bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A069: Quantification of lymph node transit times reveals differences in the trafficking behavior of regulatory versus conventional CD4+ T cells
Authors: Tong Alexander
Published: November 2016

Regulatory T cells (Tregs) express the canonical marker Foxp3 and are critical for suppressing the immune response in homeostatic and inflammatory conditions. Despite how widely studied CD4+Foxp3+ Tregs are in mice, their physiologic trafficking and scanning behavior of antigen-presenting cells in secondary lymphoid organs (SLOs) have yet to be quantitatively analyzed in the intravital setting. Our laboratory has previously described the dynamics of T cell trafficking through SLOs and scanning of dendritic cells (DCs), revealing that CD4+ T cells and CD8+ T cells exhibit different transit kinetics through lymph nodes (LNs). On average, CD4+ T cells spend less time scanning for self-peptide/MHC (self-pMHC), dwell shorter in LNs, and egress faster compared to CD8+ T cells. Here we use flow cytometry and 2-photon laser scanning microscopy (2-PLSM) to quantitate the homeostatic trafficking behavior and surveillance strategy of Tregs in LNs and to compare their behavior to conventional T cells (Tconvs). Our data reveal surprising differences in Treg versus Tconv trafficking in mesenteric versus other peripheral LNs. We find that Treg LN transit time is heavily dependent on recognition of self-pMHC in peripheral but not mesenteric LN. Furthermore, we describe a novel imaging platform which enables high resolution intravital imaging of gut SLOs, especially mesenteric LN, for up to 10 hours. Finally, we use intravital 2-PLSM to quantify the contribution of self-pMHC recognition to Treg kinetics and scanning of DCs in peripheral and mesenteric LNs. These results add quantitative data to the emerging paradigm that self-pMHC interactions dynamically control homeostatic Treg retainment and localized suppressive function in peripheral LNs.

Citation Format: Alexander Tong. Quantification of lymph node transit times reveals differences in the trafficking behavior of regulatory versus conventional CD4+ T cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A069.
Abstract A073: Epigenetic regulation of the Cd4 locus
Authors: Issuree Priya , Day Kenny , Littman Dan R.
Published: November 2016

CD4 helper and CD8 cytotoxic T cells originate from progenitor cells with binary potential. Transcriptional regulatory elements are key in establishing heritable epigenetic mechanisms to ensure gene-expression programs that maintain lineage identity. We show that, during thymocyte development, the co-receptor CD4 is regulated by a stage-specific regulatory element that ensures commitment to the CD4 lineage. Sustained CD4 expression in post-selection thymocytes requires this intronic element situated 3' to a silencer element previously shown to repress cd4 in CD8 cytotoxic T cells. Despite normal CD4 expression before selection, thymocytes lacking this intronic element failed to maintain CD4 expression following positive selection and were redirected to the CD8 lineage after MHC class II–restricted selection. We find that this regulatory element is key for propagating TET enzyme–mediated locus demethylation in CD4 T cells, a process that is likely initiated by another cis-element controlling CD4 expression at the double positive stage, before lineage commitment. This locus demethylation event is critical in ensuring heritable lineage identity and disruption of this process results in unstable CD4 expression and altered gene expression profiles. Cis and trans element interactions and molecular mechanisms governing heritable expression in response to distinct signals that dictate lineage ‘choice’ during T cell development will be presented.

Citation Format: Priya Issuree, Kenny Day, Dan R. Littman. Epigenetic regulation of the Cd4 locus [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A073.
Abstract A078: Cutaneous microbiota in development of endogenous anti-melanocyte immunity
Authors: Vujkovic-Cvijin Ivan , Davis Jacquice , Singh Rasnik , Lee Kristina , Yu Zhiya , Restifo Nicholas , Wei Maria , Belkaid Yasmine
Published: November 2016

Despite major advances in immunotherapeutic approaches designed to bolster endogenous immune responses against tumors, metastatic melanoma remains associated with high mortality and rapid progression. Development of novel approaches toward enhancing endogenous immune responses against melanoma constitutes a critical step toward the management of disease. Of note, initiating immune responses against non-mutated melanocyte-specific protein antigens has shown efficacy in augmenting immunotherapies. Vitiligo is an autoimmune disease characterized by immune-mediated destruction of melanocytes, though the etiology of this disease remains poorly understood. Microbes have been recently found to have a profound effect on immune processes of the skin, including those that are associated with vitiligo progression. Identifying skin-resident microbes that initiate or exacerbate anti-melanocyte immunity, as well as the precise pathways that mediate these processes, may provide a novel framework for the development of immunotherapies to augment anti-tumor immune responses and efficacy of existing therapies. Thus, we have recruited a cohort of individuals with vitiligo and profiled their skin microbiomes at various body sites. We have isolated numerous vitiligo-associated bacteria and have adapted a mouse model of vitiligo in order to understand mechanisms of host-microbiome interaction in this setting. Interrogation of such a relationship between the microbiome and autoimmune destruction of melanocytes in vitiligo may lead to the identification of novel targets and pathways by which to augment melanoma immunotherapies.

