Correction to: Human MDSCs derived from the bone marrow maintain their functional ability but have a reduced frequency of induction in the elderly compared to pediatric donors.

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Human MDSCs derived from the bone marrow maintain their functional ability but have a reduced frequency of induction in the elderly compared to pediatric donors.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immunosuppressive cells developing from myeloid progenitors, which are enriched in pathological conditions such as cancer, and are known to inhibit the functions of effector T cells. During aging, several changes occur both at the adaptive and innate immune system level, in a process defined as immunoscenescence. In particular, the low-grade inflammation state observed in the elderly appears to affect hematopoiesis. We previously demonstrated that the combination of GM-CSF and G-CSF drives the in vitro generation of bone marrow-derived MDSCs (BM-MDSCs) from precursors present in human bone marrow aspirates of healthy donors, and that these cells are endowed with a strong immune suppressive ability, resembling that of cancer-associated MDSCs. In the present work we investigated BM-MDSCs induction and functional ability in a cohort of pediatric versus elderly donors. To this aim, we analyzed the differences in maturation stages and ability to suppress T cell proliferation. We found that the ex vivo distribution of myeloid progenitors is similar between pediatric and elderly individuals, whereas after cytokine treatment a significant reduction in the more immature compartment is observed in the elderly. Despite the decreased frequency, BM-MDSCs maintain their suppressive capacity in aged donors. Taken together, these results indicate that in vitro induction of MDSCs from the BM is reduced with aging and opens new hypotheses on the role of age-related processes in myelopoiesis.

In Vitro Characterization of Adipose Stem Cells Non-Enzymatically Extracted from the Thigh and Abdomen.

Autologous fat grafting is a surgical technique in which adipose tissue is transferred from one area of the body to another, in order to reconstruct or regenerate damaged or injured tissues. Before reinjection, adipose tissue needs to be purified from blood and cellular debris to avoid inflammation and preserve the graft viability. To perform this purification, different enzymatic and mechanical methods can be used. In this study, we characterized in vitro the product of a closed automatic device based on mechanical disaggregation, named Rigenera®, focusing on two sites of adipose tissue harvesting. At first, we optimized the Rigenera® operating timing, demonstrating that 60 s of treatment allows a higher cellular yield, in terms of the cell number and growth rate. This result optimizes the mechanical disaggregation and it can increase the clinical efficiency of the final product. When comparing the extracted adipose samples from the thigh and abdomen, our results showed that the thigh provides a higher number of mesenchymal-like cells, with a faster replication rate and a higher ability to form colonies. We can conclude that by collecting adipose tissue from the thigh and treating it with the Rigenera® device for 60 s, it is possible to obtain the most efficient product.

Immunosuppression by monocytic myeloid-derived suppressor cells in patients with pancreatic ductal carcinoma is orchestrated by STAT3.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly devastating disease with an overall 5-year survival rate of less than 8%. New evidence indicates that PDAC cells release pro-inflammatory metabolites that induce a marked alteration of normal hematopoiesis, favoring the expansion and accumulation of myeloid-derived suppressor cells (MDSCs). We report here that PDAC patients show increased levels of both circulating and tumor-infiltrating MDSC-like cells. METHODS: The frequency of MDSC subsets in the peripheral blood was determined by flow cytometry in three independent cohorts of PDAC patients (total analyzed patients, n = 117). Frequency of circulating MDSCs was correlated with overall survival of PDAC patients. We also analyzed the frequency of tumor-infiltrating MDSC and the immune landscape in fresh biopsies. Purified myeloid cell subsets were tested in vitro for their T-cell suppressive capacity. RESULTS: Correlation with clinical data revealed that MDSC frequency was significantly associated with a shorter patients' overall survival and metastatic disease. However, the immunosuppressive activity of purified MDSCs was detectable only in some patients and mainly limited to the monocytic subset. A transcriptome analysis of the immunosuppressive M-MDSCs highlighted a distinct gene signature in which STAT3 was crucial for monocyte re-programming. Suppressive M-MDSCs can be characterized as circulating STAT3/arginase1-expressing CD14+ cells. CONCLUSION: MDSC analysis aids in defining the immune landscape of PDAC patients for a more appropriate diagnosis, stratification and treatment.

The immune suppressive microenvironment of human gliomas depends on the accumulation of bone marrow-derived macrophages in the center of the lesion.

