Elevated Levels Of Myeloid-derived Suppressor Cells Research Articles

P-075: Myeloid derived suppressor cells are not elevated in monoclonal gammopathies

<h3>Background</h3> The immunologically tolerant microenvironment may support a development of malignant multiple myeloma (MM) from precancerous monoclonal gammopathy of undetermined significance (MGUS) and blocks the efficacy of immunotherapy in existing MM as well. Myeloid derived suppressor cells (MDSC) represent heterogeneous group of cells with immunosuppressive activity and capability of promotion of tumor growth in MM. Their increased number and negative effect was described in MM but usually not in other monoclonal gammopathies (MGs). <h3>Aim</h3> Identification and enumeration of MDSC subsets in different MG subjects and comparison of their number with healthy controls. <h3>Methods</h3> Whole peripheral blood of 30 newly diagnosed and untreated MGs (2 MGUS, 7 MM with AL amyloidosis and 21 MM) and 13 healthy controls was used. Incubation with combination of MoAbs CD16/CD15/HLA-DR/CD14/CD45/CD11b/CD33/CD66b was done. Samples were analysed on BD FACSCanto II (BD Biosciences) after NH4Cl lysis and reanalysed with Infinicyt SW (Cytognos). <h3>Results</h3> Whole leukocytes were distributed into subpopulations using progressive gating strategy allowing detection of MDSC derived from granulocytes (G-MDSC) and monocytes (M-MDSC) with elimination of dendritic cells (DCs) and other contaminating cells (basophils etc.). There were found no differences in M- and G-MDSC relative and absolute count when compared whole MG group and controls. Even selection of MG with ≥95 % clonal plasma cells (PCs) or MGs with presence of circulating PCs or MGs with >30 g/l monoclonal Id protein did not show statistically different values of both MDSC subsets in comparison with control samples. The only statistically different value was decreased number of DCs in MGs. <h3>Conclusion</h3> Although elevated levels of MDSC in MM were previously published, this study did not prove it probably due to the different gating procedure. However, it was able to clearly define all leukocyte subsets in peripheral blood, thus only higher numbers of dublets may affect the final count of MDSC. Interestingly, low density neutrophils and tumor associated neutrophils should not be easily distinguish from MDSC, so their better characterization is needed in the future. The isolation and verification of MDSC immunosuppressive potential should be the next step in their analyses. Supported by MH CZ - DRO (FNBr, 65269705).

Chronic Inflammation Contributes to Tumor Growth: Possible Role of L-Selectin-Expressing Myeloid-Derived Suppressor Cells (MDSCs).

Recent data have demonstrated that chronic inflammation is a crucial component of tumor initiation and progression. We previously reported that immature myeloid-derived suppressor cells (MDSCs) with immunosuppressive activity toward effector T cellswere expanded in experimental chronic inflammation. We hypothesized that elevated levels of MDSCs, induced by chronic inflammation, may contribute to the progression of tumor growth. Using the Ehrlich carcinoma animal model, we found increased tumor growth in mice with chronic adjuvant arthritis, which was accompanied by a persistent increase in the proportion of splenic monocytic and granulocytic MDSCs expressing CD62L (L-selectin), when compared to tumor mice without adjuvant arthritis. Depletion of inflammation-induced MDSCs resulted in decreased tumor growth. In vitro studies demonstrated that increased expression of CD62L by MDSCs was mediated by TNFα, elevated concentrations of which were found in tumor mice subjected to chronic inflammation. Moreover, the addition of exogenous TNFα markedly enhanced the suppressive activity of bone marrow-derived MDSCs, as revealed by the ability to impair the proliferation of CD8+ T cells in vitro.This study provides evidence that chronic inflammation may promote tumor growth via induction of CD62L expression by MDSCs that can facilitate their migration to tumor and lymph nodes and modulation of their suppressor activity.

