Targeting LxCxE cleft pocket of retinoblastoma protein in M2 macrophages inhibits ovarian cancer progression.

Ovarian cancer remains a major health threat with limited treatment options available. It is characterized by immunosuppressive tumor microenvironment (TME) maintained by tumor- associated macrophages (TAMs) hindering anti-tumor responses and immunotherapy efficacy. Here we show that targeting retinoblastoma protein (Rb) by disruption of its LxCxE cleft pocket, causes cell death in TAMs by induction of ER stress, p53 and mitochondria-related cell death pathways. A reduction of pro-tumor Rb high M2-type macrophages from TME in vivo enhanced T cell infiltration and inhibited cancer progression. We demonstrate an increased Rb expression in TAMs in women with ovarian cancer is associated with poorer prognosis. Ex vivo, we show analogous cell death induction by therapeutic Rb targeting in TAMs in post-surgery ascites from ovarian cancer patients. Overall, our data elucidates therapeutic targeting of the Rb LxCxE cleft pocket as a novel promising approach for ovarian cancer treatment through depletion of TAMs and re-shaping TME immune landscape. Statement of significance: Currently, targeting immunosuppressive myeloid cells in ovarian cancer microenvironment is the first priority need to enable successful immunotherapy, but no effective solutions are clinically available. We show that targeting LxCxE cleft pocket of Retinoblastoma protein unexpectedly induces preferential cell death in M2 tumor-associated macrophages. Depletion of immunosuppressive M2 tumor-associated macrophages reshapes tumor microenvironment, enhances anti-tumor T cell responses, and inhibits ovarian cancer. Thus, we identify a novel paradoxical function of Retinoblastoma protein in regulating macrophage viability as well as a promising target to enhance immunotherapy efficacy in ovarian cancer.

Peroxynitrite in the tumor microenvironment changes the profile of antigens allowing escape from cancer immunotherapy.

Cancer immunotherapy often depends on recognition of peptide epitopes by cytotoxic T lymphocytes (CTLs). The tumor microenvironment (TME) is enriched for peroxynitrite (PNT), a potent oxidant produced by infiltrating myeloid cells and some tumor cells. We demonstrate that PNT alters the profile of MHC class I bound peptides presented on tumor cells. Only CTLs specific for PNT-resistant peptides have a strong antitumor effect in vivo, whereas CTLs specific for PNT-sensitive peptides are not effective. Therapeutic targeting of PNT in mice reduces resistance of tumor cells to CTLs. Melanoma patients with low PNT activity in their tumors demonstrate a better clinical response to immunotherapy than patients with high PNT activity. Our data suggest that intratumoral PNT activity should be considered for the design of neoantigen-based therapy and also may be an important immunotherapeutic target.

Distinct mechanisms govern populations of myeloid-derived suppressor cells in chronic viral infection and cancer.

Myeloid-derived suppressor cells (MDSCs) are major negative regulators of immune responses in cancer and chronic infections. It remains unclear if regulation of MDSC activity in different conditions is controlled by similar mechanisms. We compared MDSCs in mice with cancer and lymphocytic choriomeningitis virus (LCMV) infection. Chronic LCMV infection caused the development of monocytic MDSCs (M-MDSCs) but did not induce polymorphonuclear MDSCs (PMN-MDSCs). In contrast, both MDSC populations were present in cancer models. An acquisition of immune-suppressive activity by PMN-MDSCs in cancer was controlled by IRE1α and ATF6 pathways of the endoplasmic reticulum (ER) stress response. Abrogation of PMN-MDSC activity by blockade of the ER stress response resulted in an increase in tumor-specific immune response and reduced tumor progression. In contrast, the ER stress response was dispensable for suppressive activity of M-MDSCs in cancer and LCMV infection. Acquisition of immune-suppressive activity by M-MDSCs in spleens was mediated by IFN-γ signaling. However, it was dispensable for suppressive activity of M-MDSCs in tumor tissues. Suppressive activity of M-MDSCs in tumors was retained due to the effect of IL-6 present at high concentrations in the tumor site. These results demonstrate disease- and population-specific mechanisms of MDSC accumulation and the need for targeting different pathways to achieve inactivation of these cells.

Analysis of classical neutrophils and polymorphonuclear myeloid-derived suppressor cells in cancer patients and tumor-bearing mice.

