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Polymorphonuclear neutrophils (PMNs) are innate cells that may act as myeloid-derived suppressor cells (MDSCs), most often characterized by their immunosuppressive capacity towards immune cells within the tumor microenvironment. Over the last decade, many studies have tried to characterize these PMN-MDSCs. Although all human neutrophils obtain suppressive activity upon activation, specialized subsets, including low-density cells, have been proposed to exert MDSC activity without requiring prior stimulation. Single-cell RNA sequencing (scRNAseq) has created a new horizon to study possible neutrophil subsets. As such, the identification of distinct neutrophil subtypes in tumors may better characterize immunosuppressive neutrophils, in the end leading to specific targeting strategies in cancer patients. Nevertheless, scRNAseq has not yet led to a clear immunophenotypic characterization of PMN-MDSCs, and clinical relevance by functional testing is still lacking. Studies on the discrepancy in RNA abundance, protein expression and functionality in neutrophils indicate that the future for defining functional neutrophil subsets will rely on further development of single-cell techniques, like proteomics, to truly carve out neutrophil subsets and plasticity.

Whereas, neutrophils have long been considered to mainly function as efficient innate immunity killers of micro-organisms at infected sites, they are now recognized to also be involved in modulation of adaptive immune responses. Immature and mature neutrophils were reported to have the capacity to suppress T cell-mediated immune responses as so-called granulocyte-myeloid-derived suppressor cells (g-MDSCs), and thereby affect the clinical outcome of cancer patients and impact the chronicity of microbial infections or rejection reactions in organ transplantation settings. These MDSCs were at first considered to be immature myeloid cells that left the bone marrow due to disease-specific signals. Current studies show that also mature neutrophils can exert suppressive activity. In this study we investigated in a robust T cell suppression assay whether immature CD11b+ myeloid cells were capable of MDSC activity comparable to mature fully differentiated neutrophils. We compared circulating neutrophils with myeloid cell fractions from the bone marrow at different differentiation stages. Our results indicate that functional MDSC activity is only becoming detectable at the final stage of differentiation, depending on the procedure of cell isolation. The MDSC activity obtained during neutrophil maturation correlated with the induction of the well-known highly mobile and toxic effector functions of the circulating neutrophil. Although immature neutrophils have been suggested to be increased in the circulation of cancer patients, we show here that immature neutrophils are not efficient in suppressing T cells. This suggests that the presence of immature neutrophils in the bloodstream of cancer patients represent a mere association or may function as a source of mature neutrophils in the tumor environment but not a direct cause of enhanced MDSC activity in cancer.

Cancer is one of the leading causes of death worldwide. Treatment outcome is largely dictated by the tumor type, disease stage, and treatment success rates, but also by the variation among patients in endogenous anti-tumor responses. Studies indicate that the presence of neutrophils in the tumor microenvironment is associated with a worse patient outcome due to their ability to suppress local anti-tumor T cell activity. Our previous studies investigated the mechanisms by which neutrophils suppress and damage T cells to become smaller in size (small T cells), debilitating their effector activities. Several studies indicate a role for tumor-associated macrophages in scavenging damaged or dead cells. We hypothesized that the observed lack of small T cells in the TME by confocal microscopy is due to immediate uptake by macrophages. In this study, we confirmed that indeed only the smaller, damaged T cells are taken up by macrophages, once serum-opsonized. Damaged T cells opsonized with complement factor C3 fragments were phagocytosed by macrophages, resulting in almost instantaneous and highly efficient uptake of these small T cells. Inhibition of the complement receptors CR1, CR3 and CR4 expressed by macrophages completely blocked phagocytosis. By contrast, actively proliferating T cells (large T cells) were neither impaired in neutrophil-MDSC activity nor opsonized for phagocytosis by macrophages. Rapid removal of damaged T cells suggests a role of complement and macrophages within the tumor microenvironment to clear suppressed T cells in cancer patients.

Suppression of antitumor immune responses is one of the main mechanisms by which tumor cells escape from destruction by the immune system. Myeloid-derived suppressor cells (MDSCs) represent the main immunosuppressive cells present in the tumor microenvironment (TME) that sustain cancer progression. MDSCs are a heterogeneous group of immature myeloid cells with a potent activity against T-cell. Studies in mice have demonstrated that MDSCs accumulate in several types of cancer where they promote invasion, angiogenesis, and metastasis formation and inhibit antitumor immunity. In addition, different clinical studies have shown that MDSCs levels in the peripheral blood of cancer patients correlates with tumor burden, stage and with poor prognosis in multiple malignancies. Thus, MDSCs are the major obstacle to many cancer immunotherapies and their targeting may be a beneficial strategy for improvement the efficiency of immunotherapeutic interventions. However, the great heterogeneity of these cells makes their identification in human cancer very challenging. Since both the phenotype and mechanisms of action of MDSCs appear to be tumor-dependent, it is important to accurately characterized the precise MDSC subsets that have clinical relevance in each tumor environment to more efficiently target them. In this review we summarize the phenotype and the suppressive mechanisms of MDSCs populations expanded within different tumor contexts. Further, we discuss about their clinical relevance for cancer diagnosis and therapy.

