Journal articles: 'Mice trail following'

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James, Britnie, Tamara Kucaba, and Thomas Griffith. "Plasmacytoid and CD8α DC are both necessary to generate antitumor immunity following Ad5-TRAIL/CpG immunotherapy (P2078)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 132.29. http://dx.doi.org/10.4049/jimmunol.190.supp.132.29.

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Abstract It is now appreciated that there are distinct subsets of dendritic cells (DC) with specialized functions. Plasmacytoid DC (pDC) and CD8α DC can contribute to the priming, activation, and function of antitumor CD8 T cells; however, their specific roles and necessity in stimulating antitumor immunity is not clearly understood. Thus, we examined the distinct role pDC and CD8α DC play in an orthotopic model of metastatic renal cell carcinoma (RCC) treated with a recombinant adenovirus encoding TRAIL (Ad-TRAIL) combined with CpG ODN. Wild-type (WT) mice bearing RCC tumors and treated with Ad-TRAIL/CpG ODN cleared primary and metastatic tumors, and these mice survived long-term. In contrast, mice specifically depleted of either pDC (using an α-PDCA1 mAb) or CD8α DC (Batf -/- mice) had uncontrolled tumor growth and high mortality regardless of treatment. Both pDC-depleted and Batf3-/- mice exhibited significantly decreased infiltration of effector CD8 T cells in the primary tumor site, as compared to WT mice after therapy, possibly related to the reduced expression of pro-inflammatory cytokines in pDC-depleted mice and reduced uptake of apoptotic tumor cells in Batf3-/- mice. These data collectively suggest that both pDC and CD8αDC carry out independent, but complementary roles that are necessary to initiate an efficacious antitumor immune after Ad5-TRAIL/CpG ODN therapy.

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Giovarelli, Mirella, Piero Musiani, Gianni Garotta, Reinhard Ebner, Emma Di Carlo, Yunsoo Kim, Paola Cappello, et al. "A “Stealth Effect”: Adenocarcinoma Cells Engineered to Express TRAIL Elude Tumor-Specific and Allogeneic T Cell Reactions." Journal of Immunology 163, no. 9 (November 1, 1999): 4886–93. http://dx.doi.org/10.4049/jimmunol.163.9.4886.

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Abstract BALB/c mammary adenocarcinoma cells engineered to express TNF-related apoptosis-inducing ligand (TRAIL)/APO-2 ligand (APO-2L) on their membrane (TSA-TRAIL) grow with kinetics similar to that of parental cells (TSA-pc) in vitro and in nu/nu mice. In contrast, TSA-TRAIL cells grow faster than TSA-pc in normal BALB/c mice. In DBA/2 mice, which differ from BALB/c mice at minor histocompatibility Ags, they also grow faster and display a higher percentage of tumor takes than TSA-pc. In fully histoincompatible C57BL/6 (B6) mice, TSA-TRAIL cells form evident tumors that are slowly rejected by most mice, but outgrow in a few. In contrast, TSA-pc cells are rejected at once by B6 mice. Since TRAIL/APO-2L induces apoptosis by interacting with a variety of specific receptors, this rapid growth in both syngeneic and allogeneic mice may be the result of an immunosuppressive mechanism. The following evidence supports this hypothesis: 1) TSA-TRAIL cells overcome the strong immunity against TSA-pc cells elicited in BALB/c mice by preimmunization with TSA cells engineered to release IL-4; 2) their rejection by B6 mice does not prime a CTL-mediated memory; 3) thymidine uptake by T lymphocytes unstimulated or stimulated by allogeneic cells is inhibited when TSA-TRAIL cells are added as third party cells; 4) CTL kill TSA-pc but not TSA-TRAIL cells in 48-h assays; and 5) activated lymphocytes interacting with TSA-TRAIL cells in vivo and in vitro undergo apoptosis.

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Carlo-Stella, Carmelo, Cristiana Lavazza, Arianna Giacomini, Daniela Sia, Loredana Cleris, Massimo Di Nicola, Paolo Longoni, et al. "Human CD34+ Cells Expressing Membrane-Bound Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Exert a Potent Anti-Lymphoma Effects by Targeting Tumor Vasculature." Blood 110, no. 11 (November 16, 2007): 527. http://dx.doi.org/10.1182/blood.v110.11.527.527.

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Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expected to play a key role in cancer therapy due to its high cancer cell-specificity and potent antitumor activity. We have previously demonstrated that CD34+ cells transduced with an adenovirus encoding the full-length human TRAIL gene (CD34-TRAIL+) exert a potent anti-lymphoma effect in NOD/SCID mice. To investigate the mechanism of action of CD34-TRAIL+ cells, in vivo experiments were perfomed to analyze the tumor homing capacity of CD34-TRAIL+ cells and the effects of transduced cells on tumor vasculature. Tumor homing of CD34-TRAIL+ cells was investigated in NOD/SCID mice bearing subcutaneous lymphoid tumors. Following a single intravenous injection of CD34-TRAIL+ cells (5 x 106), nodules were excised at different time-points and immunostained with an anti-human CD45 antibody. Sections were digitally recorded and the total number of CD45+ cells per tissue section was then counted using the imaging software ImageJ (http://rsb.info.nih.gov/ij/). Tumor homing of CD34-TRAIL+ cells peaked 24 hours after cell injection when a mean of 200 CD34-TRAIL+ cells was recorded per 1 x 105 tumor cells (0.2% CD45+ cells per 1 x 105 tumor cells). Mice pretreatment with the anti-VCAM-1 antibody or the CXCR4 antagonist AMD3100 significantly reduced tumor homing of CD34-TRAIL+ cells, with 0.09% and 0.05% CD45+ cells being recorded per 1 x 105 tumor cells, respectively. To analyze the effects of CD34-TRAIL+ cells on tumor vasculature, mice were injected with sulfo-biotin (0.1 mg, IV, 15 min) and tumor endotelial cells (TEC) were then revealed by staining frozen sections with horseradish peroxidase (HRP)-conjugated streptavidin. As compared with CD34-mock- or soluble (s)TRAIL-treated mice, treatment with CD34-TRAIL+ cells induced within 24 hours a significant (P ≤.001) increase of the thickness of the vessell wall (3.7 ± 1 μm vs 3.4 ± 1 μm vs 6 ± 1 μm, respectively) as well as the endothelial surface (10 ± 4% vs 7 ± 4% vs 21 ± 6% of total tissue section, respectively), suggesting that CD34-TRAIL+ cells induce an early vascular disruption. Confocal microscopic imaging of tumor sections double-stained with anti-CD31 and anti-TRAIL-R2 showed that this receptor was expressed by 8–12% of large tumor vessels. Interestingly, upon treatment with CD34-TRAIL+ cells, but not sTRAIL, TUNEL staining revealed an extensive apoptosis of CD31+/TRAIL-R2+ TEC. Forty-eight hours following injection of CD34-TRAIL+ cells, a 21-fold increase of apoptotic index was detected, which was associated with extensive necrotic areas (20% to 25% of tissue section). These data show that: tumor homing of CD34-TRAIL+ cells induces extensive vascular damage, hemorrhagic necrosis and tumor destruction; the antitumor effect of CD34-TRAIL+ cells is mediated by both indirect vascular-disrupting mechanisms and direct tumor cell killing.

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Loeuillard, Emilien, Jingchun Yang, Dong Haidong, Gregory Gores, and Sumera Ilyas. "Abstract PO032: Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-TRAIL-R Mediates Cholangiocarcinoma Tumor Immune Evasion by Enhancing Myeloid-Derived Suppressive Cell Population." Clinical Cancer Research 28, no. 17_Supplement (September 1, 2022): PO032. http://dx.doi.org/10.1158/1557-3265.liverca22-po032.

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Abstract Background and Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of TNF superfamily, is predominantly expressed on immune cells. Engagement of TRAIL with its cognate receptor, TRAIL-R (TR), leads to cancer cells apoptosis. Although the anti-tumor role of TRAIL-TR has been extensively studied, TRAIL agonists have had very limited anti-cancer activity in human clinical trials. TRAIL signaling can facilitate the myeloid response. However, this potential immunosuppressive function of TRAIL has not been examined extensively in cancer biology. Cholangiocarcinoma (CCA), a highly lethal biliary tract cancer, has a dense immunosuppressive microenvironment. Accordingly, CCA provides a model to examine the potential immune regulatory function of TRAIL in cancer biology. Methods: Using syngeneic, orthotopic murine models of CCA, (PMID: 29464042), murine CCA cells (SB cells) that express both Trail and Trail receptor (Tr) were implanted into livers of WT Tr− / − and LyzcreTrf/f mice, the latter have a specific deletion of TRAIL-R on myeloid cells. Hence, in this model the host immune cells express Trail but not the receptor; therefore, they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed, and tumors were characterized using flow cytometry. Results: We observed that Tr− / − mice had a significant reduction in tumor burden compared to WT mice. Myeloid-derived suppressor cells (MDSCs) were significantly decreased in Tr− / − tumors compared to WT tumors. Implantation of SB cells into LyzcreTrf/f mice resulted in a marked reduction in tumor burden and attenuation of MDSC infiltration compared to Lyzcre mice. In vitro functional studies employing MDSCs from mice deficient in Tr (MDSC-Tr −/−) were carried out. Compared to WT MDSCs, coculture of MDSC-Tr− /- with SB cells resulted in a significant reduction in the proliferation and immunosuppressive function of MDSCs. Moreover, following cocultured with SB cells, immunofluorescence analysis demonstrated that MDSC-Tr− / − had a reduction in the nuclear translocation of the NFkB subunit P65 compared to WT MDSCs. These data suggest that TRAIL-TR augments MDSC proliferation via NFkB. In conclusion, we have demonstrated that Tr− / − mice have a significant reduction in CCA tumor burden and MDSC infiltration. These results indicate that TRAIL-TR facilitates tumor immune escape and progression by fostering MDSC immunosuppressive function and infiltration, in an NFkB-dependent manner. Direct targeting of TRAIL on CCA cells is a potential anti-tumor strategy. Citation Format: Emilien Loeuillard, Jingchun Yang, Dong Haidong, Gregory Gores, Sumera Ilyas. Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-TRAIL-R Mediates Cholangiocarcinoma Tumor Immune Evasion by Enhancing Myeloid-Derived Suppressive Cell Population [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO032.

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Griffith, Thomas, Jacqueline Unsinger, Hirotaka Kazama, Jacqueline McDonough, Richard Hotchkiss, and Thomas Ferguson. "Sepsis-induced immunosuppression is mediated by TRAIL-expressing CD8+ regulatory T cells (87.13)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 87.13. http://dx.doi.org/10.4049/jimmunol.184.supp.87.13.

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Abstract Patients who survive severe sepsis often display severely compromised immune function. One hallmark of such immune suppression in septic patients is an impaired delayed-type hypersensitivity (DTH) response, manifested by a loss of skin testing to recall antigens. Since sepsis induces significant apoptosis in lymphoid and myeloid cells and apoptotic cells are themselves tolerogenic, we tested the hypothesis that suppression of DTH is mediated by tolerogenic properties of the apoptotic cells generated during sepsis. Mice subjected to cecal ligation and puncture (CLP) demonstrated a loss of DTH for the 7 days following CLP; however, the immune response returned to normal by day 10. Blocking sepsis-induced apoptosis via Bcl-2 overexpression or Bim deficiency prevented the loss of DTH. Importantly, injection of apoptotic cells into Bim-/- mice prevented an effective DTH response, thereby suggesting a causal link between apoptotic cells and immune suppression. Surprisingly, when Trail-/- mice were examined, we found that these animals had significant apoptosis but retained their DTH responses. Further studies revealed that apoptotic cells generated during sepsis induced a CD8+ regulatory T cell (Treg) that mediated suppressed DTH by TRAIL production. These results establish a link between apoptotic cells and immune suppression during sepsis, and suggest TRAIL may be a viable therapeutic target for boosting the adaptive immune response following sepsis.

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Mercer-Smith, Alison, Wulin Jiang, Alain Valdivia, Noah Bell, Alex Woodell, Scott Floyd, and Shawn Hingtgen. "MMAP-04 CYTOTOXIC, TUMOR-HOMING INDUCED NEURAL STEM CELLS AS AN ADJUVANT TO RADIATION IN THE TREATMENT OF NON-SMALL CELL LUNG CANCER LEPTOMENINGEAL METASTASES." Neuro-Oncology Advances 4, Supplement_1 (August 1, 2022): i15. http://dx.doi.org/10.1093/noajnl/vdac078.060.

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Abstract INTRODUCTION Non-small cell lung cancer (NSCLC) is the most common cancer to spread to the brain, and spread to the leptomeninges is particularly devastating, with a median survival of only months. While radiation may offer symptomatic relief, new adjuvant therapies are needed for more durable tumor kill. Spheroidal, human induced neural stem cells (hiNeuroS) transdifferentiated from fibroblasts are inherently tumoritropic. When engineered to secrete the cytotoxic protein TRAIL, they provide the potential for a personalized, targeted approach to NSCLC leptomeningeal metastases. METHODS hiNeuroS-TRAIL in vivo efficacy was determined by tracking the progression and survival of mice with NSCLC leptomeningeal tumors treated with intracerebroventricular hiNeuroS, radiation, or both. To determine the impact of radiation on the tumor tropism of hiNeuroS, we performed 2-dimensional motion assays on hiNeuroS with and without the presence of NSCLC pre- and post-radiation. Migrational capacity in vivo was determined by infusing hiNeuroS into the lateral ventricles of mice with established NSCLC tumors and monitoring hiNeuroS accumulation using post-mortem fluorescent analysis. RESULTS/CONCLUSION Mice treated with the combination of hiNeuroS-TRAIL and 2 Gy showed a significantly reduced mean tumor signal (2.7%) compared to controls (100%) or 2 Gy-only (54.9%). Mice treated with 2 Gy alone showed no significant survival difference compared to controls. Both combination and hiNeuroS-TRAIL-only-treated mice showed a significant improvement in median survival compared to controls (36.6% and 46.3% improvement, respectively). hiNeuroS showed enhanced directionality and displacement in the presence of NSCLC in 2-dimensional motion assays, indicating directional migration, and they maintained this ability following exposure to radiation. Co-localization of hiNeuroS with NSCLC was also observed in vivo. These results suggest the potential of hiNeuroS-TRAIL as a powerful adjuvant to radiation in the treatment of leptomeningeal NSCLC.

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Mercer-Smith, Alison, Wulin Jiang, Alain Valdivia, Juli Bago, Scott Floyd, Simon Khagi, and Shawn Hingtgen. "EXTH-07. TUMOR-HOMING INDUCED NEURAL STEM CELLS SECRETING A CYTOTOXIC PAYLOAD AS AN ADJUVANT TREATMENT FOR NON-SMALL CELL LUNG CANCER BRAIN METASTASES." Neuro-Oncology 22, Supplement_2 (November 2020): ii88. http://dx.doi.org/10.1093/neuonc/noaa215.361.

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Abstract INTRODUCTION Non-small cell lung cancer (NSCLC) is the most common cancer to form brain metastases. Radiation treatment is standard-of-care, but recurrence is still observed in 40% of patients. An adjuvant treatment is desperately needed to track down and kill tumor remnants after radiation. Tumoritropic neural stem cells (NSCs) that can home to and deliver a cytotoxic payload offer potential as such an adjuvant treatment. Here we show the transdifferentiation of human fibroblasts into tumor-homing induced neural stem cells (hiNSCs) that secrete the cytotoxic protein TRAIL (hiNSC-TRAIL) and explore the use of hiNSC-TRAIL to treat NSCLC brain metastases. METHODS To determine the migratory capacity of hiNSCs, hiNSCs were infused intracerebroventricularly (ICV) into mice bearing established bilateral NSCLC H460 brain tumors. hiNSC accumulation at tumor foci was monitored using bioluminescent imaging and post-mortem fluorescent analysis. To determine synergistic effects of radiation with TRAIL on NSCLC, we performed in vitro co-culture assays and isobologram analysis. In vivo, efficacy was determined by tracking the progression and survival of mice bearing intracranial H460 treated with hiNSC-TRAIL alone or in combination with 2 Gy radiation. RESULTS/CONCLUSION Following ICV infusion, hiNSCs persisted in the brain for > 1 week and migrated from the ventricles to colocalize with bilateral tumor foci. In vitro, viability assays and isobologram analysis revealed the combination treatment of hiNSC-TRAIL and 2 Gy radiation induced synergistic killing (combination index=0.64). In vivo, hiNSC-TRAIL/radiation combination therapy reduced tumor volumes > 90% compared to control-treated animals while radiation-only and hiNSC-TRAIL-only treated mice showed 21% and 52% reduced volumes, respectively. Dual-treatment extended survival 40%, increasing survival from a median of 20 days in controls to 28 days in the treatment group. These results suggest hiNSC-TRAIL can improve radiation therapy for NSCLC brain metastases and could potentially improve outcomes for patients suffering from this aggressive form of cancer.

