106 Increasing AMPK activity in human T cells enhances memory subset formation without sacrificing in vitro expansion

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A117-A118
Author(s):  
Erica Braverman ◽  
Andrea Dobbs ◽  
Darlene Monlish ◽  
Craig Byersdorfer
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Oxidative Metabolism ◽  
Mitochondrial Density ◽  
Cell Counts ◽  
Human T Cells ◽  
In Vitro Expansion ◽  
Ampk Activity

BackgroundThe ideal adoptive cell therapy consists of memory-like T cells with enhanced oxidative potential. However, current expansion protocols drive T cells towards terminal differentiation, decreasing the number of T cells fit for the in vivo environment. AMP-activated protein kinase (AMPK), whose activity increases in memory cells, is a key regulator of mitochondrial biogenesis and oxidative metabolism, making AMPK activation an attractive candidate to improve adoptive T cell function.MethodsTo increase AMPK activity, AMPKγ, which controls the phosphorylation status of AMPKa and therefore activity of the AMPK complex, was cloned into a lentiviral plasmid downstream of the elongation factor 1a (EF1a) promoter and upstream of green fluorescent protein (GFP). An empty vector, containing GFP only, served as a negative control. Human T cells were transduced and expanded in vitro in the presence of IL-2. AMPK activity was assessed via immunoblot for phosphorylation of AMPKa on Thr172 and S555 on downstream target Unc-51-like kinase 1 (ULK1). Memory-marker expression and mitochondrial density (using Mitotracker Red) were analyzed by flow cytometry. Oxidative metabolism and spare respiratory capacity (SRC) were determined using the Seahorse Metabolic Analyzer. Fold changes of in vitro expansion were calculated by adjusting manual cell counts for GFP positivity and CD4+/CD8+ staining.ResultsAMPKγ was efficiently transduced and expressed by human T cells, which significantly increased AMPK activity (AMPKa phosphorylation 1.93 ± 0.05 vs 0.6 ± 0.09, p<0.001, ULK1 phosphorylation 1.28 ± 0.11 vs 0.67 ± 0.08, p<0.01). AMPKγ-overexpressing T cells augmented expression of memory markers CD62L, CD27, and CCR7, with an increased yield of stem cell memory-like T cells marked by co-expression of CD45RA and CD62L (figure 1). Mitochondrial density, SRC, and maximal oxygen consumption rates were similarly increased in AMPKγ-transduced cells (figure 2A,B). Further, while enhanced memory cell production is often linked with reduced proliferation, T cells with increased AMPK activity maintained and even trended towards increased rates of expansion compared to empty-transduced controls (figure 3A), with a measurable increase in CD4+ T cell percentages by flow cytometry (figure 3B).Abstract 106 Figure 1AMPK-transduced T cells increase expression of memory surface markers. Human T cells were transduced with AMPK-GFP or GFP-only control (Empty). Memory markers were assessed by flow cytometry on Days 7–14 of in vitro culture following expansion with IL-2. Plots are representative of 3 separate donorsAbstract 106 Figure 2AMPK-transduced T cells show enhanced mitochondrial density and SRC. (A) Human T cells transduced with AMPK-GFP or GFP-only (Empty) were stained with Mitotracker Red and fluorescence intensity compared between transduced cells and GFP- controls within the same culture to account for variability in Mitotracker dye staining. (B) AMPK and Empty transduced T cells were assessed via Seahorse Metabolic Analyzer using the Mito Stress Test. Results are representative of 3 separate donors. OCR = O2 consumption rateAbstract 106 Figure 3Proliferation is maintained in AMPK-transduced T cells, with enhanced recovery of CD4+ T cells. (A) Primary human T cells transduced with AMPK-GFP or GFP-only (Empty) were expanded in vitro in the presence of IL-2. Cells were manually counted and the ratio of day 7 to day 5 cell counts calculated to assess fold expansion over time. (B) At the same, CD4+ and CD8+ percentages were measured in GFP+ cells by flow cytometryConclusionsIncreasing AMPK activity endows T cells with a variety of characteristics ideal for adoptive cell therapy, including increased memory-marker expression, enhanced SRC and oxidative metabolism, equivalent to augmented in vitro expansion, and improved CD4+ T cell yields. Further studies are ongoing to assess the activity and function of AMPK-transduced CAR-T cells both in vitro and in vivo.

