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Antigen-specific immunotherapy can be limited by induced tumor immunoediting (e.g., antigen loss) or through failure to recognize antigen-negative tumor clones. Melanoma differentiation–associated gene-7/IL24 (MDA-7/IL24) has profound tumor-specific cytotoxic effects in a broad spectrum of cancers. Here we report the enhanced therapeutic impact of genetically engineering mouse tumor-reactive or antigen-specific T cells to produce human MDA-7/IL24. While mock-transduced T cells only killed antigen-expressing tumor cells, MDA-7/IL24-producing T cells destroyed both antigen-positive and negative cancer targets. MDA-7/IL24-expressing T cells were superior to their mock-engineered counterparts in suppressing mouse prostate cancer and melanoma growth as well as metastasis. This enhanced antitumor potency correlated with increased tumor infiltration and expansion of antigen-specific T cells as well as induction of a Th1-skewed immunostimulatory tumor environment. MDA-7/IL24-potentiated T-cell expansion was dependent on T-cell–intrinsic STAT3 signaling. Finally, MDA-7/IL24-modified T-cell therapy significantly inhibited progression of spontaneous prostate cancers in Hi-Myc transgenic mice. Taken together, arming T cells with tumoricidal and immune-potentiating MDA-7/IL24 confers new capabilities of eradicating antigen-negative cancer cell clones and improving T-cell expansion within tumors. This promising approach may be used to optimize cellular immunotherapy for treating heterogeneous solid cancers and provides a mechanism for inhibiting tumor escape. Significance: This research describes a novel strategy to overcome the antigenic heterogeneity of solid cancers and prevent tumor escape by engineering T lymphocytes to produce a broad-spectrum tumoricidal agent.

Cancer immunoediting is a hallmark of cancer that predicts T cells recognize and kill tumor cells expressing immunogenic mutations (neoantigens), to induce less immunogenic clones to outgrow in tumors. Although established through longitudinal studies of how tumors evolve in immune-proficient and -deficient mice, whether the human immune system naturally targets neoantigens to edit tumors remains unclear. Here, we investigate how 70 human pancreatic ductal adenocarcinomas (PDACs) – a poorly immunogenic cancer with few neoantigens and thus largely presumed to not be subject to immunoediting – evolved over 10 years. We use longitudinal tumor sampling to compare how primary tumors evolve to recurrence in rare long-term PDAC survivors previously shown to have more immunogenic tumors (n = 9 patients, n = 9 primary and 22 recurrent tumors, median survival 5.4 years), to short-term survivors with less immunogenic primary tumors (n = 6 patients, n = 6 primary and 33 recurrent tumors, median survival 1.8 years). Compared to short-term survivors, we observe that long-term survivors evolve fewer recurrent tumors with longer latency, and distinct tissue tropism. To evaluate if differential immune pressures could explain these differences, we perform whole exome sequencing to bioinformatically predict tumor clonal structures and neoantigens. We discover that despite longer times to evolve, long-term survivors evolve genetically less heterogeneous tumors with fewer clones, fewer nonsynonymous mutations, and fewer neoantigens. To identify the edited neoantigens, we sought to improve upon and apply our previously defined quality model that quantifies the immunogenic features of a neoantigen. With our quality model, we now infer a neoantigen is immunogenic (“high quality”) by two parameters – if the immune system can recognize a neoantigen as “non-self” by its similarity to known immunogenic antigens, and discriminate it from “self” by estimating if a neoantigen has sufficient antigenic distance from its wild-type peptide to differentially bind the MHC or activate a T cell. We integrate these features to estimate neoantigen quality in primary and recurrent tumors of long- and short-term survivors. With the quality model, we observe that neoantigens with greater antigenic distance (“less self”) are more depleted in primary and recurrent tumors of long- compared to short-term survivors. Furthermore, we find that long-term survivors evolve markedly fewer new neoantigens of strikingly lower quality, to indicate clones with high quality (more immunogenic) neoantigens are immunoedited. Thus, we submit longitudinal evidence that the human immune system naturally edits neoantigens in PDAC. Furthermore, we present a model that describes how cancer neoantigens evolve under immune pressure over time, with implications for cancer biology and therapy. More broadly, our results argue that immunoediting is a fundamental cancer suppressive mechanism that can be quantified to predict tumor evolution. Citation Format: Luis A. Rojas, Marta Łuksza, Zachary M. Sethna, Kevin Soares, Joanne Leung, Jayon Lihm, David Hoyos, Anton Dobrin, Rajya Kappagantula, Alvin Makohon-Moore, Amber Johns, Anthony Gill, Masataka Amisaki, Pablo Guasp, Abderezak Zebboudj, Rebecca Yu, Adrienne Kaya Chandra, Zagaa Odgerel, Michel Sadelain, Erin Patterson, Christine Iacobuzio-Donahue, Benjamin D. Greenbaum, Vinod P. Balachandran. High quality neoantigens are immunoedited in long-term pancreatic cancer survivors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-003.

Cancer immunoediting, a hallmark of cancer, predicts that lymphocytes recognize tumor-specific mutations (neoantigens) to kill immunogenic tumor cells and select less immunogenic clones to survive in immunocompetent hosts. Yet, though proven in mice, whether immunoediting governs how human tumors evolve naturally, and the principles of how the immune system edits tumors remain unclear. Here, we report that T cell rich tumors of rare long-term survivors of human pancreatic ductal adenocarcinoma longitudinally lose immunogenic features predicted by a fitness model based on immune recognition of high quality neoantigens. We find that compared to T cell poor tumors in short-term survivors, long-term survivors evolve less immunogenic recurrent tumors over 10 years with fewer clones, fewer neoantigens, and new neoantigens of markedly lower quality, to indicate that the immune system edits lower fitness clones with high-quality neoantigens. To explore if the immune system more broadly selects against non-self-like changes in host genomes, we define a minimal amino acid substitution distance sufficient for T cell receptors to discriminate self from non-self mutations. We find that both primary and recurrent tumors in long but not short-term survivors are enriched in self-like mutations, to suggest that the immune system depletes non-self-like mutations. Thus, we submit longitudinal evidence that the immune system edits neoantigens to sculpt recurrent human pancreatic ductal adenocarcinomas, a lethal tumor largely considered resistant to endogenous immune attack. Furthermore, we demonstrate that the neoantigen quality fitness model predicts how immune pressure changes tumor cell populations over time, with significant implications for cancer biology and immunotherapy. More broadly, our results argue that cancer immunoediting is a fundamental principle of how human cancers evolve in immunocompetent hosts. Citation Format: Marta Luksza, Zachary Sethna, Luis Rojas, Kevin Soares, Yuval Elhanati, Jayon Lihm, David Hoyos, Rajya Kappagantula, Alvin M. Moore, Erin Patterson, Christine Iacobuzio-Donahue, Benjamin Greenbaum, Vinod P. Balachandran. High quality neoantigens are immunoedited in long term survivors of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB005.

Patients with high-risk non muscle-invasive bladder cancer (NMIBC) frequently relapse after standard BCG immunotherapy and have a dismal outcome after progression to muscle-invasive bladder cancer (MIBC). The mechanisms of tumor resistance to such immunotherapy remain elusive. We performed functional assays of fresh bladder tumors mixed with BCG, reinforced with in vitro experiments and in situ immune profiling of a cohort of MIBC pre- and post BCG therapy. Here, we demonstrate two distinct patterns of BCG-induced immune subversion. In the first pattern, intracellular BCG infection was associated with HLA-I loss and epithelial-to- mesenchymal transition (EMT) characteristics. HLA-I deficient tumors displayed a myeloid immunosuppressive microenvironment and dismal outcomes. Conversely, HLA-I+ BCG-treated tumors generated a Th1 type of immune response associated with an upregulation of exhaustion markers. Such patients had a very favorable outcome upon radical surgery. We surmise that HLA-I expression in bladder cancers does not result from immunoediting but rather from a BCG-induced EMT process that predicts dismal prognosis. Cancer cells HLA-I scoring by immunohistochemistry (IHC) staining can be easily implemented by pathologists in routine practice in order to stratify future urothelial cancer patient treatment strategies. Citation Format: Mathieu Rouanne, Julien Adam, Camélia Radulescu, Séverine Mouraud, Delphine Bredel, Diane Letourneur, Tuan Zea Tan, Nicolas Signolle, Amélie Bigorgne, Michael Dussiot, Sandrine Susini, Lambros Tselikas, François-Xavier Danlos, Roman Chabanon, Anna K. Schneider, Sophie Vacher, Thierry Lebret, Ivan Bièche, Yves Allory, Jean-Charles Soria, Jean Paul Thiery, Laurence Zitvogel, Aurélien Marabelle. BCG can subvert patients antitumor immune response by downregulating HLA-I expression on cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 66.

