scholarly journals Myeloid-derived suppressor cells are bound and inhibited by anti-thymocyte globulin

2019 ◽  
Vol 25 (1) ◽  
pp. 46-59 ◽  
Author(s):  
Young Suk Lee ◽  
Eduardo Davila ◽  
Tianshu Zhang ◽  
Hugh P Milmoe ◽  
Stefanie N Vogel ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Suppressor Cells ◽  
Arginase Activity ◽  
T Cell Proliferation ◽  
Myeloid Derived Suppressor Cells ◽  
Tumor Bearing ◽  
And Function

Myeloid-derived suppressor cells (MDSCs) inhibit T cell responses and are relevant to cancer, autoimmunity and transplant biology. Anti-thymocyte globulin (ATG) is a commonly used T cell depletion agent, yet the effect of ATG on MDSCs has not been investigated. MDSCs were generated in Lewis Lung Carcinoma 1 tumor-bearing mice. MDSC development and function were assessed in vivo and in vitro with and without ATG administration. T cell suppression assays, RT-PCR, flow cytometry and arginase activity assays were used to assess MDSC phenotype and function. MDSCs increased dramatically in tumor-bearing mice and the majority of splenic MDSCs were of the polymorphonuclear subset. MDSCs potently suppressed T cell proliferation. ATG-treated mice developed 50% fewer MDSCs and these MDSCs were significantly less suppressive of T cell proliferation. In vitro, ATG directly bound 99.6% of MDSCs. CCR7, L-selectin and LFA-1 were expressed by both T cells and MDSCs, and binding of LFA-1 was inhibited by ATG pre-treatment. Arg-1 and PD-L1 transcript expression were reduced 30–40% and arginase activity decreased in ATG-pretreated MDSCs. MDSCs were bound and functionally inhibited by ATG. T cells and MDSCs expressed common Ags which were also targets of ATG. ATG may be helpful in tumor models seeking to suppress MDSCs. Alternatively, ATG may inadvertently inhibit important T cell regulatory events in autoimmunity and transplantation.

scholarly journals Expansion of Functional Myeloid-Derived Suppressor Cells in Controlled Human Malaria Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Carlos Lamsfus Calle ◽  
Rolf Fendel ◽  
Anurag Singh ◽  
Thomas L. Richie ◽  
Stephen L. Hoffman ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Malaria Infection ◽  
Malaria Vaccine ◽  
Suppressor Cells ◽  
T Cell Proliferation ◽  
Myeloid Derived Suppressor Cells ◽  
Controlled Human Malaria Infection

Malaria can cause life-threatening complications which are often associated with inflammatory reactions. More subtle, but also contributing to the burden of disease are chronic, often subclinical infections, which result in conditions like anemia and immunologic hyporesponsiveness. Although very frequent, such infections are difficult to study in endemic regions because of interaction with concurrent infections and immune responses. In particular, knowledge about mechanisms of malaria-induced immunosuppression is scarce. We measured circulating immune cells by cytometry in healthy, malaria-naïve, adult volunteers undergoing controlled human malaria infection (CHMI) with a focus on potentially immunosuppressive cells. Infectious Plasmodium falciparum (Pf) sporozoites (SPZ) (PfSPZ Challenge) were inoculated during two independent studies to assess malaria vaccine efficacy. Volunteers were followed daily until parasites were detected in the circulation by RT-qPCR. This allowed us to analyze immune responses during pre-patency and at very low parasite densities in malaria-naïve healthy adults. We observed a consistent increase in circulating polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in volunteers who developed P. falciparum blood stage parasitemia. The increase was independent of preceding vaccination with a pre-erythrocytic malaria vaccine. PMN-MDSC were functional, they suppressed CD4+ and CD8+ T cell proliferation as shown by ex-vivo co-cultivation with stimulated T cells. PMN-MDSC reduced T cell proliferation upon stimulation by about 50%. Interestingly, high circulating PMN-MDSC numbers were associated with lymphocytopenia. The number of circulating regulatory T cells (Treg) and monocytic MDSC (M-MDSC) showed no significant parasitemia-dependent variation. These results highlight PMN-MDSC in the peripheral circulation as an early indicator of infection during malaria. They suppress CD4+ and CD8+ T cell proliferation in vitro. Their contribution to immunosuppression in vivo in subclinical and uncomplicated malaria will be the subject of further research. Pre-emptive antimalarial pre-treatment of vaccinees to reverse malaria-associated PMN-MDSC immunosuppression could improve vaccine response in exposed individuals.