scholarly journals Increasing AMPK Activity in Human T Cells Enhances Memory Subset Formation without Sacrificing in Vitro Expansion

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 38-39
Author(s):  
Erica Lynne Braverman ◽  
Andrea Dobbs ◽  
Darlene A. Monlish ◽  
Craig Byersdorfer
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Human T Cells ◽  
Car T ◽  
Ampk Activity

BACKGROUND: While chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of relapsed/refractory acute lymphoblastic leukemia (ALL), treatment failures continue to occur. In studying therapeutic T cell function, it has become clear that achieving a memory-like phenotype is ideal for CAR-T production. This is likely related to the enhanced oxidative metabolic potential of this subset, which allows for improved persistence and enhanced anti-leukemia activity in vivo. However, current expansion protocols drive T cells towards terminal differentiation, decreasing the number of T cells fit for the in vivo environment. Finding methods to improve the yield of memory-like cells without sacrificing T cell expansion has been challenging. AMP-activated protein kinase (AMPK) is a key metabolic regulator responsible for promoting mitochondrial biogenesis and oxidative metabolism, and is more active in memory T cells at baseline. It is similarly induced by TCR ligation, making it unlikely that it would significantly detract from proliferation. These properties make activation of AMPK a potential candidate pathway for improving the yield of more functional T cells for CAR-T cell therapy. METHODS: AMPK is a heterotrimeric protein complex consisting of alpha, beta, and gamma domains. Functionally, the alpha subunit contains the kinase domain, which is activated by phosphorylation. The gamma subunit controls the phosphorylation, and therefore the activity, of the alpha domain. To increase AMPK signaling in T cells, we cloned the gamma subunit into a lentiviral plasmid containing the elongation factor 1a (EF1a) promoter and a green fluorescent protein (GFP) tag. An empty vector, containing GFP only, served as a negative control. Human T cells were isolated from three separate donors, transduced with our lentiviral construct, and expanded in vitro in the presence of IL-2. AMPK activity was assessed by phosphorylation of Thr172 on the AMPKα subunit as well as phosphorylation of S555 on downstream target Unc-51-like autophagy activating kinase (ULK1) using western blot densitometry, normalized to the total protein amounts. Memory marker expression and mitochondrial density (using Mitotracker Red) were analyzed by flow cytometry. Oxidative metabolism and spare respiratory capacity (SRC) were determined using the Seahorse Metabolic Analyzer. Fold changes for in vitro expansion were calculated by adjusting manual cell counts to reflect GFP positivity and CD4+/CD8+ surface staining. RESULTS: The AMPK gamma subunit was efficiently transduced and expressed by human T cells as measured by GFP expression, qRT-PCR, and western blot analysis. Further, AMPK activity increased in GFP+ cells as indicated by the phosphorylation of AMPKα Thr172 (1.93 +/- 0.05 vs 0.6 +/- 0.09, p&lt;0.001) and ULK1 S555 (1.28 +/- 0.11 vs 0.67 +/- 0.08, p&lt;0.01). Cells transduced with AMPK augmented expression of memory markers CD62L, CD27, and CCR7, with an increased yield of stem cell memory-like T cells marked by co-expression of CD45RA and CD62L (Figure 1). In addition, AMPK-transduced T cells showed a statistically significant increase in mitochondrial density along with notable enhancement of SRC and maximal oxygen consumption rates (Figure 2A,B). Furthermore, the rate of expansion of AMPK-transduced T cells did not differ significantly from Empty-transduced controls, and in fact trended towards increased in both CD4+ and CD8+ cells (Figure 3A). Indeed, the improved rate of expansion in AMPK-transduced CD4+ T cells led to a measurable increase in CD4+ T cell percentages by flow cytometry (Figure 3B). DISCUSSION: Here we present an efficient and direct method to increase AMPK activity in human T cells and demonstrate that increased AMPK activity endows T cells with a variety of characteristics ideal for CAR-T cell therapy. These features include increased memory-marker expression, enhanced SRC and oxidative metabolism, equivalent to augmented in vitro expansion, and improved CD4+ T cell yields. Further studies are ongoing to assess the activity and function of AMPK-transduced CAR-T cells both in vitro and in vivo. Disclosures No relevant conflicts of interest to declare.

