Granulocyte-like Myeloid Derived Suppressor Cells Research Articles

Myeloid‑derived suppressor cell accumulation induces Treg expansion and modulates lung malignancy progression.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous family of myeloid cells that suppress T cell immunity in tumor-bearing hosts. The present study aimed to examine roles of T and MDSC subsets in lung malignancy. The study analyzed 102 cases with lung malignancy and 34 healthy individuals. Flow cytometry was performed for identification of T cell and MDSC subsets and their phenotypic characteristics in peripheral blood. The lung malignancy cases exhibited lower frequencies of granulocyte-like MDSCs (G-MDSCs) expressing PD-L2 and PD-L1 than healthy controls (P=0.013 and P<0.001, respectively). Additionally, there was a higher frequency of monocyte-like MDSCs (M-MDSCs) expressing PD-L1 in the peripheral blood of patients with lung malignancy than healthy controls (P<0.001). The frequencies of G-MDSCs and M-MDSCs were positively correlated with proportions of PD-1+ and CTLA-4+ regulatory T cells (Tregs). In vitro co-culture assay demonstrated M-MDSCs of lung malignancy enhanced naive T cell apoptosis and promoted Treg subset differentiation compared with M-MDSCs of healthy controls. The findings suggested accumulation of MDSC subsets in lung malignancy and MDSCs expressing PD-L2 and PD-L1 induced Treg expansion by binding to PD-1 on the surface of Tregs.

Abstract 2369: Tumor-suppressive efficacy of let-7b microRNA against lung carcinogenesis is mediated by modulating the tumor microenvironment

Abstract Lung cancer is the leading cause of mortality worldwide. MicroRNAs (miRNAs) are potential candidates for lung cancer therapy. However, a major limitation is the lack of an efficient delivery system to directly deliver miRNA to cancer cells while limiting exposure to healthy cells. In our previous studies on aerosolized let-7b in lung cancer prevention, let-7b showed good inhibition of B[a]P-induced lung adenoma with no side effects. In this study, we found that aerosolized let-7b decreased tumor growth in the LKR13 (KRAS mutant) syngeneic mouse model. Let-7b post-transcriptionally suppresses PD-L1 and PD-1 expression in the tumor immune microenvironment, suggesting that let-7 microRNAs may promote antitumor immunity in vivo. Single cell RNA sequencing (scRNAseq) data showed that let-7b treatment decreased the expression of PD-1 in CD8+ T cells and reduced PD-L1 expression in lung tumor cells. Let-7b treatment also significantly changed the percentages of distinct CD8+ tumor-infiltrating lymphocytes (TIL) states. The proportion of CD8+ T cells mediating anti-tumor functions (EM-like CD8+ TILs) was increased significantly by let-7b treatment compared to control. In contrast, the CD8+ T cell subpopulation that has negative effects on anti-tumor immune response, exhausted CD8+ TILs, was significantly decreased by let-7b. Flow cytometry data showed that Let-7b treatment led to the accumulation of CD8+ T cells, granzyme B+ CD8+ T cells and IFN-γ+ CD8+ T cells in tumors, and a decrease of intratumoral Granulocyte-like myeloid derived suppressor cells (G-MDSC) cells. Our results suggest that the in vivo tumor-suppressive efficacy of let-7 is mediated, at least in part, by immune-promoting effects via down-regulating PD-L1 in tumors and/or PD-1 on CD8+ T cells. Citation Format: Qi Zhang, Jing Pan, Donghai Xiong, Yian Wang, Mark S. Miller, Alberto Izzotti, Ming You. Tumor-suppressive efficacy of let-7b microRNA against lung carcinogenesis is mediated by modulating the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2369.

