Myeloid Cell Function Research Articles

Arginase 1-Based Immune Modulatory Vaccines Induce Anticancer Immunity and Synergize with Anti-PD-1 Checkpoint Blockade.

Expression of the L-arginine catabolizing enzyme arginase 1 (ARG1) is a central immunosuppressive mechanism mediated by tumor-educated myeloid cells. Increased activity of ARG1 promotes the formation of an immunosuppressive microenvironment and leads to a more aggressive phenotype in many cancers. Intrinsic T-cell immunity against ARG1-derived epitopes in the peripheral blood of cancer patients and healthy subjects has previously been demonstrated. To evaluate the antitumor efficacy of ARG1-derived peptide vaccines as a monotherapy and as a combinational therapy with checkpoint blockade, different in vivo syngeneic mouse tumor models were utilized. To evaluate the antitumor effects, flow cytometry analysis and IHC were performed on tumors, and ELISPOT assays were performed to characterize immune responses. We show that ARG1-targeting therapeutic vaccines were able to activate endogenous antitumor immunity in several in vivo syngeneic mouse tumor models and to modulate the cell composition of the tumor microenvironment without causing any associated side effects or systemic toxicity. ARG1-targeting vaccines in combination with anti-PD-1 also resulted in increased T-cell infiltration, decreased ARG1 expression, reduced suppressive function of tumor-educated myeloid cells, and a shift in the M1/M2 ratio of tumor-infiltrating macrophages. These results indicated that the induced shift toward a more proinflammatory microenvironment by ARG1-targeting immunotherapy favors effective tumor control when combined with anti-PD-1 checkpoint blockade. Our data illustrate the ability of ARG1-based immune modulatory vaccination to elicit antigen-specific immunosurveillance and imply the feasibility of this novel immunotherapeutic approach for clinical translation.

Colony‐stimulating factor‐1 receptor blockade attenuates inflammation in inflamed gingival tissue explants

Colony-stimulating factor-1 receptor (CSF-1R) regulates myeloid cell function and mediates osteoclastogenesis. CSF-1R blockade has been suggested as a potential therapeutic target to halt inflammation and bone resorption; however, the expression and function of CSF-1R in human gingiva is yet unknown. Gingival tissue was collected from 22 non-periodontitis controls and 31 periodontitis (PD) patients. CSF-1R expression in gingival tissue was assessed with q-PCR, western blot, and immunohistochemistry (IHC). Cell surface expression of CSF-1R was analyzed by flow cytometry. The effects of CSF-1R inhibition on the production of inflammatory mediators by inflamed gingival tissue explants and peripheral blood mononuclear cells (PBMCs) were assessed with a bead-based multiplex array and ELISA. CSF-1R protein expression was increased in gingival tissue from PD patients compared with controls as assessed with western blot (1.5-fold increase) and IHC (4.5-fold increase). Similar proportions of HLA-DR+ CD64+ cells and comparable CSF-1R expression in this cell population were found in gingival tissue from PD patients and controls. In peripheral blood monocytes, CSF-1R was predominantly expressed by non-classical and intermediate monocytes. Targeting CSF-1R in gingival tissue explants attenuated the production of MMP-1, MMP-2, MMP-12, and MMP-13. The blocking in PBMCs attenuated the production of IL-8 and MMP-9. These results indicate that CSF-1R is elevated in PD, and its inhibition attenuates inflammatory mediators in the inflamed gingival tissue and circulating myeloid cells. Together these findings suggest that CSF-1R might be involved in regulating inflammatory processes in PD, and a potential therapeutic target to reduce the harmful inflammation.

Critical Role of Zinc Transporter (ZIP8) in Myeloid Innate Immune Cell Function and the Host Response against Bacterial Pneumonia.

Zinc (Zn) is required for proper immune function and host defense. Zn homeostasis is tightly regulated by Zn transporters that coordinate biological processes through Zn mobilization. Zn deficiency is associated with increased susceptibility to bacterial infections, including Streptococcus pneumoniae, the most commonly identified cause of community-acquired pneumonia. Myeloid cells, including macrophages and dendritic cells (DCs), are at the front line of host defense against invading bacterial pathogens in the lung and play a critical role early on in shaping the immune response. Expression of the Zn transporter ZIP8 is rapidly induced following bacterial infection and regulates myeloid cell function in a Zn-dependent manner. To what extent ZIP8 is instrumental in myeloid cell function requires further study. Using a novel, myeloid-specific, Zip8 knockout model, we identified vital roles of ZIP8 in macrophage and DC function upon pneumococcal infection. Administration of S. pneumoniae into the lung resulted in increased inflammation, morbidity, and mortality in Zip8 knockout mice compared with wild-type counterparts. This was associated with increased numbers of myeloid cells, cytokine production, and cell death. In vitro analysis of macrophage and DC function revealed deficits in phagocytosis and increased cytokine production upon bacterial stimulation that was, in part, due to increased NF-κB signaling. Strikingly, alteration of myeloid cell function resulted in an imbalance of Th17/Th2 responses, which is potentially detrimental to host defense. These results (for the first time, to our knowledge) reveal a vital ZIP8- and Zn-mediated axis that alters the lung myeloid cell landscape and the host response against pneumococcus.