Citation Format: Ivan Vujkovic-Cvijin, Jacquice Davis, Rasnik Singh, Kristina Lee, Zhiya Yu, Nicholas Restifo, Maria Wei, Yasmine Belkaid. Cutaneous microbiota in development of endogenous anti-melanocyte immunity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A078.
Abstract A100: Crosstalk between regulatory T cells and tumor-associated dendritic cells controls antitumor immunity in pancreatic cancer
Authors: Jang Jung-Eun , Hajdu Cristina H. , Miller George , Dustin Michael L. , Bar-Sagi Dafna
Published: November 2016

Pancreatic ductal adenocarcinoma (PDA) is marked by an abundant fibroinflammatory microenvironment. Regulatory T (Treg) cell infiltration constitutes a prominent feature of PDA. However, the immunomodulatory function of Treg cells in PDA remains poorly understood. Using orthotopic and autochthonous mouse models of PDA we have found that Treg cell ablation is sufficient to evoke effective anti-tumor immune response in early and advanced pancreatic neoplasia. This response is dependent on IFN-γ producing cytotoxic CD8+ T cells. We show that Treg cells engage in extended interactions with tumor-associated CD11c+ dendritic cells (DCs) and restrain their immunogenic function by suppressing the expression of costimulatory ligands necessary for CD8+ T cell activation. Consequently, tumor-associated CD8+ T cells fail to display effector activities when Treg cell ablation is combined with DC depletion. We propose that tumor-infiltrating Treg cells promote immune-tolerance by suppressing DC immunogenicity. Therapeutic manipulation this axis might provide an effective approach for the targeting of PDA.

Citation Format: Jung-Eun Jang, Cristina H. Hajdu, George Miller, Michael L. Dustin, Dafna Bar-Sagi. Crosstalk between regulatory T cells and tumor-associated dendritic cells controls antitumor immunity in pancreatic cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A100.
Abstract A108: CD103+ intraepithelial T cells in high-grade serous ovarian cancer are phenotypically diverse TCRαβ+ CD8αβ+ T cells that can be targeted for cancer immunotherapy
Authors: Nijman Hans , Komdeur Fenne , Wouters Maartje , Workel Hagma , Brunekreeft Kim , Plat Annechien , Eggink Florine , Wisman Bea , Daemen Toos , Duiker Evelien , Hollema Harry , Bruyn Marco de
Published: November 2016

Introduction: CD103+ tumor-infiltrating lymphocytes (TIL) have been linked to specific epithelial infiltration and a prolonged survival in epithelial malignancies. However, whether these cells are induced as part of an ongoing anti-tumor immune response or represent non-specifically expanded resident or mucosal lymphocytes remains largely unknown.

Methods: CD103+ TIL were quantified in a cohort of ovarian and endometrial cancer patients by immunohistochemistry. Localization of CD103+ cells and co-expression of CD103 with CD3, CD8, CD16 and FoxP3 was assessed by immunofluorescence. Further phenotyping and functional experiments of CD103+ cells was performed mainly by flow cytometry on primary tumor digests.

Summary of the Data: In this study, we first confirmed that CD103+ TIL from ovarian and uterine cancer were predominantly localized in the cancer epithelium and were strongly correlated with an improved prognosis. We further demonstrate that CD103+ TIL were almost exclusively CD3+ TCRαβ+ CD8αβ+ CD4- T cells, but heterogeneously expressed T cell memory and differentiation markers. Activation of peripheral T cells in the presence of HGSC was sufficient to trigger induction of CD103 in over 90% of all CD8+ cells in a T cell receptor (TCR)- and TGFβR1-dependent manner. Finally, CD103+ TIL isolated from primary ovarian and uterine tumors showed signs of recent activation and dominantly co-expressed key immunotherapeutic targets PD-1 and CD27.

Conclusion: Our data indicate CD103+ TIL are formed as the result of an adaptive anti-tumor immune response that might be reactivated by (dual) checkpoint inhibition.

Citation Format: Hans Nijman, Fenne Komdeur, Maartje Wouters, Hagma Workel, Kim Brunekreeft, Annechien Plat, Florine Eggink, Bea Wisman, Toos Daemen, Evelien Duiker, Harry Hollema, Marco de Bruyn. CD103+ intraepithelial T cells in high-grade serous ovarian cancer are phenotypically diverse TCRαβ+ CD8αβ+ T cells that can be targeted for cancer immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A108.
Abstract A117: CXCR1 is required for neutrophil recruitment to wounds and Kras-transformed cells in zebrafish
Authors: Powell Davalyn , Deng Qing , Huttenlocher Anna
Published: November 2016

Neutrophils are the first responder cells to sites of infection and tissue damage. Evidence from patient tumors and animal models of cancer also suggest that they are highly recruited to the tumor microenvironment, though their role there is still poorly understood. The larval zebrafish is an excellent model to study neutrophil migration and chemotaxis in vivo and is an emerging model for cancer development and progression. Here, we use the zebrafish to model Kras-driven oncogenesis in keratinocytes and glial cells. Beginning at 3 days post fertilization (dpf), we observe invasive EMT-like cell shape changes in oncogenic mutant Kras (KrasG12V) expressing cells as well as enhanced cell proliferation as marked by phospho-histone H3. Using fluorescently labeled transgenic lines, we demonstrate that neutrophils are recruited to transformed cells and that blocking this recruitment results in decreased proliferation of transformed cells, suggesting that neutrophils may play a tumor-promotional role in our model. To determine the method of neutrophil chemotaxis to transformed cells, we used Transcription Activator-like Effector Nuclease (TALEN) mutagenesis to generate zebrafish mutants for the chemokine receptor CXCR1, which is known regulate neutrophil chemotaxis. We demonstrate that CXCR1 is required for neutrophil chemotaxis to a tail-transection wound and to Kras-transformed cells in zebrafish. Together these data suggest that blocking CXCR1 activity may reduce neutrophilic inflammation in the tumor microenvironment which may in turn reduce tumor cell proliferation and slow tumor progression.

