scholarly journals P04.05 Modulating tumor microenvironment with arginase-1 specific T cells

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A38.2-A39
Author(s):  
E Martinenaite ◽  
M Aaboe Joergensen ◽  
RE Johansson Mortensen ◽  
S Munir Ahmad ◽  
SE Weis-Banke ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Therapeutic Effect ◽  
Potential Role ◽  
Myeloid Cells ◽  
Tumor Site ◽  
Arginase 1 ◽  
Part Time

BackgroundCancer progression is associated with an increased immune suppression at the tumor site. Arginase-1 is an enzyme well-known for its involvement in metabolic immune regulation. At the tumor site, arginase-1 acts by reducing availability of L-arginine to the infiltrating immune cells thus reducing T cell functionality and proliferation. While arginase-1 is expressed by some tumor cells, it has also been shown to be produced by immune inhibitory myeloid cells, such as myeloid derived suppressor cells (MDSCs), tumor associated macrophages (TAMs) and is associated with poor prognosis. Previously, we demonstrated that spontaneous CD4+ and CD8+ T-cell immune responses against arginase-derived, HLA-restricted peptides can be found in both cancer patients and healthy individuals (Martinenaite et al, 2018, DOI: 10.1080/2162402X.2017.1404215). These T cells are present in the memory T cell compartment, and that they are activated in arginase-1 inducing conditions, such as presence of TH2 cytokines IL-4 or IL-13 in vitro (Martinenaite et al, 2019, DOI: 10.1038/s41423-019-0231-3 and DOI: 10.1007/s00262-019-02425-6).Methods and ResultsIn order to explore if arginase-1-specific T cells have a potential role in modulation of immune homeostasis, human arginase-1-specific memory T cells were isolated and expanded for functional characterization. We show that arginase-1-specific T cells specifically recognize arginase-1 expressing cells, such as mRNA transfected autologous dendritic cells (DCs) and B cells as well as M2 polarized macrophages in vitro. In addition, activated arginase-1-specific T cells produce pro-inflammatory cytokines IFNγ and TNFα. Secretion of TH1 cytokines by these T cells suggests potential role as potent immune modulators in the tumor microenvironment, since many arginase-1 expressing myeloid cells are not terminally differentiated and they can be re-polarized to an immunostimulatory, M1-like phenotype. We also observed that targeting of M2-polarized arginase-1 expressing monocytic leukemia cell line THP-1 with arginase-1-specific CD4+ T cells induces upregulation of PD-L1 on the THP-1 cells. Furthermore, we demonstrate that an arginase-1-derive peptide vaccine has a therapeutic effect in syngeneic mouse tumor models (B16 and MC38), both as monotherapy and in combination with anti-PD-1 treatment. The therapeutic effect was associated with increased immune infiltration in the peptide vaccinated mice compared to the control.ConclusionsOur study provides evidence that immune modulatory vaccination targeting arginase-1 is an intriguing way of targeting the immune suppressive microenvironment.Disclosure InformationE. Martinenaite: A. Employment (full or part-time); Significant; IO Biotech. M. Aaboe Joergensen: None. R.E. Johansson Mortensen: None. S. Munir Ahmad: None. S.E. Weis-Banke: None. M. Orebo Holmström: None. A. Wakatsuki Pedersen: A. Employment (full or part-time); Significant; IO Biotech. Ö. Met: None. I.M. Svane: F. Consultant/Advisory Board; Significant; IO Biotech. M. Hald Andersen: A. Employment (full or part-time); Significant; IO Biotech.

Overcoming the breast tumor microenvironment by targeting MDSCs through CAR-T cell therapy.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 1032-1032
Author(s):  
Saisha Abhay Nalawade ◽  
Paul Shafer ◽  
Pradip Bajgain ◽  
Katie McKenna ◽  
Arushana Ali ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Nuclear Translocation ◽  
T Cell Proliferation ◽  
Tumor Site ◽  
Car T Cells ◽  
Car T