627 The DLL3-targeted half-life extended bispecific T cell engager (HLE BiTE®) immune-oncology therapy AMG 757 has potent antitumor activity in neuroendocrine cancer

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A663-A663
Author(s):  
Keegan Cooke ◽  
Juan Estrada ◽  
Jinghui Zhan ◽  
Jonathan Werner ◽  
Fei Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Mouse Models ◽  
T Cell Activation ◽  
Phase 1 ◽  
Phase 1 Study ◽  
Human T Cells

BackgroundNeuroendocrine tumors (NET), including small cell lung cancer (SCLC), have poor prognosis and limited therapeutic options. AMG 757 is an HLE BiTE® immune therapy designed to redirect T cell cytotoxicity to NET cells by binding to Delta-like ligand 3 (DLL3) expressed on the tumor cell surface and CD3 on T cells.MethodsWe evaluated activity of AMG 757 in NET cells in vitro and in mouse models of neuroendocrine cancer in vivo. In vitro, co-cultures of NET cells and human T cells were treated with AMG 757 in a concentration range and T cell activation, cytokine production, and tumor cell killing were assessed. In vivo, AMG 757 antitumor efficacy was evaluated in xenograft NET and in orthotopic models designed to mimic primary and metastatic SCLC lesions. NSG mice bearing established NET were administered human T cells and then treated once weekly with AMG 757 or control HLE BiTE molecule; tumor growth inhibition was assessed. Pharmacodynamic effects of AMG 757 in tumors were also evaluated in SCLC models following a single administration of human T cells and AMG 757 or control HLE BiTE molecule.ResultsAMG 757 induced T cell activation, cytokine production, and potent T cell redirected killing of DLL3-expressing SCLC, neuroendocrine prostate cancer, and other DLL3-expressing NET cell lines in vitro. AMG 757-mediated redirected lysis was specific for DLL3-expressing cells. In patient-derived xenograft and orthotopic models of SCLC, single-dose AMG 757 effectively engaged human T cells administered systemically, leading to a significant increase in the number of human CD4+ and CD8+ T cells in primary and metastatic tumor lesions. Weekly administration of AMG 757 induced significant tumor growth inhibition of SCLC (figure 1) and other NET, including complete regression of established tumors and clearance of metastatic lesions. These findings warranted evaluation of AMG 757 (NCT03319940); the phase 1 study includes dose exploration (monotherapy and in combination with pembrolizumab) and dose expansion (monotherapy) in patients with SCLC (figure 2). A study of AMG 757 in patients with neuroendocrine prostate cancer is under development based on emerging data from the ongoing phase 1 study.Abstract 627 Figure 1AMG 757 Significantly reduced tumor growth in orthotopic SCLC mouse modelsAbstract 627 Figure 2AMG 757 Phase 1 study designConclusionsAMG 757 engages and activates T cells to kill DLL3-expressing SCLC and other NET cells in vitro and induces significant antitumor activity against established xenograft tumors in mouse models. These preclinical data support evaluation of AMG 757 in clinical studies of patients with NET.Ethics ApprovalAll in vivo work was conducted under IACUC-approved protocol #2009-00046.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Gisele Olinto Libanio Rodrigues ◽  
Julie Hixon ◽  
Hila Winer ◽  
Erica Matich ◽  
Caroline Andrews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Cell Counts