BACKGROUND: Systemic and local immune suppression plays a significant role in glioma progression. Glioma microenvironment contains both brain-resident microglial cells (MG) and bone marrow-derived macrophages (BMDM), but the study of their functional and immune regulatory activity has been hampered until now by the lack of markers allowing a proper identification and isolation to collect pure populations. METHODS: Myeloid and lymphoid infiltrate were characterized in grade II, III and IV gliomas by multicolor flow cytometry, along with the composition of the cell subsets of circulating myeloid cells. Macrophages were sorted and tested for their immunosuppressive ability. Moreover, following preoperative administration of 5-aminolevulinic acid to patients, distinct areas of tumor lesion were surgically removed and analyzed, based on protoporphyrin IX fluorescence emission. RESULTS: The immune microenvironment of grade II to grade IV gliomas contains a large proportion of myeloid cells and a small proportion of lymphocytes expressing markers of dysfunctional activity. BMDM and resident MG cells were characterized through a combination of markers, thus permitting their geographical identification in the lesions, their sorting and subsequent analysis of the functional characteristics. The infiltration by BMDM reached the highest percentages in grade IV gliomas, and it increased from the periphery to the center of the lesion, where it exerted a strong immunosuppression that was, instead, absent in the marginal area. By contrast, MG showed little or no suppression. Functional differences, such as iron metabolism and phagocytosis, characterized resident versus blood-derived macrophages. Significant alterations in circulating monocytes were present in grade IV patients, correlating with accumulation of tumor macrophages. CONCLUSIONS: Grade IV gliomas have an alteration in both circulating and tumor-associated myeloid cells and, differently from grade II and III gliomas, show a significant presence of blood-derived, immune suppressive macrophages. BMDM and MG have different functional properties.

Methods to Measure MDSC Immune Suppressive Activity In Vitro and In Vivo.

This unit presents methods to assess the immunosuppressive properties of immunoregulatory cells of myeloid origin, such as myeloid-derived suppressor cells (MDSCs), both in vitro and in vivo in mice, as well as in biological samples from cancer patients. These methods could be adapted to test the impact of different suppressive populations on T cell activation, proliferation, and cytotoxic activity; moreover, they could be useful to assess the influence exerted by genetic modifications, chemical inhibitors, and drugs on immune suppressive pathways © 2018 by John Wiley & Sons, Inc.

Induction of immunosuppressive functions and NF-κB by FLIP in monocytes.

Immunosuppression is a hallmark of tumor progression, and treatments that inhibit or deplete monocytic myeloid-derived suppressive cells could promote anti-tumor immunity. c-FLIP is a central regulator of caspase-8-mediated apoptosis and necroptosis. Here we show that low-dose cytotoxic chemotherapy agents cause apoptosis linked to c-FLIP down-regulation selectively in monocytes. Enforced expression of c-FLIP or viral FLIP rescues monocytes from cytotoxicity and concurrently induces potent immunosuppressive activity, in T cell cultures and in vivo models of tumor progression and immunotherapy. FLIP-transduced human blood monocytes can suppress graft versus host disease. Neither expression of FLIP in granulocytes nor expression of other anti-apoptotic genes in monocytes conferred immunosuppression, suggesting that FLIP effects on immunosuppression are specific to monocytic lineage and distinct from death inhibition. Mechanistically, FLIP controls a broad transcriptional program, partially by NF-κB activation. Therefore, modulation of FLIP in monocytes offers a means to elicit or block immunosuppressive myeloid cells.

Immunosuppressive activity of tumor-infiltrating myeloid cells in patients with meningioma.

Meningiomas WHO grade I and II are common intracranial tumors in adults that normally display a benign outcome, but are characterized by a great clinical heterogeneity and frequent recurrence of the disease. Although the presence of an immune cell infiltrate has been documented in these tumors, a clear phenotypical and functional characterization of the immune web is missing. Here, we performed an extensive immunophenotyping of peripheral blood and fresh tumor tissue at surgery by multiparametric flow cytometry in 34 meningioma patients, along with immunosuppressive activity of sorted cells of myeloid origin. Four subsets of myeloid cells, phenotypically corresponding to myeloid-derived suppressor cells (MDSCs) are detectable in the blood and in the tumor tissue of patients and three of them are significantly expanded in the blood of patients, but show no evidence of suppressive activity. At the tumor site, a large leukocyte infiltrate is present, predominantly constituted by CD33+ myeloid cells, largely composed of macrophages endowed with suppressive activity and significantly expanded in grade II meningioma patients as compared to grade I.

