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.

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.

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.

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 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 The Role of Interleukin-10 (IL-10) in IL-15–Mediated T-Cell Responses

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4513-4521 ◽  
Author(s):  
Dieter Körholz ◽  
Ursula Banning ◽  
Halvard Bönig ◽  
Markus Grewe ◽  
Marion Schneider ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Interleukin 10 ◽  
T Cell Proliferation ◽  
Cell Production ◽  
Cd25 Expression ◽  
Tnf Α ◽  
Ifn Γ

Abstract Interleukin-15 (IL-15) is a potent T-cell stimulating factor, which has recently been used for pre-clinical in vivo immunotherapy. Here, the IL-15 effect on CD3-stimulated peripheral human T cells was investigated. IL-15 induced a significant T-cell proliferation and upregulated CD25 expression. IL-15 significantly enhanced T-cell production of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-10. Between 10- and 100-fold greater concentrations of IL-15 were necessary to reach a biological effect equivalent to that of IL-2. Blockade of IL-2 binding to the high-affinity IL-2 receptor did not affect the IL-15 effects, suggesting that IL-15 did not act by inducing endogenous IL-2. Exogenously administered IL-10 significantly reduced the IL-15 and IL-2–mediated IFN-γ and TNF-α production, whereas T-cell proliferation and CD25 expression were not affected. The inhibitory effects of exogenously administered IL-10 on T-cell cytokine production appeared indirect, and are likely secondary to decreased IL-12 production by accessory cells. Inhibition of endogenous IL-10 binding to the IL-10 receptor significantly increased IFN-γ and TNF-α release from T cells. These data suggest that endogenous IL-10 can regulate activated T-cell production of IFN-γ and TNF-α via a paracrine negative feedback loop. The observations of this study could be of relevance for the therapeutic use of IL-15 in vivo.

Chimeric Regulatory T cells Enhance HSC Engraftment in An Antigen-Specific Fashion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2424-2424
Author(s):  
Yiming Huang ◽  
Larry D Bozulic ◽  
Thomas Miller ◽  
Hong Xu ◽  
Yujie Wen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Third Party ◽  
T Cell Proliferation ◽  
Hematopoietic Stem ◽  
Mixed Chimeras