Immunosurveillance constitutes the first step of cancer immunoediting in which developing malignant lesions are eliminated by antitumorigenic immune cells. However, the mechanisms by which neoplastic cells induce an immunosuppressive state to evade the immune response are still unclear. The transcription factor STAT3 has been implicated in breast carcinogenesis and tumor immunosuppression in advanced disease, but its involvement in early disease development has not been established. Here, we genetically ablated Stat3 in the tumor epithelia of the inducible PyVmT mammary tumor model and found that Stat3-deficient mice recapitulated the three phases of immunoediting: elimination, equilibrium, and escape. Pathologic analyses revealed that Stat3-deficient mice initially formed hyperplastic and early adenoma-like lesions that later completely regressed, thereby preventing the emergence of mammary tumors in the majority of animals. Furthermore, tumor regression was correlated with massive immune infiltration into the Stat3-deficient lesions, leading to their elimination. In a minority of animals, focal, nonmetastatic Stat3-deficient mammary tumors escaped immune surveillance after a long latency or equilibrium period. Taken together, our findings suggest that tumor epithelial expression of Stat3 plays a critical role in promoting an immunosuppressive tumor microenvironment during breast tumor initiation and progression, and prompt further investigation of Stat3-inhibitory strategies that may reactivate the immunosurveillance program. Cancer Res; 76(6); 1416–28. ©2015 AACR.

Why are some early neoplastic lesions identified through immunosurveillance and subsequently destroyed, whereas other tumors evade this clearance step and progress to clinically-relevant tumors? The cancer immunoediting hypothesis highlights that the innate and adaptive immune responses work together to “flag” early neoplastic lesions for immune-mediated elimination. An early mediator of this “elimination” process is activation of type I interferon signaling. Thus, suppression of type I interferon signaling may help developing tumors evade the critical early steps of immune recognition and clearance. Preliminary data from our lab suggest that progesterone, working through the progesterone receptor (PR), may be a key player in tumor immune evasion through downregulation of interferon signaling. In breast cancer cells, we have shown that presence/activation of PR can attenuate interferon-signaling at multiple steps within the signaling cascade: decreased STAT1 phosphorylation, decreased STAT1/STAT2/IRF9 transcriptional complex formation (called ISGF3, required for interferon-alpha signaling), decreased recruitment of ISGF3 to ISG promoter sequences, and decreased transcriptional activation of interferon-stimulated genes (ISG, end point of interferon signaling). These data suggest a concerted effort aimed at reducing interferon-signaling through activation of PR. These data translate to the immune microenvironment in the murine mammary gland. In mice treated with progesterone for 21 days, we see immunosuppressive changes in the mammary gland: increased T-regulatory cells (immunosuppressive), decreased frequency of antigen-presenting cells (decreased ability to present potential tumor-associated antigens to CD8 T-cells), and a decrease in MHC class I presentation in non-lymphoid cells (decreased ability for epithelial cells to present potential tumor-associated antigens). We observe similar phenotypes in mice with transgenic overexpression of PR. These changes in the immune system suggest that PR can promote immunosuppressive changes in the mammary gland. Moreover, 80% of these mice in a multiparous cohort develop mammary gland tumors. Therefore, early changes in the immune microenvironment in the mammary gland may translate to the development of mammary gland tumors in PR transgenic mice. Cumulatively, these data suggest a mechanism through which PR aids early breast cancer lesions in escaping immune surveillance. These data have significant implications for the use of progesterone-containing hormone replacement therapy, as well as underscore the importance of studying anti-progestins as novel chemo-preventative agents for breast cancer. Citation Format: Christy Hagan, Merit Goodman, Gloria Trinca, Katherine Walter, Katelin Gibson, Prabhakar Chalise, Mary Markiewicz. Progesterone promotes immunosuppression in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-20.

Pancreatic adenocarcinoma (PDAC) carries a dismal prognosis and efforts to harness immune-based therapies are in high demand. Using retrograde pancreatic duct instillation of lentiviral particles, we have established and characterized a preclinical autochthonous mouse model for immunogenic PDAC. Specifically, pancreatic tumors are initiated in KRASLSL-G12D/+; p53flox/flox (KP) mice using lentiviral particles expressing Cre recombinase and a tumor-specific antigen (TSA) to track the endogenous antigen-specific T cell response in both precursor lesions (PanIN) and adenocarcinoma (PDAC). Using this model system, we demonstrate profound immunoediting in the autochthonous pancreas, which occurs primarily at the preinvasive (PanIN) stage. In parallel, we derived a biologic library of immunogenic or nonimmunogenic pancreatic organoids and developed an ex vivo co-culture system of pancreatic organoids with antigen-specific CD8 T cells that recapitulates the phenomena of immunoediting. Additionally, we examined the effect of TSA expression in established adenocarcinomas and performed preclinical trials in which the CD8 T-cell compartment was transiently depleted and then restored, to evaluate at what point during tumor progression pancreatic tumors gain the ability to evade immune clearance. Intriguingly, we identified both “regressor” tumors as well as “progressor” tumors in response to CD8 restoration. Longitudinal imaging was paired with flow cytometric analysis to track both clinical and antigen-specific T-cell responses. Excitingly, we found that a subset of “regressor” tumors undergo a complete pathologic response following CD8 T-cell restoration, highlighting the translational potential for novel insights regarding immune escape in PDAC. In addition, we demonstrate that both the magnitude and kinetics of the antigen-specific T-cell response tracks with the clinical response of these tumors, with “progressor” tumors eliciting both delayed kinetics and a blunted response compared with “regressor” tumors. Additionally, we developed an immunogenic genetically defined organoid system in which KP organoids were engineered to express a defined neoantigen from a safe harbor locus. In line with prior studies, a subset of immunogenic organoids was completely rejected upon orthotopic transplantation and this clearance was dependent upon CD8 T cells. However, in contrast to observations using two-dimensional monolayer cell lines, we also identified a significant subset of orthotopically transplanted immunogenic pancreatic organoids that successfully evade immune clearance, despite eliciting an antigen-specific immune response. Immunophenotyping the tumor microenvironment revealed that antigen-specific CD8 T cells within these “escaper” tumors display hallmarks of T-cell exhaustion. These complementary systems now provide a robust preclinical platform to rapidly interrogate potential therapeutic interventions. This abstract is also being presented as Poster C17. Citation Format: William A. Freed-Pastor, Laurens Lambert, Ana P. Garcia, George Eng, Omer Yilmaz, Tyler Jacks. Preclinical models to dissect immune escape in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr PR14.

Immune escape is a hallmark of cancer, but whether it relies upon extrinsic immune-selective pressure or is inherently orchestrated by oncogenic pathways is unresolved. To address this question, we took advantage of an in vitro model of sequentially transformed human mesenchymal stem cells (hMSC). Neoplastic transformation in this model increased the natural immune-evasive properties of hMSC, both by reducing their immunogenicity and by increasing their capacity to inhibit mitogen-driven T-cell proliferation. We also found that IFNγ signaling was globally affected in transformed hMSC. As a consequence, the natural inhibitory effect of hMSC on T-cell proliferation switched from an inducible mechanism depending on IFNγ signaling and mediated by indoleamine 2,3-dioxygenase to a constitutive mechanism that relied upon IL1β involving both secreted and membrane-expressed molecules. After transformation, increased IL1β expression both sustained the immunosuppressive properties of hMSC and increased their tumorigenicity. Thus, in this model system, IL1β acted as intrinsic inflammatory mediator that exerted an autocrine influence on tumor growth by coordinately linking immune escape and tumorigenicity. Collectively, our findings show how oncogenes directly orchestrate inflammation and immune escape to drive the multistep process of cancer progression, independently of any need for immunoediting in the tumor microenvironment. Cancer Res; 75(15); 3032–42. ©2015 AACR.

