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Immune checkpoint therapy (ICT) using antibody blockade of programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) can provoke T cell–dependent antitumor activity that generates durable clinical responses in some patients. The epigenetic and transcriptional features that T cells require for efficacious ICT remain to be fully elucidated. Herein, we report that anti–PD-1 and anti–CTLA-4 ICT induce upregulation of the transcription factor BHLHE40 in tumor antigen–specific CD8+ and CD4+ T cells and that T cells require BHLHE40 for effective ICT in mice bearing immune-edited tumors. Single-cell RNA sequencing of intratumoral immune cells in BHLHE40-deficient mice revealed differential ICT-induced immune cell remodeling. The BHLHE40-dependent gene expression changes indicated dysregulated metabolism, NF-κB signaling, and IFNγ response within certain subpopulations of CD4+ and CD8+ T cells. Intratumoral CD4+ and CD8+ T cells from BHLHE40-deficient mice exhibited higher expression of the inhibitory receptor gene Tigit and displayed alterations in expression of genes encoding chemokines/chemokine receptors and granzyme family members. Mice lacking BHLHE40 had reduced ICT-driven IFNγ production by CD4+ and CD8+ T cells and defects in ICT-induced remodeling of macrophages from a CX3CR1+CD206+ subpopulation to an iNOS+ subpopulation that is typically observed during effective ICT. Although both anti–PD-1 and anti–CTLA-4 ICT in BHLHE40-deficient mice led to the same outcome—tumor outgrowth—several BHLHE40-dependent alterations were specific to the ICT that was used. Our results reveal a crucial role for BHLHE40 in effective ICT and suggest that BHLHE40 may be a predictive or prognostic biomarker for ICT efficacy and a potential therapeutic target.

Supplementary Figure from BHLHE40 Regulates the T-Cell Effector Function Required for Tumor Microenvironment Remodeling and Immune Checkpoint Therapy Efficacy

Antibody blockade of programmed death-1 (PD-1) or its ligand, PD-L1, has led to unprecedented therapeutic responses in certain tumor-bearing individuals, but PD-L1 expression's prognostic value in stratifying cancer patients for such treatment remains unclear. Reports conflict on the significance of correlations between PD-L1 on tumor cells and positive clinical outcomes to PD-1/PD-L1 blockade. We investigated this issue using genomically related, clonal subsets from the same methylcholanthrene-induced sarcoma: a highly immunogenic subset that is spontaneously eliminated in vivo by adaptive immunity and a less immunogenic subset that forms tumors in immunocompetent mice, but is sensitive to PD-1/PD-L1 blockade therapy. Using CRISPR/Cas9-induced loss-of-function approaches and overexpression gain-of-function techniques, we confirmed that PD-L1 on tumor cells is key to promoting tumor escape. In addition, the capacity of PD-L1 to suppress antitumor responses was inversely proportional to tumor cell antigenicity. PD-L1 expression on host cells, particularly tumor-associated macrophages (TAM), was also important for tumor immune escape. We demonstrated that induction of PD-L1 on tumor cells was IFNγ-dependent and transient, but PD-L1 induction on TAMs was of greater magnitude, only partially IFNγ dependent, and was stable over time. Thus, PD-L1 expression on either tumor cells or host immune cells could lead to tumor escape from immune control, indicating that total PD-L1 expression in the immediate tumor microenvironment may represent a more accurate biomarker for predicting response to PD-1/PD-L1 blockade therapy, compared with monitoring PD-L1 expression on tumor cells alone. Cancer Immunol Res; 5(2); 106–17. ©2017 AACR.

Supplementary Figure S1. Genomic sequence of the PD-L1 locus and expression of immunogenic neoantigens in T3deltaPDL1 lines. Supplementary Figure S2. Expression of PD-L1 and neoantigens in the escape T3deltaPDL1 cells. Supplementary Figure S3. PD-L1 but not PD-L2 is upregulated on MCA sarcomas in vitro and in vivo following exposure to IFNgamma. Supplementary Figure S4. T9-PDL1 cells form progressively growing tumors in WT mice. Supplementary Figure S5. T9-PDL1ovr but not T9-PDL1phy cells inhibit antigen-specific T cell killing in vitro. Supplementary Figure S6. Extrinsic IFNgamma stimulation controls upregulation and maintenance of tumor PD-L1 expression. Supplementary Figure S7. Sixteen immune subsets in MCA sarcomas by flow cytometry. Supplementary Figure S8. Comprehensive analysis of cell types expressing PD-L1 in T3 tumors.

Supplementary Table S1. Staining panels for comprehensive analysis of cell types expressing PD-L1.

Figure Legends for Supplementary Data

The induction of proinflammatory T cells by dendritic cell (DC) subtypes is critical for antitumor responses and effective immune checkpoint blockade (ICB) therapy. Here, we show that human CD1c + CD5 + DCs are reduced in melanoma-affected lymph nodes, with CD5 expression on DCs correlating with patient survival. Activating CD5 on DCs enhanced T cell priming and improved survival after ICB therapy. CD5 + DC numbers increased during ICB therapy, and low interleukin-6 (IL-6) concentrations promoted their de novo differentiation. Mechanistically, CD5 expression by DCs was required to generate optimally protective CD5 hi T helper and CD8 + T cells; further, deletion of CD5 from T cells dampened tumor elimination in response to ICB therapy in vivo. Thus, CD5 + DCs are an essential component of optimal ICB therapy.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)–mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4–mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs.

Basic science breakthroughs in T-cell biology and immune–tumor cell interactions ushered in a new era of cancer immunotherapy. Twenty years ago, cancer immunoediting was proposed as a framework to understand the dynamic process by which the immune system can both control and shape cancer and in its most complex form occurs through three phases termed elimination, equilibrium, and escape. During cancer progression through these phases, tumors undergo immunoediting, rendering them less immunogenic and more capable of establishing an immunosuppressive microenvironment. Therefore, cancer immunoediting integrates the complex immune–tumor cell interactions occurring in the tumor microenvironment and sculpts immunogenicity beyond shaping antigenicity. However, with the success of cancer immunotherapy resulting in durable clinical responses in the last decade and subsequent emergence of immuno-oncology as a clinical subspecialty, the phrase “cancer immunoediting” has recently, at times, been inappropriately restricted to describing neoantigen loss by immunoselection. This focus has obscured other mechanisms by which cancer immunoediting modifies tumor immunogenicity. Although establishment of the concept of cancer immunoediting and definitive experimental evidence supporting its existence was initially obtained from preclinical models in the absence of immunotherapy, cancer immunoediting is a continual process that also occurs during immunotherapy in human patients with cancer. Herein, we discuss the known mechanisms of cancer immunoediting obtained from preclinical and clinical data with an emphasis on how a greater understanding of cancer immunoediting may provide insights into immunotherapy resistance and how this resistance can be overcome.