1032 Background: Successful targeting of solid tumors such as breast cancer (BC) using CAR T cells (CARTs) has proven challenging, largely due to the immune suppressive tumor microenvironment (TME). Myeloid derived suppressor cells (MDSCs) inhibit CART’s function and persistence within the breast TME. We generated CAR T cells targeting tumor-expressed mucin 1 (MUC1) (Bajgain P et al, 2018) for BC. To potentiate expansion and persistence of MUC1 CARTs and modulate the suppressive TME, we developed a novel chimeric co-stimulatory receptor, TR2.4-1BB, encoding a ScFv derived from a TNF-related apoptosis-inducing ligand receptor 2 (TR2) mAb followed by a 4-1BB endodomain. We hypothesize that engagement with TR2 expressed on TME-resident MDSCs, will lead to both MDSC apoptosis and CART co-stimulation, promoting T cell persistence and expansion at tumor site. Methods: Function of the novel TR2.4-1BB receptor, was assessed by exposing non-transduced (NT) and TR2.4-1BB transduced T cells to recombinant TR2 and nuclear translocation of NFκB was measured by ELISA. Functionality of in vitro generated MDSCs was determined by the suppression assay. In vitro CART/costimulatory receptor T cell function was measured by cytotoxicity assays using MUC1+ tumor targets in presence or absence of MDSCs. In vivo anti-tumor activity was assessed using MDSC enriched tumor-bearing mice using calipers to assess tumor volume and bioluminescence imaging to track T cells. Results: Nuclear translocation of NFκB was detected only in TR2.4-1BB T cells. MDSCs significantly attenuated T cell proliferation by 50±5% and IFNγ production by half compared with T cells cultured alone. Additionally, presence of MDSCs, diminished cytotoxic potential of MUC1 CARTs against MUC1+ BC cell lines by 25%. However, TR2.4-1BB expression on CAR.MUC1 T cells induced MDSC apoptosis thereby restoring the cytotoxic activity of CAR.MUC1 against MUC1+ BC lines in presence of TR2.4-1BB (67±8.5%). There was an approximate two-fold increase in tumor growth due enhanced angiogenesis and fibroblast accumulation in mice receiving tumors + MDSCs compared to tumors alone. Treatment of these MDSC-enriched tumors with MUC1.TR2.4-1BB CARTs led to superior tumor cell killing and significant reduction in tumor growth (24.54±8.55 mm3) compared to CAR.MUC1 (469.79.9±81.46mm3) or TR2.4-1BB (434.86±64.25 mm3) T cells alone (Day 28 after T cell injection). The treatment also improved T cell proliferation and persistence at the tumor site. Thereby, leading to negligible metastasis demonstrating ability of CARTs to eliminate tumor and prevent dissemination. We observed similar results using HER2.TR2.4-1BB CARTs in a HER2+ BC model. Conclusions: Our findings demonstrate that CARTs co-expressing our novel TR2.4-1BB receptor have higher anti-tumor potential against BC tumors and infiltrating MDSCs, resulting in TME remodeling and improved T cell proliferation at the tumor site.

scholarly journals 778 Modulating tumor microenvironment with arginase-1 specific T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A813-A813
Author(s):  
Evelina Martinenaite ◽  
Mia Aaboe Jørgensen ◽  
Rasmus Erik Johansson Mortensen ◽  
Shamaila Munir Ahmad ◽  
Stine Emilie Weis-Banke ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Memory T Cells ◽  
Ethics Committee ◽  
Mouse Tumor ◽  
Immune Modulators ◽  
Arginase 1 ◽  
Mouse Tumor Models

BackgroundIO112 is an immune modulatory cancer therapy under preclinical development to target arginase-1-expressing tumor cells and immune inhibitory myeloid cells, such as myeloid derived suppressor cells (MDSCs), and tumor associated macrophages (TAMs). Arginase-1 acts as a metabolic immune regulator at the tumor site by reducing availability of L-arginine to the infiltrating immune cells thus reducing T cell functionality and proliferation. Previously, we demonstrated that IO112 triggers activation of spontaneous CD4+ and CD8+ T-cell responses against arginase-1, found in both cancer patients and healthy individuals.1 These T cells are present in the memory T cell compartment, and are activated in arginase-1 inducing conditions, such as presence of TH2 cytokines IL-4 or IL-13 in vitro.2 3 In this study we aimed to explore the role of arginase-1-specific T cells as immune modulators in immune homeostasis and tumor microenvironment for the development of IO112 immunomodulatory therapy.MethodsHuman arginase-1-specific T cells were isolated and expanded for functional characterization of reactivity against arginase-1 expressing target cells as well as subsequent phenotyping of the targeted arginase-1 positive cells. Syngeneic C57BL/6 mouse tumor models were used to assess the therapeutic efficacy of IO112.ResultsWe show that arginase-1-specific memory T cells specifically recognize arginase-1 expressing cells, such as mRNA transfected autologous dendritic cells (DCs) and B cells as well as M2 polarized macrophages in vitro. In addition, activated arginase-1-specific T cells produce pro-inflammatory cytokines IFNγ and TNFα. Secretion of TH1 cytokines by these T cells suggests that they may act as potent immune modulators in the tumor microenvironment, since many arginase-1 expressing myeloid cells are not terminally differentiated and they can be re-polarized to an immunostimulatory, M1-like phenotype. We also observed that targeting of M2-polarized arginase-1 expressing monocytic leukemia cell line THP-1 with arginase-1-specific CD4+ T cells induces upregulation of PD-L1 on the THP-1 cells. Furthermore, we demonstrate anti-tumor activity of IO112 in syngeneic mouse tumor models (B16 and MC38), both as monotherapy and in combination with anti-PD-1 treatment. The therapeutic effect was associated with increased immune infiltration in the IO112-treated mice compared to the control.ConclusionsWe demonstrate that arginase-1 specific T cells can influence the polarization of arginase-1-expressing immune cells. Our study provides evidence that IO112 immune therapy against arginase-1 is an attractive way of modulating the immune suppressive tumor microenvironment for therapeutic benefit. With this rationale, we are currently undertaking Investigational New Drug (IND) application enabling studies to explore this approach in a clinical setting.ReferencesMartinenaite E, Mortensen REJ, Hansen M, Holmström MO, Ahmad SM, Jørgensen NGD, Met Ö, Donia M, Svane IM, Andersen MH. Frequent adaptive immune responses against arginase-1. Oncoimmunology 2018;7(3):e1404215.Martinenaite E, Ahmad SM, Svane IM, Andersen MH. Peripheral memory T cells specific for Arginase-1. Cell Mol Immunol 2019;16(8):718–719.Martinenaite E, Ahmad SM, Bendtsen SK, Jørgensen MA, Weis-Banke SE, Svane IM, Andersen MH. Arginase-1-based vaccination against the tumor microenvironment: the identification of an optimal T-cell epitope. Cancer Immunol Immunother 2019;68(11):1901–1907.Ethics ApprovalThis study was approved by the Scientific Ethics Committee for The Capital Region of Denmark and Danish Ethics Committee on experimental animal welfare.