Abstract 555: Prostate cancer cell-induced differentiation of human monocytes into MDSCs ex vivo is inhibited by targeting STAT3

Abstract Background: The transcription factor Signal Transducer and Activator of Transcription Factor 3 (STAT3) is implicated in acquired drug resistance, metastases and immune suppression in prostate cancer and is a potential target in drug-resistant prostate cancer. Today, the treatment options for metastatic drug-resistant prostate cancer are very limited and existing immunotherapies have so far shown low efficacy. Cancer cells may avoid immune responses by expressing immunosuppressive markers or activating immunosuppressive cells. Myeloid-derived suppressor cells (MDSC) play a major role in the suppression of antitumor immunity and active STAT3 signaling has been shown to be involved in the immunosuppressive functions of these cells. Elevated levels of MDSC have been found in the peripheral blood and at the tumor site of prostate cancer patients and to correlate with disease progression. Aims: In this study we aimed to investigate if human monocytes could be induced to differentiate into immunosuppressive cells in the presence of prostate cancer cells and if this effect could be blocked by the STAT3 inhibitor GPA500 (galiellalactone). Methods: Monocytes were isolated from peripheral blood mononuclear cells from healthy human donors using a magnetic monocyte enrichment kit. Monocytes were co-cultured with the prostate cancer cell lines DU145, LNCaP or longterm IL6 treated LNCaP cells (LNCaP-IL6+) in transwells allowing cancer cells and monocytes to share culture medium. The co-cultures were grown for three days with or without the STAT3 inhibitor GPA500 (5 uM or 10 uM) with subsequent analysis by flow cytometry for cell surface markers of MDSC differentiation (CD14+ HLA-DRlo). The presence of active STAT3 (pSTAT3) in the prostate cancer cell lines was determined by Western blot. Results: Monocytes in the presence of DU145 and LNCaP-IL6+ cells showed a significantly increased population of CD14+ HLA-DRlo cells. In the presence of GPA500 the induction of this population was inhibited in a dose dependent manner compared to untreated cells. Co-culture with LNCaP cells that lack pSTAT3 did not induce this population in monocytes after three days in culture. Furthermore, GPA500 induced a dendritic cell-like population in monocytes in the absence of prostate cancer cell co-culture. This population was also observed in monocytes when co-cultured with LNCaP-IL6+ cells, but not in DU145 co-cultures, with GPA500 present. Conclusion: The prostate cancer cell lines DU145 and LNCaP-IL6+ were able to induce a monocyte population displaying MDSC markers and this induction could be inhibited by the STAT3 inhibitor GPA500. Impact: This study demonstrates that inhibiting the transcription factor STAT3 in prostate cancer could potentially reverse the immunosuppressive mechanisms caused by MDSC activation and that STAT3 inhibitors, alone or in combination with other therapies, could be a new treatment option for prostate cancer. Citation Format: Rebecka Hellsten, Karin Leandersson, Anders Bjartell, Martin E. Johansson. Prostate cancer cell-induced differentiation of human monocytes into MDSCs ex vivo is inhibited by targeting STAT3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 555.

Myeloid-derived suppressor cells contribute to maintaining allogeneic pregnancies (IRC4P.461)

Abstract In pregnant women the fetus is an allograft and successful human pregnancies require maternal tolerance to fetal alloantigens. Myeloid-derived suppressor cells (MDSC) are potent inducers of T cell tolerance in tumor-bearing hosts. MDSC levels also increase during normal pregnancy; however, whether they mediate maternal tolerance is unknown. To investigate if MDSC are involved, female BALB/c (H-2d) mice were caged overnight with allogeneic C57BL/6 (H-2b) males. The following morning, females with vaginal plugs indicative of copulation, were started on an MDSC depletion regimen. MDSC-depleted mice produced no offspring, while 46% of control-treated mice had healthy litters. One of the mechanisms MDSC use to inhibit anti-tumor immunity is to down-regulate CD62L (L-selectin) on T cells and prevent T cell entry into lymph nodes and subsequent activation. To determine if a similar mechanism is operative during pregnancy, mice were treated with granulocyte colony stimulating factor (G-CSF), an inducer of MDSC. G-CSF-treated, but not control-treated, mice contained elevated levels of MDSC, and elevated levels of naïve, circulating T cells with reduced expression of CD62L. MDSC-depletion of G-CSF-treated mice restored CD62L expression. These results suggest that MDSC promote maternal-fetal tolerance, and that tolerance during successful pregnancies may be due to the failure of T cells to enter lymph nodes where they could become activated to fetal alloantigens.

