Proliferation Of Myeloid-derived Suppressor Cells Research Articles

Yu-Ping-Feng Formula Exerts Antilung Cancer Effects by Remodeling the Tumor Microenvironment through Regulating Myeloid-Derived Suppressor Cells.

Yu-Ping-Feng (YPF) formula is a classical prescription used for enhancing the body's immunity function in traditional Chinese medicine (TCM). In clinical practice, the YPF formula has been reported to exhibit antilung cancer and immunomodulatory effect. However, the relationship between them remains unclear. The present study aimed to investigate the antilung cancer effect of the YPF formula and its immune-related mechanisms. The C57BL/6 tumor-bearing mice model was established and randomly divided into the YPF group and the control group. Tumor volume, spleen weight, and survival in both groups were measured and evaluated during 28 days of consecutive intervention. Flow cytometry was used to detect the proportion of immune cell subsets. Myeloid-derived suppressor cells (MDSCs) were induced in vitro from bone marrow cells. After intervention by the YPF formula, CCK-8 and flow cytometry analyses were performed to detect proliferation and apoptosis of MDSCs. A coculture system containing T cells and MDSCs was established to further study the role of MDSCs in the regulation of T-cell subsets proportion by the YPF formula. The expressions of MDSCs-related genes and proteins were detected by RT-PCR and Western blotting. The results showed the YPF formula inhibited tumor growth, reduced spleen weight, and prolonged the survival of mice. Besides, the proportions of MDSCs subsets and Regulatory T (Treg) in the YPF group decreased, whereas those of CD4+T and CD8+T increased both in vitro and in vivo. CCK-8 and flow cytometry demonstrated that the YPF formula could inhibit proliferation and promote apoptosis of MDSCs. The coculture experiments further confirmed that MDSCs served a critical role in regulating the tumor microenvironment by the YPF formula. RT-PCR and Western blotting indicated that the levels of MDSCs' activation and proliferation-related proteins and genes were downregulated in the YPF group. Therefore, our results demonstrated that the YPF formula could promote apoptosis and inhibit the proliferation of MDSCs. As a result, the negative regulatory effect on the positive immune cells induced by MDSCs was weakened, thus achieving the antilung cancer effect by remodeling the tumor microenvironment.

Single-Cell RNA-seq of Human Myeloid-Derived Suppressor Cells in Late Sepsis Reveals Multiple Subsets With Unique Transcriptional Responses: A Pilot Study.

Increased circulating myeloid-derived suppressor cells (MDSCs) are independently associated with poor long-term clinical outcomes in sepsis. Studies implicate subsets of MDSCs having unique roles in lymphocyte suppression; however, characterization of these cells after sepsis remains incomplete. We performed a pilot study to determine the transcriptomic landscape in MDSC subsets in sepsis using single-cell RNAseq (scRNA-seq). A mixture of whole blood myeloid-enriched and Ficoll-enriched PBMCs from two late septic patients on post-sepsis day 21 and two control subjects underwent Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq). We successfully identified the three MDSC subset clusters-granulocytic (G-), monocytic (M-), and early (E-) MDSCs. Sepsis was associated with a greater relative expansion of G-MDSCs versus M-MDSCs at 21 days as compared to control subjects. Genomic analysis between septic patients and control subjects revealed cell-specific and common differential expression of genes in both G-MDSC and M-MDSC subsets. Many of the common genes have previously been associated with MDSC proliferation and immunosuppressive function. Interestingly, there was no differential expression of several genes demonstrated in the literature to be vital to immunosuppression in cancer-induced MDSC. This pilot study successfully demonstrated that MDSCs maintain a transcriptomic profile that is immunosuppressive in late sepsis. Interestingly, the landscape in chronic critical illness is partially dependent on the original septic insult. Preliminary data would also indicate immunosuppressive MDSCs from late sepsis patients appear to have a somewhat unique transcriptome from cancer and/or other inflammatory diseases.

Increased Galectin-9 expression, a prognostic biomarker of aGVHD, regulates the immune response through the Galectin-9 induced MDSC pathway after allogeneic hematopoietic stem cell transplantation

Galectin-9 (Gal-9) is a β-galactoside-binding soluble lectin family member that exerts its primary biological functions via specific glycoconjugate interactions. Gal-9 expression is closely related to tumor occurrence, development, metastasis and prognosis. In transplant immunology, a high level of Gal-9 expression has been shown to markedly reduce the severity of acute graft rejection and effectively prolong survival time in organ and bone marrow transplantation (BMT) models. The main mechanism of Gal-9-mediated immunoregulation involves the Tim-3/Gal-9 axis in T cells. However, myeloid-derived suppressor cell (MDSC) accumulation in transgenic mice with persistently high Gal-9 expression was observed in a model of lung inflammation, indicating that a potential immunosuppressive mechanism distinct from the Gal-9/Tim-3 axis might exist. In the present study, increased Gal-9 expression and MDSC frequencies before acute graft-versus-host disease (aGVHD) onset were observed in patients who developed aGVHD. Patients with higher Gal-9 expression (≥14.8417 ng/ml) exhibited reduced overall survival and increased cumulative incidences of GVHD at +100 day. We considered the elevated Gal-9 expression before aGVHD onset a secondary inflammatory response. This increase might be part of a negative feedback pathway corresponding to aGVHD pathogenesis. Additionally, a high Gal-9 concentration induced MDSC proliferation in vivo and in vitro. Gal-9-induced MDSCs (G9-MDSCs) suppressed T cell proliferation and activation. An infusion of G9-MDSCs into a graft contributed to the successful control of severe aGVHD and long-term survival in an allogeneic (allo)-BMT mouse model. Thus, we speculated that increased Gal-9 expression after allo-hematopoietic stem cell transplantation is a potential prognostic biomarker of aGVHD. The Gal-9-associated immunosuppressive effects on aGVHD development might occurr through G9-MDSCs and were independent of the Gal-9/Tim-3 axis.