scholarly journals Hepatic Stellate Cells Enhance Liver Cancer Progression by Inducing Myeloid-Derived Suppressor Cells through Interleukin-6 Signaling

2019 ◽  
Vol 20 (20) ◽  
pp. 5079 ◽  
Author(s):  
Ching-Chuan Hsieh ◽  
Chien-Hui Hung ◽  
Meihua Chiang ◽  
Yu-Chin Tsai ◽  
Jie-Teng He
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Hepatic Stellate Cells ◽  
Suppressor Cells ◽  
Stellate Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Term Survival ◽  
Arginase 1

The tumor microenvironment, which consists of fibroblasts, smooth muscle cells, endothelial cells, immune cells, epithelial cells, and extracellular matrices, plays a crucial role in tumor progression. Hepatic stellate cells (HSCs), a class of unique liver stromal cells, participate in immunomodulatory activities by inducing the apoptosis of effector T-cells, generation of regulatory T-cells, and development of myeloid-derived suppressor cells (MDSCs) to achieve long-term survival of islet allografts. This study provides in vitro and in vivo evidences that HSCs induce the generation of MDSCs to promote hepatocellular carcinoma (HCC) progression through interleukin (IL)-6 secretion. HSC-induced MDSCs highly expressed inducible nitric oxide synthase (iNOS) and arginase 1 mRNA and presented potent inhibitory T-cell immune responses in the tumor environment. Wild-type HSC-induced MDSCs expressed lower levels of CD40, CD86, and MHC II, and a higher level of B7-H1 surface molecules, as well as increased the production of iNOS and arginase I compared with MDSCs induced by IL-6-deficient HSCs in vitro. A murine-transplanted model of the liver tumor showed that HCCs cotransplanted with HSCs could significantly enhance the tumor area and detect more MDSCs compared with HCCs alone or HCCs cotransplanted with HSCs lacking IL-6. In conclusion, the results indicated that MDSCs are induced mainly by HSCs through IL-6 signaling and produce inhibitory enzymes to reduce T-cell immunity and then promote HCC progression within the tumor microenvironment. Therapies targeting the pathway involved in MDSC production or its immune-modulating pathways can serve as an alternative immunotherapy for HCC.

scholarly journals P01.22 Extending CAR T cell therapy applications via drug inducible control of transgene expression

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A18.2-A19
Author(s):  
B Kotter ◽  
N Werchau ◽  
W Krueger ◽  
A Roy ◽  
J Mittelstaet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Transgene Expression ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Part Time ◽  
Anti Cd20

