Anti-tumor Effector Functions Research Articles

Iron Induces Anti-tumor Activity in Tumor-Associated Macrophages.

Tumor-associated macrophages (TAMs) frequently help to sustain tumor growth and mediate immune suppression in the tumor microenvironment (TME). Here, we identified a subset of iron-loaded, pro-inflammatory TAMs localized in hemorrhagic areas of the TME. The occurrence of iron-loaded TAMs (iTAMs) correlated with reduced tumor size in patients with non-small cell lung cancer. Ex vivo experiments established that TAMs exposed to hemolytic red blood cells (RBCs) were converted into pro-inflammatory macrophages capable of directly killing tumor cells. This anti-tumor effect could also be elicited via iron oxide nanoparticles. When tested in vivo, tumors injected with such iron oxide nanoparticles led to significantly smaller tumor sizes compared to controls. These results identify hemolytic RBCs and iron as novel players in the TME that repolarize TAMs to exert direct anti-tumor effector function. Thus, the delivery of iron to TAMs emerges as a simple adjuvant therapeutic strategy to promote anti-cancer immune responses.

Tumor-associated myeloid cells: new understandings on their metabolic regulation and their influence in cancer immunotherapy.

Tumor-associated myeloid cells (TAMCs), mainly represented by tumor-associated macrophages and myeloid-derived suppressor cells, can promote tumor growth directly, by favoring tumor cell proliferation and survival, and indirectly, by creating an immunosuppressive microenvironment. Myeloid cells are characterized by an extreme phenotypical and functional plasticity. Immunometabolism is now emerging as a crucial aspect of TAMCs skewing toward pro-tumoral activities. The metabolic re-education of myeloid cells is a new strategy to boost their antitumor effector functions. Several anticancer therapies targeting TAMCs are already under investigation. Nowadays, the hot topic of cancer immunotherapy is represented by immune checkpoint inhibitors. These drugs unrestrain T-cell-mediated tumor elimination by removing suppressive signals delivered by tumor-associated cells. The efficacy of immune checkpoint blockade can be enhanced using coordinated strategies to counteract the TAMCs-dependent impairment of immune adaptive responses. In the first part of the review, we will describe the association between metabolic reprogramming and TAMCs biological activities. In the second part, we will illustrate the potential of combination therapies associating TAMC-targeting drugs with immune checkpoint inhibitors.

Molecular mechanisms of programmed cell death-1 dependent T cell suppression: relevance for immunotherapy.

Programmed cell death-1 (PD1) has become a significant target for cancer immunotherapy. PD1 and its receptor programmed cell death 1 ligand 1 (PDL1) are key regulatory physiological immune checkpoints that maintain self-tolerance in the organism by regulating the degree of activation of T and B cells amongst other immune cell types. However, cancer cells take advantage of these immunosuppressive regulatory mechanisms to escape T and B cell-mediated immunity. PD1 engagement on T cells by PDL1 on the surface of cancer cells dramatically interferes with T cell activation and the acquisition of effector capacities. Interestingly, PD1-targeted therapies have demonstrated to be highly effective in rescuing T cell anti-tumor effector functions. Amongst these the use of anti-PD1/PDL1 monoclonal antibodies are particularly efficacious in human therapies. Furthermore, clinical findings with PD1/PDL1 blockers over several cancer types demonstrate clinical benefit. Despite the successful results, the molecular mechanisms by which PD1-targeted therapies rescue T cell functions still remain elusive. Therefore, it is a key issue to uncover the molecular pathways by which these therapies exert its function in T cells. A profound knowledge of PDL1/PD1 mechanisms will surely uncover a new array of targets susceptible of therapeutic intervention. Here, we provide an overview of the molecular events underlying PD1-dependent T cell suppression in cancer.

Abstract 3024: CD4+ outperform CD8+ central memory-derived CAR T cells, mediating persistent antitumor responses and long-term eradication of glioblastoma

