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Supplementary Figure from Holistic Characterization of Tumor Monocyte-to-Macrophage Differentiation Integrates Distinct Immune Phenotypes in Kidney Cancer

The tumor immune microenvironment (TIME) is commonly infiltrated by diverse collections of myeloid cells. Yet, the complexity of myeloid-cell identity and plasticity has challenged efforts to define bona fide populations and determine their connections to T-cell function and their relationship to patient outcome. Here, we have leveraged single-cell RNA-sequencing analysis of several mouse and human tumors and found that monocyte–macrophage diversity is characterized by a combination of conserved lineage states as well as transcriptional programs accessed along the differentiation trajectory. We also found in mouse models that tumor monocyte-to-macrophage progression was profoundly tied to regulatory T cell (Treg) abundance. In human kidney cancer, heterogeneity in macrophage accumulation and myeloid composition corresponded to variance in, not only Treg density, but also the quality of infiltrating CD8+ T cells. In this way, holistic analysis of monocyte-to-macrophage differentiation creates a framework for critically different immune states.

Supplementary Table from Holistic Characterization of Tumor Monocyte-to-Macrophage Differentiation Integrates Distinct Immune Phenotypes in Kidney Cancer

The tumor immune microenvironment (TIME) is commonly infiltrated by diverse collections of myeloid cells. Yet, the complexity of myeloid-cell identity and plasticity has challenged efforts to define bona fide populations and determine their connections to T-cell function and their relationship to patient outcome. Here, we have leveraged single-cell RNA-sequencing analysis of several mouse and human tumors and found that monocyte–macrophage diversity is characterized by a combination of conserved lineage states as well as transcriptional programs accessed along the differentiation trajectory. We also found in mouse models that tumor monocyte-to-macrophage progression was profoundly tied to regulatory T cell (Treg) abundance. In human kidney cancer, heterogeneity in macrophage accumulation and myeloid composition corresponded to variance in, not only Treg density, but also the quality of infiltrating CD8+ T cells. In this way, holistic analysis of monocyte-to-macrophage differentiation creates a framework for critically different immune states.

The tumor immune microenvironment (TIME) is commonly infiltrated by diverse collections of myeloid cells. Yet, the complexity of myeloid cell identity and plasticity has challenged efforts to define bona fide populations and determine their connections to T cell function and their relation to patient outcome. Here we leverage single-cell RNA-sequencing (scRNA-seq) analysis of several mouse and human tumors and find that monocyte-macrophage diversity is characterized by a combination of conserved lineage states as well as transcriptional programs accessed along the differentiation trajectory. Using mouse models, we also find that tumor monocyte-to-macrophage progression is profoundly tied to regulatory T cell (Treg) abundance. Importantly, in human kidney cancer, heterogeneity in macrophage accumulation and myeloid composition corresponded to variance in, not only Treg density, but also the quality of infiltrating CD8+ T cells. In this way, holistic analysis of monocyte-to-macrophage differentiation creates a framework for critically different immune states in kidney tumors.

BackgroundThe tumor microenvironment (TME) often contains high levels of suppressive myeloid cells that contribute to innate checkpoint inhibitor (CPI) resistance. Pionyr’s Myeloid Tuning approach involves altering the composition and/or the function of myeloid cells in the TME. Myeloid reprogramming alters the function of immunosuppressive myeloid cells to acquire an immunostimulatory phenotype. Triggering receptor expressed on myeloid cells-1 (TREM1) is an immunoglobulin superfamily cell surface receptor enriched on tumor-associated myeloid cells. To investigate the potential of TREM1 modulation as an anti-cancer therapeutic strategy, Pionyr developed an afucosylated humanized anti-TREM1 monoclonal antibody termed PY159 and characterized it in pre-clinical and translational biomarker assays described below.MethodsPY159 responses in human whole blood and dissociated primary tumor cells in vitro were evaluated by flow cytometry and measurement of secreted cytokines and chemokines by MSD. TREM1 expression in human tumors was validated by scRNAseq, flow cytometry, and immunohistochemistry (IHC). In vivo efficacy and pharmacodynamic studies of a surrogate anti-mouse TREM1 antibody, termed PY159m, were evaluated using syngeneic mouse tumor models, either as a single-agent or in combination with anti-PD-1. To select tumor types and patients most likely to benefit from PY159 therapy, Pionyr developed qualitative and quantitative monoplex and multiplex IHC assays that detect TREM1 expression levels in human tumor tissues.ResultsPY159 treatment in vitro induced signaling, upregulated monocyte activation markers, and induced proinflammatory cytokines. In human tumors, TREM1 was detected on tumor-associated neutrophils, tumor-associated macrophages, and monocytic myeloid-derived suppressive cells. The surrogate PY159m anti-mouse TREM1 antibody exhibited anti-tumor efficacy in several syngeneic mouse tumor models, both as single-agent and in combination with anti-PD-1. Screening for TREM1 expression in tumor tissues demonstrated that TREM1+ tumor associated myeloid cells were highly enriched in the TME of multiple solid tumor indications. The monoplex and multiplex IHC assays offered insights into the localization of TREM1+ myeloid cells and their spatial relationship with other immune cells present in the TME to determine what immune composition will be more favorable for response to PY159 therapy.ConclusionsCollectively, the available nonclinical data support PY159 as a TREM1 agonist that reprograms myeloid cells and unleashes anti-tumor immunity. PY159 safety and efficacy are currently being evaluated in first-in-human clinical trial (NCT04682431) involving select advanced solid tumors patients resistant and refractory to standard of care therapies alone and in combination with a CPI. The TREM1 IHC assay is successfully being used on FFPE archival tumor tissues from enrolled patients to determine TREM1 expression levels.