BackgroundAdoptive transfer of chimeric antigen receptor (CAR)-modified T cells has emerged as a promising treatment modality for a broad range of cancers highlighted by the approval of Kymriah™ and Yescarta™ for the treatment of B cell malignancies. However, lack of control of CAR T cell function and consequent excessive inflammation in patients can result in severe side effects especially when targeting tumor-associated rather than tumor-specific antigens. Thus, temporal and tunable control of CAR activity is of major importance for the clinical translation of innovative CAR designs. While the activation of suicide switches results in the apoptotic elimination of the transferred cells, other strategies, e.g. anti-tag CARs or small molecule-gated CARs, enable the reversible control of CAR-mediated function at the protein level but are restricted to a particular CAR design. Focusing on the control of expression rather than CAR signaling, transcriptional regulators represent a versatile tool facilitating a wide range of CAR T cell applications.Materials and MethodsTo maintain control over the infused CAR T cell product and mitigate risks for the patient, we describe here the development of an inducible switch system for the transcriptional regulation of transgene expression in primary, human T cells. Chemically regulated synthetic transcription factors composed of a zinc finger DNA-binding domain, an inducible control domain and a transcription activation domain were designed, screened for functionality, and evaluated in T cells regarding their potential to control CAR expression both in vitro and in vivo.ResultsBy screening, we identified a synthetic transcription factor, which shows high transcriptional output in T cells in the presence of a clinically relevant inducer drug and absence of background activity in the non-induced state. Using this system we were able to control the expression of a CAR recognizing the CD20 antigen present on B cells and B cell leukemic blasts. The addition of the inducer drug resulted in rapid expression of the anti-CD20 CAR on the T cell surface. Moreover, inducible anti-CD20 CAR T cells executed cytolytic activity against CD20 positive target cells and secreted cytokines upon stimulation in vitro. Effectivity in co-cultures was thereby comparable to T cells expressing the anti-CD20 CAR under a conventional constitutive promoter. Furthermore, we could fine-tune CAR activity by titrating the inducer concentration. By defining the time-point of induction, modulation of the onset of therapy was achieved. Upon inducer drug discontinuation, inducible CD20 CAR T cells lost CAR expression and concurrently all CAR-related functions, indicating that the ‘on’ and ‘off’ status can be tightly controlled by the administration of the drug. After pausing of CAR T cell-mediated activity, we could re-induce CAR expression suggesting complete reversibility of effector function. Finally, we were able to show that inducible CD20 CAR T cells mediate a significant, strictly inducer-dependent antitumor activity in a well-established mouse model of B cell lymphoma.ConclusionsThe zinc-finger-based transcriptional control system investigated in this study provides small molecule-inducible control over a therapeutically relevant anti-CD20 CAR in primary T cells in a time- and dose-dependent manner. The tight regulation of CAR expression will pave the way for safer cellular therapies.Disclosure InformationB. Kotter: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. N. Werchau: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. W. Krueger: A. Employment (full or part-time); Significant; Lentigen Technology Inc. A. Roy: A. Employment (full or part-time); Significant; Lentigen Technology Inc. J. Mittelstaet: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. A. Kaiser: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG.

Myeloid-Derived Suppressor Cells Increase in Chronic Myeloid Leukemia and Exert Immune Suppressive Activity.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2779-2779
Author(s):  
Cesarina Giallongo ◽  
Nunziatina Parrinello ◽  
Daniele Tibullo ◽  
Piera La Cava ◽  
Alessandra Cupri ◽  
...  
Keyword(s):  
T Cell ◽  
Chronic Myeloid Leukemia ◽  
T Lymphocytes ◽  
Enzymatic Activity ◽  
Myeloid Leukemia ◽  
Myeloid Cells ◽  
Suppressor Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Arginase 1

Abstract Abstract 2779 Background: Tumor cells are able to develop immune evasion mechanisms which induce a state of immune tolerance and inactivate tumor-specific T cells. In this context, in some solid tumors it has been demonstrated that a subpopulation of myeloid cells, defined as “myeloid-derived suppressor cells” (MDSCs), plays an important role in inducing T cell tolerance by production of arginase that depletes microenvironment of arginine, an essential aminoacid for T cell function. Since chronic myeloid leukemia (CML) patients have high levels of immature myeloid cells it is of interest to investigate if these cells have MDSCs phenotype and activity. Aim: The aim of this study was to analyze MDSCs and investigate their involvement in T-cell anergy of CML patients. Methods: MDSCs were analyzed in peripheral blood (PB) of 13 CML patients (at diagnosis and during therapy) and healthy donors (HD; n=20) by cytofluorimetric analysis (CD14+DR- for monocytic MDSCs and CD11b+CD33+CD14-DR- for granulocytic MDSCs). Arginase 1 expression was assessed in PB of HD and CML patient using real time PCR. Purification of granulocytes, monocytes and lymphocytes from PB was performed by a positive magnetic separation kit (EasySep, STEMCELL Technologies). Arginase activity was measured in granulocyte lysates using a colorimetric test after enzymatic activation and arginine hydrolysis. To evaluate the activation of CD3+ T lymphocytes after incubation with phytoemagglutinin, we analyzed at 24, 48, 72 h the following markers: CD69+, CD71+, DR+. Microvesicles were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation. Results: CML patients showed high levels of monocytic and granulocytic MDSCs at diagnosis in comparison to HD (63±8 and 83±12,2% respectively in CML vs 4,9±2,1 and 55,8±5,3% respectively in HD; p<0.001) while after 3–6 months of tyrosine kinase inhibitors (TKIs) therapy MDSC levels returned to normal values. Either in PB and in the purified granulocytes subpopulation, arginase1 expression showed a 30 fold increase in CML at diagnosis (CML vs HD: p<0.01) and decreased after therapy. We also evaluated arginase enzymatic activity in granulocytes and we found it increased in CML patients (n=4) compared to HD (n=5) (p<0.05). CML as well as HD T lymphocytes showed a normal activation in vitro which was significantly lost when they was incubated with CML serum (n=4). In addition, an increase of monocytic MDSCs in vitro was observed after incubation of HD monocytes with CML serum (39±6%; p<0.01) or microvescicles (9,2±1,2%; p<0.05) compared to control serum. Conclusions: Granulocytic and monocytic MDSCs are increased in CML patients at diagnosis and decrease during TKIs treatment. Their levels also correlates with Arginase 1 expression and enzymatic activity in granulocytes. CML serum as well as CML microvesicles increase the percentage of HD monocytic MDSCs. Moreover, CML serum leads to anergy of T lymphocytes, probably by Arginase 1 secretion. Disclosures: Off Label Use: Eltrombopag is a thrombopoietin receptor agonist indicated for the treatment of thrombocytopenia in patients with chronic immune (idiopathic) thrombocytopenic purpura (ITP).