In this study, using single-cell RNA-seq, cell mass spectrometry, flow cytometry, and functional analysis, we characterized the heterogeneity of polymorphonuclear neutrophils (PMNs) in cancer. We describe three populations of PMNs in tumor-bearing mice: classical PMNs, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), and activated PMN-MDSCs with potent immune suppressive activity. In spleens of mice, PMN-MDSCs gradually replaced PMNs during tumor progression. Activated PMN-MDSCs were found only in tumors, where they were present at the very early stages of the disease. These populations of PMNs in mice could be separated based on the expression of CD14. In peripheral blood of cancer patients, we identified two distinct populations of PMNs with characteristics of classical PMNs and PMN-MDSCs. The gene signature of tumor PMN-MDSCs was similar to that in mouse activated PMN-MDSCs and was closely associated with negative clinical outcome in cancer patients. Thus, we provide evidence that PMN-MDSCs are a distinct population of PMNs with unique features and potential for selective targeting opportunities.

Myeloid-Derived Suppressor Cells Are a Major Source of Wnt5A in the Melanoma Microenvironment and Depend on Wnt5A for Full Suppressive Activity.

Metastatic dissemination remains a significant barrier to successful therapy for melanoma. Wnt5A is a potent driver of invasion in melanoma and is believed to be secreted from the tumor microenvironment (TME). Our data suggest that myeloid-derived suppressor cells (MDSC) in the TME are a major source of Wnt5A and are reliant upon Wnt5A for multiple actions. Knockdown of Wnt5A specifically in the myeloid cells demonstrated a clear decrease in Wnt5A expression within the TME in vivo as well as a decrease in intratumoral MDSC and regulatory T cell (Treg). Wnt5A knockdown also decreased the immunosuppressive nature of MDSC and decreased expression of TGFß1 and arginase 1. In the presence of Wnt5A-depleted MDSC, tumor-infiltrating lymphocytes expressed decreased PD-1 and LAG3, suggesting a less exhausted phenotype. Myeloid-specific Wnt5A knockdown also led to decreased lung metastasis. Tumor-infiltrating MDSC from control animals showed a strong positive correlation with Treg, which was completely ablated in animals with Wnt5A-negative MDSC. Overall, our data suggest that while MDSC contribute to an immunosuppressive and less immunogenic environment, they exhibit an additional function as the major source of Wnt5A in the TME. SIGNIFICANCE: These findings demonstrate that myeloid cells provide a major source of Wnt5A to facilitate metastatic potential in melanoma cells and rely on Wnt5A for their immunosuppressive function.

BTN3A1 governs antitumor responses by coordinating αß and γδ T cells.

Gamma delta (γδ) T cells infiltrate most human tumors, but current immunotherapies fail to exploit their in situ major histocompatibility complex-independent tumoricidal potential. Activation of γδ T cells can be elicited by butyrophilin and butyrophilin-like molecules that are structurally similar to the immunosuppressive B7 family members, yet how they regulate and coordinate αß and γδ T cell responses remains unknown. Here, we report that the butyrophilin BTN3A1 inhibits tumor-reactive αß T cell receptor activation by preventing segregation of N-glycosylated CD45 from the immune synapse. Notably, CD277-specific antibodies elicit coordinated restoration of αß T cell effector activity and BTN2A1-dependent γδ lymphocyte cytotoxicity against BTN3A1+ cancer cells, abrogating malignant progression. Targeting BTN3A1 therefore orchestrates cooperative killing of established tumors by αß and γδ T cells and may present a treatment strategy for tumors resistant to existing immunotherapies.

Polymorphonuclear myeloid-derived suppressor cells limit antigen cross-presentation by dendritic cells in cancer.

DCs are a critical component of immune responses in cancer primarily due to their ability to cross-present tumor-associated antigens. Cross-presentation by DCs in cancer is impaired, which may represent one of the obstacles for the success of cancer immunotherapies. Here, we report that polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) blocked cross-presentation by DCs without affecting direct presentation of antigens by these cells. This effect did not require direct cell-cell contact and was associated with transfer of lipids. Neutrophils (PMN) and PMN-MDSC transferred lipid to DCs equally well; however, PMN did not affect DC cross-presentation. PMN-MDSC generate oxidatively truncated lipids previously shown to be involved in impaired cross-presentation by DCs. Accumulation of oxidized lipids in PMN-MDSC was dependent on myeloperoxidase (MPO). MPO-deficient PMN-MDSC did not affect cross-presentation by DCs. Cross-presentation of tumor-associated antigens in vivo by DCs was improved in MDSC-depleted or tumor-bearing MPO-KO mice. Pharmacological inhibition of MPO in combination with checkpoint blockade reduced tumor progression in different tumor models. These data suggest MPO-driven lipid peroxidation in PMN-MDSC as a possible non-cell autonomous mechanism of inhibition of antigen cross-presentation by DCs and propose MPO as potential therapeutic target to enhance the efficacy of current immunotherapies for patients with cancer.