1030TiP An open-label multicenter phase Ib study of AN0025, a selective inhibitor of the prostaglandin E2 receptor-type-4 receptor, in combination with pembrolizumab in patients with advanced solid tumors

AN0025 is a selective inhibitor of the prostaglandin E2 type-4 receptor, inhibiting PGE2-mediated differentiation and suppressive function of monocytic myeloid cells, which contribute to the immunosuppressive tumor microenvironment. These cells are associated with a poorer prognosis in many cancers. A previous Ph 1 study with AN0025 was conducted to assess safety, MTD and RP2D. AN0025 was well tolerated in 30 patients with advanced cancer at 125, 250, 500, 750 mg QD with no DLTs. The MTD was not reached.

Interleukin-21 in Viral Infections.

Interleukin (IL)-21 is a cytokine that affects the differentiation and function of lymphoid and myeloid cells and regulates both innate and adaptive immune responses. In addition to regulating the immune response to tumor and viral infections, IL-21 also has a profound effect on the development of autoimmune and inflammatory diseases. IL-21 is produced mainly from CD4+ T cells—in particular, follicular helper T (Tfh) cells—which have a great influence on the regulation of antibody production. It is also an important cytokine for the activation of CD8+ T cells, and its role in recovering the function of CD8+ T cells exhausted by chronic microbial infections and cancer has been clarified. Thus, IL-21 plays an extremely important role in viral infections, especially chronic viral infections. In this review, I will introduce the findings to date on how IL-21 is involved in some typical viral infections and the potential of treating viral diseases with IL-21.

Development of immunosuppressive myeloid cells to induce tolerance in solid organ and hematopoietic cell transplant recipients

AbstractReplacement of failed organs followed by safe withdrawal of immunosuppressive drugs has long been the goal of organ transplantation. We studied changes in the balance of T cells and myeloid cells in the blood of HLA-matched and -mismatched patients given living donor kidney transplants followed by total lymphoid irradiation, anti-thymocyte globulin conditioning, and donor hematopoietic cell transplant to induce mixed chimerism and immune tolerance. The clinical trials were based on a conditioning regimen used to establish mixed chimerism and tolerance in mice. In preclinical murine studies, there was a profound depletion of T cells and an increase in immunosuppressive polymorphonuclear (pmn) myeloid-derived suppressor cells (MDSCs) in the spleen and blood following transplant. Selective depletion of pmn MDSCs in mice abrogated mixed chimerism and tolerance. In our clinical trials, patients given an analogous tolerance conditioning regimen developed similar changes, including profound depletion of T cells and a marked increase in MDSCs in blood posttransplant. Posttransplant pmn MDSCs transiently increased expression of lectin-type oxidized LDL receptor-1, a marker of immunosuppression, and production of the T-cell inhibitor arginase-1. These posttransplant pmn MDSCs suppressed the activation, proliferation, and inflammatory cytokine secretion of autologous T-cell receptor microbead-stimulated pretransplant T cells when cocultured in vitro. In conclusion, we elucidated changes in receptors and function of immunosuppressive myeloid cells in patients enrolled in the tolerance protocol that were nearly identical to those of MDSCs required for tolerance in mice. These trials were registered at www.clinicaltrials.gov as #NCT00319657 and #NCT01165762.

Aging influences the cardiac macrophage phenotype and function during steady state and during inflammation.