Myeloid-Derived Suppressor Cell Subtypes Are Responsible for the Th2 Phenotype of T Cells in CLL

In chronic lymphocytic leukemia (CLL), monoclonal B cells expand and accumulate in the blood, bone marrow, spleen and secondary lymphoid organs in response to incompletely defined cellular and molecular interactions involving multiple cell types within the tumor microenvironment (TME). Bidirectional interactions of CLL cells with components of the TME - mesenchymal stromal cells, myeloid cells, T cells, and matrix - are critical for leukemic cell survival and proliferation. Here, we focused on understanding how two non-leukemic cell types within the TME, T lymphocytes and myeloid-derived suppressor cells (MDSCs), interact to create a TME supportive of a CLL clone.In CLL, it has been suggested alterations in CD4+ and CD8+ T cell ratios and functions are responsible for a non-inflammatory and immunosuppressive cytokine TME, with expansions of Th2s and Tregs. Conversely, higher numbers of Th17 and CD8+ cells are associated with longer survival. MDSCs are not well studied in CLL, although existing reports suggest that their expansion suppresses T cell proliferation in vitro. Here we studied the effects of MDSCs and CLL B cells on T cell numbers and function.First, we identified a correlation between blood T cells and MDSCs in 56 CLL patients. Absolute numbers of MDSCs correlated with total numbers of CD3+, CD4+ and CD8+ cells (P= 0.002, < 0.001, < 0.001 and Spearman r = 0.44, 0.646, = 0.61, respectively). However, the correlation of MDSC subtypes with CD4+ and CD8+ cells differed: monocyte-like MDSCs (mMDSCs) associated with CD4+ cells (P < 0.001, Spearman r = 0.73) whereas granulocyte-like MDSCs (gMDSCs) associated with CD8+ cells (P= 0.008; Spearman r = 0.45).Moreover, in a subset of 18 CLL patients tested, MDSC numbers directly correlated with the levels of Th2 (P= 0.049, Spearman r = 0.470), Th17 (P= 0.040, Spearman r = 0.488), Tc17 (P= 0.018, Spearman r = 0.550) and Tc22 (P= 0.010, Spearman r = 0.587) cells. When dividing MDSCs into subsets, gMDSCs exhibited a similar correlation except for the absence of Th2 and the presence of a correlation with Th22 (P= 0.033, Spearman r = 0.505). Conversely, mMDSCs positively associated with Th1 (P= 0.024, Spearman r = 0.549) and Tc1 (P= 0.019, Spearman r = 0.569) cells.We next addressed the functional effect of MDSCs on naïve CD4+ cell differentiation using samples from 16 CLL patients and 10 healthy, age-matched controls (HCs). CD3+CD45RO-CD62L+ CD4+ lymphocytes were FACS-sorted and stimulated in vitro with anti-CD3/CD28 beads plus IL-2, and the percentages of CD4+ T cells producing various cytokines were quantified by intracellular flow cytometry. These results were compared with those obtained in the presence of mMDSCs (HLA-DRlo/CD11b+/CD33+/CD14+), gMDSCs (HLA-DRlo/CD11b+/CD33+/CD15+), monocytes (HLA-DRhi/CD11b+/CD14+) or CLL B lymphocytes (CD19+). On day 7, CLL T cells, cultured in the absence of other cells, generated a higher percentage of cytokine-producing cells than those from HCs (22% vs. 8%; P < 0.001). This difference was due to an increase in all cytokine-producing cells except classical Th17s producing IL-17A. When CLL T cells were co-cultured with gMDSCs, a significant expansion of Th2 (IL-4+) (P= 0.044) and a trend in growth of IL-17F+ Th17cells (P= 0.101) was found. In contrast, co-culture of CLL T cells with mMDSCs led to a reduction in Th22 (IL-22+) (P= 0.005) and Treg (FoxP3+) (P= 0.006) cells and to a trend towards expansion of Th2 (IL-4+) cells. Co-cultures of CLL T cells with monocytes, similarly to mMDSCs, led to an expansion of Th2 (IL-4+) cells (P= 0.01) and a reduction in Treg (FoxP3+) cells (P= 0.002). Finally, co-culturing naïve CD4+ lymphocytes with CLL B cells had a different and unexpected effect, promoting the expansion of Th1 (IFNγ+) cells (P= 0.033) and Th22 (IL-22+) T cells (P= 0.052).In summary, the absolute number of blood T cells, CD4+ and CD8+ subsets, and MDSCs are linked, although associations of T cell subsets differ between mMDSCs and gMDSCs. MDSCs and their subsets influence naïve T cell differentiation differently, in general leading to expansion of Th2 (IL-4+) cells. In contrast, CLL B cells had a different effect on T cell polarization, preferentially expanding Th1 (IFNγ+) cells. Thus, it appears that myeloid cells and especially MDSCs are responsible for polarization of the T cell compartment to Th2 which is said to be dominant in CLL patients. DisclosuresKolitz:Gilead, Magellan, and Novartis: Honoraria; Gilead, Novartis, and Seattle Genetics: Other: Travel Support; Boehringer Ingelheim, Cantex, Erytech, and Millennium: Research Funding; Celgene, Jazz: Equity Ownership; Gilead, Magellan, Novartis, Pharmacyclics, and Seattle Genetics: Consultancy. Barrientos:Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Janssen: Consultancy; Gilead: Consultancy, Research Funding. Stamatopoulos:Novartis SA: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding.