scholarly journals Granulocytic Myeloid-Derived Suppressor Cells in Murine Models of Immune-Mediated Bone Marrow Failure

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2176-2176
Author(s):  
Xingmin Feng ◽  
Jisoo Kim ◽  
Gladys Gonzalez Matias ◽  
Zhijie Wu ◽  
Sabrina Solorzano ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Bone Marrow Failure ◽  
Suppressor Cells ◽  
T Cell Proliferation ◽  
Immune Mediated

Abstract Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immature myeloid cells with immunoregulatory function. Limited published studies have reported conflicting data concerning the effects of MDSCs on autoimmune diseases and graft-versus-host disease. MDSCs can be divided into two major subsets, more abundant granulocytic (G-MDSCs) and monocytic (M-MDSCs). We examined G-MDSCs in murine models of human bone marrow failure (BMF). We first characterized bone marrow (BM) MDSCs from C.B10 mice. CD11b +Ly6G +Ly6C low G-MDSCs suppressed in vitro proliferation of both CD4 and CD8 T cells from C57BL/6 (B6) mice, while Ly6G +Ly6C - cells had no effect and Ly6G -Ly6C + cells increased T cell proliferation (Fig. 1A). We then tested G-MDSCs in vivo utilizing antibody-mediated cell depletion. Lymph node (LN) cells from B6 donor mice were injected into sub-lethally irradiated major histocompatibility-mismatched CByB6F1 mice to induce BMF. Anti-Ly6G antibody injection worsened cytopenias and BM hypoplasia, and they increased BM CD4 and CD8 T cell infiltration. In contrast, anti-Ly6G antibody injection in the minor histocompatibility-mismatched C.B10 BMF model improved platelet counts and reduced BM CD8 T cells. The pathogenic and protective effects in the two models correlated with differential anti-Ly6G antibody modulation of G-MDSCs: in the CByB6F1 model, anti-Ly6G antibody eradicated G-MDSCs in blood and BM while in the C.B10 model the same antibody generated a novel G-MDSC cell population, of identical Ly6C lowCD11b + phenotype but intermediate Ly6G expression, which was not present in the CByB6F1 animals after antibody injection. When we examined the efficacy of G-MDSCs in C.B10 BMF: Ly6G + cells were enriched from BM of normal C.B10 donors (94%-97% Ly6C lowLy6G +CD11b +), and injected at the time of marrow failure initiation. Mice infused with Ly6G + cells had significantly higher levels of WBC, RBC, platelets, and total BM cells, decreased BM CD4 and CD8 T cell infiltration, and improved BM cellularity. These results indicated a protective role of G-MDSCs. When G-MDSCs were injected at day 3 after LN cell infusion, treated mice again had higher levels of WBC, RBC, platelets, and total BM cells at day 14, alleviating BMF. As both prophylaxis and therapy, G-MDSCs decreased Fas expression and Annexin V binding of residual BM cells, suppressed intracellular levels of gamma interferon and tumor necrosis factor alpha, as well as cell proliferation protein Ki67 levels in BM CD4 and CD8 T cells, relative to BMF control mice. TotalSeq simultaneously detecting surface proteins and mRNA expression in whole BM mononuclear cells in the therapy model showed an increased proportion of myeloid cells and reduced proportion of T cells in marrow from G-MDSC-treated mice based on cell surface markers and marker gene expression (Fig. 1B). Gene pathway analysis revealed down-regulation of Fas expression and reduced program cell death in total BM cells and decreased expression of genes related to cell cycle in infiltrating T cells from Ly6G + cell-treated mice-both results consistent with suppression by G-MDSCs of T cell proliferation and protection of target BM cells from apoptosis. In vitro culture of T cells from B6 mice with G-MDSCs which had been isolated from C.B10 BM cells showed dose-dependent suppression of T cell proliferation. In conclusion, our results demonstrate an active role of G-MDSCs in protecting BM from immune-mediated destruction, by suppression of T cell proliferation in the BM. G-MDSCs might have clinical application as treatment in human aplastic anemia and other immune-mediated and autoimmune diseases. Figure 1 Figure 1. Disclosures Young: Novartis: Research Funding.

