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Cancer immunoediting1is a hallmark of cancer2that predicts that lymphocytes kill more immunogenic cancer cells to cause less immunogenic clones to dominate a population. Although proven in mice1,3, whether immunoediting occurs naturally in human cancers remains unclear. Here, to address this, we investigate how 70 human pancreatic cancers evolved over 10 years. We find that, despite having more time to accumulate mutations, rare long-term survivors of pancreatic cancer who have stronger T cell activity in primary tumours develop genetically less heterogeneous recurrent tumours with fewer immunogenic mutations (neoantigens). To quantify whether immunoediting underlies these observations, we infer that a neoantigen is immunogenic (high-quality) by two features—‘non-selfness’ based on neoantigen similarity to known antigens4,5, and ‘selfness’ based on the antigenic distance required for a neoantigen to differentially bind to the MHC or activate a T cell compared with its wild-type peptide. Using these features, we estimate cancer clone fitness as the aggregate cost of T cells recognizing high-quality neoantigens offset by gains from oncogenic mutations. With this model, we predict the clonal evolution of tumours to reveal that long-term survivors of pancreatic cancer develop recurrent tumours with fewer high-quality neoantigens. Thus, we submit evidence that that the human immune system naturally edits neoantigens. Furthermore, we present a model to predict how immune pressure induces cancer cell populations to evolve over time. More broadly, our results argue that the immune system fundamentally surveils host genetic changes to suppress cancer.

Supplementary information and materials and methods. Supplemental Figure 1: Laser capture microdissection (LCM) successfully isolates normal vs. tumor-associated stromal compartments. Supplemental Figure 2: Identifying differentially expressed genes based on stroma-defined axes. Supplemental Figure 3: Survival curves for T, B, and E axis scores when first stratified by D axis score. Supplemental Figure 4: Survival curves for T, B, and E axis scores among patients not treated with chemotherapy when first stratified by D axis score. Supplemental Figure 5: Survival curves for T, B, and E axis scores among patients treated with chemotherapy when first stratified by D axis score. Supplemental Figure 6: Full heatmap depicting the subtype-specific performance of predictors. Supplemental Figure 7: Naive Bayes classifier predictions agree with ROI95 assignments

Triple-negative breast cancer (TNBC) is a molecularly heterogeneous cancer that is difficult to treat. Despite the role it may play in tumor progression and response to therapy, microenvironmental (stromal) heterogeneity in TNBC has not been well characterized. To address this challenge, we investigated the transcriptome of tumor-associated stroma isolated from TNBC (n = 57). We identified four stromal axes enriched for T cells (T), B cells (B), epithelial markers (E), or desmoplasia (D). Our analysis method (STROMA4) assigns a score along each stromal axis for each patient and then combined the axis scores to subtype patients. Analysis of these subtypes revealed that prognostic capacity of the B, T, and E scores was governed by the D score. When compared with a previously published TNBC subtyping scheme, the STROMA4 method better captured tumor heterogeneity and predicted patient benefit from therapy with increased sensitivity. This approach produces a simple ontology that captures TNBC heterogeneity and informs how tumor-associated properties interact to affect prognosis. Cancer Res; 77(17); 4673–83. ©2017 AACR.

The supplementary file discusses the limitations of existing tools, performance of CrosstalkNet, along with more biological case studies.

Variations in physiological conditions can rewire molecular interactions between biological compartments, which can yield novel insights into gain or loss of interactions specific to perturbations of interest. Networks are a promising tool to elucidate intercellular interactions, yet exploration of these large-scale networks remains a challenge due to their high dimensionality. To retrieve and mine interactions, we developed CrosstalkNet, a user friendly, web-based network visualization tool that provides a statistical framework to infer condition-specific interactions coupled with a community detection algorithm for bipartite graphs to identify significantly dense subnetworks. As a case study, we used CrosstalkNet to mine a set of 54 and 22 gene-expression profiles from breast tumor and normal samples, respectively, with epithelial and stromal compartments extracted via laser microdissection. We show how CrosstalkNet can be used to explore large-scale co-expression networks and to obtain insights into the biological processes that govern cross-talk between different tumor compartments.Significance: This web application enables researchers to mine complex networks and to decipher novel biological processes in tumor epithelial-stroma cross-talk as well as in other studies of intercompartmental interactions. Cancer Res; 78(8); 2140–3. ©2018 AACR.

Supplemental Table 1: TNBCType calls match between ROI95 and the online webtool Supplemental Table 2: Summary of patient characteristics for LCM cohort Supplemental table 3: List of differentially expressed genes for each axis Supplemental Table 4: Representative pathways and genes used to label the stromal axes Supplemental Table 5: Agreement between classifications in LCM dataset and matched bulk expression profiles. Supplemental Table 6: Observed frequencies of the states for each stromal axis score across the whole cohort of TNBC patients (n=1,098). Supplemental Table 7: Enrichment and depletion of some stromal subtypes List of all stromal axis score-baed assignments as well as values entered for binomial test and resultant p-value

Multiplexed imaging of 8 molecular targets with SERS nanoprobes in mice reveals spatially heterogeneous tumor responses to immunotherapy. Multichannel image segmentation enables classification of the tumor regions into responders and naïve.

The majority of JAK2V617F-negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin (CALR), resulting in a common carboxyl-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in patients with CALRMUTMPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALRMUTMPN from two independent cohorts. We observed that MHC-I alleles that present CALRMUTneoepitopes with high affinity are underrepresented in patients with CALRMUTMPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALRMUTMPN would not efficiently respond to a CALRMUTfragment cancer vaccine but would when immunized with a modified CALRMUTheteroclitic peptide vaccine approach. We found that heteroclitic CALRMUTpeptides specifically designed for the MHC-I alleles of patients with CALRMUTMPN efficiently elicited a CALRMUTcross-reactive CD8+T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALRMUTnative peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8+T cell response to the CALRMUTfragment upon immunization with a CALRMUTheteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide–based cancer vaccines in patients with CALRMUTMPN.