Abstract Purpose: To compare antitumor effector function mediated by CD4+ and CD8+ chimeric antigen receptor (CAR)-T cells targeting IL13 receptor α2 (IL13Rα2) for treatment of glioblastoma (GBM). Experimental Design: CD4+ and CD8+ IL13Rα2-specific CAR-T cells, derived from enriched central memory T cells (Tcm), were evaluated for their antitumor potential both in vitro by recursive challenge with GBM cells and in vivo in established orthotopic GBM xenograft models. Further, GBM-stimulated CD4+ and CD8+ CAR-T cells were examined for their exhaustion- and memory-associated phenotypes at transcriptional and protein levels, which would account for their differential antitumor activity. Results: While CD8+ CAR-T cells mediated robust short-term cytolytic effector function, they became rapidly exhausted and lost antitumor potential after repeated stimulation with tumor cells. CD4+ CAR-T cells, by contrast, persisted and retained effector potency even after repetitive tumor challenge. CD4-mediated cytotoxicity acted through the granzyme B/perforin pathway and was independent of the CD8+ cells. Activated CD4+ CAR-T cells augmented the proliferation of CD8+ cells, but failed to protect the CD8+ cells against stimulation-induced exhaustion. In NSG mice bearing GBM xenografts, CD4+ CAR-T cell treatment led to long-term tumor-free survival, while CD8+ CAR-T cell treated mice showed short-term tumor regression followed by antigen-positive relapse and associated T cell exhaustion. Upon GBM stimulation, CD4+ cells expressed less co-inhibitory receptors and more memory-associated markers than the CD8+ T cell subset. Further, a comprehensive transcriptional analysis revealed that when activated, CD4+ cells retained expression of genes involved in survival- and self-renewal-related pathways, indicating a less exhausted status and a better functional persistence in comparison to the CD8+ cells. Conclusion: We observed a superior anti-GBM activity mediated by CD4+ CAR-T cells over the CD8+ subset, featured by their ability to maintain long-term immune response and reduce stimulation-induced exhaustion. It is thus inferred that the frequency of CD4+ cells could be an important criteria for evaluating the efficacy of CAR-T cells as immunotherapeutic products. Citation Format: Dongrui Wang, Renate Starr, Brenda Aguilar, Alfonso Brito, Brenda Chang, Aniee Sarkissian, Behnam Badie, Michael E. Barish, Stephen J. Forman, Christine E. Brown. CD4+ outperform CD8+ central memory-derived CAR T cells, mediating persistent antitumor responses and long-term eradication of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3024. doi:10.1158/1538-7445.AM2017-3024

Abstract 647: Bortezomib enhances CD8+ T Lymphocyte antitumor effector function: Potential mechanism(s) via notch regulation

Abstract The immunosuppressive tumor microenvironment disturbs host antitumor immunity by multiple mechanisms including interference with the Notch system, which is important for various metazoan cell fate decisions and hematopoietic cell differentiation and function. We observed that treatment with the proteasome inhibitor bortezomib in mice bearing various solid tumors resulted in an upregulated expression of various Notch signaling components in lymphoid tissues, thereby increasing CD8+ T lymphocyte IFNγ secretion and expression of effector molecules, perforin and granzyme B, as well as the T-box transcription factor eomesodermin. Of note, bortezomib reversed tumor-induced downregulation of Notch receptors, Notch1 and Notch2, as well as increased the levels of cleaved Notch intracellular domain (NICD) and downstream targets Hes1 and Hey1 in tumor-draining CD8+ T cells. Moreover, bortezomib promoted CD8+ T cell nuclear factor-κB (NFκB) activity by increasing the total and phosphorylated levels of the IκB kinase and IκBα as well as the cytoplasmic and nuclear levels of phosphorylated p65. Even when we blocked NFκB activity by Bay-11-7082, or NICD cleavage by γ-secretase inhibitor, bortezomib significantly increased expression of Notch Hes1 and Hey1 genes as well as perforin, granzyme B and eomesodermin in activated CD8+ T cells. Data suggest that bortezomib can rescue tumor-induced dysfunction of CD8+ T cells by its intrinsic stimulatory effects promoting NICD-NFκB crosstalk. We are also elucidating components of microRNA regulation affecting NICD-NFκB crosstalk. Our preliminary data suggest that bortezomib is also able to positively regulate miR-155 expression in CD8+ T cells from mice bearing tumor. As well as, miR-155 suppression downregulates bortezomib-induced increase in Notch target genes in T cells. We are currently investigating alternative proteasome inhibitors in order to understand whether bortezomib’s effect on miR-155 expression in CD8+ T cells is specific to bortezomib or primarily conducive to a proteasome inhibition effect. These findings provide novel insights on using bortezomib not only as an agent to sensitize tumors to cell death, but also to provide lymphocyte-stimulatory effects, thereby overcoming immunosuppressive actions of tumor on antitumor T cell functions. Citation Format: Ariana N. Renrick, Menaka C. Thounaojam, Portia Thomas, Samuel T. Pellom, Anil Shanker. Bortezomib enhances CD8+ T Lymphocyte antitumor effector function: Potential mechanism(s) via notch regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 647. doi:10.1158/1538-7445.AM2017-647

Metabolic barriers to immunotherapy in renal cell carcinoma.

11560 Background: Cancer cells can inhibit effector T cells through both immunomodulatory receptors and alteration of the tumor microenvironment as a result of cancer metabolism. A majority of patients treated with immune checkpoint inhibitors recently approved by the FDA fail to exhibit a clinical response. The extent to which metabolic conditions within the tumor impede T cell activation and anti-tumor effector function in renal cell carcinoma (RCC) are unknown. Methods: Under the IACUC protocol M1600005-00, BALB/c or Rag mice were subcutaneously injected with 100,000 Renca cells obtained from ATCC and growth monitored by caliper measurements in 3 dimensions every 3 days. In vivoPD-1 blockade was performed by 200 mcg/i.p. injection every 3 days using purified mPD-1 (BioXcell, J43). Deidentified tissue donations from patients with RCC were collected under the IRB protocol #151549 and processed into single cell suspensions following mechanical dissociation for the functional assays indicated below. Results: Through work with Rag deficient mice lacking functional B and T cells, we have established that tumor growth is regulated in a T cell dependent manner as evidenced by earlier formation and faster tumor growth. In a syngeneic mouse model of RCC (RenCa), we find that inhibition of PD-1 delays tumor growth and size. Tumor infiltrating lymphocytes (TILs) were abundant in patient-derived RCC, but are phenotypically distinct and are impaired both functionally and metabolically from healthy control. Instead of efficient use of aerobic glycolysis, TILs fail to increase glucose metabolism, and instead display increased reactive oxygen species (ROS) and mitochondrial dysfunction. CD8 effector cells found in tumors have notable differences in mitochondrial morphology compared to healthy control CD8 T cells by electron microscopy and immunofluorescence where CD8 TIL are punctate and dispersed throughout the cell while healthy control CD8 mitochondria are fused in networks. Thus bypassing metabolic defects may partially restore TIL activation. Conclusions: Preclinical data suggests that improved understanding of metabolic dysfunction in TILs of RCC may allow for combined therapies to improve response rates of checkpoint inhibition in this disease.