HDAC6 Inhibition Synergizes with Anti-PD-L1 Therapy in ARID1A-Inactivated Ovarian Cancer.

ARID1A, encoding a subunit of the SWI/SNF complex, is the most frequently mutated epigenetic regulator in human cancers and is mutated in more than 50% of ovarian clear cell carcinomas (OCCC), a disease that currently has no effective therapy. Inhibition of histone deacetylase 6 (HDAC6) suppresses the growth of ARID1A-mutated tumors and modulates tumor immune microenvironment. Here, we show that inhibition of HDAC6 synergizes with anti-PD-L1 immune checkpoint blockade in ARID1A-inactivated ovarian cancer. ARID1A directly repressed transcription of CD274, the gene encoding PD-L1. Reduced tumor burden and improved survival were observed in ARID1Aflox/flox/PIK3CAH1047R OCCC mice treated with the HDAC6 inhibitor ACY1215 and anti-PD-L1 immune checkpoint blockade as a result of activation and increased presence of IFNγ-positive CD8 T cells. We confirmed that the combined treatment limited tumor progression in a cytotoxic T-cell-dependent manner, as depletion of CD8+ T cells abrogated these antitumor effects. Together, these findings indicate that combined HDAC6 inhibition and immune checkpoint blockade represents a potential treatment strategy for ARID1A-mutated cancers. SIGNIFICANCE: These findings offer a mechanistic rationale for combining epigenetic modulators and existing immunotherapeutic interventions against a disease that has been so far resistant to checkpoint blockade as a monotherapy.See related commentary by Prokunina-Olsson, p. 5476.

Identification of monocyte-like precursors of granulocytes in cancer as a mechanism for accumulation of PMN-MDSCs.

We have identified a precursor that differentiates into granulocytes in vitro and in vivo yet belongs to the monocytic lineage. We have termed these cells monocyte-like precursors of granulocytes (MLPGs). Under steady state conditions, MLPGs were absent in the spleen and barely detectable in the bone marrow (BM). In contrast, these cells significantly expanded in tumor-bearing mice and differentiated to polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Selective depletion of monocytic cells had no effect on the number of granulocytes in naive mice but decreased the population of PMN-MDSCs in tumor-bearing mice by 50%. The expansion of MLPGs was found to be controlled by the down-regulation of Rb1, but not IRF8, which is known to regulate the expansion of PMN-MDSCs from classic granulocyte precursors. In cancer patients, putative MLPGs were found within the population of CXCR1+CD15-CD14+HLA-DR-/lo monocytic cells. These findings describe a mechanism of abnormal myelopoiesis in cancer and suggest potential new approaches for selective targeting of MDSCs.

Plasticity of myeloid-derived suppressor cells in cancer.

In recent years, myeloid-derived suppressor cells (MDSC) have emerged as one of the major inhibitors of immune effector cell function in cancer. MDSC represent a heterogeneous population of largely immature myeloid cells that are characterized by a pathological state of activation and display potent immune suppressive activity. Two major subsets of MDSC have been identified: monocytic (M-MDSC) and polymorphonuclear (PMN-MDSC). PMN-MSDC share phenotypic and morphologic features with neutrophils, whereas M-MDSC are similar to monocytes and are characterized by high plasticity. Differentiation of M-MDSC to macrophages and dendritic cells is shaped by tumor microenvironment. In recent years, the mechanisms of this process start to emerge.

Exposure to the Epstein-Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo.

Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system (CNS). Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP)-specific antibodies from MS patients cross-react with Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1). In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo. We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20-50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs) or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading.

The introduction of mesenchymal stromal cells induces different immunological responses in the lungs of healthy and M. tuberculosis infected mice.