Citation Format: Davalyn Powell, Qing Deng, Anna Huttenlocher. CXCR1 is required for neutrophil recruitment to wounds and Kras-transformed cells in zebrafish [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A117.
Abstract A123: Modulation of immune cell trafficking into human colorectal cancer by gut microbiota
Authors: Cremonesi Eleonora , Amicarella Francesca , Garzon Jesus Francisco Glaus , Governa Valeria , Muraro Manuele Giuseppe , Mele Valentina , Padovan Elisabetta , Oertli Daniel , Zajac Paul , Spagnoli Giulio Cesare , Borsig Lubor , Iezzi Giandomenica
Published: November 2016

Colorectal cancer (CRC) is a leading cause of cancer-related death. CRC infiltration by immune cells, including cytotoxic CD8+ T cells (CTLs), IFN-gamma-producing T-helper 1 cells (Th1), Foxp3+ regulatory T cells (Tregs) and CD16+ MPO+ neutrophils, is associated with favorable prognosis. However, chemokines driving these cell populations into the tumor site, their cellular sources and their microenvironmental triggers remain to be elucidated. We investigated the chemokine/chemokine receptor network promoting CRC infiltration by immune cells associated to favorable prognosis. Analysis of freshly excised specimens of CRC and adjacent healthy colonic tissues revealed that tumor infiltration by beneficial immune cells is associated with the expression of four main chemokine patterns: 1) CCL3, CCL5, CCL8, CXCL9, CXCL10, and CXCL12 for CTLs; 2) CCL5, CCL22, CXCL9 and CXCL12 correlating with CRC infiltration by Th1; 3) CCL22 and CXCL12 attracting Tregs; 4) CXCL2 and CXCL5, promoting chemotaxis of CD16+ MPO+ myeloid cells. Most of identified chemokines were found to be expressed, although at different levels, in primary CRC cells purified from tumor specimens and from xenografts generated upon injection of CRC cells in immunodeficient mice. Notably, chemokine expression levels in orthotopic xenografts, developed upon intracecal injection of tumor cells, were significantly higher as compared to those of subcutaneous tumors. Antibiotic treatment of tumor bearing mice drastically reduced chemokine expression in orthotopic xenografts, thus suggesting a role for commensal bacteria in chemokines induction in tumor cells. Importantly, human CRC samples characterized by high expression of chemokine and immune cell markers, displayed significantly higher bacterial loads, as compared to samples showing low chemokine expression and immune cell infiltration. Furthermore, bacterial loads significantly correlated with expression of CCL3 and CCL5, and Th1 infiltration. These data suggest that stimuli derived from gut flora components may trigger chemokine production in CRC tissues thus leading to tumor infiltration by beneficial immune cells.

Note: This abstract was not presented at the conference.

Citation Format: Eleonora Cremonesi, Francesca Amicarella, Jesus Francisco Glaus Garzon, Valeria Governa, Manuele Giuseppe Muraro, Valentina Mele, Elisabetta Padovan, Daniel Oertli, Paul Zajac, Giulio Cesare Spagnoli, Lubor Borsig, Giandomenica Iezzi. Modulation of immune cell trafficking into human colorectal cancer by gut microbiota [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A123.
Abstract A129: Ablation of neuropilin 1 from glioma associated macrophages and microglia slows tumor progression
Authors: Miyauchi Jeremy T. , Chen Danling , Choi Matthew , Nissen Jillian , Shroyer Kenneth , Selwood David , Tsirka Stella E.
Published: November 2016

Gliomas are the most commonly diagnosed primary tumors of the central nervous system (CNS). Median times of survival are dismal regardless of the treatment approach, underlying the need to develop more effective therapies. Modulation of the immune system is a promising strategy as innate and adaptive immunity play important roles in cancer progression. Glioma associated microglia and macrophages (GAMs) can comprise over 30% of the cells in glioma biopsies. Gliomas secrete cytokines that suppress the anti-tumorigenic properties of GAMs, causing them to secrete factors that support the tumor's spread and growth. Neuropilin 1 (Nrp1) is a transmembrane receptor that in mice both amplifies pro-angiogenic signaling in the tumor microenvironment and affects behavior of innate immune cells. Using a Cre-lox system, we generated mice that lack expression of Nrp1 in GAMs. We demonstrate, using an in vivo orthotopic glioma model, that tumors in mice with Nrp1-deficient GAMs exhibit less vascularity, grow at a slower pace, and are populated by increased numbers of anti-tumorigenic GAMs. Moreover, glioma survival times in mice with Nrp1-deficient GAMs were significantly longer. Treating wild-type mice with a small molecule inhibitor of Nrp1's b1 domain, EG00229, which we show here is selective for Nrp1 over Nrp2, yielded an identical outcome. Moreover, transplanting bone marrow from mice with Nrp1-deficient macrophages to wild type recipient mice led to reductions in tumor growth, al-be-it not to the extent seen in knockout mice. Nrp1-deficient or EG00229-treated wild-type microglia exhibited a shift towards anti-tumorigenicity as evident by altered inflammatory marker profiles in vivo and decreased SMAD2/3 activation when conditioned in the presence of glioma-derived factors. These results provide support for the proposal that pharmacological inhibition of Nrp1 constitutes a potential strategy for suppressing glioma progression.