Hepatic Stellate Cell Conditioned Myeloid Cells Provide a Novel Therapy for Prevention of Factor VIII Antibody Formation in the Hemophilia A Mouse Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4117-4117
Author(s):  
Sumantha Bhatt ◽  
Kathleen Brown ◽  
Feng Lin ◽  
Michael P Meyer ◽  
Margaret V. Ragni ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Hemophilia A ◽  
Myeloid Cells ◽  
Gm Csf ◽  
Cox 2

Abstract Abstract 4117 Background: Hemophilia is an X-linked bleeding disorder resulting from a mutation in coagulation factor VIII (F.VIII). A major drawback of current plasma-derived or recombinant F.VIII therapy is the formation of F.VIII antibodies (inhibitors). Inhibitor formation is a T cell-dependent, B cell-mediated immune response to foreign infused F.VIII. Myeloid derived suppressor cells (MDSCs) are potent suppressors of T cell and B cell responses and are currently under study for therapeutic applications in transplantation and autoimmune diseases. However, the mechanisms of MDSC development and function remain unknown, and in vitro propagation of MDSCs has been a challenge. We hypothesized that MDSCs might be effective in inhibiting F.VIII inhibitor formation in the hemophilia A model. Methods: We developed a novel method for generating MDSCs in vitro by culturing bone marrow cells from hemophilia A mice with hepatic stellate cells (HSCs), hereafter referred to as HSC-conditioned myeloid cells (H-MCs). DCs were propagated from the bone marrow with GM-CSF and IL-4, whereas H-MCs were propagated from the bone marrow with GM-CSF and HSCs. Granulocyte contaminants were removed on day 2 and the remaining monocytic populations were harvested on day 5. Expression of cell surface antigens was analyzed by flow cytometry. Arginase1 and iNOS levels were compared by qPCR, with or without LPS stimulation. The in vitro suppressive capacity of the H-MCs was determined by a mixed leukocyte reaction culture. Splenic T cells from hemophilia A mice were stimulated by irradiated DCs (at a 1–20 ratio, APC to T cell) and recombinant F.VIII. Additional irradiated DCs or H-MCs were added in graded numbers as regulators. The proliferative response was determined by 3H-thymidine incorporation. The phenotype of cultured CD4+ T cells was characterized by intracellular staining for Foxp3 and IFN-gamma and analyzed by flow cytometry. Inhibition of B cells by H-MCs was determined by a CFSE dilution assay. Purified splenic B cells were labeled with CFSE and stimulated by Ig-M and IL-4. APCs (spleen cells) or H-MCs were added at a ratio of 1:10 (APC to B cell). The proportion of proliferating B cells was determined by CFSE dilution of B220 stained cells. In the COX-2 suppression assay, CFSE labeled B cells were treated with varying concentrations of the selective inhibitor of COX-2, NS398. The suppressive effect of H-MCs on B cells in vivo was determined by simultaneously administering H-MCs (I.V) and F.VIII (I.V.) to hemophila A mice on day 0 and rechallenging with recombinant F.VIII on days 2 and 4. WT B6 mice and hemophilia A mice without H-MC transfer served as controls. Plasma anti-F.VIII antibody titers were measured on day 12 by a modified ELISA assay. Results: H-MCs expressed low levels of costimulatory molecules but high levels of the inhibitory molecule B7-H1 and immunoregulatory enzyme arginase-1. In contrast, DCs expressed high levels of costimulatory molecules and MHC class II. In vitro studies demonstrated that the H-MCs markedly inhibited antigen specific T cell proliferation induced by dendritic cells in response to recombinant F.VIII (Fig. 1). H-MCs altered the T cell response in hemophilia A mice by promoting the expansion of regulatory T cells and inhibiting IFN-γ producing CD4+ T cells. When the H-MCs were cocultured with B cells isolated from hemophilia A mice, in the presence of Ig-M and IL-4, the H-MCs abrogated B cell activation and proliferation directly (Fig. 2). H-MCs may be modulating the B cell response through the Cox-2 pathway, as inhibition of Cox-2 through NS398 led to the restoration of B cell proliferation. More importantly, adoptive transfer of H-MCs into hemophilia Amice, at the time of F.VIII infusion, markedly suppressed anti-F.VIII antibody formation (Fig. 3). Conclusion: These results suggest that HSC conditioned myeloid cells may represent a potential therapeutic approach to induction of immune tolerance in patients with hemophilia A andother immune disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals SOD3 induces a HIF-2α-dependent program in endothelial cells that provides a selective signal for tumor infiltration by T cells