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals Sunitinib Exerts In Vitro Immunomodulatory Activity on Sarcomas via Dendritic Cells and Synergizes With PD-1 Blockade

2021 ◽  
Vol 12 ◽  
Author(s):  
Darina Ocadlikova ◽  
Mariangela Lecciso ◽  
Javier Martin Broto ◽  
Katia Scotlandi ◽  
Michele Cavo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cell Lines ◽  
Effector T Cells ◽  
Sarcoma Cell ◽  
Ifn Γ ◽  
Sarcoma Cells

BackgroundHigh-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD-1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in in vitro cocultures of sarcoma.MethodsThe human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-γ production, before and after nivolumab exposure, were analyzed.ResultsAlong with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib-treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-γ. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-γ-producing effector T cells.ConclusionsTaken together, our in vitro data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-γ-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.

scholarly journals Bisphenol A modulates the metabolic regulator oestrogen-related receptor-α in T-cells

Reproduction ◽  
2014 ◽  
Vol 147 (4) ◽  
pp. 419-426 ◽  
Author(s):  
Riccardo Cipelli ◽  
Lorna Harries ◽  
Katsuhiro Okuda ◽  
Shin'ichi Yoshihara ◽  
David Melzer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Bisphenol A ◽  
Oxidative Metabolism ◽  
Biological Effects ◽  
Control Point ◽  
White Blood Cells ◽  
Fold Increase ◽  
Metabolic Effects

Bisphenol A (BPA) is a widely used plastics constituent that has been associated with endocrine, immune and metabolic effects. Evidence for how BPA exerts significant biological effects at chronic low levels of exposure has remained elusive. In adult men, exposure to BPA has been associated with higher expression of two nuclear receptors, oestrogen receptor-β (ERβ) and oestrogen-related-receptor-α (ERRα), in peripheral white blood cells in vivo. In this study, we explore the expression of ESR2 (ERβ) and ESRRA (ERRα) in human leukaemic T-cell lymphoblasts (Jurkat cells) exposed to BPA in vitro. We show that exposure to BPA led to enhanced expression of ESRRA within 6 h of exposure (mean±s.e.m.: 1.43±0.08-fold increase compared with the control, P<0.05). After 72 h, expression of ESRRA remained significantly enhanced at concentrations of BPA ≥1 nM. Oxidative metabolism of BPA by rat liver S9 fractions yields the potent oestrogenic metabolite, 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP). Exposure of cells to 1–100 nM MBP increased the expression of both ESRRA (significantly induced, P<0.05, at 1, 10, 100 nM) and ESR2 (1.32±0.07-fold increase at 100 nM exposure, P<0.01). ERRα is a major control point for oxidative metabolism in many cell types, including T-cells. Following exposure to both BPA and MBP, we found that cells showed a decrease in cell proliferation rate. Taken together, these results confirm the bioactivity of BPA against putative T-cell targets in vitro at concentrations relevant to general human exposure.

CD26+CD4+T cell counts and attack risk in interferon-treated multiple sclerosis

2005 ◽  
Vol 11 (6) ◽  
pp. 641-645 ◽  
Author(s):  
F Sellebjerg ◽  
C Ross ◽  
N Koch-Henriksen ◽  
P Soelberg Sørensen ◽  
J L Frederiksen ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Hazard Ratio ◽  
Cd4 T Cell ◽  
Immunomodulatory Treatment ◽  
Cell Counts ◽  
Increased Risk ◽  
Insufficient Response

Biomarkers that allow the identification of patients with multiple sclerosis (MS) with an insufficient response to immunomodulatory treatment would be desirable, as currently available treatments are only incompletely efficacious. Previous studies have shown that the expression of CD25, CD26 and CCR5 on T cells is altered in patients with active MS. We studied the expression of these molecules by flow cytometry in patients followed for six months during immunomodulatory treatment. In interferon (IFN)-β-treated patients, we found that the hazard ratio for developing an attack was 2.8 in patients with CD26+CD4+T cell counts above median, and this risk was independent of the risk conferred by neutralizing anti-IFN-β antibodies. CD26+CD4+T cell counts may identify patients with MS at increased risk of attack during treatment with IFN-β.