Prolonged residence of an albumin-IL-4 fusion protein in secondary lymphoid organs ameliorates experimental autoimmune encephalomyelitis.

Interleukin-4 (IL-4) suppresses the development of multiple sclerosis in a murine model of experimental autoimmune encephalomyelitis (EAE). Here, we show that, in mice with EAE, the accumulation and persistence in the lymph nodes and spleen of a systemically administered serum albumin (SA)-IL-4 fusion protein leads to higher efficacy in preventing disease development than the administration of wild-type IL-4 or of the clinically approved drug fingolimod. We also show that the SA-IL-4 fusion protein prevents immune-cell infiltration in the spinal cord, decreases integrin expression in antigen-specific CD4+ T cells, increases the number of granulocyte-like myeloid-derived suppressor cells (and their expression of programmed-death-ligand-1) in spinal cord-draining lymph nodes and decreases the number of T helper 17 cells, a pathogenic cell population in EAE. In mice with chronic EAE, SA-IL-4 inhibits immune-cell infiltration into the spinal cord and completely abrogates immune responses to myelin antigen in the spleen. The SA-IL-4 fusion protein may be prophylactically and therapeutically advantageous in the treatment of multiple sclerosis.

An albumin-bound drug conjugate of paclitaxel and indoleamine-2,3-dioxygenase inhibitor for enhanced cancer chemo-immunotherapy

Despite the promising target of immunosuppressive enzyme indoleamine-2,3-dioxygenase (IDO) for cancer immunotherapy, IDO blockade monotherapy does not show significant benefit to cancer patients in the clinic. Recent research has focused on the combinatorial therapy of the IDO inhibitor and the immune checkpoint blockade or chemotherapy. Here, we synthesize a drug conjugate methyltryptophan-paclitaxel (MP) by linking the IDO inhibitor, D-1-methyltryptophan (D-1MT), to the chemotherapeutic agent, paclitaxel (PTX), through an ester bond. MP exhibits a similar tubulin-stabilizing effect to PTX. Like PTX, MP binds to human serum albumin to form albumin-bound MP nanoparticles (MP NPs) with a particle size of ∼115 nm in diameter. MP NPs significantly improve the tumor concentration of D-1MT due to the hydrolysis of MP in tumors. The codelivery of PTX and D-1MT offered by MP NPs in tumors significantly enhances the anti-tumor effect compared with the albumin-bound PTX NPs. Immune cell phenotyping reveals that MP NPs ameliorate the immune environment through increasing the number of the effector CD8+ T cells, and decreasing the population of regulatory T cells and granulocyte-like myeloid-derived suppressor cells. These results prove that the design of the twin drug from the IDO inhibitor and PTX synergizes the anti-tumor effect and shows promise in clinical translation.