2020 ◽  
Vol 8 (1) ◽  
pp. e000432 ◽  
Author(s):  
Lorena Carmona-Rodríguez ◽  
Diego Martínez-Rey ◽  
Maria Jesús Fernández-Aceñero ◽  
Alicia González-Martín ◽  
Mateo Paz-Cabezas ◽  
...  
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
T Lymphocytes ◽  
Adhesion Molecule ◽  
Cell Infiltration ◽  
Tumor Infiltration ◽  
T Cell Infiltration

BackgroundTumor-infiltrating lymphocytes (TILs), mainly CD8+ cytotoxic T lymphocytes (CTL), are linked to immune-mediated control of human cancers and response to immunotherapy. Tumors have nonetheless developed specific mechanisms that selectively restrict T cell entry into the tumor microenvironment. The extracellular superoxide dismutase (SOD3) is an anti-oxidant enzyme usually downregulated in tumors. We hypothesize that upregulation of SOD3 in the tumor microenvironment might be a mechanism to boost T cell infiltration by normalizing the tumor-associated endothelium.ResultsHere we show that SOD3 overexpression in endothelial cells increased in vitro transmigration of naïve and activated CD4+ and CD8+ T cells, but not of myeloid cells. Perivascular expression of SOD3 also specifically increased CD4+ and CD8+ effector T cell infiltration into tumors and improved the effectiveness of adoptively transferred tumor-specific CD8+ T cells. SOD3-induced enhanced transmigration in vitro and tumor infiltration in vivo were not associated to upregulation of T cell chemokines such as CXCL9 or CXCL10, nor to changes in the levels of endothelial adhesion receptors such as intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1). Instead, SOD3 enhanced T cell infiltration via HIF-2α-dependent induction of specific WNT ligands in endothelial cells; this led to WNT signaling pathway activation in the endothelium, FOXM1 stabilization, and transcriptional induction of laminin-α4 (LAMA4), an endothelial basement membrane component permissive for T cell infiltration. In patients with stage II colorectal cancer, SOD3 was associated with increased CD8+ TIL density and disease-free survival. SOD3 expression was also linked to a T cell–inflamed gene signature using the COAD cohort from The Cancer Genome Atlas program.ConclusionOur findings suggest that SOD3-induced upregulation of LAMA4 in endothelial cells boosts selective tumor infiltration by T lymphocytes, thus transforming immunologically “cold” into “hot” tumors. High SOD3 levels are associated with human colon cancer infiltration by CD8+ T cells, with potential consequences for the clinical outcome of these patients. Our results also uncover a cell type–specific, distinct activity of the WNT pathway for the regulation of T cell infiltration into tumors.

Bone Marrow Myeloid-Derived Suppressor Cells (MDSC) Inhibit Graft Versus Host Disease (GVHD) Via An Arginase-1 Dependant Mechanism That Is Upregulated by IL-13

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 241-241
Author(s):  
Steven L Highfill ◽  
Paulo Rodriguez ◽  
Qing Zhou ◽  
Christine A Goetz ◽  
Rachelle Veenstra ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
State University ◽  
Medical College ◽  
Louisiana State University ◽  
Arginase 1 ◽  
University Patents ◽  
Donor T Cells