Aging‐mediated immune dysregulation affects the normal cardiac immune cell phenotypes and functions, resulting in cardiac distress. During cardiac inflammation, immune activation is critical for mounting the regenerative responses to maintain normal heart function. We investigated the impact of aging on myeloid cell phenotype and function during cardiac inflammation induced by a sub‐lethal dose of LPS. Our data show that hearts of old mice contain more myeloid cells than the hearts of young mice. However, while the number of monocytic‐derived suppressor cells did not differ between young and old mice, monocytic‐derived suppressor cells from old mice were less able to suppress T‐cell proliferation. Since cardiac resident macrophages (CRMs) are important for immune surveillance, clearance of dead cells, and tissue repair, we focused our studies on CRMs phenotype and function during steady state and LPS treatment. In the steady state, we observed significantly more MHC‐IIlow and MHC‐IIhigh CRMs in the hearts of old mice; however, these populations were decreased in both young and aged mice upon LPS treatment and the decrease in CRM populations correlated with defects in cardiac electrical activity. Notably, mice treated with a liver X receptor (LXR) agonist showed an increase in MerTK expression in CRMs of both young and old mice, which resulted in the reversal of cardiac electrical dysfunction caused by lipopolysaccharide (LPS). We conclude that aging alters the phenotype of CRMs, which contributes to the dysregulation of cardiac electrical dysfunction during infection in aged mice.

Myeloid DJ-1 deficiency protects acetaminophen-induced acute liver injury through decreasing inflammatory response.

Background: DJ-1 (also known as PARK7), a noted protein implicated in modulating ROS production and immune response, has been observed to play critical roles in the pathogenesis of many forms of liver disease through multiple mechanisms. However, its role and specific mechanism in acetaminophen (APAP) -induced liver injury have not been explored.Results: In this present study, by employing an acute liver injury induced by APAP overdose mouse model, we demonstrated that DJ-1 knockout (DJ-1−/−) mice showed reduced liver injury and lower mortality. In accordance with these changes, there were also alleviating inflammatory responses in both the serum and the liver of the DJ-1−/− mice compared to those of the wild-type (WT) mice. Functional experiments showed that APAP metabolism did not affected by DJ-1 deficiency. In addition, to investigate DJ-1 modulates which kind of cell types during APAP-overdose-induced acute liver injury, hepatocyte-specific DJ-1-knockout (Alb-DJ-1−/−) and myeloid-specific DJ-1-knockout (Lysm-DJ-1−/−) mice were generated. Interestingly, hepatic deletion of DJ-1 did not protect APAP-overdose induced hepatotoxicity and inflammation, whereas Lysm-DJ-1−/− mice showed similar protective effects as DJ-1−/− mice which suggest that the protective effects of deletion of DJ-1 was through modulating myeloid cell function. Consistently, there were alleviated pro-inflammatory cells infiltration and reduced reactive oxygen species (ROS) production in the liver of Lysm-DJ-1−/− mice relative to control mice.Conclusion: our findings clearly defined that deletion of DJ-1 protects APAP-induced acute liver injury through decreasing inflammatory response, and suggest DJ-1 as a potential therapeutic and/or prophylactic target of APAP-induced acute liver injury.

Effect of HSP90AB1 and CC domain interaction on Bcr-Abl protein cytoplasm localization and function in chronic myeloid leukemia cells

BackgroundThe fusion oncoprotein Bcr-Abl is mostly located in the cytoplasm, which causes chronic myeloid leukemia (CML). After moving into the nucleus, the fusion protein can induce apoptosis of CML cells. The coiled-coil domain (CC domain) of Bcr-Abl protein plays a central role in the subcellular localization. However, how CC domain affects subcellular localization of Bcr-Abl remains unclear.MethodsHerein, the key proteins interacting with the Bcr-Abl CC domain were screened by immunoprecipitation binding mass spectrometry. The specific site of Bcr-Abl CC domain binding to target protein was predicted by Deep Viewer. Immunoprecipitation assay was used to confirmed the specific sites of protein binding. IF and western blot were used to observe the subcellular localization of target protein. Western blot was used to examine the protein changes. CCK-8, clonal formation test and FCM cycle detection were used to observe the effect of inhibitor on the proliferation ability of CML cells. FCM apoptosis detection was used to observe the level of cells apoptosis.ResultsHSP90AB1 interacts with Bcr-Abl CC domain via N-terminal domain (NTD), preventing the transport of Bcr-Abl protein to the nucleus and maintaining the activation of Bcr-Abl tyrosine kinase. The nucleus-entrapped Bcr-Abl markedly inhibits the proliferation and induces apoptosis of CML cells by activating p73 and repressing the expression of cytoplasmic oncogenic signaling pathways mediated by Bcr-Abl. Moreover, the combination of 17AAG (Tanespimycin) with Leptomycin B (LMB) considerably decreased the proliferation of CML cells.ConclusionOur study provides evidence that it is feasible to transport Bcr-Abl into the nucleus as an alternative strategy for the treatment of CML, and targeting the NTD of HSP90AB1 to inhibit the interaction with Bcr-Abl is more accurate for the development and application of HSP90 inhibitor in the treatment of CML and other Bcr-Abl-addicted malignancies.F7yTRqYkfkXw_k-2eiVFmgVideo abstract

Abstract LB071: Tuning the tumor myeloid microenvironment (TME) by targeting TREM2+ tumor-associated macrophages to overcome resistance to immune checkpoint inhibitors

Abstract 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.