DI-Leu16-IL2, an Anti-CD20-Interleukin-2 Immunocytokine, Is Safe and Active in Patients with Relapsed and Refractory B-Cell Lymphoma: A Report of Maximum Tolerated Dose, Optimal Biologic Dose, and Recommended Phase 2 Dose

Background: DI-Leu16-IL2 is a recombinant fusion immunocytokine composed of interleukin-2 and a CD20-targeting monoclonal antibody that maintains the activities of both antibody and cytokine components but is also involved in tumor targeting, engagement of the immune system, and induction of an anti-cancer vaccine effect.Methods: In this multicenter open label, dose escalation trial to determine the maximum tolerated dose (MTD), optimal biological dose (OBD), and recommended phase 2 dose (RP2D), we evaluated the safety, tolerability, and efficacy of subcutaneously (SC) administered DI-Leu16-IL2 in patients (pts) with relapsed or refractory CD20+ B-cell lymphoma (NCT01874288). Following peripheral B-cell depletion with low-dose rituximab (50mg/m2), DI-Leu16-IL2 was administered SC for 3 consecutive days every 21 days for 6 cycles. A 3+3 dose escalation trial design began at 0.5 mg/m2. Responses were evaluated by PET in pts who had received at least 2 cycles of therapy. Immunologic correlates were done including peripheral blood flow cytometry and serum cytokine levels. Responding (complete or partial response, CR or PR) and stable disease (SD) pts were permitted to continue treatment for an additional year.Results: Twenty-two pts in 5 dose cohorts have been enrolled, 3 at 0.5 and 1mg/m2, 7 at 2mg/m2 and 4mg/m2, and 2 at 6mg/m2. The median age is 64 years (40-84), median prior therapies is 4 (range 1-21), with 100% having received prior rituximab and chemotherapy, and 41% with prior autologous stem cell transplant. Dose limiting toxicities (DLT) occurred in 2 pts at 6 mg/m2, both with delayed (8-10 days) grade 3 cytokine release syndrome (CRS), suggesting effector cell expansion as the cause. None of the CRS required treatment with tocilizumab, however one required dose reduction, and the other discontinued therapy.The Gr 3-4 drug-related adverse events (AEs) that required dose reduction were: QTC prolongation (Gr 3) in one patient at 4 mg/m2, tumor lysis syndrome (Gr 3) at 4mg/m2, and diarrhea (Gr 3) at 1mg/m2. Hematologic AEs were eosinophilia (Gr 1-2 = 22.7%), anemia (Gr 1-2 = 45%, Gr 3-4 = 9%), thrombocytopenia (Gr 1-2 = 36%, Gr 3-4 = 4.5%), neutropenia (Gr 1-2 = 14%, Gr 3-4 = 14%) in the 22 pts evaluable for toxicity. Other AEs were local injection site reactions (Gr 1-2 = 82%), pruritus (Gr 1-2 = 82%), edema (Gr 1-2 = 23% Gr 3-4 = 5%), fever (Gr 1-2 = 41%, Gr 3-4 = 5%), and chills (Gr 1-2 = 59%). Six pts have been enrolled at 4mg/m2 and no DLTs have been encountered. The MTD was determined to be 4mg/m2.Patients with CR or PR had higher pre-treatment absolute lymphocyte counts (>700/mcL) than pts with progressive disease (P < 0.05). Repetitive SC dosing elicited lymphocyte expansion at all dose levels. Although T-regulatory cell expansion was observed in the blood, it was not associated with a worse response. Pts achieving CR showed a trend towards lower % of granulocyte-like myeloid derived suppressor cells. Pts showed reproducible increases in cytokine levels of IL-10, MCP-1, IFNgamma, and TNFalpha. Pharmacokinetic data showed consistent drug levels between cycle 1 and 2.Therefore, the OBD and RP2D was determined to be 2mg/m2 which produced 3 CR and 3 SD.Durable tumor regression or stabilization was noted in 14 of 16 (88%) evaluable pts. There were 4 CR: 1 diffuse large B-cell lymphoma (DLBCL), 2 follicular lymphoma and 1 marginal zone lymphoma; 1 PR (DLBCL) and 9 SD, and objective response rate of 31% [Figure 1]. The median duration of disease stabilization was 8 months (4-14). Of the responders, the median duration of response was 13 months (10-15). One CR continues 11 months after stopping therapy at cycle 6.Conclusions: Promising clinical efficacy of DI-Leu16-IL2 has been observed with long-term responses in relapsed/refractory B-cell NHL pts. SC administration has permitted higher doses than could be achieved with IV treatment, however, at the higher dose range, CRS was observed. Even though the MTD was 4mg/m2, given the responses, AEs, and biologic activity in the Phase I study, the RP2D was determined to be 2mg/m2. Responses were associated with a higher starting ALC, and lymphocyte expansion was dose dependent, with reproducible serum cytokine profiles. The protocol continues to enroll pts in the expansion cohorts of this phase I/II study. DI-Leu16-IL2 given subcutaneously is safe and effective, and induces durable responses even after discontinuation of therapy, suggesting an anti-tumor vaccination effect. [Display omitted] DisclosuresLansigan:Celgene: Consultancy; Teva: Research Funding; Pharmacyclics: Consultancy; Spectrum: Consultancy, Research Funding. Vlock:Alopexx Oncology, LLC: Employment. Gillies:Provenance Bioparmaceuticals Corp: Employment.