How the genomic landscape of a tumor shapes and is shaped by anti-tumor immunity has not been systematically explored. Using large-scale genomic datasets of solid tissue tumor biopsies, we quantified the cytolytic activity of the local immune infiltrate and identified associated properties across 18 tumor types. The number of predicted MHC Class I-associated neoantigens was correlated with cytolytic activity and was lower than expected in colorectal and other tumors, suggesting immune-mediated elimination. We identified recurrently mutated genes that showed positive association with cytolytic activity, including beta-2-microglobulin (B2M), HLA-A, -B and -C and Caspase 8 (CASP8), highlighting loss of antigen presentation and blockade of extrinsic apoptosis as key strategies of resistance to cytolytic activity. Genetic amplifications were also associated with high cytolytic activity, including immunosuppressive factors such as PDL1/2 and ALOX12B/15B. Our genetic findings thus provide evidence for immunoediting in tumors and uncover mechanisms of tumor-intrinsic resistance to cytolytic activity. Citation Format: Nir Hacohen. Genetics of immune escape. [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 SY11-04. doi:10.1158/1538-7445.AM2015-SY11-04

Immunoediting caused by antitumor immunity drives tumor cells to acquire refractory phenotypes. We demonstrated previously that tumor antigen–specific T cells edit these cells such that they become resistant to CTL killing and enrich NANOGhigh cancer stem cell-like cells. In this study, we show that synaptonemal complex protein 3 (SCP3), a member of the Cor1 family, is overexpressed in immunoedited cells and upregulates NANOG by hyperactivating the cyclin D1–CDK4/6 axis. The SCP3–cyclin D1–CDK4/6 axis was preserved across various types of human cancer and correlated negatively with progression-free survival of cervical cancer patients. Targeting CDK4/6 with the inhibitor palbociclib reversed multiaggressive phenotypes of SCP3high immunoedited tumor cells and led to long-term control of the disease. Collectively, our findings establish a firm molecular link of multiaggressiveness among SCP3, NANOG, cyclin D1, and CDK4/6 and identify CDK4/6 inhibitors as actionable drugs for controlling SCP3high immune-refractory cancer. Significance: These findings reveal cyclin D1-CDK4/6 inhibition as an effective strategy for controlling SCP3high immune-refractroy cancer. Cancer Res; 78(10); 2638–53. ©2018 AACR.

Natural killer (NK) cells are potent immune effector cells capable of mediating antitumor responses. Thus, during immunoediting, tumor cell populations evolve strategies to escape NK-cell–mediated recognition. In this study, we report a novel mechanism of immune escape involving tumor cell shedding of B7-H6, a ligand for the activating receptor NKp30 that mediates NK-cell binding and NK-cell–mediated killing. Tumor cells from different cancer entities released B7-H6 by ectodomain shedding mediated by the cell surface proteases “a disintegrin and metalloproteases” (ADAM)-10 and ADAM-17, as demonstrated through the use of pharmacologic inhibitors or siRNA-mediated gene attenuation. Inhibiting this proteolytic shedding process increased the levels of B7-H6 expressed on the surface of tumor cells, enhancing NKp30-mediated activation of NK cells. Notably, we documented elevated levels of soluble B7-H6 levels in blood sera obtained from a subset of patients with malignant melanoma, compared with healthy control individuals, along with evidence of elevated B7-H6 expression in melanoma specimens in situ. Taken together, our results illustrated a novel mechanism of immune escape in which tumor cells impede NK-mediated recognition by metalloprotease-mediated shedding of B7-H6. One implication of our findings is that therapeutic inhibition of specific metalloproteases may help support NK-cell–based cancer therapy. Cancer Res; 74(13); 3429–40. ©2014 AACR.

Why are some early neoplastic lesions identified through immunosurveillance and subsequently destroyed, whereas other tumors evade this clearance step and progress to clinically-relevant tumors? The cancer immunoediting hypothesis highlights that the innate and adaptive immune responses work together to “flag” early neoplastic lesions for immune-mediated elimination. An early mediator of this “elimination” process is activation of type I interferon signaling. Thus, suppression of type I interferon signaling may help developing tumors evade the critical early steps of immune recognition and clearance. Preliminary data from our lab suggest that progesterone, working through the progesterone receptor (PR), may be a key player in tumor immune evasion through downregulation of interferon signaling. In breast cancer cells, we have shown that presence/activation of PR can attenuate interferon-signaling at multiple steps within the signaling cascade: decreased STAT1 phosphorylation, decreased STAT1/STAT2/IRF9 transcriptional complex formation (called ISGF3, required for interferon-alpha signaling), decreased recruitment of ISGF3 to ISG promoter sequences, and decreased transcriptional activation of interferon-stimulated genes (ISG, end point of interferon signaling). These data suggest a concerted effort aimed at reducing interferon-signaling through activation of PR. These data translate to the immune microenvironment in the murine mammary gland. In mice treated with progesterone for 21 days, we see immunosuppressive changes in the mammary gland: increased T-regulatory cells (immunosuppressive), decreased frequency of antigen-presenting cells (decreased ability to present potential tumor-associated antigens to CD8 T-cells), and a decrease in MHC class I presentation in non-lymphoid cells (decreased ability for epithelial cells to present potential tumor-associated antigens). We observe similar phenotypes in mice with transgenic overexpression of PR. These changes in the immune system suggest that PR can promote immunosuppressive changes in the mammary gland. Moreover, 80% of these mice in a multiparous cohort develop mammary gland tumors. Therefore, early changes in the immune microenvironment in the mammary gland may translate to the development of mammary gland tumors in PR transgenic mice. Cumulatively, these data suggest a mechanism through which PR aids early breast cancer lesions in escaping immune surveillance. These data have significant implications for the use of progesterone-containing hormone replacement therapy, as well as underscore the importance of studying anti-progestins as novel chemo-preventative agents for breast cancer. Citation Format: Merit Goodman, Gloria Trinca, Katherine Walter, Katelin Gibson, Prabhakar Chalise, Mary Markiewicz, Christy R. Hagan. Progesterone receptor promotes immunosuppressive microenvironment in the mammary gland [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2361.

Metastatic basal-like breast cancers are believed to correspond with EpCAM-/Cd49f- cancer stem cell (CSC) enrichment. As well, basal-like breast cancers typically correspond with tumor inflammation and immunoediting phenotypes. However, the exact interplay between CSCs and the inflammatory signature of basal-like breast cancers is not well understood. To provide insight regarding the clinical overlap between breast cancer stem cells and tumor inflammation, we compared the 450K DNA methylation profile of EpCAM-/CD49f- CSCs from the isogenic MCF10A p53-/PTEN- breast cell line against the corresponding EpCAM+/CD49f+ and EpCAM-/CD49f+ subpopulations to determine whether differential DNA methylation occurred within the promoters of immune-related genes in CSCs. In addition, we also overlapped the 450K DNA methylation profile from 16 established breast cancer cell lines of varying EpCAM-/CD49f- concentrations to compare against the isolated CSCs. Based on our results, we identified 1432 differentially methylated promoter regions overall (ANOVA FDR p-value Citation Format: Emma V. Gray, Caroline E. Dyar, Maria Ouzounova, Max S. Wicha, Hasan Korkaya, Austin Y. Shull. IL32 expression is epigenetically regulated in EpCAM-/Cd49f- basal-like breast cancers and can be suppressed by the bromodomain inhibitor JQ1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3683.

The immune system can recognize and attack cancer cells and their precursors, especially those with a high load of mutation-induced neoantigens. Such neoantigens are particularly abundant in DNA mismatch repair (MMR)-deficient cancers. MMR deficiency results in microsatellite instability (MSI), which leads to multiple insertion/deletion mutations at coding microsatellites and to neoantigen-inducing translational frameshifts. The significance of immune selection and immunoediting potentially shaping the neoantigen landscape during the progression from premalignant MMR-deficient lesions into cancers has not yet been analyzed. We hypothesized that the neoantigen landscape of MSI cancers may reflect the impact of immunoediting. We developed a novel tool for quantitative analysis of microsatellite mutations to explore the neoantigen landscape of MSI colorectal (CRC, n=139) cancers. Frameshift mutations were examined in 41 coding microsatellite (cMS) regions using our new algorithm. We predicted the resulting frameshift neoantigen sequences and used the publicly available prediction tool NetMHCpan 4.0 for prediction of MHC binding sequences. Immunological scores were generated to quantify the likelihood of defined cMS mutations to generate immunogenic neoantigens in different populations with defined HLA allele distributions. Across the 41 cMS analyzed, 77% of all mutations were in the reading frame of 1 nucleotide deletions (m1). The cMS mutation frequency and FSP epitope distribution across HLA genotypes (described by a general epitope likelihood score, GELS) showed a significant negative correlation (Pearson’s r=-0.42, p=0.0149). Some cMS presented with high mutation frequencies despite a high GELS (i.e. TGFBR2: pmut = 88%, GELS = 78.9%), suggesting mutation-induced driver effects, which may outweigh the increased immunogenicity. Our results show that MSI cancers share several highly immunogenic neoantigens. Importantly, a negative correlation between the antigenic strength of neoepitopes and their mutation frequency in MMR-deficient cancers points towards continuous immunoediting during their evolution. These findings will have substantial impact on the optimization of vaccines designed to potentially prevent or treat MSI-driven cancers. Citation Format: Matthias Kloor, Alexej Ballhausen, Moritz Przybilla, Michael Jendrusch, Elisabeth Pfaffendorf, Markus Draxlbauer, Florian Seidler, Sonja Krausert, Aysel Ahadova, Simon Kalteis, Daniel Heid, Johannes Gebert, Maria Bonsack, Sarah Schott, Hendrik Bläker, Toni Seppälä, Jukka-Pekka Mecklin, Sanne Ten Broeke, Maartje Nielsen, Julia Krzykalla, Axel Benner, Angelika Riemer, Magnus von Knebel Doeberitz. The shared mutation and neoantigen landscape of MMR-deficient colorectal cancers suggests immunoediting during tumor evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 571.

