectively. In these mice, combined REGN2810 and REGN3767 therapy reduced MC38.Ova tumor growth and prolonged survival, compared to REGN2810 and REGN3767 monotherapies. To explore the underlying molecular mechanisms, we conducted RNA sequencing and RT PCR analyses of MC38.Ova tumors in PD-1/LAG-3 humanized mice following administration of one or two doses of REGN2810, REGN3767, or the combination. Both REGN3767 and REGN2810 promoted robust transcriptional changes in tumors, and combined PD-1 and LAG-3 blockade resulted in enhanced immune activation signatures. Analysis of differentially expressed genes revealed T-cell expansion and activation induced by either antibody monotherapy. While tumor immune responses to REGN3767 were delayed compared to REGN2810, both agents induced similar gene changes via shared pathways. In addition to T-cell activation, REGN2810 or REGN3767 treatments engaged other tumor-infiltrating leukocytes, including neutrophils, myeloid and NK cells. Stronger tumor growth inhibition in response to REGN2810 or REGN3767 therapy was associated with more robust immune activation signatures. Combination of REGN2810 and REGN3767 enhanced antitumor efficacy, resulting in gene expression changes not seen with either monotherapy. The combination therapy also enhanced immune responses promoted by either antibody alone, including genes associated with T-cell activation and effector function. REGN2810 and REGN3767 combination also increased the expression of other checkpoint and costimulatory molecules, including CTLA-4, CD40 and VISTA. The robust intratumoral immune activation associated with antitumor efficacy in preclinical setting supports the clinical development of REGN2810 and REGN3767 as a combination cancer immunotherapy. Citation Format: Elena Burova, Gabor Halls, Omaira Allbritton, Wen Zhang, William Olson, Markus Mohrs, Gavin Thurston, Ella Ioffe. The ","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"61 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":"125515462","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 A216: Functionally specialized subsets of exhausted CD8+ T-cells mediate tumor control and response to checkpoint blockade","authors":"Debattama R. Sen, Brian C. Miller, Rose Al Abosy, K. Bi, M. LaFleur, K. Yates, Ana Lako, K. Felt, G. S. Naik, M. Manos, E. Gjini, Yamini V. Virkud, Stephen F. Hodi, S. Rodig, A. Sharpe, W. Haining","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A216","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A216","url":null,"abstract":"T-cell dysfunction in the tumor microenvironment (TME) is a hallmark of many cancers. Reinvigoration of T-cell function by PD-1 checkpoint blockade can result in striking clinical responses, but is effective only in a minority of patients. The basis for T-cell dysfunction in the TME, as well as the mechanisms by which anti-PD-1 therapy acts on dysfunctional T-cells are not fully understood. Here we show that anti-PD-1 therapy acts on a specific subpopulation of CD8+ tumor-infiltrating lymphocytes (TILs) in melanoma mouse models as well as patients with melanoma. We find that dysfunctional CD8+ TILs possess canonical epigenetic and transcriptional features of T-cell exhaustion, mirroring those seen in chronic viral infection. Similar to chronic viral infection, exhausted CD8+ TILs contain a subpopulation of “stem-like exhausted” T-cells that have a distinct regulatory state. Stem-like exhausted TILs also have critical functional attributes that are not shared by the majority “terminally exhausted” TILs: they retain more polyfunctionality, persist following transfer into tumor-bearing mice, and differentiate to repopulate terminally exhausted TILs in the TME. As a result, stem-like exhausted CD8+ TILs are better able to control tumor growth than terminally exhausted cells. Stem-like exhausted, but not terminally exhausted, CD8+ TILs can respond to anti-PD-1 therapy without reversion of their exhausted epigenetic state. CD8+ T-cells with a stem-like exhausted phenotype can be found in human melanoma samples and patients with a higher fraction of this subpopulation in their tumors have a significantly longer duration of response to combination checkpoint blockade therapy. Responsiveness to checkpoint blockade is therefore restricted to a subpopulation of exhausted TILs that retain specific functional properties which enable them to control tumors. Approaches to expand stem-like