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Simple Summary Glioblastoma (GBM) is the most aggressive type of brain cancer and, despite standard treatments, recurrence is inevitable. The immunosuppressive microenvironment, characterized by an intense recruitment of myeloid cells and a low frequency of anti-tumor lymphocytes, hampers the success of new immunological therapies. Thus, understanding how treatments impact the tumor microenvironment is crucial to limit recurrences. In this study, we compared the immune profile in the central and in the marginal areas of resected tumors on a cohort of patients with primary or relapsing GBM and identified a different immune composition according to tumor localization. In addition, levels of four subsets of myeloid-derived suppressor cells were determined before and after standard therapy. Significant correlations were obtained by combining data collected from tumor and blood, thus reinforcing the notion that immunosuppression should be evaluated both in the circulation and in the tumor microenvironment, to circumvent this phenomenon from a therapeutic point of view. Abstract The cell composition of the glioblastoma (GBM) microenvironment depends on the recruitment of myeloid cells from the blood, promoting tumor progression by inducing immunosuppression. This phenomenon hampers immunotherapies and investigating its complexity may help to tailor new treatments. Peripheral blood and tissue specimens from the central and marginal tumor areas were collected from 44 primary and 19 recurrent GBM patients. Myeloid and lymphoid cell subsets and the levels of immunosuppressive markers were defined by multiparametric flow cytometry. Multiplexed immunohistochemistry was used to confirm the differences in the immune infiltrate and to analyze the cell spatial distribution. Relapsing GBM showed an increased presence of blood-derived macrophages in both tumor areas and a higher frequency of infiltrating lymphocytes, with a high level of exhaustion markers. The expansion of some myeloid-derived suppressor cell (MDSC) subsets in the blood was found in both primary and recurrent GBM patients. A significant inverse correlation between infiltrating T cells and an MDSC subset was also found. In patients with recurrent GBM after standard first-line therapy, the immune-hostile tumor microenvironment and the levels of some MDSC subsets in the blood persisted. Analysis of the immune landscape in GBM relapses aids in the definition of more appropriate stratification and treatment.

Background Although gliomas are strictly confined to the central nervous system, their negative influence over the immune system can extend to peripheral circulation. The immune suppression exerted by myeloid cells is capable of affecting both response to therapy and disease outcome. Here we analyzed the expansion of several myeloid parameters in the blood of low- and high-grade gliomas and assessed their relevance as biomarkers of disease and clinical outcome. Methods Peripheral blood was obtained from 134 low- and high-grade glioma patients before surgery and treatment. Myeloid cell subsets such as total CD14+, CD14+/p-STAT3+, CD14+/PD-L1+, CD15+ cells and 4 myeloid derived suppressor cell (MDSC) subsets, were evaluated by multiparametric flow cytometry. Arginase-1 (ARG1) quantity and activity was determined in the plasma. Principal component analysis was performed to define correlations between myeloid markers. Multivariable logistic regression model was used to obtain a diagnostic score to discriminate glioma patients from healthy controls, and between each glioma grade. A glioblastoma prognostic model was determined by multiple Cox regression using clinical and myeloid parameters. Results In the blood of glioma patients, changes in myeloid parameters associated with immune suppression were identified and allowed us to define a diagnostic score calculating the risk of being a glioma patient, that included CD15+ cells, MDSC1, MDSC3, p-STAT3 and ARG1 activity. Of note, the same parameters, together with age, can also be used to calculate the risk score in differentiating each glioma grade. Finally, a prognostic model for glioblastoma patients stemmed out from a Cox multiple analysis, highlighting the role of MDSC, p-STAT3 and ARG1 activity together with clinical parameters in predicting the patient outcome. Conclusions This work emphasizes the role of systemic immune suppression carried out by myeloid cells in gliomas. The identification of biomarkers associated with immune landscape, diagnosis and outcome of glioblastoma patients lays the ground for their clinical use for stratification and follow up.

An amendment to this paper has been published and can be accessed via the original article.

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.

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 toinhibit 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 onthe role of age-related processes in myelopoiesis.

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. Electronic supplementary material The online version of this article (10.1186/s40425-019-0536-x) contains supplementary material, which is available to authorized users.

Introduction: Pembrolizumab demonstrated promising results in hypermutated tumors of diverse origin. Immunohistochemical loss of mismatch repair (MMR) proteins has been suggested as a surrogate of hypermutation in high-grade gliomas (HGG). We evaluated the efficacy and safety of pembrolizumab in relapsing HGGs with immunohistochemical loss of at least 1 MMR protein. Molecular biomarkers of pembrolizumab activity were also analyzed. Methods: Consecutive patients with recurrent HGG and partial or complete loss of MMR protein expression were prospectively enrolled, they received pembrolizumab 200 mg once every 3 weeks until disease progression. The primary endpoint was disease control rate (DCR). Post hoc exploratory analyses included next-generation sequencing to assess tumor mutational burden (TMB), and immunostaining for CD8+ T-cells and CD68+ macrophages. Results: Among 310 HGG patients screened, 13 cases with MMR loss were enrolled: eight glioblastoma, four anaplastic astrocytoma, and one anaplastic oligodendroglioma. Median age was 43 years. DCR was 31%: four patients had stable disease and no patient had complete or partial response. TMB ranged between 6.8 and 23.4 mutations/megabase. Neither TMB nor gene mutations, nor CD8+ T-cell and CD68+ macrophage content, were associated with pembrolizumab activity. Conclusions: pembrolizumab showed no apparent benefit in these patients. No molecular biomarker was found to be associated with pembrolizumab activity.

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.