Low dose gemcitabine-loaded lipid nanocapsules target monocytic myeloid-derived suppressor cells and potentiate cancer immunotherapy.

Tumor-induced expansion of myeloid-derived suppressor cells (MDSCs) is known to impair the efficacy of cancer immunotherapy. Among pharmacological approaches for MDSC modulation, chemotherapy with selected drugs has a considerable interest due to the possibility of a rapid translation to the clinic. However, such approach is poorly selective and may be associated with dose-dependent toxicities. In the present study, we showed that lipid nanocapsules (LNCs) loaded with a lauroyl-modified form of gemcitabine (GemC12) efficiently target the monocytic (M-) MDSC subset. Subcutaneous administration of GemC12-loaded LNCs reduced the percentage of spleen and tumor-infiltrating M-MDSCs in lymphoma and melanoma-bearing mice, with enhanced efficacy when compared to free gemcitabine. Consistently, fluorochrome-labeled LNCs were preferentially uptaken by monocytic cells rather than by other immune cells, in both tumor-bearing mice and human blood samples from healthy donors and melanoma patients. Very low dose administration of GemC12-loaded LNCs attenuated tumor-associated immunosuppression and increased the efficacy of adoptive T cell therapy. Overall, our results show that GemC12-LNCs have monocyte-targeting properties that can be useful for immunomodulatory purposes, and unveil new possibilities for the exploitation of nanoparticulate drug formulations in cancer immunotherapy.

MDSCs in cancer: Conceiving new prognostic and therapeutic targets.

The incomplete clinical efficacy of anti-tumor immunotherapy can depend on the presence of an immunosuppressive environment in the host that supports tumor progression. Tumor-derived cytokines and growth factors induce an altered hematopoiesis that modifies the myeloid cell differentiation process, promoting proliferation and expansion of cells with immunosuppressive skills, namely myeloid derived suppressor cells (MDSCs). MDSCs promote tumor growth not only by shaping immune responses towards tumor tolerance, but also by supporting several processes necessary for the neoplastic progression such as tumor angiogenesis, cancer stemness, and metastasis dissemination. Thus, MDSC targeting represents a promising tool to eliminate host immune dysfunctions and increase the efficacy of immune-based cancer therapies.

Activated T cells sustain myeloid-derived suppressor cell-mediated immune suppression.

The expansion of myeloid derived suppressor cells (MDSCs), a suppressive population able to hamper the immune response against cancer, correlates with tumor progression and overall survival in several cancer types. We have previously shown that MDSCs can be induced in vitro from precursors present in the bone marrow and observed that these cells are able to actively proliferate in the presence of activated T cells, whose activation level is critical to drive the suppressive activity of MDSCs. Here we investigated at molecular level the mechanisms involved in the interplay between MDSCs and activated T cells. We found that activated T cells secrete IL-10 following interaction with MDSCs which, in turn, activates STAT3 phosphorylation on MDSCs then leading to B7-H1 expression. We also demonstrated that B7-H1+ MDSCs are responsible for immune suppression through a mechanism involving ARG-1 and IDO expression. Finally, we show that the expression of ligands B7-H1 and MHC class II both on in vitro-induced MDSCs and on MDSCs in the tumor microenvironment of cancer patients is paralleled by an increased expression of their respective receptors PD-1 and LAG-3 on T cells, two inhibitory molecules associated with T cell dysfunction. These findings highlight key molecules and interactions responsible for the extensive cross-talk between MDSCs and activated T cells that are at the basis of immune suppression.

Complexity and challenges in defining myeloid-derived suppressor cells.

Study of myeloid cells endowed with suppressive activity is an active field of research which has particular importance in cancer, in view of the negative regulatory capacity of these cells to the host's immune response. The expansion of these cells, called myeloid-derived suppressor cells (MDSCs), has been documented in many models of tumor-bearing mice and in patients with tumors of various origin, and their presence is associated with disease progression and reduced survival. For this reason, monitoring this type of cell expansion is of clinical importance, and flow cytometry is the technique of choice for their identification. Over the years, it has been demonstrated that MDSCs comprise a group of immature myeloid cells belonging both to monocytic and granulocytic lineages, with several stages of differentiation; their occurrence depends on tumor-derived soluble factors, which guide their expansion and determine their block of differentiation. Because of their heterogeneous composition, accurate phenotyping of these cells requires a multicolor approach, so that the expansion of all MDSC subsets can be appreciated. This review article focuses on identifying MDSCs and discusses problems associated with phenotyping circulating and tumor-associated MDSCs in humans and in mouse models.