Background Tumor-associated myeloid cells can impede productive anti-tumor immunity. One strategy for targeting immunosuppression is myeloid reprogramming, which drives immunosuppressive myeloid cells to acquire an immunostimulatory phenotype. Triggering receptor expressed on myeloid cells-1 (TREM1) is an immunoglobulin superfamily cell surface receptor expressed on neutrophils and subsets of monocytes and tissue macrophages. TREM1 associates with DAP12 adaptor and induces proinflammatory signaling, amplifies innate immune responses, and is implicated in the development of acute and chronic inflammatory diseases. TREM1 is also enriched in tumors, specifically on tumor-associated myeloid cells. To investigate the potential of TREM1 modulation as an anti-cancer therapeutic strategy, we developed PY159, an afucosylated humanized anti-TREM1 monoclonal antibody, and characterized it in the pre-clinical assays described below. Materials and Methods An FcγR binding ELISA and a Jurkat TREM1/DAP12 NFAT-luciferase reporter cell line were used to assess PY159 binding to human FcγRs and TREM1 signaling, respectively. PY159 responses in human whole blood in vitro were evaluated by flow cytometry, transcriptional analysis of sorted leukocyte subsets, and measurement of secreted cytokines/chemokines by MSD. A Transwell system was used to evaluate PY159 effects on neutrophil chemotaxis. TREM1 expression in human tumors was validated by scRNAseq, immunohistochemistry, and flow cytometry. Anti-tumor efficacy of a surrogate anti-mouse TREM1 antibody, PY159m, was evaluated using syngeneic mouse tumor models, either as a single agent or in combination with anti-PD-1. Results PY159 afucosylation increased its binding affinity for FcγR and its ability to activate TREM1/DAP12 signaling. In human blood assays, PY159 treatment did not induce depletion of TREM1-expressing cells. Rather, it upregulated monocyte activation markers, promoted neutrophil chemotaxis, and induced proinflammatory cytokines and chemokines, which was dependent on PY159 afucosylation. In human tumors, TREM1 was detected on tumor-associated neutrophils, tumor-associated macrophages, and monocytic myeloid-derived suppressive cells. PY159 induced proinflammatory cytokines and chemokines in dissociated human tumors in vitro, demonstrating that PY159 can reprogram tumor-associated myeloid cells. A surrogate anti-mouse TREM1 antibody, PY159m, exhibited anti-tumor efficacy in several syngeneic mouse tumor models, both as single-agent and in combination with anti-PD-1. Conclusions These results show that PY159 is a TREM1 agonist that reprograms myeloid cells and unleashes anti-tumor immunity. PY159 safety and efficacy are currently being evaluated in first-in-human clinical trial (NCT04682431) involving patients resistant and refractory to standard of care therapies. Disclosure Information V. Juric: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. E. Mayes: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. M. Binnewies: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. P. Canaday: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. T. Lee: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. S. Dash: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. J.L. Pollack: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. J. Rudolph: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. V. Huang: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. X. Du: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. N. Jahchan: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. A.J. Ramoth: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. S. Mankikar: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. M. Norng: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. C. Santamaria: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. K.P. Baker: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. L. Liang: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc.

Summary: Converting checkpoint inhibitor (CPI)-resistant individuals to being responsive requires identifying suppressive mechanisms. We identify TREM2+ tumor-associated macrophages (TAMs) as being correlated with exhausted CD8+ tumor-infiltrating lymphocytes (TILs) in mouse syngeneic tumor models and human solid tumors of multiple histological types. Fc domain-enhanced anti-TREM2 monoclonal antibody (mAb) therapy promotes anti-tumor immunity by elimination and modulation of TAM populations, which leads to enhanced CD8+ TIL infiltration and effector function. TREM2+ TAMs are most enriched in individuals with ovarian cancer, where TREM2 expression corresponds to disease grade accompanied by worse recurrence-free survival. In an aggressive orthotopic ovarian cancer model, anti-TREM2 mAb therapy drives potent anti-tumor immunity. These results highlight TREM2 as a highly attractive target for immunotherapy modulation in individuals who are refractory to CPI therapy and likely have a TAM-rich tumor microenvironment.

Converting checkpoint inhibitor (CPI) resistant patients to responsive requires identifying novel suppressive mechanisms. We identified TREM2 + tumor associated macrophages (TAMs) as being correlated with exhausted CD8 + tumor infiltrating lymphocytes (TILs) in both mouse syngeneic tumor models and human solid tumors of multiple histological types. Fc-domain enhanced anti-TREM2 monoclonal antibody (mAb) therapy promoted anti-tumor immunity by elimination and modulation of TAM populations which led to enhanced CD8 + TIL infiltration and effector function. TREM2 + TAMs were most enriched in human ovarian cancer patients, where TREM2 expression corresponded to disease grade accompanied by worse recurrence-free survival. In an aggressive orthotopic ovarian cancer model, anti-TREM2 mAb therapy drove potent anti-tumor immunity. Together, these results highlight TREM2 as a highly attractive target for immunotherapeutic modulation in patients who are refractory to CPI therapy and likely have a TAM-rich tumor microenvironment.