Umbilical cord blood-derived mesenchymal stromal cells promote myeloid-derived suppressor cell proliferation by secreting HLA-G to reduce acute graft-versus-host disease after hematopoietic stem cell transplantation

Background aimsMesenchymal stem cells (MSCs) use multiple mechanisms to constrain both innate and adaptive immune responses to prevent graft-versus-host disease (GVHD). Myeloid-derived suppressor cells (MDSCs), as a heterogeneous population of early myeloid progenitor cells originating from bone marrow, are a naturally occurring immune regulatory population associated with inhibition of ongoing inflammatory responses, indicating their potential for GVHD therapy. There is accumulating evidence that MSCs and MDSCs do not act independently, but rather establish crosstalk. However, the role of MSCs in MDSC expansion and activation in GVHD remains unexplored. MethodsIn vitro experiments included 2 groups: peripheral blood mononuclear cells (PBMCs) after mobilization and human umbilical cord blood-derived MSCs (UCB-MSCs) co-cultured with PBMCs. The number and functional difference of MDSCs in PBMCs were determined by flow cytometry. The culture supernatants of co-cultured cells were analyzed to identify cytokines involved in MDSC proliferation. The relationship between MSCs and MDSCs was clarified in GVHD and graft-versus-leukemia (GVL) animal models. ResultsIn vitro experiments confirmed that UCB-MSCs secreted HLA-G protein to promote and maintain the proliferation of MDSCs in peripheral blood after granulocyte colony-stimulating factor mobilization, and UCB-MSCs mediated the function of MDSCs to inhibit the proliferation of T cells and promote the proliferation of regulatory T cells. UCB-MSCs overexpressing HLA-G induced MDSC production in recipient mice, improved the ability of MDSCs to suppress T cells and further reduced acute GVHD (aGVHD) symptoms and survival time without influencing GVL effects. ConclusionsUCB-MSCs expanded MDSCs via HLA-G/Ig-like transcript 4, reducing the severity of aGVHD without affecting GVL. The immunosuppressive potential of MSCs for the treatment of aGVHD significantly affects the development of MDSCs, thereby consolidating the position of MSCs in the prevention and treatment of aGVHD.

Anticancer Effects of ACT001 via NF-κB Suppression in Murine Triple-Negative Breast Cancer Cell Line 4T1.

PurposeACT001 is a novel sesquiterpene lactone derivative with anticancer effects, including the reversal of tamoxifen resistance in estrogen receptor-positive breast cancer cells. However, few studies have investigated the anticancer effects of ACT001 in triple-negative breast cancer (TNBC), a highly aggressive cancer with a poor prognosis. This study aimed to investigate the effects of ACT001 on TNBC and the potential mechanism underlying these effects.Materials and MethodsThe anticancer effects of ACT001 on the murine TNBC cell line 4T1 were evaluated by Cell Counting Kit-8 assay, animal experiments, TUNEL staining, flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay, and Western blotting analysis.ResultsACT001 induced apoptosis in 4T1 cells by upregulating B cell lymphoma 2-associated X protein expression. Moreover, ACT001 markedly decreased levels of secretory granulocyte-macrophage colony stimulating factor (GM-CSF) in 4T1 tumors, decreased the number of myeloid-derived suppressor cells (MDSCs), and reduced angiogenesis. Furthermore, GM-CSF promoted angiogenesis and the proliferation of MDSCs in a dose-dependent manner. Finally, ACT001 suppressed phospho-NF-κB and IκB-α levels in 4T1 cells, thereby further decreasing GM-CSF levels.ConclusionOur results suggest that ACT001 exerts its anticancer effects by inducing apoptosis in murine TNBC cell line 4T1 and regulates the tumor microenvironment by attenuating angiogenesis and accumulation of MDSCs in 4T1 tumors. The underlying mechanism may involve the suppression of NF-κB activity.

Hypofractionated Irradiation Suppressed the Off-Target Mouse Hepatocarcinoma Growth by Inhibiting Myeloid-Derived Suppressor Cell-Mediated Immune Suppression.