Interleukin-15 enhances the proliferation, stimulatory phenotype, and antitumor effector functions of human gamma delta T cells.

BackgroundAdoptive immunotherapy is gaining momentum to fight malignancies, whereby γδ T cells have received recent attention as an alternative cell source as to natural killer cells and αβ T cells. The advent of γδ T cells is largely due to their ability to recognize and target tumor cells using both innate characteristic and T cell receptor (TCR)-mediated mechanisms, their capacity to enhance the generation of antigen-specific T cell responses, and their potential to be used in an autologous or allogeneic setting.MethodsIn this study, we explored the beneficial effect of the immunostimulatory cytokine interleukin (IL)-15 on purified γδ T cells and its use as a stimulatory signal in the ex vivo expansion of γδ T cells for adoptive transfer. The expansion protocol was validated both with immune cells of healthy individuals and acute myeloid leukemia patients.ResultsWe report that the addition of IL-15 to γδ T cell cultures results in a more activated phenotype, a higher proliferative capacity, a more pronounced T helper 1 polarization, and an increased cytotoxic capacity of γδ T cells. Moreover γδ T cell expansion starting with peripheral blood mononuclear cells from healthy individuals and acute myeloid leukemia patients is boosted in the presence of IL-15, whereby the antitumor properties of the γδ T cells are strengthened as well.ConclusionsOur results support the rationale to explore the use of IL-15 in clinical adoptive therapy protocols exploiting γδ T cells.

Immunotherapeutic strategies targeting natural killer T cell responses in cancer.

Natural killer T (NKT) cells are a unique subset of lymphocytes that bridge the innate and adaptive immune system. NKT cells possess a classic αβ T cell receptor (TCR) that is able to recognize self and foreign glycolipid antigens presented by the nonclassical class I major histocompatibility complex (MHC) molecule, CD1d. Type I NKT cells (referred to as invariant NKT cells) express a semi-invariant Vα14Jα18 TCR in mice and Vα24Jα18 TCR in humans. Type II NKT cells are CD1d-restricted T cells that express a more diverse set of TCR α chains. The two types of NKT cells often exert opposing effects especially in tumor immunity, where type II cells generally suppress tumor immunity while type I NKT cells can enhance anti-tumor immune responses. In this review, we focus on the role of NKT cells in cancer. We discuss their effector and suppressive functions, as well as describe preclinical and clinical studies utilizing therapeutic strategies focused on harnessing their potent anti-tumor effector functions, and conclude with a discussion on potential next steps for the utilization of NKT cell-targeted therapies for the treatment of cancer.

Abstract A07: Gene analysis maps HCC heterogeneity and orientates personalized therapy

Abstract Hepatocellular carcinoma (HCC) has variable prognosis depending both on the severity of underlying liver disease and of its biological characteristic. We recently identified, in a prospective series of HCCs at first presentation, a 5-genes transcriptomic signature, which accurately and significantly predicts growth speed and survival (Gut. 2015 Feb 9. pii:gutjnl-2014-308483. doi: 10.1136/gutjnl-2014-308483). This signature encounters 5 up-regulated genes (ANGPT2, DLL4, NETO2, ESM1, NR4A1), all associated with neo-angiogenesis. Such gene analysis prompted us to characterize further the molecular pathways underlying tumor aggressiveness and consequently clinical outcome. The cohort of 78 prospectively identified HCCs was further characterized. Transcriptomic analysis was performed by microarray experiments (Agilent Technologies, Palo Alto, CA; Genomics Service Department of Miltenyi Biotec GmbH Bergisch Gladbach, Germany). Circulating cytokines were measured using human Quantikine® ELISA kit (R&D Systems, Minneapolis, MN, USA). For PD-1 and PD-L1 immunohistochemistry, samples were incubated with mouse monoclonal antibody (Ventana), for 24 minutes at 37°C. Biological data were analyzed according to HCC growth speed and patients’ survival. Genes down- or up-regulated affected prognosis and survival. In particular, the worst survival was correlated with down-regulation of CLEC4G and CLEC1B and up-regulation of MMP1, MMP10, MMP12 and WNT11. Next, we also measured a large panel of circulating cytokines tested, and among them TGF-β1 was the only significantly and positively associated with HCC aggressiveness. PD-1 and PD-L1 were strongly co-expressed especially at the boundary between tumoral and non-tumoral cirrhotic tissue but only in the most aggressive type. In slow-growing HCCs, PD-1/PD-L1 were only faintly expressed. Bringing together all these results, the most aggressive and associated with a worst clinical outcome aggressively HCCs were characterized by genes regulating different molecular pathways, including neo-angiogenesis (ANGPT2, DLL4, NETO2, ESM1, NR4A1and, indirectly, CLEC4G and CLEC1B), tumoral spread (MMP1, MMP10, MMP12), tissue remodelling, loss of immune local control (hyperactivated PD-1 and PD-1L). Aberrant PD-L1 expression in cancers facilitates escape from immune attack. PD-1 ligation by PD-L1 down-modulates anti-tumor immune effector functions allowing the development of a subset of hyper-aggressive HCCs. All such different pathways contribute to explain HCC heterogeneity, leading to therapeutic failures, but at the same time, stimulate a better stratification of patients directing the options for personalized therapies. Citation Format: Rosina Maria Critelli, Fabiola Milosa, Barbara Lei, Luca Marzi, Rosario Condello, Elena Turola, Nicola De Maria, Antonino Maiorana, Gianluigi Giannelli, Erica Villa. Gene analysis maps HCC heterogeneity and orientates personalized therapy. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A07.