The thymus is responsible for generating a diverse yet self-tolerant pool of T cells1. Although the thymic medulla consists mostly of developing and mature AIRE+ epithelial cells, recent evidence has suggested that there is far greater heterogeneity among medullary thymic epithelial cells than was previously thought2. Here we describe in detail an epithelial subset that is remarkably similar to peripheral tuft cells that are found at mucosal barriers3. Similar to the periphery, thymic tuft cells express the canonical taste transduction pathway and IL-25. However, they are unique in their spatial association with cornified aggregates, ability to present antigens and expression of a broad diversity of taste receptors. Some thymic tuft cells pass through an Aire-expressing stage and depend on a known AIRE-binding partner, HIPK2, for their development. Notably, the taste chemosensory protein TRPM5 is required for their thymic function through which they support the development and polarization of thymic invariant natural killer T cells and act to establish a medullary microenvironment that is enriched in the type 2 cytokine, IL-4. These findings indicate that there is a compartmentalized medullary environment in which differentiation of a minor and highly specialized epithelial subset has a non-redundant role in shaping thymic function.

Myeloid cells present in the tumor microenvironment can exist in immunosuppressive states that impede productive anti-tumor immunity. One strategy for targeting these immunosuppressive mechanisms is reprogramming of myeloid cells from immunosuppressive to immunostimulatory, resulting in the removal of the immune inhibition and unleashing of anti-tumor immunity. Triggering receptor expressed on myeloid cells-1 (TREM1) is an immunoglobulin superfamily cell surface receptor expressed primarily on neutrophils and subsets of monocytes and tissue macrophages. TREM1 signals through the association with DAP12 adaptor protein and mediates proinflammatory signaling, amplifies the host immune response to microbial pathogens, and has been implicated in the development of acute and chronic inflammatory diseases. TREM1 is also enriched in tumors, specifically on tumor-associated myeloid cells. To investigate the potential of TREM1 modulation as an anti-cancer therapeutic strategy, we developed PY159, an afucosylated humanized anti-TREM1 monoclonal antibody. We found that PY159 does not deplete TREM1-expressing cells, but rather acts as a TREM1 agonist. In vitro human blood assays showed that PY159 treatment upregulated activation markers on monocytes and stimulated neutrophil chemotaxis, as assayed by flow cytometry, transcriptional analysis, and in vitro migration assays. Furthermore, PY159 induced a selective set of proinflammatory cytokines and chemokines, which was dependent on PY159 afucosylation. We validated TREM1 expression in human tumors by single-cell RNAseq, immunohistochemistry, and flow cytometry, and found that it is expressed on myeloid populations, including tumor-associated neutrophils (TAN), tumor-associated macrophages (TAM), and monocytic myeloid-derived suppressive cells (mMDSC). We showed that PY159 can also induce proinflammatory cytokines and chemokines in dissociated human tumors in vitro, demonstrating that PY159 can reprogram tumor-associated myeloid cells. Finally, in vivo treatment of mice with a surrogate anti-mouse TREM1 antibody, PY159m, promoted anti-tumor efficacy in several syngeneic mouse tumor models, both as single-agent and in combination with checkpoint inhibitors, such as anti-PD-1 antibody. Together, these results demonstrate that therapeutic targeting of TREM1 with a TREM1 agonist antibody, PY159, promotes myeloid cell reprogramming and anti-tumor immunity. PY159 safety and tolerability have been demonstrated in non-human primates, and safety and efficacy of PY159 are currently being evaluated in first-in-human clinical trial (NCT04682431) including solid tumors that are resistant and refractory to standard of care therapies. Citation Format: Erin Mayes, Vladi Juric, Mikhail Binnewies, Pamela Canaday, Tian Lee, Subhadra Dash, Joshua L. Pollack, Joshua Rudolph, Vicky Huang, Xiaoyan Du, Nadine Jahchan, Asa J. Ramoth, Shilpa Mankikar, Manith Norng, Carlos Santamaria, Kevin P. Baker, Linda Liang. Therapeutic targeting of TREM1 with PY159 promotes myeloid cell reprogramming and unleashes anti-tumor immunity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P104.

RationaleQuantification of type 2 inflammation provided a molecular basis for heterogeneity in asthma. Non-type 2 pathways that contribute to asthma pathogenesis are not well understood.ObjectivesTo identify dysregulated pathways beyond type 2 inflammation.MethodsWe applied RNA sequencing to airway epithelial brushings obtained from subjects with stable mild asthma not on corticosteroids (n = 19) and healthy control subjects (n = 16). Sequencing reads were mapped to human and viral genomes. In the same cohort, and in a separate group with severe asthma (n = 301), we profiled blood gene expression with microarrays.Measurements and main resultsIn airway brushings from mild asthma on inhaled corticosteroids, RNA sequencing yielded 1,379 differentially expressed genes (false discovery rate

Differentiation of proinflammatory CD4(+) conventional T cells (T(conv)) are critical for productive antitumor responses yet their elicitation remains poorly understood. We exhaustively characterized myeloid cells in tumor draining lymph nodes (tdLN) of mice and identified two subsets of conventional type-2 dendritic cells (cDC2) that traffic from tumor to tdLN and present tumor-derived antigens to CD4(+) T(conv), but then fail to support antitumor CD4(+) T(conv) differentiation. Regulatory T cell (T(reg)) depletion enhanced their capacity to elicit strong CD4(+) T(conv) responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice their abundance relative to T(reg) predicts protective ICOS(+) PD-1(lo) CD4(+) T(conv) phenotypes and survival. Further, in melanoma patients with low T(reg) abundance, intratumoral cDC2 density alone correlates with abundant CD4(+) T(conv) and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway which restrains cDC2, and whose reversal enhances CD4(+) T(conv) abundance and controls tumor growth.