Cancer immunoediting, the process by which the immune system controls tumor growth and shapes tumor immunogenicity, consists of 3 stages: elimination, equilibrium, and escape. The molecular mechanisms that underlie the equilibrium phase, during which the immune system maintains tumor dormancy, remain incompletely defined. Here, we investigated the length of the equilibrium phase during immune control of methycholanthrene (MCA)-induced or p53 mutant cancers and showed the critical and opposing roles of interleukin (IL)-23 and IL-12 in maintaining cancer cells in a state of immune-mediated dormancy. Inhibition of IL-23p19 was shown to reduce the malignant potential of lesions established by MCA inoculation, whereas inhibition of IL-12/23p40 enhanced tumor outgrowth. Furthermore, agonistic anti-CD40 antibody treatment mimicked the effects of anti-IL-23p19 monoclonal antibody treatment. Other cytokines such as IL-4, IL-17, TNF, and IFNαβ, which are known to play important roles either in MCA tumorigenesis or in the elimination phase of cancer immunoediting, did not play critical roles in maintaining the equilibrium phase. Taken together, our findings show opposing roles for IL-23 and IL-12 in determining the outgrowth versus dormancy of occult neoplasia and suggest a potential long-term danger in using IL-12/23p40 antibodies for treating human autoimmune inflammatory disorders. Cancer Res; 72(16); 3987–96. ©2012 AACR.

Deficiencies in immune function that accumulate during cancer immunoediting lead to a progressive escape from host immunosurveillance. Therapies that correct or overcome these defects could have a powerful impact on cancer management, but current knowledge of the types and mechanisms of immune escape is still incomplete. Here, we report a novel mechanism of escape from T-cell immunity that is caused by reduction in levels of the Delta family Notch ligands DLL1 and DLL4 in hematopoietic microenvironments. An important mediator of this effect was an elevation in the levels of circulating VEGF. Selective activation of the DLL1-Notch signaling pathway in bone marrow precursors enhanced T-cell activation and inhibited tumor growth. Conversely, tumor growth led to inhibition of Delta family ligand signaling through Notch in the hematopoietic environment, resulting in suppressed T-cell function. Overall, our findings uncover a novel mechanism of tumoral immune escape and suggest that a soluble multivalent form of DLL1 may offer a generalized therapeutic intervention to stimulate T-cell immunity and suppress tumor growth. Cancer Res; 71(19); 6122–31. ©2011 AACR.

Targeting relapsed acute myeloid leukemia (AML) with engineered T cells holds exceptional promise, but obstacles to efficacy exist. Understanding resistance mechanisms is essential to improve therapy and may broadly inform cancer immune escape. We and others have developed transgenic T cell receptor (TCR) therapies targeting the intracellular WT1 oncoprotein that promotes growth of most high-risk AMLs. TCR therapies generally target epitopes processed by the proteasome for Class I presentation. As mutation or loss of WT1 or loss of HLA expression is infrequent in AML, we hypothesized resistant clones may have defective antigen processing. A 25-year-old man with AML recurrent after a 2nd allogeneic stem cell transplant received reinduction chemotherapy and transgenic donor-derived T cells recognizing the HLA-A2 restricted WT1126-134 epitope (NCT01640301). The patient experienced a 12-month remission followed by relapse despite robust T cell persistence (>6% of CD8+ T cells). The relapsed leukemia expressed WT1 and HLA-A2 but was refractory to retreatment with additional WT1126-134-restricted T cells. Single cell RNA sequencing (scRNAseq) was performed on cryopreserved PBMCs from remission (n=4976 cells) and relapsed (n=2780) time points (10x Genomics platform). During apparent remission, transgenic T cells had a transcriptome distinct from native CD8s, expressing activation markers including HLA-DR, IL32, CD69 and CTLA4, suggesting recent TCR triggering from antigen encounter. However, at the time AML progression was observed, transgenic cells clustered with native quiescent CD8s, implying they were no longer encountering the targeted epitope. Transcriptome analysis of relapsed AML demonstrated downregulation of a single subunit of the immune proteasome (β1i), which we recently demonstrated in cell lines to be critical for processing the targeted WT1126-134 epitope. This patient's refractory AML was killed by T cells restricted to an alternate HLA-A2 epitope of WT1 processed independently of β1i, confirming the functional impact of immune proteasome downregulation. At an early responding timepoint, scRNAseq detected clinically unrecognized rare circulating AML cells already β1i-low, suggesting β1i loss represented immunoediting/outgrowth of a pre-existing clone as a consequence of T cell immunotherapy pressure. ScRNAseq is poised to become an important diagnostic in immunotherapy, with sufficient depth to profile both transgenic T cells and low burden/minimal residual AML, and, in our case, successfully identified a potentially targetable mechanism of immune escape. Our data suggest T cell therapy in AML and other tumors may be improved by in depth analysis of individual leukemic cells, and from selecting target epitopes processed by both standard and immunoproteasomes and/or early combination therapy combining TCRs recognizing differentially processed epitopes. Citation Format: Kelly G. Paulson, Thomas M. Schmitt, Daniel Egan, Valentin Voillet, Miranda C. Lahman, Felecia D. Wagener, Daniel S. Hunter, Petri R. Muhlhauser, Paul C. Hendrie, Cecilia Yeung, Nathalie Vigneron, Benoit Van den Eynde, Jason H. Bielas, Merav Bar, Raphael Gottardo, Aude G. Chapuis, Philip D. Greenberg. Single cell RNA sequencing reveals AML immunoediting under pressure from engineered T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2980.

Overexpression of HER-2/neu (c-erbB2) is associated with increased risk of recurrent disease in ductal carcinoma in situ (DCIS) and a poorer prognosis in node-positive breast cancer. We therefore examined the early immunotherapeutic targeting of HER-2/neu in DCIS. Before surgical resection, HER-2/neupos DCIS patients (n = 13) received 4 weekly vaccinations of dendritic cells pulsed with HER-2/neu HLA class I and II peptides. The vaccine dendritic cells were activated in vitro with IFN-γ and bacterial lipopolysaccharide to become highly polarized DC1-type dendritic cells that secrete high levels of interleukin-12p70 (IL-12p70). Intranodal delivery of dendritic cells supplied both antigenic stimulation and a synchronized preconditioned burst of IL-12p70 production directly to the anatomic site of T-cell sensitization. Before vaccination, many subjects possessed HER-2/neu–HLA-A2 tetramer-staining CD8pos T cells that expressed low levels of CD28 and high levels of the inhibitory B7 ligand CTLA-4, but this ratio inverted after vaccination. The vaccinated subjects also showed high rates of peptide-specific sensitization for both IFN-γ–secreting CD4pos (85%) and CD8pos (80%) T cells, with recognition of antigenically relevant breast cancer lines, accumulation of T and B lymphocytes in the breast, and induction of complement-dependent, tumor-lytic antibodies. Seven of 11 evaluable patients also showed markedly decreased HER-2/neu expression in surgical tumor specimens, often with measurable decreases in residual DCIS, suggesting an active process of “immunoediting” for HER-2/neu–expressing tumor cells following vaccination. DC1 vaccination strategies may therefore have potential for both the prevention and the treatment of early breast cancer. [Cancer Res 2007;67(4):1842–52]

Tumors cells can escape cytotoxic CD8+ T cells by preventing MHC I display of tumor antigens. It is unknown how tumors evade CD4+ T-cell responses, but because many tumor cells lack MHC II expression, novel mechanisms would be required. We have investigated this issue in a model in which MHC IINEG myeloma cells secrete a monoclonal Ig containing a V region L chain (VL) epitope recognized by CD4+ T cells. Infiltrating macrophages process and present the secreted tumor antigen to Th1 cells, resulting in induction of macrophage cytotoxicity and apparent rejection of the tumor. Despite long-term tumor protection in VL-specific T-cell receptor transgenic mice, we here describe that some myeloma cells persisted in a dormant state and, eventually, formed expanding tumors. Escape tumor cells maintained their secretion of complete (H+L) monoclonal Ig with unchanged sequence, while secretion of surplus free L chain was severely diminished. Although free L chains were efficiently processed and presented by tumor-infiltrating macrophages to CD4+ T cells, complete (H+L) monoclonal Ig was not. Forced overexpression of free L chain secretion reinstated tumor rejection. These results show that tumors can escape CD4+ T-cell–mediated rejection by impairing indirect presentation of tumor antigen by infiltrating macrophages. This occurs through a novel mechanism of immunoediting, in which modulation of the quaternary structure of the secreted tumor-specific antigen reduces its immunogenicity. Cancer Res; 75(16); 3268–78. ©2015 AACR.