Abstract Abstract 2424 Poster Board II-401 We previously reported that CD8+TCR- facilitating cells (FC) induce the generation of chimeric regulatory T cells (Treg) in vivo. Transplantation of a mixture of CD8+/TCR- FC and hematopoietic stem cells (HSC) into ablated recipients results in chimerism and tolerance. Treg harvested from the spleen of chimeras (chimeric Treg) potently increase long-term donor chimerism in secondary NOD recipient mice. Here, we evaluated whether chimeric Treg enhance engraftment of hematopoietic stem cells (HSC) in an antigen-specific manner. To prepare mixed chimeras (B6 → NOD), NOD recipients were conditioned with 950 cGy TBI and transplanted with 10,000 B6 HSC and 1,000 NOD HSC plus 45,000 CD8+TCR- B6 FC. At 5 weeks, CD8-CD4+CD25bright chimeric Treg were sorted from spleens of the mixed chimeras (B6 → NOD). 100,000 chimeric Treg were then mixed with 10,000 B6 HSC (donor-specific) + 10,000 B10.BR HSC (third-party) and transplanted into conditioned NOD recipients in competitive repopulation assays. NOD mice given HSC plus nonchimeric naïve B6 Treg or HSC alone served as controls. Two of the four animals that received HSC alone engrafted and exhibited an average of 6.7% donor B6 chimerism at 30 days, 11.2% at 60 days, and 10.6% at 90 days. Three of five animals given HSC plus naïve B6 Treg engrafted with 21.3% donor B6 chimerism at 30 days, 28.8% at 60 days, and 28.9% at 90 days. In contrast, eight of nine recipients of HSC + chimeric Treg engrafted. These animals exhibited a significantly higher level of donor B6 chimerism, ranging from 56.3% at 30 days, 75.4% at 60 days to 85% at 90 days (P = 0.034). None of the recipients engrafted with the MHC-disparate third-party B10.BR HSC. We then assessed the suppressive function of chimeric Tregin vitro by using MLR suppressor cell assays. CD8-/CD4+/CD25bright Treg were sorted from chimeric spleens 5 wks to 12 wks after HSC + FC transplantation. As shown in the Figure 1, Treg from naïve B6 mice resulted in 1.9 fold; 1.3 fold and 1.1 fold inhibition of proliferation at 1:1, 1:0.25, 1:0.125 responder/Treg ratios (n = 3). In contrast, chimeric Treg potently suppressed T cell proliferation by 10.5 fold; 3.2 fold; and 1.7 fold at responder/Treg ratios of 1:1, 1:0.25, 1:0.125 (n = 4). Chimeric Treg significantly suppressed T cell proliferation at responder/Treg ratios of 1:1 and 1:0.25 compared with naïve B6 Treg (P < 0.05). NOD responder splenocytes remained hypoproliferative in response to B6 stimulator and chimeric Treg compared with stimulator plus B6 Treg, suggesting that chimeric Treg are significantly more potent than naïve B6 Treg in suppressing effector T cell proliferation in vitro. These data show that chimeric Treg enhance donor B6 HSC engraftment but not third-party B10.BR HSC, demonstrating that chimeric Treg function in vivo in an antigen-specific fashion. These data also show that the mechanism of FC function in vivo is associated with the establishment of an antigen-specific regulatory feedback loop. Figure 1 Figure 1. Disclosures: Bozulic: Regenerex: Employment. Ildstad:Regenerex: Equity Ownership.