Abstract 1721: TAC-001, a toll-like receptor 9 (TLR9) agonist antibody conjugate targeting B cells, promotes anti-tumor immunity and favorable safety profile following systemic administration in preclinical models

Abstract Novel therapies capable of engaging both innate and adaptive immune responses may engender durable anti-tumor immunity. Activation of toll-like receptor 9 (TLR9) by unmethylated oligonucleotides promotes innate inflammatory responses and the induction of adaptive immunity. Recently, several CpG-based TLR9 agonists have demonstrated clinical benefit in melanoma patients when administered intra-tumorally. Immune cells expressing TLR9 include B cells, plasmacytoid dendritic cells and myeloid cells. In B cells, TLR9 activation in human and mouse drives B cell proliferation and differentiation. Recent studies have described the presence and potential role of B cells in the response to checkpoint inhibitors in multiple solid tumor types. Specifically, enrichment of memory B cells, plasma cells and tertiary lymphoid organs have been observed in the tumors of responders to checkpoint inhibitors. We developed a novel Toll-like Receptor Agonist Antibody Conjugate (TRAAC) platform to deliver a potent and differentiated TLR9 agonist (T-CpG) for targeted immune activation via systemic administration. TAC-001 is comprised of T-CpG conjugated to an anti-CD22 antibody, a receptor restricted to B cells. It is designed to systemically deliver T-CpG to B cells by binding CD22 receptor on B cells, leading to TLR9 signaling, B cell activation and a cascade of immune reactions. We show that the innate and adaptive immune responses triggered by TAC-001 leads to potent anti-tumor activity. In vitro stimulation of B cells with TAC-001 leads to increase expression of co-stimulatory molecules, cross-presentation leading to T cell proliferation and induction of immunoglobulin secretion. In mice, systemic delivery of a murine reactive anti-CD22-CpG conjugate shows durable single agent activity in PD-1 refractory and PD-1 resistant tumors. Mice with eradicated tumors acquire memory immune response upon tumor rechallenge. The cellular response in mice treated with TAC-001 exhibit B cell differentiation with significant decrease in IL-10+Bregs, along with increased in T cell effector function and modulation in suppressive myeloid cells within the tumor microenvironment. Systemic intravenous administration of TAC-001 in monkeys showed favorable tolerability, PK and PD profiles. These results demonstrate the unique properties of TAC-001 which integrates TLR9 activation and B cells to employ both innate and adaptive immune responses to promote anti-tumor activity. These data support the development of TAC-001 for a broad range of solid tumor malignancies. Citation Format: Tracy C. Kuo, Ons Harrabi, Amy Chen, Emma R. Sangalang, Laura Doyle, Danielle Fontaine, Min Li, Bora Han, Jaume Pons, Janet Sim, Hong I. Wan. TAC-001, a toll-like receptor 9 (TLR9) agonist antibody conjugate targeting B cells, promotes anti-tumor immunity and favorable safety profile following systemic administration in preclinical models [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 1721.

Abstract 1486: Inhibition of host PI3K-gamma modulates anti-tumor immunity in poorly immunogenic HNSCC

Abstract HNSCC is the 6th most common cancer, with around 650,000 new cases yearly. Gain of function mutations in the PI3K pathway are common in HNSCC, and inhibition of the PI3K p110γ subunit has shown promise in HNSCC treatment. However, given that PI3K p110γ plays an important role in myeloid and lymphoid immune cell function, it is essential to understand how PI3K p110γ inhibition affects the anti-tumor immune response independent of tumor cells. To elucidate PI3K p110γ function in HNSCC, we employed an orthotopic mouse model using poorly immunogenic and aggressive cell line MOC2 on Pik3cg-/- mice. We observed that wild-type and Pik3cg-/- mice displayed similar rates of HNSCC tumor growth and metastasis after 20 days post tumor injection. T cell infiltration and intrinsic T cell responses to MOC2 oral tumors were comparable between wild-type and Pik3cg-/- mice. Interestingly, the immune response of tumor-bearing Pik3cg-/- mice was marked by increased anti-tumor cytotoxic molecules (IFN-γ, IL-17, PD-1) by T cells, and immune checkpoint marker (PD-L1) expression by myeloid cells compared to tumor bearing wild type mice. Taken together, our findings demonstrate that inhibition of PI3K p110γ modulates tumor associated immune cells, which likely potentiates HNSCC treatment when used in combination with selective PD-L1 inhibitors. Citation Format: Kelvin Anderson, Nathan Ryan, Anastasia Alkhimovitch, Arham Siddiqui, Steve Oghumu. Inhibition of host PI3K-gamma modulates anti-tumor immunity in poorly immunogenic HNSCC [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 1486.