Single-cell motility and gene expression signature as predictors of the overall survival of act in melanoma patients

Meeting abstracts Adoptive cell therapy (ACT) of tumor infiltrating lymphocytes (TIL) has shown the ability to induce complete regression of stage IV melanoma. Two challenges associated TIL ACT are that (i) T cells are capable of different anti-tumor effector functions including cytotoxicity,

Abstract B45: Mitochondrial Ca2+ transport fine-tunes functional cross-talk between antitumor CD8+T lymphocytes and natural killer cells

Abstract Tumor antigen-specific T cell and natural killer (NK) cell collaboration is indispensable for the elimination of tumor cells, including antigen-deficient tumor escape variants before metastasis. While mechanistic details are available for the innate instruction of T lymphocyte responses, little is known for the adaptive control of NK cell activity. We observed in a mouse model of mastocytoma expressing a self tumor antigen P1A that effector CD8+T cells provided a necessary “help” to dormant NK cells in eliciting their antitumor effector function. Bioluminescence imaging of mastocytoma tumors following adoptive transfer of P1A-specific T cells in RAG-/- and RAG-/-gamma chain-/- mice showed that NK cell anti-tumor activity requires cytolytic T cells, whereas T cells can function independent of NK cells. The goal of present study was to dissect the molecular mechanisms underlying functional cross-talk between activated CD8+T lymphocytes and naïve NK cells. In 2D and 3D co-culture systems, we observed that activated CD8+T cells form multiple intercellular contacts with naïve NK lymphocytes. Moreover, co-culture of activated T cells with naïve NK cells results in the selective down-regulation of CD25 molecule in T cells while elevating CD25 and CD69 expression on naïve NK cells. Further, CD8+T and NK cells cross-regulate their mitochondrial homeostasis including calcium transport. This effect is dependent on both cytokines and intercellular contacts, and partially involves natural killer group 2 member D (NKG2D) receptor activation. Data also indicate that activated CD8+T cells may exchange mitochondria and activation molecules such as CD25 and CD69 with naïve NK cells. Alterations in phosporylation status of multiple signaling proteins during CD8+T-NK interaction suggest a cellular remodeling whereby NK cells shift activated CD8+T cells towards T central-memory (TCM) phenotype and activated CD8+T lymphocytes alter the naïve state of NK cells towards effector/regulatory phenotype. Inhibition of mitochondrial Ca2+ uptake (mCU) or Na+/Ca2+ exchanger (mNCE) with Ru360 and CGP37157, respectively, mimicked observed alterations in CD8+ T and NK cell phenotypes upon their interaction. These results highlight a novel role of mitochondrial Ca2+ homeostasis in the re-modeling of memory differentiation and activation signaling of interacting CD8+T cells and NK cells, respectively. Improved mechanistic understanding of CD8+T-NK interplay will help refine cellular immunotherapy approach against cancer. Citation Format: Roman V. Uzhachenko, J Shawn Goodwin, Lino Costa, Alexander Terekhov, Menaka C. Thounaojam, William H. Hofmeister, Anil Shanker. Mitochondrial Ca2+ transport fine-tunes functional cross-talk between antitumor CD8+T lymphocytes and natural killer cells. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B45.