Myeloid Leukemias Directly Suppress T Cell Proliferation Through STAT3 and Arginase Pathways

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3885-3885 ◽  
Author(s):  
Samantha Miner ◽  
Sawa Ito ◽  
Kazushi Tanimoto ◽  
Nancy F. Hensel ◽  
Fariba Chinian ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd4 T Cells ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Suppressor Cells ◽  
T Cell Proliferation ◽  
Immune Editing

Abstract The immune-editing effect of myeloid leukemia has recently been reported in several studies. We previously demonstrated that the K562 leukemia-derived cell line suppresses T cell proliferation, which suggests that myeloid leukemia may function in a similar way to myeloid derived suppressor cells (MDSC). While the mechanism of suppression in leukemia is not fully understood, recent murine and human studies suggest that the STAT3 and arginase pathways play a key role in the immunosuppressive function of MDSC. We hypothesized that myeloid leukemia utilizes the MDSC STAT3 and arginase pathway to evade immune control, and block anti-leukemic immune responses. To evaluate the suppressive capacity of myeloid leukemia on T cell proliferation, we isolated CD34+ blasts and myeloid derived suppressor cells (MDSC: CD11b+CD14+) from blood of primary leukemia samples by FACS sorting (n=5). These cells were co-cultured with CFSE-labeled CD4+ T cells (n=9), previously isolated from healthy donor PBMCs using an automated cell separator (RoboSep). After stimulating with CD3/CD28 Dynabeads (Invitrogen, New York, USA) for 72 hours, proliferation was measured by CFSE dilution of the viable cell population. In three myeloid leukemias studied, CD4+ T cell proliferation was significantly suppressed in the presence of primary CD34 blasts and MDSC cells (p<0.001). Interestingly, CD34 blasts demonstrated a greater suppressive effect on T cells compared to MDSC cells for these samples (not statistically significant p=0.61). Next we repeated the proliferation assay using five leukemia cell lines: THP-1 and AML1 (derived from AML), K562 and CML1 (derived from CML), and the Daudi lymphoid-derived leukemia cell line. After staining with cell tracer dye and irradiating 100Gy, the cells were co-incubated with CFSE-labeled CD4+ T cells from healthy volunteers (n=6). We found that CD4+ T cell proliferation in the presence of the myeloid leukemia cell lines was significantly suppressed (mean proliferation 5.7±0.9% to 26.1±10.7%: p<0.0001 to 0.05) compared to lymphoid cell lines (mean proliferation 76.3±8.2%: p>0.05), consistent with the results obtained with the primary leukemia samples. To evaluate the impact of STAT3 and arginase on the immunosuppressive function of myeloid leukemia, the five cell lines were primed overnight with either arginase inhibitor (N(ω)-Hydroxy-nor-L-arginine; EMD Biosciences, Inc., California, USA) or two STAT3 inhibitors (STAT3 Inhibitor VI or Cucurbitacin I; EMD Millipore, Massachusetts, USA). Then, CD4+ T cells from healthy donors (n=3) were cultured with either (1) leukemia without any inhibitor (2) leukemia in the presence of inhibitor (3) leukemia primed with inhibitor. Priming leukemia with arginase inhibitor and STAT3 inhibitors almost completely abrogated their suppressive effect of T cell proliferation (p<0.001). We conclude that myeloid leukemia, like MDSC, directly immunosuppresses T cells, through STAT-3 and arginase. This finding may underlie the immune-editing of T cells by myeloid leukemia. Our results suggest that STAT3 inhibitors could be used to augment leukemia-targeted immunotherapy. Further investigation of T cell biology within the leukemia microenvironment is needed to further define immune editing mechanisms in myeloid leukemia. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures: No relevant conflicts of interest to declare.