The tumor microenvironment (TME) often contains high levels of suppressive myeloid cells that may contribute to innate checkpoint inhibitor (CPI) resistance. Pionyr's Myeloid Tuning approach involves altering the composition and/or the function of myeloid cells in the TME. To this end, therapeutic targeting of tumor-associated macrophages (TAMs) is a promising strategy to increase CPI response rates in solid tumor indications, as well as to overcome resistance to CPI therapies. Pionyr and others identified the transmembrane protein triggering receptor expressed on myeloid cells-2 (TREM2) as a highly enriched TAMs target. Furthermore, TREM2 mRNA expression negatively correlates with patient survival in a variety of tumor types, supporting the involvement of TAMs in tumor progression. Pionyr developed a lead anti-TREM2 monoclonal antibody (mAb), termed PY314, as well as a murinized version of PY314, termed PY314m. PY314m demonstrated significant anti-tumor activity either as single agent in CPI-sensitive syngeneic tumor models or in combination with anti-PD-1 in CPI-resistant syngeneic tumor models. Mechanistically, PY314m reduced the pro-tumorigenic MHC class II-low, M2-like TAMs, induced pro-inflammatory cytokine production, significant increased CD8+ T cell infiltration into the TME. These findings suggest that PY314 therapy could be used to overcome CPI resistance in humans. To select patients most likely to benefit from PY314 therapy, Pionyr developed a qualitative IHC assay that detects TREM2 expression levels in formalin-fixed, paraffin-embedded human tumor tissues. Screening for TREM2 expression in tumor tissues demonstrated that TREM2+ TAMs were present in multiple solid tumor indications and their number increased with disease grade in a selected set of indications. Ongoing efforts are aimed at better understanding localization of TREM2+ TAMs within the TME, and spatial relationship of the TREM2+ TAMs to other immune cells present in the TME. The TREM2 IHC assay will be used to test our hypothesis that patients with tumors with high level of TREM2+ TAMs are most likely to benefit from PY314 treatment. Citation Format: Nadine S. Jahchan, Mikhail Binnewies, Joshua L. Pollack, Ranna Mehta, Subhadra Dash, Christine Tun, Erick Lu, Xiaoyan Du, Kevin P. Baker, Len Reyno, Venkataraman Sriram. Tuning the tumor myeloid microenvironment (TME) by targeting TREM2+ tumor-associated macrophages to overcome resistance to immune checkpoint inhibitors [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 LB071.

Group 2 innate lymphoid cells (ILC2s) and CD4+ type 2 helper T cells (TH2 cells) are defined by their similar effector cytokines, which together mediate the features of allergic immunity. We found that tissue ILC2s and TH2 cells differentiated independently but shared overlapping effector function programs that were mediated by exposure to the tissue-derived cytokines interleukin 25 (IL-25), IL-33 and thymic stromal lymphopoietin (TSLP). Loss of these three tissue signals did not affect lymph node priming, but abrogated the terminal differentiation of effector TH2 cells and adaptive lung inflammation in a T cell-intrinsic manner. Our findings suggest a mechanism by which diverse perturbations can activate type 2 immunity and reveal a shared local-tissue-elicited checkpoint that can be exploited to control both innate and adaptive allergic inflammation.

SummaryInitiation of immune responses requires innate immune sensing, but immune detection of the helminths, protists, and allergens that stimulate type 2 immunity remains poorly understood. In the small intestine, type 2 immune responses are regulated by a tuft cell-ILC2 signaling circuit. Tuft cells express components of a canonical taste transduction pathway, including the membrane channel TRPM5, but the ligands and receptors that activate tuft cells in the small intestine are unknown. Here we identify succinate as the first ligand that activates intestinal tuft cells to initiate type 2 immune responses. Using mRNA-Seq on tuft cells from different tissues, we show that all tuft cells express the intracellular taste transduction pathway, but expression of upstream receptors is tissue-specific. In the small intestine, tuft cells express the succinate receptor SUCNR1. Remarkably, providing succinate in drinking water is sufficient to induce a multifaceted type 2 immune response in the murine small intestine, involving all known components of the tuft-ILC2 circuit. The helminthNippostrongylus brasiliensissecretes succinate as a metabolite, and sensing of both succinate andN. brasiliensisrequires tuft cells and TRPM5, suggesting a novel paradigm in which type 2 immunity monitors microbial metabolism. Manipulation of succinate sensing may have therapeutic benefit in numerous intestinal diseases.

Intratumoral stimulatory dendritic cells (SDCs) play an important role in stimulating cytotoxic T cells and driving immune responses against cancer. Understanding the mechanisms that regulate their abundance in the tumor microenvironment (TME) could unveil new therapeutic opportunities. We find that in human melanoma, SDC abundance is associated with intratumoral expression of the gene encoding the cytokine FLT3LG. FLT3LG is predominantly produced by lymphocytes, notably natural killer (NK) cells in mouse and human tumors. NK cells stably form conjugates with SDCs in the mouse TME, and genetic and cellular ablation of NK cells in mice demonstrates their importance in positively regulating SDC abundance in tumor through production of FLT3L. Although anti-PD-1 ‘checkpoint’ immunotherapy for cancer largely targets T cells, we find that NK cell frequency correlates with protective SDCs in human cancers, with patient responsiveness to anti-PD-1 immunotherapy, and with increased overall survival. Our studies reveal that innate immune SDCs and NK cells cluster together as an excellent prognostic tool for T cell–directed immunotherapy and that these innate cells are necessary for enhanced T cell tumor responses, suggesting this axis as a target for new therapies.