exhausted CD8+ T-cells in the tumor microenvironment may be an important component of improving checkpoint blockade response. Citation Format: Debattama R. Sen, Brian C. Miller, Rose Al Abosy, Kevin Bi, Martin W. LaFleur, Kathleen B. Yates, Ana Lako, Kristen D. Felt, Girish S. Naik, Michael Manos, Evisa Gjini, Yamini V. Virkud, Stephen Hodi, Scott J. Rodig, Arlene H. Sharpe, W. Nicholas Haining. Functionally specialized subsets of exhausted CD8+ T-cells mediate tumor control and response to checkpoint blockade [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 A216.","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"39 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":"127061727","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 A192: Non-IL-2 blocking anti-CD25-targeting antibodies: depletion of regulatory T-cells driving optimal effector response for rejection of established tumors","authors":"A. Goubier, I. Solomon, F. Vargas, D. Zervas, C. Qing, Josephine Salimu, Mark A. Brown, P. Merchiers, K. Peggs, S. Quezada","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A192","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A192","url":null,"abstract":"The accumulation of regulatory T-cells (Tregs) in the tumor hampers effector antitumor activity and correlates with a poor prognosis in several human cancers. Increasing the effector T-cell (Teff) to regulatory T-cell (Treg) ratio is known to result in improved control of established tumors. Studies demonstrating high levels of CD25 expression on Tregs and only low levels on Teff in human tumors have underscored its relevance as a target for Treg depletion. This supported the development of anti-CD25 depleting monoclonal antibodies (mAbs) as a promising monotherapy as well as combination partner in cancer immunotherapies. To date, anti-CD25 antibodies for clinical use have been designed to block IL-2 binding and signaling through the IL-2 receptor complex and also depletes CD25 positive cells. Since IL-2 is a critical cytokine involved in T-cell activation and proliferation, we hypothesized that a depleting antibody targeting CD25 but unable to block IL-2 signaling would promote a more potent anti-tumor response by preferentially depleting Tregs while still allowing IL-2 to stimulate effector T-cells expressing low levels of CD25 on their surface. We therefore compared the functional activity of anti-mouse CD25 depleting mAbs with and without IL-2 blocking activity. After having confirmed their differential impact on IL-2 signaling in vitro and in vivo, we evaluated the therapeutic activity of these mAbs in various syngeneic tumor models. While both the IL-2 blocking and non-IL-2 blocking mAbs showed equivalent Treg-depleting activity, the antibody sparing IL-2 signaling (αCD25NIB) promoted stronger antitumor effect than the IL-2 blocking mAb, with complete tumor regression observed in 70-100% of the mice after a single administration of the antibody. We demonstrated that the αCD25NIB would be an ideal partner for combination against several tumor types including “immune cold” tumors and αPD-L1 resistant tumors. Our data demonstrate that targeting CD25 with ADCC enabled antibodies preserving IL-2 signaling is a novel and powerful strategy for rejection of established tumors via depletion of Treg cells and enhanced, cell intrinsic, IL-2-driven effector T-cell activation. Citation Format: Anne Goubier, Isabelle Solomon, Frederick Arce Vargas, Dimitrios Zervas, Chen Qing, Josephine Salimu, Mark Brown, Pascal Merchiers, Karl S. Peggs, Sergio A. Quezada. Non-IL-2 blocking anti-CD25-targeting antibodies: depletion of regulatory T-cells driving optimal effector response for rejection of established 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 A192.","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":"125420156","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 A184: T-cell rejuvenation is associated with vorinostat-induced immune response in combination with immune checkpoint blockade","authors":"Nur Pradani Damayanti, Justin A. Budka, Josue D. Ordaz, A. Orillion, K. Ahmed, Xi Chu, Yue Wang, Yunlong Liu, R. Pili","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A184","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A184","url":null,"abstract":"Background: Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown clinical benefit in solid tumor patients, including renal cell carcinoma (RCC). However, the