In CLL, Myeloid-Derived Suppressor Cells and Their Monocytic and Granulocytic Varieties Differ in T-Cell Subset Association and Polarization Induction

In chronic lymphocytic leukemia (CLL) monoclonal B cells expand and progressively accumulate in the bone marrow, eventually migrating to secondary lymphoid organs for even greater proliferation. At both sites, CLL cells engage in complex, incompletely defined cellular and molecular interactions involving multiple cell types such as T cells, myeloid cells, mesenchymal stromal cells, and matrix, collectively referred to as the "tumor microenvironment". This microenvironment is critical for the survival and proliferation of CLL cells, and data indicate that T cells and myeloid cells have an important role in these processes. In this study, we focus on two cells types: CD4+ T lymphocytes and myeloid-derived suppressor cells (MDSCs). In CLL patients, these populations are altered and impact on clinical outcome. CD4+ T cells comprise several subtypes, and CLL patients often have expanded Th2 and Tregs populations, consistent with the immunosuppressive status of these patients. Moreover, patients with higher numbers of another CD4+ subset, Th17 cells that produce IL-17 and other pro-inflammatory cytokines, can have longer survival times. Although studied minimally in CLL, MDSCs are known suppressors of T cell proliferation in vitro, and expand along with malignant cells in several cancers. However, no information is available about their effects on CD4+ T cell differentiation or on B-cell biology in CLL.In a cohort of 56 untreated CLL patients, we first explored correlation of the numbers of MDSCs and autologous T cells, using flow cytometry. CD3+ cell numbers significantly paralleled total MDSCs and monocyte-like MDSCs (mMDSCs) (P = 0.002, Spearman r = 0.44; P = 0.004, Spearman r = 0.41, respectively). Interestingly, MDSCs correlated with CD4+ and CD8+ T-cells (P < 0.001, Spearman r = 0.646; P < 0.001, Spearman r = 0.61, respectively). However, the correlation of MDSC subpopulations with CD4+ and CD8+ cells differed: mMDSCs associated significantly with CD4+ cells (P < 0.001, Spearman r = 0.73) and granulocyte-like MDSCs (gMDSCs) with CD8+ cells (P= 0.008; Spearman r = 0.45). Furthermore, although gMDSCs did not correlate with the numbers of CD4+ T-cells, we observed that they positively paralleled Tregs defined as CD3+/CD4+/CD25+/CD127-/FoxP3+ cells (P = 0.020, Spearman r = 0.44). Other subpopulations are currently under study.To address the effect of MDSCs on CD4+ cell differentiation, we FACS sorted CD3+/CD45RO- naïve CD4+ lymphocytes and stimulated them in vitro with anti-CD3/CD28 beads and IL2 in the presence or absence of mMDSCs (HLA-DRlo/CD11b+/CD33+/CD14+), gMDSCs (HLA-DRlo/CD11b+/CD33+/CD15+) or monocytes (HLA-DRhi/CD11b+/CD14+); these studies involved samples from 3 CLLs and 3 healthy controls (HCs). On day 7, cells were harvested and cytokine production was quantified by intracellular flow cytometry as the percentages of the following populations: Th1 (INFγ), Th2 (IL-4), Tregs (FoxP3), Th17 (IL-17A and IL-17F), Th9 (IL-9) and Th22 (IL-22). Culturing CLL or HC T cells in the absence of MDSCs revealed a lower percentage of cytokine-producing cells (24% vs. 55%; P = 0.017) in CLL, which was mainly due to a reduction in IL-4+ cells (P = 0.066). However, when analyzing the effects of MDSC subsets on the polarization of CLL or HC T cell, gMDSCs promoted significantly more FoxP3+ and less IL-22+ cells in CLL than in HC (P = 0.025 and P = 0.048, respectively). When analyzing only CLL T cells, supplementation with mMDSCs induced a reduction in IL-22+ cells (P = 0.027) and an insignificant increase of IL-4+ and IL-17+ cells. Conversely monocytes supported an expansion of INFγ+ T-cells (P=0.066), and gMDSCS promoted an increase of IL-9+ cells (P = 0.046) and a reduction of FoxP3+ cells (P = 0.019).In summary, in CLL the absolute numbers of total MDSCs and T cells are tightly linked. There is a significant correlation between CD4+ T cells and mMDSCs, and between CD8+ T cells and gMDSCs. Additionally, in CLL naïve CD4+ differentiation appears reduced compared to HC, in concordance with lower T-cell responses previously reported. Moreover, the preliminary aspects of the study suggest that CLL mMDSCs promote an expansion of Th2, Th17 cells and a reduction of Th22 cells, and monocytes enhance Th1s. Unexpectedly, since we observed a significant positive correlation in the PBMCs, gMDSCs may reduce Tregs and augment Th9. These findings depict differential consequences of CLL T cell - MDSC / mMDSC / gMDSC interactions. DisclosuresStamatopoulos:Abbvie: Honoraria, Other: Travel expenses; Gilead: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

