Differential expansion of circulating human MDSC subsets in patients with cancer, infection and inflammation

Background Myeloid-derived suppressor cells (MDSC) are a functional myeloid cell subset that includes myeloid cells with immune suppressive properties. The presence of MDSC has been reported in the peripheral blood of patients with several malignant and non-malignant diseases. So far, direct comparison of MDSC across different diseases and Centers is hindered by technical pitfalls and a lack of standardized methodology. To overcome this issue, we formed a network through the COST Action Mye-EUNITER (www.mye-euniter.eu) with the goal to standardize and facilitate the comparative analysis of human circulating MDSC in cancer, inflammation and infection. In this manuscript, we present the results of the multicenter study Mye-EUNITER MDSC Monitoring Initiative, that involved 13 laboratories and compared circulating MDSC subsets across multiple diseases, using a common protocol for the isolation, identification and characterization of these cells. Methods We developed, tested, executed and optimized a standard operating procedure for the isolation and immunophenotyping of MDSC using blood from healthy donors. We applied this procedure to the blood of almost 400 patients and controls with different solid tumors and non-malignant diseases. The latter included viral infections such as HIV and hepatitis B virus, but also psoriasis and cardiovascular disorders. Results We observed that the frequency of MDSC in healthy donors varied substantially between centers and was influenced by technical aspects such as the anticoagulant and separation method used. Expansion of polymorphonuclear (PMN)-MDSC exceeded the expansion of monocytic MDSC (M-MDSC) in five out of six solid tumors. PMN-MDSC expansion was more pronounced in cancer compared with infection and inflammation. Programmed death-ligand 1 was primarily expressed in M-MDSC and e-MDSC and was not upregulated as a consequence of disease. LOX-1 expression was confined to PMN-MDSC. Conclusions This study provides improved technical protocols and workflows for the multi-center analysis of circulating human MDSC subsets. Application of these workflows revealed a predominant expansion of PMN-MDSC in solid tumors that exceeds expansion in chronic infection and inflammation

Проблемы увеличения продуктивности АПК в Украине и пути повышения его потенциала

Целью статьи является изучение причин снижения показателей продуктивности в агропромышленном комплексе и путей повышения продуктивности сельскохозяйственных культур

Nuclear receptor Nur77 deficiency alters dendritic cell function

Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system. Proper function of DCs is crucial to elicit an effective immune response against pathogens and to induce antitumor immunity. Different members of the nuclear receptor (NR) family of transcription factors have been reported to affect proper function of immune cells. Nur77 is a member of the NR4A subfamily of orphan NRs that is expressed and has a function within the immune system. We now show that Nur77 is expressed in different murine DCs subsets in vitro and ex vivo, in human monocyte-derived DCs (moDCs) and in freshly isolated human BDCA1+ DCs, but its expression is dispensable for DC development in the spleen and lymph nodes. We show, by siRNA-mediated knockdown of Nur77 in human moDCs and by using Nur77-/- murine DCs, that Nur77-deficient DCs have enhanced inflammatory responses leading to increased T cell proliferation. Treatment of human moDCs with 6-mercaptopurine, an activator of Nur77, leads to diminished DC activation resulting in an impaired capacity to induce IFNγ production by allogeneic T cells. Altogether, our data show a yet unexplored role for Nur77 in modifying the activation status of murine and human DCs. Ultimately, targeting Nur77 may prove to be efficacious in boosting or diminishing the activation status of DCs and may lead to the development of improved DC-based immunotherapies in, respectively, cancer treatment or treatment of autoimmune diseases

Elevated levels of polymorphonuclear myeloid-derived suppressor cells in patients with glioblastoma highly express S100A8/9 and arginase and suppress T cell function