Citation Format: Jeremy T. Miyauchi, Danling Chen, Matthew Choi, Jillian Nissen, Kenneth Shroyer, David Selwood, Stella E. Tsirka. Ablation of neuropilin 1 from glioma associated macrophages and microglia slows tumor progression [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A129.
Abstract A138: Increased CD3(+) cell infiltration during pancreatic intraepithelial neoplasia progression in KrasG12D/Pdx1-Cre transgenic mouse model
Authors: Hong Xiafei , Dai Hongmei , Wang Xianze , Tian Feng , Wu Wenming , Zhao Yupei
Published: November 2016

This study aim to quantify the dynamic number changes of CD3(+) lymph cells in a pre-malignant stage of pancreatic cancer, namely, pancreatic intraepithelial neoplasia (PanIN). The experiment was conducted using a well-established KrasG12D/Pdx1-Cre transgenic mouse model. The animal model expressed a pancreas-specific active KrasG12D gene, resulting in PanIN progression and acinar structure loss in a time dependent manner. Experiment mice were sacrificed at 5, 7, 9, and 11 months (n = 3 for each time point). The pancreas tissues were fixed and stained by a rabbit monoclonal [EPR4517] to CD3G (Abcam Cat#: ab134096, 1:500 dilution). The CD3(+) lymph cell number were quantified by Leica optical system and the CD3(+) lymph cells were manually counted (ten fields for each mouse). All experimental conducted were approved by the ethical committee. The CD3(+) lymph cell number was calculated by averaging all field in each time point (thirty fields per time point). The number was then presented as mean ± SEM. Quantitatively, the CD3(+) cell were 11.73 ± 2.83, 14.23 ± 2.07, 14.90 ± 1.99, and 29.13 ± 2.04 per field in 5, 7, 9, and 11 month-old mice. The CD3(+) cell number was not significantly different among 5, 7, and 9 month-old mice. However, the number significantly increase in the 11 month-old mice compared with the 9-month ones (P< 0.0001, unpaired t-test). We observed that CD3(+) lymph cells were predominately present in PanIN area, while almost not observable in the remaining normal acinar area. The phenomenon that CD3(+) lymph cells infiltrate in PanIN area indicates that this type of immune cells may involve in PanIN progression.

Citation Format: Xiafei Hong, Hongmei Dai, Xianze Wang, Feng Tian, Wenming Wu, Yupei Zhao. Increased CD3(+) cell infiltration during pancreatic intraepithelial neoplasia progression in KrasG12D/Pdx1-Cre transgenic mouse model [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A138.
Abstract B014: Improved therapeutic activity of agonistic, human anti-CD40 monoclonal Abs by selective Fc receptor-engagement
Authors: Dahan Rony , Ravetch Jeffrey V.
Published: November 2016

The use of immunomodulatory mAbs to stimulate effective therapeutic anticancer immunity has shown clinical successes for the treatment of an increasing number of cancer types. An approach that is currently under clinical development uses agonistic anti-CD40 mAbs to induce APC activation, thereby harnessing the potency of the immune response to eradicate tumors. While engagement of the inhibitory Fcγ Receptor(FcγR)IIB is required for the in vivo agonistic activity of anti-mouse CD40 mAbs, a similar requirement for human mAbs has been disputed based on in vitro studies and in vivo studies performed either in wild-type or mouse FcγR knockout strains and thus fails to address the unique binding affinities and tissue distribution of human FcγRs. To overcome these limitations we generated and used unique mouse model fully humanized for its FcγRs and CD40, and revealed that FcγRIIB engagement is also essential for the activity of the human CD40 agonistic mAbs, including CP-870,893, while engagement of the activating FcγRIIA inhibits their activity. By engineering Fc variants of human mAbs with selective enhancement to FcγRIIB but not to FcγRIIA, significant improvement of antitumor immunity was observed in multiple tumor models. These findings highlight the necessity of optimizing the Fc domain for this class of therapeutic antibodies by using an appropriate pre-clinical model that accurately reflects the unique affinities and cellular expression of human FcRs, thus providing a development approach for therapeutic human antibodies based on their requirement for selective FcγR interactions. Here we describe the selection of optimized next-generation Fc-engineered agonistic CD40 mAbs.