Dendritic Cell Vaccination in Acute Leukemia Patients Induces Reduction of Myeloid-Derived Suppressor Cells : Immunological Analysis of a Pilot Study

Background and Purpose: Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population that have an immunesuppresive function and play an important role in tumor immunology and self tolerance. Elevated levels of MDSCs in peripheral blood (PB) are reported in various cancers and hematological malignancies such as MDS, NHL and MM. We reported that vaccination with Wilms’ tumor 1 (WT1) peptide-pulsed dendritic cells (DC) in NHL patients induced not only WT1 specific immune responses but also innate immunity which correlated with clinical efficacy (ASH 2013). In the present study, we focused on MDSCs and investigated the effects of DC vaccination on the alteration of MDSCs which have negative influence on disease progression.Patients and Methods: 8 patients with acute leukemia (6 AML and 2ALL patients; aged 16-77) who were treated with WT1 peptide-loaded DC vaccination were enrolled in the present study. MDSCs were analyzed by FACS. Linage negative, CD11b+CD33+HLA-DR- cells were defined as MDSC. Monocytic and granulocytic MDSC were defined as CD14+HLA-DRlow/- and CD15+CD11b+CD33+HLA-DR-, respectively. For proliferation assay, CD14+DR- cells and CD3+ T cells were isolated by magnetic beads method. CFSE-labeled CD3+ T cells were stimulated with anti-CD3 and anti-CD28 moAb together with IL-2 in the presence or absence of CD14+DR-cells for 4 days and cell division was analyzed by FACS. Cytoplasmic staining of indoleamine 2,3-deoxygenase (IDO) and arginase 1 were performed by permeabilization using saponin followed by moAb staining. Arginase 1 protein levels in plasma was assessed using a commercially available ELISA kit.Results: 3 patients were in complete remission (responder) and 5 others were in disease progression (non-responder) following one course of vaccination. WT1 specific T cell responses evaluated by a HLA-tetramer assay and a delayed type hypersensitivity (DTH) test were observed in all the responders and 2 non-responders. Mean percentage of MDSCs, monocytic and granulocytic MDSCs in PB was higher in acute leukemia patients than in healthy donors (1.60±0.72%, 16.56±6.07%, 1.34±0.69%, respectively in patients; 0.60±0.31%, 13.1±4.1%, 0.37±0.1%, respectively in healthy donors). Absolute number of circulating monocytic and granulocytic MDSCs before and after vaccination in responders was 359.4±117.8/μl and 35.2±14.8/μl, 282.8±119.2 /μl and 14.4±12.0/μl, respectively. On the other hand, those in non-responders were 126.3±60.2/μl and 36.3±21.7/μl, 181.3±124.0/μl and 65.1±12.0/μl, respectively. While DC vaccination resulted in reduction of both monocytic and granulocytic MDSCs by 21.3% and 59.1%, respectively in responders, it induced augmentation of these MDSCs by 43.5% and 79.3%, respectively in non-responders. A portion of MDSCs expressed IDO and arginase 1 in cytoplasm. While the percentage of IDO positive cells in monocytic MDSCs increased by 2.2 and 2.3 times in responders and non-responders, respectively following vaccination, the positivity of arginase 1 decreased by 59% in responders. On the other hand, the positivity of IDO and arginase 1 in granulocytic MDSCs decreased at the same level in both responders and non-responders following vaccination. CD14+DR-cells inhibited the proliferation of CD3+ T cells in a dose dependent manner, indicating that these cells share characteristics of MDSCs. The inhibition of the proliferation of CD3+T cells was partially restored by the addition of 1-methyl-DL-tryptophan (IDO inhibitor) and Nω-hydroxy-nor-arginine (arginase 1 inhibitor), suggesting that the inhibition was mediated at least in part by IDO and arginase 1. Amount of arginase 1 in the plasma before and after vaccination decreased by 25% in responders, not in non-responders.Conclusions: These results demonstrated that DC vaccination resulted in the reduction of the absolute number of MDSCs which seems to be correlated with clinical efficacy. DC vaccination may have inhibitory effects on several functions of MDSCs that can suppress immune surveillance against leukemia, which is favorable for regression of leukemia cells. DisclosuresNo relevant conflicts of interest to declare.