Myeloid Suppressive Cells Mobilized by GM-CSF in Non-Tumor Bearing Mice Are Dependent On Interferon Gamma for Function

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 832-832
Author(s):  
Mark A. Schroeder ◽  
Julie Ritchey ◽  
John F. DiPersio
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Proliferation ◽  
Suppressive Function ◽  
Gm Csf ◽  
Tumor Bearing

Abstract Abstract 832 Myeloid-derived-suppressor cells (MDSCs) are enriched in tumors, and exist to a lesser extent in the blood, spleen and bone marrow of tumor bearing mice. Monocytic MDSCs (monoMDSCs) suppress CD8+ T-cells via expression of Arginase 1 (ARG1) and inducible nitric oxide synthase (iNOS). Tumor derived factors are critical to the maintenance of MDSCs and preventing differentiation to mature macrophages and dendritic cells. GM-CSF is a hematopoietic cytokine that can be secreted by tumors and promotes MDSC generation. Cells phenotypically similar to MDSCs can be isolated from blood of normal individuals but lack suppressive function. Hematopoietic peripheral blood stem cell mobilization with G-CSF and GM-CSF enriches for cells phenotypically similar to MDSCs. There is limited data on the role and function of these cells isolated from non-tumor bearing, normal individuals. Recent evidence suggests that graft-versus-host disease (GvHD) can be abrogated in mice by ex vivo expanded, bone marrow derived, MDSCs generated in the presence of GM-CSF, G-CSF and IL-13 (Highfill et al. Blood 2010 116:5738). It remains to be shown whether phenotypic MDSCs identified in non-tumor bearing mice are capable of immune suppression; and, the mechanism by which an immature myeloid cell becomes a functional MDSC remains unknown. We have observed an increase (up to 8 fold) in a population of cells phenotypically resembling monoMDSCs (CD11b+/Ly6C+/Ly6G-) in the spleens and blood of mice mobilized with pegylated-murine-GM-CSF (peg-mGM-CSF). We hypothesized that this population of cells would have suppressive function similar to MDSCs in vitro and in vivo, and may have the potential to abrogate graft-versus-host disease (GvHD). To investigate the function of MDSCs found in spleens of C57/Bl6 (B6) mice treated with peg-mGM-CSF we performed CFSE based anti-CD3/CD28 antibody stimulated T-cell proliferation assays, mixed leukocyte reactions and transwell assays. We observed that CD11b+Ly6C+Ly6G- cells isolated from spleens of mice treated with peg-mGM-CSF have potent suppressive function in vitro that is contact dependent and abrogated by blocking ARG1 or iNOS. This suppressive effect was lost in APC stimulated MLRs using B6 T-cells and Balb/C stimulators (confirmed in two separate experiments). Furthermore, the in vivo potential of these putative MDSCs to abrogate murine GvHD was investigated using a B6 to Balb/C donor leukocyte infusion GvHD model. Adoptive transfer of purified splenic CD11b+Ly6C+Ly6G- cells from peg-mGM-CSF mobilized B6 donors along with an equivalent number of congenic T-cells failed to abrogate GvHD. We investigated timing of MDSC infusion in the B6 to Balb/C GvHD model and found no improvement in weight loss, GvHD score or survival in mice receiving 5×105 monoMDSCs IV on day 1, 6 or 10 after transplant compared to T-cells alone control (n = 5 – 10/group, Log rank, p= NS). To address in vivo function further in a bioluminescent imaging (BLI) tumor model. Balb/C recipients were injected SC with A20 cells mixed +/− monoMDSCs at a 1:10 ratio after lethal irradiation and T-cell deplete bone marrow on day 0. Donor T-cells were infused at day +11. The rate of tumor growth measured by photon flux was the same between subcutaneous tumors either with or without monoMDSCs. (two separate experiments, 5 mice/group). This in vivo data suggested that a critical factor present in vitro might be lacking or insufficient in vivo. To investigate the critical factor(s) present in vitro we performed T-cell proliferation assays in the presence of blocking antibodies against IFNy, TNFalpha, IL-10, GM-CSF and CD154. Only neutralization of IFNy resulted in negation of the suppressive effects of these cells. To investigate the source of IFNy production we used transgenic IFNy knockout mice as T-cell and MDSC donors. Proliferation of IFNy deficient T-cells was suppressed efficiently by wild-type (WT) MDSCs, and, neutralizing IFNy using a blocking antibody negated suppression. This suggested IFNy production by a cell within the putative MDSC sorted population might be critical for MDSC function. IFNy deficient peg-mGM-CSF mobilized CD11b+Ly6C+Ly6G- spleen cells failed to suppress WT or IFNy deficient T-cell proliferation. These results suggest a critical role for IFNy production by CD11b+Ly6C+Ly6G- myeloid cells in maintaining their suppressive phenotype in vitro and perhaps in vivo. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4018-4018
Author(s):  
Cesarina Giallongo ◽  
Nunziatina L Parrinello ◽  
Daniele Tibullo ◽  
Claudia Bellofiore ◽  
Piera La Cava ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
T Cells ◽  
Mesenchymal Stem Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Suppressor Cells ◽  
T Cell Proliferation ◽  
Myeloid Derived Suppressor Cells ◽  
Healthy Donors