The ability of the immune system to eliminate and shape the immunogenicity of tumours defines the process of cancer immunoediting1. Immunotherapies such as those that target immune checkpoint molecules can be used to augment immune-mediated elimination of tumours and have resulted in durable responses in patients with cancer that did not respond to previous treatments. However, only a subset of patients benefit from immunotherapy and more knowledge about what is required for successful treatment is needed2–4. Although the role of tumour neoantigen-specific CD8+ T cells in tumour rejection is well established5–9, the roles of other subsets of T cells have received less attention. Here we show that spontaneous and immunotherapy-induced anti-tumour responses require the activity of both tumour-antigen-specific CD8+ and CD4+ T cells, even in tumours that do not express major histocompatibility complex (MHC) class II molecules. In addition, the expression of MHC class II-restricted antigens by tumour cells is required at the site of successful rejection, indicating that activation of CD4+ T cells must also occur in the tumour microenvironment. These findings suggest that MHC class II-restricted neoantigens have a key function in the anti-tumour response that is nonoverlapping with that of MHC class I-restricted neoantigens and therefore needs to be considered when identifying patients who will most benefit from immunotherapy., National Institutes of Health Cancer Center Support Grant (P30CA14051)

Immunotherapies such as anti-CTLA-4 immune checkpoint blockade (ICB) have revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by off-target tissue damage or immune-related adverse events (irAEs). At present, there is limited understanding of irAE mechanisms, hampering development of approaches to mitigate their damage. We addressed this problem by generating animal models of intestinal irAE. Our results show that disruption of homeostatic immunity by genetic predisposition to intestinal inflammation or acute gastrointestinal infection sensitizes mice to anti-CTLA-4-mediated intestinal toxicity. Inflammation-prone mice treated with anti-CTLA-4 showed neutrophil accumulation, systemic interleukin-6 (IL-6) release, and dysbiosis. Significantly, IL-6 blockade combined with antibiotic treatment improved anti-CTLA-4 therapeutic efficacy and reduced intestinal irAEs. Immune signatures were validated in biopsies from patients who developed colitis during ICB, supporting the utility of our models. This study provides new pre-clinical models, mechanistic insight into irAEs, and potential approaches to enhance ICB efficacy while mitigating irAEs. Citation Format: Yifan Zhou, Yusra B. Medik, Bhakti Patel, Daniel B. Zamler, Sijie Chen, Thomas Chapman, Sarah Schneider, Rachel L. Babcock, Taylor T. Chrisikos, Laura M. Kahn, Allison M. Dyevoich, Elizabeth M. Park, Alexandria P. Cogdill, Daniel H. Johnson, Sarah B. Johnson, Khalida M. Wani, Debora A. Ledesma, Courtney W. Hudgens, Jingjing Wang, Md Abdul Wadud Khan, Aron Y. Joon, Weiyi Peng, Haiyan S. Li, Reetakshi Arora, Ximing Tang, Maria Gabriela Raso, Xuegong Zhang, Wai Chin Foo, Michael T. Tetzlaff, Gretchen E. Diehl, Karen Clise-Dwyer, Elizabeth M. Whitley, Matthew M. Gubin, James P. Allison, Patrick Hwu, Nadim J. Ajami, Adi Diab, Jennifer A. Wargo, Stephanie S. Watowich. Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5545.

The success of immune checkpoint therapy (ICT) in generating durable clinical responses is remarkable but not all cancer patients respond for reasons that are incompletely understood. In our previous studies of immune responses to ICT in the mouse T3 sarcoma model, we found strong upregulation of the BHLHE40 transcription factor in a number of immune cell populations. It was particularly prominent in the tumor antigen-specific CD8+ and CD4+ T cells, which are crucial for ICT-induced tumor rejection and elimination. We used global and cell-type specific Bhlhe40 knockout (KO) mice to investigate its role in tumor rejection. Both global Bhlhe40-/- and CD4-Cre+ Bhlhe40f/f (CD4+ and CD8+ T cell-specific Bhlhe40 deletion) mice bearing the 1956 methylcholanthrene-induced sarcoma line failed to respond to either anti-PD-1 or anti-CTLA-4 ICT, in stark contrast to wild type (WT) mice. At the same time, Bhlhe40 deletion in macrophages and granulocytes (LysM-Cre+ Bhlhe40f/f mice) did not have an apparent effect on ICT-mediated tumor rejection. To gain insight into the functional changes orchestrated by BHLHE40 in response to ICT, we used single-cell RNA sequencing (scRNAseq) to analyze the transcriptional profiles of the CD45+ tumor-infiltrating immune cells from 1956 tumor-bearing Bhlhe40+/+ and Bhlhe40-/- mice treated with control, anti-PD-1 or anti-CTLA-4 mAb. While no major changes in the relative fractions of effector T cell clusters were observed, ICT induced BHLHE40-dependent remodeling within individual CD4+ and CD8+ clusters. Most notably, Bhlhe40-/- mice were strongly deficient in ICT-induced Ifng expression by CD8+ T cells, with intracellular cytokine staining revealing low IFNγ production by both CD4+ and CD8+ T cells. GSEA and Ingenuity Pathway analysis indicated changes in energy metabolism, hypoxia adaptation, NF-κB signaling, IFNγ response, and actin cytoskeleton remodeling in a number of Bhlhe40-/- T cell clusters. This was accompanied by upregulation of the inhibitory receptor Tigit mRNA, altered expression of chemokine/chemokine receptor genes, as well as granzyme gene family members. In addition to changes in the transcriptional profile shared between anti-PD-1 and anti-CTLA-4 treatments, each checkpoint inhibitor induced its own specific alterations both in WT and KO mice. Analysis of macrophage populations also revealed a notable difference between Bhlhe40+/+ and Bhlhe40-/- mice. The shift of macrophages from the CX3CR1+ CD206+ to the iNOS+ phenotype, typically observed during effective ICT, was dramatically reduced in the Bhlhe40-/- mice, which was confirmed by flow cytometry. Our results provide the basis for understanding the role of BHLHE40 in the ICT-induced tumor rejection, the pathways and effector mechanisms under its control, and the potential ways for therapeutic targeting of this regulatory network to improve the efficacy of cancer immunotherapy. Citation Format: Alexander S. Shavkunov, Avery J. Salmon, Qi Miao, Sunita Keshari, Charmelle D. Williams, Josué E. Pineda, Ken Chen, Brian T. Edelson, Matthew M. Gubin. BHLHE40 orchestrates remodeling of the intratumoral immune cell populations in response to immune checkpoint therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1365.