Immune checkpoint blockade therapy has revolutionized cancer care, including the treatment of advanced metastatic disease. However, most patients derive little or no clinical benefit from these therapies and many cancer types are notoriously non-responsive. Motivated by (1) the correlation between tumor neoantigen abundance and anti-tumor immunity and (2) that most cancers are characterized by widespread dysregulation of RNA processing, we reasoned that pharmacologic modulation of RNA splicing might increase cancer cell immunogenicity via the generation of splicing-derived neoantigens. We demonstrated that two compounds which modulate RNA splicing via distinct mechanisms, inhibited tumor growth and enhanced response to immune checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Critical for their clinical translatability, therapeutic doses of splicing inhibitors were non-toxic, tolerated by the host immune system, and did not affect T cell activation, proliferation, and anti-cancer killing activities. Mechanistically, splicing modulation induced stereotyped, dose-dependent “splicing failure” — dramatic intron retention, alternative exon skipping, etc. — that was consistent across multiple mouse and human tumor types. By combining RNA-seq-based peptide predictions and mass spectrometry of the MHC I-bound immunopeptidome, we identified drug-induced, splicing-derived peptides that promote the expansion of antigen-specific CD8+ T cells and trigger anti-tumor T cell responses in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic. Citation Format: James D. Thomas, Sydney X. Lu, Emma De Neef, Erich Sabio, Benoit Rousseau, Mathieu Gigoux, David A. Knorr, Benjamin Greenbaum, Yuval Elhanati, Simon J. Hogg, Andrew Chow, Arnab Ghosh, Abigail Xie, Dmitriy Zamarin, Daniel Cui, Caroline Erickson, Michael Singer, Hana Cho, Eric Wang, Bin Lu, Benjamin H. Durham, Harshal Shah, Diego Chowell, Austin M. Gabel, Yudao Shen, Jing Liu, Jian Jin, Matthew C. Rhodes, Richard E. Taylor, Henrik Molina, Jedd D. Wolchok, Taha Merghoub, Luis A. Diaz Jr, Omar Abdel-Wahab, Robert K. Bradley. Pharmacologic modulation of RNA splicing enhances anti-tumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5742.

Transcription factors ThPOK and Runx3 regulate the differentiation of “helper” CD4+ and “cytotoxic” CD8+ T cell lineages respectively, inducing single positive (SP) T cells that enter the periphery with the expression of either the CD4 or CD8 co-receptor. Despite the expectation that these cell fates are mutually exclusive and that mature CD4+CD8+ double positive (DP) T cells are present in healthy individuals and augmented in the context of disease, yet their molecular features and pathophysiologic role are disputed. Here, we show DP T cells in murine and human tumors as a heterogenous population originating from SP T cells which re-express the opposite co-receptor and acquire features of the opposite cell type’s phenotype and function following TCR stimulation. We identified distinct clonally expanded DP T cells in human melanoma and lung cancer by scRNA sequencing and demonstrated their tumor reactivity in cytotoxicity assays. Our findings indicate that antigen stimulation induces SP T cells to differentiate into DP T cell subsets gaining in polyfunctional characteristics.

Summary Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic.

Intratumoral therapy with oncolytic viruses is increasingly being explored as a strategy to potentiate an immune response against cancer, but it remains unknown whether such therapy should be restricted to cancers sensitive to virus-mediated lysis. Using Newcastle Disease Virus (NDV) as a model, we explore immunogenic potential of an oncolytic virus in bladder cancer, where existing immunotherapy with PD-1 and PD-L1-targeting antibodies to date has shown suboptimal response rates. Infection of human and mouse bladder cancer cells with NDV resulted in immunogenic cell death, activation of innate immune pathways, and upregulation of MHC and PD-L1 in all tested cell lines, including the cell lines completely resistant to NDV-mediated lysis. In a bilateral flank NDV-lysis-resistant syngeneic murine bladder cancer model, intratumoral therapy with NDV led to an increase of immune infiltration in both treated and distant tumors and a shift from an inhibitory to effector T cell phenotype. Consequently, combination of intratumoral NDV with systemic PD-1 or CTLA-4 blockade led to improved local and abscopal tumor control and overall survival. These findings encourage future clinical trials combining intratumoral NDV therapy with systemic immunomodulatory agents and underscore the rationale for such treatments irrespective of tumor cell sensitivity to NDV-mediated lysis.

Variations in physiological conditions can rewire molecular interactions between biological compartments, which can yield novel insights into gain or loss of interactions specific to perturbations of interest. Networks are a promising tool to elucidate intercellular interactions, yet exploration of these large-scale networks remains a challenge due to their high dimensionality. To retrieve and mine interactions, we developed CrosstalkNet, a user friendly, web-based network visualization tool that provides a statistical framework to infer condition-specific interactions coupled with a community detection algorithm for bipartite graphs to identify significantly dense subnetworks. As a case study, we used CrosstalkNet to mine a set of 54 and 22 gene-expression profiles from breast tumor and normal samples, respectively, with epithelial and stromal compartments extracted via laser microdissection. We show how CrosstalkNet can be used to explore large-scale co-expression networks and to obtain insights into the biological processes that govern cross-talk between different tumor compartments. Significance: This web application enables researchers to mine complex networks and to decipher novel biological processes in tumor epithelial-stroma cross-talk as well as in other studies of intercompartmental interactions. Cancer Res; 78(8); 2140–3. ©2018 AACR.