Background: Stereotactic radiotherapy treats hepatocellular carcinoma (HCC) at different stages effectively and safely. Besides its direct killing of cancer cells, radiotherapy stimulates host immunity against hepatoma. However, the role of myeloid-derived suppressor cells (MDSCs) in on-target and off-target anti-HCC effects induced by hypofractionated irradiation (IR) is unclear.Methods and Materials: Hepa1-6 and H22 allogeneic transplanted tumors on hind limbs of C57BL/6 and Institute of Cancer Research (ICR) mice, respectively, were irradiated with 0, 2.5, 4, 6, or 8 Gy/fraction until the total dose reached 40 Gy. The off-target effect induced by the IR was investigated by subsequently inoculating the same HCC cells subcutaneously on the abdomen. MDSCs in peripheral blood and tumor tissues were measured by flow cytometry or immunofluorescence microscopy analysis. IL-6, regulated on activation normal T cell expressed and secreted (RANTES), and granulocyte colony-stimulating factor (G-CSF) in irradiated mouse plasma and hepatoma cell cultures were measured with ELISA kits. Conditioned media (CM) from irradiated HCC cell cultures on bone marrow cell differentiation and MDSC proliferation were examined by co-culture and flow cytometry.Results: Our study showed that the IR of primarily inoculated HCC on hind limbs created an “in situ tumor vaccine” and triggered the antitumor immunity. The immunity was capable of suppressing the growth of the same type of HCC subcutaneously implanted on the abdomen, accompanied with reduced MDSCs in both blood and tumors. The decreased MDSCs were associated with low plasma levels of IL-6, RANTES, and G-CSF. The cytokines IL-6 and RANTES in the CM were lower in the high single IR dose group than in the control groups, but G-CSF was higher. The CM from high single-dose IR-Hepa1-6 cell culture reduced the differentiation of C57BL/6 mouse bone marrow cells into MDSCs, whereas CM from high single-dose IR-H22 cells reduced the proliferation of MDSCs, which might be due to the decreased p-STAT3 in bone marrow cells.Conclusions: The hypofractionated IR on transplanted tumors at the primary location exerted a strong antitumor effect on the same tumor at a different location (off target). This abscopal effect is most likely through the reduction of MDSCs and decrease of IL-6, RANTES, and G-CSF.

Immune function of myeloid-derived suppressor cells and its mechanism in obstructive sleep apnea syndrome

Objective: To investigate the immune function of myeloid-derived suppressor cells (MDSC) and its mechanism in obstructive sleep apnea syndrome (OSAS). Methods: Twenty OSAS patients who were diagnosed by polysomnography (Apnea Hyponea Index>30 events/h) from Sleep Disorders Center at First Affiliated Hospital between January 2015 and December 2016 were selected. The percent of CD14(+) low expression or lack of human leukocyte antigen DR (HLA-DR(-/low)) MDSC in the CD14(+) monocyte from both OSAS patients and healthy people were analyzed by flow cytometry. In vitro assay, MDSC from OSAS patients and health people were sorted by flow cytometry and T cells were sorted with negative isolation kit. For T-cell proliferation assays, the carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled T cells were respectively incubated with autologous MDSC. CFSE fluorescence intensity of T cells was detected by flow cytometry. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis were used to evaluate the concentrations of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), IL-10, transforming growth factor-β(1) (TGF-β(1)), positive rate of programmed death ligand-L1 (PD-L1), relative transcript level of Arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), hypoxic inducible factor-1α (HIF-1α) expressed by MDSC. Results: Comparing to healthy people, the percentage of CD14(+)HLA-DR(-/low) MDSC in CD14(+) monocyte was significantly elevated [(12.5±1.5)% vs (3.5±0.4)%, P<0.05]. In vitro, OSAS patient-derived MDSC exhibited a stronger suppressive effect on T-cell proliferation [(23.2±1.1)% vs (53.7±3.2)%, P<0.05]. Further analysis revealed that OSAS patient-derived MDSC secreted much higher concentrations of IL-6, TNF-α, IL-10 and TGF-β(1) [(1 316±163) vs (642±72) ng/L, (316±35) vs (167±18) ng/L, (385±42) vs (108±26) ng/L and (44 276±4 589) vs (9 557±1 124) ng/L] (all P<0.05). The percentage of membrane molecule PD-L1-positive cells in OSAS patient-derived MDSC was obviously higher than that in healthy people-derived MDSC [(75.6±7.9)% vs (30.6±2.5)%, P<0.05]. Compared with healthy people-derived MDSC, the relative transcript level of Arg1, iNOS and HIF-1α in OSAS patient-derived MDSC was also increased by (4.6±0.5), (2.8±0.3) and (4.3±0.4) fold, respectively (all P<0.05). Conclusions: OSAS may be capable of inducing the proliferation of MDSC and its expression of immunosuppressive molecules by activating HIF-1α signal, thereby enhancing the immunosuppressive ability of MDSC.