Effective cancer immunotherapy, such as immune checkpoint therapy (ICT) [e.g., anti-CTLA-4 and anti-PD-1/PD-L1], is dependent on T cell recognition of tumor antigens presented on major histocompatibility complex (MHC) and leads to durable responses in certain cancer patients. Recent advances have facilitated identification of tumor mutant neoantigens and led to efforts to develop effective personalized neoantigen cancer vaccines. However, little is known about how expression of multiple neoantigens with variable peptide-MHC (pMHC) binding or TCR affinity/avidity affects T cell responses to both discrete neoantigens and shared non-mutant antigens, and if the localization of T cells with distinctive pMHC specificity differs within the tumor. To address this, we genetically introduced multiple combinations of MHC-I and MHC-II neoantigens in the poorly immunogenic Brafv600e Pten-/- Cdkn2a-/- YUMM1.7 (Y1.7) mouse melanoma line that contains a paucity of neoantigens. We expressed neoantigens previously found in a mouse sarcoma line of the same MHC haplotype as Y1.7. The MHC-I neoantigens used were formed by G1254V mutation in Laminin alpha subunit-4 (Lama4) and A506T mutation in the Asparagine-linked glycosylation 8 (Alg8) and the MHC-II neoantigen was formed by A710T mutation in Integrin beta-1 (Itgb1). We generated Y1.7 lines expressing minigenes for mLama4, mAlg8, or mLama4 + mAlg8 MHC-I neoantigens. All three lines also expressed the mItgb1 MHC-II neoantigen. Whereas ICT-treated mice bearing the parental Y1.7 melanoma line showed rapid tumor growth, modified Y1.7 lines expressing mLama4 and/or mAlg8 plus mItgb1 were rendered sensitive to ICT. Tumors expressing mLama4 and/or mAlg8 contained mLama4- and/or mAlg8-specific CD8+ T cells, respectively. Preliminary analysis revealed expression of either MHC-I neoantigen alters the frequency of CD8+ T cells specific for non-mutant endogenous antigens in Y1.7. Interestingly, therapeutic synthetic long-peptide (SLP) vaccines incorporating mLama4 and/or mAlg8 neoantigens plus poly I:C induced tumor rejection comparable to ICT. Moreover, SLP vaccines amplified intratumoral mLama4- and mAlg8-specific CD8+ T cells, as well as in tumor-draining lymph nodes, where these neoantigen-specific CD8+ T cells were found to express IFN-γ. During either vaccination or ICT, CD8+ T cell response to mLama4 was higher than that to mAlg8 when both neoantigens were co-expressed. Altogether, these melanoma models will enable us to determine whether expression of one or more MHC-I neoantigens influences CD8+ T cell responses to both distinct neoantigens and non-mutant antigens (e.g., gp100, Trp2) during ICT and SLP vaccination. Furthermore, high-dimensional analyses approaches including scRNA-seq. and CODEX imaging are ongoing and will facilitate development of improved immunotherapies and may help uncover novel biomarkers of response. Citation Format: Sunita Keshari, Charmelle D. Williams, Elizabeth D. Sanchez, Alexander S. Shavkunov, Matthew M. Gubin. High-dimensional analysis of T cell responses to mutant neoantigens and nonmutant antigens in mouse models of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1387.

Immune checkpoint therapy (ICT) using antibody blockade of programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) can provoke T cell–dependent antitumor activity that generates durable clinical responses in some patients. The epigenetic and transcriptional features that T cells require for efficacious ICT remain to be fully elucidated. Herein, we report that anti–PD-1 and anti–CTLA-4 ICT induce upregulation of the transcription factor BHLHE40 in tumor antigen–specific CD8+ and CD4+ T cells and that T cells require BHLHE40 for effective ICT in mice bearing immune-edited tumors. Single-cell RNA sequencing of intratumoral immune cells in BHLHE40-deficient mice revealed differential ICT-induced immune cell remodeling. The BHLHE40-dependent gene expression changes indicated dysregulated metabolism, NF-κB signaling, and IFNγ response within certain subpopulations of CD4+ and CD8+ T cells. Intratumoral CD4+ and CD8+ T cells from BHLHE40-deficient mice exhibited higher expression of the inhibitory receptor gene Tigit and displayed alterations in expression of genes encoding chemokines/chemokine receptors and granzyme family members. Mice lacking BHLHE40 had reduced ICT-driven IFNγ production by CD4+ and CD8+ T cells and defects in ICT-induced remodeling of macrophages from a CX3CR1+CD206+ subpopulation to an iNOS+ subpopulation that is typically observed during effective ICT. Although both anti–PD-1 and anti–CTLA-4 ICT in BHLHE40-deficient mice led to the same outcome—tumor outgrowth—several BHLHE40-dependent alterations were specific to the ICT that was used. Our results reveal a crucial role for BHLHE40 in effective ICT and suggest that BHLHE40 may be a predictive or prognostic biomarker for ICT efficacy and a potential therapeutic target.

BackgroundBackground: Radiotherapy of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)-expressing CRC cells systemically; however, incidences of abscopal tumor remission are extremely rare. We sought to unravel the post-irradiation immune escape mechanisms in CRC.MethodsMethodsFlow cytometry, gene knockdown, RNA and T cell receptor sequencing, and multiple murine syngeneic CRC models were used to interrogate mechanisms of CRC immune evasion following radiotherapy. Comparison of immunohistochemistry staining between pretreatment biopsy and post-irradiation surgical specimens was performed in rectal patients who underwent neoadjuvant radiotherapy with 5 Gy for 5 fractions.ResultsResultsWe find that CRC cells utilize a common DNA repair signaling pathway — ATR/Chk1/STAT3 — to upregulate both CD47 and PD-L1 in response to radiotherapy, which through engagement of SIRPα and PD-1 suppresses the capacity of antigen-presenting cells (APCs) to phagocytose them thereby preventing TAA cross-presentation. This post-irradiation CD47 and PD-L1 upregulation can be observed in CRC cells treated with either photon or proton radiotherapy and across a wide variety of human solid tumor cells. Concordantly, rectal cancer patients who responded poorly (tumor regression grade 4–5, n = 10) to neoadjuvant radiotherapy exhibited significantly elevated post-irradiation CD47 levels (P = 0.005). In murine CRC models, the combination of radiotherapy, αSIRPα, and αPD-1 (RSP) profoundly enhances TAA uptake, activation of innate immune sensors, and TAA cross-priming across various antigen-presenting myeloid populations in the irradiated tumor microenvironment and facilitates TAA-presenting APC migration to secondary lymphoid organs. Furthermore, we observed robust production of TAA-specific CD8 T cells, functional activation of effector T cells, and increased tumor-infiltrating T cell clonality and clonal diversity in mice treated with RSP. Importantly, radiotherapy coupled with phagocytosis checkpoint blockade significantly improves complete response rates in both irradiated and abscopal tumors and prolongs survival in three distinct murine CRC models, including a cecal orthotopic model. In addition, αSIRPα exerts superior tumoricidal efficacy than αCD47 in combination with RT and αPD-1. We find RSP efficacy to be STING dependent as knockout animals lose most benefit of phagocytosis checkpoint blockade.ConclusionATR-mediated CD47 and PD-L1 upregulation restrains radiation-induced immune priming in CRC. Blockade of the phagocytosis checkpoints SIRPα and PD-1 during radiotherapy promotes vigorous anti-CRC immune priming leading to systemic tumor regression.AcknowledgementsThis study is supported in part by NIH grant P30 CA16672, the MD Anderson Andrew Sabin Family Fellowship, and Chang Gung Memorial Hospital grant CMRPG3K1751. RCH was supported by the CPRIT Research Training Grant (RP170067) and Ralph B. Arlinghaus Ph.D. Scholarship. The authors are grateful to the members of the Advanced Cytometry & Sorting Facility at South Campus, Tissue Bank of Chang Gung Memorial Hospital at Linkou, and MHC Tetramer Core Facility at Baylor College of Medicine for their invaluable help.Ethics ApprovalThis study was approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taiwan; approval number: 202001191B0C601.