BackgroundThe majority of JAK2V617F-negative myeloproliferative neoplasms (MPN) have disease-initiating frameshift mutations in calreticulin (CALR) resulting in a common novel C-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in CALRMUT MPN patients, but the underlying reasons for this phenomenon are unknown. We speculate that this is due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifests. As a consequence of this MHC-I allele restriction, we reasoned that CALRMUT MPN patients would not efficiently respond to cancer vaccines composed of the CALRMUT neoantigen, but could do so when immunized with a properly epitope-optimized CALRMUT heteroclitic peptide vaccine approach.MethodsWe examined MHC-I allele frequency in CALRMUT MPN patients from two independent cohorts to identify under-represented MHC-I alleles. These MHC-I alleles were assessed for their ability to bind to CALRMUT-derived peptides using NetMHC and were subsequently validated experimentally in healthy donors and in CALRMUT MPN patients having received a CALRMUT cancer vaccine (clinical trial NCT03566446) to determine if these MHC-I were potentiating immunogenicity against the CALRMUT antigen. Epitope-optimized heteroclitic variants of the CALRMUT neoantigen were identified and tested experimentally in vitro in human PBMCs and in vivo in mice for their ability to mount an immune response against the non-modified CALRMUT neoantigen.ResultsWe observed that MHC-I alleles that present CALRMUT neoepitopes with high affinity are under-represented in CALRMUT MPN patients. Heteroclitic CALRMUT peptides specifically designed for CALRMUT MPN patient MHC-I alleles efficiently elicited a cross-reactive CD8+ T cell response in human PBMC samples otherwise unable to respond to the matched weakly immunogenic CALRMUT native peptides. We also modeled this effect in mice and observed that C57BL/6J mice, which are unable to mount an immune response to the human CALRMUT fragment, can mount a cross-reactive CD8+ T cell response against a CALRMUT-derived peptide upon heteroclitic peptide immunization and this was further amplified by combining the heteroclitic peptide vaccine with blockade of the immune checkpoint molecule PD-1.ConclusionsOur study shows that MHC-I alleles able to present CALRMUT neoepitopes are under-represented in CALRMUT MPN patients, demonstrating that MHC-I haplotype is a major mechanism of passive immune-evasion in CALRMUT MPN. However, we show that a cancer vaccine composed of heteroclitic variants of the CALRMUT antigen could overcome this limitation.Ethics ApprovalApproval was obtained for the use of patient-derived specimens and access to clinical data extracted from patient charts by the Institutional Review Boards at Memorial Sloan Kettering Cancer Center, the Dana-Farber Cancer Institute and the Massachusetts General Hospital, as well as by the Danish Regional Science Ethics Committee.

Targeted immune therapies such as adoptive T cell transfer (ACT) are often ineffective because tumors evolve over time under selective pressure to display antigen loss variant. A classic example is de-differentiation and loss of expression of antigenic proteins. Therapies that activate multiple branches of the immune system may eliminate escape variants. Here we show that melanoma-specific CD4+ ACT therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate melanomas that contain clonal escape variants. As expected, early on-target recognition of melanoma antigens by adoptively transferred tumor-specific CD4+ T cells was required. Surprisingly, complete tumor eradication was partially dependent on neutrophils. Supporting these findings, extensive neutrophil extracellular traps were found in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade. Our findings uncover a novel interplay between T cells mediating the initial tumor- and tissue-specific immune response, and neutrophils mediating tumor destruction of antigen loss variants.

Vascular-targeted photodynamic therapy (VTP) induces rapid destruction of targeted tissues and is a promising therapy for prostate cancer. However, the resulting immune response, which may play an important role in either potentiating or blunting the effects of VTP, is still incompletely understood. Myeloid cells such as myeloid-derived suppressor cells (MDSCs) and macrophages are often found in tumors and are widely reported to be associated with cancer angiogenesis, tissue remodeling, and immunosuppression. These cells are also known to play a critical role in wound-healing, which is induced by rapid tissue destruction. In this study, we investigated the effects of VTP on the recruitment of tumor-infiltrating myeloid cells, specifically MDSCs and tumor-associated macrophages (TAMs), in the Myc-Cap and TRAMP C2 murine prostate cancer models. We report that VTP increased the infiltration of myeloid cells into the tumors, as well as their expression of CSF1R, a receptor required for myeloid differentiation, proliferation, and tumor migration. As anti-CSF1R treatment has previously been used to deplete these cells types in other murine models of prostate cancer, we hypothesized that combining anti-CSF1R with VTP therapy would lead to decreased tumor regrowth and improved survival. Importantly, we found that targeting myeloid cells using anti-CSF1R in combination with VTP therapy decreased the number of tumor MDSCs and TAMs, especially M2 macrophages, as well as increased CD8(+) T cell infiltration, decreased tumor growth and improved overall survival. These results suggest that targeting myeloid cells via CSF1R targeting is a promising strategy to potentiate the anti-tumor effects of VTP.