Mesenchymal Stem Cells (MSC) Regulate Activation of Granulocyte-Like Myeloid Derived Suppressor Cells (G-MDSC) in Chronic Myeloid Leukemia Patients.

It is well known that mesenchymal stem cells (MSC) have a role in promotion of tumor growth, survival and drug-resistance in chronic myeloid leukemia (CML). Recent reports indicated that a subpopulation of myeloid cells, defined as granulocyte-like myeloid-derived suppressor cells (G-MDSC) is increased in these patients. So far, the role of MSC in MDSC expansion and activation into the BM microenvironment remains unexplored. To address this question, here we use a specific experimental model in vitro, co-culturing MSC with peripheral blood mononucleated cells (PBMC) from normal individuals, in order to generate MSC-educated G-MDSC. Although MSC of healthy donors (HD) and CML patients were able to generate the same amount of MDSC, only CML-MSC-educated G-MDSC exhibited suppressive ability on autologous T lymphocytes. In addition, compared with HD-MSC, CML-MSC over-expressed some immunomodulatory factors including TGFβ, IL6 and IL10, that could be involved in MDSC activation. CML-MSC-educated G-MDSC expressed higher levels of ARG1, TNFα, IL1β, COX2 and IL6 than G-MDSC isolated from co-culture with HD-MSC. Our data provide evidence that CML-MSC may play a critical role in tumor microenvironment by orchestrating G-MDSC activation and regulating T lymphocytes-mediated leukemia surveillance, thus contributing to CML immune escape.

Granulocyte-like myeloid derived suppressor cells (G-MDSC) are increased in multiple myeloma and are driven by dysfunctional mesenchymal stem cells (MSC).

Granulocytic-Myeloid-derived suppressor cells (G-MDSC) are increased in Multiple Myeloma (MM) patients but the mechanisms of G-MDSC generation are still unknown. There are many evidences of the role of mesenchymal stem cells (MSC) in promoting MM cell growth, survival and drug-resistance. We here used a specific experimental model in vitro to evaluate the ability of MSC to induce G-MDSC. We found that although MSC derived from healthy donors (HD), MGUS and MM were able to generate the same amount of MDSC, only MM-MSC-educated G-MDSC exhibited suppressive ability. In addition, in comparison with MSC derived from HD, MM-MSC produce higher amount of immune-modulatory factors that could be involved in MDSC induction. Compared to G-MDSC obtained from co-culture models with MSC from healthy subjects, both MGUS and MM-MSC-educated G-MDSC showed increase of immune-modulatory factors. However, only MM-MSC educated G-MDSC 1) up-regulated immune-suppressive factors as ARG1 and TNFα, 2) expressed higher levels of PROK2, important in angiogenesis and inflammatory process, and 3) showed ability to digest bone matrix.Our data demonstrate that MM-MSC are functionally different from healthy subjects and MGUS-MSC, supporting an evolving concept regarding the contribution of MM-MSC to tumor development and progression.