Deciphering the Crosstalk Between Myeloid-Derived Suppressor Cells and Regulatory T Cells in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with rising incidence and a remarkable resistance to current therapies. The reasons for this therapeutic failure include the tumor's extensive infiltration by immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). By using light sheet fluorescent microscopy, we identified here direct interactions between these major immunoregulatory cells in PDAC. The in vivo depletion of MDSCs led to a significant reduction in Tregs in the pancreatic tumors. Through videomicroscopy and ex vivo functional assays we have shown that (i) MDSCs are able to induce Treg cells in a cell-cell dependent manner; (ii) Treg cells affect the survival and/or the proliferation of MDSCs. Furthermore, we have observed contacts between MDSCs and Treg cells at different stages of human cancer. Overall our findings suggest that interactions between MDSCs and Treg cells contribute to PDAC immunosuppressive environment.

The Interaction of Tumor Cells and Myeloid-Derived Suppressor Cells in Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease characterized by the formation of the BCR-ABL fusion gene. At present, basic studies of the pathogenesis of relapse after stopping tyrosine kinase inhibitors (TKIs) treatment have mainly concentrated on two main aspects: the leukemia stem cells (LSCs) and the tumor microenvironment. However, whether relapse or non-relapse patients who discontinued TKIs therapy, LSCs are still exist. Among a variety of factors that compose the CML microenvironment, myeloid-derived suppressor cells (MDSC) are considered to be a strong contributor to the immunosuppressive tumor microenvironment. Here, we designed the study to investigate the potential relation between tumor cells and MDSC in CML and find risk factors for relapse after discontinuation. We detected the percentage of MDSC and the BCR-ABL (IS) transcript levels in bone marrow of 50 CML patients in chronic phase at our center. The data indicated that the frequency of MDSC had significant positive correlation with BCR-ABL (IS) transcript levels (Figure 1A). In addition, the counts of MDSC had significant difference at different response stages (Figure 1B), especially the M-MDSC, a subtype of MDSC. The percentage of M-MDSC was significantly higher in patients with newly diagnosed or complete hematological response (CHR) or major molecular response (MMR) compared with those of CML patients obtained complete molecular response (CMR) (Figure 1C). When K562 cells or CD34+ cells were cocultured with M-MDSC at a 1:10 ratio, K562 cells or CD34+ cells proliferated significantly at day 3 (Figure 1D and E). K562 subcutaneous tumor formation in BALB/c node mice confirmed that tumors weight and volume of the coculture group were higher than control. Then, we further investigated whether tumor cells have an impact on MDSC through microvesicles (MV). After adding K562-MV to peripheral blood mononuclear cells (PBMCs) from healthy donors, MDSC counts appeared significantly elevated in the different K562 cells counts group compared to the control group (Figure 1F).To analyze the roles of K562-MV collected before and after TKIs discontinuation on MDSC, we established a TKIs discontinuation model using the K562 cell, which emulates the cessation of TKIs treatment of CML patients in some extent. The results showed that regardless of the Imatinib or Dasatinib treatment, a significant increase was observed in the proportion of MDSC after TKIs treatment cessation compared with the TKIs treatment groups (Figure 1G). Experiments in vivo also proved K562-MV after different treatments promoted the proliferation of MDSC (Figure 1H and I). In conclusion, our study introduces the notion of the role of MDSC as mediators in the cross talk between tumor cells and the microenvironment. MDSC would provide a novel and useful model to predict the relapse of CML by establishing a type of new risk stratification system. MDSC could be also act as a promising target in the relapse of CML. In addition, we found a mutual promotion of proliferation of tumor cells and MDSC, this bidirectional interaction results in a vicious cycle by providing a protective niche against immune attacks. Therapeutic interventions modulating this interaction might accelerate the success of treatment-free remission. Figure 1 Disclosures No relevant conflicts of interest to declare.

Contribution of Myeloid-Drived Suppressor Cells to Leukemia Relapse in Patients with B-ALL Who Underwent Allo-HSCT