Cancer immuno-editing is a process that describes the interaction between immune cells and tumor cells. This interaction can result in elimination of a developing tumor, tumor dormancy, or tumor cells that are capable of surviving in an immune-competent host. We have recently uncovered a novel mechanism of immunoediting by the tetraspan protein Epithelial membrane protein or EMP2. We propose that EMP2 serves as a bridge between innate and adaptive immunity via tumor mediated type I interferon expression. Most tumor cells evade the immune system through suppression or ignorance. EMP2 levels promote type I interferon expression which was found to correlate with PDL1 expression in a number of cell lines. Concordantly, in vivo, in multiple breast cancer cell lines, EMP2 promoted tumor growth. In contrast, reduction in EMP2 levels correlated with a reduction in tumor size and a corresponding increase in tumor associated macrophages. Given that tumors with lower levels of EMP2 grow poorly, we hypothesized that targeting EMP2 may provide a therapeutic benefit. Recently, we have developed a panel of novel IgG1 monoclonal antibodies to EMP2 and tested their ability to treat both xenograft and syngeneic mouse models. Our results show that anti-EMP2 IgG1 significantly reduces tumor load in both models in part through the recruitment of M1 macrophages. To explore this observation more fully, experiments using murine syngeneic models were employed. Anti-EMP2 therapy elicits a robust ADCC response, producing an immune infiltrate rich in macrophages, NK cells, and lymphocytes. In addition, we show that CD8 cells show reduced exhaustion in the presence of anti-EMP2 antibodies. Conclusions: EMP2 is a protein upregulated in a number of cancers in women including breast, ovarian, and endometrial tumors. Our work collectively show that EMP2 is a novel immune regulator where its expression creates an immunosuppressive tumor microenvironment. We thus predict that targeting EMP2 may improve therapeutic outcomes for cancers in women. Citation Format: Jessica Tsui, Negin Ashki, Yuling Chang, Deven Patel, Jasmine Sjarif, Madhuri Wadehra. Epithelial membrane protein-2 is a novel regulator of immune editing in breast cancer [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 3965. doi:10.1158/1538-7445.AM2017-3965

According to the missing-self hypothesis, natural killer (NK) cells survey for target cells that lack MHC-I molecules. The Ly49 receptor family recognizes loss of MHC-I and is critical for educating NK cells, conferring the ability to eliminate transformed or infected cells. In this study, we evaluated their requirement in innate immune surveillance of cancer cells using genetically manipulated mice with attenuated expression of Ly49 receptors (NKCKD) in several models of carcinoma and metastasis. We found that NKCKD mice exhibited uncontrolled tumor growth and metastases. Expression of two MHC-I alleles, H-2Kb and H-2Db, was decreased in tumors from NKCKD mice in support of the likelihood of NK-mediated tumor immunoediting. These tumor cells exhibited directed alterations to their cell surface expression in response to the genetically altered immune environment to evade host recognition. Immunoediting in NKCKD mice was restricted to MHC-I molecules, which are ligands for Ly49 receptors, while expression of Rae-1 and Mult1, ligands for another NK cell receptor, NKG2D, were unaffected. Restoring NK cell education in NKCKD mice with a transgene for the inhibitory self-MHC-I receptor Ly49I restored suppression of cancer onset and growth. Interestingly, immune surveillance mediated by activating Ly49 receptors remained intact in NKCKD mice, as demonstrated by the ability to stimulate the NKG2D receptor with tumor cells or splenocytes expressing Rae-1. Together, our results genetically establish the integral role of Ly49 in NK cell-mediated control of carcinogenesis through MHC-I–dependent missing-self recognition. Cancer Res; 74(14); 3684–94. ©2014 AACR.

Cancer cells gain fitness advantage over normal cells by accumulating mutations over time. Next Generation DNA sequencing technologies have made the detection of genetic alterations in tumor genomes affordable. Powerful data analysis and interpretation tools enable extraction of disease-relevant information from diverse genomics data to guide diagnosis and treatment decisions. We have built a robust cancer analytics platform OncoMD that combines tumor mutation profiles, expression signatures, copy number variations, epigenetic alterations and drug sensitivity to create a holistic view of human cancer enabling discovery of new targets for therapy and prognosis. We have analyzed the rich mutational data captured in OncoMD to identify potential T-cell neo-epitopes in different cancers and examined their prevalence in the context of the tumor microenvironment. The presence of T-cell neo-epitopes alone is not sufficient to sensitize tumors to immunotherapy drugs and requires cooperation from the tumor microenvironment. Although mutational burden was a strong predictor of T-cell neo-epitopes in our analysis, other genetic alterations such as overexpression, splicing, gene-fusions and insertions/deletions can produce altered peptides exhibiting high affinity binding to HLA class I and II molecules in cancers with low burden of missense mutations. We determined the number of potential T-cell neo-epitopes after classifying tumors based on their epithelial, stromal and immune cell content using gene expression signatures. We observed that the normalized T-cell neo-epitope burden was strongly anti-correlated with the T-cell content of the tumors, compared to their epithelial or stromal content. Together, our findings suggest that tumors enriched in T-cells may be subjected to immunoediting processes and carry relatively fewer T-cell neo-epitopes to avoid elimination. These tumors are likely to respond to immunotherapy treatments. Citation Format: Ravi Gupta, Kiran V. Paul, Kartik Kumaramangalam, Sam Santhosh, Amitabha Chaudhuri. OncoMD: A powerful genomics data analysis and interpretation platform for cancer discovery research. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5291.

The immunosurveillance/immunoediting theory postulates that tumor growth is repressed in healthy individuals through the activation of adaptive and innate immune mechanisms. The clinical success of antibodies that target inhibitory checkpoint pathways of the immune system (i.e. CTLA-4, PD-1) has provided compelling evidence in support of this theory and raises the question of whether antibodies targeting these, or other inhibitory immune cell modulators (ICM), are present in apparently healthy individuals. The present studies were designed to answer this question by probing the circulating memory B-cell compartment that represents a historic record of an individual's lifelong adaptive immune response. Methods: Blood from donors, with no known cancer history, was screened for the presence of anti-ICM binding memory B-cells targeting LAG-3, TIM-3, B7-H3, KIR, and VISTA, using Trellis's established CellSpotTM technology that can detect the presence of antigen specific memory B-cells at a frequency of 0.1 per 100,000 memory B-cells. Results: Circulating memory B-cells targeting ICM's were present in all apparently healthy individuals tested (20 total). There was substantial variation as to which ICM's were recognized within each individual across this sample population. Antibodies cloned from these rare B-cells were of IgG class and exhibited low to sub-nM affinity toward the specific ICM. These high affinity antibodies bound their respective antigens on activated T-cells and were able to augment immune stimulatory cytokine release in cellular assays. Data from ongoing studies will be presented. Conclusion: These studies demonstrate that apparently healthy individuals produce high affinity, pharmacologically active anti-ICM antibodies. Thus, healthy individuals represent a previously unrecognized source of antibodies that have undergone natural selection and hold particular promise as potential therapeutic agents for the treatment of cancer. Citation Format: Angeles Estelles, Robert Stephenson, Keyi Liu, Evelene Lomongsod, Da Ngyen, Jianzhong Zhzng, Yifan Yang, Manfred Heideker, Lawrence Kauvar, Stefan Ryser, Mikhail L. Gishizky. Antibodies targeting LAG-3, TIM-3, KIR cloned from healthy human donors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 594.