Donor Requirements For CD4+CD25+FoxP3+ Regulatory T Cells Capable Of Suppressing CD4+ and CD8+ Conventional T Cell Proliferation and Graft Versus Host Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4484-4484 ◽  
Author(s):  
Antonio Pierini ◽  
Lucrezia Colonna ◽  
Maite Alvarez ◽  
Dominik Schneidawind ◽  
Byung-Su Kim ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Animal Models ◽  
Graft Versus Host Disease ◽  
Third Party ◽  
T Cell Proliferation ◽  
Graft Versus Host

Adoptive transfer of CD4+CD25+FoxP3+ regulatory T cells (Tregs) prevents graft versus host disease (GvHD) in several animal models and following allogeneic hematopoietic cell transplantation (HCT) in clinical trials. In these models donor derived Tregs have been mainly used as they share the same major histocompatibility complex (MHC) with conventional CD4+ and CD8+ T cells (Tcons) that are primarily responsible for GvHD onset and persistence. Third-party derived Tregs are a promising alternative tool for cellular therapy as they can be prepared in advance, screened for pathogens and activity and banked. In this study we explored MHC disparities between Tregs and Tcons in HCT to evaluate the impact of these different cell populations in GvHD prevention and survival after transplant. Methods and Results We evaluated the ability of highly purified Treg to suppress proliferation of C57BL/6 (H-2b) Tcons following exposure to irradiated splenocytes from BALB/C (H-2d) mice in vitro in a mixed lymphocyte reaction (MLR). Either donor derived C57BL/6 (H-2b) or third party FVB (H-2q) Tregs suppressed Tcon proliferation at the Treg/Tcon ratios of 1:2 and 1:4. The same Treg population effectively suppressed different MHC derived Tcons where BALB/C (H-2d) or FVB (H-2q, third-party) Tcons were incubated with irradiated splenocytes from C57BL/6 (H-2b) mice and were effectively suppressed with BALB/C (H-2d) Tregs. In the MLR, third-party Tregs present the same activation molecule expression patterns as MHC matched Tregs: CTLA4 and LAG3 expression is enhanced after stimulation with interleukin-2 (IL-2) and anti-CD3/CD28 beads, while MHC class II molecule expression is increased after 3-4 days of culture with Tcons and irradiated splenocytes. Furthermore third-party and MHC matched Tregs express the same levels of interleukin-10 (IL-10). We translated these results to in vivo studies in animal models. In these studies T cell depleted bone marrow (TCD BM) from C57BL/6 (H-2b) mice was injected into lethally irradiated (total body irradiation, 8 Gy) BALB/C (H-2d) recipient mice. 2 days later GvHD was induced by injecting luc+ donor derived Tcons (1x106/mouse). Using this model GvHD was evaluated following the adoptive transfer of freshly isolated CD4+CD25+FoxP3+ Tregs derived from BALB/C (H-2d, host type), C57BL/6 (H-2b, donor type), FVB (H-2q, third-party) or BALB/B (H-2b, minor mismatched with the donor, major mismatched with the host) mice at the different Treg/Tcon ratios of 1:1, 1:2 and 1:4. As expected, donor Tregs exerted the strongest dose dependent GvHD protection (p = 0.028), while host Tregs did not improve mouse survival (p = 0.58). Third-party and minor mismatched with the donor Tregs improved mouse survival (third-party and minor mismatched with the donor respectively, p = 0.028 and p = 0.17) but mice had worse GvHD score profiles (both p< 0.001) and could not recover their weight as well as mice treated with donor Tregs (both p< 0.001). In vivoTcon bioluminescent imaging confirmed these results showing a reduced Tcon proliferation in mice treated with donor, third-party and minor mismatched with the donor Tregs, the first exerting the strongest effect (after 6 weeks of observation, p< 0.001). Conclusions Our studies indicate that MHC disparities between Tregs and Tcons do not represent an insurmountable barrier for Treg function. In vitro and in vivo data strongly suggest that Tregs can suppress Tcon proliferation without requiring MHC matching. In vivo GvHD prevention efficiency was affected by MHC disparities with donor derived Treg being the most effective, however, third party Treg also resulted in GvHD attenuation. These studies indicate that both donor and third party Treg could be effective in clinical application raising the possibility of screening and banking Treg for use. Further, these studies highlight the need for activation of the Treg on host tissues to effectively suppress conventional T cell proliferation and GvHD induction. Disclosures: No relevant conflicts of interest to declare.

Expanded CD4+Foxp3+ Regulatory T Cells through DR3 Signaling Have a Distinct Immunophenotype and Abrogate the Lethal Acute-Graft and Host Disease after Allogeneic Transplantation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1876-1876
Author(s):  
Hidekazu Nishikii ◽  
Byung-Su Kim ◽  
Yasuhisa Yokoyama ◽  
Jeanette Baker ◽  
Antonio Pierini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
T Cell Proliferation ◽  
Agonistic Antibody ◽  
Host Disease ◽  
Acute Graft