Abstract 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. Disclosures Palumbo: Novartis: Honoraria, Other: Advisory Board.

Chronic Lymphocytic Leukemia Patients and Eµ-TCL1 Mice Share a Phenotype of Functional Granulocyte-like and Dysfunctional Monocyte-like Myeloid Derived Suppressor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 614-614
Author(s):  
Gerardo Ferrer ◽  
Xiao-Jie Yan ◽  
Brendan Franca ◽  
Jacqueline C. Barrientos ◽  
Jonathan E. Kolitz ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Suppressor Cells ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
T Cell Proliferation ◽  
Myeloid Derived Suppressor Cells ◽  
Cell Responses

Abstract Immune imbalance is a common characteristic of patients with chronic lymphocytic leukemia (CLL). This feature is shared with Eμ-TCL1 transgenic mice that, like CLL patients, exhibit an expansion of CD5+ B cells with associated non-B-cell defects. In patients and in mice, T-cell responses are often ineffective. This alteration is generally considered due to the direct effects of the leukemic cells. The expansion of myeloid derived suppressor cells (MDSCs), which play a major role in helping tumor cells escape immune surveillance by inhibiting T-cell responses, is promoted by many cancers. MDSCs are a heterogeneous population of cells that are subdivided into monocyte-like (m-MDSC) and granulocyte-like (g-MDSC) subsets, both in humans and mice. There we have investigated the extent that patients with CLL have expansions of MDSCs, what are their types and functions, and how these correlate with the Eμ-TCL1 mice model. Using flow cytometry on cryopreserved PBMCs, we found that the absolute numbers of MDSCs (HLA-DRlo/CD11b+/CD33+) in 49 untreated CLL patients were significantly higher than 15 healthy controls (HCs) (966 446 vs. 163 578 cells/ml, P<0.001). Moreover, we observed that the absolute numbers of MDSCs significantly correlated with CLL B-cell counts in the blood (P=0.005, Spearman r=0.423). Of note, the distribution between m-MDSCs (CD14+) and g-MDSCs (CD15+) was dramatically different, with CLL patients exhibiting significantly higher numbers and percentages of g-MDSCs than HCs (702 296 vs. 26 818 cells/ml, P<0.001; 50.89 vs. 16.98%, P<0.001).In line with these results, when we explored the MDSC populations (CD11b+/GR1+) in Eμ-TCL1 mice of 5-16 months of age with leukemia cell blood counts ranging from 0.1 to 100 x 106 cell/ml. This analysis indicated a positive correlation between MDSCs and leukemic CD19+ CD5+ cells (P=0.003; Spearman r=0.328). Furthermore, the dot-plot analysis of GR1 and CD11b showed three well defined cell populations: one monocytic (Ly6-C+) and two granulocytic (Ly6-G+ CD11blo and Ly6-G+ CD11bhi). As