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Carlo-Stella, Carmelo, Cristiana Lavazza, Massimo Di Nicola, Loredana Cleris, Paolo Longoni, Marco Milanesi, Michele Magni, et al. "Antitumor Activity of Human CD34+ Cells Expressing Membrane-Bound Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (mTRAIL)." Blood 108, no. 11 (November 16, 2006): 233. http://dx.doi.org/10.1182/blood.v108.11.233.233.

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Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expected to play a key role in anti-cancer therapy due to its high cancer cell-specificity and potent antitumor activity. The clinical development of soluble (s)TRAIL is however hampered by several limitations, includingshort plasma half-life;liver toxicity;tumor cell resistance. To overcome these limitations, we used CD34+ cells transduced with an adenovirus encoding the full-length human TRAIL gene (CD34−TRAIL+) as vehicles for intra-tumor delivery of membrane-bound (m)TRAIL. The mean (±SD) transduction efficiency of CD34+ cells exposed to a multiplicity of infection (MOI) of 500 was 83 ± 8% (range 70 – 95%) with a cell viability ≥85%. In vitro, exposure of the sTRAIL-sensitive KMS-11 cell line to CD34−TRAIL+, but not mock-transduced CD34+ cells consistently resulted in caspase-3, -8, and -9 activation and in PARP cleavage, as well as in potent induction of apoptosis (up to 80% after a 48-hour co-culture). Exposure of the sTRAIL-resistant JVM-2 cell line to CD34−TRAIL+ cells resulted in significant levels of tumor cell death (up to 50% after a 48-hour co-culture). Studies in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografted with KMS-11 cell line showed that CD34−TRAIL+ cells significantly increased the median survival of mice bearing early-stage (92 vs 55 days, P ≤ 0.0001) and advanced-stage (83 vs 55 days, P ≤ 0.0001) disease, as compared with controls. Additionally, CD34−TRAIL+ cells significantly prolonged the median survival of mice xenografted with the sTRAIL-resistant JVM-2 (40 vs 31 days, P ≤ 0.0001) and SU-DHL-4V (38 vs 30 days, P ≤ 0.0001) cell lines. No obvious toxicity was observed upon administration of CD34−TRAIL+ cells. Histological analysis of subcutaneous lymphoma revealed an efficient tumor homing of transduced cells and high level expression of the agonistic TRAIL-R2 receptor by tumor endothelial cells. Following injection of CD34−TRAIL+ cells, but not mock-transduced CD34+ cells, TUNEL staining revealed increasing amounts of apoptotic cells with a 21-fold increase of the apoptotic index at 120 hour post-injection. Additionally, CD34−TRAIL+ cells induced signs of vascular damage leading to a progressive disintegration of the vascular bed, suggesting that tumor endothelial cells represent an early target of CD34−TRAIL+ cells. Our experiments show that:in vitro, the co-culture of tumor cells and CD34−TRAIL+ cells resulted in a marked apoptosis of both sTRAIL-sensitive and sTRAIL-resistant tumor cells;in vivo, injection of CD34−TRAIL+ cells in mice bearing advanced-stage tumors as well as sTRAIL-resistant tumors was associated with a significant prolongation of survival. These results show that CD34−TRAIL+ cells might be an efficient vehicle for mTRAIL delivery to tumors, where they exert a potent antitumor effect possibly mediated by both direct tumor cell killing and indirect vascular-disrupting mechanisms.

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Cheng, Chieh-Fang, I.-Huang Lu, Hsiang-Wen Tseng, Chung-Yuan Sun, Li-Tsen Lin, Zong-Keng Kuo, I.-Horng Pan, and Ching-Huai Ko. "Antitumor Effect of Periplocin in TRAIL-Resistant Human Hepatocellular Carcinoma Cells through Downregulation of IAPs." Evidence-Based Complementary and Alternative Medicine 2013 (2013): 1–11. http://dx.doi.org/10.1155/2013/958025.

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Cortex periplocae is the dried root bark ofPeriploca sepiumBge., a traditional Chinese herb medicine. It contains high amounts of cardiac glycosides. Several cardiac glycosides have been reported to inhibit tumor growth or induce tumor cell apoptosis. We extracted and purified cortex periplocae and identified periplocin as the active ingredient that inhibited the growth of TNF-related apoptosis-inducing ligand-(TRAIL-) resistant hepatocellular carcinoma cells. The antitumor activity of periplocin was further increased by TRAIL cotreatment. Periplocin sensitized TRAIL-resistant HCC through the following two mechanisms. First, periplocin induced the expression of DR4 and FADD. Second, the cotreatment of TRAIL and periplocin suppressed several inhibitors of apoptosis (IAPs). Both mechanisms resulted in the activation of caspase 3, 8, and 9 and led to cell apoptosis. In addition, intraperitoneal injection (IP) of periplocin repressed the growth of hepatocellular carcinoma (HCC) in xenograft tumor model in mice. In summary, periplocin sensitized TRAIL-resistant HCC cells to TRAIL treatment and resulted in tumor cell apoptosis and the repression of tumor growthin vivo.

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Stuart, Patrick, and Megan Rogge. "Soluble FasL therapy reduces Herpetic Stromal Keratitis (154.23)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 154.23. http://dx.doi.org/10.4049/jimmunol.186.supp.154.23.

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Abstract Fas ligand (FasL) signals apoptosis in Fas+ cells, and plays a vital role in controlling the entrance of inflammatory cells and neovascularization of ocular tissue. We tested the therapeutic value of soluble FasL in both an acute and recurrent model of HSK using BALB/c and BALB-lpr mice. sFasL was administered in a topical ointment and by subconjunctival injection following infection or reactivation. The controls were treatment with ointment alone or with soluble TRAIL (sTRAIL). These mice were evaluated for corneal disease by a masked observer who scored the corneas for opacity and neovascularization. Following acute infection, wild-type BALB/c mice treated with sFasL displayed reduced incidence of opacity and neovascularization when compared to the sTRAIL and ointment groups post infection. When the Fas deficient BALB-lpr mice were used, results showed no difference in HSK between sFasL and sTRAIL treated groups, as expected. When BALB/c mice undergoing recurrent HSK were treated with sFasL, we saw a decrease in both opacity and neovascularization as compared to the sTRAIL treated mice. These data we believe are due to the apoptotic signaling by sFasL of the Fas molecule found on inflammatory cells and vascular endothelium of newly growing vessels which are attempting to invade the cornea following infection or reactivation.

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Zou, YX, XD Zhang, Y. Mao, GC Lu, M. Huang, and BJ Yuan. "Acute toxicity of a single dose DATR, recombinant soluble human TRAIL mutant, in rodents and crab-eating macaques." Human & Experimental Toxicology 29, no. 8 (January 6, 2010): 645–52. http://dx.doi.org/10.1177/0960327109357214.

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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to possess activity of inducing apoptosis in variety of tumor cells in preclinical models. Several mutational versions of TRAIL have been studied as promising agents for cancer therapy and the recombinant soluble human TRAIL mutant (DATR) is one of them. The objective of the present study was to provide possible toxic target organs and proposal non-toxic dose level of DATR for clinical usage. Rodents and crab-eating macaques were used to estimate potential adverse effects of DATR following a single dose administration. The median lethal dose (LD50 ) of intravenous injection to rats and mice was determined as 262.0 and 1018.0 mg/kg b.w., respectively. The LD50 of intraperitoneal administration to mice was found to be 1432.1 mg/kg b.w. The main changes in macaques were found in the following aspects. Hematology analysis revealed an obvious decrease of red blood cell count (RBC), hemoglobin (HB) and hematocrit (HCT) after injection. Serum biochemical analysis showed an apparent increase of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), blood urea nitrogen (BUN) and creatinine (Crea). Furthermore, inflammatory cell infiltrate in liver and kidney was found by microscope. All the disorders suggested that liver, renal and hematological systems might be the target effectors of toxic effect induced by DATR. Based on the results of this study, the no observed-adverse-effect level (NOAEL) and the lowest observed-adverse-effect level of DATR in macaques are 90.0 and 135.0 mg/kg b.w., respectively.

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Yee, L., M. Fanale, K. Dimick, S. Calvert, C. Robins, J. Ing, J. Ling, W. Novotny, A. Ashkenazi, and H. Burris. "A phase IB safety and pharmacokinetic (PK) study of recombinant human Apo2L/TRAIL in combination with rituximab in patients with low-grade non-Hodgkin lymphoma." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 8078. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.8078.

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8078 Background: Recombinant human Apo2L/TRAIL (rhApo2L/TRAIL) induces apoptosis (programmed cell death) through binding to the pro-apoptotic receptors DR4 and DR5. Preclinical studies show that rhApo2L/TRAIL selectively induces apoptosis in many cancer cell lines derived from various malignancies including NHL, while sparing most normal cells. In vivo, rhApo2L/TRAIL and Rituximab cooperate to shrink or attenuate the growth of various NHL tumor xenografts in SCID mice. RhApo2L/TRAIL is being co-developed by Genentech and Amgen as a targeted therapy for solid tumors and hematologic malignancies. Methods: Subjects were eligible to participate if they had CD20+ follicular NHL or small lymphocytic lymphoma or marginal zone B-cell lymphoma that had progressed following stable disease or an objective response lasting > 6 months duration to the most recent rituximab-contain regimen. RhApo2L/TRAIL is administered intravenously over 1 hour for 5 consecutive days every three weeks up to 4 cycles at dose levels of 4 and 8 mg/kg. Rituximab is administered intravenously at 375 mg/m2 weekly for up to eight doses. Results: Six subjects with low grade NHL (4 with follicular NHL and 2 with small cell NHL) have been enrolled and treated with 4 mg/kg rhApo2L/TRAIL and rituximab, and one subject (with follicular NHL) has been enrolled and treated with 8 mg/kg rhApo2L/TRAIL and rituximab. The enrolled subjects range in age from 39–82 years; there are 6 male and 1 females. The number of prior therapies for NHL range from 1–8. Four subjects have received all protocol specified therapy. There have been no DLTs or SAEs or Grade 3/4 adverse events reported to date. To date, five subjects have undergone tumor response assessment: there have been 2 patients with complete response, 1 with partial response and 2 with stable disease. Conclusion: The combination of rhApo2L/TRAIL at 4 mg/kg/day and rituximab appears safe and shows evidence of activity in subjects with low grade NHL that has relapsed following previous rituximab-containing therapy. Enrollment is continuing to test rhApo2L/TRAIL at 8 mg/kg plus rituximab for expanded safety data and further dose optimization. No significant financial relationships to disclose.

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Turbitt, William J., Shannon K. Boi, Justin T. Gibson, Rachael M. Orlandella, and Lyse A. Norian. "Diet-Induced Obesity Impairs Outcomes and Induces Multi-Factorial Deficiencies in Effector T Cell Responses Following Anti-CTLA-4 Combinatorial Immunotherapy in Renal Tumor-Bearing Mice." Cancers 13, no. 10 (May 11, 2021): 2295. http://dx.doi.org/10.3390/cancers13102295.

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Associations between modifiable factors and the efficacy of cancer immunotherapies remain uncertain. We found previously that diet-induced obesity (DIO) reduces the efficacy of an immunotherapy consisting of adenovirus-encoded TRAIL plus CpG oligonucleotide (AdT/CpG) in mice with renal tumors. To eliminate confounding effects of diet and determine whether outcomes could be improved in DIO mice, we evaluated AdT/CpG combined with anti-CTLA-4 in diet-matched, obese-resistant (OB-RES) versus DIO tumor-bearing mice. Therapy-treated OB-RES mice displayed effective renal tumor control and sustained CD4+ and CD8+ T cell responses. In contrast, therapy-treated DIO mice exhibited progressive tumor outgrowth and blunted T cell responses, characterized by reduced intratumoral frequencies of IFNγ+ CD4+ and CD8+ T cells. Weak effector T cell responses in therapy-treated DIO mice were accompanied by low intratumoral concentrations of the T cell chemoattractant CCL5, heightened concentrations of pro-tumorigenic GM-CSF, and impaired proliferative capacity of CD44+CD8+ T cells in tumor-draining lymph nodes. Our findings demonstrate that in lean mice with renal tumors, combining in situ T cell priming upstream of anti-CTLA-4 enhances outcomes versus anti-CTLA-4 alone. However, host obesity is associated with heightened immunotherapy resistance, characterized by multi-factorial deficiencies in effector CD4+ and CD8+ T cell responses that extend beyond the tumor microenvironment.

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Lundqvist, Andreas, Kristy Greeneltch, Maria Berg, Shivani Srivastava, Nanae Harashima, Hisayuki Yokoyama, Aleah Smith, Scott Abrams, and Richard Childs. "In Vitro and In Vivo Sensitization of Malignant Cells to Autologous Natural Killer Cell Cytotoxicity Following Exposure to Bortezomib." Blood 108, no. 11 (November 16, 2006): 925. http://dx.doi.org/10.1182/blood.v108.11.925.925.

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Abstract Killer IgG like receptor (KIR) inactivation of NK cells by self HLA molecules has been proposed as a mechanism through which malignant cells evade host NK cell-mediated immunity. To overcome this limitation, we sought to develop a method to sensitize the patient’s tumor to autologous NK cell cytotoxicity. The proteasome inhibitor bortezomib has recently been shown to enhance the activity of tumor death receptors. We found that exposure of a variety of different leukemia, lymphoma and solid tumor cancer cell lines to sub-apoptotic doses of bortezomib sensitized tumor cells in vitro to lysis by allogeneic NK cells. Importantly, this sensitizing effect also occurs with autologous NK cells normally rendered inactive via tumor KIR ligands; NK cells expanded from patients with metastatic renal cell carcinoma were significantly more cytotoxic against the patient’s own autologous tumor cells when pretreated with bortezomib compared to untreated tumors. This sensitization to autologous NK cell killing was also observed in vivo in two different murine tumor models. A significant delay in tumor growth in C57BL/6 mice bearing LLC1 tumors (figure) and a delay in tumor growth and a significant prolongation (p<0.01) in survival were observed in RENCA tumor bearing Balb/c mice treated with bortezomib and syngeneic NK cell infusions compared to untreated mice or animals treated with bortezomib alone or NK cells alone. An investigation into the mechanism through which NK cell cytotoxicity was potentiated revealed bortezomib enhanced the activity of tumor death receptor-dependent and -independent apoptotic pathways. More specifically, bortezomib sensitized human and murine tumor cells to TRAIL and perforin/granzyme mediated NK cell cytotoxicity respectively. These observations suggest that pretreatment of malignant cells with bortezomib could be used as a strategy to override NK cell inhibition via tumor KIR ligands, thus potentiating the activity of adoptively infused autologous NK cells. A clinical trial evaluating the safety and anti-tumor efficacy of adoptively infused autologous NK cells in patients with advanced malignancies with and without tumor sensitization using bortezomib is currently being explored. Figure: Tumor growth in LLC1 bearing C57BL/6 mice. Fourteen days following s.c. injection of 3x105 LLC1 tumor cells, mice received 15μg (i.p) bortezomib and/or an adoptive infusion of 1x106 NK cells from C57BL/6 mice (i.v) given on day 15. Each dot represents the tumor volume of individual mice measured on day 28 post tumor injection. Tumors were significantly smaller in mice treated with bortezomib followed by NK cells compared to controls or mice that received either NK cells alone or bortezomib alone (p<0.04 for all groups). Figure:. Tumor growth in LLC1 bearing C57BL/6 mice. . / Fourteen days following s.c. injection of 3x105 LLC1 tumor cells, mice received 15μg (i.p) bortezomib and/or an adoptive infusion of 1x106 NK cells from C57BL/6 mice (i.v) given on day 15. Each dot represents the tumor volume of individual mice measured on day 28 post tumor injection. Tumors were significantly smaller in mice treated with bortezomib followed by NK cells compared to controls or mice that received either NK cells alone or bortezomib alone (p<0.04 for all groups).