Leishmaniasis is a parasitic disease caused by flagellated protozoa of the genus Leishmania. It is transmitted by infection from the bite of an infected sandfly. The 3 main clinical forms of the disease are cutaneous leishmaniasis (CL), visceral leishmaniasis (VL) and mucocutaneous leishmaniasis (MCL). Prior to 1996, all leishmaniasis cases were infected during the visit to the endemic areas. Thereafter, autochthonous leishmaniasis cases have been reported in Thailand. During 1996 to the present, at least 21 cases of autochthonous leishmaniasis have been confirmed in Thailand. Leishmania siamensis, a novel species of Leishmania, was suspected of being the causative pathogens in some of those cases, although the data supporting the existence of this new species is limited. Until recently, in-depth investigation using molecular characterization and isoenzyme analysis revealed that a suspected novel species consists of 2 different, but closely related strains: L. siamensis and L. martiniquensis. L. martiniquensis, a rare species firstly discovered on Martinique Island, is the cause of leishmaniasis in the majority of cases. Meanwhile, L. siamensis, a true novel species first and only reported from Thailand, was confirmed as the cause of leishmaniasis in two autochthonous cases. Two clinical forms (CL and VL) have been observed in both L. martiniquensis and L. siamensis infection. DNA of L. martiniquensis was found in black rats, suggesting their role as a natural reservoir. The presence of L. martiniquensis DNA in two sandfly species (Sergentomyia gemmea and Sergentomyia barraudi) that are commonly found in affected areas may also suggest their role as potential vectors. Here, we update the status of leishmaniasis in Thailand and its emergence as a potential public health concern.

The analysis of neoantigen-specific CD8+ T cells in tumour-bearing individuals is challenging due to the small pool of tumour antigen-specific T cells. Here we show that mass cytometry with multiplex combinatorial tetramer staining can identify and characterize neoantigen-specific CD8+ T cells in mice bearing T3 methylcholanthrene-induced sarcomas that are susceptible to checkpoint blockade immunotherapy. Among 81 candidate antigens tested, we identify T cells restricted to two known neoantigens simultaneously in tumours, spleens and lymph nodes in tumour-bearing mice. High-dimensional phenotypic profiling reveals that antigen-specific, tumour-infiltrating T cells are highly heterogeneous. We further show that neoantigen-specific T cells display a different phenotypic profile in mice treated with anti-CTLA-4 or anti-PD-1 immunotherapy, whereas their peripheral counterparts are not affected by the treatments. Our results provide insights into the nature of neoantigen-specific T cells and the effects of checkpoint blockade immunotherapy.Immune checkpoint blockade (ICB) therapies can unleash anti-tumour T-cell responses. Here the authors show, by integrating MHC tetramer multiplexing, mass cytometry and high-dimensional analyses, that neoantigen-specific, tumour-infiltrating T cells are highly heterogeneous and are subjected to ICB modulations.

Posttranscriptional gene regulation by RNA-binding proteins, such as HuR (elavl1), fine-tune gene expression in T cells, leading to powerful effects on immune responses. HuR can stabilize target mRNAs and/or promote translation by interacting with their 3′ untranslated region adenylate and uridylate–rich elements. It was previously demonstrated that HuR facilitates Th2 cytokine expression by mRNA stabilization. However, its effects upon IL-2 homeostasis and CD4+ Th2 differentiation are not as well understood. We found that optimal translation of Il2ra (CD25) required interaction of its mRNA with HuR. Conditional HuR knockout in CD4+ T cells resulted in loss of IL-2 homeostasis and defects in JAK–STAT signaling, Th2 differentiation, and cytokine production. HuR-knockout CD4+ T cells from OVA-immunized mice also failed to proliferate in response to Ag. These results demonstrate that HuR plays a pivotal role in maintaining normal IL-2 homeostasis and initiating CD4+ Th2 differentiation.

The Keystone Symposia conference on Cancer Immunology and Immunotherapy: Taking a Place in Mainstream Oncology was held at the Fairmont Chateau in Whistler, British Columbia, Canada, on March 19–23, 2017. The conference brought together a sold-out audience of 654 scientists, clinicians, and others from both academia and industry to discuss the latest developments in cancer immunology and immunotherapy. This meeting report summarizes the main themes that emerged during the four-day conference. Cancer Immunol Res; 5(6); 434–8. ©2017 AACR.

Summary Checkpoint immunotherapy unleashes T cell control of tumors, but is undermined by immunosuppressive myeloid cells. TREM2 is a myeloid receptor that transmits intracellular signals that sustain microglial responses during Alzheimer’s disease. TREM2 is also expressed by tumor-infiltrating macrophages. Here, we found that Trem2–/– mice are more resistant to growth of various cancers than wild-type mice and are more responsive to anti-PD-1 immunotherapy. Furthermore, treatment with anti-TREM2 mAb curbed tumor growth and fostered regression when combined with anti-PD-1. scRNA-seq revealed that both TREM2 deletion and anti-TREM2 are associated with scant MRC1+ and CX3CR1+ macrophages in the tumor infiltrate, paralleled by expansion of myeloid subsets expressing immunostimulatory molecules that promote improved T cell responses. TREM2 was expressed in tumor macrophages in over 200 human cancer cases and inversely correlated with prolonged survival for two types of cancer. Thus, TREM2 might be targeted to modify tumor myeloid infiltrates and augment checkpoint immunotherapy.

Obesity is associated with an increased risk and progression in a variety of cancers. In melanoma, obesity is associated with an increased risk of recurrence following surgical resection which may reflect immune escape of cancer surveillance. We sought to examine the impact of obesity on antitumor immunity in an immunogenic preclinical melanoma model.Male C57/Bl6 mice were fed a 45% fat diet or 10% fat diet for 12 months resulting in obese (body weight 52.8g ±1.3) vs control mice (body weight 38.8g ±2.2). For these studies, we utilized the YUMMER cell line, a C57BL/6J syngeneic melanoma cell line with three driver mutation/deletions (BrafV600E, Pten-/-, Cdkn2a-/-), irradiated to mimic the high mutation burden of human melanoma. 0.5x106 YUMMER cells were subcutaneously injected into the flank of aged (13 month old) control and obese mice. After 14 days post injection, tumors in control mice either had growth arrest or regression. In contrast, all obese mice exhibited progressively growing tumors with 6-fold greater tumor weights after 21 days as compared to control (1.3g ±0.4 vs 0.23g ±0.06). CyTOF analysis of tumor-infiltrating immune cells at day 21 showed 2-fold decrease in the percentage of CD4 T cells in tumors from obese mice (9.8% vs 21.1% of CD45). In addition, obesity resulted in 4-fold increase in the frequency of intratumoral Treg (26.1% vs 8.3% of CD3CD4 cells) and CD8/Treg ratio was 2-fold reduced in obese mice. In contrast to prior findings in obese mice bearing B16 melanomas, the percentage of CD8 T cells expressing PD1 was similar (80.4% vs 74.3%), but we found 2-fold higher frequency of CD8PD1 T cells co-expressing TIM3 and LAG3 in tumors from obese mice (43.3% vs 23.0%). Moreover, obesity also attenuated the frequency of tumor-infiltrating cDC2 (1.4% vs 3.5% of CD45) but did not affect NK cells or B cell proportions. Furthermore, the ratio of tumor infiltrating myeloid cells to T cells was higher in obese mice (1.94:1 vs 0.82:1). Moreover, myeloid cells from obese mouse tumors had higher expression of PDL1, CD206, and CD38 markers.Our findings provide evidence that obesity impairs antitumor immunity by modulating the relative abundance of intratumoral immune cell populations and their phenotypes to promote immunosuppressive microenvironment. These findings will be validated in subsequent studies and examined in human specimens. Citation Format: Barbara Pazdrak, Duncan Mak, William Damsky, Marcus Bosenberg, Brooklyn Lochmann, Matthew Gubin, Jennifer McQuade. Obesity-induced impairment of antitumor immunity is associated with an immunosuppressive tumor immune landscape [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 70.