in patients, the g-MDSC population was larger than the m-MDSC population (884 100 vs. 454 700,P=0.016). However in this case, the m-MDSCs correlated with the numbers of circulating leukemic cells (P<0.001; Spearman r=0.463) and the g-MDSCs did not. The latter was the case even when they were subdivided into both CD11blo and CD11bhi subgroups. A similar pattern was observed when analyzing single cell suspensions from murine spleens. When we evaluated the ability of MDSCs to inhibit autologous T-cell proliferation in CLL patients (n=7), we observed a consistent reduction of proliferation only when co-culturing with g-MDSCs(P=0.034). In contrast, the effects of m-MDSCs on T-cell expansion were varied and insignificant statistically. In 5 CLL samples, we induced m-MDSCs (im-MDSCs) from purified CD33+ cells in vitro with GM-CSF, IL10, and IL6; the im-MDSCs effectively suppressed T-cell proliferation in 4 of 5 cases at an average inhibition of 33% (range: 10-79%). Thus, dysfunctional m-MDSC suppression was not inherent and functional suppression could be achieved by stimulation of CLL precursor cells. Similarly in 3 independent experiments performed with MDSCs from Eμ-TCL1 mice (12-14 months of age), we observed a reduction of in vitro proliferation with g-MDSCs (P=0.049) and not with m-MDSCs. In addition, for those Eμ-TCL1 animals for which sufficient sample was available, we subdivided the g-MDSC population into the two subpopulations based on CD11b density; the CD11blo subset present less nuclear segmentation and higher suppressive activity. In summary, absolute numbers of MDSCs in the blood of CLL patients and Eμ-TCL1 mice are elevated and correlate with the levels of expansion of the leukemia. The major subtype in both situations was g-MDSCs.These g-MDSCs were functionally competent suppressors, whereas m-MDSCs were impaired in this function. In CLL patients, this m-MDSC suppressor defect could be corrected by in vitro stimulation with growth factors that support monocyte differentiation. The high similarity between CLL patients and Eμ-TCL1 mice in relation to MDSC number and function suggest that an imbalance in g-MDC vs. m-MDSC function may affect CLL development and expansion, altering interactions with members of the microenvironment such as T cells. Disclosures No relevant conflicts of interest to declare.

Pegylated Murine GM-CSF Increases Myeloid Derived Suppressor Cells In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2967-2967
Author(s):  
Mark A. Schroeder ◽  
Julie Ritchey ◽  
Brian K Dieckgraefe ◽  
John F. DiPersio
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Suppressor Cells ◽  
Fold Increase ◽  
T Cell Proliferation ◽  
Gm Csf ◽  
Tumor Bearing ◽  
Proliferation Assays