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Ghosh, Arnab, Yildirim Dogan, Amanda M. Holland, Odette M. Smith, Lauren F. Young, Mallory L. West, Natalie V. Singer, et al. "Over-Expression of TRAIL on Donor T Cells Enhances GVT and Suppresses Gvhd Via Elimination of Alloreactive T Cells and Host APC." Blood 118, no. 21 (November 18, 2011): 817. http://dx.doi.org/10.1182/blood.v118.21.817.817.

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Abstract Abstract 817 Strategies to suppress GVHD are often associated with broader suppression of the immune system leading to a compromised GVT effect. Using experimental models, we have demonstrated a novel strategy to enhance GVT effects and explicitly suppress GVHD using genetically engineered T lineage cells over-expressing TNF-Related Apoptosis Inducing Ligand (TRAIL). TRAIL can induce apoptotic signals through death receptor (DR) 4 and 5 molecules (only DR5 in mice) expressed on target cells. Expression of DR5 is higher on certain tumors and can be enhanced on others using small molecules rendering them susceptible to TRAIL mediated killing. TRAIL is therefore an attractive candidate for genetic engineering of donor T cells to enhance their GVT potential. We evaluated the effect of TRAIL over-expression (TRAIL+) in donor T cells (mature and precursor) on GVHD and GVT. Mature T cells derived from donor B6 splenocytes were transduced with a lentiviral TRAIL expression vector. The transduced TRAIL+ T cells were adoptively transferred on day 0 into lethally irradiated CBF1 recipients of T cell depleted allografts and LB27.4 tumor (B6 ^ CBF1+LB27.4) to assess their GVHD and GVT activity. TRAIL+ T cells displayed significantly enhanced antitumor immunity compared to T cells transduced with a control vector against LB27.4 tumor cell lines in vitro and upon transfer into tumor bearing allo-BMT recipients (p<0.01, 100% survival in TRAIL+ T cell group) (Fig 1A, also shown at the annual meeting last year). Precursor (pre)T cells have the benefit of regenerating the T cell compartment without causing GVHD and being available for “off the shelf” use. We generated TRAIL+ preT cells from transduced B6 hematopoietic stem cells and expanded them using the OP9-DL1 co-culture system. Adoptive transfer of B6 TRAIL+ preT cells into syngeneic-transplanted BALB/c mice could reconstitute the T cell compartment with TRAIL-expressing T cells and caused enhanced antitumor activity (p<0.05) compared to mock (GFP)-transduced controls. Interestingly, in addition to enhanced GVT, the recipients treated with TRAIL+ T cells had significantly less GVHD lethality and morbidity (Fig1B). This was observed across multiple GVHD models (B6 ^ CBF1, B6^ BALB/c and B10.BR^ B6). To explore the factors contributing to TRAIL-mediated suppression of GVHD, we used animals deficient in DR5 (DR5ko) in our models of GVHD. We found that GVHD suppressive effects of TRAIL were lost when hosts were DR5ko or when DR5ko TRAIL+ T cells were adoptively transferred indicating that TRAIL+ T cells suppress GVHD by targeting both host and donor compartments. We observed a higher DR5 expression in host MHC-IIhi antigen presenting cells (APC) following total body irradiation, suggesting that TRAIL+ donor T cells could potently eliminate host APC, resulting in less GVHD. Further, on transferring wild type T cells into irradiated hosts, we found that alloreactive CD25+ T cells had a significantly higher DR5 expression compared to CD25− T cells. This indicates that TRAIL+ T cells can specifically target the alloreactive CD25+ T cells in order to suppress GVHD. Collectively, our data demonstrate that donor T cells genetically engineered to express TRAIL can enhance GVT effects and suppress the development of lethal GVHD in recipients of allo-HSCT. Our data suggests that his suppression of GVHD is mediated by the elimination the alloreactive donor T cells and the elimination of GVHD-promoting residual APC. Furthermore, we demonstrated that allogeneic ex vivo generated preT cells expressing TRAIL could mediate a strong protection against tumor challenge in syngeneic HSCT recipients. TRAIL over-expression thus represents a potential off the shelf approach to enhancing GVT in both allogeneic and autologous transplantation. Despite elimination of alloreactive donor T cells, TRAIL+ T cells demonstrated enhanced GVT by directly targeting DR5+ tumors in the absence of alloreactivity. Disclosures: No relevant conflicts of interest to declare.

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Nasholm, Nicole, Brynn Duncan, Christian Capitini, and Terry Fry. "Graft versus host disease impairs vaccine responses via decreased CD4+ and CD8+ T cell proliferation and increased CD8+ T cell apoptosis through the perforin pathway (126.27)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 126.27. http://dx.doi.org/10.4049/jimmunol.188.supp.126.27.

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Abstract Graft versus leukemia (GVL) responses contribute to the curative potential of allogeneic blood or marrow transplantation (alloBMT) but can be associated with graft versus host disease (GVHD). Tumor-targeted vaccines have the potential to enhance the potency and specificity of GVL, but we have shown that GVHD can limit responses. Using a minor histocompatibility-mismatched BMT (B6 ->C3H.SW) with donor lymphocyte infusions (DLI), we examined the mechanism of reduced vaccine responses. The proliferative capacity, persistence and survival of vaccine-responding T cells were monitored following adoptive transfer of HY-specific T cells and HY-expressing dendritic cells in mice with GVHD. Both CD4+ and CD8+ HY specific T cells undergo less vaccine-driven proliferation in recipients with GVHD. Furthermore, HY-specific CD8+ T cells demonstrated increased apoptosis, whereas HY-specific CD4+ T cells did not. To determine a mechanism of apoptosis, we used a Fas Ligand-, TRAIL-, or perforin-deficient DLI to induce GVHD. Fas ligand- and TRAIL-deficient DLI did not affect apoptosis of the CD8+ HY-specific T cell population. However, a perforin-deficient DLI resulted in significantly less apoptosis. Finally, perforin-deficient DLI allowed for enhanced HY-expressing tumor protection. Thus, diminished vaccine responses seen during GVHD result from reduced proliferation and perforin-mediated CD8+ T cell apoptosis, factors to be considered when optimizing vaccine strategies following alloBMT.

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Lubart, Alisa, Amit Benbenishty, Hagai Har-Gil, Hadas Laufer, Amos Gdalyahu, Yaniv Assaf, and Pablo Blinder. "Single Cortical Microinfarcts Lead to Widespread Microglia/Macrophage Migration Along the White Matter." Cerebral Cortex 31, no. 1 (September 21, 2020): 248–66. http://dx.doi.org/10.1093/cercor/bhaa223.

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Abstract Loss of cognitive function with aging is a complex and poorly understood process. Recently, clinical research has linked the occurrence of cortical microinfarcts to cognitive decline. Cortical microinfarcts form following the occlusion of penetrating vessels and are considered to be restricted to the proximity of the occluded vessel. Whether and how such local events propagate and affect remote brain regions remain unknown. To this end, we combined histological analysis and longitudinal diffusion tensor imaging (DTI), following the targeted-photothrombotic occlusion of single cortical penetrating vessels. Occlusions resulted in distant tissue reorganization across the mouse brain. This remodeling co-occurred with the formation of a microglia/macrophage migratory path along subcortical white matter tracts, reaching the contralateral hemisphere through the corpus callosum and leaving a microstructural signature detected by DTI-tractography. CX3CR1-deficient mice exhibited shorter trail lengths, differential remodeling, and only ipsilateral white matter tract changes. We concluded that microinfarcts lead to brain-wide remodeling in a microglial CX3CR1-dependent manner.

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Huntington, Kelsey E., Anna Louie, Young Lee, Jared Mompoint, Isacco Ferrarini, Aakash Jhaveri, Varun V. Prabhu, Allen Melemed, Seulki Lee, and Wafik S. El-Deiry. "Abstract PO-064: ONC212 stimulates cytotoxic T-cell killing, increases tumor-immune cell interactions, and promotes tumor regression in combination with TLY012 in a PDAC murine model." Cancer Research 81, no. 22_Supplement (November 15, 2021): PO—064—PO—064. http://dx.doi.org/10.1158/1538-7445.panca21-po-064.

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Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer with a 4.2% 5-year survival rate. There is an urgent need for innovative treatments for patients suffering from PDAC. Patients with PDAC often have immunosuppressed tumors that are apoptosis-resistant and have limited immune cell infiltration and/or activation. Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) selectively induces cancer cell apoptosis during innate immunity, while ONC212 is a potent fluorinated second-generation imipridone currently in IND-enabling studies that induces TRAIL signaling and the integrated stress response (ISR). TLY012 is a novel PEG’ylated trimeric TRAIL receptor agonist being clinically developed to overcome the short half-life of TRAIL. We hypothesized that combining a TRAIL pathway inducer and a TRAIL receptor agonist may be efficacious in PDAC and may activate immune cell killing based on prior work with imipridone ONC201. Immune cell co-culture experiments were conducted using PANC1 PDAC cells and TALL-104 CD8+ cytotoxic T-cells at a 1:1 effector-to-target cell ratio with or without ONC212. We observed a significant increase in T-cell killing of PDAC cells in the co-culture assay following treatment with ONC212 at several doses as compared to controls. We noted an increase in tumor-immune cell interactions as measured by the number of T-cells that were directly touching tumor cells at each timepoint. The BxPC-3 immunodeficient murine PDAC model revealed an increase in natural killer (NK) cell tumor infiltration by immunohistochemistry (IHC) staining for NK1.1 after 30 days of 50 mg/kg ONC212 treatment three times a week. The combination of ONC212 and TLY012 was evaluated using subcutaneously-implanted KPC-Luc PDAC cells in a syngeneic immunocompetent C57BL/6 mouse model. Mice treated with 25 mg/kg ONC212 alone twice a week, 10 mg/kg TLY012 alone twice a week, or combination therapy with ONC212 and TLY012 showed statistically significant decreases in tumor growth compared to controls after 5 weeks of treatment (n=6 mice per treatment condition) by tumor volume and in vivo bioluminescent imaging. We are analyzing murine plasma cytokine profiles, changes in tumor-infiltrating NK- and T-cells by flow cytometric and IHC analysis of murine spleen and KPC-Luc tumor samples. We report a novel immune stimulation of the TRAIL pathway and T-cell activation by ONC212 plus TLY012 leading to cytotoxic anti-PDAC responses in vivo. Citation Format: Kelsey E. Huntington, Anna Louie, Young Lee, Jared Mompoint, Isacco Ferrarini, Aakash Jhaveri, Varun V. Prabhu, Allen Melemed, Seulki Lee, Wafik S. El-Deiry. ONC212 stimulates cytotoxic T-cell killing, increases tumor-immune cell interactions, and promotes tumor regression in combination with TLY012 in a PDAC murine model [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-064.

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Lundqvist, Andreas, Sheila Rao, Aleah Smith, Maria Berg, Su Su, Hisayuki Yokoyama, Shivani Srivastava, and Richard Childs. "Bortezomib Enhances the Antitumor Activity of Adoptively Infused Natural Killer Cells In Vivo: A Novel Approach To Override KIR-Mediated Inhibition of NK Cell Cytotoxicity." Blood 110, no. 11 (November 16, 2007): 1786. http://dx.doi.org/10.1182/blood.v110.11.1786.1786.

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Abstract Natural killer (NK) cell killer immunoglobulin-like receptor (KIR) interactions with self MHC class I molecules can regulate NK cell function; such interactions typically inactivate NK cells potentially providing a dominant mechanism through which malignant cells evade host NK cell-mediated immunity. Recently we found that the proteasome inhibitor bortezomib up-regulated surface expression of tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) on a variety of different human malignant cells rendering them susceptible to NK cell-mediated apoptosis in vitro; this effect appears to override KIR ligand-mediated NK cell inactivation, overcoming tumor resistance to both allogeneic KIR ligand-matched and autologous NK cell cytotoxicity. We also found that murine tumors were sensitized by bortezomib to the cytotoxic effects of syngeneic NK cells; the killing of RENCA and LLC1 tumors in vitro by syngeneic BALB/c and C57BL/6 NK cells respectively was enhanced when tumors were exposed to 10nM of bortezomib for 18h. Here, we show that the combined treatment of bortezomib followed by syngeneic NK cell infusions significantly delays tumor growth in tumor bearing animals. While treatment with bortezomib or interleukin-2 activated syngeneic NK cells alone had little effect on tumor growth, the combined treatment significantly delayed growth of RENCA tumors in BALB/c mice and LLC1 in C57BL/6 mice (p<0.01;figure). In contrast to human tumor cell lines where an increase in expression of TRAIL-R2 was observed following bortezomib exposure, no change in expression of death receptors was observed in either murine tumor line. Flow cytometry analysis showed caspase-8 activity was significantly enhanced in bortezomib-treated murine tumor cells upon co-culture with NK cells compared to untreated tumor cells. Concanamycin A treatment significantly reduced NK cell-mediated apoptosis (but not neutralizing antibodies to Fas ligand or TRAIL) demonstrating that the sensitizing effect was mediated through perforin. Moreover, bortezomib-treated tumor cells were resistant to killing by perforin-deficient NK cells in vitro and the reduction in tumor growth observed in tumor bearing animals treated with bortezomib and wild-type NK cells was not observed in animals treated with bortezomib and perforin-deficient NK cells. These findings demonstrate that bortezomib-induced tumor sensitization to NK cell perforin and/or TRAIL could be used as a novel strategy to potentiate anti-tumor effects of adoptively infused NK cells in patients with cancer. Figure. Left - BALB/c mice were injected with RENCA tumor cells (100.000 cells i.v) and treated with bortezomib (5ug/mouse i.v) on days 5, 12 and 19 followed by injection of sygeneric NK cells (2×106 i.v) on days 6,13 and 20. All animals received IL-2 (100.000 U i.p on days 6–9,13–16 and 20–23). Animals were euthanized on day 25 and evaluated for pulmonary metastasies. Right - C57BL/6 mice were injected with LLC1 tumor cells (500.000 s.c) and treated with bortezomib (15ug/mouse i.p) on day 14 followed by a single injection of syngeneic NK cells (1×106 i.v) on day 15. All mice were treated with IL-2 (100.000U i.p on days 15–18). Data depicts tumor sizes on day 28 post tumor injection. Figure. Left - BALB/c mice were injected with RENCA tumor cells (100.000 cells i.v) and treated with bortezomib (5ug/mouse i.v) on days 5, 12 and 19 followed by injection of sygeneric NK cells (2×106 i.v) on days 6,13 and 20. All animals received IL-2 (100.000 U i.p on days 6–9,13–16 and 20–23). Animals were euthanized on day 25 and evaluated for pulmonary metastasies. Right - C57BL/6 mice were injected with LLC1 tumor cells (500.000 s.c) and treated with bortezomib (15ug/mouse i.p) on day 14 followed by a single injection of syngeneic NK cells (1×106 i.v) on day 15. All mice were treated with IL-2 (100.000U i.p on days 15–18). Data depicts tumor sizes on day 28 post tumor injection.

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Berg, Maria, Andreas Lundqvist, Dawn Betters, and Richard W. Childs. "In Vitro Expanded NK Cells Have Increased Natural Cytotoxity Receptors, TRAIL and NKG2D Expression, and Superior Tumor Cytotoxicity Compared to Short-Term IL-2 –Activated NK Cells." Blood 114, no. 22 (November 20, 2009): 463. http://dx.doi.org/10.1182/blood.v114.22.463.463.