Cysteine cathepsins are key regulators of the innate and adaptive arms of the immune system. Their expression, activity, and subcellular localization are associated with the distinct development and differentiation stages of immune cells. They promote the activation of innate myeloid immune cells since they contribute to toll-like receptor signaling and to cytokine secretion. Furthermore, they control lysosomal biogenesis and autophagic flux, thus affecting innate immune cell survival and polarization. They also regulate bidirectional communication between the cell exterior and the cytoskeleton, thus influencing cell interactions, morphology, and motility. Importantly, cysteine cathepsins contribute to the priming of adaptive immune cells by controlling antigen presentation and are involved in cytotoxic granule mediated killing in cytotoxic T lymphocytes and natural killer cells. Cathepins'aberrant activity can be prevented by their endogenous inhibitors, cystatins. However, dysregulated proteolysis contributes significantly to tumor progression also by modulation of the antitumor immune response. Especially tumor-associated myeloid cells, such as tumor-associated macrophages and myeloid-derived suppressor cells, which are known for their tumor promoting and immunosuppressive functions, constitute the major source of excessive cysteine cathepsin activity in cancer. Since they are enriched in the tumor microenvironment, cysteine cathepsins represent exciting targets for development of new diagnostic and therapeutic moieties.

Interleukin-1β (IL-1β)–mediated inflammation suppresses antitumor immunity, leading to the generation of a tumor-permissive environment, tumor growth, and progression. Here, we demonstrate that nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in melanoma is linked to IL-1β production, inflammation, and immunosuppression. Analysis of cancer genome datasets (TCGA and GTEx) revealed greater NLRP3 and IL-1β expression in cutaneous melanoma samples (n = 469) compared to normal skin (n = 324), with a highly significant correlation between NLRP3 and IL-1β (P < 0.0001). We show the formation of the NLRP3 inflammasome in biopsies of metastatic melanoma using fluorescent resonance energy transfer analysis for NLRP3 and apoptosis-associated speck-like protein containing a CARD. In vivo, tumor-associated NLRP3/IL-1 signaling induced expansion of myeloid-derived suppressor cells (MDSCs), leading to reduced natural killer and CD8⁺ T cell activity concomitant with an increased presence of regulatory T (Treg) cells in the primary tumors. Either genetic or pharmacological inhibition of tumor-derived NLRP3 by dapansutrile (OLT1177) was sufficient to reduce MDSCs expansion and to enhance antitumor immunity, resulting in reduced tumor growth. Additionally, we observed that the combination of NLRP3 inhibition and anti–PD-1 treatment significantly increased the antitumor efficacy of the monotherapy by limiting MDSC-mediated T cell suppression and tumor progression. These data show that NLRP3 activation in melanoma cells is a protumor mechanism, which induces MDSCs expansion and immune evasion. We conclude that inhibition of NLRP3 can augment the efficacy of anti–PD-1 therapy.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are implicated in the progression and outcome of a variety of pathological states, from cancer to infection. Our previous work has identified three antimicrobial peptides differentially expressed by PMN-MDSCs compared to conventional neutrophils isolated from dogs, mice, and human patients with cancer. We therefore hypothesized that PMN-MDSCs in dogs with cancer possess antimicrobial activity. In the current work, we observed that exposure of PMN-MDSCs to Gram-negative bacteria ( ) increased the expression of reactive oxygen species by the PMN-MDSCs, indicating that they are capable of initiating an anti-microbial response. Electron microscopy revealed that the PMN-MDSCs phagocytosed Gram-negative and Gram-positive ( ) bacterial species. Lysis of bacteria within some of the PMN-MDSCs suggested bactericidal activity, which was confirmed by the recovery of significantly lower numbers of bacteria of both species following exposure to PMN-MDSCs isolated from tumor-bearing dogs. Our data therefore indicate that PMN-MDSCs isolated from dogs with cancer, in common with PMNs, have phagocytic and bactericidal activity. This nexus of immunosuppressive and antimicrobial activity reveals a hitherto unrecognized function of MDSCs.