Citation Format: Rony Dahan, Jeffrey V. Ravetch. Improved therapeutic activity of agonistic, human anti-CD40 monoclonal Abs by selective Fc receptor-engagement [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B014.
Abstract B019: LAG3 is an immunotherapeutic target in murine triple negative breast cancers whose activity is significantly enhanced in combination with phosphatidylserine targeting antibodies
Authors: Gray Michael J. , Gong Jian , Hutchins Jeff , Freimark Bruce
Published: November 2016

Previous pre-clinical modeling demonstrated that phosphatidylserine (PS) targeting antibodies are capable of significantly enhancing the effectiveness of anti- programmed cell death ligand 1 (PD-1) therapy in murine breast cancers (BC). These results prompted our hypothesis that PS blocking antibodies should also be capable of enhancing the activity of additional immunotherapies in BC, including triple negative breast cancer (TNBC). PS normally resides in the inner leaflet of the plasma membrane in most cell types. Conditions that promote cellular stress in the tumor microenvironment, such as reactive oxygen species (ROS), hypoxia, and irradiation, cause PS externalization and exposure on tumor associated endothelial cells and tumor cells, where it is recognized by specific receptors, including members of the TIM and TAM family. This PS recognition promotes an innate system driven immunosuppressive condition by promoting an influx of myeloid derived suppressor cells (MDSCs), an M2-macrophage shift, and suppression of dendritic cell maturation, while inducing anti-inflammatory cytokines. To identify additional immuno-therapeutic targets that may be potential targets for combinational treatment with PS-targeting antibodies, we employed gene expression analysis on tumors from murine TNBC treated with single and combinations of PS and PD-1 targeting antibodies. Of the genes assayed, expression of the lymphocyte activation gene 3 (LAG3) increased in each treatment arm, suggesting it may be a potential target for combinational therapy in breast cancers. To test our hypothesis, C57BL/6 mice hosting the murine TNBC line E0771 were treated with a PS targeting antibody or a LAG3 targeting antibody alone or in combination. Our data demonstrate that while PS-blocking and anti-LAG3 therapies each have moderate efficacy as single agents, combinational treatment significantly impeded the growth of TNBC, and was capable of increasing tumor infiltrating lymphocytes (TILs), including CD8 + and CD3 + T-cells, and reducing the percentage of MDSCs over that observed by single treatments. Combined, our data demonstrate that LAG3 targeting also represents a viable option for the treatment of TNBC, and that the addition of PS targeting antibodies to LAG3 therapy can effectively increase the anti-tumor and immune-activating effects mediated by multiple checkpoint therapies.

Citation Format: Michael J. Gray, Jian Gong, Jeff Hutchins, Bruce Freimark. LAG3 is an immunotherapeutic target in murine triple negative breast cancers whose activity is significantly enhanced in combination with phosphatidylserine targeting antibodies [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B019.
Abstract B044: Patient-specific immunotherapy through TCR gene transfer
Authors: Fanchi Lorenzo F. , Bies Laura , Song Ji-Ying , Linnemann Carsten , Schumacher Ton
Published: November 2016

Adoptive cell transfer (ACT) of autologous ex vivo expanded tumor-infiltrating lymphocytes has shown to mediate significant tumor regression in metastatic melanoma. This finding has demonstrated the capacity of endogenous T cells to mount an immune response against and eliminate cancer cells. Two clinically relevant approaches to adoptive cell transfer are ACT of expanded TIL and ACT of gene-modified peripheral blood lymphocytes (PBLs). The former approach consists of isolation of tumor-infiltrating lymphocytes from tumor biopsies, ex vivo expansion and re-infusion after treatment with a lymphodepleting regimen. In the latter, PBLs are engineered to express T-cell receptors (TCRs) recognizing common melanoma tumor antigens. While both approaches have shown different extents of clinical benefit, several limitations hamper clinical efficacy.

T cell specificity, phenotype and differentiation state of ex vivo expanded TIL are largely unknown, while ACT of gene-modified lymphocytes is limited by the use of TCRs recognizing common tumor antigens, which can be differentially expressed between patients. Furthermore, the use of these TCRs is often limited to a handful of HLA types. These factors can lead to reduced therapeutic efficacy and applicability.

Here we aim to compare the tumor control of T cells expressing ‘personalized’ tumor-reactive TCRs and expanded tumor-infiltrating lymphocytes after adoptive cell transfer in a xenograft mouse model. To this end, highly tumor-reactive T cell receptors are isolated from TIL on a per-patient basis. These TCRs are introduced into donor peripheral blood lymphocytes, forming a population of tumor-reactive cells tailored to the patient tumor. These cells are subsequently tested for efficacy in a xenograft mouse model bearing autologous (patient-derived) tumor.

Using the outlined approach, we aim to identify determinants of therapy efficacy. Preliminary results have shown dramatic tumor regression after ACT of patient-specific gene-modified T cells, leading to complete remission of tumors in all treated mice. In contrast, tumors of mice treated with TIL or control T cells did not show significant reduction of tumor burden, when compared to untreated controls.