Chronic intake of high fish oil diet induces myeloid-derived suppressor cells to promote tumor growth

Omega-3 polyunsaturated fatty acids enriched fish oil exerts beneficial anti-inflammatory effects in animal models with acute and chronic inflammatory diseases. Myeloid-derived suppressor cells (MDSCs), comprised of myeloid progenitors and precursors of myeloid cells, play vital roles in cancer. How fish oil affects the generation of MDSCs and the tumor development remains largely unexplored. Here, we show that dietary intake of high fish oil diet suppresses CD8(+) T cells activation and proliferation in vivo via elevated levels of MDSCs. Mechanistically, high fish oil diet induces the expression of immunosuppressive cytokine IL-10 and promotes myelopoiesis in the spleen as well as other peripheral tissues. The immature myeloid cells in the spleen exhibit morphological and functional characteristics of MDSCs with the capability to downregulate CD8(+) T cells activation. Depletion of MDSCs using anti-Gr-1 antibody decreases the growth of subcutaneously transferred B16 melanoma in mice on high fish oil diet. Interestingly, diet-induced production of MDSCs is not solely dependent of the spleen, as splenectomy has no effect on the tumor progress. Our data show that the liver functions as an alternative extramedullary hematopoiesis organ to support MDSCs differentiation and maintain tumor growth. Taken together, our study provides a novel insight into the physiological effects of fish oil and points to MDSCs as a possible mediator linking dietary fish oil intake and immunosuppression in cancer immunosurveillance.

Increased frequencies of CD11b+CD33+CD14+HLA‐DRlow myeloid‐derived suppressor cells are an early event in melanoma patients

Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population characterized by immunosuppressive activity. Elevated levels of MDSC in peripheral blood are found in inflammatory diseases as well as in malignant tumors where they are supposed to be major contributors to mechanisms of tumor-associated tolerance. We investigated the frequency and function of MDSC in peripheral blood of melanoma patients and observed an accumulation of CD11b(+) CD33(+) CD14(+) HLA-DR(low) MDSC in all stages of disease (I-IV), including early stage I patients. Disease progression and enhanced tumor burden did not result in a further increase in frequencies or change in phenotype of MDSC. By investigation of specific MDSC-associated cytokines in patients' sera, we found an accumulation of IL-8 in all stages of disease. T-cell proliferation assays revealed that MDSC critically contribute to suppressed antigen-specific T-cell reactivity and thus might explain the frequently observed transient effects of immunotherapeutic strategies in melanoma patients.