rate of clinical response remains modest. Growing evidence suggests that epigenetic modifying agents may have an immunomodulatory role. Our group has previously demonstrated that the selective class I histone deacetylase (HDAC) inhibitor entinostat decreases the function of regulatory T-cells (Treg) and myeloid-derived suppressor cells (MDSC), and synergizes with PD-1 blockade. In this study, we assessed the immunomodulatory activity and efficacy of combining PD-1 blockade with the pan-HDAC inhibitor vorinostat in a RCC model. Methods: To test the efficacy of a combination therapy with a PD-1 inhibitor, mDX-400 (10 and 20 mg/kg I.P) (Merck & Co., Inc.) and a pan-HDAC inhibitor, vorinostat (100 and 150 mg/kg I.P) (Merck & Co., Inc.), we utilized a syngeneic mouse model of metastatic RCC following orthotopic implantation of luciferase expressing RENCA cells in immunocompetent BALB/c mice. Antitumor activity was assessed by bioluminescence technique as well as end point measurements of tumor weights. Immune landscape profiling of tumor infiltrating lymphocytes (TILs) was performed by flow cytometry, immunohistochemistry, and immunofluorescence. Survival analysis was performed by Kaplan–Meier estimates and log-rank statistic. Differences in chromatin accessibility in peripheral blood mononuclear cells (PBMC) were assessed by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq). Results: Statistically significant reductions in end point tumor weights, as well as lung metastases nodules, were observed in mice treated with the combination of vorinostat (100 mg/kg P=0.0391; 150 mg/kg P=0.0165) and mDX-400 (20 mg/kg) compared to vehicle, while no statistical significant reduction was observed in those treated with single-agent mDX-400. Combination therapy also significantly lengthened the survival of the mice (median survival = 60 days; P=0.009) compared to treatment with the single agent mDX-400 (median survival=42 days). Immune landscape profiling did not demonstrate a significant increase in CD8+ tumor infiltration (P=0.479), but a statistically significant increase in natural killer cell infiltration (P=0.048) was observed. Though the CD8+ tumor infiltration was unchanged, a significant reduction (P=0.049) of exhausted CD8+ T-cells (CD8+PD1+) was observed in the combination treatment compared to mDX400 alone. Furthermore, a decrease was observed in the immunosuppressive Tregs (CD4+FOXP4+) and MDSC (CD11b+Gr1+) in the combination group compared to mDX400 alone. Bioinformatic analyses of ATAC-seq data from the PBMC cells of mice in the combination treatment and mDX400 alone showed increased chromatin accessibility between the two conditions. Pathway analysis of genes associated with more accessible chromatin in the combination treatment than mDX400 ","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"8 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":"116073623","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 A195: Mechanosensory mechanisms and in vivo tissue topology contribute to rheology of circulating leukocytes resulting in efficient post-capillary vessel wall adhesion and recruitment","authors":"A. Huang, Bryan L. Benson, Luis Correa, Lucy Li, Jay T. Myers, U. Gurkan, R. Ransohoff","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A195","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A195","url":null,"abstract":"Efficient recruitment of circulating immune cells to various tissues plays a critical role in homeostasis and immune surveillance, a process that serves as the basis of any successful cell-based immunotherapeutic strategies in cancer, particularly for solid tumors and cancers residing in body sites outside of blood vessels and sinusoid network. Clinical and experimental observations suggest that in vivo leukocyte adhesion and extravasation are maximal near the transition from capillary to post-capillary venule, through a multistep process that includes the intravascular capture, rolling, arrest, crawling of cells through interactions of adhesion molecules (selectins, integrins, chemokines/receptors, for example), eventually leading to transcellular or paracellular transmigration through intact endothelium. These cellular and molecular processes are strongly influenced by a confluence of scale-dependent physical effects. Mimicking the scale of physiologic vessels using in vitro microfluidic systems allows the capture and investigation of these