Functional characterization of noncoding sequences is crucial for understanding the human genome and learning how genetic variation contributes to disease. 3' untranslated regions (UTRs) are an important class of noncoding sequences, but their functions remain largely uncharacterized. We developed a method for massively parallel functional annotation of sequences from 3' UTRs (fast-UTR) and used this approach to measure the effects of a total of >450 kilobases of 3' UTR sequences from >2,000 human genes on steady-state mRNA abundance, mRNA stability and protein production. We found widespread regulatory effects on mRNA that were coupled to effects on mRNA stability and protein production. Furthermore, we discovered 87 novel cis-regulatory elements and measured the effects of genetic variation within known and novel 3' UTR motifs. This work shows how massively parallel approaches can improve the functional annotation of noncoding sequences, advance our understanding of cis-regulatory mechanisms and quantify the effects of human genetic variation.

The tumor microenvironment contains diverse types of myeloid cells, including tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs) and myeloid-derived suppressor cells (MDSCs). TAMs and TANs exhibit a spectrum of functional phenotypes ranging from immunosuppressive that promote tumor growth, to pro-inflammatory that unleash anti-tumor immunity. Therapies that shift the inhibitory myeloid cell phenotypes toward more pro-inflammatory function are expected to positively impact anti-tumor immune responses and convert checkpoint inhibitor (CPIs)-resistant tumors into CPI-sensitive tumors. TREM1 (triggering receptor expressed on myeloid cells-1) is a transmembrane protein enriched in myeloid cells, including intratumoral TAMs, TANs and MDSCs. In a number of tumor types, TREM1 expression negatively correlated with patient survival. Furthermore, a survey of the immune infiltrates in a variety of human solid tumor types by flow cytometry and gene expression analysis revealed a high frequency of intratumoral TREM1+ myeloid cells in all tested tumors. We developed anti-human and anti-mouse TREM1 monoclonal antibodies (mAbs), termed PY159 and PY159m, respectively, to target TREM1+ myeloid cells. These mAbs triggered signaling pathways downstream of TREM1 and induced a highly selective pro-inflammatory cytokine signature in ex vivo assays. Additionally, PY159 treatment increased the myeloid cell expression of HLA-DR and the costimulatory molecule CD40, indicating an enhanced potential for costimulation and immune activation. Consistent with ex vivo results, in vivo treatment with PY159m promoted both innate and adaptive immune pathways in syngeneic mouse tumors revealed by gene expression profiling. These findings suggest that anti-TREM1 therapy re-educates TREM1+ myeloid cells into pro-inflammatory cells. Furthermore, PY159m was sufficient to drive anti-tumor activity as a single agent in a number of syngeneic tumor models. Strikingly, PY159m also converted anti-PD-1 mAb-resistant tumors into treatment-sensitive tumors with combination therapy, demonstrating the utility of targeting TREM1+ myeloid cells as a combination approach to improve CPI therapy responses. Mice cured of their tumors by PY159m/PD-1 mAb combination therapy were resistant to tumor re-challenge, demonstrating that targeting TREM1+ myeloid cells also supports adaptive immunity and induces long-term immunological memory. Based on these preclinical findings, we are developing PY159 as a therapeutic agent for monotherapy and/or CPI combination therapy for solid tumors. Citation Format: Vladi Juric, Chris Chan, Erin Mayes, Manith Norng, Tiep Le, Subhadra Dash, Venkataraman Sriram, Erick Lu, Joshua L. Pollack, Mikhail Binnewies, Joanna Waszczuk, Xiaoyan Du, Shilpa Mankikar, Aritra Pal, Kevin P. Baker, Linda Liang. Targeting of TREM1+ myeloid cells to promote antitumor immunity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C105. doi:10.1158/1535-7163.TARG-19-C105