Chronic Progressive HIV-1 Infection Is Associated With Elevated Levels of Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSC) consist of a heterogeneous population of progenitor cells and immature myeloid cells that are potent suppressors of T-cell function in many tumor models. The purpose of this study was to evaluate the possibility that MDSC play a role in HIV-induced immunosuppression.Ninety-seven patients participated in this study: 16 healthy controls; 20 HIV-infected individuals with suppressed viral loads; 49 HIV-infected treatment-naive individuals; 2 individuals with hepatitis C co-infection; 4 HIV-infected individuals failing to respond to treatment; and 8 individuals with lung cancer as positive controls.Flow cytometry analysis was performed on freshly isolated peripheral blood mononuclear cells. MDSC were defined as CD11b+CD14-CD33+CD15+. CD4+CD25+, FoxP3+ regulatory T cells were also analyzed. Mixed lymphocyte reactions and proliferation assays were used to measure suppressor function.Subjects with progressive or chronic uncontrolled HIV infection had significantly higher levels of MDSC than healthy controls. Subjects with lung cancer, as expected, had elevated MDSC. In HIV-infected individuals, percent MDSC correlated positively with elevated viral load (>50 000/mL) and low CD4 count (<250/mm3). In addition, effective antiretroviral therapy was associated with normalization of MDSC number; individuals with elevated MDSC before antiviral therapy had significant reductions in MDSC after initiation of therapy. MDSC number was correlated with generation of CD4+CD25+FoxP3+ regulatory T cells. Finally, MDSC directly impaired the proliferative capacity of T cells of healthy donors and HIV controllers.Uncontrolled HIV infection is associated with elevated levels of MDSC. These cells potentially contribute to the impaired T-cell responses characteristic of progressive HIV disease.The variety of hematopoietic cells seems endless, and as new cell types are defined, they routinely seem to be affected by HIV infection. MDSC are a case in point. The relevance of these cells in tumor biology has recently been better defined. It has long been known that chronic inflammation creates a tumor-inducing environment. It is now speculated that infiltrating MDSC inhibits effective immunologic control of evolving malignant cells. The current study suggests that MDSC may contribute to the immunopathogenesis of HIV. Are these cells contributing to the immunologic suppression associated with HIV, or are they a regulatory response to HIV-associated persistent immune activation? As described in an accompanying Editorial Comment (Macatangay BJ, et al. AIDS. 2012;26[12]:1567–1569), dampening the effects of MDSC could be important in purging latent HIV reservoirs in approaches aimed at curing HIV disease. However, understanding the immunoregulatory networks involved in HIV pathogenesis is likely critical for the design of effective HIV immune-based therapies.

Chronic progressive HIV-1 infection is associated with elevated levels of myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) have been described as suppressors of T-cell functions in many tumor models. However, MDSC in HIV-1 infection have not been studied to date. As impaired T-cell function is a hallmark of chronic progressive HIV-1 infection, we hypothesized that MDSC also play a role here. Surface staining and flow cytometry analysis were performed on freshly isolated peripheral blood mononuclear cells (PBMC) of HIV-infected individuals and compared to healthy controls and individuals with lung carcinoma. MDSC of late-stage HIV-infected individuals were isolated using magnetic beads and cocultured with the respective CD8 T cells for evaluation of proliferative capacity. We found that chronically HIV-infected HAART-naive individuals had significantly higher CD11bCD14CD33CD15 MDSC levels than healthy controls (P = 0.01). MDSC frequencies showed a positive correlation with viral load (r = 0.24, P = 0.0002) and a negative correlation with CD4 cell count (r = 0.29, P < 0.0001). Initiation of HAART led to a rapid drop in MDSC levels. MDSC from HIV-infected progressors restricted the proliferative capacity of CD8 T cells from healthy donors and of Gag/Nef-specific CD8 T cells from HIV-controllers in vitro. Furthermore, CD11bCD14CD33CD15 MDSC induced the expansion of CD4CD25FoxP3 regulatory T cells when coincubated with PBMC from controllers in vitro. We conclude that chronic uncontrolled HIV-infection is associated with elevated levels of MDSC, which potentially contribute to the impaired T-cell responses characteristic for the progressive disease stage.

SNX9 as a novel biomarker for chronic inflammation-induced immunosuppression: implications for cancer therapy (112.16)