Posttranscriptional gene regulation by RNA-binding proteins, such as HuR (elavl1), fine-tune gene expression in T cells, leading to powerful effects on immune responses. HuR can stabilize target mRNAs and/or promote translation by interacting with their 3′ untranslated region adenylate and uridylate–rich elements. It was previously demonstrated that HuR facilitates Th2 cytokine expression by mRNA stabilization. However, its effects upon IL-2 homeostasis and CD4+ Th2 differentiation are not as well understood. We found that optimal translation of Il2ra (CD25) required interaction of its mRNA with HuR. Conditional HuR knockout in CD4+ T cells resulted in loss of IL-2 homeostasis and defects in JAK–STAT signaling, Th2 differentiation, and cytokine production. HuR-knockout CD4+ T cells from OVA-immunized mice also failed to proliferate in response to Ag. These results demonstrate that HuR plays a pivotal role in maintaining normal IL-2 homeostasis and initiating CD4+ Th2 differentiation.

SummaryFailure of T cells to protect against cancer is thought to result from lack of antigen recognition, chronic activation, and/or suppression by other cells. Using a mouse sarcoma model, we show that glucose consumption by tumors metabolically restricts T cells, leading to their dampened mTOR activity, glycolytic capacity, and IFN-γ production, thereby allowing tumor progression. We show that enhancing glycolysis in an antigenic “regressor” tumor is sufficient to override the protective ability of T cells to control tumor growth. We also show that checkpoint blockade antibodies against CTLA-4, PD-1, and PD-L1, which are used clinically, restore glucose in tumor microenvironment, permitting T cell glycolysis and IFN-γ production. Furthermore, we found that blocking PD-L1 directly on tumors dampens glycolysis by inhibiting mTOR activity and decreasing expression of glycolysis enzymes, reflecting a role for PD-L1 in tumor glucose utilization. Our results establish that tumor-imposed metabolic restrictions can mediate T cell hyporesponsiveness during cancer.

The posttranscriptional mechanisms by which RNA binding proteins (RBPs) regulate T-cell differentiation and cytokine production in vivo remain unclear. The RBP HuR binds to labile mRNAs, usually leading to increases in mRNA stability and/or translation. Previous work demonstrated that HuR binds to the mRNAs encoding the Th2 transcription factor trans-acting T-cell-specific transcription factor (GATA-3) and Th2 cytokines interleukin (IL)-4 and IL-13, thereby regulating their expression. By using a novel conditional HuR knockout (KO) mouse in which HuR is deleted in activated T cells, we show that Th2-polarized cells from heterozygous HuR conditional (OX40-Cre HuR(fl/+)) KO mice had decreased steady-state levels of Gata3, Il4 and Il13 mRNAs with little changes at the protein level. Surprisingly, Th2-polarized cells from homozygous HuR conditional (OX40-Cre HuR(fl/fl)) KO mice showed increased Il2, Il4 and Il13 mRNA and protein via different mechanisms. Specifically, Il4 was transcriptionally upregulated in HuR KO T cells, whereas Il2 and Il13 mRNA stabilities increased. Additionally, when using the standard ovalbumin model of allergic airway inflammation, HuR conditional KO mice mounted a robust inflammatory response similar to mice with wild-type HuR levels. These results reveal a complex differential posttranscriptional regulation of cytokines by HuR in which gene dosage plays an important role. These findings may have significant implications in allergies and asthma, as well as autoimmune diseases and infection.

Antibody blockade of programmed death-1 (PD-1) or its ligand, PD-L1, has led to unprecedented therapeutic responses in certain tumor-bearing individuals, but PD-L1 expression's prognostic value in stratifying cancer patients for such treatment remains unclear. Reports conflict on the significance of correlations between PD-L1 on tumor cells and positive clinical outcomes to PD-1/PD-L1 blockade. We investigated this issue using genomically related, clonal subsets from the same methylcholanthrene-induced sarcoma: a highly immunogenic subset that is spontaneously eliminated in vivo by adaptive immunity and a less immunogenic subset that forms tumors in immunocompetent mice, but is sensitive to PD-1/PD-L1 blockade therapy. Using CRISPR/Cas9-induced loss-of-function approaches and overexpression gain-of-function techniques, we confirmed that PD-L1 on tumor cells is key to promoting tumor escape. In addition, the capacity of PD-L1 to suppress antitumor responses was inversely proportional to tumor cell antigenicity. PD-L1 expression on host cells, particularly tumor-associated macrophages (TAM), was also important for tumor immune escape. We demonstrated that induction of PD-L1 on tumor cells was IFNγ-dependent and transient, but PD-L1 induction on TAMs was of greater magnitude, only partially IFNγ dependent, and was stable over time. Thus, PD-L1 expression on either tumor cells or host immune cells could lead to tumor escape from immune control, indicating that total PD-L1 expression in the immediate tumor microenvironment may represent a more accurate biomarker for predicting response to PD-1/PD-L1 blockade therapy, compared with monitoring PD-L1 expression on tumor cells alone. Cancer Immunol Res; 5(2); 106–17. ©2017 AACR.