Homeostatic functions of monocytes and interstitial lung macrophages are regulated via collagen domain-binding receptor LAIR1

Myeloid cells encounter stromal cells and their matrix determinants on a continual basis during their residence in any given organ. Here, we examined the impact of the collagen receptor LAIR1 on myeloid cell homeostasis and function. LAIR1 was highly expressed in the myeloid lineage and enriched in non-classical monocytes. Proteomic definition of the LAIR1 interactome identified stromal factor Colec12 as a high-affinity LAIR1 ligand. Proteomic profiling of LAIR1 signaling triggered by Collagen1 and Colec12 highlighted pathways associated with survival, proliferation, and differentiation. Lair1-/- mice had reduced frequencies of Ly6C- monocytes, which were associated with altered proliferation and apoptosis of non-classical monocytes from bone marrow and altered heterogeneity of interstitial macrophages in lung. Myeloid-specific LAIR1 deficiency promoted metastatic growth in a melanoma model and LAIR1 expression associated with improved clinical outcomes in human metastatic melanoma. Thus, monocytes and macrophages rely on LAIR1 sensing of stromal determinants for fitness and function, with relevance in homeostasis and disease.

Abstract 1636: CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy limiting tumor cells phagocytosis and tumor antigen cross-presentation

Abstract Myeloid cells represent one of the most abundant immune cell types in solid tumors that impede myeloid phagocytosis by triggering “don't eat me” and “don't find me” signals. Recent literature demonstrates that C-type lectin receptors (CLRs) are powerful pattern recognition receptors shaping immune cell-mediated tissue damage by positively or negatively regulating myeloid cell functions and hence tumor elimination or evasion. We previously reported that the orphan CLR CLEC-1 expressed by dendritic cells (DCs) and macrophages (MPs) is enhanced by TGFβ and tempers downstream T cells responses. Furthermore, we observed that CLEC-1 is highly expressed by myeloid cells purified from the human tumor microenvironment, in particular, tumor-associated MPs. We evaluated whether CLEC-1 could also be a receptor for DAMPs and influences phagocytosis. We found that CLEC-1 fusion protein binds specifically to secondary necrotic healthy or tumor cells induced by chemotherapy, radiation (UV, X-ray), or culture stress conditions suggesting that ligands of CLEC-1 are generated upon stress and programmed cell death. Importantly, further to promising results in KO CLEC-1 mice, we observed in vivo that CLEC-1 deficient mice, in contrast to wild-type littermates, eradicate MC38 colorectal tumors in combination with cytotoxic and immunogenic chemotherapy. Importantly, disruption of CLEC-1 signaling by Fc-CLEC-1 fusion protein also promotes tumor eradication. We then generated and identified different anti-human CLEC-1 antagonist monoclonal antibodies (mAbs) with the capacity to block CLEC-1/CLEC-1L interaction. We developed innovative antagonist CLEC-1 mAbs which, in contrast to non-antagonist CLEC-1 control mAb, increase the phagocytosis of CLEC-1L-positive human tumor cells by human TGFβ-polarized DCs or MPs. Indeed, TGFβ-polarized DCs phagocytose more efficiently a NSCLC cell line (A549) as well as Rituximab (anti-CD20 mAb)-opsonized Burkitt lymphoma cells (Raji) when CLEC-1 is antagonized by Abs. Similarly, macrophages significantly more efficiently engulfed human tumors in the presence of CLEC-1 antagonist Abs, in particular when tumor cells were opsonized such as Rituximab-opsonized Raji cells, Cetuximab opsonized colon carcinoma cells (DLD-1; EGFR+) or Trastuzumab opsonized mammary carcinoma cells (SK-BR-3; Her2+). Importantly, we observed both in vitro and in vivo that DCs from Clec1a deficient mice cross-present more efficiently dead cell-associated antigens to CD8+ T cells (OT-1). We generated hCLEC-1 knock-in mice and in vivo preclinical evaluation of CLEC-1 blocking mAbs is ongoing. Altogether, these data illustrate that CLEC-1 broadly notably inhibits tumor-cell phagocytosis and synergized with tumor-targeted cytotoxic monoclonal antibodies in both solid and hematological tumors, and hampers DC antigen cross-presentation. Citation Format: Vanessa Gauttier, Sabrina Pengam, Marion Drouin, Javier Saenz, Bérangère Evrard, Kevin Biteau, Caroline Mary, Géraldine Teppaz, Ariane Desselle, Virginie Thépénier, Emmanuelle Wilhelm, Elise Chiffoleau, Nicolas Poirier. CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy limiting tumor cells phagocytosis and tumor antigen cross-presentation [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 1636.