Abstract Abstract 241 Background: Myeloid-derived suppressor cells (MDSC) are a well-defined population of cells that accumulate in the tissue of tumor-bearing animals and are known to inhibit immune responses. Here, we have developed a novel method by which we can generate MDSC from the BM of wild type mice. Our goals were to determine the effectiveness of these cells in inhibiting an allogeneic T-cell reaction, the mechanism by which this occurs, and the impact on the graft-versus-tumor (GVT) activity of donor T-cells. Results: Our results show that the incubation of WT BM with G-CSF and GM-CSF for a period of 4 days results in a population of cells predominately co-expressing CD11b and Gr1 (50%). Most of these cells have a monocytic phenotype of Ly6GloLy6C+ (73%) and also express other markers associated with MDSCs such as IL4Ra (64%), F4/80 (63%) and CD115 (43%). Such MDSC potently inhibited in vitro allogeneic T-cell responses and the addition of IL-13 to the MDSC culture enhanced their suppressive capacity (60% suppression by MDSC at the peak of the response, day 5 vs. 75% suppression by MDSC-IL13, P<0.001). Suppression was dependent on L-arginine depletion because only the addition excess L-arginine, but not tryptophan, resulted in a significant reduction in suppression (60% reduction at day 4, P=0.001). Adding IL-13 to the MDSC cultures resulted in arginase-1 upregulation (20 fold increase in expression over control BM for MDSC vs. 350 fold increase in MDSC-IL13, P<0.001). The arginase inhibitor, nor-NOHA, significantly reduced MDSC suppression in vitro. GNC2 kinase knockout T cells were resistant to suppression, consistent with their inability to sense L-arginine depletion. Although iNOS was upregulated 5-fold in MDSCs vs BM, IL13 addition did not further upregulate iNOS. Suppression was mostly contact-independent. In vivo, MDSC-IL13 derived from GFP transgenic BM migrated to sites of allopriming. MDSC-IL13 from luciferase transgenic donors increased over a 3 week period of time as assessed by bioluminescent imaging. Although both MDSC and MDSC-IL13 inhibited GVHD lethality, MDSC-IL13 were more effective at enhancing survival (MDSC vs. MDSC-IL13, P=0.001). When compared with untreated mice, GVHD inhibition in MDSC-IL13 treated mice was associated with: 1) Limited donor T-cell proliferation (CD4+CFSE−, 40% vs. 17%, P=0.01; CD8+CFSE−, 51% vs. 16%, P=0.01); 2) Decreased donor T-cell activation (CD4+CD62L−, 74% vs. 50%, P<0.001; CD8+CD62L−, 77% vs. 41%, P<0.001); 3) Decreased proinflammatory cytokine production (CD4+IFNg+, 37% vs. 24%, P=0.04; CD8+IFNg+, 30% vs. 15%, P=0.007); 4) Decreased expression of intracellular CD3z chain (CD4+CD3z+, 86% vs. 56%, P<0.001; CD8+CD3z+, 85% vs. 68%, P<0.001). On day 14, donor MDSC-IL13 cells, distinguishable from donor BM and the host by a CD45 congenic marker, were re-isolated from the spleen of murine GVHD recipients. MDSC-IL13 cells retained their initial phenotype and were potent ex vivo suppressors of an allogeneic T cell response. Arginase-1 knockout MDSC-IL13 did not have a significant effect on reducing GVHD, indicating that arginase-1 expression was vital to the in vivo suppressive effect of MDSCs. MDSC-IL13 did not abrogate the graft-versus-tumor effect of donor T-cells. In MDSC-IL13 treated mice, donor T-cells retained their ability to eliminate A20 lymphoma cells, and at the same time, had improved survival when compared to mice receiving A20 cells plus T-cells all of which succumbed to GVHD (BM+T+A20 vs. BM+T+A20+MDSC-IL13, P<0.001). Since arginase-1 expression was critical for suppression, we exploited this mechanism of action by administering a pegylated form of human arginase-1 (PEG-arg1) to systemically deplete L-arginine. We find that GVHD survival is significantly improved (BM+T vs. BM+T+PEG-arg1, P=0.003). Conclusions: We can conclude that BM-derived MDSC-IL13 have the ability to dampen GVHD and enhance survival when using a fully-mismatched murine model of BMT. Arginase-1 expression was found to play a critical role in MDSC-mediated suppression. A GVT effect was not ablated by MDSC-IL13. In vivo administration of PEG-arg1 resulted in L-arginine depletion and significant GVHD reduction. Both MDSC infusion and PEG-arg1 administration are promising strategies that warrant further preclinical studies to prevent GVHD that may be readily translatable in the clinic. Disclosures: Highfill: UIniversity of Minnesota: Patents & Royalties; Louisiana State University: Patents & Royalties; Medical College of Georgia: Patents & Royalties. Rodriguez:University of Minnesota: Patents & Royalties; Louisiana State University: Patents & Royalties; Medical College of Georgia: Patents & Royalties. Ochoa:University of Minnesota: Patents & Royalties; Louisiana State University: Patents & Royalties; Medical College of Georgia: Patents & Royalties. Blazar:University of Minnesota: Patents & Royalties; Louisiana State University: Patents & Royalties; Medical College of Georgia: Patents & Royalties.