Abstract Chronic inflammation is the cause for immunosuppression observed in human and mouse with developing tumors; myeloid derived suppressor cells (MDSCs) accumulate and induce T and NK cell dysfunction associated with z down-regulation. Moreover, we discovered that z chain could serve as a biomarker to evaluate the efficacy of a given therapy; whether the tumor regresses or reappears, based on the developing chronic inflammatory environment. Recently, we identified SNX9, which is also expressed in B cells, as an additional biomarker playing a role in a pathway different from that of the z chain, for evaluating the immune status under chronic inflammatory conditions. Using the mouse melanoma model system (B16-F10.9), we found that SNX9 expression was significantly decreased, correlating with the tumor induced inflammatory environment. Our results also identify down-regulation of SNX9 expression in additional pathologies characterized by chronic inflammation such as during autoimmune disorders and infectious diseases, which correlates with elevated levels of MDSCs. Moreover, 5FU chemotherapy treatment, led to the recovery of SNX9 expression levels by eliminating the MDSCs population, indicating that SNX9 down-regulation is a reversible phenomenon and reflects the host’s chronic inflammatory state and immune status. These results may lead to a well-controlled personalized treatment of cancer-bearing hosts’ immune status by combining measurements of both z and SNX9.

Psychological stress is associated with altered levels of myeloid-derived suppressor cells in breast cancer patients

Our group has shown in a randomized clinical trial that psychological intervention to reduce stress in patients with stages II and III breast cancer led to enhanced immune function, fewer recurrences and improved overall survival. We hypothesized that patients with high levels of stress would have alterations in myeloid-derived suppressor cells (MDSC) compared to patients with lower stress. PBMC from 16 patients with high stress ( n = 8) or with low stress ( n = 8) after surgery as measured by the Impact of Event Scale (IES) questionnaire were evaluated for the presence of MDSC. Patients with higher IES scores had significantly elevated salivary cortisol levels ( P = 0.013; 13 μg/dl vs. 9.74 μg/dl). Levels of IL-1Rα were also significantly elevated in the higher IES group (45.09 pg/ml vs. 97.16 pg/ml; P = 0.010). IP 10, G-CSF, and IL-6 were all higher in the high stress group although not to a significant degree. Flow cytometric analysis for CD33+/HLA-DR-neg/CD15+/CD11b+ MDSC revealed increased MDSC in patients with lower IES scores ( P = 0.009). CD11b+/CD15+ cells constituted 9.4% of the CD33+/HLA DR-neg cell population in patients with high IES, vs. 27.3% in patients with low IES scores. Additional analyzes of the number of stressful events that affected the patients in addition to their cancer diagnosis revealed that this type of stress measure correlated with elevated levels of MDSC ( P = 0.064). These data indicate the existence of a complex relationship between stress and immune function in breast cancer patients.

Myeloid-derived suppressor cells down-regulate L-selectin expression on CD4+ and CD8+ T cells.

Effective cell-mediated antitumor immunity requires the activation of tumor-reactive T cells and the trafficking of activated T cells to tumor sites. These processes involve the extravasation of lymphocytes from the blood and lymphatics, and their homing to lymph nodes and tumors. L-selectin (CD62L) is an important molecule in these processes. It directs naive lymphocytes to peripheral lymph nodes where they become activated and it traffics naive lymphocytes to inflammatory environments, such as tumors. Individuals with advanced cancer are immune suppressed due to myeloid-derived suppressor cells (MDSC), a population of immature myeloid cells that accumulate to high levels in response to tumor-secreted and proinflammatory factors. We now demonstrate that the reduction in T cell levels of L-selectin that is commonly seen in individuals with cancer inversely correlates with MDSC levels. Three lines of evidence demonstrate that MDSC directly down-regulate L-selectin on naive T cells: 1) naive T cells cocultured with tumor-induced MDSC have reduced L-selectin; 2) T cells in tumor-free aged mice with elevated levels of MDSC have reduced L-selectin, and 3) peritoneal exudate T cells of tumor-free mice treated with plasminogen activator urokinase to elevate MDSC have reduced levels of L-selectin. MDSC are likely to down-regulate L-selectin through their plasma membrane expression of ADAM17 (a disintegrin and metalloproteinase domain 17), an enzyme that cleaves the ectodomain of L-selectin. Therefore, MDSC down-regulate L-selectin levels on naive T cells, decreasing their ability to home to sites where they would be activated. This is another mechanism by which MDSC inhibit antitumor immunity.