Background:B cell acute lymphoblastic leukemia (B-ALL) is a clonal hematopoietic stem cell neoplasm. Despite advances in its therapeutic strategies, including induction chemotherapy followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT), relapse remains the major barrier to the improvement of survival. Unfortunately, the underlying mechanism of relapse in B-ALL is largely unknown. Myeloid-derived suppressor cell (MDSC), a relatively novel population of immunomodulatory cells, has been identified to be associated with the onset of solid tumor and poor prognosis. However, the role of MDSCs played in prompting B-ALL relapse is vague. Aims:The present study was performed to explore the association between the percentage of MDSCs in bone marrow (BM) and B-ALL relapse and to investigate the effects of MDSCs on relapse of B-ALL. Methods:Firstly, twenty relapsed B-ALL patients and 10 non-relapsed patients after allo-HSCT as well as 14 healthy donors were enrolled. The percentage of CD33+HLA-DR-CD11b+ MDSCs in BM was analyzed by flow cytometry (FCM). Secondly, we co-cultured GFP+ Nalm-6 cells (a B cell precursor leukemia cell line initiated from an adolescent male) with MDSCs depleted bone marrow mononuclear cells (BMMCs) or replete of MDSCs from relapsed B-ALL patients post-transplantation. Then, we tested the direct effects of MDSCs on leukemia cells proliferation by co-culturing Nalm-6 cells with MDSCs. In addition, the effects of MDSCs on the functions of T cells and natural killer (NK) cells were also investigated. Briefly, CD3+ T cells or CD3-CD56+ NK cells purified from BMMCs via FCM were co-cultured with the same donor's CD11b+HLA-DR-CD33+ MDSCs using anti-CD3/CD28 beads, PMA or K562 cells as activators. The functional indexes of T cells or NK cells, including proliferation, IFN-γ, CD107a, Granzyme B, and perforin were detected by FCM. Furthermore, we analyzed the expression levels of immune response regulating proteins, including Foxp3 and PD-1 in T cells co-cultured with MDSCs. Finally, we detected the cytokines regulating MDSCs proliferation and activation in the sera of BM from relapsed B-ALL patients after allo-HSCT . Results:We demonstrated an increased percentage of MDSCs in BM of relapsed patients compared with paired non-relapsed cases (n=10 vs. 10, 28.76% vs. 5.18%, P=0.0068) and healthy donors (n=20 vs. 14, 22.76% vs. 6.57%, P=0.0007). In vitro experiments showed that the MDSCs sorted from relapsed patients' BM enhanced the proliferation of tumor cells (n=3, the mean number of GFP+Nalm-6 cells in co-culture with MDSCs group 2.2×104, vs. without MDSCs group 1.2×104, P=0.0447) in a BMMCs condition rather than a co-culture of tumor cells with MDSCs directly. MDSCs obtained from relapsed B-ALL patients' BM could suppress T cell proliferation and IFN-γ production in a cell dose-dependent manner. The percentages of proliferated CD4+T cells were 89.5%, 61.2%, 49.7%, 7.1% at a ratio of T: MDSC 1:0, 1:0.5, 1:1, 1:2 respectively (n=3, P&lt;0.05); The percentages of proliferated CD8+T cells were 74.7%, 57.6%, 37.8%, 10.6% at a ratio of T: MDSC 1:0, 1:0.5, 1:1, 1:2 respectively (n=3, P&lt;0.05). MDSCs from relapsed patients suppressed the cytotoxicity of NK cells. The percentages of IFN-γ+,CD107a+ andGranzyme B+ cells among NK cells were significantly lower in co-culture with MDSCs group than in without MDSCs group (n=4, IFN-γ+ cells, mean 0.6 fold, P=0.0237; CD107a+ cells, mean 0.8 fold, P=0.0242; Granzyme B+ cells, mean 0.8 fold, P=0.0326). We revealed that MDSCs sorted from relapsed patients' BMs induced PD-1 expression on T cells. The percentages of PD-1+ cells among CD4+ cells and CD8+ cells were both significantly higher in co-culture with MDSCs group than in without MDSCs group (n=6, among CD4+ cells, mean 1.8 fold, P=0.0342; among CD8+ cells, mean 1.4 fold, P=0.0041), which may contribute to the dysfunction of T cells. In addition, we found that the mean levels (ng/L) of IL-17A, IL-21, and IL-22 were significantly higher in the sera of BM from relapsed patients than in non-relapsed patients (n=5 vs. 4, IL-17A, 46.9 vs. 6.1, P=0.0077; IL-21, 31.8 vs. 5.6, P=0.0078;IL-22, 132.7 vs. 20.7, P=0.0050). Conclusions:Our results suggest the increased MDSCs might contribute to the relapse of B-ALL via promoting leukemia proliferation, suppressing the function of T cells and NK cells. Our findings suggest that MDSC could be served as a potential therapeutic target for this disease. Disclosures No relevant conflicts of interest to declare.

The interaction of tumor cells and myeloid-derived suppressor cells in chronic myelogenous leukemia

Myeloid-derived suppressor cells (MDSCs) are considered to be a strong contributor to the immunosuppressive tumor microenvironment. In our study, the counts of MDSCs were correlated with the remission status of CML patients, especially the M-MDSCs. M-MDSCs promoted the proliferation of K562 cells or CD34+ cells from newly diagnosed CML patients, no matter in cells or mice experiments. We also established a TKI discontinuation model using the K562 cell line for examining the effect of microvesicles (MVs) derived from K562 cells before and after TKI discontinuation on MDSCs. We found a mutual promotion of proliferation of tumor cells and MDSCs. Moreover, MVs derived from K562 cells after TKI discontinuation significantly improved the proliferation of MDSCs compared with MVs from before TKI discontinuation. The bidirectional interaction results in a vicious cycle, by providing a protective niche against immune attacks. Therapeutic interventions modulating this interaction might accelerate the success of TFR.