The posttranscriptional mechanisms whereby RNA-binding proteins (RBPs) regulate T cell differentiation remain unclear. RBPs can coordinately regulate the expression of functionally related genes via binding to shared regulatory sequences, such as the adenylate-uridylate–rich elements (AREs) present in the 3′ untranslated region (UTR) of mRNA. The RBP HuR posttranscriptionally regulates IL-4, IL-13, and other Th2 cell-restricted transcripts. We hypothesized that the ARE-bearing GATA-3 gene, a critical regulator of Th2 polarization, is under HuR control as part of its coordinate posttranscriptional regulation of the Th2 program. We report that in parallel with stimulus-induced increase in GATA-3 mRNA and protein levels, GATA-3 mRNA half-life is increased after restimulation in the human T cell line Jurkat, in human memory and Th2 cells, and in murine Th2-skewed cells. We demonstrate by immunoprecipitation of ribonucleoprotein complexes that HuR associates with the GATA-3 endogenous transcript in human T cells and found, using biotin pulldown assay, that HuR specifically interacts with its 3′UTR. Using both loss-of-function and gain-of-function approaches in vitro and in animal models, we show that HuR is a critical mediator of stimulus-induced increase in GATA-3 mRNA and protein expression and that it positively influences GATA-3 mRNA turnover, in parallel with selective promotion of Th2 cytokine overexpression. These results suggest that HuR-driven posttranscriptional control plays a significant role in T cell development and effector function in both murine and human systems. A better understanding of HuR-mediated control of Th2 polarization may have utility in altering allergic airway inflammation in human asthmatic patients.

Although current immune-checkpoint therapy (ICT) mainly targets lymphoid cells, it is associated with a broader remodeling of the tumor micro-environment. Here, using complementary forms of high-dimensional profiling, we define differences across all hematopoietic cells from syngeneic mouse tumors during unrestrained tumor growth or effective ICT. Unbiased assessment of gene expression of tumor-infiltrating cells by single-cell RNA sequencing (scRNAseq) and longitudinal assessment of cellular protein expression by mass cytometry (CyTOF) revealed significant remodeling of both the lymphoid and myeloid intratumoral compartments. Surprisingly, we observed multiple subpopulations of monocytes/macrophages, distinguishable by the markers CD206, CX3CR1, CD1d, and iNOS, that change over time during ICT in a manner partially dependent on IFNγ. Our data support the hypothesis that this macrophage polarization/activation results from effects on circulatory monocytes and early macrophages entering tumors, rather than on pre-polarized mature intratumoral macrophages.

Interactions between RNA binding proteins (RBPs) and genes are not well understood, especially in regulation of angiogenesis. The RBP HuR binds to the AU-rich (ARE) regions of labile mRNAs, facilitating their translation into protein and has been hypothesized to be a tumor-maintenance gene. Elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR controls the expression of multiple genes involved in angiogenesis including VEGFα, HIF1α and thrombospondin 1 (TSP1). We investigated the role of HuR in estrogen receptor negative (ER(-)) breast cancer. MDA-MB-231 cells with higher levels of HuR have alterations in cell cycle kinetics and faster growth. Unexpectedly, HuR overexpression significantly interfered with tumor growth in orthotopic mouse models. The putative mechanism seems to be an anti-angiogenetic effect by increasing expression of TSP1 but also surprisingly, downregulating VEGF, a target which HuR normally increases. Our findings reveal that HuR may be regulating a cluster of genes involved in blood vessel formation which controls tumor angiogenesis. An approach of modulating HuR levels may overcome limitations associated with monotherapies targeting tumor vessel formation.

Definitive experimental evidence from mouse cancer models and strong correlative clinical data gave rise to the Cancer Immunoediting concept that explains the dual host-protective and tumor-promoting actions of immunity on developing cancers. Tumor-specific neoantigens can serve as targets of spontaneously arising adaptive immunity to cancer and thereby determine the ultimate fate of developing tumors. Tumor-specific neoantigens can also function as optimal targets of cancer immunotherapy against established tumors. These antigens are derived from nonsynonymous mutations that occur during cellular transformation and, because they are foreign to the host genome, are not subject to central tolerance. In this review, we summarize the experimental evidence indicating that cancer neoantigens are the source of both spontaneously occurring and therapeutically induced immune responses against cancer. We also review the advances in genomics, bioinformatics, and cancer immunotherapy that have facilitated identification of neoantigens and have moved personalized cancer immunotherapies into clinical trials, with the promise of providing more specific, safer, more effective, and perhaps even more generalizable treatments to cancer patients than current immunotherapies.

It is now well established that the immune system can recognize developing cancers and that therapeutic manipulation of immunity can induce tumor regression. The capacity to manifest remarkably durable responses in some patients has been ascribed in part to T cells that can (a) kill tumor cells directly, (b) orchestrate diverse antitumor immune responses, (c) manifest long-lasting memory, and (d) display remarkable specificity for tumor-derived proteins. This specificity stems from fundamental differences between cancer cells and their normal counterparts in that the former develop protein-altering mutations and undergo epigenetic and genetic alterations, resulting in aberrant protein expression. These events can result in formation of tumor antigens. The identification of mutated and aberrantly expressed self-tumor antigens has historically been time consuming and laborious. While mutant antigens are usually expressed in a tumor-specific manner, aberrantly expressed antigens are often shared between cancers and, therefore, in the past, have been the major focus of therapeutic cancer vaccines. However, advances in next-generation sequencing and epitope prediction now permit the rapid identification of mutant tumor neoantigens. This review focuses on a discussion of mutant tumor neoantigens and their use in personalizing cancer immunotherapies.

Due to poor correlation between steady state mRNA levels and protein product, purely transcriptomic profiling methods may miss genes posttranscriptionally regulated by RNA binding proteins (RBPs) and microRNAs (miRNAs). RNA immunoprecipitation (RIP) methods developed to identify in vivo targets of RBPs have greatly elucidated those mRNAs which may be regulated via transcript stability and translation. The RBP HuR (ELAVL1) and family members are major stabilizers of mRNA. Many labs have identified HuR mRNA targets; however, many of these analyses have been performed in cell lines and oftentimes are not independent biological replicates. Little is known about how HuR target mRNAs behave in conditional knock-out models. In the present work, we performed HuR RIP-Seq and RNA-Seq to investigate HuR direct and indirect targets using a novel conditional knock-out model of HuR genetic ablation during CD4+ T activation and Th2 differentiation. Using independent biological replicates, we generated a high coverage RIP-Seq data set (>160 million reads) that was analyzed using bioinformatics methods specifically designed to find direct mRNA targets in RIP-Seq data. Simultaneously, another set of independent biological replicates were sequenced by RNA-Seq (>425 million reads) to identify indirect HuR targets. These direct and indirect targets were combined to determine canonical pathways in CD4+ T cell activation and differentiation for which HuR plays an important role. We show that HuR may regulate genes in multiple canonical pathways involved in T cell activation especially the CD28 family signaling pathway. These data provide insights into potential HuR-regulated genes during T cell activation and immune mechanisms.

The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell immunogenicity1,2,3,4,5,6, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion1,2,7. Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells8,9. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits—including durable responses—to patients with different malignancies10,11,12,13. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.

Cancer immunotherapy has advanced to the forefront of molecular medicine as a consequence of the success of monoclonal antibodies (mAbs) that block immune checkpoints. Such antibodies, like ipilimumab, reverse cancer-induced immunosuppression and induce durable therapeutic responses in certain cancer patients ( 1 ). However, because only some patients respond to checkpoint blockade therapy, there is a need for reliable biomarkers that identify individuals most likely to respond to such treatment. On page 207 of this issue, Van Allen et al. ( 2 ) report the genomic analyses of tumors from 110 melanoma patients prior to ipilimumab therapy. The study not only validates features of responsive melanomas suggested in smaller-scale analyses, but also refutes claims that associate responsiveness to ipilimumab with tumor antigens that show putative similarities to microbial proteins ( 3 ).