Mesenchymal STEM CELLS Favor Tumor Growth By Generating Granulocyte-like Myeloid Derived Suppressor CELLS in CML Patients

INTRODUCTION. The complex interplay between cancer cells and immune system allows neoplastic cells to evade immune surveillance and expand. Recently, our and another group have demonstrated that a subpopulation of myeloid cells, defined as "granulocytic myeloid-derived suppressor cells" (G-MDSC), plays an important role for immune escape in chronic myeloid leukemia (CML) patients by reducing T cell activation. The aim of this study was to evaluate the influence of Mesenchymal stem cells (MSC) on generation of MDSCs by comparing CML MSCs (n=10) with healthy donors (HD) MSC (n=8).METHODS. G-MDSC (CD11b+CD33+CD14-HLADR- cells) were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis by cytofluorimetric analysis. Immuno-suppressive activity was tested through incubation of G-MDSC with autologous CFSE-labeled T cells and stimulation with phytohaemagglutinin (PHA). Controls included a positive T cell proliferation control (T cells plus PHA) and a negative one (T cells only). After three days, T cell proliferation was analyzed by flow cytometry. For G-MDSC generation, human peripheral blood mononucleated cells (PBMC) from HD were cultured alone and with MSC of CML (n=10) or HD (n=8) (1:100 ratio). After one week, G-MDSC were isolated using anti-CD66b magnetic microbeads and the phenotype was confirmed by cytofluorimetric analysis. Expression of ARG1, NOS2, PTGS2, TNFα, TGFβ, IL6, IL10, IL1β was also evaluated using real time PCR.RESULTS. Percentage of cells with a G-MDSC phenotype was greater in PB obtained from CML patients than HD (82.5±9.6% vs 56,2±5.4%, p<0.0001). G-MDSC were able to inhibit T cell proliferation compared to positive control (25±5% vs 48±7.6%, p=0.0057). To investigate if CML MSC may be involved in G-MDSC generation, we incubated HD PBMC with CML or HD MSC for one week. After magnetic isolation, we found that only CML MSC-educated G-MDSC acquired immune-suppressive ability, inhibiting T cell proliferation compared to G-MDSCs control (isolated from PBMC cultured in medium alone) (32±12% vs 63±5.9%, p=0.003). On the contrary, HD MSC-educated G-MDSC did not show any suppressive effect. We also found that CML MSC-educated G-MDSC expressed higher level of the following immune modulatory factors: TNFα (20.8±19.3, p=0.006), IL1β (47.3±25.2, p=0.001), PTGS2 (20.7±10.9, p=0.002) and IL6 (33.8±13.9, p=0.004) compared to HD MSC-educated G-MDSCs (arbitrarily 2-ΔΔCt value: 1). MSC WE also observed ane an up-regulation of PTGS2 (19±4.4, p=0.04), TGFβ (6±3, p=0.01) and IL6 (5±2.8, p=0.04) in CML MSCs at time 0 with a great variability among the patients (calculated value of 2-ΔΔCt in HD MSC was 1). After 48 h of co-culture with PBMC, CML MSC showed statistically significant up-regulation of ARG1 (23.5±11.9, p=0.02), TGFβ (4.8±3, p=0.04), IL10 (5.6±2.8, p=0.03) and IL6 (54.3±23, p=0.02) expression, suggesting that multiple mechanisms are involved in MDSC induction by CML MSC.CONCLUSION. Our work demonstrates that CML MSCs are able to activate MDSCs favoring cancer immune evasion in CML patients. DisclosuresPalumbo:Novartis: Honoraria, Other: Advisory Board.