Pancreatic ductal adenocarcinoma (PDAC) is often refractory to treatment with gemcitabine (GEM) and immune checkpoint inhibitors including anti-programmed cell death ligand 1 (PD-L1) antibody. However, the precise relationship between GEM-resistant PDAC and development of an immunosuppressive tumor microenvironment (TME) remains unclear. In this study, we investigated the immunosuppressive TME in parental and GEM-resistant PDAC tumors and assessed the therapeutic potential of combination therapy with the telomerase-specific replication-competent oncolytic adenovirus OBP-702, which induces tumor suppressor p53 protein and PD-L1 blockade against GEM-resistant PDAC tumors. Mouse PDAC cells (PAN02) and human PDAC cells (MIA PaCa-2, BxPC-3) were used to establish GEM-resistant PDAC lines. PD-L1 expression and the immunosuppressive TME were analyzed using parental and GEM-resistant PDAC cells. A cytokine array was used to investigate the underlying mechanism of immunosuppressive TME induction by GEM-resistant PAN02 cells. The GEM-resistant PAN02 tumor model was used to evaluate the antitumor effect of combination therapy with OBP-702 and PD-L1 blockade. GEM-resistant PDAC cells exhibited higher PD-L1 expression and produced higher granulocyte–macrophage colony-stimulating factor (GM-CSF) levels compared with parental cells, inducing an immunosuppressive TME and the accumulation of myeloid-derived suppressor cells (MDSCs). OBP-702 significantly inhibited GEM-resistant PAN02 tumor growth by suppressing GM-CSF-mediated MDSC accumulation. Moreover, combination treatment with OBP-702 significantly enhanced the antitumor efficacy of PD-L1 blockade against GEM-resistant PAN02 tumors. The present results suggest that combination therapy involving OBP-702 and PD-L1 blockade is a promising antitumor strategy for treating GEM-resistant PDAC with GM-CSF-induced immunosuppressive TME formation.

Pancreatic cancer (PC) mobilizes myeloid cells from the bone marrow to the tumor where they promote tumor growth and proliferation. Cancer stem cells (CSCs) are a population of tumor cells that are responsible for tumor initiation. Aldehyde dehydrogenase-1 activity in PC identifies CSCs, and its activity has been correlated with poor overall prognosis in human PC. Myeloid cells have been shown to impact tumor stemness, but the impact of immunosuppressive tumor-infiltrating granulocytic and monocytic myeloid-derived suppressor cells (Mo-MDSC) on ALDH1 Bright CSCs and epithelial to mesenchymal transition is not well understood. In this study, we demonstrate that Mo-MDSC (CD11b + /Gr1 + /Ly6G − /Ly6C hi ) significantly increase the frequency of ALDH1 Bright CSCs in a mouse model of PC. Additionally, there was significant upregulation of genes associated with epithelial to mesenchymal transition. We also found that human PC converts CD14 + peripheral blood monocytes into Mo-MDSC (CD14 + /HLA-DR low/− ) in vitro, and this transformation is dependent on the activation of the STAT3 pathway. In turn, these Mo-MDSC increase the frequency of ALDH1 Bright CSCs and promote mesenchymal features of tumor cells. Finally, blockade of STAT3 activation reversed the increase in ALDH1 Bright CSCs. These data suggest that the PC tumor microenvironment transforms monocytes to Mo-MDSC by STAT3 activation, and these cells increase the frequency of ALDH1 Bright CSCs. Therefore, targeting STAT3 activation may be an effective therapeutic strategy in targeting CSCs in PC.

For decades, tumor-bearing murine models established using tumor cell lines have been the most commonly used models to study human cancers. Even though there are several studies reported that implant sites caused disparities in tumor behaviors, few of them illuminated the positional effect on immunotherapy. Herein, we describe surgical techniques for a novel orthotopic implantation of syngeneic pancreatic ductal adenocarcinoma (PDAC) tissue slices. This method has a high success modeling rate and stable growth kinetics, which makes it useful for testing novel therapeutics. Pathological examination indicated that the orthotopic tumor displayed poor vascularization, desmoplastic stromal reaction, and a highly immunosuppressive tumor microenvironment. This unique microenvironment resulted in limited response to PD1/CTLA4 blockade therapy and anti-MUC1 (αMUC1) CAR-T transfer treatment. To reverse the suppressive tumor microenvironment, we developed gene modified T-cells bearing a chimeric receptor in which activating receptor NKG2D fused to intracellular domains of 4-1BB and CD3ζ (NKG2D CAR). The NKG2D CAR-T cells target myeloid-derived suppressor cells (MDSCs), which overexpress Rae1 (NKG2D ligands) within the TME. Results indicated that NKG2D CAR-T cells eliminated MDSCs and improved antitumor activity of subsequently infused CAR-T cells. Moreover, we generated a bicistronic CAR-T, including αMUC1 CAR and NKG2D CAR separated by a P2A element. Treatment with the dual targeted bicistronic CAR-T cells also resulted in prolonged survival of orthotopic model mice. In summary, this study describes construction of a novel orthotopic PDAC model through implantation of tissue slices and discusses resistance to immunotherapy from the perspective of a PDAC microenvironment. Based on the obtained results, it is evident that elimination MDSCs by NKG2D CAR could rescue the impaired CAR-T cell activity.