The principles of cancer immunoediting have set the foundations for understanding the dual host-protective and tumour sculpting actions of immunity on cancer and establishing the basis for novel individualized cancer immunotherapies. During cancer immunoediting, the host immune system shapes tumour fate in three phases through the activation of innate and adaptive immune mechanisms. In the first phase, Elimination, transformed cells are destroyed by a competent immune system. Sporadic tumour cells that manage to survive immune destruction may then enter an Equilibrium phase where editing occurs. The Escape phase represents the third and final phase of the process, where immunologically sculpted tumours begin to grow progressively, become clinically apparent and establish an immunosuppressive tumour microenvironment. This review focuses on important recent developments that have enhanced our understanding of each phase of the cancer immunoediting process, summarizes the discovery of new predictive and prognostic biomarkers and discusses development of novel and objectively effective cancer immunotherapies.

IL-17 is a proinflammatory cytokine produced by activated Th17 cells and other immune cells. IL-17–producing Th17 cells are major contributors to chronic inflammatory and autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Although the transcriptional regulation of Th17 cells is well understood, the posttranscriptional regulation of IL-17 gene expression remains unknown. The RNA-binding protein HuR positively regulates the stability of many target mRNAs via binding the AU-rich elements present in the 3′ untranslated region of many inflammatory cytokines including IL-4, IL-13, and TNF-α. However, the regulation of IL-17 expression by HuR has not been established. CD4+ Th17 cells from HuR knockout mice had decreased IL-17 steady-state mRNA and protein levels compared with wild-type Th17 cells, as well as decreases in frequency of IL-17+ cells. Moreover, we demonstrated that HuR directly binds to the IL-17 mRNA 3′ untranslated region by using RNA immunoprecipitation and biotin pulldown assays. In addition, the knockout of HuR decreased cellular proliferation of CD4+ T cells. Mice with adoptively transferred HuR KO Th17 cells had delayed initiation and reduced disease severity in the onset of experimental autoimmune encephalomyelitis compared with wild-type Th17 cells. Our results reveal a HuR-induced posttranscriptional regulatory mechanism of Th17 differentiation that influences IL-17 expression. These findings may provide novel therapeutic targets for the treatment of Th17-mediated autoimmune neuroinflammation.

Monoclonal antibody (mAb) blockade of immune checkpoints such as PD-1 and CTLA-4 can stimulate therapeutic, T cell-dependent anti-tumor activity in mice and humans. We asked whether exome sequencing and epitope prediction algorithms could identify antigens in progressively growing tumors that render them susceptible to anti-PD-1- or anti-CTLA-4-induced immune elimination. Expressed mutations in progressively growing, T3 methylcholanthrene (MCA)-sarcomas were identified by exome sequencing and prioritized based primarily on potential MHC class I binding affinity and predicted peptide processing. The two best candidate tumor-specific mutant antigens (TSMA) were identified from mutations in Laminin α subunit 4 (Lama4) and a glucosyltransferase called Alg8. When tested in vitro, mutant Lama4 (mLama4) and mutant Alg8 (mAlg8) peptides were the only top predicted TSMA that stimulated T3-specific T cells. The relevance of these findings was validated by four in vivo studies: (i) mLama4 and mAlg8 peptides bound to H-2Kb were biochemically detected on the surface of T3 tumors, (ii) as shown by tetramer staining, CD8+ tumor infiltrating lymphocytes (TIL) expressing TCRs for mLama4 and mAlg8 accumulated over time in T3 tumors in checkpoint blockade treated, tumor-bearing mice, reaching maximum levels just prior to tumor rejection, (iii) vaccination of naïve wild type mice with mutant but not WT forms of Lama4 or Alg8 induced strong CD8+ T cell responses and (iv) prophylactic or therapeutic vaccination with mLama4 plus mAlg8 synthetic long peptides (SLP) resulted in rejection of T3 cells. The therapeutic protection provided by the SLP vaccine was equal to that afforded by checkpoint blockade therapy. Since we did not see evidence of T cell responses to any other TSMA, we then explored whether the immunodominant antigens masked responses to other TSMA. Using CRISPR/Cas9, we introduced point mutations to revert mLama4 and mAlg8 in T3 back to their nonimmunogenic wild type counterparts. T3 tumor cells with either mLama4 or mAlg8 reverted to wild type were still susceptible to anti-PD-1 or anti-CTLA-4 immune-mediated control with skewing of the T cell response in the TIL towards the remaining TSMA. Additionally, we have obtained T3 tumor cells with both TSMA reverted to wild type and are currently testing whether these “fixed” T3 tumor cells are still subject to anti-PD-1 or anti-CTLA-4 and whether SLP vaccines comprised of subdominant TSMA still present in fixed T3 tumor cells can protect mice against tumor growth. This system will allow us to address the many questions surrounding poorly understood phenomenon of immunodominance and how this may influence patient-specific personalized cancer immunotherapy directed against TSMA. Citation Format: Matthew M. Gubin, Jeffrey P. Ward, Takuro Noguchi, Xiuli Zhang, Cora Arthur, Willem-Jan Krebber, Gwenn E. Mulder, Cornelis J.M. Melief, William E. Gillanders, Maxim Artyomov, Elaine R. Mardis, Robert D. Schreiber. Tumor-specific mutant antigens in cancer immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A001.

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.

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

Purpose Although situational risk factors for incisional hernia formation are known, the methods used to determine who would be most susceptible to develop one are unreliable. We hypothesized that patients with recurrent incisional hernias may possess unique gene expression profiles. Methods Skin and intact fascia were collected from 15 normal control (NC) patients with no hernia history and 18 patients presenting for recurrent incisional hernia (RH) repair. Microarray analysis was performed using whole genome microarray chips on NC (n = 8) and RH (n = 9). These samples were further investigated using a pathway-specific PCR array containing fibrosis-related genes. Results Microarray data revealed distinct differences in the gene expression profiles between RH and NC patients. One hundred and sixty-seven genes in the skin and 7 genes in the fascia were differentially expressed, including 8 directly involved in collagen synthesis. In particular, GREMLIN1, or bone morphogenetic protein antagonist 1, was under expressed in skin (fold = 0.49, p

Background The discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs). Methods The ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE) of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established. Results We used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, CD9 and CALM2 mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis. Conclusion This is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular environment.