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Abstract Abstract 463 IL-2 activates NK cell enhancing their capacity to lyse tumor cells. To date, most clinical studies of adoptive NK cell transfer have utilized short-term (12-16 hours) IL-2-activated NK cells. Because IL-2 alone is ineffective in expanding NK cells in vitro, the relatively low numbers of NK cells obtained for infusion following short term IL-2 activation may limit the full therapeutic impact of this approach. To obtain larger numbers of NK cells, novel ex vivo expansion protocols that utilize irradiated EBV-LCL or K562 feeder cells have recently been developed. However, concerns exist that extensive ex vivo expansion might significantly reduce the in vivo proliferative potential and long-term viability of adoptively transferred NK cells. Here we investigated for differences in phenotype, tumor cytotoxicity and in vivo persistence between short-term IL-2 activated and long-term expanded NK cells. CD56+/CD3- NK cells were isolated from normal donors by immuno-magnetic bead selection and were either activated with IL-2 (500U/ml) for 12-16 hours or were expanded in vitro over 14 days using irradiated EBV-LCL feeder cells in IL-2 containing media (500U/ml). Short-term IL-2 activated NK cells did not expand in number in contrast to EBV-LCL stimulated NK cells which expanded 400-1000 fold by culture day 14. Flow cytometry analysis revealed no differences in expression of DNAM-1, 2B4, LFA-1 or granzyme B between short-term activated and expanded NK cells. However, expanded NK cells had significantly higher expression of TRAIL, NKG2D, and the natural cytotoxicity receptors NKp30, NKp44 and NKp46 and a slight increase in KIR3DL1 and KIR2DL2/3. A 4-hour 51Cr-release assay showed expanded NK cells were significantly more cytotoxic against K562 cell compared to short-term IL-2 activated NK cells; at a 1:1 effector to target ratio, 67±6%, 26±1%, and 9±1% of K562 cells were killed by expanded, short term IL-2 activated and fresh NK cells respectively (p<0.05). Increased TRAIL expression on expanded NK cells was also associated with increased lysis of TRAIL-sensitive tumor cells (RCC tumors treated with bortezomib); at a 1:1 E:T ratio, 55±3% and 5±2% of bortezomib-treated RCC tumors were killed by expanded and short-term IL-2 activated NK cells respectively (p<0.05). We next assessed for differences in the in vivo longevity of these NK cell populations when transferred into immuno-deficient mice. Two million NK cells were labeled with a near infrared-dye (DiR; 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) and injected intra-peritoneal (i.p.) into CB.17 SCID-beige mice. Mice were administered IL-2 (100,000U/ml bid i.p.) for five days then underwent bioluminescent imaging using the IVIS100 system. Although FACS analysis of NK cells performed immediately prior to injection showed increased DiR fluorescent intensity in short-term IL-2 activated vs. expanded NK cells, fluorescence signal in vivo was slightly higher in the first 24-96 hours in mice that received expanded NK cells; fluorescence intensity was 5-41% (p=0.003) stronger in recipients of expanded NK cells compared to mice receiving short-term IL-2 activated NK cells. We next evaluated the in vivo anti-tumor effects of activated vs. expanded NK cells. CB.17 SCID-beige mice were injected i.p. with luciferase transduced 526 human melanoma cells three days prior to receiving an i.p. injection of short term IL-2 activated vs. expanded NK cells (+ bid i.p. IL-2). Bioluminescent imaging measuring tumor flux to calculate tumor burden and tumor doubling time showed no difference in tumor progression between both NK cell cohorts. In conclusion, these results demonstrate that ex vivo expanded NK cells are phenotypically and functionally different than short-term IL-2 activated NK cells. Expanded NK cells have increased expression of natural cytotoxicity receptors, NKG2D and TRAIL and have greater TRAIL-mediated tumor cytotoxicity compared to IL-2 activated NK cells. Importantly, despite extensive ex vivo proliferation, expanded NK cells appear maintain similar longevity in vivo as non-expanded short term IL-2 activated NK cells. Disclosures: No relevant conflicts of interest to declare.

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Furutani, Elissa, Su Su, Aleah Smith, Maria Berg, and Richard Childs. "siRNA Inactivation of the Inhibitory Receptor NKG2A Augments the Anti-Tumor Effects of Adoptively Transferred NK Cells In Tumor-Bearing Hosts." Blood 116, no. 21 (November 19, 2010): 1015. http://dx.doi.org/10.1182/blood.v116.21.1015.1015.

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Abstract Abstract 1015 Natural killer (NK) cells are a component of the innate immune system that target both tumors and virally infected cells. NK cell killing of tumors is regulated by a delicate balance of activating and inhibitory receptors. These inhibitory receptors bind HLA ligands which prevent NK cell targeting of normal “self” cells. Up regulation of surface expression of HLA molecules has been utilized by tumors as a method to evade NK cell cytotoxicity. Disrupting the function or expression of inhibitory receptors on NK cells could potentially be used as a method to overcome this effect. While most inhibitory receptors are present in only a subset of NK cells, NK cells universally express the HLA-E binding inhibitory receptor NKG2A. We hypothesized that siRNA inactivation of NK cell NKG2A would could be used as a therapeutic approach to enhance NK cell tumor cytotoxicity in vivo. The human natural killer cell line NKL was transduced with lentiviral vectors encoding shRNA targeting various regions of the NKG2A transcript. Following lentiviral transduction, knockdown of receptor expression was confirmed by flow cytometry and RT-qPCR. Compared to wild type (WT) and GFP-transduced NKL controls, NKG2A silenced NKL cells had increased secretion of IFN-gamma and Fas-L by ELISA and increased granzymes A and B and Nkp30 expression by flow cytometry. In contrast, expression of NKG2D, Nkp44, Nkp46, LFA-1, DNAM, and TRAIL was not altered by NKG2A silencing. Chromium-based cytotoxicity assays showed shRNA knockdown of NKG2A significantly enhanced NK cell cytotoxicity of tumor cells: at a 20:1 effector to target ratio, NKG2A knockdown NKLs, WT NKLs and GFP-transduced NKLs induced 68.9%, 8.2% and 8.3% lysis respectively of 721.221 EBV-LCL tumor targets (p=0.001). Remarkably, NKG2A silencing enhanced NKL killing of both HLA-E positive (721.221 EBV-LCL and 526 melanoma cells) and HLA-E negative (K562) tumor cell lines, suggesting NKG2A inactivation increased NK cell cytotoxicity through both HLA-E dependent and independent mechanisms. Using a xenogeneic model, we next explored the in vivo effects of transferring NKG2A silenced NK cells in tumor bearing mice. Immunodeficient NSG mice were injected with 1 million human luciferase transduced 721.221 HLA-E expressing EBV-LCL tumor cells. Twenty-four hours later, tumor-bearing mice were injected with 2–5 million WT NKL cells, GFP-control-transduced NKL, or NKG2A silenced NKL cells, then received IL-2 sq for 10 days to induce in vivo NK cell proliferation. NKL numbers in blood were subsequently analyzed by flow cytometry and tumor burden was assessed by luciferase-based bioluminescence imaging (BLI). At 16 and 21 days following adoptive NK cell transfer, BLI showed that recipients of NKG2A silenced NKL cells had slower tumor growth and significantly smaller tumor burden compared to NKL wt and NKL-GFP transduced controls (figure). Importantly, no toxicity related to infusing NKG2A inactivated NK cells was observed. These in vitro and in vivo data suggest shRNA knockdown of the NKG2A inhibitory receptor could be used as a method to augment NK cell tumor cytotoxicity in patients with hematological malignancies. Figure: Tumor burden in mice Luciferase-tagged 721.221 HLA-E EBV LCLs were injected into mice and imaged using a bioluminescence imager at days 10, 16, and 22 following NKL injection. 5 mice were followed in each group. Figure:. Tumor burden in mice . / Luciferase-tagged 721.221 HLA-E EBV LCLs were injected into mice and imaged using a bioluminescence imager at days 10, 16, and 22 following NKL injection. 5 mice were followed in each group. Disclosures: No relevant conflicts of interest to declare.

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Kokkinos, John, George Sharbeen, Janet Youkhana, Cyrille Boyer, David Goldstein, Koroush S. Haghighi, Joshua A. McCarroll, and Phoebe A. Phillips. "Abstract B076: βIII-Tubulin is a brake on extrinsic cell-death in pancreatic cancer." Cancer Research 82, no. 22_Supplement (November 15, 2022): B076. http://dx.doi.org/10.1158/1538-7445.panca22-b076.

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Abstract Background: Pancreatic Ductal Adenocarcinoma (PDAC) urgently needs more effective therapies, with a 5-year survival rate <11%. We previously demonstrated βIII-tubulin knockdown in PDAC cells reduced tumor growth and metastasis in vivo, increased chemosensitivity, and induced apoptosis, making it a potent therapeutic target [McCarroll et al, Oncotarget, 6, 2235 (2015)]. However, mechanisms driving these effects are unknown. Apoptosis consists of intrinsic (caspase-9) and extrinsic (caspase-8) pathways. To better understand mechanism and inform therapeutic combinations, our study investigated apoptotic pathways controlled by βIII-tubulin. Aims: 1) Evaluate apoptotic pathways controlled by βIII-tubulin in PDAC cells a) in vitro and b) in vivo; 2) Assess the effect of silencing βIII-tubulin combined with tumor-specific extrinsic apoptosis inducer (TRAIL) in a patient-derived 3D PDAC model. Methods: 1a) PDAC cells were transfected with control-siRNA or βIII-tubulin-siRNA ± caspase-8 or -9 inhibitors and apoptosis measured. The effect of βIII-tubulin knockdown on PDAC cell sensitivity to extrinsic apoptosis inducers (TRAIL, TNFα, FasL) was tested (n≥3). TRAIL induces extrinsic cell death by binding to the TRAIL-receptor DR5, inducing clustering and intracellular activation of caspase 8. DR5 localization following βIII-tubulin knockdown was assessed by confocal microscopy (n=4). Live cell imaging was used to assess trafficking of GFP-tagged DR5 at the membrane of PDAC cells (n≥17 cells/treatment). 1b) Mice with orthotopic PDAC tumors (n=10/group) were treated (5-weeks post-implantation) with nanoparticle+βIII-tubulin-siRNA or control-siRNA for 3.5-weeks. Tumor volume and cleaved caspase-8 were measured. 2) Explants [validated patient-derived 3D tumor model with complete intact stroma, as described in: Kokkinos et al, Sci Rep, 11,1941 (2021); Sharbeen et al, Cancer Res, 81,3461 (2021)] from surgically resected PDAC tumors (5 patients) were cultured for 12 days and treated with nanoparticle+βIII-tubulin-siRNA or control-siRNA every 3 days and TRAIL once weekly. Tumor explants were fixed and assessed for tumor and viability markers via immunohistochemistry. Results: 1a) βIII-tubulin knockdown-induced apoptosis in PDAC cells was blocked by inhibiting caspase-8, but not caspase 9. Knockdown of βIII-tubulin sensitized PDAC cells to extrinsic apoptosis inducers. βIII-tubulin knockdown increased the movement of TRAIL-receptor DR5 through the cell membrane and triggered its clustering in PDAC cells. 1b) βIII-tubulin knockdown in orthotopic PDAC mouse tumors decreased tumor growth and activated extrinsic apoptosis. 2) In patient-derived tumor explants, βIII-tubulin-siRNA plus TRAIL reduced tumor cell frequency. Conclusions: 1) Silencing βIII-tubulin in PDAC cells sensitizes them to extrinsic apoptosis inducers and facilitates increased membrane clustering of the TRAIL receptor DR5. 2) βIII-tubulin knockdown represents an innovative therapeutic strategy to unleash a suicide signal in PDAC cells and render them sensitive to a tumor-specific therapeutic. Citation Format: John Kokkinos, George Sharbeen, Janet Youkhana, Cyrille Boyer, David Goldstein, Koroush S. Haghighi, Joshua A. McCarroll, Phoebe A. Phillips. βIII-Tubulin is a brake on extrinsic cell-death in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B076.

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Seal, Sudeshna, Daniella B. Kerbauy, Vladimir Lesnikov, Nissa Abbasi-Shafer, and H. Joachim Deeg. "FLIPLong(L) and FLIPShort(S) Overexpression in the Human Myeloid Leukemia Cell Line ML-1 and Its Role in TRAIL [Tumor Necrosis Factor(TNF)-Related Apoptosis-Inducing Ligand] and TNFa Induced Apoptosis." Blood 106, no. 11 (November 16, 2005): 4378. http://dx.doi.org/10.1182/blood.v106.11.4378.4378.

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Abstract TRAIL initiates activation of Caspase-8, which is blocked by the FLICE inhibitory protein (FLIP), resulting in resistance to apoptosis. Here we show that overexpression of FLIPL and FLIPS in ML1 cells, with low constitutive levels of FLIP, protects these cells against apoptosis induced by TRAIL, not only via Caspase-8 inhibition, but also via upregulation of anti-apoptotic molecules. Methods: 1) Apoptosis was determined by Annexin V/ 7-AAD following treatment with TRAIL (100–500ng/ml), or TNFa (20–100ng/ml) in ML1 cells transduced with FLIPL.GFP (green fluorescent protein), FLIPS.GFP or Neo.GFP (control). 2) Caspase-8, Caspase-3, Bid, Bcl-xL, XIAP, phosphorylated (P)-IKBa and P-Akt were determined by western blots. 3) Active Caspase-3 was determined using EnzChek Caspase-3 assay kit. Results: Both FLIPL and FLIPS transduction protected ML1 cells against apoptosis induced by TRAIL (300ng/ml), while no protection was observed in Neo.GFP cells. FLIPL had a more profound protective effect than FLIPS (Fig.1A). Both FLIPL and FLIPS, but not Neo.GFP, blocked Caspase-8 and Caspase-3 activation (Fig.1B); FLIPS cells showed two-fold higher levels of active Caspase-3 than FLIPL cells, consistent with higher apoptosis in FLIPS cells. Caspase-3 can be activated through Caspase-8 (extrinsic pathway) or via Caspase-8/Bid (intrinsic pathway). The latter was responsible for high active Caspase-3 levels in FLIPS cells as shown by the presence of cleaved Bid (t-Bid) (Fig.1B); cleavage of Bid was inhibited by combination of TRAIL and Z-IETD-FMK (Caspase-8 inhibitor). Anti-apoptotic molecules, including Bcl-xL, XIAP and FLIP are regulated by NF-kB and FLIP participates in an NF-kB auto-amplification loop. While Neo.GFP cells showed little Bcl-xL after 4h of TRAIL exposure and there was a twofold reduction in FLIPS cells, only a slight reduction of Bcl-xL was noted in FLIPL cells. FLIPL cells showed the lowest rates of apoptosis when exposed to TNFa and BMS543541, a specific inhibitor of IkB kinase (Fig. 1C). In the presence of BMS543541, phosphorylation of IkBa and levels of Bcl-xL and XIAP decreased in Neo.GFP and FLIPS but not in FLIPL cells. Additional data suggest that the PI3-kinase/Akt pathway is involved in constitutive NF-kB activation and differentially affected by FLIPL and FLIPS (Fig. 1D). Preliminary results in immunodeficient mice transplanted with transduced ML1 cells indicated the in vivo relevance of the differences between FLIPL and FLIPS with FLIPL cells engrafting earlier and showing earlier signs of sickness. Conclusions: FLIPL and FLIPS conferred resistance to TRAIL induced apoptosis but showed differential effects: Caspase-8/Bid was involved in the apoptosis pathway in FLIPS, but not in FLIPL cells. FLIPL cells’ resistance was due not only to caspase inhibition but to the recruitment of downstream anti-apoptotic pathways such as NF-kB and PI3K/Akt. In vivo data further substantiated the antiapoptotic/pro-survival behavior of FLIPL cells. Figure Figure

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Zimmerman, Zachary F., and Robert B. Levy. "Both Recipient TNaive and TMemory Cells Mediate Efficient Resistance Against Marrow Allografts in the Absence of Major Cytotoxic Effector Pathways." Blood 104, no. 11 (November 16, 2004): 2130. http://dx.doi.org/10.1182/blood.v104.11.2130.2130.