effects on leukocyte adhesion assays, but imposes practical limits on reproducibility and reliable quantification. We have developed a microfluidic platform that provides multiple (54-512) technical replicates within a 15-minute sample collection time, coupled with an automated computer vision analysis pipeline that captures leukocyte adhesion probabilities as a function of not only shear stress imposed on leukocytes within the vessels as conventional wisdom dictates, but also of the extensional stresses imposed by the topology of post-capillary venules and the rheology of circulating leukocytes in these vessels. We identified that in post-capillary channels of physiologic scale, efficient leukocyte adhesion requires erythrocytes forcing leukocytes against the wall, a phenomenon that is promoted by the transitional flow in post-capillary venule expansions and highly dependent on the adhesion molecule ICAM-1. These studies help identified a mechanosensory mechanism that determines the increased likelihood of leukocyte adhesion in post-capillary venules, and further suggests a significant role of mechanosensory channel(s) in influencing leukocyte integrin affinity for cellular capture to the vessel wall. Through a series of truncation mutants, we have narrowed down a small region of putative interacting domain between integrin and a candidate mechanosensory channel—PIEZO1—on leukocytes. These ongoing investigations offer new insights into immune cellular recruitment and new molecular targets to enhance leukocyte recruitment to peripheral tumor sites. Citation Format: Alex Y. Huang, Bryan L. Benson, Luis Correa, Lucy Li, Jay T. Myers, Umut A. Gurkan, Richard Ransohoff. Mechanosensory mechanisms and in vivo tissue topology contribute to rheology of circulating leukocytes resulting in efficient post-capillary vessel wall adhesion and recruitment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR Interna","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"97 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":"122448027","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 A224: Bone marrow T-cells are tumor-infiltrating T-cells in pediatric patients with acute leukemia and their phenotype reflects immune evasion of leukemic blasts","authors":"Semjon Willier, Paula Rothaemel, Jonas Wilhelm, D. Stenger, Theresa Käuferle, I. Schmid, M. Albert, V. Binder, F. Blaeschke, T. Feuchtinger","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A224","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A224","url":null,"abstract":"Object: Acute leukemia is the most common malignancy in children. Despite recent therapeutic advances patients with relapsed or refractory disease require new treatment options. While synthetic immunotherapies such as chimeric antigen receptor (CAR) T-cells have shown impressive efficacy in B-precursor acute lymphoblastic leukemia (BCP-ALL) patients, the interaction between leukemic blasts and bone marrow T-cells remains largely unknown. Therefore, the role for immune response amplifiers in leukemia patients is currently unclear. Leukemia outgrowth leads to low frequency of physiologic bone marrow populations such as T-cells. Those T-cells are consequently within the site of tumor development and can thus be defined as tumor-infiltrating lymphocytes (TILs). Dysfunction of TILs has been described in a variety of solid and in some hematologic malignancies. To determine the changes driven by leukemia blasts we analyzed T-cells in bone marrow samples from pediatric patients with BCP-ALL, T-precursor ALL (TCP-ALL) and acute myelogenous leukemia (AML) at the time of diagnosis and relapse in comparison to healthy bone marrow donors. Material and Methods: In pilot experiments, any artificial changes in marker expression due to cryopreservation and thawing were excluded (n=5). Then, cryopreserved bone marrow samples from both pediatric patients with acute leukemia (n= 77; BCP-ALL: 18, TCP-ALL: 23, AML: 36) and age-matched healthy bone marrow donors (n=23) were identified in our local biobank. Multicolor flow cytometry was performed to quantify co-inhibitory markers on CD4 and CD8 T-cells in primary (n=49) and relapse leukemia samples (n=28). Results: The frequency of bone marrow T-cells was reduced in patients with acute leukemia in comparison with healthy controls (5.9% vs. 24.4%, mean values, p primary > healthy). These findings could reflect insufficient immune