PD-1 Mediates Lineage Fate Determination and Function of Myeloid Cells in Response to Tumor-Mediated “Emergency” Myelopoiesis

PD-1 is an inhibitory receptor for which blocking agents have achieved significant success as anti-cancer therapeutics. The mechanism(s) of how PD-1 compromises anti-tumor function and how such effect is reversed by PD-1 blockade remain poorly understood. The rapid change in hematopoietic output that occurs in response to immunologic stress is known as “emergency” myelopoiesis. This process is co-opted by tumors to evade the immune system by inducing potent immunosuppression. Cancer-driven granulo-monocytopoiesis modifies the composition of the hematopoietic output by stimulating the expansion of tumor promoting immature myeloid populations, defined as myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). Monocytic (M-) and granulocytic (PMN-) MDSCs and TAMs differentiate from granulocyte/macrophage progenitors (GMP) in the bone marrow. We analyzed the myeloid compartment of tumor-bearing mice to determine the expression and function of PD-1 in myeloid cells during cancer-driven emergency myelopoiesis. Using three different mouse tumor models (MC38, B16 and MC17), we determined that PD-1 and its natural ligand PD-L1, were expressed at high levels in all subsets of myeloid cells isolated from the spleen of tumor-bearing mice, including CD11b+Ly6C+ M-MDSC, CD11b+Ly6G+ PMN-MDSC, CD11b+F/480+ macrophages and CD11b+HLA-DR+ dendritic cells (DC). This was in contrast to the significantly lower or undetectable levels of PD-1 expressed in these cellular counterparts isolated from non-tumor bearing mice. High expression of PD-1 and PD-L1 was also identified in these myeloid subsets isolated from the tumor site and at sites of secondary myelopoiesis in the gut. Because these myeloid cell populations differentiate from GMP upon cancer-mediated emergency myelopoiesis, we examined bone marrow myeloid progenitors in tumor bearing and control, non-tumor bearing mice. In non-tumor bearing mice, PD-1 was detected in Lin-/Sca+/Kit+ (LSK) myeloid progenitors, whereas PD-L1 was expressed in Lin-/Sca-/Kit+ (LK), common myeloid progenitors (CMP) and GMP. In tumor-bearing mice, both PD-1 and PD-L1 expression was significantly higher in all myeloid progenitors compared to tumor-free control mice. To determine whether PD-1-mediated signaling in myeloid cells might affect the magnitude and composition of the myeloid output that is altered by cancer-mediated emergency response, we used PD-1 deficient mice. PD-1 ablation, which decreased tumor growth and metastasis, stimulated the differentiation of hematopoietic progenitor cells and resulted in increase of Ly6Chi monocytes and monocyte-derived DC (mo-DC). Abrogation of PD-1 signaling enhanced the maturation of MDSCs by inducing the lineage commitment transcription factors IRF8, IRF4 and RORC1. IRF8 and IRF4 promote monocyte/macrophage and hinder granulocyte differentiation, whereas RORC1 is required for expansion of Ly6Chi monocytes and resolution of inflammation. To examine whether the effects of PD-1 on cancer-mediated emergency myelopoiesis and on myeloid lineage commitment and differentiation were mediated by myeloid cell-intrinsic mechanisms, we generated mice with conditional targeting of the PD-1 gene in the myeloid compartment by crossing PD-1fl/fl with LysM-Cre mice. Myeloid-specific ablation of PD-1 signaling in PD-1fl/fl/LysM-Cre mice impaired tumor growth to a similar extent as global PD-1 ablation in PD1-KO mice and induced a myeloid cell fate switch from MDSCs to effector-like myeloid cells that could be identified in spleen and tumor. Further, abrogation of PD-1 signaling in the myeloid compartment prevented the expansion of tumor-stress associated GMP in PD-1fl/fl/LysM-Cre mice and resulted in increased differentiation of committed monocyte progenitors (CMOP) and monocyte progenitors (MP) in the bone marrow. Moreover, PD-1 deletion significantly diminished NO production by M-MDSC and reversed their ability to suppress antigen-specific T cell responses. Our results reveal a previously unidentified role of the PD-1:PD-L1 pathway in the differentiation of lineage-committed myeloid progenitors during tumor-mediated emergency myelopoiesis to promote MDSCs and suppress effector monocytes and mo-DCs. Switch of myeloid progenitor fate commitment might be a key mechanism by which PD-1 blockade mediates its anti-tumor function. DisclosuresNo relevant conflicts of interest to declare.