CD4+ Foxp3+ regulatory T cell-mediated immunomodulation by anti-depressants inhibiting acid sphingomyelinase

2018 ◽  
Vol 399 (10) ◽  
pp. 1175-1182 ◽  
Author(s):  
Jürgen Schneider-Schaulies ◽  
Niklas Beyersdorf
Keyword(s):  
T Cells ◽  
T Cell ◽  
Treg Cells ◽  
Tricyclic Antidepressant ◽  
Acid Sphingomyelinase ◽  
Human T Cells ◽  
And Function ◽  
Rate Limiting ◽  
Deficient Mice

AbstractAcid sphingomyelinase (ASM) is the rate-limiting enzyme cleaving sphingomyelin into ceramide and phosphorylcholin. CD4+Foxp3+regulatory T (Treg) cells depend on CD28 signaling for their survival and function, a receptor that activates the ASM. Both, basal and CD28-induced ASM activities are higher in Treg cells than in conventional CD4+T (Tconv) cells. In ASM-deficient (Smpd1−/−) as compared to wt mice, membranes of T cells contain 7–10-fold more sphingomyelin and two- to three-fold more ceramide, and are in a state of higher order than membranes of T cells from wt mice, which may facilitate their activation. Indeed, the frequency of Treg cells among CD4+T cells in ASM-deficient mice and their suppressive activityin vitroare increased. Moreover,in vitrostimulation of ASM-deficient T cells in the presence of TGF-β and IL-2 leads to higher numbers of induced Treg cells. Pharmacological inhibition of the ASM with a clinically used tricyclic antidepressant such as amitriptyline in mice or in tissue culture of murine or human T cells induces higher frequencies of Treg cells among CD4+T cells within a few days. This fast alteration of the balance between T cell populationsin vitrois due to the elevated cell death of Tconv cells and protection of the CD25highTreg cells by IL-2. Together, these findings suggest that ASM-inhibiting antidepressants, including a fraction of the serotonin re-uptake inhibitors (SSRIs), are moderately immunosuppressive and should be considered for the therapy of inflammatory and autoimmune disorders.

Mesothelin Targeting Bites for Pediatric AML: In Vivo Efficacy and Specificity

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3925-3925
Author(s):  
Anilkumar Gopalakrishnapillai ◽  
Colin Correnti ◽  
Anne Kisielewski ◽  
Allison Kaeding ◽  
Soheil Meshinchi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Median Survival ◽  
Single Chain ◽  
Pediatric Leukemia ◽  
Effector Cells ◽  
Cell Counts ◽  
Human T Cells ◽  
Pediatric Aml