BACKGROUND: Gliomas are primary brain tumors that are associated with a poor prognosis. The introduction of new treatment modalities (including immunotherapy) for these neoplasms in the last 3 decades has resulted in only limited improvement in survival. Gliomas are known to create an immunosuppressive microenvironment that hampers the efficacy of (immuno)therapy. One component of this immunosuppressive environment is the myeloid-derived suppressor cell (MDSC). METHODS: We set out to analyze the presence and activation state of MDSCs in blood (n = 41) and tumor (n = 20) of glioma patients by measuring S100A8/9 and arginase using flow cytometry and qPCR. Inhibition of T cell proliferation and cytokine production after stimulation with anti-CD3/anti-CD28 coated beads was used to measure in vitro MDSC suppression capacity. RESULTS: We report a trend toward a tumor grade-dependent increase of both monocytic (M-) and polymorphonuclear (PMN-) MDSC subpopulations in the blood of patients with glioma. M-MDSCs of glioma patients have increased levels of intracellular S100A8/9 compared with M-MDSCs in healthy controls (HCs). Glioma patients also have increased S100A8/9 serum levels, which correlates with increased arginase activity in serum. PMN-MDSCs in both blood and tumor tissue demonstrated high expression of arginase. Furthermore, we assessed blood-derived PMN-MDSC function and showed that these cells have potent T cell suppressive function in vitro. CONCLUSIONS: These data indicate a tumor grade-dependent increase of MDSCs in the blood of patients with a glioma. These MDSCs exhibit an increased activation state compared with MDSCs in HCs, independent of tumor grade

Elevated levels of polymorphonuclear myeloid-derived suppressor cells in patients with glioblastoma highly express S100A8/9 and arginase and suppress T cell function

Background Gliomas are primary brain tumors that are associated with a poor prognosis. The introduction of new treatment modalities (including immunotherapy) for these neoplasms in the last 3 decades has resulted in only limited improvement in survival. Gliomas are known to create an immunosuppressive microenvironment that hampers the efficacy of (immuno)therapy. One component of this immunosuppressive environment is the myeloid-derived suppressor cell (MDSC). Methods We set out to analyze the presence and activation state of MDSCs in blood (n = 41) and tumor (n = 20) of glioma patients by measuring S100A8/9 and arginase using flow cytometry and qPCR. Inhibition of T cell proliferation and cytokine production after stimulation with anti-CD3/anti-CD28 coated beads was used to measure in vitro MDSC suppression capacity. Results We report a trend toward a tumor grade-dependent increase of both monocytic (M-) and polymorphonuclear (PMN-) MDSC subpopulations in the blood of patients with glioma. M-MDSCs of glioma patients have increased levels of intracellular S100A8/9 compared with M-MDSCs in healthy controls (HCs). Glioma patients also have increased S100A8/9 serum levels, which correlates with increased arginase activity in serum. PMN-MDSCs in both blood and tumor tissue demonstrated high expression of arginase. Furthermore, we assessed blood-derived PMN-MDSC function and showed that these cells have potent T cell suppressive function in vitro. Conclusions These data indicate a tumor grade-dependent increase of MDSCs in the blood of patients with a glioma. These MDSCs exhibit an increased activation state compared with MDSCs in HCs, independent of tumor grade

Steering siglec–sialic acid interactions on living cells using bioorthogonal chemistry

Sialic acid sugars that terminate cell‐surface glycans form the ligands for the sialic acid binding immunoglobulin‐like lectin (Siglec) family, which are immunomodulatory receptors expressed by immune cells. Interactions between sialic acid and Siglecs regulate the immune system, and aberrations contribute to pathologies like autoimmunity and cancer. Sialic acid/Siglec interactions between living cells are difficult to study owing to a lack of specific tools. Here, we report a glycoengineering approach to remodel the sialic acids of living cells and their binding to Siglecs. Using bioorthogonal chemistry, a library of cells with more than sixty different sialic acid modifications was generated that showed dramatically increased binding toward the different Siglec family members. Rational design reduced cross‐reactivity and led to the discovery of three selective Siglec‐5/14 ligands. Furthermore, glycoengineered cells carrying sialic acid ligands for Siglec‐3 dampened the activation of Siglec‐3+ monocytic cells through the NF‐κB and IRF pathways