Abstract Abstract 2967 Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells initially identified in tumor bearing mice that have potent immunosuppressive capabilities. Recent evidence suggests that graft-versus-host disease (GvHD) can be abrogated by ex vivo expanded, bone marrow derived, MDSCs generated in the presence of GM-CSF, G-CSF and IL-13 (Highfill et al. Blood 2010 116 :5738). It remains to be shown whether phenotypic MDSCs identified in non-tumor bearing mice are capable of immune suppression. In addition, the mechanism by which an immature myeloid cell becomes a functional MDSC remains unknown. We hypothesized that pegylated murine GM-CSF (peg-mGM) may be protective from acute GvHD in MHC mismatched murine models by increasing regulatory T-cells (Treg) and MDSCs. Previously, we reported that peg-mGM increased circulating and splenic Tregs by 2–3 fold and they were functional in mixed leukocyte reactions (MLRs). We have also reported on the in vivo potential of mobilized splenocytes to abrogate murine GvHD. B6D2F1 mice receiving C57/Bl6 GM treated splenocytes had improved survival and less weight loss compared to G-CSF and PBS controls (3 independent experiments, n=15-19/group, GM vs. G p = 0.0005, GM vs. PBS p = 0.0005, G vs. PBS p = 0.5 (Log rank test)). In an attempt to identify cellular mediators of the reduced incidence of GvHD we investigated the impact of peg-mGM on putative MDSCs. We have observed an ∼8 fold increase in putative monocytic MDSCs (monoMDSCs) (CD11b+Ly6C+Ly6G-) and an ∼18 fold increase in putative granulocytic MDSCs (granMDSCs) (CD11b+Ly6C+Ly6G+) in the spleens and blood of mice mobilized with peg-mGM. To investigate the function of MDSCs we performed bead stimulated tritiated thymidine and CFSE based proliferation assays. We observed that granMDSCs and monoMDSCs isolated from spleens of mice treated with peg-mGM have potent suppressive function on bead stimulated T-cell proliferation exceeding that of na•ve Tregs at equal suppressor :Tcell ratios (Fold suppression of CD4+ T-cells: granMDSCs = 4.5, monoMDSCs = 2.3, Tregs = 1.08. Fold suppression of CD8+ T-cells: granMDSCs = 2.26, monoMDSCs = 1.4, Tregs = 1.05). To investigate mechanism we performed a transwell experiment using bead stimulated T-cells separated from MDSCs by a permeable membrane. Sorted monoMDSCs and granMDSCs were not suppressive in this assay suggesting the dependence on contact for inhibition of T-cell proliferation. In addition, we observed that in bead stimulated proliferation assays wells containing putative MDSCs had more dispersed beads suggesting possible sequestration of beads by the suppressor cells. To determine if all subsets were suppressive in an alternative non-bead based proliferation assay we coated plates with CD3/CD28 antibodies. Only the putative monoMDSCs were suppressive in this assay. We observed that suppression of bead stimulated T-cells was abrogated by adding an arginase-1 inhibitor, nor-NOHA, to cultures containing putative monoMDSCs. When attempting to validate these results in a MLR using MHC mismatched antigen-presenting cell (APC) stimulation, the suppressive effect was decreased or lost suggesting that the magnitude of stimulation by APC, bead or antibodies may affect activation and function of MDSCs; or, a critical factor produced in bead and antibody stimulated T-cell proliferation assays is lacking in the APC setting. We are currently functionally characterizing the monoMDSCs generated by treatment with peg-mGM and investigating potential secondary factors critical to the development of MDSCs such as IL-13 and IFN-gamma. In addition, future studies will evaluate the in vivo function of monoMDSCs generated by peg-mGM mobilization on GvHD and GVL outcomes. In summary, treatment with peg-mGM results in enrichment in functional MDSCs in the spleens of non-tumor bearing mice. The mechanism by which immature myeloid cells generated by peg-mGM become MDSCs is under investigation but appears to be contact dependent. This work is currently being translated in a clinical trial investigating the combination of GM-CSF and plerixafor for the mobilization of peripheral blood stem cells for allogeneic stem cell transplantation from matched sibling donors. Correlative studies to characterize stem cell subsets and evaluate the content of Tregs and MDSCs in the blood and apheresis product are ongoing. Disclosures: No relevant conflicts of interest to declare.

473 Mavrilimumab, a human monoclonal antibody targeting GM-CSFRα, inhibits polarization to myeloid-derived suppressor cells (MDSCs) that express PD-L1 and restores T-cell proliferation in vitro

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A504-A504
Author(s):  
Luis Carvajal ◽  
Luciana Gneo ◽  
Carmela De Santo ◽  
Matt Perez ◽  
Tracy Garron ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Proliferation ◽  
T Cell ◽  
Cancer Cell ◽  
Suppressor Cells ◽  
T Cell Proliferation ◽  
Myeloid Derived Suppressor Cells ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