Citation Format: Lorenzo F. Fanchi, Laura Bies, Ji-Ying Song, Carsten Linnemann, Ton Schumacher. Patient-specific immunotherapy through TCR gene transfer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B044.
Abstract B051: Super cross-presentation of tumor antigens by synthetic design of an anti-phosphatidylserine bridge protein
Authors: Corey Daniel , Ring Aaron , McCracken Melissa , Miyanishi Masanori , Gordon Sydney , Weissman Irving
Published: November 2016

Cell loss by apoptosis is a common feature in tumors. Dying tumor cells induce immune tolerance within the tumor microenvironment largely through highly conserved homeostatic clearance programs that restore tissue immune homeostasis and contribute to the formation of an immunosuppressive niche. The translocation of phosphatidylserine (PS) on cellular membranes, during the initial phases of apoptosis, functions as a recognition and removal signal that limits the immunogenicity of cell death. We examined whether altering clearance of dying cancer cells to elicit inflammatory turnover can allow for and potentiate immune responses against tumor cells. To remove inhibitory signals in the homeostatic clearance pathway we utilized a molecular bridge scaffold to engineer a modified phosphatidylserine bridge protein (FA58C2-hIgG1 or C2-hIgG1) that works as a bridge between apoptotic cells expressing aminophospholipids and phagocytes bearing Fc receptors. In vivo administration of C2-hIgG1 partially restores immune responses to dead tumor cells in antigen cross presentation assays and promotes recruitment and retention of tumor antigen specific CD8+ T cells, dendritic cells, and natural killer cells into tumors. These effects combine to elicit anti-lymphoma immunity, improve responses to immune checkpoint inhibitors, and enhance the effectiveness of adoptive T cell transfers using engineered T Cell Receptors (TCRs) but not chimeric antigen receptor engineered (CAR-T) T cells.

Citation Format: Daniel Corey, Aaron Ring, Melissa McCracken, Masanori Miyanishi, Sydney Gordon, Irving Weissman. Super cross-presentation of tumor antigens by synthetic design of an anti-phosphatidylserine bridge protein [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B051.
Abstract B062: Immune activation by LMP1 CD40 pathway and LMP1-IPS-1 STING pathway activators
Authors: Kornbluth Richard S. , Gupta Sachin , Termini James M. , Guirado Elizabeth , Stone Geoffrey W.
Published: November 2016

The strongest CD8+ T cell responses ever recorded in humans occur as a result of infection by the Epstein-Barr Virus (EBV). Following a massive CD8+ T cell response during acute EBV infection, ∼5.5% of the total CD8+ T cell repertoire in blood is thereafter committed to the life-long suppression of viral replication. The key to this response is the EBV Latent Membrane Protein-1 (LMP1) which functions as a constitutively activated form of the CD40 receptor. LMP1 consists of an N-terminal domain that clusters in cell membranes and a C-terminal domain that engages the proteins of the CD40 signalosome. Studies to determine if LMP1 could be used for immunotherapy led to the following conclusions: (1) LMP1 mRNA directly activated human dendritic cells (DCs), showing its potential as an adjuvant for mRNA vaccines; (2) Adenovirus (Ad)-delivered LMP1 strongly enhanced vaccine-induced CD8+ T cell responses in mice, indicating that LMP1 may enhance Ad-based oncolytic therapies; (3) lentiviruses incorporating LMP1 became strong activators of DCs in vitro; and (4) DNA vaccines incorporating LMP1 protected mice from tumor challenge in a B16F10 melanoma metastasis model. These data show that the LMP1 nucleic acid sequence is a portable genetic adjuvant with broad applicability to tumor immunotherapy. In related studies, the membrane clustering N-terminal domain of LMP1 was fused to Interferon-beta Promoting Stimulator-1 (IPS-1, also called MAVS, VISA, or Cardif), a protein that requires membrane clustering to become activated. This fusion protein acts independently of cyclic dinucleotides (CDN) to activate downstream mediators of the STING pathway. Vaccination of mice with Ad expressing antigen plus LMP1-IPS-1 resulted in complete protection from challenge with Vaccinia-antigen virus (>1 million-fold protection). These data suggest that LMP1-IPS-1 combined with gene delivery technologies may supplant the need for small molecule CDNs for STING pathway activation in immuno-oncology.

Citation Format: Richard S. Kornbluth, Sachin Gupta, James M. Termini, Elizabeth Guirado, Geoffrey W. Stone. Immune activation by LMP1 CD40 pathway and LMP1-IPS-1 STING pathway activators [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B062.
Abstract B065: Use of simultaneous detection of externalized CD107a and CD137 for evaluation of specificity and effector functions of polyclonal T-cells produced for adoptive cellular immunotherapy of prostate cancer
Authors: Taborska Pavla , Stakheev Dmitry , Vavrova Katerina , Vrabcova Petra , Bartunkova Jirina , Smrz Daniel
Published: November 2016