Increase in Both CD14-Positive and CD15-Positive Myeloid-Derived Suppressor Cell Subpopulations in the Blood of Patients with Glioma but Predominance of CD15-Positive Myeloid-Derived Suppressor Cells in Glioma Tissue

Myeloid-derived suppressor cells (MDSCs), defined as CD33-positive major histocompatibility complex class II-negative cells, are increased in a variety of human tumors and are associated with immunosuppression. Myeloid-derived suppressor cells can be further subdivided into CD14-positive monocytic MDSC and CD15-positive granulocytic MDSC (polymorphonuclear MDSC) subpopulations. Here we analyzed MDSC subsets in the blood and tumor tissue of patients with glioma, including the most malignant variant, glioblastoma multiforme (GBM). CD33-positive major histocompatibility complex class II-negative MDSCs in blood from 21 patients with glioma and 12 healthy individuals were phenotyped and quantified by flow cytometry. Myeloid populations of the monocytic MDSC and polymorphonuclear MDSC phenotypes were both significantly increased in the blood of patients with GBM versus healthy controls. The myeloid activation markers CD80 and PD-L1 could not be detected on either of these MDSC subsets; CD124, CD86, and CD40 were detected at similar levels on MDSCs in patients with glioma and healthy donors. By contrast, in tumor cell suspensions, the MDSC population consisted almost exclusively of CD15-positive cells. Immunohistochemistry confirmed infiltration of CD15-positive major histocompatibility complex class II-negative cells in glioma tissue samples. These data support a role for cells with an MDSC phenotype in the blood and tumor microenvironment of patients with GBM

Type I and type III IFN responses are induced in mDC subsets upon encounter of CVB-infected cells.

<p><b>A</b>) DCs were co-cultured with mock- or CVB-infected Min6 cells (M6/M or M6/CVB), infected with CVB3 (MOI 50), stimulated with poly I:C, or left unstimulated (medium; Med) and after o/n incubation supernatant was harvested and analyzed for production of IFN-α2 (left panel) or IFN-λ1 (right panel). Shown are data from 4 (IFN-α2) and 3 (IFN-λ1) different donors. <b>B</b>) DCs were stimulated as in A) and after 6 hours mRNA expression was analyzed by qPCR. Shown are data from 4 donors (B, corresponding symbols represent the same donor). * p<0.05, ** p<0.01, *** p<0.001 determined by ANOVA and post-hoc Tukey test.</p

BDCA1⁺ mDCs more efficiently phagocytose murine Min6 cells compared to BDCA3⁺ mDCs.

<p><b>A</b>) BDCA1⁺ mDCs or <b>B</b>) BDCA3⁺ mDCs were co-cultured overnight (o/n) with PKH67-labeled mock- or CVB-infected Min6 cells (M6/M and M6/CVB, respectively) stained for the CD11c or BDCA3 and analyzed by flow cytometry on viable, single cells (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121670#pone.0121670.s001" target="_blank">S1 Fig</a>). Percentages in upper right corner represent the percentage of DCs that has engulfed Min6 material. This was calculated as follows: percentage PKH⁺ DCs/total DCs (i.e. total CD11c positive cells or total BDCA3 positive cells) <b>C</b>) DCs were analyzed as in A) and B), shown is average + SEM for >3 donors. * p<0.05, *** p<0.001 determined by ANOVA and post-hoc Tukey test.</p

Cytokine and chemokine production in BDCA1⁺ and BDCA3⁺ mDCs upon encounter of mock- or virus-infected cells.

<p>DCs were stimulated o/n as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121670#pone.0121670.g002" target="_blank">Fig. 2A</a> and supernatant was analyzed for production of pro-inflammatory cytokines and chemokines. Data represent average of 4 independent experiments using different donors + SEM. * P<0.05, ** P<0.01, ***p<0.001 comparing M6/CVB versus M6/M or poly I:C versus medium; # p<0.05 Min6/CVB versus poly I:C; $ p<0.05 in BDCA1⁺ versus BDCA3⁺ mDCs with the same stimulus as determined by ANOVA and post-hoc Tukey test.</p