Phosphatidylserine (PS) is a phospholipid that is exposed on surface of apoptotic cells and has been shown to promote immunosuppressive signals in the tumor microenvironment (TME). Antibodies that block PS interaction with its receptors have been shown to repolarize tumor associated macrophages (TAMs) into a pro-inflammatory (M1) state, reduce the number of myeloid derived suppressor cells (MDSCs) in tumors and promote maturation of dendritic cells (DCs). Radiation therapy (RT) is an effective focal treatment of isolated solid tumors but is less effective in controlling metastatic cancers as a monotherapy. Tumor-directed RT is known to induce immunogenic cell death and enhance tumor-specific T cell infiltration into treated tumors. We found that a single dose of 15 Gy of tumor-directed RT on mouse B16 melanoma causes an increase in expression of PS on the surface of viable immune infiltrates. We hypothesize that expression of PS on immune cells may act similar to immune checkpoint molecules and provide a negative feedback mechanism to immune cells. Therefore, we posit that blocking PS may enhance the efficacy of tumor-directed RT. Indeed, we found that treatment with an antibody that targets PS (mch1N11) enhanced the anti-tumor efficacy of tumor-directed RT and improved overall survival. This combination led to an increase in M1 TAMs and a corresponding decrease in M2 TAMs which translated to a more pro-inflammatory TME. The addition of anti-PD-1 to RT and mch1N11 led to even greater anti-tumor efficacy and overall survival. Anti-PD-1 exerted its effects primarily by enhancing CD8+ T cell infiltration, activation, and effector function in the triple combination. Consistent with the mouse studies, we found increased PS expression on several immune subsets in the blood in patients with metastatic melanoma after receiving tumor-directed RT. These findings highlight the potential of combining PS targeting with RT and PD-1 pathway blockade to improve outcomes in patients with advanced-stage cancers and may inform the design of clinical studies combining PS-targeting antibodies with RT and/or checkpoint blockade.

Immune checkpoint blockade (ICB) has been shown to convey significant clinical activity across a spectrum of malignancies, yet there is now recognition that multiple mechanisms of resistance can impair response. The catabolism of tryptophan into metabolites known as kynurenines (Kyn) by enzymes such as indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO) plays a major suppressive role. Recently it was shown that Kyn acts as an endogenous agonist of the Aryl hydrocarbon receptor (AhR). In order to gain a better understanding of this pathway, we sought to characterize the mechanisms of immunosuppression associated with AhR and evaluate its potential as therapeutic target. Gene-expression analysis of IDO-overexpressing melanomas (B16-IDO vs. B16-WT) demonstrated reduced expression levels of Type 1 inflammatory genes, including IFNy, TNF, GzmB, and CD40. In addition, B16-IDO presents higher infiltration of tumor-associated macrophages TAMs, which upregulate the AHR as well as classic AhR-regulated genes (Cyp1a1 and Cyp1b1) and are differentially skewed towards an immunosuppressive M2 phenotype. Tumor-antigen specific CD8+T cells show reduced expression of activation markers (GzmB and CD44) and proliferation rate when primed by Kyn-treated antigen-presenting cells. In addition, TAMs from B16-IDO tumors suppressed activation of CD8+T cells in vitro and their depletion delayed tumor growth. When B16-IDO cells are implanted in mice depleted of Foxp3 expressing cells, TAMs do not accumulate. Treatment of B16-IDO tumors with an AhR-specific antagonist (CH-223191) upregulates MHC II in APCs, activation markers in CD8s, and reduced frequency of T-regs in B16-IDO tumors. AhR inhibition slows tumor growth and prolongs survival of tumors with active IDO/TDO/Kyn pathway (B16-IDO and B16-TDO), and this is enhanced when PD-1 blockade is used in combination. In summary, our findings demonstrate that targeting the Kyn pathway through AhR inhibition could overcome key suppressive mechanisms and sensitize tumors to ICB. This abstract is also being presented as Poster A57. Citation Format: Luis F. Campesato, Sadna Budhu, Mathieu Gigoux, Jeremy Tchaicha, Stephane Pourpe, Cailian Liu, Dmitriy Zamarin, Mark G. Manfredi, Karen McGovern, Jedd D. Wolchok, Taha Merghoub. Blockade of AHR activation by IDO/TDO-derived kynurenine restricts cancer immune suppression [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR05.

Intralesional therapy with oncolytic viruses (OVs) leads to the activation of local and systemic immune pathways, which may present targets for further combinatorial therapies. Here, we used human tumor histocultures as well as syngeneic tumor models treated with Newcastle disease virus (NDV) to identify a range of immune targets upregulated with OV treatment. Despite tumor infiltration of effector T lymphocytes in response to NDV, there was ongoing inhibition through programmed death ligand 1 (PD-L1), acting as a mechanism of early and late adaptive immune resistance to the type I IFN response and T cell infiltration, respectively. Systemic therapeutic targeting of programmed cell death receptor 1 (PD-1) or PD-L1 in combination with intratumoral NDV resulted in the rejection of both treated and distant tumors. These findings have implications for the timing of PD-1/PD-L1 blockade in conjunction with OV therapy and highlight the importance of understanding the adaptive mechanisms of immune resistance to specific OVs for the rational design of combinatorial approaches using these agents.

Understanding the tumor immune microenvironment (TIME) promises to be key for optimal cancer therapy, especially in triple-negative breast cancer (TNBC). Integrating spatial resolution of immune cells with laser capture microdissection gene expression profiles, we defined distinct TIME stratification in TNBC, with implications for current therapies including immune checkpoint blockade. TNBCs with an immunoreactive microenvironment exhibited tumoral infiltration of granzyme B(+)CD8(+) T cells (GzmB(+)CD8(+) T cells), a type 1 IFN signature, and elevated expression of multiple immune inhibitory molecules including indoleamine 2,3-dioxygenase (IDO) and programmed cell death ligand 1 (PD-L1), and resulted in good outcomes. An “immune-cold” microenvironment with an absence of tumoral CD8(+) T cells was defined by elevated expression of the immunosuppressive marker B7-H4, signatures of fibrotic stroma, and poor outcomes. A distinct poor-outcome immunomodulatory microenvironment, hitherto poorly characterized, exhibited stromal restriction of CD8(+) T cells, stromal expression of PD-L1, and enrichment for signatures of cholesterol biosynthesis. Metasignatures defining these TIME subtypes allowed us to stratify TNBCs, predict outcomes, and identify potential therapeutic targets for TNBC.