Acute myeloid leukemia (AML) remains the type of pediatric leukemia with poorest outcome. Despite maximally intensive therapy, approximately 20% of patients experience recurrent disease. Novel targeted therapies are needed to improve survival. We recently identified that mesothelin, a well-validated target in some cancers, is also highly expressed in a subset of pediatric AML samples (Tarlock et al., Blood, 128:2873, 2016). Considering that it is not expressed in normal tissues in children (Fan et al., Blood, 130:3792, 2017), MSLN is a viable target for immunotherapies such as Bispecific T-cell Engaging antibodies (BiTEs) that combine antibody single chain variable (scFv) regions targeting a cancer antigen and the T-cell co-receptor CD3. We designed and tested the efficacy and specificity of BiTEs targeting MSLN in disseminated xenograft models of pediatric AML. Using scFv sequences derived from Amatuximab, which recognizes the N-terminal domain of the GPI-linked ectodomain of MSLN, targeting region 1 of MSLN, and from Blinatumomab/AMG-330 targeting CD3, we engineered and expressed two kinds of BiTE molecules - a canonical BiTE and an IgG BiTE, a larger molecule with improved serum half life in vivo. To evaluate the specificity and efficacy of canonical BiTEs, MV4;11-MSLN cell line was generated by lentiviral transduction of parental MV4;11 cells which do not constitutively express MSLN (Fig. 1A, B). These two cell lines were injected i.v. into NSG-SGM3 mice. Once engraftment was confirmed, purified human T cells (3 x 106) were injected to act as effector cells. Mice were then treated with the canonical αMSLN-αCD3 BiTE at a dose of 3 mg/kg/day daily for 6 days. A cohort of mice that were untreated or received BiTE or T-cell infusion only served as controls. Mice from both treated and untreated groups had to be euthanized when they presented with distended abdomens due to myeloid sarcomas and no significant differences in survival were observed. Post euthanasia, bone marrows were flushed and evaluated for the percentage of AML cells (human CD45+CD33+) and T cells (human CD45+CD3+). We observed that the αMSLN-αCD3 BiTE was effective in promoting T-cell activation (based on high T-cell counts compared to mice injected with T-cells alone) and greatly reducing leukemic burden in mice injected with MV4;11 cells engineered to express MSLN (Fig. 1C, D). Similar results were obtained using BiTEs targeting a different MSLN epitope. No T-cell expansion and anti-leukemic effect was observed in mice engrafted with parental MV4;11 cells. Although, there were no significant differences between the median survival of untreated and treated miceThese data highlight the specificity and efficacy of the aMSLN-CD3 BiTEs. Among a panel of 8 AML patient-derived xenograft (PDX) lines generated in the laboratory, NTPL-146 bearing MLL-ENL fusion was found to have endogenous MSLN expression (Fig. 1E). We evaluated the efficacy of αMSLN-αCD3 canonical BiTE (3 mg/Kg Qdx6) against NTPL-146 PDX line in NSG-B2m mice by transfusing human CD3+ T-cells to act as effector cells. A Kaplan-Meier survival plot based on the time when each mouse reached experimental end-point (reduced body weight greater than 20%, impaired mobility, hind limb paralysis) showed that the survival benefit for mice receiving BiTE in the presence of human T-cells (4/6 mice survived at the end of experiment) greatly exceeded the efficacy of T-cells alone (22-day improvement in median survival with no surviving mice), or BiTE treatment alone (no improvement in survival) compared to untreated mice (Fig. 1F, P<0.001). These data validate the efficacy of MSLN targeting BiTEs in a PDX model with endogenous MSLN expression. The efficacy of canonical vs IgG BiTEs was evaluated in MV4;11-MSLN xenografted mice. Mice were dosed Qd5x3 for canonical BiTE and Q7dx3 for IgG BiTE as shown (Fig. 1G). IgG BiTE treatment along with T-cell infusion significantly prolonged survival in mice transplanted with MV4;11-MSLN (Fig. 1H), suggesting that IgG BiTE was far more efficacious than canonical BiTEs (P<0.01). Taken together, these data indicate that MSLN-targeting BiTEs could be used as novel immunotherapy for pediatric AML with MSLN expression. Figure 1 Disclosures Kaeding: Celgene: Employment.