Tumor-associated macrophages (TAMs) are a major sources of innate checkpoint inhibitor (CPI) resistance, as they subvert anti-tumor immunity through immunosuppression and supporting tumor growth. In patients, high levels of TAMs predict poor prognosis across multiple solid tumor indications. Therefore, therapeutic targeting of TAMs by impacting their survival and/or modulating their suppressive function is a promising strategy to augment response rates in solid tumor indications, as well as overcome resistance to CPI therapies. We and others have identified the transmembrane protein triggering receptor expressed on myeloid cells-2 (TREM2) as a highly enriched TAM target. TREM2 is comprised of one IgV domain but lacks any known intracellular signaling motifs. However, TREM2 associates with TYRO protein tyrosine kinase-binding protein (TYROBP), which contains an intracellular immunoreceptor tyrosine-baased activation motif (ITAM), allowing for TREM2/TYROBP-mediated signaling to occur. Screening of human tumor tissue microarrays for TREM2 protein demonstrated that TREM2+ TAMs were present in multiple human solid tumors and correlated with disease severity in some indications. Furthermore, in a variety of tumor types, TREM2 mRNA expression negatively correlated with patient survival. To target TAMs, we developed an anti-TREM2 monoclonal antibody (mAb), termed PY314m, that demonstrated compelling anti-tumor activity either as single agent in CPI-sensitive syngeneic tumor models or in combination with anti-PD-1 in CPI-resistant mouse syngeneic tumor models. Mechanistically, PY314m reduced a subset of TAMs, namely the MHCIILow ArginaseHigh M2-like TAMs, expanded MHCIIHigh M1-like TAMs, and induced both innate and adaptive pro-inflammatory cytokine production. By depleting M2-like TAMs and inducing expansion of M1-like TAMs, PY314m treatment removes and modulates multiple immunosuppressive mechanisms. The combination treatment of PY314m and anti-PD-1 mAb produced long-term immunological memory as evidenced by the resistance to tumor rechallenge in mice cured of their tumors. Collectively these findings suggest that anti-TREM2 mAb therapy could be used to overcome CPI resistance in humans. To this end, we developed a humanized version of PY314m termed PY314, which was well-tolerated and showed good pharmacokinetic properties in pilot, repeat dose non-GLP studies in non-human primates. Our ongoing efforts are aimed at better defining CPI-resistant patient populations that will maximally benefit from PY314 therapy. Citation Format: Mikhail Binnewies, Marwan Abushawish, Tian Lee, Tiep Le, Joshua L. Pollack, Erick Lu, Amanda Chen, Ranna Mehta, Nadine Jahchan, Vicky Huang, Xiaoyan Du, Subhadra Dash, Manith Norng, Aritra Pal, Kevin P. Baker, Venkataraman Sriram. Therapeutic targeting of TREM2+ tumor-associated macrophages as a means of overcoming checkpoint inhibitor resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C104. doi:10.1158/1535-7163.TARG-19-C104

Messenger RNAs (mRNAs) contain prominent untranslated regions (UTRs) that are increasingly recognized to play roles in mRNA processing, transport, stability, and translation. 3′ UTRs are believed to harbor recognition sites for a diverse set of RNA-binding proteins that regulate gene expression as well as most active microRNA target sites. Although the roles of 3′ UTRs in the normal and diseased lung have not yet been studied extensively, available evidence suggests important roles for 3′ UTRs in lung development, inflammation, asthma, pulmonary fibrosis, and cancer. Systematic, genome-wide approaches are beginning to catalog functional elements within 3′ UTRs and identify the proteins and microRNAs that interact with these elements. Application of new data sets and experimental approaches should provide powerful insights into how 3′ UTR-mediated regulatory events contribute to disease and may inspire novel therapeutic approaches.

In the small intestine, type 2 responses are regulated by a signaling circuit that involves tuft cells and group 2 innate lymphoid cells (ILC2s). Here, we identified the microbial metabolite succinate as an activating ligand for small intestinal (SI) tuft cells. Sequencing analyses of tuft cells isolated from the small intestine, gall bladder, colon, thymus, and trachea revealed that expression of tuft cell chemosensory receptors is tissue specific. SI tuft cells expressed the succinate receptor (SUCNR1), and providing succinate in drinking water was sufficient to induce a multifaceted type 2 immune response via the tuft-ILC2 circuit. The helminth Nippostrongylus brasiliensis and a tritrichomonad protist both secreted succinate as a metabolite. Invivo sensing of the tritrichomonad required SUCNR1, whereas N.brasiliensis was SUCNR1 independent. These findings define a paradigm wherein tuft cells monitor microbial metabolites to initiate type 2 immunity and suggest the existence of othersensing pathways triggering the response to helminths.

The gene genealogies of two linked loci in three species are analyzed using a series of Markov chain models. We calculate the probability that the gene tree of one locus is concordant with the species tree, given that the gene tree of the other locus is concordant. We define a threshold value of the recombination rate, r*, to be the rate for which the difference between the conditional probability of concordance and its asymptotic value is reduced to 5% of the initial difference. We find that, although r* depends in a complicated way on the times of speciation and effective population sizes, it is always relatively small

The maintenance of immune tolerance requires the deletion of self-reactive T cells in the thymus. The expression of tissue-specific antigen genes (TSAs) by thymic epithelial cells is critical for this process and depends on the activity of the Autoimmune Regulator (Aire) protein, however, the molecular mechanism(s) Aire uses to target TSA gene loci are unknown. Here we identified two Aire-interacting proteins – activating transcription factor 7 interacting protein (ATF7ip) and methyl CpG binding protein 1 (MBD1) –that are required for Aire’s targeting of TSA geneloci. Moreover, Mbd1−/− mice developed pathological autoimmunity and had a defect in Aire-dependent thymic TSA gene expression underscoring the critical importance of Aire’s interaction with the ATF7ip-MBD1 protein complex in maintaining central tolerance.

The medial ganglionic eminence (MGE) is an embryonic forebrain structure that generates the majority of cortical interneurons. MGE transplantation into specific regions of the postnatal central nervous system modifies circuit function and improves deficits in mouse models of epilepsy, Parkinson's disease, pain, and phencyclidine-induced cognitive deficits. Herein, we describe approaches to generate MGE-like progenitor cells from mouse embryonic stem (ES) cells. Using a modified embryoid body method, we provided gene expression evidence that mouse ES-derived Lhx6(+) cells closely resemble immature interneurons generated from authentic MGE-derived Lhx6(+) cells. We hypothesized that enhancers that are active in the mouse MGE would be useful tools in detecting when ES cells differentiate into MGE cells. Here we demonstrate the utility of enhancer elements [422 (DlxI12b), Lhx6, 692, 1056, and 1538] as tools to mark MGE-like cells in ES cell differentiation experiments. We found that enhancers DlxI12b, 692, and 1538 are active in Lhx6-GFP(+) cells, while enhancer 1056 is active in Olig2(+) cells. These data demonstrate unique techniques to follow and purify MGE-like derivatives from ES cells, including GABAergic cortical interneurons and oligodendrocytes, for use in stem cell-based therapeutic assays and treatments.