TRAF3 is a critical regulator of the expansion of myeloid-derived suppressor cells in mice

Abstract TRAF3 is an adaptor protein and E3 ligase that regulates the signal transduction pathways of the TNFR superfamily, pattern recognition receptors, and cytokine receptors. We previously showed that specific deletion of TRAF3 in myeloid cells leads to spontaneous inflammation and tumor development in aging M-TRAF3−/− mice, which is consistently associated with a striking expansion of CD11b+Gr1+ myeloid cells. Here we report that these CD11b+Gr1+ cells were myeloid-derived suppressor cells (MDSCs), as they exhibited a strong suppressive activity on CD8 T cell proliferation and also displayed a nuclear morphology of immature myeloid cells. Using a chronic inflammation model induced by repeated injections of heat-killed Bacillus Calmette-Guérin (BCG), we showed that young adult M-TRAF3−/− mice also exhibited hyperexpansion of MDSCs after BCG injections. Our in vivo BrdU labeling experiments revealed that MDSC hyperexpansion in M-TRAF3−/− mice was mainly due to the increased proliferation of MDSCs or progenitor cells in the spleen but not in the bone marrow (BM). Interestingly, TRAF3 does not appear to be an intrinsic regulator of the development, survival or proliferation of MDSCs, as suggested by the results of cultured BM progenitor cells and purified splenic MDSCs, in the absence or presence of CSFs or agonists of TLRs or CD40. Furthermore, we identified environmental factors that induced the aberrant expansion of MDSCs in M-TRAF3−/− mice using cytokine protein array analyses, which were further verified by macrophage cultures and in vivo neutralization studies. Taken together, our findings identify TRAF3 as a new regulator of MDSC expansion, and therefore also a critical regulator of chronic inflammation and immunosuppression.

Novel modulation on myeloid-derived suppressor cells (MDSCs) by methionine encephalin (MENK)

The purpose of this study was to identify the modulatory effect of MENK on MDSCs and to investigate the relationship between this modulation and the expression of opioid receptors. Our results showed that MENK could inhibit the proliferation of MDSCs from both marine bone marrow and spleen. MENK also could promote the expression of opioid receptors MOR and DOR. MENK suppressed the PMN-MDSCs generated from splenocyte while up-regulated M-MDSCs. The stimulation of MENK increased the production of IL-4 secreted by MDSCs from slpenocytes. Our currently data indicated that MENK could suppress the accumulation of MDSCs in tumor-bearing mice via binding to and up-regulating expressions of subunits of opioid receptors. Therefore, it is concluded that MENK, through triggering opioid receptors could exert inhibiting modulation on MDSCs.

Analysis of the Expression and Regulation of PD-1 Protein on the Surface of Myeloid-Derived Suppressor Cells (MDSCs).

Myeloid-derived suppressor cells (MDSCs) that are able to suppress T cell function are a heterogeneous cell population frequently observed in cancer, infection, and autoimmune disease. Immune checkpoint molecules, such as programmed death 1 (PD-1) expressed on T cells and its ligand (PD-L1) expressed on tumor cells or antigen-presenting cells, have received extensive attention in the past decade due to the dramatic effects of their inhibitors in patients with various types of cancer. In the present study, we investigated the expression of PD-1 on MDSCs in bone marrow, spleen, and tumor tissue derived from breast tumor-bearing mice. Our studies demonstrate that PD-1 expression is markedly increased in tumor-infiltrating MDSCs compared to expression in bone marrow and spleens and that it can be induced by LPS that is able to mediate NF-κB signaling. Moreover, expression of PD-L1 and CD80 on PD-1+ MDSCs was higher than on PD-1− MDSCs and proliferation of MDSCs in a tumor microenvironment was more strongly induced in PD-1+ MDSCs than in PD-1− MDSCs. Although we could not characterize the inducer of PD-1 expression derived from cancer cells, our findings indicate that the study on the mechanism of PD-1 induction in MDSCs is important and necessary for the control of MDSC activity; our results suggest that PD-1+ MDSCs in a tumor microenvironment may induce tumor development and relapse through the modulation of their proliferation and suppressive molecules.

CCL9/CCR1 induces myeloid‑derived suppressor cell recruitment to the spleen in a murine H22 orthotopic hepatoma model.

Myeloid‑derived suppressor cells(MDSCs) are the major negative regulators of immune responses and expand in numerous tumor models. They contribute to tumor progression and metastasis, and are involved in limiting the effects of cancer immunotherapy. To selectively target MDSCs, it is required to understand the molecular mechanisms that drive MDSC expansion. The mechanisms of their accumulation in tumor tissue have been extensively studied, while the mechanisms of their expansion in lymphoid organs have been rarely explored. The spleen is the largest lymphoid organ in the human body. A previous study by our group reported that a negative immune status in the spleen facilitated tumor growth, with MDSCs being the major immunosuppressive cells. In the present study, a murine H22 orthotopic hepatoma model was established and the mechanisms of splenic MDSC accumulation were studied, including MDSC proliferation, apoptosis and chemotaxis. The proliferation and apoptosis of splenic MDSCs did not differ between normal and tumor‑bearing(TB) mice. Cytokine array and ELISA of splenic tissues indicated elevated chemokine(C‑C motif) ligand9(CCL9) levels in TB mice. Furthermore, splenic macrophages were able to secrete CCL9. Flow cytometric analysis revealed that splenic MDSCs from TB mice also overexpressed C‑C motif chemokine receptor1(CCR1), the receptor for CCL9. Taken together, the present results indicate that CCL9 secreted by splenic macrophages induces a CCR1‑dependent accumulation of MDSCs in the spleen in a murine H22 hepatoma model.