Diabetes With Multiple Autoimmune and Inflammatory Conditions Linked to an Activating SKAP2 Mutation.

Multiple genome-wide association studies have identified a strong genetic linkage between the SKAP2 locus and type 1 diabetes (T1D), but how this leads to disease remains obscure. Here, we characterized the functional consequence of a novel SKAP2 coding mutation in a patient with T1D to gain further insight into how this impacts immune tolerance. We identified a 24-year-old individual with T1D and other autoimmune and inflammatory conditions. The proband and first-degree relatives were recruited for whole-exome sequencing. Functional studies of the protein variant were performed using a cell line and primary myeloid immune cells collected from family members. Sequencing identified a de novo SKAP2 variant (c.457G>A, p.Gly153Arg) in the proband. Assays using monocyte-derived macrophages from the individual revealed enhanced activity of integrin pathways and a migratory phenotype in the absence of chemokine stimulation, consistent with SKAP2 p.Gly153Arg being constitutively active. The p.Gly153Arg variant, located in the well-conserved lipid-binding loop, induced similar phenotypes when expressed in a human macrophage cell line. SKAP2 p.Gly153Arg is a gain-of-function, pathogenic mutation that disrupts myeloid immune cell function, likely resulting in a break in immune tolerance and T1D. SKAP2 plays a key role in myeloid cell activation and migration. This particular mutation in a patient with T1D and multiple autoimmune conditions implicates a role for activating SKAP2 variants in autoimmune T1D.

Ovarian cancer modulates the immunosuppressive function of CD11b+Gr1+ myeloid cells via glutamine metabolism

ObjectiveImmature CD11b + Gr1+ myeloid cells that acquire immunosuppressive capability, also known as myeloid-derived suppressor cells (MDSCs), are a heterogeneous population of cells that regulate immune responses. Our study's objective was to elucidate the role of ovarian cancer microenvironment in regulating the immunosuppressive function of CD11b+Gr1+ myeloid cells. MethodsAll studies were performed using the intraperitoneal ID8 syngeneic epithelial ovarian cancer mouse model. Myeloid cell depletion and immunotherapy were carried out using anti-Gr1 mAb, gemcitabine treatments, and/or anti-PD1 mAb. The treatment effect was assessed by a survival curve, in situ luciferase-guided imaging, and histopathologic evaluation. Adoptive transfer assays were carried out between congenic CD45.2 and CD45.1 mice. Immune surface and intracellular markers were assessed by flow cytometry. ELISA, western blot, and RT-PCR techniques were employed to assess the protein and RNA expression of various markers. Bone marrow-derived myeloid cells were used for ex-vivo studies. ResultsThe depletion of Gr1+ immunosuppressive myeloid cells alone and in combination with anti-PD1 immunotherapy inhibited ovarian cancer growth. In addition to the adoptive transfer studies, these findings validate the role of immunosuppressive CD11b+Gr1+ myeloid cells in promoting ovarian cancer. Mechanistic investigations showed that ID8 tumor cells and their microenvironments produced recruitment and regulatory factors for immunosuppressive CD11b+Gr1+ myeloid cells. CD11b+Gr1+ myeloid cells primed by ID8 tumors showed increased immunosuppressive marker expression and acquired an energetic metabolic phenotype promoted primarily by increased oxidative phosphorylation fueled by glutamine. Inhibiting the glutamine metabolic pathway reduced the increased oxidative phosphorylation and decreased immunosuppressive markers’ expression and function. Dihydrolipoamide succinyl transferase (DLST), a subunit of α-KGDC in the TCA cycle, was found to be the most significantly elevated gene in tumor-primed myeloid cells. The inhibition of DLST reduced oxidative phosphorylation, immunosuppressive marker expression and function in myeloid cells. ConclusionOur study shows that the ovarian cancer microenvironment can regulate the metabolism and function of immunosuppressive CD11b + Gr1+ myeloid cells and modulate its immune microenvironment. Targeting glutamine metabolism via DLST in immunosuppressive myeloid cells decreased their activity, leading to a reduction in the immunosuppressive tumor microenvironment. Thus, targeting glutamine metabolism has the potential to enhance the success of immunotherapy in ovarian cancer.