BackgroundAsthma is characterized by increased airway narrowing in response to nonspecific stimuli. The disorder is influenced by both environmental and genetic factors. Exosomes are nanosized vesicles of endosomal origin released from inflammatory and epithelial cells that have been implicated in asthma. In this study we characterized the microRNA (miRNA) content of exosomes in healthy control subjects and patients with mild intermittent asthma both at unprovoked baseline and in response to environmental challenge.ObjectiveTo investigate alterations in bronchoalveolar lavage fluid (BALF) exosomal miRNA profiles due to asthma, and following subway air exposure.MethodsExosomes were isolated from BALF from healthy control subjects (n=10) and patients with mild intermittent asthma (n=10) after subway and control exposures. Exosomal RNA was analyzed by using microarrays containing probes for 894 human miRNAs, and selected findings were validated with quantitative RT-PCR. Results were analyzed by using multivariate modeling.ResultsThe presence of miRNAs was confirmed in exosomes from BALF of both asthmatic patients and healthy control subjects. Significant differences in BALF exosomal miRNA was detected for 24 miRNAs with a subset of 16 miRNAs, including members of the let-7 and miRNA-200 families, providing robust classification of patients with mild nonsymptomatic asthma from healthy subjects with 72% cross-validated predictive power (Q(2)=0.72). In contrast, subway exposure did not cause any significant alterations in miRNA profiles.ConclusionThese studies demonstrate substantial differences in exosomal miRNA profiles between healthy subjects and patients with unprovoked, mild, stable asthma. These changes might be important in the inflammatory response leading to bronchial hyperresponsiveness and asthma.

We consider gene trees in three species for which the species tree is known. We show that population subdivision in ancestral species can lead to asymmetry in the frequencies of the two gene trees not concordant with the species tree and, if subdivision is extreme, cause the one of the nonconcordant gene trees to be more probable than the concordant gene tree. Although published data for the human-chimp-gorilla clade and for three species of Drosophila show asymmetry consistent with our model, sequencing error could also account for observed patterns. We show that substantial levels of persistent ancestral subdivision are needed to account for the observed levels of asymmetry found in these two studies.

Summary Comparisons of genetic variation between humans and great apes are hampered by the fact that we still know little about the demographics and evolutionary history of the latter species [1–4]. In addition, characterizing ape genetic variation is important because they are threatened with extinction, and knowledge about genetic differentiation among groups may guide conservation efforts [5]. We sequenced multiple intergenic autosomal regions totaling 22,400 base pairs (bp) in ten individuals each from western, central, and eastern chimpanzee groups and in nine bonobos, and 16,000 bp in ten Bornean and six Sumatran orangutans. These regions are analyzed together with homologous information from three human populations and gorillas. We find that whereas orangutans have the highest diversity, western chimpanzees have the lowest, and that the demographic histories of most groups differ drastically. Special attention should therefore be paid to sampling strategies and the statistics chosen when comparing levels of variation within and among groups. Finally, we find that the extent of genetic differentiation among "subspecies" of chimpanzees and orangutans is comparable to that seen among human populations, calling the validity of the "subspecies" concept in apes into question.

We have analyzed partial DNA sequences of the mitochondrial cytochrome b gene from extant striped, brown, and spotted hyenas as well as from Pleistocene cave hyenas. Sequences of the Pleistocene cave hyenas from Eurasia and modern spotted hyenas from Africa are intermixed in phylogenetic analyses, questioning any taxonomic delineation between the two groups. Contrary to cave hyenas in Eurasia, spotted hyenas in Africa show a phylogeographic pattern with little geographical overlap between two mitochondrial DNA (mtDNA) clades, suggesting two Pleistocene refugia in the north and south of Africa. Our results, furthermore, suggest three waves of migration from Africa to Eurasia for spotted hyenas, around 3, 1, and 0.3 MYA. A recent emigration of striped hyenas from Africa to Eurasia took place less than 0.1 MYA, resulting in a dramatic expansion of the geographical range of striped hyenas. In striped hyenas and within the geographical range of mtDNA clades in spotted hyenas, we found identical sequences several thousand kilometers apart, indicating a high rate of migration during the Pleistocene as well as the Holocene. Both striped and brown hyenas show low amounts of genetic diversity, with the latter ones displaying just a single haplotype.

Despite remarkable progress in the study of ancient DNA, the amount of continuous sequence retrievable from Late Pleistocene organisms is still limited. Segments of about 1,000 base pairs have been reconstructed for mammoths and a few other species, but that's often not much to go on when it comes to studying inter-species relationships. Now a new PCR-based method that makes clever use of sequence overlaps and multiple amplifications has made it possible to produce long DNA sequences from minute amounts of bone material. Starting from just 200 mg of bone, this method has generated the complete mitochondrial genome sequence of a Pleistocene woolly mammoth preserved in permafrost, a total of over 16,000 base pairs. The sequence resolves a long-standing controversy over mammoth phylogeny, showing the woolly mammoth to be more closely related to the Asian elephant than to its African cousin. In studying the genomes of extinct species, two principal limitations are typically the small quantities of endogenous ancient DNA and its degraded condition1, even though products of up to 1,600 base pairs (bp) have been amplified in rare cases2. Using small overlapping polymerase chain reaction products, longer stretches of sequences or even whole mitochondrial genomes3,4 can be reconstructed, but this approach is limited by the number of amplifications that can be performed from rare samples. Thus, even from well-studied Pleistocene species such as mammoths, ground sloths and cave bears, no DNA sequences of more than about 1,000 bp have been reconstructed5,6,7. Here we report the complete mitochondrial genome sequence of the Pleistocene woolly mammoth Mammuthus primigenius. We used about 200 mg of bone and a new approach that allows the simultaneous retrieval of multiple sequences from small amounts of degraded DNA. Our phylogenetic analyses show that the mammoth was more closely related to the Asian than to the African elephant. However, the divergence of mammoth, African and Asian elephants occurred over a short time, corresponding to only about 7% of the total length of the phylogenetic tree for the three evolutionary lineages.