Abstract Background : CD4+Foxp3+ regulatory T cells (Treg) are a subpopulation of T cells which regulate the immune system, maintain self-tolerance and enhance immune tolerance after transplantation. Several groups have demonstrated that donor-derived Treg prevent the development of lethal acute graft and host disease (GVHD) in murine allogeneic transplant models. However, the low frequency of Treg limits clinical translation. To overcome the paucity of Treg, several strategies have been developed for Treg expansion. However, the activation of other immune cells and the instability of Foxp3 expression in ex vivo culture are problematic for widescale clinical usage. Recently, we showed that a single dose of agonistic antibody to DR3 (Death receptor 3, also called tumor necrosis factor super family 25; TNFSF25) into donor mice resulted in the expansion of donor derived Treg and prevented acute GVHD (Blood. 2015). Although the treatment with DR3 antibodies can preferentially expand Treg in vivo, the precise role of DR3 signaling in Treg has not been fully elucidated. In this study, we investigated the immune phenotype, gene expression profiles, and function of Treg after activation with DR3 signaling. Methods: To analyze the heterogeneous immunophenotype of Treg after DR3 signal activation, we comprehensively analyzed multicolor cytometry data using viSNE (visualization of stochastic neighbor embedding algorithm). For gene expression analysis using microarray (Affymetrix GeneChip 2.0 ST Array), CD4+Foxp3+ cells from Foxp3-GFP mice with or without DR3 activation were sorted by FACS. Normalized expression data was analyzed using TIGR Multi Experiment Viewer (MeV, version 4.9). To investigate the function of Treg after DR3 activation, CD4+CD25+Treg from wild type (WT) C57BL/6 mice (H2kb) with or without treatment of agonistic antibody to DR3 were isolated by FACS and then injected into lethally irradiated (8Gy in total) BALB/c mice (H2kd) together with 5x106 T cell depleted bone marrow (from WT C57BL/6 mice) and 1x106 T cells (C57BL/6-luciferase mice). The transplanted mice were monitored by clinical GVHD score, weight, bioluminescence imaging (BLI) for donor T cell trafficking and survival. Results: The results of viSNE showed the heterogenic elevated expression level of Nrp1, Helios (natural occurring Treg marker/transcription factor), CD103, KLRG1, CD44, ICOS, PD-1, Lag3, TIGIT (effector or inhibitory molecules), and Ki67 (proliferation marker) in Treg after DR3 activation. On the other hand, the expression of CD25, the receptor for IL-2 was down regulated. In the microarray data, a significant elevated level (>2 fold relative expression levels in DR3 activated Treg) of chemokine/cytokine (ccr3, cxcl10) and effector molecules (CD74, Gzmb) were observed. These data suggest that the effect of DR3 signaling in Treg results in not only the expansion of Treg but also their activation. In transplantation experiments, the mice that received DR3 activated Treg (5X105/mouse) showed significantly lower donor T cell proliferation compared with the mice that received non-activated Tregs (n=5 in each group, P<0.01 on day 7 and 10 after transplant). Interestingly, even a smaller number (1x105/mouse) of DR3 treated Treg suppressed donor T cell proliferation in host mice (n=5 in each group, P<0.05 on day7 and day10), and the survival of the mice in the DR3 activated Treg group was also improved compared with control GVHD group (n=10 in each group, P<0.01 in Log-rank test). These data suggested that Treg isolated after DR3 activation were more functional for the prevention in GVHD. Conclusion: In conclusion, our data demonstrate that the activation of DR3 signaling can induce Treg populations with enhanced function in vivo. These observations support for future clinical testing using human DR3 signal modulation. Disclosures No relevant conflicts of interest to declare.

Combination of Anti-PD-L1 Antibody with IMiD® Immunomodulatory Drugs, HDAC Inhibitor ACY-1215, Bortezomib, or Toll-like Receptor 9 Agonist Enhances Anti-Tumor Immunity and Cytotoxicity in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3014-3014 ◽  
Author(s):  
Arghya Ray ◽  
Deepika Sharma DAS ◽  
Yan Song ◽  
Dharminder Chauhan ◽  
Kenneth C Anderson
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Tumor Immunity ◽  
Immune Suppression ◽  
Nk Cell ◽  
Fold Increase ◽  
T Cell Proliferation ◽  
Toll Like Receptor ◽  
Ctl Activity