Abstract IA08: Using genomics to define the antigenic targets of checkpoint blockade cancer immunotherapy

Abstract Cancer Immunoediting is the process by which the immune system controls and shapes cancer. We originally envisaged and subsequently showed that, in its most complex form, cancer immunoediting occurs in three phases: Elimination (also known as cancer immunosurveillance, the host protective phase of the process), Equilibrium (the phase in which tumor cells that survive immune elimination remain under immunologic growth control resulting in a state of functional tumor dormancy) and Escape (the phase where clinically apparent tumors emerge because immune sculpting of the tumor cells has produced variants that display either reduced immunogenicity or enhanced immunosuppressive activity) (1-3). Strong experimental data have been obtained using mouse cancer models to demonstrate the existence of each phase of the cancer immunoediting process and compelling clinical data suggests that a similar process also occurs during the evolution of certain types of human cancer. Our efforts now focus on elucidating the molecular and cellular mechanisms that underlie each phase of cancer immunoediting and identifying the critical checkpoints that regulate progression from one phase of the process to the next. We recently used a combination of exome sequencing and epitope prediction algorithms to show that mutant proteins in highly immunogenic tumor cells derived from methylcholanthrene treated immunodeficient mice represent immunodominant, tumor specific antigens for CD8+ T cells and that immunoselection is a major mechanism of immunoediting (4). More recently, we asked whether our approach could identify antigens in progressively growing tumors that render them susceptible to checkpoint blockade immunotherapy. T cell lines generated from anti-PD-1 treated mice that had rejected d42m1-T3 progressor sarcoma cells displayed restriction to H-2Kb but not to H-2Db, suggesting that anti-PD-1 promotes T cell responses to only a limited number of antigens. We then identified expressed nonsynonymous mutations in d42m1-T3 cells using exome sequencing and generated a prioritized list of potential H-2Kb binding epitopes. This analysis predicted two unequivocal “best candidates”—a mutant form of Laminin a subunit 4 (mLama4) and a mutant glucosyltransferase (mAlg8). When tested in vitro, these two epitopes were the only ones among the 61 top predicted H-2Kb binding sequences that stimulated either d42m1-T3 specific T cell lines or CD8+ tumor infiltrating T cells freshly isolated from d42m1-T3 tumors growing in mice treated with anti-PD-1. These findings were further validated by showing that: (i) mLama4 and mAlg8 epitopes were detected in association with H-2Kb on d42m1-T3 tumor cells; (ii) CTLs expressing TCRs for mLama4 and mAlg8 accumulated over time in d42m1-T3 tumors in anti-PD-1-treated, tumor-bearing mice; (iii) vaccination of naïve WT mice with mutant but not WT forms of Lama4 or Alg8 induced strong CD8+ T cell responses; and (iv) naïve mice vaccinated against mLama4 plus mAlg8 controlled outgrowth of d42m1-T3 tumors when vaccination was performed not only prophylactically but also therapeutically. Interestingly, we found that mLama4 and mAlg8 CD8+ T cells were also present in d42m1-T3 tumors growing progressively in WT mice treated with control mAb. However, as determined by RNA Seq and immunostaining/flow cytometry, the functional profiles of antigen specific T cells from tumor bearing mice treated with control versus anti-PD-1 mAb were very different. Antigen specific CD8+ TILs from tumor bearing mice treated with control mAb displayed a gene expression pattern and surface markers suggesting an “exhausted” T cell phenotype. In contrast, antigen specific CD8+ TILs from mice treated with anti-PD-1 displayed patterns suggesting a reversal of the “exhausted” phenotype and acquisition of anti-tumor effector functions. These patterns were only observed in antigen specific CD8+ TILs and not in CD8+ TILs that showed no specificity for tumor antigens. These results thus reveal that our genomics approach is useful in (a) developing personalized cancer immunotherapies, (b) identifying individuals who would best benefit from checkpoint blockade cancer immunotherapy and (c) identifying tumor antigen specific T cells that function as a predictive biomarker of successful immunotherapy. Citation Format: Robert D. Schreiber. Using genomics to define the antigenic targets of checkpoint blockade cancer immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA08.

Abstract 4059: Crosstalk between CD8+ T and NK cells: fine-tuning of antitumor immune response

Abstract Local tumor antigen-specific T cell-NK cell collaboration is indispensable for the elimination of tumor cells, including antigen-deficient tumor escape variants before metastasis. While mechanistic details are available for the innate instruction of the T cell responses, little is known for the adaptive control of NK cell activity. We observed in a mouse model of mastocytoma expressing a self tumor antigen P1A that effector CD8+ T cells provided a necessary “help” to dormant NK cells in eliciting their antitumor effector function. Bioluminescence imaging of mastocytoma tumors following adoptive transfer of P1A-specific T cells in RAG-/- and RAG-/-γc-/- mice showed that NK cell anti-tumor activity requires cytolytic T cells, whereas T cells can function independent of NK cells. In 2D and 3D co-culture systems, we observed that PMA/ionomycin-stimulated CD8+ T cells form multiple contacts with naïve NK lymphocytes. Data show that NK cells interacting with activated CD8+ T cells show an up-regulation of CD25 and CD69 expression mediated by intercellular contacts, and activation of NKG2D receptors and Stat2, Stat6, Jak1, Jak3, Tyk2, and PTEN signaling molecules with a decrease in the phosphorylation of Stat1, PKB/Akt, SAPK/JNK, p38. On the other hand, interacting NK cells down-regulate CD25 molecule expression on CD8+ T cells and promote differentiation of central memory CD44+CD62L+ T cells. CD8+ T cells display an elevation in the phosphorylation of Stat1 and down-regulation of Stat5 with stimulated PKB/Akt, Lck, mTOR, and p42/p44. Moreover, significant changes in the cytosolic and mitochondrial Ca2+, production of mitochondrial ROS, mitochondrial membrane potential, mitochondrial permeability transition pore, and synthesis of nitric oxide and non-protein thiols (mostly, reduced glutathion) were observed in a reciprocal T cell-NK cell interaction. These results highlight the importance of mitochondrial activity in the re-modeling of activation signaling and memory differentiation of interacting CD8 T cells and NK cells. These results will help refine cancer immunotherapeutic strategies. Citation Format: Roman V. Uzhachenko, J Shawn Goodwin, Lino Costa, Alexander Terekhov, Menaka C. Thounaojam, William H. Hofmeister, Anil Shanker. Crosstalk between CD8+ T and NK cells: fine-tuning of antitumor immune response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4059. doi:10.1158/1538-7445.AM2015-4059