BackgroundMyeloid-derived suppressor cells (MDSCs) accumulate in the blood and tumor microenvironment (TME) and suppress anti-tumor immune responses.1 Cancer cells express the granulocyte-macrophage colony-stimulating factor (GM-CSF), which drives MDSC differentiation and function.2 3 4 It is upregulated in several cancers, including mesothelioma, pancreatic and colorectal, and it is linked to higher levels of intra-tumoral MDSCs and poorer overall survival.2 4 5 In animal models, knockdown of GM-CSF in pancreatic epithelium or pancreatic mesenchymal stem cells inhibits tumorigenesis, reduces intra-tumor MDSCs and enhances CD8+ T cell accumulation.6 7 8 Therefore, targeting the GM-CSF receptor alpha (GM-CSFRα) on MDSCs is an attractive strategy to restore anti-tumor immunity. Mavrilimumab is a clinical stage fully human monoclonal antibody that blocks GM-CSFRα. It has demonstrated efficacy and acceptable safety profile in patients with rheumatoid arthritis, and it’s currently undergoing investigation in phase II studies in giant cell arteritis and in patients with severe COVID-19 pneumonia and hyper-inflammation (NCT03827018, NCT04397497, respectively). The present study investigates its potential as a therapeutic strategy to target MDSCs in the TME as an adjuvant to immunotherapy.MethodsCancer cell supernatants were collected when cells reached confluency. Human GM-CSF was measured by ELISA. Healthy donor CD14+ monocytes were incubated (± mavrilimumab) with cancer cell supernatants for either 3 or 6 days followed by phenotypic analysis (CD14, CD33, HLA-DR, CD11b, CD206, CD80, PD-L1, Arginase-1) by flow cytometry. On day 3, autologous CD3+ T cells were stimulated with CD3/CD28 and IL-2 and co-cultured with putative MDSCs for 5 days. T-cell proliferation was evaluated by measuring carboxyfluorescein succinimidyl ester (CFSE) dilution in CD4+ and CD8+ T cells by flow cytometry.ResultsGM-CSF is expressed in the supernatant of cancer cell lines (HCT116, SW-480, Panc-1, Capan-1). Human monocytes cultured with conditioned medium from colorectal carcinoma (SW-480) or pancreatic adenocarcinoma (Capan-1) show downregulation of HLA-DR, increased expression of PD-L1, Arg-1, CD206, and can suppress T-cell proliferation in-vitro. Similarly, peripheral blood monocytes purified from pancreatic cancer patients suppress T-cell proliferation ex-vivo. Notably, Mavrilimumab inhibits the polarization of healthy donor monocytes to M-MDSCs and restores T-cell proliferation.ConclusionsTargeting of GM-CSFRα with mavrilimumab may alleviate the pro-tumorigenic and immunosuppressive functions of MDSCs in the TME. Future clinical studies should evaluate whether targeting of the GM-CSFRα in combination with immune checkpoint inhibitors is a viable therapeutic option to bolster their efficacy.Ethics ApprovalThe study was approved by the Institute of Immunology and Immunotherapy, University of Birmingham, UK Ethics Board. Healthy volunteer human material was obtained from commercial sources and approved by Stemexpress Institutional Review Board (IRB).ReferencesLaw AMK, Valdes-Mora F, Gallego-Ortega D. Myeloid-Derived Suppressor Cells as a Therapeutic Target for Cancer. Cells 2020;9(3):561.Khanna S, Graef S, Mussai F, et al. Tumor-Derived GM-CSF Promotes Granulocyte Immunosuppression in Mesothelioma Patients. Clin Cancer Res 2018;24(12):2859–2872.Dolcetti L, Peranzoni E, Ugel S, et al. Hierarchy of immunosuppressive strength among myeloid-derived suppressor cell subsets is determined by GM-CSF. Eur J Immunol 2010;40(1):22–35.Takeuchi S, Baghdadi M, Tsuchikawa T, et al. Chemotherapy-derived inflammatory responses accelerate the formation of immunosuppressive myeloid cells in the tissue microenvironment of human pancreatic cancer. Cancer Res 2015;75(13):2629–2640.Chen Y, Zhao Z, Chen Y, et al. An epithelial-to-mesenchymal transition-inducing potential of granulocyte macrophage colony-stimulating factor in colon cancer. Sci Rep 2017;7(1):8265.Bayne LJ, Beatty GL, Jhala N, et al. Tumor-derived granulocyte-macrophage colony-stimulating factor regulates myeloid inflammation and T cell immunity in pancreatic cancer. Cancer Cell 2012;21(6):822–835.Pylayeva-Gupta Y, Lee KE, Hajdu CH, Miller G, Bar-Sagi D. Oncogenic Kras-induced GM-CSF production promotes the development of pancreatic neoplasia. Cancer Cell 2012;21(6):836–847.Waghray M, Yalamanchili M, Dziubinski M, et al. GM-CSF mediates mesenchymal-epithelial cross-talk in pancreatic cancer. Cancer Discov 2016;6(8):886–899.