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Abstract Allogeneic hematopoietic stem/progenitor cell (allo-HSPC) transplant is a potentially curative therapy for hematopoietic malignancies/disorders, however host resistance can lead to delayed reconstitution or graft rejection, especially following non-myeloablative conditioning. Our laboratory has been investigating the effector pathways utilized by host cell populations(NK, Tnaive, Tmemory), which mediate resistance to hematopoietic grafts. The effector molecules used by these host populations appear to be varied and include presently undefined pathways. This study examined the dependence of resistance on cytotoxic pathway function mediated by naïve versus memory T cells. Peripheral donor chimerism was assessed following transplant of MHC matched, minor histocompatibility antigen (mHag) mismatched C3H.SW(H-2b)àB6(H-2b) unsensitized, i.e. naive non-myeloablated (5.5 Gy TBI) cytotoxically normal (B6-wt) or defective(B6-pko and B6-gld) recipients. In wild-type recipients, transient peripheral blood donor chimerism was detected which peaked on day 7-10 post-transplant. Donor chimerism was generally undetectable 3–4 weeks post-BMT. Following a secondary transplant in these recipients, no chimerism was detected indicating that the resistance was T cell mediated and engendered a memory response. To determine the cellular requirements for resistance in naïve recipients, CD4−/− and CD8−/− mice were examined. Interestingly, although CD8 mediated B6 anti-C3H.SW responses are well documented, resistance in this model was abrogated in CD4−/−, but not CD8−/− recipients. This indicates that CD4 T cells may play a role in this resistance at the efferent phase, afferent phase, or both. To examine the requirement of cytotoxic pathways in this model, C3H.SW BM was transferred into B6-perforin−/− or B6-gld (FasL functionally deficient mice) recipients. The resistance observed was not appreciably different (ie rejection day 21–28) than that in wild-type mice. CD8 T memory cells primed to donor antigens prior to transplant were analyzed in B6-cdd (H-2b) recipients, which cannot mediate perforin and FasL dependent cytotoxicity. Anti-donor (H-2d) primed B6-cdd mice were transplanted with BALB/cTNFR1−/− bone marrow(BM) and injected with excess levels of blocking mAbs against 3 TNF family apoptosis inducing ligands, i.e. αTL1a, TRAIL and TWEAK as we previously reported. Donor CFU were assessed as a measure of early resistance(D+5). Sensitized B6-cdd mice receiving allo BM lacking TNFR1 and the 3 mAb cocktail exhibited rejection, indicating memory CD8 T cell mediated resistance remained intact despite the disruption of six candidate cytotoxic pathways. Interestingly, rejection in cytotoxically impaired (B6-cdd) recipients was a rapidly occurring event, complete within 48hrs post-BMT, an observation indistinguishable from the kinetics occurring in cytotoxically normal recipients. In total, these findings highlight the potential importance of alternative T cell effector mechanism(s) distinct from classical cytotoxic mechanisms active in allogeneic resistance against hematopoietic stem/progenitor cell grafts mediated by effector cells derived from TNaive and TMemory populations.

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Lopez, Rebecca, Andreas Lundqvist, Stephanie Sellers, Maria Berg, Muthalagu Ramanathan, Sumithira Vasu, Aleah Smith, Cynthia E. Dunbar, and Richard W. Childs. "A Rhesus Macaque Model to Optimize Adoptive NK Cell Therapy." Blood 112, no. 11 (November 16, 2008): 3905. http://dx.doi.org/10.1182/blood.v112.11.3905.3905.

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Abstract NK cell based immunotherapy represents a promising treatment approach for patients with cancer. Although preliminary clinical trials in humans suggest NK cell infusions can mediate anti-tumor effects, animal models are needed to provide insight into methods to enhance both the function and in vivo longevity of adoptively infused NK cells. Research conducted in our laboratory has shown that ex vivo expanded human NK cells are highly activated, up-regulating NKG2D, Granzyme B, TRAIL and Fas-ligand expression making them much more cytotoxic to tumor cells compared to freshly isolated NK cells. However, important questions remain regarding whether in vitro expansion alters the capacity of these cells to replicate, and traffic to tissues in vivo following their adoptive infusion into recipients. Differences in the genotype and phenotype of mouse NK cells compared to human NK cells limit the value of murine animal models to address these questions. In contrast to mice, Rhesus macaques have orthologues to most of the human MHC class I and II genes and possess NK cells expressing KIRs that are phenotypically and functionally similar to human NK cells, thus providing an excellent model system for evaluating questions related to adoptive NK cell therapy. We developed an in vitro method to expand macaque NK cells to characterize their in vivo longevity and tissue trafficking following adoptive infusion. Macaque NK cells were enriched from peripheral blood mononuclear cells by depleting CD3+ cells using immunomagnetic beads and were then expanded in vitro with autologous plasma and a human EBV-LCL feeder cell line using culture conditions identical to those used to expand NK cells from humans. NK cell cultures expanded 50- to 100-fold over 7 to 20 days, were greater than 99% CD3 negative, and had a similar phenotype to human NK cells including a large proportion of CD16/CD56 double positive cells, and ubiquitous expression of NKG2D, KIR2D, LFA-1, granzyme B, and CXCR3. In contrast to mice but analogous to human NK cells, macaque expanded NK cells upregulated surface expression of TRAIL and were highly cytotoxic to K562 cells and other human tumor lines (Figure). CFSE labelling of expanded NK cells did not alter their phenotype or tumor cytotoxic function. Data characterizing the longevity, proliferative capacity, and tissue trafficking patterns in the blood, bone marrow and lymph node of in vitro expanded and adoptively infused CFSE labeled NK cells (up to 1 × 108 NK Cells/kg i.v.) in macaque recipients will be presented from this analysis. Figure Figure

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Chai, Yihuan, Huiying Qiu, and Hui Lv. "The Role of Selective Depletion of Alloreactive Donor T Cells in Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation." Blood 108, no. 11 (November 16, 2006): 5167. http://dx.doi.org/10.1182/blood.v108.11.5167.5167.

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Abstract One of the main goals in allogeneic bone marrow(BM) transplantation is the abrogation of graft-versus-host disease (GVHD) with the preservation of antileukemia and antiviral activity. The Study present a selective T cell depletion strategy based on the physical separation of the alloreactive T cells, which were identified by expression of two activation-induced antigens (CD25 and CD69). T cells from C57BL/6(H-2b) mice were first activated with BALB/c (H-2d) recipient spleen cells in a 2-day mixed-lymphocyte-culture (MLC). Following this activation, this compound is selectively depleted based on expression of two activation-induced antigens CD25 and CD69 using magnetic cell sorting. The depleted cells or the untreated cells were then rechallenged respectively in a secondary MLC, with the same stimulator cells or a third-party (DBAH-2k) or tumor- specific (SP2/0, BALB/c-origin myeloma) cells. Cells proliferation were assayed at the indicated time points(1, 2, 3, 4, 5 days). These treated cells or control-cultured cells (2.0×106) mixed with 5.0×106 BM cells from C57BL/6 were transfused respectively by the trail vain into the lethally irradiated BALB/c to observe the survival time, GVHD incidence and pathological analysis. MLC assays demonstrated that this technique led to a significant decrease in alloreactivity of donor cells(29.02~64.17%), which at the same time preserved reactivity against third party cells(49.61~75.69%)and anti-tumor cells(61.14~68.62%). The mice in the group of control-coclutured were died of acute GVHD within 24days. The 7 recipient mice in the treated group were free of acute GVHD, and 3 mice were died of acute GVHD (aGVHD) within 23 days. MACS-based ex-vivo depletion of alloreactive donor T cells based on expression of two activation-induced antigens (CD25 and CD69) could inhibit anti-host responses, by contrast, anti-SP2/O and anti-third-party responses were preserved. Cotransplantation of these selected depleted cells and BM cells could reduce aGVHD.

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Mercer-Smith, Alison, Wulin Jiang, Juli Bago, Simon Khagi, Carey Anders, and Shawn Hingtgen. "THER-15. DEVELOPING TUMOR-HOMING CYTOTOXIC HUMAN INDUCED NEURAL STEM CELL THERAPY FOR BRAIN METASTASES." Neuro-Oncology Advances 1, Supplement_1 (August 2019): i13—i14. http://dx.doi.org/10.1093/noajnl/vdz014.058.

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Abstract INTRODUCTION: Non-small cell lung cancer (NSCLC) and breast cancer are the most common cancers that metastasize to the brain. New therapies are needed to seek out and eradicate metastases. Genetically engineered neural stem cells (NSCs) have shown unique tumor-homing capacity, allowing them to deliver cytotoxic proteins directly to tumors. An ideal NSC drug carrier would be readily available and autologous. We have transdifferentiated human fibroblasts into induced NSCs (hiNSCs) that home to tumors and engineered the hiNSCs to release the cytotoxic protein TRAIL. Here we used intracerebroventricular (ICV) injections to deliver hiNSCs to metastatic foci. METHODS: We performed an in vitro efficacy co-culture assay, used in vivo studies to determine the migration, persistence, and efficacy of therapeutic hiNSCs against H460 NSCLC and triple-negative breast cancer MB231-Br tumors in the brain. Following the establishment of tumors in the brains of nude mice, hiNSCs were injected directly into the tumor or the ventricle contralateral to the site of tumor. The migration and persistence of hiNSCs was investigated by following the bioluminescence of the hiNSCs. The therapeutic efficacy of the hiNSCs was determined by following the bioluminescece of the tumor. RESULTS/CONCLUSION: Co-culture results demonstrated that hiNSC therapy reduced the viability of H460 and MB231-Br up to 75% and 99.8% respectively compared to non-treated controls. ICV-administered hiNSC serial imaging show that cells persisted for more than one week. Fluorescent analysis of tissue sections showed that hiNSCs co-localized with lateral and a contralateral tumors within 7 days. Using H460 and MB231-Br models, kinetic tracking of intracranial tumor volumes showed intratumoral or ICV-injected therapeutic hiNSCs reduced the growth rate of brain tumors by 31-fold and 3-fold, respectively. This work demonstrates for the first time that we can effectively deliver personalized cytotoxic tumor-homing cells through the ventricles to target brain metastases.

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Martelli, M. P., V. Pettirossi, N. Manes, A. Liso, F. Mezzasoma, F. Cecchetti, M. F. De Marco, et al. "Selective Silencing of the NPM1 Mutant Protein and Apoptosis Induction upon ATRA In Vitro Treatment of AML Cells Carrying NPM1 Mutations." Blood 110, no. 11 (November 16, 2007): 868. http://dx.doi.org/10.1182/blood.v110.11.868.868.

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Abstract We previously identified a new AML category carrying NPM1 mutations which lead to aberrant cytoplasmic expression of the nucleolar protein NPM1, hence the term NPMc+ AML[Falini et al, NEJM 2005]. This leukemia accounts for about one-third of adult AML and shows distinctive biological and clinical features[Falini et al, Blood 2007]. Notably, AML carrying NPM1 mutations in the absence of FLT3-ITD are characterized by a favourable prognosis. However, still a proportion of NPMc+ AML cannot be cured by conventional treatments and new therapeutic strategies need to be explored. We previously identified OCI/AML3 as the only human AML cell line carrying cytoplasmic mutated NPM (type A) in the absence of FLT3-ITD[Quentmeier et al, Leukemia 2005]. Because of these features and the ability to engraft in NOD/SCID mice, the OCI-AML3 represents a remarkable tool for the study of NPMc+ AML. Previous findings that ATRA exerts growth inhibitory effects on the OCI/AML3 prompt us to investigate the molecular mechanisms underlying the response to ATRA, with focus on the NPM mutant protein. As cellular model for our studies, we also used primary leukemia cells originated from a patient with NPMc+ AML (mutation A) bearing FLT3-ITD (Mont1) that have been propagated in NOD/SCID mice for 5 years without loss of initial characteristics. Early cell cycle arrest and proapoptotic effects of pharmacological doses of ATRA were confirmed in both cellular models in vitro. Morphological signs of differentiation were not evident. Western blot analysis using specific antibodies showed marked downregulation of the leukemic NPM1 mutant protein upon ATRA treatment, preceding apoptosis activation. On the other hand, wild-type NPM1 protein levels remained unchanged, leading to a condition of NPM1 haploinsufficiency. Semi-quantitative RT-PCR for NPM mutant A showed no change in mRNA expression following treatment, suggesting a regulation of the NPM mutant protein expression at post-transcriptional level. Indeed, concomitant treatment with proteasome-inhibitors partly reverted this effect. Downregulation of NPM mutant protein preceded activation of caspase-8 and caspase-3, PARP-cleavage and Bax activation. No NF-kB activation was observed upon ATRA treatment. Activation of the p53-dependent pathway was a later event, as expected in conditions of NPM1 haploinsufficiency. Importantly, these results were confirmed in the primary NPMc+ AML cells from patient Mont1. Activation of caspase-8 suggests that the response to ATRA in NPMc+ AML cells may be mediated through the death receptor pathway. Although protein levels of TRAIL, TRAIL receptors and TNF-alpha receptors seem to be unaffected, it might be possible that the NPM1 mutant protein modulates the signalling through death cell receptors. Analysis of ATRA-induced transcriptome and proteome modifications in NPMc+ AML is ongoing and will be also presented, as well as further pre-clinical studies on patients’ primary AML cells and in NOD/SCID mice. In conclusion, our data suggest that NPM mutant protein might be involved in the in vitro response to ATRA in AML cells carrying NPM1 mutations. Figure Figure

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Jiang, Wulin, Alison Mercer-Smith, Juli Bago, Simon Khagi, Carey Anders, and Shawn Hingtgen. "SCIDOT-22. INTRACEREBROVENTRICULAR DELIVERY OF TUMOR-HOMING CYTOTOXIC HUMAN INDUCED NEURAL STEM CELLS FOR TREATMENT OF BRAIN METASTASES." Neuro-Oncology 21, Supplement_6 (November 2019): vi276. http://dx.doi.org/10.1093/neuonc/noz175.1158.

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Abstract INTRODUCTION Non-small cell lung cancer (NSCLC) and breast cancer are the most common cancers that metastasize to the brain. New therapies are needed to target and eradicate metastases. We have developed genetically-engineered induced neural stem cells (hiNSCs) derived from human fibroblasts that selectively home to tumors and release the cytotoxic protein TRAIL. Building on these results, we explored the efficacy of hiNSC therapy delivered via intracerebroventricular (ICV) injections for the treatment of metastatic foci in the brain for the first time. METHODS We performed in vitro efficacy and migration assays in conjunction with in vivo studies to determine the migration, persistence, and efficacy of therapeutic hiNSCs against H460 NSCLC and triple-negative breast cancer MB231-Br tumors in the brain. Following the establishment of tumors in the brains of nude mice, hiNSCs were injected directly into the tumor or the ventricle contralateral to the tumor. The migration and persistence of hiNSCs were investigated by following the bioluminescence of the hiNSCs. The therapeutic efficacy of the hiNSCs was determined by following the bioluminescence of the tumor. RESULTS/ CONCLUSION Co-culture results demonstrated that hiNSC therapy reduced the viability of H460 and MB231-Br up to 75% and 99.8% respectively compared to non-treated controls. In vitro migration assays showed significant directional migration toward both lung and breast cancer cells within 4 days. ICV-administered hiNSC serial imaging shows that cells persisted for >1 week in the brain. Fluorescent analysis of tissue sections showed that hiNSCs co-localized with lateral and contralateral tumors within 7 days. Using H460 and MB231-Br models, kinetic tracking of intracranial tumor volumes showed intratumoral or ICV-injected therapeutic hiNSCs suppressed the growth rate of brain tumors by 31-fold and 3-fold, respectively. This work demonstrates for the first time that we can effectively deliver personalized cytotoxic tumor-homing cells through the ventricles to target brain metastases.

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Smith, Aleah, Hahn Khuu, Dawn M. Betters, Lisa Cook, Catalina Ramos, Sophia Grasmeder, Maria Berg, et al. "Adoptive Transfer of Escalating Doses of Ex Vivo Expanded Autologous Natural Killer (NK) Cells In Patients with Advanced Malignancies Following Bortezomib Treatment to Sensitize to NK-TRAIL Cytotoxicity." Blood 116, no. 21 (November 19, 2010): 4296. http://dx.doi.org/10.1182/blood.v116.21.4296.4296.