Resveratrol protects experimental Staphylococcus aureus enterotoxin B (SEB)-induced acute liver injury (ALI) by regulating microRNAs associated with myeloid-derived suppressor cells

Abstract Resveratrol (RES) possesses anti-inflammatory, anticancer, and antioxidant properties. Because of anti-inflammatory property of RES, we investigated whether RES can attenuate Staphylococcal enterotoxin B (SEB)-induced acute liver injury (ALI) in mice. SEB exposure causes food poisoning as well as toxic shock syndrome leading to mortality. We observed that SEB caused ALI in female mice and there was an increase in enzyme aspartate transaminase (AST) levels, induction of inflammatory cytokines, and massive infiltration of immune cells into the liver. RES (50 mg/kg body weight) treatment attenuated SEB-induced ALI and significantly decreased AST levels, inflammatory cytokines and chemokines, and cellular infiltration in the liver. Interestingly, RES treatment led to an increase in myeloid derived suppressor cells (MDSC) in SEB-immunized mice. Upon examination of microRNAs (miRs) profile of liver infiltrating mononuclear cells (MNCs), there was marked change in microRNA expression profile in mice exposed to SEB and treated with RES. Specifically, there was a decrease in the expression of miR-130a and miR-185 in MNCs from SEB+RES treated mice when compared to SEB+VEH treated group. Furthermore, there was an inverse relationship between miR-130a and miR-185 with CSF-1 gene expression in liver MNCs. The data presented demonstrate for the first time that RES can effectively attenuate SEB-induced ALI by inducing the proliferation of MDSC. In summary, the beneficial effects of RES may be mediated by changes in miRs regulating pathways leading to the generation of immunosuppressive cells and/or production of anti-inflammatory cytokines suppressing SEB-induced ALI.

Myeloid derived suppressor cells (MDSC) and anti-MUC1 immunosurveillance in pre-malignancy and cancer

Abstract Recent work from our lab showed that in a colon cancer prevention trial of a tumor antigen MUC1 vaccine in patients with premalignant colon adenomas, those who did not make anti-MUC1 IgG, had higher pre-vaccination levels of circulating MDSC compared to vaccine responders. This was the first observation of increased MDSC in premalignancy. MDSC in premalignancy have not been compared to MDSC in cancer and our hypothesis is that there might be differences in the composition of various MDSC subpopulation and their immunosuppressive functions due to the shorter time of exposure to disease and differences in the disease microenvironment. We are testing this hypothesis on two cohorts of patient samples, premalignant and malignant colon and pancreas. In both cohorts we saw an increase in MDSC in premalignant and malignant disease over healthy controls. The colon premalignant group could be divided into MDSChighAnti-MUC1 IgGlow and MDSClowAnti-MUC1 IgGhigh confirming our previous observations that MDSC are impeding anti-MUC1 immune response in premalignancy as well therefore potentially facilitating progression to cancer. The pancreas cohort also showed increase in MDSC in patients with pancreatic cysts (IPMN), precursors to pancreatic cancer, as well as their suppression of anti-tumor immunity. Furthermore, we found increased levels of PGE2 and its metabolite in patients’ serum from both cohorts, which is closely related to MDSC proliferation and mechanism of suppression. While we do not see much difference in the MDSC phenotype and subpopulation composition, the immunosuppressive capacity of tumor MDSC appears to be greater. We are currently testing this observation in vitro in T cell suppression assays.

Dual negative roles of C/EBP\u03b1 in the expansion and pro-tumor functions of MDSCs

Myeloid-derived suppressor cells (MDSCs) are greatly expanded in cancer patients and tumor-bearing mice. They infiltrate into tumors and modulate the tumor microenvironment. In an effort to identify molecular mediators responsible for expansion and the tumor-promoting function of MDSCs, we discovered CCAAT/enhancer binding protein alpha (C/EBPα) expression was significantly reduced in MDSCs from tumor-bearing mice compared to non-tumor-bearing hosts. Tumor-conditioned medium down-regulated C/EBPα expression, suggesting tumor secreted factors inhibiting the gene expression. Consistent with the function of C/EBPα in regulating the balance between proliferation and growth arrest in hematopoietic progenitors, myeloid lineage specific deletion of C/EBPα resulted in significantly enhanced MDSC proliferation and expansion, as well as an increase of myeloid progenitors and a decrease of mature cells. In addition, deletion of C/EBPα in MDSCs enhanced the pro-angiogenic, immune suppressive and pro-tumorigenic behavior of these cells by upregulating the production of iNOS and arginase, as well as MMP-9 and VEGF. Accordingly, tumors growing in C/EBPα conditional null mice displayed greater MDSC infiltration, increased vascularization and accelerated tumor growth. Taken together, this study reveals dual negative roles of C/EBPα in the expansion as well as pro-angiogenic and immune suppressive functions in MDSCs.

HMGB1 promotes myeloid-derived suppressor cells and renal cell carcinoma immune escape.