Abstract Introduction Dysfunctional T cells and Natural Killer (NK) cells in MM, together with functionally defective plasmacytoid dendritic cells (pDCs), contribute to the immune suppression in MM (Chauhan et al, Cancer Cell 2009, 16:309-323; Ray et al, Leukemia 2014, 28: 1716-1724). The mechanism and the role of immunoregulatory molecules mediating pDC-T cell and pDC-NK cell interactions in MM are now defined. Programmed cell death protein 1 (PD-1) is highly expressed on MM patient T cells and NK cells; and both pDCs and MM cells express PD-1 ligand PD-L1 (B7-H1). PD-L1 interaction with PD-1 results in bidirectional inhibitory responses in T cells. Our study showed that pDCs confer T cell and NK cell immune suppression in the MM BM milieu by engaging immune checkpoints via PD-L1/PD-1 signaling axis (Ray et al, Leukemia 2015, 29:1441-1444). Importantly, blockade of PD-L1-PD-1 using anti-PD-L1 Ab generates MM-specific CD8+ CTL activity, as well as enhances NK-cell-mediated MM cell cytolytic activity. Anti-MM therapies may modulate MM-host immune responses, which raises the possibility that efficacy of anti-PD-Ll Ab can be improved by combining these therapies with immune-stimulating agents. Here we examined the impact of combining immune checkpoint blockade with lenalidomide, pomalidomide, bortezomib, HDAC inhibitor ACY-1215, or Toll-Like Receptor 9 agonists on anti-tumor immunity and cytotoxicity in MM. Methods For combination studies, we utilized low concentrations of various drugs (pomalidomide, lenalidomide, ACY-1215, or bortezomib) that do not significantly decrease viability of MM cells. As in our prior studies, anti-PD-L1 Ab and TLR9 agonist are not cytotoxic against MM cells. T cell proliferation assay: MM patient pDCs were co-cultured with autologous T cells (pDC:T ratio; 1:10) in the presence of anti-PD-L1 Ab (5 μg/ml) alone, drug alone, or anti-PD-L1 Ab plus drug for 5-6 days, and proliferation was quantified with CellTrace Violet Cell proliferation Kit using FACS. CTL activity assays: MM patient CD8+ T cells were cultured with autologous pDCs (1:10 pDC/T ratio) with anti-PD-L1 Ab, drug alone, or anti-PD-L1 plus drug for 5 days; cells were washed to remove drug, and GFP+MM.1S cells (20:1 E/T ratio) were added for another 2-3 days, followed by quantification of viable GFP+MM.1S cells using FACS. NK-cell mediated cytotoxic activity was assessed using flow-based CFSE-stained K562 lysis assays, as well as degranulation assay quantifying cell surface CD107a. All statistical parameters were calculated using GraphPad Prism 6. Anti-PD-L1 Ab was purchased from eBiosciences, USA; and ACY-1215, bortezomib, lenalidomide, and pomalidomide were purchased from Selleck chemicals, USA. Results Combination of anti-PD-L1 Ab (5 μg/ml) with lenalidomide (50-100 nM) or pomalidomide (100 nM) triggered a more robust MM-specific CD8+ CTL activity than anti-PD-L1 Ab alone (1.5-2 and 2-3 fold increase in CTL activity for lenalidomide and pomalidomide combinations, repectively). Anti-PD-L1 Ab combination with lenalidomide or pomalidomide also significantly increased NK-cell-mediated MM cell cytotoxicity (p < 0.05). We next determined whether anti-PD-L1 Ab can be combined with histone deacetylase inhibitors ACY-1215 (250 nM) or Panobinostat (2 nM). Combination of anti-PD-L1 Ab with ACY-1215 or panobinostat enhanced MM-specific CD8+ CTL activity versus anti-PD-L1 Ab alone (1.5 and 2 fold increase in CTL activity for panobinostat and ACY-1215 combinations, respectively). Assessment of surface CD107a as a marker of NK cell functional activity showed that anti-PD-L1 Ab plus ACY-1215 markedly increased CD107a expression (>10 fold) versus anti-PD-L1 Ab alone. Our prior studies showed that TLR9 agonists can restore pDCs ability to trigger T cell proliferation. We found that a combination of anti-PD-L1 Ab and TLR9 agonists (1 μM) enhances MM-specific pDC-induced CTL activities (2-3 fold increase in CTL activity in combination regimen versus anti-PD-L1 Ab alone). Finally, a combination of bortezomib (2 nM) with anti-PD-L1 Ab increased the MM-specific CTL activity (1.5-2 fold increase). Conclusions Our study provides the basis for combining novel immunotherapies targeting PD-1/PD-L1 pathway with current anti-MM agents or pDC-activating TLR agonists, to both restore immune function and enhance cytotoxicity in MM. Corresponding Author: Dharminder Chauhan, PhD Disclosures Chauhan: Stemline Therapeutics: Consultancy.

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