Complexity and challenges in defining myeloid-derived suppressor cells.

Study of myeloid cells endowed with suppressive activity is an active field of research which has particular importance in cancer, in view of the negative regulatory capacity of these cells to the host's immune response. The expansion of these cells, called myeloid-derived suppressor cells (MDSCs), has been documented in many models of tumor-bearing mice and in patients with tumors of various origin, and their presence is associated with disease progression and reduced survival. For this reason, monitoring this type of cell expansion is of clinical importance, and flow cytometry is the technique of choice for their identification. Over the years, it has been demonstrated that MDSCs comprise a group of immature myeloid cells belonging both to monocytic and granulocytic lineages, with several stages of differentiation; their occurrence depends on tumor-derived soluble factors, which guide their expansion and determine their block of differentiation. Because of their heterogeneous composition, accurate phenotyping of these cells requires a multicolor approach, so that the expansion of all MDSC subsets can be appreciated. This review article focuses on identifying MDSCs and discusses problems associated with phenotyping circulating and tumor-associated MDSCs in humans and in mouse models. This article is protected by copyright. All rights reserved.

Myeloid-derived suppressor cell heterogeneity in human cancers.

The dynamic interplay between cancer and host immune system often affects the process of myelopoiesis. As a consequence, tumor-derived factors sustain the accumulation and functional differentiation of myeloid cells, including myeloid-derived suppressor cells (MDSCs), which can interfere with T cell-mediated responses. Since both the phenotype and mechanisms of action of MDSCs appear to be tumor-dependent, it is important not only to determine the presence of all MDSC subsets in each cancer patient, but also which MDSC subsets have clinical relevance in each tumor environment. In this review, we describe the differences between MDSC populations expanded within different tumor contexts and evaluate the prognostic significance of MDSC expansion in peripheral blood and within tumor masses of neoplastic patients.

Highlights on molecular mechanisms of MDSC-mediated immune suppression: paving the way for new working hypotheses.

MDSCs have been recognized in the last years as tolerogenic cells, potentially dangerous in the context of neoplasia, since they are able to induce tolerance to a variety of anti-tumor effectors, including CD4(+) and CD8(+) T cells. It is currently believed that the origin of MDSCs is due to an arrest of the myeloid differentiation process caused by tumor-secreted factors released in the tumor microenvironment that are able to exert an effect on myeloid progenitors, rendering them unable to terminally differentiate into dendritic cells, granulocytes and macrophages. As a consequence, these immature myeloid cells acquire suppressive activity through the activation of several mechanisms, controlled by different transcription factors. The lack of consensus about the phenotypical characterization of human MDSCs is the result of the existence of different MDSC subsets, most likely depending on the tumor in which they expand and on the tumor specific cytokine cocktail driving their activation. This, in turn, might also influence the mechanisms of MDSC-mediated immune suppression. In this review article we address the role of tumor-derived factors (TDFs) in MDSC-recruitment and activation, discuss the complex heterogeneity of MDSC phenotype and analyze the crosstalk between activated T cells and MDSCs.

A human promyelocytic-like population is responsible for the immune suppression mediated by myeloid-derived suppressor cells.

We recently demonstrated that human BM cells can be treated in vitro with defined growth factors to induce the rapid generation of myeloid-derived suppressor cells (MDSCs), hereafter defined as BM-MDSCs. Indeed, combination of G-CSF + GM-CSF led to the development of a heterogeneous mixture of immature myeloid cells ranging from myeloblasts to band cells that were able to suppress alloantigen- and mitogen-stimulated T lymphocytes. Here, we further investigate the mechanism of suppression and define the cell subset that is fully responsible for BM-MDSC-mediated immune suppression. This population, which displays the structure and markers of promyelocytes, is however distinct from physiologic promyelocytes that, instead, are devoid of immuosuppressive function. In addition, we demonstrate that promyelocyte-like cells proliferate in the presence of activated lymphocytes and that, when these cells exert suppressive activity, they do not differentiate but rather maintain their immature phenotype. Finally, we show that promyelocyte-like BM-MDSCs are equivalent to MDSCs present in the blood of patients with breast cancer and patients with colorectal cancer and that increased circulating levels of these immunosuppressive myeloid cells correlate with worse prognosis and radiographic progression.