Superior Immunogenicity of Idiotype Fab Fragments As Compared to Entire Immunoglobulin for Active Lymphoma Immunotherapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1633-1633
Author(s):  
Marcelo A. Navarrete ◽  
Benjamin Kisser ◽  
Hendrik J. Veelken
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
B Cell Lymphomas ◽  
Cd8 Cells ◽  
Fab Fragments ◽  
The Individual

Abstract Abstract 1633 Introduction: The individual collection of epitopes within the variable regions of the unique immunoglobulin expressed by every mature B-cell lymphoma (idiotype, or Id) represents a tumor-specific antigen and lends itself as a target for therapeutic vaccination strategies. Immunization with tumor Id has the capacity to elicit polyclonal antibody responses as well as CD8+ and CD4+ T cells recognizing Id-derived peptides presented on class I and class II HLA molecules, respectively. Due to a perceived low immunogenicity of lymphoma-derived Id, most Id vaccines tested in clinical trials so far have been formulated as conjugates with the strongly immunogenic carrier keyhole limpet hemocyanin (KLH). In contrast, we have consistently observed high rates of humoral and cellular anti-Id immune responses in consecutive trials of active immunization with unconjugated recombinant Fab fragments of Id in indolent B-cell lymphomas (Bertinetti et al., Cancer Res. 2006; Navarrete et al., BLOOD 2011). We therefore hypothesized that Id Fab fragment might be intrinsically more immunogenic than entire Id Ig and tested this hypothesis by comparative in vitro experiments. Methods: Monocyte-derived dendritic cells (DC) where loaded with human monoclonal IgG, papain-digested Fab fragments, Fc fragments, or recombinant lymphoma-derived Fab fragments. Functional DC phenotypes were assessed by flow cytometry of crucial maturation and activation markers. IL-10 and IL-12 was measured in DC culture supernatants by ELISA. Antigen-loaded DC where subsequently used for priming of CFSE-labeled autologous peripheral blood mononuclear cells. Stimulated T cell populations were analyzed by multicolor flow cytometry. Results: Loading of DC with Fab, Fc, IgG, or mixtures of Fab and Fc fragments did not alter surface expression of CD11c, CD80, CD83, CD86, HLA-DR, PDL-1 and PDL-2 on DC. Likewise, the various antigens did not influence the cytokine release by DC during the loading or maturation process. DC loaded with isolated Fab fragments induced significantly higher proliferation of both CD4+ and CD8+ T cells than undigested IgG. The mean proliferation rate of CD4+ cells stimulated with Fab fragments was 18.5% versus 5.6% for undigested IgG stimulation (p=0.021); proliferation rates of CD8+ cells were 14.2% versus 6.2% (p=0.034). These results were reproduced for 4 different monoclonal IgGs tested on 4 different donors. The addition of Fc fragments to Fab reduced the proliferation rates of CD4+ and CD8+ cells to 10.2% and 8.6% respectively. In addition, DC loaded with undigested IgG induced a relative increase in the number of CD25high/FoxP3+ regulatory T cells compared with Fab stimulation (8.2% versus 1.4%; p<0.01). Conclusions: Isolated Fab fragments, i.e. the Id portions that contain the individual candidate antigenic epitopes of B-cell lymphomas, prime autologous T cells in vitro more efficiently than entire IgG. This finding is consistent with the high immune response rate against recombinant unconjugated Fab fragments observed in vivo in our clinical vaccination trials. Peptide sequences shared between Ig molecules that are predominantly located in the IgG Fc fragment appear to exert an inhibitory effect on T-cell priming. In accordance with our recent in vivo data in a syngeneic mouse model of Id vaccination (Warncke et al., Cancer Immunol. Immunother. 2011), this effect may be mediated by effective activation of Treg. Fab fragments therefore appear to be the more immunogenic and therefore preferable Ig antigenic format for active anti-Id immunotherapy. Furthermore, the inhibitory effects of IgG Fc offers a potential explanation for the recently reported lack of efficacy of Id vaccination in IgG-expressing follicular lymphomas in a randomized phase III trial, in which patients with IgM-expressing lymphomas, in contrast, had a significant benefit from Id vaccination in intention-to-treat analyses (Schuster et al., JCO 2011). Disclosures: No relevant conflicts of interest to declare.

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