surveillance of pediatric leukemia by bone marrow T-cells and may provide a rationale for future therapeutic interventions. Citation Format: Semjon Manuel Willier, Paula Rothaemel, Jonas Wilhelm, Dana Stenger, Theresa Kauferle, Irene Schmid, Michael H. Albert, Vera Binder, Franziska Blaeschke, Tobias Feuchtinger. Bone marrow T-cells are tumor-infiltrating T-cells in pediatric patients with acute leukemia and their phenotype reflects immune evasion of leukemic blasts [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 A224.","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"78 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":"128607522","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 A182: T-cell exhaustion assessment using a fully automated sequential chromogenic multiplex assay","authors":"Aurélie Collignon, Assil Benchaaben, Anna R Martirosyan, M. Duval, Emilie Bonzom, Emmanuel Prestat, C. Haond, J. Fieschi","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A182","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A182","url":null,"abstract":"Cancer immunotherapy by anti-PD-1 or anti-PD-L1 antibodies is now established as an efficient approach to restore the cytotoxic activity of exhausted T-cells. However, the strong and durable response observed in non-small cell lung cancer (NSCLC) patients is limited to a fraction of the treated population. Biomarkers, such as PD-L1 expression on tumor cells or tumor mutational burden, are approved as predictive markers of the response to anti-PD-1 or anti-PD-L1 antibodies. However, they are far from perfect to select patients eligible to immunotherapy. Since exhausted T-cells express more than one checkpoint inhibitor, it is hypothesized that an anti-PD1 antibody may not be sufficient to restore the activity of exhausted cells. Multiplex detection of the major immune checkpoints, i.e., PD-1, LAG-3 and TIM-3, on T-cells within the tumor microenvironment could, on one hand, predict the poor response to a single agent and could, on the other hand, predict the best combination of antibodies targeting more than one immune checkpoint. Here we present an automated sequential chromogenic multiplex assay allowing the assessment of the expression of PD-1, LAG-3 and TIM-3 on CD3+/CD8+ cells on a single FFPE tumor tissue section. Briefly, a tissue section is sequentially stained, digitized, unstained and restained with antibodies targeting the five markers. Images of the whole slide are then analyzed by digital pathology. First, a newly developed software is used to co-register the 5 virtual slides and perform colors deconvolution. Detection of positive cells is performed for each marker independently, using Indica Lab’s HALO software. Then, individual cells can be analyzed to identify complex phenotypes and assess their density on the entire tissue section. In addition, tissue segmentation tools are used to assess densities of exhausted T-cells expressing the major immune checkpoints in parenchyma, tumor stroma and invasive margin regions. The quantitative evaluation of T-cells’ exhaustion based on the expression of more than one immune checkpoint could improve patient stratification and lead to individualized combinations of immunotherapy agents. Citation Format: Aurelie Collignon, Assil Benchaaben, Anna Martirosyan, Matthieu Duval, Emilie Bonzom, Emmanuel Prestat, Christophe Haond, Jacques Fieschi. T-cell exhaustion assessment using a fully automated sequential chromogenic multiplex assay [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 A182.","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","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":"126586772","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 A189: Targeting T-cell epigenetic programs to enhance the efficacy of immune checkpoint blockade","authors":"Hazem E. Ghoneim, Yiping Fan, A. Moustaki, Hossam A. Abdelsamed, P. Dash, P. Dogra, R. Carter, Walid Awad, G. Neale, P. Thomas, Ben Youngblood","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A189","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A189","url":null,"abstract":"Immune checkpoint blockade (ICB)-mediated rejuvenation of exhausted T-cells has emerged as a promising approach for treating various cancers and chronic infections. However, T-cells that become fully exhausted during prolonged antigen exposure remain refractory to ICB-mediated rejuvenation. Given that many of the impaired