Granulocyte-like Myeloid Derived Suppressor Cells (G-MDSCs) are increased in Multiple Myeloma due to immunological dysregulation of mesenchymal stem cells (MSCs)

Granulocyte-like Myeloid Derived Suppressor Cells (G-MDSCs) are increased in Multiple Myeloma due to immunological dysregulation of mesenchymal stem cells (MSCs)

Chronic Lymphocytic Leukemia Patients Exhibit Expanded Functional Granulocyte-like Myeloid Derived Suppressor Cells

Chronic lymphocytic leukemia (CLL) is a disease commonly associated with an immune disturbance. These alterations of the immune system have been generally considered due to the direct effects of CLL cells. In the last years, myeloid derived suppressor cells (MDSCs) have been found to be expanded in several cancers and to play a major role in helping tumor cells escape from immune surveillance. MDSCs represent a heterogeneous population of HLA-DRlo/CD11b+/CD33+ cells that are subdivided into monocyte-like (CD14+, m-) or granulocyte-like (CD15+, g-) subsets. Here we have investigated the extent that patients with CLL have expansions of MDSCs, their types and functions, and how these correlate with clinical and laboratory characteristics.Using flow cytometry on cryopreserved PBMCs, we did not observe differences in the percentages of HLA-DRlo/CD11b+/CD33+ cells between 17 untreated CLL patients and 10 healthy controls (HC) (3.3% vs. 3.1%). However, the distribution between m-MDSCs and g-MDSCs was dramatically different, with CLL patients exhibiting significantly higher levels of g-MDSCs (79.7% vs. 4.1%, p<0.001). When analyzing the phenotypes of g-MDSCs and m-MDSCs from CLL patients, we found that the latter exhibited significantly higher levels of CD80, an immune modulator that can promote Tregs and the immune suppressor molecule IDO. As in other cancers, the ratio of CLL T cells compared to MDSCs was significantly lower than in HC (p=0.030). In vitro studies revealed that the ratio between MDSCs and T cells was important when studying the induction of suppression in the latter. Furthermore, when evaluating the ability of MDSCs to inhibit autologous T cell proliferation in 5 patients, we observed a consistent reduction of proliferation only when co-culturing with g-MDSCs. The results on m-MDSCs were varied and insignificant statistically. To address this, we induced m-MDSCs (im-MDSCs) from purified CD33+ cells in vitro with GM-CSF, IL10 and IL6. Notably, the im-MDSC population suppressed autologous T cell proliferation in 4 of 5 cases at an average of 33% (range: 10-79%). Significance analysis of microarrays (SAM) of im-MDSCs, m-MDSCS, and HLA-DRhi monocytes indicated that im-MDSCs exhibit a lower expression of pro-inflammatory genes (e.g., TNF, IL-1α/β, CCR7, CCL3, CXCL10, CCL5) and higher levels of genes related to the ability to suppress and to MDSCs (e.g., HMOX1, CD40, FN1). Thus, these two sets of studies of m-MDSC function indicate that the poor suppressor activity of the m-MDSCs in patients is induced and not inherent. Finally we looked for clinicobiological correlations with the above laboratory findings. In this cohort we did not observed that the frequency of g-MDSCs was associated with good or bad prognostic markers, but we found several correlations with the phenotype of the g-MDSCs and m-MDSCS. Notably, those patients that required therapy expressed significantly more CD80 and iNOS in the g-MDSCs (p=0.038 and p=0.009), as well as higher iNOS, IDO and S100A9 in the m-MDSCs (p=0.035, p=0.075 and p=0.047). Highlighting an immune suppressor/modulator phenotype, that could facilitate the progression of the CLL by a reduction of the immune surveillance.In summary, CLL patients demonstrate an altered HLA-DRlo/CD11b+/CD33+ population with significantly more g-MDSCs than m-MDSCs. CLL g-MDSCs are functional suppressors, whereas m-MDSCs are not, even though they exhibit increased levels of inhibitory molecules and can perform this function when induced from CD33+ cells. The latter suggest that that the poor suppressor activity of patient derived m-MDSCs is induced in vivo. Finally, different clinicobiological characteristics associate with the phenotype of MDSCs, especially an increase in suppressor molecules and need to treat. DisclosuresNo relevant conflicts of interest to declare.