Abstract 5009: NKG2D receptor stimulation of effector CD8+ T cells alters the transcriptome and activation of mTOR to enhance anti-tumor immunity

Abstract To induce strong immune responses, naïve CD8+ T cells require concurrent stimulation through the TCR and costimulatory receptors. Each costimulatory molecule can have a unique effect on the function of T cells due to differential activation of downstream signaling pathways and gene expression. However, the effect of stimulating costimulatory receptors on effector T cells is still unclear. One costimulation receptor that is likely to be engaged in the tumor microenvironment is NKG2D since the ligands for this receptor are expressed on over 80% of tumors. To determine how activation of costimulatory receptors in the tumor microenvironment alters CD8+ T cell functions, this study investigated the differential activation of signaling pathways by two costimulatory receptors, CD28 and NKG2D. To obtain an in-depth view of how stimulation through the NKG2D receptor alters T cell function, the transcriptome of NKG2D- and CD28- costimulated murine CD8+ T cells was analyzed using RNA-sequencing. These analyses showed that NKG2D costimulation of effector CD8+ T cells altered expression of over 5200 genes compared to CD3 stimulation alone. Some genes that were increased in NKG2D-stimulated cells include IFNγ, and granzymes A and C, suggesting the NKG2D stimulation enhances the anti-tumor functions of CD8+ T cells. When comparing NKG2D and CD28 costimulation, many genes were differentially regulated, including genes in the mTOR pathway. To determine if the differential activation of mTORc1 and mTORc2 signaling pathways altered T function, murine effector CD8+ T cells were activated through CD3 in combination with CD28 or NKG2D and treated with no, low, or high amounts of rapamycin to deactivate either mTORc1 or both mTORc1 and mTORc2. NKG2D-stimulated T cells increased expression of Eomes, a transcription factor that is involved in the differentiation of central memory T cells, while CD28 stimulated cells increased expression of TCF1. Rapamycin inhibition studies showed that stronger activation of mTORc1 in CD28 stimulated T cells inhibited Eomes expression and also increased TCF1 expression compared to NKG2D-stimulated T cells. In addition, NKG2D stimulation lead to differential expression of memory markers including CD127, CCR7, CD62L, and KLRG1, with NKG2D stimulation inducing a central memory phenotype compared to CD28 stimulation which caused an effector memory phenotype. Together, these data suggest that differential activation of mTORc1 by the NKG2D receptor alters CD8+ T cell differentiation to become central memory T cells. Thus, NKG2D stimulation likely enhances CD8+ T cell anti-tumor effector functions. Citation Format: Kelsey Trace, Sara Jacobson, Amorette E. Barber. NKG2D receptor stimulation of effector CD8+ T cells alters the transcriptome and activation of mTOR to enhance anti-tumor immunity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5009. doi:10.1158/1538-7445.AM2015-5009

Abstract IA23: Using genomics to identify tumor specific antigens and inform cancer immunotherapy