The inducible costimulator (ICOS) is highly expressed in follicular helper T (Tfh) cells, a subset of CD4 T cells that migrate into the B cell zone and facilitate germinal center reactions. Although ICOS is known to play a critical role in forming the Tfh cell population during immune responses, its contribution to the effector functions of Tfh cells remains unclear. Using activated mouse splenic CD4 T cells we demonstrate that ICOS assists TCR-mediated signal transduction by potentiating the PI3K-AKT-mTOR signaling cascade that leads to hyper-phosphorylation of p70S6K and 4E-BP1, events that are known to augment cap-dependent mRNA translation. Consequently, ICOS costimulation promotes the formation of polysomes on IL-4 mRNA in a PI3K-dependent manner. Furthermore, we show that the supply of IL-4 becomes a limiting factor for T-dependent B cell activation during in vitro co-culture when the ICOS-PI3K signaling axis is disrupted in T cells. This ICOS costimulation-dependent translational control may ensure targeted delivery of IL-4 to cognate B cells during T–B collaborations in the germinal center.

Objectifs Les cellules myeloides infiltrant les tumeurs (tim) forment un microenvironnement protumoral pouvant induire des resistances aux therapies focales. Le recepteur au csf1 (csf1r) favorise le recrutement tumoral des tim. Le but de cette etude etait de demontrer que l’ajout d’un inhibiteur du csf1r ameliorait l’efficacite de la phototherapie dynamique au wst11 (vtp) pour le traitement d’un cancer de prostate dans un modele murin. Methodes Nous avons injecte des cellules tumorales prostatiques murines myccap dans le flanc de souris immunocompetentes fvb. Les souris ont ete randomisees en 4 groupes : controle, traitement par anti-csf1r, traitement par vtp, traitement par anti-csf1r + vtp. La survie et la croissance tumorale ont ete evaluees tous les 7 jours. L’infiltration tumorale par les tim a ete evaluee par immunohistochimie (iba1) et par cytometrie de flux (cd45, csf-1r, f4/80, cd8a, cd11b, cd11c, ly6c, ly6g). Resultats Comparee au groupe controle, la vtp a significativement augmente l’infiltration tumorale par les tim a j10 ainsi que l’expression de csf1r (p Fig. 1 , Fig. 2 ). L’association vtp + anti-csf1r a significativement ameliore la survie specifique des souris traitees (survie moyenne dans les groupes vtp + anti-csf1r, vtp, anti-csf1r et controle respectivement de 37,5 jours, IC95 % : 35,5–39,5 ; 21,8 jours, IC95 % : 17,2–26,4 ; 16,3 jours, IC95 % : 13,4–19,2 ; 14,5 jours, IC95 % : 13,5–15,5 ; p Fig. 3 ). L’association vtp + anti-csf1r a significativement diminue l’infiltration tumorale par les tim a j10 post-vtp (resultat valide en immunohistochimie et cytometrie de flux, Fig. 1 , Fig. 2 ). Conclusion La phototherapie dynamique au wst11 induit le recrutement de tim pouvant induire une resistance au traitement. L’association de la vtp avec un anti-csf1r ameliorait la survie specifique et diminuait la croissance tumorale chez un modele murin immunocompetent en diminuant l’infiltration tumorale par les tim. Ces resultats demontrent l’interet de l’immunotherapie ciblant les cellules myeloides en complement des therapies focales.

In this issue of JEM, Marijt et al. (https://doi.org/10.1084/jem.20180577) report their discovery of 16 novel human TAP-independent TEIPP peptides, whereas only one had been previously identified. This opens the door to new therapeutic options for patients with TAP-deficient tumors.

Immune checkpoint blockade (ICB) results in clinical benefit for a subset of cancer patients, yet multiple mechanisms of resistance can impair optimal response. The catabolism of tryptophan into metabolites known as kynurenines (Kyn) by the expression of enzymes such as IDO or TDO is a frequent phenomenon that plays a suppressive role in tumor immunity. Recently it was shown that Kyn acts as agonist of the aryl hydrocarbon receptor (AHR). Here we sought to characterize the mechanisms of immune suppression associated with the AHR pathway and to evaluate its potential as therapeutic target. RNAseq analysis of human cancers revealed a correlation between the expressions of AHR-related genes with markers associated with immunotherapy resistance (PD-1, FOXP3, CD206). By using IDO or TDO-overexpressing variants of a melanoma cell model (B16-F10), we found that myeloid cells, such as tumor-associated macrophages (TAMs) and dendritic cells (DCs), present up-regulation of the AHR. IDO-expressing tumors (B16-IDO) show higher myeloid cell infiltration, which present a tolerogenic phenotype. Tumor-antigen specific CD8T cells present reduced expression of activation markers and proliferation rate when primed by Kyn-treated BMDCs. Treatment of B16-IDO-bearing mice with an AHR-specific antagonist (CH-223191) leads to an increase of MHC II in TAMs, of activation markers in CD8 T cells and reduced frequency of T-regs. AHR inhibition delays progression of tumors with an active IDO/TDO/Kyn pathway (B16-IDO and B16-TDO), and efficacy is further improved when ICB is used in combination. In summary, our findings demonstrate that targeting the Kyn pathway through AHR inhibition could overcome key suppressive mechanisms and sensitize tumors to ICB.