The ex vivo production of T-cells that can recognize tumor-associated antigens (TAAs) and through this recognition eliminate cancer cells is the goal of T-cell-based adoptive cellular immunotherapy (ACI). The technological approaches that use whole cancer cells or their pertinent lysates as a source of TAAs for production of these T-cells lead to generation of polyclonal T-cells their antigen specificity of which is often unknown. Evaluation of such polyclonal T-cells is therefore challenging because their target and off-target performance might be difficult to predict. In our study we used human dendritic cells (DCs) pulsed with inactivated prostate cancer cell line LNCaP and matured with polyinosinic:polycytidylic acid (poly(I:C)) to induce expansion of autologous lymphocytes. The expanded lymphocytes were then challenged with live LNCaP cells and analyzed by simultaneous detection of a marker of T-cell cytotoxicity and antigen-induced T-cell stimulation, the surface externalization of, respectively, CD107a and CD137. Our data showed that lymphocytes expanded with LNCaP-pulsed and poly(I:C)-matured DCs contained a notable fraction of CD137 and/or CD107a positive T-cells after their challenge with live LNCaP cells. No such notable fraction was however observed in resting lymphocytes, or when the lymphocytes were challenged with ovarian cancer cell line SKOV-3. Importantly, autologous lymphocytes that were expanded with poly(I:C)-matured DCs not previously pulsed with the inactivated LNCaP cells did not show a notable population of the marker positive cells. Collectively, our data showed that simultaneous detection of externalized CD107a and CD137 on the surface of polyclonal T-cells may allow for evaluation of polyclonal T-cell specificity and effector functions after their challenge with complex antigens.

Citation Format: Pavla Taborska, Dmitry Stakheev, Katerina Vavrova, Petra Vrabcova, Jirina Bartunkova, Daniel Smrz. Use of simultaneous detection of externalized CD107a and CD137 for evaluation of specificity and effector functions of polyclonal T-cells produced for adoptive cellular immunotherapy of prostate cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B065.
Abstract B076: PIN-2: A novel immunopriming peptide with immunomodulatory activity linking the innate and adaptive immune systems
Authors: Goldberg Joshua B. , Hanscom Sophie , Bier Colin B.
Published: November 2016

PIN-2 is a novel immunomodulatory agent derivatized from a transactivator protein. We have demonstrated its activity to both stimulate and induce maturation of monocytes into dendritic cells and translational proof of principle using an aggressive metastatic murine mammary cancer model.PIN-2 initiates activity by enhancement of innate immune signaling de novo. Next Generation Sequencing of CD14+ primary human monocytes after 3-hour exposure to PIN-2 in vitro, reveals a unique profile of gene expression indicative of innate immune activation which is necessary to drive adaptive immunity towards killer T-cell immune responses. Genetic programs in the monocyte impacted by PIN-2 exposure include cytokine signaling pathways, co-stimulatory ligands, and coding and non-coding RNAs.Understanding the genetic signature of the changes by PIN-2 through the innate immune response supports the rational development of immune based combination therapies such as cell based immunotherapies and checkpoint blockade that function to modulate adaptive immunity in T-cells in a clinical setting.

Citation Format: Joshua B. Goldberg, Sophie Hanscom, Colin B. Bier. PIN-2: A novel immunopriming peptide with immunomodulatory activity linking the innate and adaptive immune systems [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B076.
Abstract B080: Noninvasive imaging of antitumor responses as a possible predictive tool
Authors: Rashidian Mohammad , Ploegh Hidde
Published: November 2016

Immunotherapy has produced durable remissions across a spectrum of malignancies. However, predicting and monitoring therapeutic outcomes remains a challenge. Techniques to achieve these goals without multiple biopsies or other invasive methods will be critical to further improve on immunotherapy. Noninvasive imaging of immune responses is therefore a highly desirable goal. The abundance, movement and expansion of relevant cell types may yield reproducible patterns in responders and nonresponders that could help understand, if not predict, the failure or success of a therapy. To enable noninvasive imaging of the targeted cell populations, we isolated camelid-derived single domain antibody fragments (VHHs) against CD8, PD-L1, CTLA, Class II MHC, and CD11b markers. We also developed a method to site-specifically label VHHs with 18F, 64Cu or 89Zr. Radiolabeled VHHs rapidly clear the circulation (t1/2≈15-30 min) and clearly visualize their targets. Through PET imaging using radiolabeled VHHs, we can monitor and explore patterns of responses in the tumor microenvironment, as such or in response to therapy. Early results suggest that infiltration of a tumor by CD8 T cells may be used as a prognostic criterion. Tumors homogenously infiltrated by CD8-T cells appear more likely to respond than tumors that have heterogeneous CD8-T cell infiltration. Important next questions to be explored by PET imaging include the following. What is the role of regulatory T cells (Tregs) and macrophages? How does the CD8-T cell/Treg-cell ratio change in response to treatment? Is it possible to stratify responders from non-responders soon after starting therapy, and how would this affect treatment strategies? Because the basic methodology proposed here is in principle translatable to a human setting, this approach may ultimately find clinical application.