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Abstract Abstract 4296 The proteosome inhibitor bortezomib (bort) augments NK cell TRAIL and perforin/granzyme-mediated caspase-8 activity in tumor cells. We have observed that bort sensitizes human tumors to autologous NK cell cytotoxicity in vitro and enhances the antitumor effects of adoptively transferred NK cells in vivo in tumor bearing mice (Lundqvist A et al, Blood 2009). Based on these data, we initiated a clinical trial to explore the safety and antitumor efficacy of infusing escalating doses of adoptively infused ex vivo expanded autologous NK cells following bort treatment in patients with advanced malignancies. Patients (pts) age 18–70 with hematological malignancies or metastatic tumors with progressive disease refractory to conventional therapy were eligible for study. Pts underwent a 15–20 liter apheresis to isolate NK cells that were enriched using immuno-magnetic beads to deplete CD3+ T-cells followed by CD56+ selection. Enriched NK cells (5-8 × 107cells) were expanded in vitro over 2 weeks using an irradiated clinical grade EBV-LCL feeder cell line. Three days prior to NK cell infusion, subjects received a single injection of pentostatin (4mg/m2) to deplete regulatory T-cells followed by a single injection of bort (1.3 mg/m2) 24 hours prior to the infusion of autologous ex vivo expanded NK cells given on day 0. To maintain NK cell viability and TRAIL surface expression, 2 million IU/m2 of IL-2 was given s.c. for 7 days (daily for cohorts 1–2 and q 12 hours for cohorts 3–4). Pts were restaged at 3 weeks and those with disease regression or stable disease were eligible to receive additional cycles. Cohorts of 3–6 pts were enrolled into escalating NK cell dose levels (5 × 106, 1× 107, 5×107, and 1×108 NK cells/kg). To date, 4 dose levels have been completed with a total of 43 adoptive NK cell infusions given to 14 pts. Following a 2 week culture, NK cells expanded a median 184 fold (range 58–683) with 42/43 cultures expanding successfully to achieve the target NK cell dose. NK cells harvested a median 14 days after expansion (range 14–16) contained a median 99.7% (range 96–100) CD3-/CD56+ NK cells, 0% CD3+ T-cells and had a median 87% (range 71–93) viability by 7AAD/Annexin V staining. The absolute lymphocyte count nadired on day +1 at a median 160 cells/ul (range 0–430) and peaked on day 7 at a median 2060 cells/ul (range760-6090). For patients in cohorts 3 and 4, circulating numbers of CD3-/CD56+ NK cells in the blood peaked at day 7 at a median of 382cells/uL (range 60–1851 cells/uL-Figure 1A). Despite an initial decline in circulating Treg (CD3+CD4+CD25+CD127-FoxP3+) numbers following pentostatin therapy, IL-2 administration in these pts was associated with an increase in Treg cells which peaked on day 7 at 112 cells/uL (range 0–700 cells/uL-Figure 1B). The most common adverse events related to treatment were attributable to IL-2 therapy and included grades I-II fever, renal insufficiency, edema and hypotension. Two pts had unexplained and transient grade I and II elevations in hepatic enzymes and one patient developed zoster. Two pts developed elevated free T4 levels and low TSH levels following NK cell therapy (one in dose level 2 and one in dose level 4) consistent with acute thyroiditis; both subsequently developed hypothyroidism with one developing myopathy requiring thyroid replacement therapy. Best clinical response to date includes 6 pts with progressive disease, 7 pts with stable disease (including two pts with metastatic tumors who had more than a 30% decline in serum tumor markers), and 1 minor response in a patient with metastatic kidney cancer. 9/14 (64%) pts received more than 1 NK cell infusion including 3/14 pts who received 3 cycles, 2/14 pts who received 4 cycles, 3/14 pts who received 5 cycles, and 1 patient who received 6 cycles of NK cells before going off study for progressive disease. The trial continues to accrue pts and is currently evaluating the effects of infusing increasing numbers of expanded NK cells given sequentially on days 0 and +5. Conclusions: With the exception of thyroiditis, infusions of up to 1 × 108 ex vivo expanded NK cells/kg have been well tolerated and have provided preliminary clinical evidence for mediating anti-tumor immunity in pts with cancer. Disclosures: No relevant conflicts of interest to declare.

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Heinrichs, Jessica Lauren, Hung Nguyen, David Bastian, Yongxia Wu, Anusara Daenthanasanmak, and Xue-Zhong Yu. "CD8 Tregs Promote Gvhd Prevention and Restore Impaired GVL Effect Mediated By CD4 Tregs in Mice." Blood 126, no. 23 (December 3, 2015): 1873. http://dx.doi.org/10.1182/blood.v126.23.1873.1873.

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Abstract Adoptive regulatory T-cell (Treg) therapy has enhanced the outcome of patients suffering from graft-versus-host (GVH) disease following allogeneic hematopoietic stem cell transplantation (allo-HCT); however, fear of broad immune suppression and subsequent dampening of the beneficial graft-versus-leukemic (GVL) responses remains a challenge. In order to subvert broad immune suppression, we generated alloantigen-specific induced Tregs (iTregs) from resting CD4 or CD8 T cells and tested the ability of iTregs to suppress GVH and maintain GVL responses. We utilized a clinically relevant murine model of haploidentical-HCT with the addition of host-original leukemia cell line to evaluate the effects of CD4 and CD8 iTregs in GVH and GVL responses. While alloantigen-specific CD4 iTregs were effective in preventing GVHD (Fig. 1 A and C), they completely abrogated the GVL effect against aggressive leukemia resulting in 100% tumor mortality (Fig. 1 B and D). Mechanistically, these CD4 iTregs were found to potently suppress the expansion of effector T cells (Teffs) and their ability to secrete IFNγ and granzyme B in the recipient spleen and liver, which may contribute to the impaired GVL activity. Using similar approach, we generated alloantigen-specific CD8 iTregs and found they express higher levels of granzyme B and CTLA-4 compared to nTreg and CD4 iTregs. In vivo studies showed these CD8 iTregs moderately attenuated GVHD (Fig. 1 A and C)while completely sparing the GVL effect (Fig. 1 B and D). We thus further reasoned that the combination of CD4 and CD8 iTregs could achieve the optimal goal of allo-HCT: GVHD suppression with GVL preservation. Indeed, the combination therapy potently suppressed GVHD resulting in increased survival and decreased pathological injury to target organs than either CD4 or CD8 iTreg singular therapy (Fig. 1 A and C). More importantly, the combination therapy maintained potent GVL responses reflected by significantly decreased tumor mortality and load (Fig. 1 B and D).Mechanistically, we observed addition of CD8 iTregs maintained the suppression of Teff expansion but restored the ability of Teffs in producing inflammatory cytokines (e.g. IFNγ and TFNα) and cytolytic effector molecules (e.g. granzyme B and TRAIL). To our knowledge the current findings are the first to support the use of combinational iTreg therapy to achieve optimal suppression of GVHD while maintaining GVL responses. This work was supported by NIH grants: R01 CA118116 and R01 CA169116 Disclosures No relevant conflicts of interest to declare.

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Lundqvist, Andreas, M. Berg, A. Smith, L. Cook, R. Goodwin, C. Ramos, S. Vasu, et al. "Adoptive Infusion of Ex Vivo Expanded Autologous Natural Killer (NK) Cells in Cancer Patients Treated with Bortezomib to Sensitize to NK-TRAIL Cytotoxicity." Blood 114, no. 22 (November 20, 2009): 4080. http://dx.doi.org/10.1182/blood.v114.22.4080.4080.

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Abstract Abstract 4080 Poster Board III-1015 Ligation of inhibitory receptors renders natural killer (NK) cells inactive against autologous tumors. Recently, bortezomib (bort) was shown to sensitize a variety of hematological malignancies and solid tumors to NK cell cytotoxicity in vitro and in tumor bearing mice by augmenting TRAIL and perforin/granzyme-mediated caspase-8 activity. Based on these findings, we initiated a clinical trial to explore the anti-tumor effects of escalating doses of adoptively infused ex vivo expanded autologous NK cells against malignancies in pts treated with bortezomib to sensitize to NK Cell TRAIL cytotoxicity. Pts age 18-70 with treatment refractory metastatic tumors or hematological malignancies with progressive disease refractory to conventional therapy were eligible for study. Pts underwent a 15-20 liter apheresis that was enriched for NK cells using immuno-magnetic beads to deplete CD3+ T-cells followed by CD56+ selection. Enriched NK cells (5 × 107) were expanded in vitro over 2 weeks using irradiated EBV-LCL feeder cells. Three days prior to NK cell infusion, subjects received a single injection of pentostatin (4mg/m2) to deplete lymphocytes followed by a single injection of bort (1.3 mg/m2) 24 hours prior to the infusion of autologous ex vivo expanded NK cells given on day 0. To maintain NK cell viability and TRAIL surface expression, IL-2 (2 million IU/m2) was given s.c. for 7 days (initially daily, subsequently increased to b.i.d.) after each NK cell infusion. Pts were restaged at 3 weeks and those with a disease responses or stable disease were eligible to receive additional cycles. Cohorts of 3-6 pts are currently being enrolled into escalating NK cell dose levels (5 × 106, 1× 107, 5×107 and 1×108 NK cells/kg). The study is currently accruing pts into NK cell dose level 3, with 9 pts having received a total of 19 NK cell infusions. Following a 2 week culture, NK cells expanded a median 191 fold (range 61-502) with 19/20 cultures expanding successfully to achieve the target NK cell dose. At harvest, expanded NK cell cultures contained a median 99% (range 96-100) CD3-/CD56+ NK cells that had a median 86% (range 71-92) viability by 7AAD/Annexin V staining. To date, no grade III/IV toxicities attributable to NK cell infusions have occurred. The most common adverse events were related to IL-2 and included grades I-II fever, renal insufficiency, edema and hypotension. Two pts had unexplained and transient grade I and II elevations in hepatic enzymes. One pt (dose level I) developed zoster leading to a protocol amendment for subsequent pts to receive valacyclovir prophylaxis. The absolute lymphocyte count nadired on day 0 at a median 0 cells/ul (range 0-150) and peaked on day 10 at a median 1700 cells/ul (range 646-7530). On day 10, PBMCs contained a median 48% of CD3+ T-cells, 13% CD3-/CD56+ NK cells and 10% CD3+/CD4+/CD25+/CD127-/Foxp3 + T-regs. An ELISA analysis of IL-2 levels in sequential serum samples from 2 patients given daily IL-2 (2 million IU/m2) showed levels peaked at 2-4 hours in the range of 300-800 pg/ml but declined rapidly to undetectable levels by 24 hours after each injection. Subsequently the protocol was amended to increase IL-2 dosing to q12 hours. Best clinical response to date includes 4 pts with progressive disease and 5 pts with stable disease. All 5 pts with stable disease have received subsequent cycles of NK cell therapy (range 2-5 cycles). Although no pt met criteria for a PR using RECIST criteria, two pts with metastatic tumors (colon cancer and non small cell lung cancer) who had stable disease had a >30% fall in CEA levels following each NK cell infusion. Conclusions: Using EBV-LCL feeder cells, pure populations of highly activated NK cells can be successfully expanded ex vivo under GMP conditions to evaluate the anti-tumor effects of escalating numbers of adoptively infused NK cells. This regimen utilizing bortezomib to potentiate the antitumor effects of adoptively transferred autologous NK cells continues to enroll patients and to date has been well tolerated and has provided early clinical evidence for anti-tumor activity. Disclosures: No relevant conflicts of interest to declare.

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Omidvar, Nader, Stephanie Beurlet, Carole Le Pogam, Anne Janin, Christophe Leboeuf, Annie Soulie, Niclas Setterblad, et al. "ABT-737 Targets Intrinsic Apoptosis during Cooperation of BCL-2 and Oncogenic NRAS in An in Vivo Progression Model of Myelodysplasia/Acute Myeloid Leukaemia." Blood 112, no. 11 (November 16, 2008): 848. http://dx.doi.org/10.1182/blood.v112.11.848.848.

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Abstract OBJECTIVES: Activating RAS mutations and over-expression of BCL-2 are prognostic features of myelodysplastic syndromes/acute myeloid leukemia (MDS/AML) transformation. Using NRASD12 and BCL-2, we created two distinct transplantable in vivo models of MDS and AML. Expression of hBCL-2 in a primitive compartment by MMTV-LTR results in a disease resembling human MDS with bone marrow blasts of 15% with increased apoptosis assayed by TUNEL on liver sections, whilst the myeloid MRP8 promoter induces a disease with characteristics of human AML with marrow blasts of up to 90% with liver apoptosis patterns similar to wild type. In MDS mice RAS and BCL-2 do not co-localize in the mitochondria, but localize to the plasma membrane, where active pro-apoptotic RAS is normally located, whereas in the AML disease RAS and BCL-2 co-localize in the mitochondria, where BCL-2 is normally found; consistent with its anti-apoptotic properties. We next examine the mouse hematopoietic FDCP-1 in vitro model with stable exogenous expression of NRASD12, hBCL-2 or NRASD12 and hBCL-2. Furthermore, we report the in vivo effects of the BH-3 mimetic inhibitor ABT-737. RESULTS: FDCP-1 lines demonstrate that whilst hBCL-2 alone prevents staurosporine-induced mitochondrial-dependent (caspase-9-mediated intrinsic) apoptosis, both NRAS-D12 and NRASD12/hBCL-2 cell lines were pro-apoptotic. No significant difference was observed in cell-death receptor (caspase-8-mediated extrinsic) apoptosis between the cell lines following pharmacological induction with Fas-ligand or TRAIL. Annexin-V/Propidium Iodide flow cytometry and caspase activity assays on hematopoietic primary cells from the mouse models recapitulated the findings; namely the MDS-model demonstrates increased caspase-9-mediated apoptosis (within the Lin−/Sca-1+/KIT+ (LSK) sub-population), whilst the AML-model illustrate anti-apoptotic activity. This is concordant with the signaling profile where phosphorylated AKT is reduced in the RAS-mediated MDS mice and active-AKT is increased in the BCL-2 mediated AML disease. Expanded leukemic stem cell LSK populations had increased hBCL-2 expression in the RAS-GTP complex in both MDS/AML diseases. Thus we suggest that this complex can be a specific target for therapy. When hBCL-2 is switched off with doxycycline in the conditional MDS mice, only partial reversal of the phenotype was observed as RAS recruits endogenous mouse BCL-2 to remain active; thus demonstrating the role of the complex in the disease. In order to target both human and mouse BCL-2 the efficacy of in vivo treatment with the specific BH-3 mimetic inhibitor ABT-737 was determined. We show that the treatment significantly reduced the progression of the disease, increased the peripheral blood platelet counts, decreased the bone marrow blast and cleared the tissue invasion in the MDS mice or reduced tissue infiltration of the AML. In vivo imaging by single-photon emission computed tomography (SPECT) using 99mTc-labelled Annexin-V shows that ABT-737 induces apoptosis of the blast cells that infiltrate the liver and the spleen of the treated mice, which was confirmed by TUNEL on liver sections. Additionally, ABT-737 can reduce the myeloid colony growth and the LSK expansion in these mice; whilst abrogating BCL-2:RAS-GTP complex formation illustrated via biochemical and confocal assays. CONCLUSION: This represents the first in vivo progression model of MDS/AML dependent on the formation of a BCL-2:RAS-GTP complex rescued by ABT-737 via intrinsic apoptosis and thus support the case for BH-3 mimetic therapy.

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Ueno, Niina, Shikiko Ueno, Shinya Endo, Nao Nishimura, Hiro Tatetsu, Shinya Hirata, Hiroaki Mitsuya, and Yutaka Okuno. "PU.1-Induced IRF4 Down-Regulation and Subsequent IRF7 up-Regulation in Myeloma Cells." Blood 126, no. 23 (December 3, 2015): 2957. http://dx.doi.org/10.1182/blood.v126.23.2957.2957.