effector properties of terminally exhausted CD8 T-cells appear to be heritably maintained even in the absence of antigen, we investigated the role of de novo DNA methylation programming as a T-cell-intrinsic mechanism for establishing the ICB-nonresponsive state of T-cell exhaustion. Whole-genome bisulfite sequencing of antigen-specific murine CD8 T-cells at the effector and exhaustion stages of an immune response identified progressively acquired heritable de novo DNA methylation programs. Blocking de novo DNA methylation in activated CD8 T-cells allowed them to retain their effector functions despite persistent stimulation during chronic viral infection. We found that these de novo epigenetic programs are not only critical for establishing T-cell exhaustion, but also restrict T-cell expansion and clonal diversity during PD-L1 blockade treatment. Moreover, these exhaustion-associated DNA methylation programs were acquired in tumor-infiltrating PD-1hi CD8 T-cell. Therapeutic approaches to reverse these programs enhanced antitumor CD8 T-cell responses and tumor control during PD-L1 blockade therapy. These data establish de novo DNA methylation programming as a major T-cell-intrinsic barrier of ICB therapy. Targeting T-cell epigenetic programs provides great potential for fostering more powerful T-cell immunotherapies. Reference: Ghoneim, et al. Cell 2017. Citation Format: Hazem E. Ghoneim, Yiping Fan, Ardiana Moustaki, Hossam Abdelsamed, Pradyot Dash, Pranay Dogra, Robert Carter, Walid Awad, Geoff Neale, Paul Thomas, Ben Youngblood. Targeting T-cell epigenetic programs to enhance the efficacy of immune checkpoint blockade [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 A189.","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"27 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":"131731088","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 A204: A protective GD3-based vaccine increases NKT-cells in a C57BL/6 murine model","authors":"R. Milner, B. Sahay, Matt Cascio, M. Salute","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A204","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A204","url":null,"abstract":"The disialyl gangliosides GD2/GD3 have been implicated in the enhancement of malignancy in a number of human and animal cancers and as a tumor antigen target for immunotherapy. Earlier we reported on a GD3-based vaccine, which protects canine melanoma patients when provided as an adjunct to standard of care (1). Despite a significant increase in the median survival time, the mechanism by which vaccine works remains unclear; therefore, we vaccinated C57BL/6 mice with the GD3-vaccine formulation and appropriate controls and evaluated changes in the immune cells. Methods: C57BL/6 mice were vaccinated weekly for 4 weeks followed by a week of rest. Mice were then euthanized to collect blood, liver, and spleen for evaluation of NKT-cells. Liver lymphocytes were collected by making mono-cellular suspension of liver obtained after enzymatic digestion. Collected lymphocytes were stained for CD3, CD4, TCRβ, NK1.1, CD49b and dead cells. In these experiments untreated and α-galactosylceramide (α-GalCer) treated mice were used as controls. Results: Mice treated with the subcutaneous injections of the GD3 vaccine showed an increase in NKT-cells (NK1.1+CD49b+CD3+CD4+TCRb+) in the liver but failed to show an increase in the spleen and blood when compared with the untreated mice. The mice treated with α-GalCer also showed an accumulation of NKT-cells in the liver; however, the increase in the livers obtained from vaccine-treated mice was 5-10-fold higher compared to the α-GalCer treated mice. Discussion: NKT-cells are the Swiss army knife of the immune system which are capable of producing different cytokines and chemokines to regulate the overall immune system. After their discovery two decades ago, activation of NKT-cells have shown to have a crucial protective role in various infectious, and non-infectious diseases. These cells form a bridge between the innate and adaptive immune cells. The activation and maintenance of these cells are dependent upon the presentation of lipid molecules on CD1 receptors by dendritic cells. Since the target antigen in the vaccine is a lipid (GD3) it is very likely it would be presented on CD1 receptors for the activation of NKT-cells. Due to the lack of validated and suitable canine CD1 receptor reagents, we were unable to define the