Myeloid Derived Suppressor Cells (MDSCs) Are Increased and Exert Immunosuppressive Activity In CML Patients At Diagnosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2711-2711
Author(s):  
Cesarina Giallongo ◽  
Nunziatina Parrinello ◽  
Daniele Tibullo ◽  
Piera La Cava ◽  
Alessandra Romano ◽  
...  
Keyword(s):  
T Cell ◽  
Western Blot ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Suppressor Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Immunosuppressive Activity ◽  
Arginase 1

Abstract Introduction In some solid tumors it has been demonstrated that a subpopulation of myeloid cells, defined as “myeloid-derived suppressor cells” (MDSCs), plays an important role in inducing T cell tolerance by production of arginase 1 (arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function. Since chronic myeloid leukemia (CML) patients have high levels of immature myeloid cells it is of interest to investigate if these cells have MDSCs phenotype and activity. The aim of this study was to analyze MDSCs and investigate their activity in CML patients. Methods MDSCs were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis. In 21 patients MDSCs were also measured during TKI treatment. Granulocytic MDSCs (G-MDSCs) were identified as CD11b+CD33+CD14-HLADR- cells, while the monocytic MDSCs (Mo-MDSCs) as CD14+HLADR by cytofluorimetric analysis. Arg1 expression was assessed using real time PCR and Western Blot. Arg activity was measured in granulocyte lysates using a colorimetric test after enzymatic activation and arginine hydrolysis. Microvesicles (MV) were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation. Results CML patients showed high levels of Mo- and G-MDSCs at diagnosis in comparison to HD (41±8 and 82,5±12,2% respectively for CML vs 9±2,1 and 55±5,3% for HD; p<0.001), while after TKIs therapy both subpopulations decreased, returning to normal values. T-reg (CD4+ CD25high Foxp3+ cells) were also significantly increased in CML patients at diagnosis in respect to HD (9±2% vs 6,1±0,8%, p<0.001) with a significant correlation with the percentage of Gr-MDSCs (r=0,6254; p<0.001). Both in PB and purified granulocytic cells, Arg1 expression showed a 30 fold increase in CML at diagnosis compared to HD (p<0.001) and decreased after therapy. The same data were confirmed by Western Blot analysis. Arg enzymatic activity in granulocytes resulted also increased in CML (n=10) compared to HD (n=10) (p<0.001). The suppressive function of CML G-MDSCs was demonstrated by their ability to inhibit the proliferation of CFSE+ HD T cells (p<0.001). In addition, an increase of Mo-MDSCs in vitro was observed after incubation of HD monocytes with CML sera (29±13%; p<0.0001) or MV (8±2,8%; p<0.05). Conclusions MDSCs are increased in CML patients at diagnosis and decrease during TKIs treatment. CML granulocytes have high arg1 activity and immunosuppressive activity. Moreover, CML serum as well as CML microvesicles increase the percentage of HD Mo-MDSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Anti-EGFR Fibronectin Bispecific Chemically Self-Assembling Nanorings (CSANs) Induce Potent T cell Mediated Anti-Tumor Response and Downregulation of EGFR Signaling and PD-1/PD-L1 Expression

2020 ◽  
Author(s):  
Ozgun Kilic ◽  
Marcos R. Matos de Souza ◽  
Abdulaziz A. Almotlak ◽  
Jill M. Siegfried ◽  
Carston R. Wagner
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Tumor Antigens ◽  
Cancer Therapeutics ◽  
High Avidity ◽  
Egfr Signaling ◽  
Single Chain

ABSTRACTNumerous approaches have targeted the Epidermal Growth Factor Receptor (EGFR) for the development of anti-cancer therapeutics, since it is over-expressed on a variety of cancers. Recently, αEGFR chimeric antigen receptor (CAR)-T cells have shown potential promise for the immunological control of tumors. Our laboratory has recently demonstrated that bispecific chemically self-assembled nanorings (CSANs) can modify T cell surfaces and function as prosthetic antigen receptors (PARs). This technology allows selective targeting of tumor antigens due to high avidity of the multimeric rings, while incorporating a mechanism to dissociate the rings to prevent further T cell stimulation. Previously, PARs with single-chain variable fragments (scFvs) have been successful in vitro and in vivo, activating T cells selectively at the tumor site. Alternatively, here we report fibronectin (FN3)-based PARs with improved properties such as increased protein yield, rapid protein production, increased protein stability and predicted low immunogenicity due to the human origin of fibronectins. We examined the cytotoxicity of EGFR-targeting PARs in vitro in which the affinities of the αEGFR fibronectins, the αEGFR/ αCD3 valency of the CSANs and the antigen expression levels were varied. Based on these selective in vitro cytotoxicity results, we conducted an in vivo study of FN3-PARs using an orthotopic breast cancer model. The FN3-PARs demonstrated potent tumor growth suppression with no adverse effects. Furthermore, these results demonstrated that FN3-PARs modulated the tumor microenvironment by downregulating EGFR signaling resulting in decreased PD-L1 expression. In addition, the expression of PD-1 was also found to be reduced. Collectively, these results demonstrate that FN3-PARs have the potential to direct selective T cell targeted tumor killing and that αEGFR FN3-PARs may enhance anti-tumor T cell efficacy by modulating the tumor microenvironment.

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