Breast cancer is one of the most prevalent cancers worldwide. Distance metastasis is responsible for patient mortality. Therefore, understanding the mechanisms underlying tumor pathogenesis and metastasis is crucial for the development of novel therapies as well as preventive strategies for those who are prone to breast cancer. In contrast to transcriptional gene regulation, posttranscriptional control mechanisms of gene expression are poorly understood. Yet, many metastasis genes are regulated by RNA binding proteins (RBPs) at the levels of mRNA stability and translation. The paraneoplastic antigen, HuR, is an RBP shown to regulate multiple genes that are significantly related to breast cancer metastasis by stabilizing target mRNAs and facilitating translation into proteins. Using novel techniques developed in our lab of RNA immunoprecipitation applied to microarrays (RIP-Chip) we identified novel discrete HuR-associated mRNAs in triple negative breast cancer and estrogen receptor positive breast cancer. Many of these HuR-associated mRNA transcripts were metastasis related. We investigated the role of HuR in an aggressive triple-negative breast cancer metastatic cell line, LM2. The LM2 cells were retrovirally transduced with triple fusion reporter construct encoding thymidine kinase1, GFP and firefly luciferase to obtain in vivo and in vitro tracking capabilities. The cells were further transfected with plasmid containing HA-HuR or empty vector control to demonstrate the function of HuR in LM2. HuR over-expressing clones showed greater metastatic capability by in vitro matrigel invasion assay. Furthermore, athymic mice that were intravenously injected with HuR over-expressing LM2 cells had greater (335-fold more) tumor metastasis to the lungs than empty vector control injected group as measured by IVIS imaging. Mice injected with HuR over-expressing LM2 cells were more moribund and had greater mortality as compared with mice injected with empty vector control cells. These results suggest that HuR may play a role in breast cancer metastasis by stabilizing various pro-metastatic genes. The implications of this work are twofold. First, HuR RIP-Chip can be used to identify novel cancer relevant genes and second, interference with HuR function may ameliorate distant metastasis in breast cancer, potentially providing new therapeutic approaches for treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A27.

Breast cancer is the second most common cancer among women worldwide. It is estimated that approximately one million women are diagnosed with breast cancer annually. Furthermore, more than 410,000 women will die each year from the disease primarily due to tumor metastasis. Currently, even the most effective treatments often result in recurrence and metastasis, in part due to genetic factors. Therefore, understanding the mechanism of breast cancer related gene regulation is crucial for the development of novel treatments and preventive strategies to those who are predisposed to developing breast cancer. Previously, we have shown that the RNA-binding protein HuR posttranscriptionally regulates various proto-oncogenes by stabilizing their mRNAs and facilitating their translation into proteins. Additionally, HuR upregulation and cytoplasmic localization has been associated with invasive cancer progression and poor prognosis. HuR has been described to control genes in multiple areas of the acquired capabilities model of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established. HuR regulates genes involved in angiogenesis, cell growth and cell cycle regulation including VEGFα, TSP1, HIF1α, CDKN1A(p21) and β-catenin. In this study, we investigated the role of HuR in an aggressive triple-negative (estrogen receptor ER-, human epidermal growth factor receptor 2 (HER2)- and progesterone receptor (PR)-) breast cancer cell subline LM-2. The LM-2 cells were derived from triple-negative breast cancer cells MDA-MB-231 which were isolated from two rounds of in vivo selection of the cancer cells which metastasized to the lungs. The LM-2 cells were retrovirally transduced with triple-fusion protein reporter construct encoding thymidine kinase1, green fluorescent protein (GFP) and firefly luciferase in order to obtain nuclear imaging, fluorescent and bioluminescent properties for in vivo and in vitro tracking. LM2 cells were further transfected with a plasmid containing HA-HuR or empty vector control to investigate the function of HuR in LM2 cells. Two clones of HuR overexpressing LM2 cells were shown to grow faster in vitro compared to the empty vector control. Also, HuR overexpression significantly facilitates tumor invasion in vitro by matrigel invasion assay. Further analysis of HuR overexpressing LM2 cells using in vivo imaging system (IVIS) will reveal the role of HuR in breast cancer growth and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3084. doi:10.1158/1538-7445.AM2011-3084

Due to the poor correlation between steady state mRNA levels and protein products, traditional microarray analysis may miss many genes which are regulated primarily at the level of mRNA stability and translation. Posttranscriptional gene regulation mediated by microRNAs and RNA binding proteins (RBPs) is being recognized as an important form of gene regulation. The elav (embryonic lethal abnormal vision) family of RBPs, are paraneoplastic antigens, over-expressed in a variety of malignancies, including breast cancer. Antibodies against elav family members are believed to be cancer-protective. The elav family binds to the AU-rich elements (AREs) found in the 3’ untranslated regions (UTRs) of many early-response genes, including proto-oncogenes and cell cycle regulators. HuR, the ubiquitously expressed family member, has been described to play a role in cancer progression by stabilizing and translationally up regulating expression of its target mRNAs. Elevated levels of cytoplasmic HuR directly correlate with increased invasiveness of malignancy and poor prognosis for many cancers, including those of the breast. HuR has been described to positively control the expression of multiple genes in the acquired capabilities model, such as VEGF and HIF1α. Hence, it has been suggested that HuR may serve as a tumor maintenance gene which allows for cancers to proliferate. Therefore, it is of interest to discover in vivo HuR targets, as these genes may play vital roles in transformed cells. We have developed methods called RNA immunoprecipitations applied to microarrays, RIP-Chip. We used RIP-Chip to identify distinct subsets of HuR associated mRNAs in MDA-MB-231 and MCF-7 breast cancer cell lines and validated several novel targets. To further investigate the role of HuR in triple negative breast cancer, we over expressed HuR in MDA-MB-231 cells, which results in accelerated growth and alterations in cell cycle kinetics. Surprisingly, when employed in orthotopic mouse models of cancer, HuR over expression significantly inhibited growth of triple negative tumors by 90%. Putative mechanisms appear to be anti-angiogenic, as HuR over expression increases anti-angiogenic factors, but surprisingly, also down regulates pro-angiogenic factors such as VEGF. These results are highly significant because they implicate HuR as a master regulator of angiogenesis in triple negative breast cancer tumor formation and suggest potentially novel treatment methods for this aggressive form of breast cancer. 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 3271.

Traditional microarray analysis has uncovered a variety of genes involved in diseases such as asthma. However, it may potentially miss important genes due to the discordance between steady state mRNA levels and protein products. Posttranscriptional events conferred by RNA binding proteins (RBPs) contribute to this phenomenon. We have developed novel techniques known as RIP-on-Chip (microchip) which allow for the immunoprecipitation and identification of in vivo mRNA targets bound to RBPs. We study the RBP HuR, which regulates many early response genes including cytokines and chemokines. Using RIP-on-Chip, HuR has been demonstrated to associate and regulate many asthma relevant genes including recently identified genes IL-4 and IL-13. We have extended this assay to solid organ tissues. Employing the standard ovalbumin model of allergic airway inflammation, coupled to genome-wide arrays (47,000 genes) we have identified in vivo HuR targets in lungs of naïve, alum only or ova challenged mice. Remarkably, there are only 12 HuR targets (2 fold or greater) up regulated in ova and alum immunized over alum alone mice. Some are known HuR target genes such as TLR4 and IL-13; others are potentially novel asthma genes. Furthermore, we have created a transgenic mouse which over expresses HuR in CD4+ T cells. In the allergic airway inflammation model these mice have an increase in lung lymphocyte infiltration when compared to wild-type littermates, further implicating coordinate posttranscriptional gene regulation and HuR in allergic airway inflammation.