mechanism behind the hypothetical protection found in the canine GD3 based vaccine (1). In the murine model NKT-cells are well characterized, and provided us with an opportunity to understand the possible selective activation of NKT-cells by the GD3 based vaccine. Our data found an increase in NKT-cells in the liver for mice vaccinated with GD3 and α-GalCer, but no discernible differences were found in blood and spleen between vaccinated mice and normal controls. Further investigation using the B16 melanoma cell line in C57BL/6 mice vaccinated with the GD3 vaccine and suitable controls may identify the NKT response in the tumor microenvironment. Conclusions: While the GD3-based vaccine and α-GalCer ","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"23 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":"134010029","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 A207: PD-1-functionality and CD28 molecule expression in CD8+ T-cells of cancer patients","authors":"B. Palermo, O. Franzese, M. Panetta, V. Ferraresi, G. Alessandrini, F. Facciolo, G. Ciliberto, P. Nisticò","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A207","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A207","url":null,"abstract":"The main objective of cancer immunotherapy is an efficacious control over tumor progression through the generation of a strong and persistent T-cell mediated immune response. T-cells constantly exposed to tumor-associated antigens experience phenotypic and functional changes, acquiring a dysfunctional state due, at least in part, to the expression of co-inhibitory receptors including Programmed Death 1 (PD-1). Nevertheless, recently it has been demonstrated that the main downstream target of PD-1-mediated signaling is CD28, opening an important implication for the immune checkpoint blockade in cancer immunotherapy. Recently we have demonstrated that CD8+ T-cell anti-tumor functional advantage induced by combined chemotherapy before peptide-vaccination is determined by tumor antigen nature, immune-checkpoint phenotype, TCR repertoire diversity and antitumor lytic capability. In particular, in accordance with several recent studies, we have observed that in Ag-specific CD8+ T-cells the co-expression of PD-1 with CD28 confers an exhausted phenotype and a defective antitumor functionality, that may be reverted by the blockade of PD-1, while a subset of Ag-specific CD8+ T-cell clones characterized by high levels of PD-1 in the absence of CD28, and the presence of ICOS, showed high proliferative capability and an AKT-dependent antitumor functionality sustained by ICOS (1-3). Herein, to better elucidate the role of PD-1 in CD8 T-cell differentiation and function, we show that, differently from functional PD1-positive/CD28-negative T-cells, Ag- specific CD8+ T-cell clones are not polyfunctional, not able to lyse tumor cells and did not possess an active AKT pathway when PD-1 and CD28 were co-expressed. This suggests that the AKT kinase was activated in these T-cells expressing PD-1 but not CD28. To clarify the complex role of PD-1 in regulating T-cell functionality on the basis of our observations obtained in tumor Ag-specific CD8+ T-cell clones, we have analyzed the phenotypic and functional distribution of CD8+ T-cells, with respect to PD-1 and CD28 expression, in patients with different types of cancer. Preliminary results indicate that functional distinct PD-1/CD28 CD8+ T-cell subsets can be found in peripheral blood of cancer patients with a pattern of functionality similar to that identified in CD8 T-cell clones. Furthermore, to identity whether the lung tumor microenvironment may influence the frequency and functionality of these T-cell populations, we have compared these subsets in the periphery and tumor site in lung cancer. These results may clarify the complex role of PD-1 in regulating T-cell functionality and may provide a significant perspective for exploiting the functional significance of T-cell subsets defined by PD-1/CD28 expression, to predict and monitor tumor responsive T-cells during immunotherapy treatments based on PD-1 blockade.§ B.P. and O.F. contributed equally to this work. References: 1. Palermo B, Franzese O, et al. OncoImmu","PeriodicalId":170885,"journal":{"name":"Regulating T-cells and Their Response to Cancer","volume":"75 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":"127389626","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}