Abstract Cancer Immunoediting is the process by which the immune system controls and shapes cancer. We originally envisaged and subsequently showed that, in its most complex form, cancer immunoediting occurs in three phases: Elimination (also known as cancer immunosurveillance, the host protective phase of the process), Equilibrium (the phase in which tumor cells that survive immune elimination remain under immunologic growth control resulting in a state of functional tumor dormancy) and Escape (the phase where clinically apparent tumors emerge because immune sculpting of the tumor cells has produced variants that display either reduced immunogenicity or enhanced immunosuppressive activity) (1-3). Strong experimental data have been obtained using mouse cancer models to demonstrate the existence of each phase of the cancer immunoediting process and compelling clinical data suggests that a similar process also occurs during the evolution of certain types of human cancer. Our efforts now focus on elucidating the molecular and cellular mechanisms that underlie each phase of cancer immunoediting and identifying the critical checkpoints that regulate progression from one phase of the process to the next. We recently used a combination of exome sequencing and epitope prediction algorithms to show that mutant proteins in highly immunogenic tumor cells derived from methylcholanthrene treated immunodeficient mice represent immunodominant, tumor specific antigens for CD8+ T cells and that immunoselection is a major mechanism of immunoediting (4). More recently, we asked whether our approach could identify antigens in progressively growing tumors that render them susceptible to checkpoint blockade immunotherapy. T cell lines generated from anti-PD-1 treated mice that had rejected d42m1-T3 progressor sarcoma cells displayed restriction to H-2Kb but not to H-2Db, suggesting that anti-PD-1 promotes T cell responses to only a limited number of antigens. We then identified expressed nonsynonymous mutations in d42m1-T3 cells using exome sequencing and generated a prioritized list of potential H-2Kb binding epitopes. This analysis predicted two unequivocal “best candidates”—a mutant form of Laminin α subunit 4 (mLama4) and a mutant glucosyltransferase (mAlg8). When tested in vitro, these two epitopes were the only ones among the 61 top predicted H-2Kb binding sequences that stimulated either d42m1-T3 specific T cell lines or CD8+ tumor infiltrating T cells freshly isolated from d42m1-T3 tumors growing in mice treated with anti-PD-1. These findings were further validated by showing that: (i) mLama4 and mAlg8 epitopes were detected in association with H-2Kb on d42m1-T3 tumor cells; (ii) CTLs expressing TCRs for mLama4 and mAlg8 accumulated over time in d42m1-T3 tumors in anti-PD-1-treated, tumor-bearing mice; (iii) vaccination of naïve WT mice with mutant but not WT forms of Lama4 or Alg8 induced strong CD8+ T cell responses; and (iv) naïve mice vaccinated against mLama4 plus mAlg8 controlled outgrowth of d42m1-T3 tumors when vaccination was performed not only prophylactically but also therapeutically. Interestingly, we found that mLama4 and mAlg8 CD8+ T cells were also present in d42m1-T3 tumors growing progressively in WT mice treated with control mAb. However, as determined by RNA Seq and immunostaining/flow cytometry, the functional profiles of antigen specific T cells from tumor bearing mice treated with control versus anti-PD-1 mAb were very different. Antigen specific CD8+ TILs from tumor bearing mice treated with control mAb displayed a gene expression pattern and surface markers suggesting an “exhausted” T cell phenotype. In contrast, antigen specific CD8+ TILs from mice treated with anti-PD-1 displayed patterns suggesting a reversal of the “exhausted” phenotype and acquisition of anti-tumor effector functions. These patterns were only observed in antigen specific CD8+ TILs and not in CD8+ TILs that showed no specificity for tumor antigens. These results thus reveal that our genomics approach is useful in (a) developing personalized cancer immunotherapies, (b) identifying individuals who would best benefit from checkpoint blockade cancer immunotherapy and (c) identifying tumor antigen specific T cells that function as a predictive biomarker of successful immunotherapy. Note: This abstract was not presented at the conference. Citation Format: Robert Schreiber. Using genomics to identify tumor specific antigens and inform cancer immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr IA23.

NK cell effector functions are differentially regulated by hypoxic conditions (TUM9P.1014)

Abstract NK cells provide the first line of defense against tumor and viruses, without the requirement for prolonged pre-activation. However, anti-tumor function of NK cells is progressively dampened in the terminal stages of cancer patients, largely associated with the tumor-induced immune evasion mechanism. Hypoxic tumor environment provides once such mechanism providing NK cell tolerance, and become a potential therapeutic problem to overcome in the patients of solid cancer for NK immunotherapy. To better understand the effect of hypoxia on NK cells, we assessed the anti-tumor function of NK cells in response to decreasing concentration of O2 concentration. As expected, NK cells cultured in severe hypoxia, 0.5% O2, demonstrated diminished cytotoxicity and IFNg against A375 melanoma targets and failed to expand to a larger number ex vivo. However, to our surprise, NK cells cultured in mild hypoxia, 1.5% O2, were expanded to a much higher level and mounted increased anti-tumor effector function, both in normoxic and hypoxic conditions. NK cells grown in mild hypoxia demonstrate upregulation of major activating NK cell receptors, NCRs, while inhibiting apoptosis during ex-vivo expansion. Thus, NK cells respond differentially to O2 concentration, and mild hypoxic conditions can indeed facilitate the expansion and function of NK cells that can overcome NK cell intolerance in the patients undergoing NK cell immunotherapy.

Despite KLRG1 expression, highly cytotoxic ThEO/TcEO polarized T cells efficiently establish immunologic memory (LYM5P.662)

Abstract Targeting immune inhibitory (CTLA-4, PD-1) or stimulatory (OX-40, 4-1BB) co-receptors has proven a potent anti-cancer therapeutic strategy, eliciting robust anti-tumor T cell responses and compelling pre-clinical and clinical data in a variety of cancers. We have previously shown in pre-clinical models that 4-1BB agonist antibodies engender strong anti-tumor T cell responses, imparting enhanced survival benefit in addition to evoking durable tumor regression. This impressive anti-tumor effect is due, in part, to the unique ability of 4-1BB co-stimulation to differentiate a novel subset of T cells that acquires a highly cytotoxic effector profile typically only seen in NK cells. This subset-dubbed ThEO (CD4+) or TcEO (CD8+)-is driven by the T-box transcription factor Eomesodermin, and is further characterized by expression of the inhibitory receptor KLRG1. We show here that, despite their strong effector function and expression of several co-inhibitory receptors (PD-1, TIM-3, KLRG1), the ThEO/TcEO phenotype establishes immunological memory and recalls with high frequency upon secondary tumor challenge with potent anti-tumor effector function. Further, these cells manifest characteristics of central memory (Tcm), effector memory (Tem), as well as stem cell memory (Tscm), and reside in multiple memory niches. These findings provide mechanistic insight into the process of memory formation and give evidence for the capacity of ThEO/TcEO cells to prevent tumor recurrence.