Background:Over the last several years, we have witnessed the metamorphosis of network biology from being a mere representation of molecular interactions to models enabling inference of complex biological processes. Networks provide promising tools to elucidate intercellular interactions that contribute to the functioning of key biological pathways in a cell. However, the exploration of these large-scale networks remains a challenge due to their high-dimensionality.Results:CrosstalkNet is a user friendly, web-based network visualization tool to retrieve and mine interactions in large-scale bipartite co-expression networks. In this study, we discuss the use of gene co-expression networks to explore the rewiring of interactions between tumor epithelial and stromal cells. We show how CrosstalkNet can be used to efficiently visualize, mine, and interpret large co-expression networks representing the crosstalk occurring between the tumour and its microenvironment.Conclusion:CrosstalkNet serves as a tool to assist biologists and clinicians in exploring complex, large interaction graphs to obtain insights into the biological processes that govern the tumor epithelial-stromal crosstalk. A comprehensive tutorial along with case studies are provided with the application.Availability:The web-based application is available at the following location: http://epistroma.pmgenomics.ca/app/. The code is open-source and freely available from http://github.com/bhklab/EpiStroma-webapp.Contact:bhaibeka@uhnresearch.ca

Triple-negative breast cancer (TNBC) is a molecularly heterogeneous cancer that is difficult to treat. Despite the role it may play in tumor progression and response to therapy, microenvironmental (stromal) heterogeneity in TNBC has not been well characterized. To address this challenge, we investigated the transcriptome of tumor-associated stroma isolated from TNBC (n = 57). We identified four stromal axes enriched for T cells (T), B cells (B), epithelial markers (E), or desmoplasia (D). Our analysis method (STROMA4) assigns a score along each stromal axis for each patient and then combined the axis scores to subtype patients. Analysis of these subtypes revealed that prognostic capacity of the B, T, and E scores was governed by the D score. When compared with a previously published TNBC subtyping scheme, the STROMA4 method better captured tumor heterogeneity and predicted patient benefit from therapy with increased sensitivity. This approach produces a simple ontology that captures TNBC heterogeneity and informs how tumor-associated properties interact to affect prognosis. Cancer Res; 77(17); 4673–83. ©2017 AACR.

Background: Triple negative breast cancer (TNBC) are especially difficult to treat effectively. While only 20-30% of TNBC patients respond to chemotherapy in the neoadjuvant setting, overall outcome remains poor for non-responding patients. Engaging the immune system promises optimal personalized cancer therapy as mounting evidence suggests that immune-checkpoint inhibitor immunotherapies may become a therapeutic option for TNBC patients. The presence of CD8+ T cells, a crucial component of the cytotoxic arm of the adaptive immune response, is associated with good clinical outcome in TNBC patients. Specifically, it is the efficient CD8+ T cell invasion and infiltration in the tumor that is associated with good outcome. On the other hand, some tumors accumulate CD8+ T cells in the tumor-associated stroma with poor infiltration in the tumor epithelium. These patients show poor outcome. As CD8+ T cell infiltration in the tumor is a crucial step to mount an efficient anti-tumor response, we thus wondered how the tumor microenvironment affects CD8+ T cell invasion into the tumor epithelial compartment of the TNBC tumors. Methods: To identify potential stroma-dependent mechanisms that potentiate or inhibit CD8+ T cells invasion into the tumor epithelium, we coupled analysis of spatial patterns of CD8+ T cell localization by Immunohistochemistry (IHC) andperformed gene expression profiling of laser-capture microdissected tumor-associated stroma (as well as matched epithelium and bulk tumor) from 38 TNBC chemotherapy-naive primary cases. GSEA-based Metasignatures were derived from bulk tumor gene expression data from our cohort. To investigate the compartment of origin of the pathways identified via the Metasignatures, the (LCM)-derived tumor stromal and epithelial gene expression were analyzed. Results: CD8+ T cell quantification in different compartments of the tumor identify 3 main subgroups of TNBC based on CD8+ T cell localization. Importantly we developed a 2-step classification scheme based on CD8+ T cell localization. We developed metasignatures following our 2 steps classification and identified key bulk tumor metasignatures that showed prognostic value in an independent cohort. In addition the matched LCM gene expression from the tumor epithelium and stromal compartments allowed us to identify the compartment of origin. Importantly, while 1 group of TNBC tumor was showing a significant anti-tumor response, the 2 other groups showed absence of such environment. The 2 non inflamed immune subtypes showed distinct phenotypes and biologies associated with poor anti-tumor response that we validated by immunohistochemistry and fluorescence. These results highlight different potential mecanisms that lead to immune evasion and allow us to stratify TNBC into immune subgroups. Citation Format: Gruosso T, Gigoux M, Bertos N, Manem VS, Zuo D, Saleg SM, Souleimanova M, Zhao H, Johnson RM, Monette A, Muñoz Ramos V, Hallett MT, Stagg J, Lapointe R, Omeroglu A, Meterissian S, Buisseret L, Van den Eyden G, Salgado R, Guiot M-C, Haibe-Kains B, Park M. Distinct tumor microenvironments stratify triple negative breast cancer into immune subtypes [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD6-05.