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Abstract PU.1 is an Ets family transcription factor, which is necessary for differentiation of both myeloid and lymphoid lineages. It was previously reported that conditional knockout of the upstream regulatory element (URE) located in 14 kb 5' of the PU.1 gene resulted in down-regulation of PU.1 expression in granulocytes and B lymphocytes by 80% compared to that of wild type and induced acute myeloid leukemia and CLL-like diseases in mice. Since the URE contains a suppressor region for PU.1 expression in T cells, such mice express PU.1 in T cells and develop T cell lymphoma. Thus, the failure of proper expression of PU.1 in certain differentiation stages in certain cell lineages appears to result in hematological malignancies. We previously reported that PU.1 is down-regulated in various myeloma cell lines. In addition, PU.1 is expressed in normal plasma cells and PU.1 is down-regulated in myeloma cells of certain myeloma patients, who appear to have poor prognosis. In those myeloma cell lines, the promoter and URE of the PU.1 gene are highly methylated. A demethylation agent, 5-aza-2'-deoxycytidine, induced PU.1 up-regulation, growth arrest, and apoptosis in myeloma cell lines, KMS12PE and KHM11. In addition, conditionally expressed PU.1 induced cell growth arrest and apoptosis in PU.1-low-negative myeloma cell lines, U266 and KMS12PE, suggesting that PU.1 is a tumor suppressor for myeloma cells. To elucidate the mechanisms of the cell growth arrest and apoptosis in myeloma cells induced by PU.1, we performed DNA microarray analysis to compare gene expression levels before and after PU.1 expression. Among cell-cycle related genes, p21WAF1/CIP1 was found up-regulated in U266 cells, while among apoptosis related genes, TRAIL was highly up-regulated in both U266 and KMS12PE cell lines. With further investigation, we concluded that PU.1 directly transactivated the TRAIL gene in myeloma cells, leading to apoptosis. Based on the DNA microarray data generated, we found that IRF4 is downregulated in U266 myeloma cells after PU.1 induction. It has been reported that knockdown of IRF4 induces apoptosis in myeloma cell lines. Therefore, we examined whether IRF4 was down-regulated in three myeloma cell lines, U266, KMS12PE, and KHM11 following PU.1 induction. Conditional expression of PU.1 by tet-off system induced IRF4 down-regulation in U266and KMS12PE cells. With lentiviral transduction method, ectopic expression of PU.1 also induced IRF4 down-regulation, cell-cycle arrest, and apoptosis in KHM11 cells. To investigate the role of IRF4 in PU.1-expressing U266 cells, we stably expressed IRF4, partially rescuing U266 cells from apoptosis. IRF4 is known to directly bind to the IRF7 promoter and down-regulate IRF7 expression in activated B cell-like (ABC) subtype of diffuse large B-cell lymphoma cells. Therefore, we examined whether IRF4 bound to the IRF7 promoter in KMS12PEand U266cells using chromatin immunoprecipitation assays. We found that IRF4 directly bound to the IRF7 promoter in both myeloma cell lines. When we overexpressed PU.1, IRF4 levels were decreased and the IRF4 binding to the IRF7 promoter was significantly reduced in those cell lines. Moreover, knockdown of IRF7 significantly rescued PU.1-expressing U266cells from apoptosis. These data strongly suggest that PU.1-induced apoptosis is associated with IRF4 down-regulation and subsequent IRF7 up-regulation in myeloma cells. Since IRF4 is essential transcription factor for myeloma cell survival, up-regulation of PU.1 by demethylation agents, including 5-aza-2'-deoxycytidine may serve as a promising therapeutic modality of multiple myeloma by inducing down-regulation of IRF4. Disclosures No relevant conflicts of interest to declare.

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Yokoyama, Hisayuki, Andreas Lundqvist, Maria Berg, Muthalagu Ramanathan, Rebecca Lopez, Aleah Smith, Nicole Gormley, Su Su, J. Phillip McCoy, and Richard W. Childs. "Adoptively-Infused NK Cells Maintain Their Antitumor Effects in Vivo in the Presence of CyclosporineA (CSA)." Blood 112, no. 11 (November 16, 2008): 2563. http://dx.doi.org/10.1182/blood.v112.11.2563.2563.

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Abstract Animal models show infusions of donor NK cells given after allogeneic HCT can prevent GVHD while simultaneously mediating a graft-vs-tumor effect. However, it is unclear whether adoptively infused NK cells can mediate these beneficial effects in the presence of CSA, which is commonly given after HCT to prevent GVHD. In this study, we analyzed the in vitro effects of pharmacological concentrations of CSA on NK cell phenotype, cell proliferation, and tumor cytotoxicity. We also evaluated in vivo whether CSA administration would reduce the anti-tumor effects of adoptively infused NK cells in tumor bearing mice. PBMCs collected from healthy donors were labeled with CFSE then were stimulated in vitro with IL-2 for 7 days in the presence or absence of CSA (1000ng/ml). CFSE proliferation assays on fresh PBMC showed CSA inhibited IL-2 stimulated CD3+ T-cell proliferation more than CD3−/CD56+ NK cell proliferation (mean percentage inhibition of proliferation 49.4% vs. 22.2% for T cells and NK cells respectively; p<0.05). CD3−/CD56+ NK cells were then isolated from PBMCs of healthy donors and expanded in vitro with irradiated EBV-LCL and IL-2 for 10 days. In contrast to T-cells, CSA only minimally inhibited IL-2 induced proliferation of expanded NK cells (mean 9.5% inhibition of proliferation by CFSE staining). T cells and NK cells were next isolated from PBMCs and stimulated with either OKT3, PMA-Ionomycin (PI), or IL-2. A [3H] TdR uptake assay showed T cell proliferation was inhibited at a substantially higher level by CSA (mean stimulation index for OKT3: 0.03, for PI: 0.35, for IL-2: 0.55) compared to that of expanded NK cells (mean stimulation index for IL-2: 0.82, p<0.05). Furthermore, an ELISA assay showed CSA treatment reduced IL-2 induced secretion of INF-g by T cells more than expanded NK cells (mean reduction in INF-g secretion in T cells of 94.7 % vs. 36.5 % in NK cells, p<0.05). Compared to controls, culturing in vitro expanded NK cells in CSA did not alter surface expression of the activating receptors NKp30, NKp42, and NKG2D but did reduce surface expression of NKp44 and TRAIL (mean reduction in surface expression 36% and 36.3% respectively). Cytotoxic granule release assessed by CD107a staining was inhibited by CSA in CD8+ melanoma specific T cells co-cultured with melanoma cells (mean 12.2 % inhibition) in contrast to NK cells co-cultured with K562 cells where CSA increased CD107a expression a mean 29.7% (p<0.05). Furthermore, at a 20:1 E:T ratio, 51Cr cytotoxity assays showed CSA did not reduce the cytotoxicity of in vitro expanded NK cells against renal cell carcinoma (RCC) cells (58% mean lysis) compared to NK cells cultured in control media (55% mean; p=n.s.). In contrast, melanoma specific T-cell killing of tumor targets was significantly lower in CTL cultures containing CSA compared to control media (38.0% vs. 50.2% respectively P<0.05). Next, we assessed the impact of CSA administration on the anti-tumor effects of adoptive NK cell infusions in tumor bearing animals where syngeneic NK cell infusions following bortezomib treatment have been shown to delay tumor progression and prolong survival. BALB/c mice injected with 100,000 luciferase transfected RENCA tumor cells i.v. received 3 weekly treatments with the combination of bortezomib (5ug/mouse i.v.) and 2×106 syngeneic NK cells i.v. with or without daily administration of CSA (15mg/kg sc). Bioluminescence imaging in controls that did not receive CSA showed tumor growth was slower and survival was prolonged in mice receiving adoptive NK cell infusions (median survival 53 days) compared to mice that did not receive NK cells (median survival 30.2 days; p<0.05). CSA administration did not impair the anti-tumor effects of adoptive NK cell infusions; mice receiving CSA and adoptive NK cell infusions had similar tumor growth and survival as recipients of NK cells without CSA (median survival 47 vs. 53 days; p=n.s.). These results show that CSA is significantly more immunosuppressive to T cells compared to NK cells and provide evidence that the anti-tumor effects of adoptively infused in vitro expanded NK cells are maintained even in the presence CSA.

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Fontaine, Martina, Benjamin Demoulin, Simon Bornschein, Susanna Raitano, Steve Lenger, Hidevaldo Machado, Jonathan D. Moore, Panagiota A. Sotiropoulou, and David E. Gilham. "Next Generation NKG2D-based CAR T-cells (CYAD-02): Co-expression of a Single shRNA Targeting MICA and MICB Improves Cell Persistence and Anti-Tumor Efficacy in vivo." Blood 134, Supplement_1 (November 13, 2019): 3931. http://dx.doi.org/10.1182/blood-2019-129998.

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Background The Natural Killer Group 2D (NKG2D) receptor is a NK cell activating receptor that binds to eight different ligands (NKG2DL) commonly over-expressed in cancer, including MICA and MICB. The product candidate CYAD-01 are chimeric antigen receptor (CAR) T-cells encoding the full length human NKG2D fused to the intracellular domain of CD3ζ. Data from preclinical models have shown that CYAD-01 cells specifically target solid and hematological tumors. Encouraging preliminary results from the Phase I clinical trial THINK, assessing CYAD-01 safety, showed initial signals of objective clinical responses in patients with r/r AML and MDS. The clinical development of CAR T-cells has been limited by several challenges including achieving sufficient numbers of cells for clinical application. We have previously shown that NKG2D ligands are transiently expressed on activated T cells and that robust cell yields are generated through the addition of a blocking antibody and a PI3K inhibitor during cell manufacture. Here, we investigated the ability of an optimized short hairpin RNA (shRNA) technology to modulate NKG2DL expression on CYAD-01 cells and to determine if there is an increase in the anti-tumor activity of NKG2D-based CAR T-cells (termed CYAD-02). Methods Molecular and cellular analyses identified MICA and MICB as the key NKG2DL expressed on activated T-cells and highly likely to participate in driving fratricide. In silico analysis and in vitro screening allowed the identification of a single shRNA targeting the conserved regions of MICA and MICB, thus downregulating both MICA and MICB expression. The selected shRNA was incorporated in the NKG2D-based CAR vector, creating the next-generation NKG2D-based CAR T-cell candidate, CYAD-02. In addition, truncated versions of the NKG2D receptor were generated to explore the mechanisms of action of NKG2D receptor activity in vivo. The in vivo persistence and anti-tumor activity of CYAD-02 cells was evaluated in an aggressive preclinical model of AML. Results Injection of CAR T-cells bearing truncated forms of the NKG2D-CAR in immunosuppressed mice resulted in similar persistence to the control T-cells. In contrast, CYAD-01 cells had reduced persistence, suggesting that the recognition of the NKG2DL by the NKG2D receptor could contribute to this effect. Analysis of cell phenotype upon CAR T-cell activation showed that MICA and MICB were transiently expressed on T-cells during manufacturing. These results collectively suggested that downregulating MICA and MICB expression in CYAD-01 cells could be a mean to increase CAR T-cell persistence in vivo. Candidate shRNA were screened for efficient targeting of both MICA and MICB at the mRNA and protein level. T-cells transduced with a single vector encoding for the NKG2D-based CAR and the selected shRNA targeting MICA and MICB (CYAD-02) demonstrated 3-fold increased expansion during in vitro culture in the absence of the blocking antibody used to increase cell yield during manufacture. When injected into immunosuppressed mice, CYAD-02 cells generated with the Optimab process showed 10-fold higher engraftment one week after injection and potent anti-tumor activity resulting in 2.6-fold increase of mouse survival in an aggressive AML model. Conclusions By using a single vector encoding the NKG2D-based CAR next to a shRNA targeting MICA and MICB and combined with improved cell culture methods, CYAD-02, the next-generation of NKG2D-based CAR T-cells, demonstrated enhanced in vivo persistence and anti-tumor activity. Following FDA acceptance of the IND application, a Phase 1 dose-escalation trial evaluating the safety and clinical activity of CYAD-02 for the treatment of r/r AML and MDS is scheduled to start in early 2020. Disclosures Fontaine: Celyad: Employment. Demoulin:Celyad: Employment. Bornschein:Celyad: Employment. Raitano:Celyad: Employment. Machado:Horizon Discovery: Employment. Moore:Avvinity Therapeutics: Employment, Other: Relationship at the time the work was performed; Horizon Discovery: Employment, Equity Ownership, Other: Relationship at the time the work was performed; Centauri Therapeutics: Consultancy, Other: Current relationship. Sotiropoulou:Celyad: Employment. Gilham:Celyad: Employment.

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Blinova, Ekaterina, Egor Turovsky, Elena Eliseikina, Alexandra Igrunkova, Elena Semeleva, Grigorii Golodnev, Rita Termulaeva, et al. "Novel Hydroxypyridine Compound Protects Brain Cells against Ischemic Damage In Vitro and In Vivo." International Journal of Molecular Sciences 23, no. 21 (October 26, 2022): 12953. http://dx.doi.org/10.3390/ijms232112953.

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A non-surgical pharmacological approach to control cellular vitality and functionality during ischemic and/or reperfusion-induced phases of strokes remains extremely important. The synthesis of 2-ethyl-6-methyl-3-hydroxypyridinium gammalactone-2,3-dehydro-L-gulonate (3-EA) was performed using a topochemical reaction. The cell-protective effects of 3-EA were studied on a model of glutamate excitotoxicity (GluTox) and glucose-oxygen deprivation (OGD) in a culture of NMRI mice cortical cells. Ca2+ dynamics was studied using fluorescent bioimaging and a Fura-2 probe, cell viability was assessed using cytochemical staining with propidium iodide, and gene expression was assessed by a real-time polymerase chain reaction. The compound anti-ischemic efficacy in vivo was evaluated on a model of irreversible middle cerebral artery (MCA) occlusion in Sprague-Dawley male rats. Brain morphological changes and antioxidant capacity were assessed one week after the pathology onset. The severity of neurological disorder was evaluated dynamically. 3-EA suppressed cortical cell death in a dose-dependent manner under the excitotoxic effect of glutamate and ischemia/reoxygenation. Pre-incubation of cerebral cortex cells with 10–100 µM 3-EA led to significant stagnation in Ca2+ concentration in a cytosol ([Ca2+]i) of neurons and astrocytes suffering GluTox and OGD. Decreasing intracellular Ca2+ and establishing a lower [Ca2+]i baseline inhibited necrotic cell death in an acute experiment. The mechanism of 3-EA cytoprotective action involved changes in the baseline and ischemia/reoxygenation-induced expression of genes encoding anti-apoptotic proteins and proteins of the oxidative status; this led to inhibition of the late irreversible stages of apoptosis. Incubation of brain cortex cells with 3-EA induced an overexpression of the anti-apoptotic genes BCL-2, STAT3, and SOCS3, whereas the expression of genes regulating necrosis and inflammation (TRAIL, MLKL, Cas-1, Cas-3, IL-1β and TNFa) were suppressed. 3-EA 18.0 mg/kg intravenous daily administration for 7 days following MCA occlusion preserved rats’ cortex neuron population, decreased the severity of neurological deficit, and spared antioxidant capacity of damaged tissues. 3-EA demonstrated proven short-term anti-ischemic activity in vivo and in vitro, which can be associated with antioxidant activity and the ability to target necrotic and apoptotic death. The compound may be considered a potential neuroprotective molecule for further pre-clinical investigation.

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Carroll, Scott F., Erin I. Lafferty, Adam Flaczyk, T. Mary Fujiwara, Robert Homer, Kenneth Morgan, J. C. Loredo-Osti, and Salman T. Qureshi. "Susceptibility to Progressive Cryptococcus neoformans Pulmonary Infection Is Regulated by Loci on Mouse Chromosomes 1 and 9." Infection and Immunity 80, no. 12 (September 17, 2012): 4167–76. http://dx.doi.org/10.1128/iai.00417-12.

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ABSTRACTGenetic factors that regulate the pathogenesis of pneumonia caused by the fungusCryptococcus neoformansare poorly understood. Through a phenotypic strain survey we observed that inbred C3H/HeN mice develop a significantly greater lung fungal burden than mice of the resistant CBA/J strain 4 weeks following intratracheal infection withC. neoformansATCC 24067. The aim of the present study was to characterize the inflammatory response of C3H/HeN mice followingC. neoformanspulmonary infection and to identify genetic loci that regulate host defense. Following cryptococcal infection, C3H/HeN mice demonstrated a Th2 immune response with heightened airway and tissue eosinophilia, goblet cell metaplasia, and significantly higher lung interleukin-5 (IL-5) and IL-13 protein expression relative to CBA/J mice. Conversely, CBA/J mice exhibited greater airway and tissue neutrophilia that was associated with significantly higher pulmonary expression of gamma interferon, CXCL10, and IL-17 proteins than C3H/HeN mice. Using the fungal burden at 4 weeks postinfection as a phenotype, genome-wide quantitative trait locus (QTL) analysis among 435 segregating (C3H/HeN × CBA/J)F2 (C3HCBAF2) hybrids identified two significant QTLs on chromosomes 1 (Cnes4) and 9 (Cnes5) that control susceptibility to cryptococcal pneumonia in an additive manner. Susceptible C3H/HeN mice carry a resistance allele atCnes4and a susceptibility allele atCnes5. These studies reveal additional genetic complexity of the host response toC. neoformansthat is associated with divergent patterns of pulmonary inflammation.

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Zahedi, Payam, Raquel De Souza, Micheline Piquette-Miller, and Christine Allen. "Docetaxel Distribution Following Intraperitoneal Administration in Mice." Journal of Pharmacy & Pharmaceutical Sciences 14, no. 1 (February 28, 2011): 90. http://dx.doi.org/10.18433/j3qw26.