Antigen specificity of immune suppression by myeloid-derived suppressor cells.

Among the mechanisms set in motion by the tumor to escape the control of the immune system, MDSCs play a central role in inducing tolerance to a variety of anti-tumor effectors, including T lymphocytes. It has been demonstrated that MDSCs expand in tumor-bearing mice and in cancer patients, leading to an impairment of T cell reactivity against the tumor. However, as the presence of MDSCs is not correlated with a general immune suppression, it was advanced that a mechanism regulating the specificity of MDSC inhibition must be present. In this article, we review the literature showing that MDSCs exert their immune-suppressive function on Ag-specific T cell responses but at times, also on mitogen-activated T lymphocytes, therefore bypassing the Ag dependency. We propose that the features of MDSC-mediated immune suppression might be influenced not only by the specific microenvironment in which MDSCs expand and by the tumor characteristics but also by the levels of activation of the target lymphocytes.

Myeloid cell diversification and complexity: an old concept with new turns in oncology.

Tumour development is accompanied by an enhanced haematopoiesis. This is not a widespread activation since only cells belonging to the myelo-monocytic compartment are expanded and mobilized from primary sites of haematopoiesis to other organs, reaching also the tumour stroma. This process occurs early during tumour formation but becomes more evident in advanced disease. Far from being a simple, unwanted consequence of cancer development, accumulation of myelo-monocytitc cells plays a role in tumour vascularization, local spreading, establishment of metastasis at distant sites, and contribute to create an environment unfavourable for the adoptive immunity against tumour-associated antigens. Myeloid populations involved in these process are likely different but many cells, expanded in primary and secondary lymphoid organs of tumour-bearing mice, share various levels of the CD11b and Gr-1 (Ly6C/G) markers. CD11b(+)Gr-1(+) cells are currently named myeloid-derived suppressor cells for their ability to inhibit T lymphocyte responses in tumour-bearing hosts. In this manuscript, we review the recent literature on tumour-conditioned myeloid subsets that assist tumour growth, both in mice and humans.

Tumor-induced tolerance and immune suppression depend on the C/EBPbeta transcription factor.

Tumor growth is associated with a profound alteration in myelopoiesis, leading to recruitment of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs). We showed that among factors produced by various experimental tumors, the cytokines GM-CSF, G-CSF, and IL-6 allowed a rapid generation of MDSCs from precursors present in mouse and human bone marrow (BM). BM-MDSCs induced by GM-CSF+IL-6 possessed the highest tolerogenic activity, as revealed by the ability to impair the priming of CD8(+) T cells and allow long term acceptance of pancreatic islet allografts. Cytokines inducing MDSCs acted on a common molecular pathway and the immunoregulatory activity of both tumor-induced and BM-derived MDSCs was entirely dependent on the C/EBPbeta transcription factor. Adoptive transfer of tumor antigen-specific CD8(+) T lymphocytes resulted in therapy of established tumors only in mice lacking C/EBPbeta in the myeloid compartment, suggesting that C/EBPbeta is a critical regulator of the immunosuppressive environment created by growing cancers.

IL4Ralpha+ myeloid-derived suppressor cell expansion in cancer patients.

Myeloid-derived suppressor cells (MDSC) contribute to immune dysfunctions induced by tumors both in experimental models and patients. In mice, MDSC are phenotypically heterogeneous cells that vary in their surface markers, likely depending on soluble factors produced by different tumors. We recently described a subset of inflammatory monocytes with immunosuppressive properties that can be found within the tumor mass, blood, and lymphoid organs of tumor-bearing mice. These cells expressed the alpha-chain of the receptor for IL-4 (IL4Ralpha) that was critical for their negative activity on CD8(+) T cells. In cancer patients, the nature of MDSC is still poorly defined because evidence exists for both monocytic and granulocytic features. We show in this study that myeloid cells with immunosuppressive properties accumulate both in mononuclear and polymorphonuclear fractions of circulating blood leukocytes of patients with colon cancer and melanoma, thus unveiling a generalized alteration in the homeostasis of the myeloid compartment. Similarly to mouse MDSC, IL4Ralpha is up-regulated in both myeloid populations but its presence correlates with an immunosuppressive phenotype only when mononuclear cells, but not granulocytes, of tumor-bearing patients are considered.