The Met receptor tyrosine kinase is activated or genetically amplified in some gastric cancers, but resistance to small-molecule inhibitors of Met often emerges in patients. We found that Met abundance correlated with a proliferation marker in patient gastric tumor sections, and gastric cancer cell lines that have MET amplifications depended on Met for proliferation and anchorage-independent growth in culture. Inhibition of Met induced temporal changes in gene expression in the cell lines, initiated by a rapid decrease in the expression of genes encoding transcription factors, followed by those encoding proteins involved in epithelial-mesenchymal transition, and finally those encoding cell cycle-related proteins. In the gastric cancer cell lines, microarray and chromatin immunoprecipitation analysis revealed considerable overlap between genes regulated in response to Met stimulation and those regulated by signal transducer and activator of transcription 3 (STAT3). The activity of STAT3, extracellular signal-regulated kinase (ERK), and the kinase Akt was decreased by Met inhibition, but only inhibitors of STAT3 were as effective as the Met inhibitor in decreasing tumor cell proliferation in culture and in xenografts, suggesting that STAT3 mediates the pro-proliferative program induced by Met. However, the phosphorylation of ERK increased after prolonged Met inhibition in culture, correlating with decreased abundance of the phosphatases DUSP4 and DUSP6, which inhibit ERK. Combined inhibition of Met and the mitogen-activated protein kinase kinase (MEK)-ERK pathway induced greater cell death in cultured gastric cancer cells than did either inhibitor alone. These findings indicate combination therapies that may counteract resistance to Met inhibitors.

We and others have previously shown that ICOS plays an important role in inducing acute graft-versus-host disease (GVHD) in murine models of allogeneic bone marrow transplantation. ICOS potentiates TCR-mediated PI3K activation and intracellular calcium mobilization. However, ICOS signal transduction pathways involved in GVHD remain unknown. In this study, we examined the contribution of ICOS-PI3K signaling in the pathogenic potential of T cells using a knock-in mouse strain, ICOS-YF, which selectively lost the ability to activate PI3K. We found that when total T cells were used as alloreactive T cells, ICOS-YF T cells caused less severe GVHD compared with ICOS wild-type T cells, but they induced much more aggressive disease than ICOS knockout T cells. This intermediate level of pathogenic capacity of ICOS-YF T cells was correlated with similar levels of IFN-γ–producing CD8 T cells that developed in the recipients of ICOS-WT or ICOS-YF T cells. We further evaluated the role of ICOS-PI3K signaling in CD4 versus CD8 T cell compartment using GVHD models that are exclusively driven by CD4 or CD8 T cells. Remarkably, ICOS-YF CD8 T cells caused disease similar to ICOS wild-type CD8 T cells, whereas ICOS-YF CD4 T cells behaved very similarly to their ICOS knockout counterparts. Consistent with their in vivo pathogenic potential, CD8 T cells responded to ICOS ligation in vitro by PI3K-independent calcium flux, T cell activation, and proliferation. Thus, in acute GVHD in mice, CD4 T cells heavily rely on ICOS-PI3K signaling pathways; in contrast, CD8 T cells can use PI3K-independent ICOS signaling pathways, possibly through calcium.

Breast cancer heterogeneity is one of the principal obstacles both to predicting outcome and to determining an effective course of treatment for this disease. Although genomic technologies have been used to gain a better understanding, by identifying gene expression signatures associated with clinical outcome and breast cancer subtypes, relatively little is known about heterogeneity in the tumor microenvironment. It is now accepted that changes in the normal cells that constitute the tumor microenvironment (TME) play important roles in determining cancer progression and ultimate outcome. We and others have established that an immune gene expression signature correlates with good outcome in triple negative breast cancer (TNBC), yet fails to accurately predict outcome in all patients. Examining stromal heterogeneity in TNBC has identified four distinct stromal clusters, three of which are prognostic, contain distinct immune signatures and a signature of fibrosis. Lymphocytic infiltration and access to tumor parenchyma is not well understood due to high levels of spatial heterogeneity within tumors. We show that location of CD8+T cells is strongly influenced by TME subtypes and identify gene expression signatures predictive of distinct CD8+T cell localization and patient outcome. Since mounting evidence suggests that immune-checkpoint inhibitor immunotherapies may be promising for only a subset of TNBC patients, highlights the importance of understanding how the TME influences CD8+T cell location. Citation Format: Sadiq Saleh, Tina Gruosso, Mathieu Gigoux, Nicholas Bertos, Atilla Omeroglu, Dongmei Zuo, Sarkis Meterissian, Michael Hallett, Morag Park. Deconvolution of the triple-negative breast cancer microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA23.

Triple negative breast cancer (TNBC), defined as tumors lacking expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), are especially difficult to treat effectively. While ER+ and HER2+ breast cancer subtypes can be treated with Tamoxifen and Herceptin, respectively, there are no targeted therapies for TNBC patients. Furthermore, while only 20-30% of TNBC patients respond to chemotherapy in the neoadjuvant setting, overall outcome remains poor for non-responding patients. However, mounting evidence suggests that immune-checkpoint inhibitor immunotherapies may be especially promising for TNBC patients. We and others have shown that the presence of CD8+ T cells, a crucial component of the cytotoxic arm of the adaptive immune response, is a sign of good clinical outcome in TNBC patients. However, good outcome only correlates with CD8+ T cell invasion of the tumor parenchyma. Here we show that some patients have an accumulation of CD8+ T cells in the surrounding tumor-associated stroma, but not the tumor epithelium, and these patients responded as poorly as patients with no CD8+ T cells at all. Yet how cancer associated fibroblasts (CAFs), a dominant cell type of the tumor-associated stroma, affects CD8+ T cell invasion into the tumor epithelium is still poorly understood. To identify potential stroma-dependent mechanisms that potentiate or inhibit CD8+ T cells invasion into the tumor epithelium, we performed gene expression profiling of laser-capture microdissected tumor-associated stroma (and matched epithelium) from 56 TNBC cases. Here we identify several key stromal features that may explain the accumulation of CD8+ T cells outside of the tumor epithelium. Preliminary data by immunohistochemistry and immunofluorescence validate some key stromal features and decipher the implication of other immune cell types in CD8+ T cells lack of tumor epithelium infiltration. These key stromal features that impair CD8+ T cell infiltration into the tumor in some patients might explain the relative low efficiency of immunotherapies in TNBC patients (20% of patients respond). One could speculate that targeting these key stromal features would allow a significant CD8+ T cell infiltration into the tumor and thus sensitize patients to immunotherapies. Citation Format: Tina Gruosso, Mathieu Gigoux, Nicholas Bertos, Sadiq Saleh, Atilla Omeroglu, Dongmei Zuo, Hong Zhao, Margarita Souleimanova, Valerie Weaver, Sarkis Meterissian, Michael Hallett, Morag Park. Mechanisms of CD8+ T cell immunosuppression in triple negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A15.