Thymic epithelial cells (TECs) support T cell development in the thymus. Cortical thymic epithelial cells (cTECs) facilitate positive selection of developing thymocytes whereas medullary thymic epithelial cells (mTECs) facilitate the deletion of self-reactive thymocytes in order to prevent autoimmunity. The mTEC compartment is highly dynamic with continuous maturation and turnover, but the genetic regulation of these processes remains poorly understood. MicroRNAs (miRNAs) are important regulators of TEC genetic programs since miRNA-deficient TECs are severely defective. However, the individual miRNAs important for TEC maintenance and function and their mechanisms of action remain unknown. Here, we demonstrate that miR-205 is highly and preferentially expressed in mTECs during both thymic ontogeny and in the postnatal thymus. This distinct expression is suggestive of functional importance for TEC biology. Genetic ablation of miR-205 in TECs, however, neither revealed a role for miR-205 in TEC function during homeostatic conditions nor during recovery from thymic stress conditions. Thus, despite its distinct expression, miR-205 on its own is largely dispensable for mTEC biology.

SummaryIt is well understood that antigen-presenting cells (APCs) within tumors typically do not maintain cytotoxic T cell (CTL) function, despite engaging them. Across multiple mouse tumor models and human tumor biopsies, we have delineated the intratumoral dendritic cell (DC) populations as distinct from macrophage populations. Within these, CD103+ DCs are extremely sparse and yet remarkably capable CTL stimulators. These are uniquely dependent on IRF8, Zbtb46, and Batf3 transcription factors and are generated by GM-CSF and FLT3L cytokines. Regressing tumors have higher proportions of these cells, T-cell-dependent immune clearance relies on them, and abundance of their transcripts in human tumors correlates with clinical outcome. This cell type presents opportunities for prognostic and therapeutic approaches across multiple cancer types.

Corrigendum: The transcriptional regulator Aire coopts the repressive ATF7ip-MBD1 complex for the induction of immunotolerance

We have sequenced the complete mitochondrial genome of the extinct American mastodon (Mammut americanum) from an Alaskan fossil that is between 50,000 and 130,000 y old, extending the age range of genomic analyses by almost a complete glacial cycle. The sequence we obtained is substantially different from previously reported partial mastodon mitochondrial DNA sequences. By comparing those partial sequences to other proboscidean sequences, we conclude that we have obtained the first sequence of mastodon DNA ever reported. Using the sequence of the mastodon, which diverged 24–28 million years ago (mya) from the Elephantidae lineage, as an outgroup, we infer that the ancestors of African elephants diverged from the lineage leading to mammoths and Asian elephants approximately 7.6 mya and that mammoths and Asian elephants diverged approximately 6.7 mya. We also conclude that the nuclear genomes of the African savannah and forest elephants diverged approximately 4.0 mya, supporting the view that these two groups represent different species. Finally, we found the mitochondrial mutation rate of proboscideans to be roughly half of the rate in primates during at least the last 24 million years., Author Summary We determined the complete mitochondrial genome of the mastodon (Mammut americanum), a recently extinct relative of the living elephants that diverged about 26 million years ago. We obtained the sequence from a tooth dated to 50,000–130,000 years ago, increasing the specimen age for which such palaeogenomic analyses have been done by almost a complete glacial cycle. Using this sequence, together with mitochondrial genome sequences from two African elephants, two Asian elephants, and two woolly mammoths (all of which have been previously sequenced), we show that mammoths are more closely related to Asian than to African elephants. Moreover, we used a calibration point lying outside the Elephantidae radiation (elephants and mammoths), which enabled us to estimate accurately the time of divergence of African elephants from Asian elephants and mammoths (about 7.6 million years ago) and the time of divergence between mammoths and Asian elephants (about 6.7 million years ago). These dates are strikingly similar to the divergence time for humans, chimpanzees, and gorillas, and raise the possibility that the speciation of mammoth and elephants and of humans and African great apes had a common cause. Despite the similarity in divergence times, the substitution rate within primates is more than twice as high as in proboscideans., The first complete mitochondrial DNA genome for the mastodon extends the age range for genomic analyses by almost a complete glacial cycle and resolves the relationships among African and Asian elephants and mammoths.

In the absence of interaction with genetically distinct populations, changes in frequencies of distinct mitochondrial DNA (mtDNA) sequences (haplotypes) within a population are caused by mutations and genetic drift, both of which are comparatively slow processes. By contrast, interactions between populations may lead to rapid changes in haplotype frequencies [1–3], for instance through extinction and recolonialisation. So far, the only case of a direct replacement of mtDNA sequences within a continuous population has been described in historical mouse populations [2].