Despite high immunogenicity and marked presence of immune cells in the RCC(renal cell carcinoma), immunotherapy fails to develop effective anti-tumor immune responses. This is due to the negative regulatory factors in the tumor microenvironment. As the main contributor of immunosuppression, myeloid-derived suppressor cells (MDSCs) inhibited anti-tumor immunity and promoted tumor progression. Meanwhile, it is confirmed that high mobility group box-1 protein (HMGB1) shows a high expression in many solid tumors and HMGB1 with high expression is involved in tumor immune escape. However, the mechanisms linking HMGB1 with tumor immune escape are unclear. The present study aimed to explore whether HMGB1 can promote RCC immune escape by inducing the generation of MDSCs. In this study, Renca mouse model was established and the influence of HMGB1 on MDSCs was investigated by using HMGB1 antibody to downregulate the expression of HMGB1 in tumor-bearing mice. The result showed that with the down-regulation of HMGB1, the tumor growth was inhibited significantly and the mice survival was prolonged greatly. Furthermore, the differentiation and proliferation of MDSCs were inhibited both in vitro and in vivo, and the inhibition rate showed a positive correlation with the degree of down-regulation of HMGB1. When MDSCs were eliminated with Gr-1 antibody in vivo, the ability of the HMGB1 to promote tumor growth was severely impaired. Thus, our findings indicated that HMGB1 might mediate tumor immune escape by promoting MDSCs cell proliferation, which provided a novel theoretical basis for preventing RCC using HMGB1 as the target.

Pak2 Regulates MDSC Development and Function

Myeloid derived suppressor cells (MDSCs) are a heterogeneous cell population at various stages of differentiation that can increase under various pathologic conditions such as cancer, infection or inflammation, displaying suppressive function. It is well recognized that MDSCs contribute to tumor evasion by suppressing cell-mediated immunity. Based on the differential expression of Ly6C and/or Ly6G in mice, MDSCs are characterized as granulocytic (CD11b+Ly6G+Ly6Clow) or monocytic MDSCs (CD11b+Ly6Glow/−Ly6Chi). These subsets induce T-cell hyporesponsiveness and can have various functions and distribution depending on their environment. Although much research has focused on the tumorigenic effects of MDSCs, studies on the regulation of their development during hematopoiesis remain limited.p21-activated kinases (Paks) are serine/threonine kinases that regulate diverse cellular activities including cytoskeletal remodeling, cell motility, proliferation, apoptosis and mitosis. Despite active research on pharmacological inhibition of group I Paks in treating solid tumors, few studies have examined the role of Paks in modulating normal hematopoiesis. Knowledge of the role of Pak2 in regulating long-term hematopoiesis and lineage commitment remains limited. Utilizing a conditional Pak2-KO murine model, we have previously demonstrated that Pak2 disruption in hematopoietic stem/progenitor cells (HSPCs) induces myeloid lineage skewing and CD11b+Gr1+ cell expansion in mice. Compared to mice reconstituted with wild type (WT) bone marrow (BM), mice transplanted with Pak2-KO BM displayed a significantly higher percentage of granulocyte-monocyte progenitors (GMPs) in the BM and higher numbers of CD11b+Gr1+ cells in the spleen.In this study, we demonstrated that CD11b+Gr1high cells isolated from the spleens of mice with Pak2-KO BM displayed significantly greater suppressive function on T cell proliferation in vitro, consistent with MDSC phenotype. There was a near 2-fold increase in the numbers of both granulocytic and monocytic splenic MDSCs in mice reconstituted with Pak2-KO BM. At HSPC level, Pak2-KO BM yielded greater than 3-fold more colonies in response to GM-CSF but not G-CSF or M-CSF when compared to WT cells, indicating selective hypersensitivity to GM-CSF. In parallel experiments, Pak2-KO and WT BM C-kit+ cells that were enriched for hematopoietic progenitor cells (HPCs) were cultured in liquid culture in the presence of GM-CSF. Pak2-KO BM C-kit+ cells yielded greater than 2-fold higher numbers of CD11b+Gr1+ MDSCs that displayed potent suppression on CD8+ T cell proliferation. These data demonstrate that Pak2 disruption increases HPC sensitivity to GM-CSF signaling and drives lineage commitment toward granulocyte-monocyte lineage thus promoting MDSC development. In addition, we have also found that Pak2 deficient MDSCs are more proliferative and more resistant to apoptosis when compared to WT CD11b+Gr1+cells, thus contributing to expansion of this population in vivo. Loss of Pak2 decreases MDSC sensitivity to apoptosis through differential regulation of multiple pro- and anti-apoptotic gene expression. Furthermore, Pak2 disruption down regulates the expression of IRF8, a well-described myeloid transcription factor. Together, our data indicate that loss of Pak2 promotes HPC myeloid lineage commitment and CD11b+Gr1+ MDSC proliferation while suppressing apoptotic cell death in these cells. Further studies are ongoing to determine the interaction between Pak2 and IRF8. DisclosuresNo relevant conflicts of interest to declare.