Citation Format: Mohammad Rashidian, Hidde Ploegh. Noninvasive imaging of antitumor responses as a possible predictive tool [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B080.
Abstract B089: High-throughput profiling of HLA allele-specific peptides by MS for improved epitope prediction
Authors: Rooney Michael S. , Abelin Jennifer G. , Keskin Derin B. , Sarkizova Siranush , Hartigan Christina , Zhang Wandi , Sidney John , Stevens Jonathan , Lane William J. , Zhang Guang L. , Clauser Karl R. , Hacohen Nir , Carr Steven A. , Wu Catherine J.
Published: November 2016

Cancer mutations yield neo-antigens, which are instrumental to immune-mediated recognition and control of cancer. Vaccine-based therapies targeting neo-antigens will require accurate prediction of which mutations yield peptides presented on polymorphic HLA class I. While in vitro methods have produced increasingly accurate predictors of peptide:MHC binding, there remains a need to define rules for endogenous antigen presentation. Here, we use rapid, high-resolution liquid chromatography mass spectrometry (LC-MS/MS) to identify >24,000 peptides associated with 16 HLA alleles in B cell lines that each express a single HLA allele. The elution of peptides from single HLA alleles allowed us to develop improved rules for endogenous peptide presentation based on the physicochemical properties of binding peptides, patterns of peptide cleavage and abundance of cognate transcripts. Finally, we trained models that integrated MS-derived peptide data and gene expression and demonstrate improved prediction of endogenous peptide presentation in independent datasets. Our strategy thus improves the performance of current predictive algorithms and provides a rapid and scalable method to generate rules for the massive and diverse set of human HLA alleles.

Citation Format: Michael S. Rooney, Jennifer G. Abelin, Derin B. Keskin, Siranush Sarkizova, Christina Hartigan, Wandi Zhang, John Sidney, Jonathan Stevens, William J. Lane, Guang L. Zhang, Karl R. Clauser, Nir Hacohen, Steven A. Carr, Catherine J. Wu. High-throughput profiling of HLA allele-specific peptides by MS for improved epitope prediction [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B089.
Abstract B093: In vitro testing solid cancers immunotherapy protocols through impedance technology allows dynamic and label-free evaluation of immune response and reagent efficacy
Authors: Cerignoli Fabio , Xi Biao , Guenther Garret , Rector Trent , Muir Lincoln , Zhao Leyna , Abassi Yama
Published: November 2016

In vitro characterization of reagents efficacy in the context of cancer immunotherapy for solid tumors is a necessary step before moving to more expensive animal models and clinical studies. However, current in vitro assays are difficult to implement in high throughput environment and are mainly based on end point methodologies that are unable to capture the full dynamic of the immune response. Here we present the adaptation of an impedance-based platform to monitoring cytotoxic activity of immune cells activated trough different means. The technology detects cell death and proliferation of adherent cells by measuring changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates. We utilized adherent cell lines and primary tumor cells with different expression levels of the EpCAM antigen, which is currently under evaluation as a target for cancer immunotherapy. Using PBMCs as effector cells and combinations of EpCAM/CD3 BiTE we demonstrated the suitability of such impedance-based approach to quantitatively monitor the efficacy of immune cells-mediated cancer cell killing both under different effector:target ratios and antibody concentrations. Combination treatments with checkpoint inhibitors were also effective when evaluated with the same approach. Finally, we validated the technology with engineered Car-T cells directed against common antigens expressed in tumors. Overall, our results demonstrate the value of such approach in measuring the cytotoxic response across the temporal scale, an aspect that is otherwise very difficult to assess with more canonical end point assays like Chromium 51 release or ATP-based luminescence. Furthermore, the availability of 384-wells format and minimal sample handling place the technology in an ideal spot for applications in personalized medicine, like therapeutic protocol validation directly on patient samples.

Citation Format: Fabio Cerignoli, Biao Xi, Garret Guenther, Trent Rector, Lincoln Muir, Leyna Zhao, Yama Abassi. In vitro testing solid cancers immunotherapy protocols through impedance technology allows dynamic and label-free evaluation of immune response and reagent efficacy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B093.
Abstract B096: Optogenetics-based dissection of chemotactic signaling in neutrophils
Authors: Graziano Brian R. , Gong Delquin , Anderson Karen E. , Weiner Orion D.
Published: November 2016

Neutrophils comprise a crucial component of the innate immune system and rely on chemotaxis (directed migration) to track down, engulf, and destroy pathogens. A variety of tumor-produced chemokines also recruit neutrophils to sites of tumor growth where, through mechanisms that remain unclear, they exhibit both beneficial and detrimental effects on tumor clearance. Effective chemotaxis requires that neutrophils restrict activity of the GTPase Rac to a single location to properly form a leading edge. However, multiple GEFs function redundantly to activate Rac and it has been difficult to determine where within the signaling cascade each specific GEF is required. Here, we develop an optogenetic tool to drive PIP3 production, which itself is sufficient to activate Rac in human neutrophil-like HL-60 cells. In cells where we used CRISPR-mediated editing to knockout the Rac GEF PREX1, we find that optogenetic stimulation of PIP3 production fails to activate Rac. In contrast, when these PREX1null neutrophils are instead treated with chemoattractant, other GEFs that do not explicitly rely on PIP3 production can still activate Rac. While signaling cascades often feature multiple redundant pathways functioning in parallel, our optogenetics-based approach reveals the location within such a network where a particular factor is necessary for the transmission of information. A more nuanced understanding of chemotactic signaling in neutrophils should lead to the development of treatments that can more precisely modulate the interactions between neutrophils and the tumor microenvironment.

Citation Format: Brian R. Graziano, Delquin Gong, Karen E. Anderson, Orion D. Weiner. Optogenetics-based dissection of chemotactic signaling in neutrophils [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B096.