Breast cancer is a heterogeneous disease in terms of presentation, morphology, molecular profile and response to therapy. Gene expression profiling has identified intrinsic molecular subtypes that are associated with clinical markers (ER, PR, HER2) as well as prognosis and survival. However, it is well established that the intrinsic molecular profiles of breast tumors are not sufficient to perfectly predict disease outcome. Increasing evidence indicates that characteristics of the breast stroma influence tumor progression and response to therapy. Previous work in our lab has demonstrated that gene expression signatures in human stroma can predict outcome of breast cancer patients independently of clinical parameters and molecular subtypes. In this study, we expand our findings by focusing on a previously underrepresented subset of breast tumors that have no detectable ER, PR or HER2 (termed Triple-Negative, TN). TN tumors, which represent approximately 15% of all breast cancers, are typically associated with poor outcome. However, the contribution of the stroma to the underlying heterogeneity of TN breast cancer and its corresponding influence on therapeutic response is not well understood. To address this, we isolated TN tumor epithelial and stromal tissues by laser capture microdissection and subjected them to gene expression profiling. Class discovery revealed distinct gene-clusters (stromal properties) which are associated with prognosis in TNBC whole tumor samples. Analysis of the genes comprising each stromal property suggests that the properties primarily represent the prevalence of distinct cell types, namely T cells, B cells, activated fibroblasts, and myoepithelial cells. Importantly, these properties are not mutually exclusive, i.e. some tumors are associated with multiple stromal properties. While confirming the heterogeneity of TN-associated stroma, this also indicates that a multi-parameter classification better reflects the true nature of the tumor microenvironment. This project provides the first integrated in-depth analysis of the contribution of tumor stromal processes to TN disease heterogeneity, and positions the tumor microenvironment for therapeutic intervention. Citation Format: Crista Thompson, Sadiq M. Saleh, Nicholas Bertos, Mathieu Gigoux, Tina Gruosso, Margarita Souleimanova, Hong Zhao, Michael T. Hallett, Morag Park. Novel prognostic stromal subtypes in triple-negative breast cancer. [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 720.

The T-cell costimulatory receptors, CD28 and the inducible costimulator (ICOS), are required for the generation of follicular B helper T cells (TFH) and germinal center (GC) reaction. A common signal transducer used by CD28 and ICOS is the phosphoinositide 3-kinase (PI3K). Although it is known that CD28-mediated PI3K activation is dispensable for GC reaction, the role of ICOS-driven PI3K signaling has not been defined. We show here that knock-in mice that selectively lost the ability to activate PI3K through ICOS had severe defects in TFHgeneration, GC reaction, antibody class switch, and antibody affinity maturation. In preactivated CD4+T cells, ICOS delivered a potent PI3K signal that was critical for the induction of the key TFHcytokines, IL-21 and IL-4. Under the same settings, CD28 was unable to activate PI3K but supported a robust secondary expansion of T cells. Thus, our results demonstrate a nonredundant function of ICOS-PI3K pathway in the generation of TFHand suggest that CD28 and ICOS play differential roles during a multistep process of TFHdifferentiation.

It is now accepted that changes in the normal cells that constitute the tumor microenvironment (TME) play important roles in determining cancer progression and ultimate outcome. We and others have established that an immune signature, and more recently that the ability of CD8+ T cells to infiltrate the tumor bed, are correlated with good outcome in triple negative breast cancer. Importantly, since the cytotoxic effector functions of CD8+ T cells are mainly contact-dependent, the tumor microenvironment may act to inhibit access to the epithelial tumor bed, leading to uncontrolled tumor growth. Therefore, in order to determine the role that the TME plays on mediating the entry of CD8+ T cells into the epithelial tumor bed, our group performed laser-capture microdissection to separate the tumor epithelial compartment from the surrounding tumor-associated stroma from the primary tumor of 56 triple negative breast cancer patients and followed by gene expression profiling. Our aims are to provide clinical validation of predicted CD8+ T cell localisation based on our bioinformatic analysis, test our predictions that the identified stroma signature correlates with CD8+ T cell retention in other cohort of TN breast cancer patients and develop suitable markers of this stroma, and develop a muliplex based assay integrating CD8+ T cell localisation with stromal markers suitable for a retrospective study to validate predictions with outcome. Using gene expression profiling, our data have identified a canonical gene expression signature which correlates with retention of CD8+ T cells in the stroma and poor outcome. Thus, our results demonstrate that gene expression analysis of clinical triple negative breast cancer samples can predict the location of CD8+ T cells within the tumor and in turn the outcome. Citation Format: Mathieu Gigoux, Tina Gruosso, Nicholas Bertos, Sadiq Saleh, Atilla Omeroglu, Hong Zhao, Margarita Souleimanova, Sarkis Meterissian, Michael Hallett, Morag Park. The immunological environment in triple negative breast cancer; impact on clinical outcomes towards a prognostic clinical test. [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 A69.