Host immune surveillance as a mechanism to promote tumor growth or to suppress tumor development is well studied. Less is known how immuno-selective pressure in the form of immunotherapies can instruct the immune system during this process. We hypothesized that the application of a T cell-dependent selection pressure in the form of a whole tumor vaccine would alter the immunogenic architecture in our transplantable murine melanoma model SB-3123. We generated an escape variant to test this hypothesis. We performed whole-exome and RNA-sequencing of the tumor line SB-3123 and two fresh tumors generated by subcutaneous implantation of SB-3123 to identify mutations and to assess levels of expression. There were 349 mutations shared between the tumor line and fresh tumors and less than 5 mutations were unique to each sample. We then investigated whether tumors that had undergone an additional round of host immunoediting would acquire different mutations. To test this, we generated a re-derived tumor cell line from a fresh SB-3123 tumor. Remarkably, the re-derived tissue culture line retained 333 mutations in common with the prior samples and produced only 2 unique mutations. To test the immunogenicity of SB-3123, we vaccinated mice with irradiated SB-3123 and administered a live tumor challenge two weeks after vaccination. This resulted in a profound vaccination response with mice being completely protected from tumor for over 200 days. However, one vaccinated mouse developed a recrudesced tumor at the site of implantation 40 days after challenge and a tumor line was created (SB-3123-esc). We asked whether SB-3123-esc represented an escape variant by immunizing mice with SB-3123 and then challenging with either SB-3123 or SB-3123-esc. Vaccination with SB-3123 did not protect against SB-3123-esc. We also performed sequencing on SB-3123-esc and discovered that 441 nonsynonymous mutations were common to both tumor lines while 80 were unique to SB-3123 and 40 to SB-3123-esc. Furthermore, we were able to determine that SB-3123-esc lost 2 immunogenic neoantigens but had acquired 5. In addition, we identified a murine T cell receptor with specific reactivity for the escape variant but not the parental tumor. Our results demonstrate that the expressed mutations of SB-3123 tumor did not change, regardless of whether we sequenced the tumor line, fresh tumors, or a re-derived tumor cell line. However, the escape tumor had new unique mutations and at least one of these was demonstrably immunogenic; we were able to develop a murine T cell receptor with specific reactivity for the escape variant but not the parental tumor. In conclusion, we posit that these observations offer a proof of concept that the process of cancer immune evasion can create new opportunities to treat escape tumors with T cell based therapies. Citation Format: Jenny H. Pan, Suman Vodnala, Robert L. Eil, Zhiya Yu, Jared Gartner, David Clever, Rahul Roychoudhuri, Shashank J. Patel, Christopher A. Klebanoff, Madhu Sukumar, Tori Yamamoto, Nicholas P. Restifo. Immunotherapy generates selective pressure to create an escape tumor with increased susceptibility to treatment with T cell based therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4034.

Tumor cells can escape immune destruction via engaging the PD-1/PD-L1 inhibitory pathway. However since PD-L1 is induced on many different cell types by IFN-ã, the importance of PD-L1 on tumor versus host cells in mediating immune escape remains unclear. In this study, we assessed the role of PD-L1 expression on two closely-related but distinct clones derived from the same d42m1 methylcholanthrene (MCA)-induced sarcoma line. Edited d42m1-T3 sarcoma cells grow progressively in wild type (WT) mice but are rejected in mice treated with anti-PD-L1. Upregulation of PD-L1 was observed on both immune and tumor cells in vivo. To assess the role of PD-L1 specifically on tumor cells, we generated d42m1-T3 cells lacking PD-L1 (T3ÄPDL1) using CRISPR-Cas9. T3ÄPDL1 cells were spontaneously rejected in naïve syngeneic WT mice but formed progressively growing tumors in Rag2-/- mice. To explore the role of host-cell expressed PD-L1, we challenged WT mice with 3-fold higher numbers of T3ÄPDL1 cells and found that the tumor cells formed progressively growing tumors in WT mice. However, when tumor bearing mice injected with the high dose of T3ÄPDL1 cells were treated with blocking PD-L1 mAb, they all rejected their tumors. Thus, PD-L1 expression on both tumor and host cells plays a critical role in preventing immune elimination of edited MCA sarcoma cells. Alternatively highly immunogenic, unedited d42m1-T9 sarcoma cells were spontaneously rejected in WT mice due to the presence of the strong rejection antigen, mutant Spectrin-â2. Enforced expression of PD-L1 on unedited d42m1-T9 cells to levels similar to those induced under physiologic in vivo conditions did not alter the regressor phenotype of these cells. In contrast, enforced overexpression of PD-L1 on d42m-T9 cells to levels that were 20 fold higher than physiologic led to formation of progressively growing tumors in WT mice. Thus, physiologic levels of PD-L1 expression on highly immunogenic unedited tumor cells are not sufficient to counteract immune effector mechanisms driven by “strong” tumor antigens. This result shows that adaptive immune resistance occurs only on tumor cells that have undergone immunoediting. Citation Format: Takuro Noguchi, Jeffrey P. Ward, Matthew M. Gubin, Cora Arthur, Robert D. Schreiber. IFN-ã induced PD-L1 on tumor and host cells co-operatively prevents tumor immune elimination after cancer immunoediting. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 903.

There is increasing evidence that T cells are able to control tumor growth and survival in cancer patients, both in early and late stages of the disease. However, tumor-specific T-cell responses are difficult to mount and sustain in cancer patients, and are limited by numerous immune escape mechanisms of tumor cells selected during immunoediting. An alternative approach to engage T cells for cancer therapy are antibodies, which are bispecific for a surface target antigen on cancer cells, and for CD3 on T cells. These are capable of connecting any kind of cytotoxic T cell to a cancer cell, independently of T-cell receptor specificity, costimulation, or peptide antigen presentation. Here, we review the principle of a new class of bispecific antibodies called BiTE (for “bispecific T-cell engager”) antibodies. Recent results from clinical studies with a CD19/CD3-bispecific BiTE antibody suggest that this therapeutic paradigm is finally showing promise for treatment of both bulky and minimal residual disease. [Cancer Res 2009;69(12):4941–4]

Immunosurveillance is an extrinsic mechanism of cancer suppression that eliminates nascent tumors. However, the selection imposed by immunosurveillance can drive tumor evolution and the emergence of clinically apparent neoplasms. Mechanisms of immune escape acquired by less immunogenic variants during this process, termed immunoediting, may contribute significantly to malignant progression. In this review, we summarize the evidence that up-regulation of the nonclassic human leukocyte antigen (HLA) class I molecule HLA-G in tumor cells plays an important role in cancer and immune escape. [Cancer Res 2008;68(3):627–30]

Immunoediting of tumor-associated antigens occurs in response to immune pressure. We show that the mutation of residues within epitopes of HER-2/neu leads to the outgrowth of autochthonous tumors after immunizing HER-2/neu transgenic mice with Listeria monocytogenes therapeutic vaccines expressing fragments of HER-2/neu. Three of these vaccines target the extracellular domain (LmLLO-EC1, LmLLO-EC2, and LmLLO-EC3), and two of these vaccines target the intracellular domain (Lm-LLO-IC1 and Lm-LLO-IC2). Mutations occurred in the regions of the HER-2/neu molecule targeted by the Listeria strain expressing that region, which suggests that the rate of generation of escape mutants was a significant factor in the efficacy of each vaccine. A longer delay in the onset of tumors after immunotherapy occurred with the vaccine that targeted the kinase domain. We verified that the mutations in this domain occurred within novel CD8+ T-cell epitopes, and that the mutation of these residues abrogated CTL responses to these epitopes. The long delay in the onset of tumors after immunotherapy targeting the kinase domain may be because this region of HER-2/neu cannot undergo extensive mutations without impairing its ability to signal cell growth. [Cancer Res 2007;67(5):1887–92]

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.

The tryptophan-catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase 1) has been implicated as a mediator of immune tolerance in the reproductively essential process of shielding the ‘foreign’ fetus from maternal immunity. By extrapolation, we and others hypothesized that tumors might elevate IDO1 under selective pressure by the immune system. Our finding that loss of the Bin1 tumor suppressor gene potentiates the superinduction of IDO1 provided the first discreet example of a molecular pathway through which this immune escape process can occur. However, the involvement of IDO1 in tumorigenesis has turned out to be far more complex. Tumors that inherently lack IDO1 expression have been demonstrated to induce IDO1 expression in antigen presenting cells of the host, providing an alternative mechanism for immune escape. We have also found from studies in the classical DMBA/TPA skin carcinogenesis model that IDO1 can be induced by the inflammatory tumor-promoting process itself independent of the presence of an initiated tumor. Thus, IDO1 can be a factor in tumor promotion throughout the entire immunoediting process. With the rapid pace of development of IDO inhibitors, which are currently being evaluated in clinical trials, we are interested in determining whether mouse tumor models might provide additional insight into the optimal therapeutic application of these agents based on the underlying biology. In current studies, we have found that IDO1-nullizygous mice are resistant to both KRAS-induced lung adenocarcinomas and pulmonary breast carcinoma metastases. Micro-computed tomographic imaging confirmed that lung tumor burden was correspondingly lower in IDO1-nullizygous mice. Surprisingly, this analysis also revealed a significantly reduced pulmonary blood vessel density in IDO1-nullizygous mice. Elevation of the inflammatory cytokine IL6 (interleukin 6) was greatly attenuated in conjunction with the loss of IDO1, consistent with in vitro evidence that IDO1 potentiates IL6 production. MDSCs (myeloid derived suppressor cells) from IDO1-nullizygous animals exhibited reduced T cell suppressive activity that could be rescued by IL6. IL6 could likewise reverse the pulmonary metastasis resistance exhibited by IDO1-nullizygous mice. Together, our findings provide support for the emerging concept of IDO1 as a prototypical, integrative immune modifier that bridges inflammation, vascularization and immune escape to foster the establishment of a pathogenic, tumor-promoting environment. Citation Format: Alexander J. Muller, Courtney Smith, Mee Young Chang, James DuHadaway, Arpita Mondal, Hollie Flick, Katherine Parker, Daniel Beury, Suzanne Ostrand-Rosenberg, George C. Prendergast. IDO1 is an integrative determinant of tumor-promoting, pathogenic inflammation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3665. doi:10.1158/1538-7445.AM2014-3665