Increasing Th1 phenotype in Th17 cells improves anti-tumor T cells function (VAC7P.1032)

Abstract Immunotherapy of cancer is a promising approach for successful eradication of large and established tumor. While cytolytic CD8+ T cells have been extensively investigated and used for adoptive cell therapy (ACT), accumulating evidence indicates that CD4+ T helper (Th) cells including Th1 and Th17 subsets are also effective in tumor immunotherapy. Although Th1 cells possess key anti-tumor effector functions as IFNγhi, CD107ahi, T-bethi, Granzyme Bhi, but their poor persistence due to lack of stem-cell like characteristic effect long-term tumor control and reduce their potential in ACT. However, Th17 cells programmed ex vivo in TGFβ exhibit increased stem-cell like features and reduced effector function as compared to Th1 cells, which corelates to their increased persistence but reduced long-term tumor control. Herein we identified the ex vivo programming conditions that combine potent Th1 effector conditions with key stemness feature of Th17 to generate hybrid Th17/Th1 cells that exhibit improved control of murine melanoma (B16-F10) and human melanoma (624-MEL), as compared to Th1 or Th17 cells alone. The hybrid Th1/Th17 cells were less susceptible to activation induced cell death, exhibited increased persistence along with potent effector function, and have potential in future translational studies.

Optimization and validation of processes related to the GMP compliant manufacture of genetically modified CAR expressing NK-92 cells

Emerging evidences show immunotherapy by enhancing our own immune system has become an attractive approach for cancer treatment due to the rapid drug resistance of chemotherapy. Fusion cytokines derived from GM-CSF and common g-chain interleukins have potent gain-of-function properties to alter host immune response.Wehave previously demonstrated that coupling ofGM-CSF at the N-terminus of common g-chain interleukins leads to meaningful gain-of function activity in interleukin-responsive lymphomyeloid cells. Recently, wehave generated a newly bioengineered fusokine GIFT4 that is derived from GM-CSF and IL-4. Here we tested the bioactivity of the novel fusion cytokine GIFT4 as a potential immunotherapeutic agent against caner. We observed that GIFT4 directly elicits anti-tumor adaptive B-cell immune response and consequent T cell immunity in vitro and in vivo. Murine GIFT4 expression suppresses melanoma tumor growth in immunologically intact mouse and administration of GIFT4 protein inhibits murine melanoma growth in vivo. GIFT4-B cell primed cytotoxic T cells frommelanoma patients specifically kill humanmelanoma cells in vitro and in immunedeficientNSGmice.Thus,GIFT4defines a novel engineered cytokine that mediates endogenous expansion of B-cells with potent anti-tumor effector function. We propose that GIFT4 protein could serve as a novel immunotherapeutic agent and defines a previously unrecognized potential of B-cells for personalized cancer cell immunotherapy. We expect that GIFT4 as an anti-melanomaagentwill provideanovel strategyandopens anewavenue forhumanBcellbased immunotherapy against melanoma. We propose that GIFT4-B cells could serve as a potent immunotherapeutic agent and define a previously unrecognized potential of B-cells for personalized cancer cell immunotherapy.

Engineering monocyte-derived dendritic cells to secrete interferon-α enhances their ability to promote adaptive and innate anti-tumor immune effector functions.

Dendritic cell (DC) vaccination has demonstrated potential in clinical trials as a new effective cancer treatment, but objective and durable clinical responses are confined to a minority of patients. Interferon (IFN)-α, a type-I IFN, can bolster anti-tumor immunity by restoring or increasing the function of DCs, T cells and natural killer (NK) cells. Moreover, type-I IFN signaling on DCs was found to be essential in mice for tumor rejection by the innate and adaptive immune system. Targeted delivery of IFN-α by DCs to immune cells could boost the generation of anti-tumor immunity, while avoiding the side effects frequently associated with systemic administration. Naturally circulating plasmacytoid DCs, major producers of type-I IFN, were already shown capable of inducing tumor antigen-specific T cell responses in cancer patients without severe toxicity, but their limited number complicates their use in cancer vaccination. In the present work, we hypothesized that engineering easily generated human monocyte-derived mature DCs to secrete IFN-α using mRNA electroporation enhances their ability to promote adaptive and innate anti-tumor immunity. Our results show that IFN-α mRNA electroporation of DCs significantly increases the stimulation of tumor antigen-specific cytotoxic T cell as well as anti-tumor NK cell effector functions in vitro through high levels of IFN-α secretion. Altogether, our findings mark IFN-α mRNA-electroporated DCs as potent inducers of both adaptive and innate anti-tumor immunity and pave the way for clinical trial evaluation in cancer patients.