scholarly journals Siplizumab, an Anti-CD2 Monoclonal Antibody, Induces a Unique Set of Immune Modulatory Effects Compared to Alemtuzumab and Rabbit Anti-Thymocyte Globulin In Vitro

2020 ◽  
Vol 11 ◽  
Author(s):  
Christian Binder ◽  
Felix Sellberg ◽  
Filip Cvetkovski ◽  
Erik Berglund ◽  
David Berglund
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mixed Lymphocyte Reaction ◽  
T Cell Proliferation ◽  
Allogeneic Mixed Lymphocyte Reaction ◽  
Dependent Cell

Antibodies are commonly used in organ transplant induction therapy and to treat autoimmune disorders. The effects of some biologics on the human immune system remain incompletely characterized and a deeper understanding of their mechanisms of action may provide useful insights for their clinical application. The goal of this study was to contrast the mechanistic properties of siplizumab with Alemtuzumab and rabbit Anti-Thymocyte Globulin (rATG). Mechanistic assay systems investigating antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis and complement-dependent cytotoxicity were used to characterize siplizumab. Further, functional effects of siplizumab, Alemtuzumab, and rATG were investigated in allogeneic mixed lymphocyte reaction. Changes in T cell activation, T cell proliferation and frequency of naïve T cells, memory T cells and regulatory T cells induced by siplizumab, Alemtuzumab and rATG in allogeneic mixed lymphocyte reaction were assessed via flow cytometry. Siplizumab depleted T cells, decreased T cell activation, inhibited T cell proliferation and enriched naïve and bona fide regulatory T cells. Neither Alemtuzumab nor rATG induced the same combination of functional effects. The results presented in this study should be used for further in vitro and in vivo investigations that guide the clinical use of immune modulatory biologics.

In Vitro Effects of Cyclosporine A and Tacrolimus on Regulatory T-Cell Proliferation and Function

2012 ◽  
Vol 94 (2) ◽  
pp. 123-131 ◽  
Author(s):  
Céline Miroux ◽  
Olivier Morales ◽  
Khaldoun Ghazal ◽  
Samia Ben Othman ◽  
Yvan de Launoit ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
Cyclosporine A ◽  
Regulatory T Cell ◽  
T Cell Proliferation ◽  
In Vitro Effects ◽  
And Function

A Nanobody Against Cytotoxic T-Lymphocyte Associated Antigen-4 Increases the Anti-Tumor Effects of Specific CD8+ T Cells

2019 ◽  
Vol 15 (11) ◽  
pp. 2229-2239 ◽  
Author(s):  
Zhuoran Tang ◽  
Fengzhen Mo ◽  
Aiqun Liu ◽  
Siliang Duan ◽  
Xiaomei Yang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cytotoxic T Lymphocyte ◽  
Ex Vivo ◽  
T Cell Proliferation ◽  
Tumor Cell Apoptosis ◽  
Xenograft Models

Adoptive cell-based immunotherapy typically utilizes cytotoxic T lymphocytes (CTLs), expanding these cells ex vivo. Such expansion is traditionally accomplished through the use of autologous APCs that are capable of interactions with T cells. However, incidental inhibitory program such as CTLA-4 pathway can impair T cell proliferation. We therefore designed a nanobody which is specific for CTLA-4 (CTLA-4 Nb 16), and we then used this molecule to assess its ability to disrupt CTLA-4 signaling and thereby overcome negative costimulation of T cells. With CTLA-4 Nb16 stimulation, dendritic cell/hepatocellular carcinoma fusion cells (DC/HepG2-FCs) enhanced autologous CD8+ T cell proliferation and production of IFN-γ in vitro, thereby leading to enhanced killing of tumor cells. Using this approach in the context of adoptive CD8+ immunotherapy led to a marked suppression of tumor growth in murine NOD/SCID hepatocarcinoma or breast cancer xenograft models. We also observed significantly increased tumor cell apoptosis, and corresponding increases in murine survival. These findings thus demonstrate that in response to nanobody stimulation, DC/tumor cells-FC-induced specific CTLs exhibit superior anti-tumor efficacy, making this a potentially valuable means of achieving better adoptive immunotherapy outcomes in cancer patients.

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