The incidence of metastatic melanoma, a very aggressive type of human skin cancer, has steadily increased over the last years and is associated with high mortality. Activating mutations in the N-Ras proto-oncogene are characteristic for cutaneous melanoma. The most frequent mutation found in N-Ras is a lysine substitution of glutamine at codon 61 (Q61K) constitutively activating the protein. Together with other mutations, especially inactivation of the CDKN2A locus, it is sufficient for spontaneous melanoma formation and subsequent metastasis formation in mice. STAT3 proteins are highly phosphorylated in melanoma. We identified strong STAT3 expression and activation in 70% of patients that show melanoma brain metastases. Here, we investigated whether deletion of this transcription factor in a mouse model expressing mutant N-RasQ61K transgene under the control of the tyrosinase promoter in combination with a homozygous CDKN2A deletion, leads to modified melanoma formation. Interestingly, we found decreased survival in male mice that do not express STAT3 in melanoma cells. We analyzed primary tumor development, metastasis, and survival using histo-pathological examination between groups of STAT3 positive and negative mice. To further assess whether the enhanced tumor progression upon loss of STAT3 is a cell autonomous effect we established melanoma cell lines derived from mouse tumors with or without STAT3. These novel cell lines will be used to analyze growth and survival properties. We will also test the influence of a functional immune system to question immunoediting by xenotransplantation experiments. Citation Format: Alexander Swoboda, Isabel J. Sobieszek, Michaela Schlederer, Safia Zahma, Jelena Marjanovic, Valeria Poli, Lukas Kenner, Markus Hengstschläger, Mario Mikula, Richard Moriggl. Deletion of STAT3 in a mouse model for metastatic melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 79. doi:10.1158/1538-7445.AM2014-79

Despite intense efforts to design immunotherapeutic strategies, the success of cancer immunotherapy remains extremely limited. Major factors limiting immune responses to tumors include insufficient activation of tumor-reactive effector T cells, poor intra-tumor recruitment of effector T cells, and/or suppression of their functions within the tumor. One mediator involved in effector T cell activation and recruitment to inflammatory sites is the T cell chemoattractant chemokine CXCL9/Mig. Our recent studies in murine models of aggressive skin cancer (cutaneous sarcoma and melanoma) have indicated that these tumors cease the production of CXCL9/Mig as a result of immunoediting mediated by T cells and IFN-g. The consequence of Mig deficiency in the tumor microenvironment is the increased resistance of immunoedited Mig-deficient tumor variants to T cell-mediated immunity. Here we demonstrated that inducing constitutive expression of Mig within Mig-deficient tumors by delivery of Mig-encoding retroviral vector promotes protective anti-tumor immune responses and results in rejection of cutaneous tumors. This modulation of intra-tumor Mig expression induced potent tumor-specific CD4 and CD8 T cell-mediated responses capable of complete tumor elimination and subsequent protection to tumor re-challenge. Furthermore, the delivery of Mig-overexpressing tumor cells as a vaccine into mice with established cutaneous tumors induced systemic anti-tumor immune responses that suppressed growth of established tumors. This therapeutic effect correlated with the presence of dendritic cells (DC) in the tumor-draining lymph nodes (TDLN) that were very potent in the activation of tumor antigen-specific IFN-g producing T cells. These results suggest that besides the recruitment of effector T cells into the tumor, tumor-derived Mig enhances activation of these T cells possibly by affecting tumor-infiltrating DC that acquire tumor antigens and then migrate into TDLN to prime tumor-specific effector T cells. Therefore, Mig might be a potent component of vaccines aimed to boost anti-tumor immunity in patients with aggressive skin cancers deficient in the expression of this chemokine. Citation Format: Anton Gorbachev, Danielle Kish, Robert Fairchild. Induction of CXCL9/Mig expression in the tumor microenvironment promotes protective antitumor immune responses. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2831. doi:10.1158/1538-7445.AM2013-2831

An investigator attempting to become informed about the field of tumor immunology encounters pre-clinical and clinical observations that do not easily form the basis of an overarching set of principles that explain and predict the interaction of the tumor with the host immune system. Pre-clinical studies have led to one widely held view termed cancer immunoediting as the basis for the failure of host immunosurveillance. This proposition holds that during the evolution of a cancer, the immune system responds to and eliminates the highly immunogenic cancer cells, but leaves behind non-immunogenic cancer cells that escape immune recognition. Immunoediting, the evidence for which has been obtained almost entirely with murine models of sarcoma, predicts that vaccination with the antigens expressed by these residual cancer cells that are not spontaneously immunogenic will alone lead to control of tumor growth, at least transiently. A second view explains that cancers escape immunosurveillance because they establish microenvironments that are immune suppressive and blunt the capacity of effector T cells to kill cancer cells. In contrast to immunoediting, the occurrence of immune suppressive predicts that spontaneous or vaccine-induced immune responses will be effective only when combined with a therapeutic intervention that interrupts whatever it is in the microenvironment that interferes with the functions of effector T cells. In a pre-clinical spontaneous tumor model of adenocarcinoma of the lung, immunosuppression was the basis of the failure of immunosurveillance, at least inferentially, because antigen-expressing, immunogenic cancer cells continued to grow despite a systemic immune response. Interestingly, careful consideration of these two views reveals that in their strictest interpretation, they are incompatible as explanations for a how a cancer avoids immunosurveillance, since stringent immune suppression would prevent cancer immunoediting, and the occurrence of immunoediting denotes unsuccessful immune suppression. Clinical studies of immunological interventions have not clearly supported either process as being the better explanation for the tumor-host interaction. Generally, therapeutic cancer vaccines (i.e., vaccines given after cancers have been established) have not been successful, even with virus-induced epithelial cancers, such as that caused by HPV, an observation that does not fulfil the prediction of immunoediting. On the other hand, studies of monoclonal antibodies that promote T cell function, such as anti-CTLA-4 (Ipilimumab), anti-PD-1, and anti-PD-L1, have been effective only in a minority of patients with melanoma, renal cell cancer, and lung cancer, and ineffective in all patients with pancreatic ductal adenocarcinoma (PDA). These results indicate either that spontaneously immunogenic cancer cells had been eliminated in the non-responsive tumors, as in immunoediting, or that these antibodies had not targeted the critical immune suppressive mechanism. That is, these clinical studies do not necessarily support either proposed explanation. In 2010, we published a study of the effects of depleting a tumoral stromal cell type that can be identified by its expression of the membrane protein, Fibroblast Activation Protein-alpha (FAP), from a transplanted, immunogenic tumor model, the Lewis Lung carcinoma line expressing ovalbumin (LL2/OVA). The FAP+ stromal cell had been identified twenty years ago in essentially all human adenocarcinomas, and had subsequently been found in several examples of chronic inflammatory tissue lesions. These findings suggested to us that the presence of the FAP+ cell in tumors reflected their inflammatory character, and that the function of the cell in this circumstance was related to tissue protection, which included immune suppression. Remarkably, we found that conditionally depleting this cell from established LL2/OVA tumors led to immune control of their growth, without any intervention that directly affected T cells. Perhaps surprisingly, therefore, a stromal cell of mesenchymal origin, had immune suppressive activity that blocked the function of pre-existing, cancer cell-specific T cells. This tumor model, however, was highly artificial, so the relevance of this study to the question of why immunosurveillance fails in human cancers was uncertain. The conditional depletion of the FAP+ stromal cell in the LL2/OVA tumor was based on its expression of the human diphtheria toxin receptor (DTR), which had been programmed by a BAC transgene in which a cassette encoding the DTR was inserted into the Fap gene. Therefore, it was possible to assess the immunological role of the FAP+ stromal cell in a spontaneous model of PDA by crossing the BAC DTR transgene into the KPC mouse line. This model replicates the immunological phenotype of human PDA in that its growth is unaffected by treatment of mice with anti-CTLA-4. We demonstrated that PDA-bearing mice had spontaneous immune responses to cancer cells, in that tumor cells induced the secretion of IFN-gamma by their CD8+ T cells. Therefore, this model exemplified escape from immune surveillance by tumoral immune suppression rather than immunoediting, since antigen-expressing cancer cells and immune T cells co-existed. Consistent with this surmise was our finding that diminishing the number of tumoral FAP+ stromal cells by giving diphtheria toxin led to T cell-dependent control of PDA growth. This finding then prompted the question of whether the FAP+ stromal cell accounted for the absence of an anti-tumor effect of anti-CTLA-4. This query was answered in the affirmative by observing that depleting the FAP+ cell and anti-CTLA-4 synergistically slowed the growth of the PDA. Thus, there is a hierarchy of tumoral immune suppression, with the FAP+ cell being dominant over at least one T cell-directed intervention. One would predict that immune suppression by the FAP+ cell also would interfere with the efficacy of vaccine-induced immune responses. We have also conducted studies to determine the relationship of the tumoral FAP+ cell to fibroblast-like stromal cells, such as the carcinoma/cancer-associated fibroblast (CAF) and the alpha-smooth actin+ (aSMA+) “myofibroblast”, and to FAP+ cells that we have found in many normal tissues and organs. The transcriptomes of FAP+ cells from PDA, normal pancreas (likely the “pancreatic stellate cell”), adipose tissue and skeletal muscle were found to be highly similar, and to form a distinct cluster from FAP-negative mouse embryonic fibroblasts when assessed by Principle Component Analysis. All FAP+ cells expressed the inflammatory gene signature reported by Hanahan and colleagues for the CAF, suggesting that this signature identifies this “fibroblast-like” lineage rather than being unique to stromal cells of the tumor microenvironment. FAP+ cells in skeletal muscle are required for the maintenance of this tissue, and FAP+ cells of the bone marrow are necessary for normal hematopoiesis, and are identical to the so-called “CXCL12-associated reticular” cell. Mouse models of cancer-induced cachexia are associated with a loss of FAP-expressing cells from these and many other tissues, a circumstance that may contribute to cachexia and anemia in cancer. In summary, the FAP+ cell mediates dominant immune suppression in mouse PDA, and is closely related to FAP+ cells of normal tissues that are also affected by cancer. Defining the molecular means of its immune suppression may uncover new immunotherapies of cancer. Citation Format: Douglas T. Fearon. The FAP+ stromal cell and escape from immunosurveillance. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr IA27.