Mesenchymal stromal cells (MSC) have strong immunomodulatory properties and therefore can be used to control inflammation and tissue damage. It was suggested recently that MSC injections can be used to treat multi-drug resistant tuberculosis (TB). However, MSC trafficking and immunomodulatory effects of MSC injections during Mycobacterium tuberculosis (Mtb) infection have not been studied. To address this issue we have analyzed MSC distribution in tissues and local immunological effects of MSC injections in Mtb infected and uninfected mice. After intravenous injection, MSC accumulated preferentially in the lungs where they were located as cell aggregates in the alveolar walls. Immunological analysis of MSC effects included detection of activated, IFN-γ and IL-4 producing CD4+ lymphocytes, the frequency analysis of dendritic cells (CD11c+F4/80) and macrophages (CD11c-F4/80+) located in the lungs, the expression of IA/IE and CD11b molecules by these cells, and evaluation of 23 cytokines/chemokines in lung lysates. In the lungs of uninfected mice, MSC transfer markedly increased the percentage of IFN-γ+ CD4+ lymphocytes and dendritic cells, elevated levels of IA/IE expression by dendritic cells and macrophages, augmented local production of type 2 cytokines (IL-4, IL-5, IL-10) and chemokines (CCL2, CCL3, CCL4, CCL5, CXCL1), and downregulated type 1 and hematopoietic cytokines (IL-12p70, IFN-γ, IL-3, IL-6, GM-CSF). Compared to uninfected mice, Mtb infected mice had statistically higher "background" frequency of activated CD69+ and IFN-γ+ CD4+ lymphocytes and dendritic cells, and higher levels of cytokines in the lungs. The injections of MSC to Mtb infected mice did not show statistically significant effects on CD4+ lymphocytes, dendritic cells and macrophages, only slightly shifted cytokine profile, and did not change pathogen load or slow down TB progression. Lung section analysis showed that in Mtb infected mice, MSC could not be found in the proximity of the inflammatory foci. Thus, in healthy recipients, MSC administration dramatically changed T-cell function and cytokine/chemokine milieu in the lungs, most likely, due to capillary blockade. But, during Mtb infection, i.e., in the highly-inflammatory conditions, MSC did not affect T-cell function and the level of inflammation. The findings emphasize the importance of the evaluation of MSC effects locally at the site of their predominant post-injection localization and question MSC usefulness as anti-TB treatment.

The Nature of Myeloid-Derived Suppressor Cells in the Tumor Microenvironment.

Myeloid-derived suppressor cells (MDSC) are one of the major components of the tumor microenvironment. The main feature of these cells is their potent immune suppressive activity. MDSC are generated in the bone marrow and, in tumor-bearing hosts, migrate to peripheral lymphoid organs and the tumor to contribute to the formation of the tumor microenvironment. Recent findings have revealed differences in the function and fate of MDSC in the tumor and peripheral lymphoid organs. We review these findings here and, in this context, we discuss the current understanding as to the nature of these differences, the underlying mechanisms, and their potential impact on the regulation of tumor progression.

Lectin-type oxidized LDL receptor-1 distinguishes population of human polymorphonuclear myeloid-derived suppressor cells in cancer patients.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) are important regulators of immune responses in cancer and have been directly implicated in promotion of tumor progression. However, the heterogeneity of these cells and lack of distinct markers hampers the progress in understanding of the biology and clinical importance of these cells. Using partial enrichment of PMN-MDSC with gradient centrifugation we determined that low density PMN-MDSC and high density neutrophils from the same cancer patients had a distinct gene profile. Most prominent changes were observed in the expression of genes associated with endoplasmic reticulum (ER) stress. Surprisingly, low-density lipoprotein (LDL) was one of the most increased regulators and its receptor oxidized LDL receptor 1 OLR1 was one of the most overexpressed genes in PMN-MDSC. Lectin-type oxidized LDL receptor 1 (LOX-1) encoded by OLR1 was practically undetectable in neutrophils in peripheral blood of healthy donors, whereas 5-15% of total neutrophils in cancer patients and 15-50% of neutrophils in tumor tissues were LOX-1+. In contrast to their LOX-1- counterparts, LOX-1+ neutrophils had gene signature, potent immune suppressive activity, up-regulation of ER stress, and other biochemical characteristics of PMN-MDSC. Moreover, induction of ER stress in neutrophils from healthy donors up-regulated LOX-1 expression and converted these cells to suppressive PMN-MDSC. Thus, we identified a specific marker of human PMN-MDSC associated with ER stress and lipid metabolism, which provides new insight to the biology and potential therapeutic targeting of these cells.