1126-P: Activating SKAP2 Mutation Linked to Diabetes with Multiple Autoimmune and Inflammatory Conditions

Recent insights into human autoimmunity and immune tolerance have come from studies of rare monogenic disorders. For example, type 1 diabetes (T1D) and thyroid autoimmunity are frequent features in patients with mutations in AIRE and FOXP3. Dissecting the consequences of these mutations has improved our understanding of the role of central T cell tolerance and T regulatory cells in controlling these organ-specific autoimmune diseases. Over the last decade, rapid advances in genetic sequencing have now allowed for a deeper exploration and discovery of rare variants that are linked to disease with techniques like whole exome sequencing. Using this approach in both kindreds and outlier patients with de novo germline mutations, there has been expanding discovery of mutations in other pathways with a link to organ-specific autoimmunity that include STAT3, STAT1, CTLA4, and LRBA. Here, we utilized a similar approach on a single patient with T1D and other autoimmune and inflammatory conditions. We identified a de novo heterozygous c.457G>A/p.Gly153Arg mutation in the SKAP2 gene. SKAP2 encodes a critical mediator of integrin signaling pathways that regulate myeloid immune cell function. Monocyte-derived macrophages from the individual displayed enhanced activity of integrin pathways and a migratory phenotype in the absence of chemokine stimulation, consistent with SKAP2 p.Gly153Arg being constitutively active. The p.Gly153Arg variant, located in the well-conserved lipid-binding loop, induced similar phenotypes when expressed in a human macrophage cell line. We propose that SKAP2 p.Gly153Arg is a gain-of-function, pathogenic mutation that disrupts myeloid immune cell function, resulting in a break in immune tolerance and T1D. This particular mutation in a patient with T1D and multiple autoimmunity implicates a role for activating SKAP2 variants in autoimmune T1D. Disclosure C. Chamberlain: None. N. Rutsch: None. W. Dixon: None. L. R. Letourneau-freiberg: None. L. H. Philipson: Advisory Panel; Self; Nevro Corp., Research Support; Self; Provention Bio, Inc. M. German: Stock/Shareholder; Self; Viacyte, Inc. M. S. Anderson: Consultant; Self; Provention Bio, Inc., VielaBio, Stock/Shareholder; Self; Medtronic, Merck & Co., Inc. C. A. Lowell: None. Funding The Leona M. and Harry B. Helmsley Charitable Trust (G-2018PG-T1D018); Larry L. Hillblom Foundation (2014-D-004-NET); University of California San Francisco Parnassus Flow Core (RRID:SCR_018206); National Institutes of Health (P30 DK063720, S101S10OD021822-01); German Research Foundation (ZA428/11-1, ZA428/18-1, INST211/604-1)

The IL-10GFP (VeRT-X) mouse strain is not suitable for the detection of IL-10 production by granulocytes during lung inflammation.

The clear and unequivocal identification of immune effector functions is essential to understand immune responses. The cytokine IL-10 is a critical immune regulator and was shown, for example, to limit pathology during various lung diseases. However, the clear identification of IL-10-producing cells is challenging and, therefore, reporter mouse lines were developed to facilitate their detection. Several such reporter lines utilize GFP, including the IL-10GFP (VeRT-X) reporter strain studied here. In line with previous reports, we found that this IL-10GFP line faithfully reports on the IL-10 production of lymphoid cells. However, we show that the IL-10GFP reporter is not suitable to analyse IL-10 production of myeloid cells during inflammation. During inflammation, the autofluorescence of myeloid cells increased to an extent that entirely masked the IL-10-specific GFP-signal. Our data illustrate a general and important technical caveat using GFP-reporter lines for the analysis of myeloid cells and suggest that previous reports on effector functions of myeloid cells using such GFP-based reporters might require re-evaluation.

Immunosuppressive Effects of Myeloid-Derived Suppressor Cells in Cancer and Immunotherapy.

The primary function of myeloid cells is to protect the host from infections. However, during cancer progression or states of chronic inflammation, these cells develop into myeloid-derived suppressor cells (MDSCs) that play a prominent role in suppressing anti-tumor immunity. Overcoming the suppressive effects of MDSCs is a major hurdle in cancer immunotherapy. Therefore, understanding the mechanisms by which MDSCs promote tumor growth is essential for improving current immunotherapies and developing new ones. This review explores mechanisms by which MDSCs suppress T-cell immunity and how this impacts the efficacy of commonly used immunotherapies.