Cancer Immunoediting is the process by which the immune system controls and shapes cancer. We originally envisaged that cancer immunoediting would occur 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). Strong experimental data has now been obtained using mouse models of cancer to demonstrate the existence of each phase of the cancer immunoediting process and compelling clinical data suggests that a similar process may also occur 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. This approach has helped identify the nature of antigens seen by immunity in nascent developing cancers and has further shown that immunoselection is a major mechanism of immunoediting. Moreover, we have found that edited tumors can still be controlled by the immune system if natural mechanisms that prevent autoimmunity are suspended. As reported by others, we have confirmed that inhibition of CTLA-4 induces ejection of edited MCA sarcomas. However, we have also found that inhibition of PD-L1 does the same, although by perhaps different mechanisms. These differences will be discussed. Citation Format: Robert D. Schreiber. The molecular basis of tumor immunogenicity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA2.

Cancer immunoediting is a dynamic process composed of three phases: elimination (EL), equilibrium (EQ), and escape (ES), that encompasses the potential host-protective and tumor-sculpting functions of the immune system throughout tumor development. Inbred mice models provide an interesting tool for identifying genes that control susceptibility to breast cancer. Inbred CBi/L FS mice were artificially selected by body conformation from CBi mice. The M-406 mammary adenocarcinoma appeared spontaneously in CBi female mouse and it is maintained in vivo in mice of the same line. When CBi/L FS mice are s.c. challenged with M-406 they show the three phases: 51.6% in ES, 18.5% in EQ en 29.8% in EL. The aim of this study was to evaluate in CBi/L FS mice in the three phases of M-406 tumor growth: 1) the profile of cytokines Th1(IL-2; INF-γ); Th2(IL-4; IL-10) in serum and 2) the effect of conditioned media (CM) from CBi/L FS mononuclear cells on tumor cells proliferation. The serum and CM cytokines were evaluated by ELISA. CMs were obtained after 24h incubation of mononuclear cells from CBi/L FS seeded at 4x105 cells/well, in RPMI + 10% BFS. Tumor cells (5x104 cells/well) were incubated 24h with different CMs and proliferation evaluated (WST-1). In serum we observed the presence of high levels of INF-γ in the ES condition, compared with EQ and EL (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3552. doi:1538-7445.AM2012-3552

Cancer immunoediting is the process whereby the immune system protects against cancer formation and sculpts the immunogenicity of developing tumors cells. Although the full process is known to depend on innate and adaptive immunity, it remains unclear whether innate immunity alone is capable of immunoediting. By comparing the incidence and immunogenicity of 3′methylcholanthrene (MCA)-induced sarcomas in syngeneic wild-type, RAG2-/-, and RAG2-/- x γc-/- mice, we show that the innate immune system can edit the immunogenicity of primary tumors. Specifically, RAG2-/-x γc-/- mice displayed the highest incidence of MCA sarcomas, followed by RAG2-/- mice, and WT mice have the lowest incidence at four separate doses of MCA. Moreover, when cell lines were generated from MCA sarcomas, the cell lines derived from MCA sarcomas growing in RAG2-/-x γc-/- mice had the highest immunogenicity, followed by cell lines from RAG2-/- mice, and cell lines from WT mice had the lowest immunogenicity. These results show that the innate immune system can edit tumors in the absence of adaptive immunity. To examine the cellular basis of editing by the innate immune system, we analyzed the innate cells that infiltrate tumors formed from transplanting highly immunogenic MCA sarcoma cell lines into RAG2-/- or RAG2-/-x γc-/- mice. We found that M1 macrophages were detected at high levels in immunogenic tumors in RAG2-/- but not RAG2-/- x γc-/- mice. The accumulation of M1 macrophages required IFNγ and was associated with effective tumor editing. In the absence of γc and IFNγ, M2 macrophages were abundant, and editing was impaired. Notably, M1 macrophages can be elicited by administration of CD40 agonistic antibodies, thereby restoring cancer editing activity in RAG2-/-x γc-/- mice. Our results suggest that crosstalk between natural killer cells and M1 macrophages, through IFNγ, can be elicited in highly immunogenic tumors, leading to cancer immunoediting. Our highly immunogenic cell lines will provide a system to dissect the cellular and molecular basis of cancer immunoediting by the innate immune system. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 526. doi:1538-7445.AM2012-526

Conditions that inhibit IFN-induced signal transduction and gene transcription can dampen the host immune system and its ability to recognize and eliminate tumors leading to impaired immunoediting. We hypothesized that in the setting of cancer, myeloid-derived suppressor cells (MDSC) could impair the responsiveness of immune effector cells to interferons. We used the murine adenocarcinoma cell line C26 which induces MDSC generation when implanted in C2DF1 mice. C26-bearing mice had significantly elevated levels of GR1+CD11b+ MDSC (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5311.

Immune cells establish dynamic adhesive cell-cell interactions at a specific contact region, termed the immunological synapse (IS). Important features of the IS are the formation of cell-cell membrane fusions and bridges and the intercellular transfer of selected proteins. In previous studied we found that upon IS formation, a variety of Ras super-family members transfer intact from B cells to T and NK cells, and consequently transmit signals across cell boundaries. Mutations in Ras proto-oncogenes are rather common in human cancers, including melanoma, and moreover immunosurveillance is particularly important for the elimination of malignant melanoma. Thus, it was of interest to study whether mutated constitutively active Ras proteins transfer from HLA-2+ melanoma cells (MEL526) to patients-derived tumor infiltrating lymphocytes (TILs), and whether they can serve as a stimulatory ‘danger’ signal. Using an ex vivo co-culture system we found that TILs_as opposed to polyclonal T cells_efficiently and rapidly acquired intact H-Ras-G12V-GFP from MEL526 transfectants. Importantly, the adopted Ras was incorporated into the plasma membrane of the TILs in the correct in-out orientation. To study the functional consequences of the ‘non-autonomously translated’ H-Ras-G12V in the adopting TILs, we analyzed its effect on ERK1/2 activation and a variety of T cell effector functions. We found that the adopted H-Ras-G12V increased p-ERK1/2 levels in the adopting lymphocyte. Moreover, H-Ras-G12V-GFP+ TILs specifically produced more IFN-g and showed increased cytotoxic activity. In conclusion, our findings show a hitherto unobserved mode of intercellular signal transfer between T cells and cancer cells that may play an important role in the complex process of cancer immunoediting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4770.

Cancer Immunoediting is the process by which the immune system controls and shapes cancer. We originally envisaged that cancer immunoediting would occur 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). Strong experimental data has now been obtained using mouse models of cancer to demonstrate the existence of each phase of the cancer immunoediting process and compelling clinical data suggests that a similar process may also occur 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. This approach has led to the discovery of a new mouse cancer model that shows remarkable developmental, molecular and functional similarity to human estrogen receptor alpha positive (ERα+) luminal breast cancer. We have found that the cancers in our mice arise as a result of dysfunctional JAK-STAT pathway signaling and, interestingly, this observation has led to the discovery of a heretofore-unrecognized similar phenotype in a subset of human ERα+ luminal breast cancers. The therapeutic implications of our work will be discussed. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr BS3-1.