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Purpose: Intraperitoneal (IP) chemotherapy with high molecular weight lipophilic antineoplastic agents such as the taxanes has shown promise in clinical trial evaluation for treatment of localized peritoneal cancers. We have previously developed an IP injectable hydrogel formulation (PoLigel) for sustained peritoneal delivery of docetaxel (DTX), and observed significant efficacy in murine models of ovarian cancer when compared to Taxotere®, the FDA approved formulation of DTX. In order to understand the relationship between drug distribution and efficacy, the current study compares the tissue distribution and pharmacokinetics of DTX administered IP in the PoLigel or Taxotere® formulations. Methods: The PoLigel was prepared by blending a water-soluble chitosan derivative, egg phosphatidylcholine and lauric aldehyde with DTX (drug to material ratio 1:8 w/w). DTX concentrations in plasma, heart, liver, spleen, stomach, intestine, kidney and peritoneal muscle were measured over a five day period following IP administration of the PoLigel and Taxotere® formulations in CD-1 female mice. Results: Three days after Taxotere® administration, no detectable levels of DTX were seen in plasma, while sustained DTX plasma levels of 0.06 ug/ml ± 0.01 per day were observed with PoLigel. At five days post Taxotere® administration, only intestine, stomach and peritoneal muscle showed detectable DTX concentrations whereas all tissues and plasma showed sustained DTX levels in mice that received PoLigel. DTX concentrations that resulted from PoLigel administration were significantly higher in the peritoneal cavity and 200 fold higher than concentrations found in plasma. Conclusions: Overall, the PoLigel formulation increases tissue and plasma drug retention and provides sustained DTX levels compared to the clinically used Taxotere® formulation. The sustained DTX levels seen in the peritoneal cavity following IP administration of the PoLigel may be responsible for the improvement in efficacy that has been observed in our previous studies.

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Kozono, Y., B. L. Kotzin, and V. M. Holers. "Resting B cells from New Zealand Black mice demonstrate a defect in apoptosis induction following surface IgM ligation." Journal of Immunology 156, no. 11 (June 1, 1996): 4498–503. http://dx.doi.org/10.4049/jimmunol.156.11.4498.

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Abstract New Zealand Black (NZB) mice spontaneously develop autoimmune disease, usually characterized by an autoimmune hemolytic anemia, and NZB genes are essential for a severe systemic lupus-like disease in (NZB x NZW)F1 mice. We have found that resting B cells from NZB mice demonstrate a pronounced defect, compared with five normal strains, in apoptosis induction after cross-linking with anti-IgM Abs. In contrast, spontaneous apoptosis of NZB B cells in culture was similar to normal strains. B cells from young (NZB x SM/J)F1 and (NZB x NZW)F1 mice underwent apoptosis normally, indicating that the NZB defect in apoptosis is a recessive trait. However, older (8-32 wk) predisease (NZB x NZW)F1 mice manifested a similar defect in apoptosis induction. The analysis of NXSM recombinant inbred mice derived from NZB and SM/J, in addition to backcross mice, suggested that the NZB apoptosis defect is a multigenic trait. Interestingly, resting B cells form B6.lpr and B6gld mice underwent apoptosis following anti-IgM treatment at a level similar to that of the C57BL/6 parental strain. Thus, the induced apoptosis of resting B cells and the NZB defect are likely not related to either Fas or Fas ligand. We propose that this phenotypic defect in apoptosis induction, or the biochemical alteration that underlies the defect, may be casually related to autoimmune disease in NZB mice and its contribution to lupus-like disease in (NZB x NZW)F1 mice.

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McKenzie, Christopher G. J., Michael Kim, Dana Safavian, Ori D. Rotstein, and John W. Semple. "A Murine Model Of Hemorrhagic Shock/Resuscitation Demonstrates Transfusion Related Acute Lung Injury (TRALI) Induced By Stored Platelet Transfusions." Blood 122, no. 21 (November 15, 2013): 789. http://dx.doi.org/10.1182/blood.v122.21.789.789.

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Abstract Transfusion related acute lung injury (TRALI) is one of the leading causes of transfusion fatalities. There have been several animal models of TRALI developed including ex vivo lung models demonstrating the importance of human anti-neutrophil antibodies in TRALI and in vivo transfusion models showing how biological response modifiers can induce recipient lung damage. In addition, an in vivo antibody-mediated TRALI model has also been established and has shown close similarities with human TRALI. Most of the animal models have a two-hit paradigm where the first hit is delivered in the form of a noxious substance such as lipopolysaccharide and the second hit is a transfusion of either a blood product or an antibody. Here we describe a novel clinically relevant two-hit model where the first hit is hemorrhagic shock followed by a second hit in the form of a platelet transfusion. Severe combined immunodeficient (SCID) mice were anesthetized and had half of their total blood volume was removed via a carotid cannula to instigate hypovolemic shock. After one hour of shock, the mice were reperfused with either Ringer’s lactate solution or fresh or three day aged platelets. 34-1-2s, a monoclonal anti-mouse MHC class I antibody known to induce TRALI was used as a positive reperfusion control. Two hours following reperfusion, the mice were sacrificed and blood was obtained via cardiac puncture in order to measure serum levels of the neutrophil chemoattractant macrophage inflammatory protein-2 (MIP-2). Lungs were removed to determine the percentage of accumulated neutrophils and to measure edema. Compared with control mice reperfused with Ringer’s lactate solution or mice administered fresh platelets, serum MIP-2 levels in mice administered with three day aged platelets were significantly elevated and were comparable to those observed in mice infused with 34-1-2s. Pulmonary neutrophil accumulation in the mice transfused with 3 day stored platelets was also elevated to levels observed in 34-1-2s treated mice. Pulmonary edema as measured by lung Wet/Dry ratios was elevated in mice receiving the 3 day aged platelets. In vivo administration of a MIP-2 antagonist (SB225002, 4mg/kg) intraperitoneally immediately before reperfusion significantly reduced all TRALI symptoms. These results show that an animal model of hypovolemic shock exhibits TRALI upon reperfusion with stored platelets and is dependent on MIP-2 production. This clinically-relevant model will be valuable in studying potential therapeutic interventions to prevent TRALI following transfusions, Disclosures: No relevant conflicts of interest to declare.

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Okwan-Duodu, Derick, Fahim Atif, David S. Yu, Seema Yousuf, Deborah Bruner, Walter John Curran, and Donald G. Stein. "Progesterone to improve neurocognitive outcomes following cranial irradiation." Journal of Clinical Oncology 34, no. 3_suppl (January 20, 2016): 128. http://dx.doi.org/10.1200/jco.2016.34.3_suppl.128.

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128 Background: Neurocognitive functional decline is a common sequalae of cranial irradiation (CI) that significantly impacts quality of life. Preclinical studies and randomized clinical trials show that following traumatic brain injury and cerebrovascular accidents, premenopausal women demonstrate decreased mortality and improved neurocognitive function, with these benefits presumed to be derived from progesterone. We hypothesized that progesterone may serve similar role in neuroprotection following cranial irradiation. Methods: Adult non-tumor bearing wild type C57BL/6 male mice were treated with two separate fractionated radiation therapy regimen (9 Gy and 15 Gy) to the brain. Cohorts of these mice were administered progesterone (16mg/kg daily) as a pretreatment for 3 days and concurrent with the radiotherapy for a total of 14 days with tapering during the last two days. The animals were then tested using different behavioral measures for cognitive function including morris water maze (MWM) for assessing spatial and related forms of learning and memory, elevated plus maze (EPM), , and spontaneous locomotor activity (SLA) tests. Mice were tested for cognitive function on day 10 and after 30 days of treatment for short and long-term effects of (CI) on memory function. Results: All irradiated mice showed statistically significant decline in MWM, EPM, and SLA measures. There were no significant differences in the 9 Gy versus 15 Gy cohorts. Progesterone administration produced a statistically significant group effect (F (4, 25) = 8.553; P<0.001) in the improvement of long-term memory function over 5 days of learning process. Progesterone administration also demonstrated a significant group effect (F (4, 25) = 8.613; P<0.001) in the probe trial, and a significant beneficial effect (F (4, 25)= 7.993; P<0.001) in short-term memory functional latency to reach the platform. Conclusions: The preclinical data show that progesterone improves radiation-induced deficits in short-and long-term memory functions in adult mice. Further work is required to show if progesterone may show similar clinical benefit in neuroprotection for adults undergoing prophylactic CI or definitive CI for brain metastases or benign intracranial processes such as AVM.

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Lopez, Rebecca, Stephanie Sellers, Cynthia E. Dunbar, and Richard Childs. "IL-15 Administration to In-Vivo Expand Adoptively Transferred NK Cells In Rhesus Macaques." Blood 116, no. 21 (November 19, 2010): 960. http://dx.doi.org/10.1182/blood.v116.21.960.960.

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Abstract Abstract 960 Immunotherapy using natural killer (NK) cells is currently being explored as a treatment option for patients with advanced malignant diseases. Although pilot clinical trials have shown adoptive NK cell transfer can result in tumor regression in humans with cancer, additional insight from animal models is needed to optimize methods to enhance the function and in vivo persistence of these adoptively infused lymphocytes. In contrast to mice, rhesus macaques have orthologues to most of the human MHC class I and II genes and possess NK cells expressing KIRs that are phenotypically and functionally similar to human NK cells, thus providing an excellent model to evaluate adoptive NK cell therapy. To characterize their in vivo longevity and tissue trafficking following adoptive infusion, we developed a method to expand large numbers of rhesus NK cells in vitro. NK cells enriched from peripheral blood mononuclear cells by depleting CD3+ cells using immunomagnetic beads were expanded in vitro with autologous plasma and a human EBV-LCL feeder cell line using culture conditions identical to those used for human NK cell expansion. Expanded rhesus NK cells were both phenotypically and genotypically similar to their human counterparts; NK cell cultures expanded up to 1000 fold within 2–3 weeks, were greater than 99% CD3 negative, and had a large proportion of CD16/CD56 double positive cells. In addition, expanded NK cells up-regulated receptors involved in tumor killing, including NKG2D, Granzyme B, TRAIL and Fas-ligand and were highly cytotoxic to K562 cells. Adoptive transfer of (3.2×107 – 1×108) CFSE-labeled ex vivo expanded rhesus NK cells has been well tolerated without any overt toxicities noted to date. Remarkably, despite the infusion of large cell numbers, CFSE labeled NK cells were detectable in the peripheral blood, lymph nodes, and bone marrow compartments at very low levels for only a few hours following infusion. Combining adoptive transfer of ex vivo expanded NK cells with IL-15 administration (rhesus recombinant IL-15 10 ug/kg s.c. × 5 days) resulted in only a minimal and transient 24 hour increase in the number of detectable CFSE labeled NK cells in the circulation and bone marrow. Although IL-15 administration did not substantially expand the number of circulating CFSE labeled NK cells that were adoptively transferred, it did result in a substantial increase in circulating numbers of endogenous NK and T-cells (4.74 fold and 5.2 fold increase in CD3-/CD56+ NK cells and CD3+ T-cells respectively). Surprisingly, IL-15 administration also resulted in a significant expansion of circulating T-regs (CD4-/CD25+/CD127Dim/FOXP3 +) which have previously been shown to suppress NK cell effector function in vitro and vivo; T-cells with a regulatory phenotype expanded 4.54 fold. Expansion of circulating T-regs occurred both when IL-15 was administered alone or in conjunction with adoptive NK cell transfer. Conclusions: IL-15 administration in macaques at the doses used in this study did not expand circulating numbers of adoptively transferred ex-vivo expanded NK cells, although it did significantly expand the numbers of circulating endogeneous NK cells. Remarkably, IL-15 administration was also associated with a significant expansion of T-cells with a regulatory phenotype. We are currently evaluating whether lympho-depletion followed by adoptive NK cell transfer can be used as a method to prevent the expansion of T-regs associated with IL-15 administration. Disclosures: No relevant conflicts of interest to declare.

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Sugane, Kazuo, and Tadashi Matsuura. "Eosinophilia in chimeric mice infected withToxocara canis." Journal of Helminthology 61, no. 2 (June 1987): 157–62. http://dx.doi.org/10.1017/s0022149x00009925.

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ABSTRACTA marked strain variation in eosinophilia following oral infection withToxocara caniseggs was observed in mice. Mutual radiation chimeras between high and low responder mice in terms of eosinophilia were made and compared with the respective donor and recipient for eosinophilia after the infection. As a result, the degree and time course of eosinophilia in chimeric mice were similar to those in donors. The result suggested that genes which regulate inheritance of the trait, marked eosinophilia inT. canis-infected mice, might be expressed in bone marrow derived cells.

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Dokun, Ayotunde O., Lingdan Chen, Mitsuharu Okutsu, Charles R. Farber, Surovi Hazarika, W. Schuyler Jones, Damian Craig, et al. "ADAM12: a genetic modifier of preclinical peripheral arterial disease." American Journal of Physiology-Heart and Circulatory Physiology 309, no. 5 (September 2015): H790—H803. http://dx.doi.org/10.1152/ajpheart.00803.2014.

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In prior studies from multiple groups, outcomes following experimental peripheral arterial disease (PAD) differed considerably across inbred mouse strains. Similarly, in humans with PAD, disease outcomes differ, even when there are similarities in risk factors, disease anatomy, arteriosclerotic burden, and hemodynamic measures. Previously, we identified a locus on mouse chromosome 7, limb salvage-associated quantitative trait locus 1 (LSq-1), which was sufficient to modify outcomes following experimental PAD. We compared expression of genes within LSq-1 in Balb/c mice, which normally show poor outcomes following experimental PAD, with that in C57Bl/6 mice, which normally show favorable outcomes, and found that a disintegrin and metalloproteinase gene 12 ( ADAM12) had the most differential expression. Augmentation of ADAM12 expression in vivo improved outcomes following experimental PAD in Balb/c mice, whereas knockdown of ADAM12 made outcomes worse in C57Bl/6 mice. In vitro, ADAM12 expression modulates endothelial cell proliferation, survival, and angiogenesis in ischemia, and this appeared to be dependent on tyrosine kinase with Ig-like and EGF-like domain 2 (Tie2) activation. ADAM12 is sufficient to modify PAD severity in mice, and this likely occurs through regulation of Tie2.

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HendricKson, Jeanne, David R. Archer, Jennifer R. Perry, Christopher D. Hillyer, and James C. Zimring. "Berkeley Mice with Sickle Cell Disease Are Not More Immunizable to Transfused HOD Red Blood Cells Than Sickle Trait Control Mice." Blood 112, no. 11 (November 16, 2008): 292. http://dx.doi.org/10.1182/blood.v112.11.292.292.

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Abstract Background: Patients with sickle cell disease have higher rates of red blood cell (RBC) alloimmunization following transfusion than any other patient population. However, it is unclear if they are inherently more immunizable, or if the high rates of RBC alloimmunization are simply due to antigenic differences between donors/recipients and heightened transfusion frequency. Utilizing a murine model, we have previously shown that rates of alloimmunization are influenced by the inflammatory status of the recipient at the time of the transfusion. Thus, we hypothesized that sickle patients may be more immunizable, due in part to the baseline inflammation associated with their disease. Herein, we present a reductionist murine sickle model of RBC alloimmunization, and investigate the hypothesis that mice with sickle cell disease are more likely to become alloimmunized following transfusion of RBCs containing a foreign antigen than sickle trait controls. Materials/Methods: Berkeley mice (with human α and βS-globin) were determined to be homozygous (sickle cell disease) or heterozygous (sickle cell trait) by cellulose acetate electrophoresis. We generated the HOD mouse, with RBC specific expression of the model humoral antigen hen egg lysozyme (HEL) fused to the model minor histocompatibility antigen ovalbumin (OVA), linked to the cell membrane by a human blood group antigen (Duffy). The inclusion of OVA in the HOD construct allows presentation of HOD peptides from H-2b MHC; thus, Berkeley mice are able to process and present the HOD antigen. 100 microliters of packed HOD RBCs were transfused IV into mice with sickle cell disease or sickle cell trait; alloimmunization to the HEL antigen was assessed by anti-HEL IgG ELISA 2 weeks following transfusion. Given that co-stimulatory molecule expression on antigen presenting cells in part determines the response of the CD4+ T cell to the antigen being presented, we examined baseline expression of B7-1, B7-2, Ox40L, CD70, 41BBL, CD30L, and CD40 on macrophages (F480 high) and dendritic cells (CD11c high) in sickle cell disease and sickle trait mice. Results: Data for a standard curve was fit to one phase exponential association, with a non-linear best fit regression analysis (R2 of 0.96). Utilizing the OD 415 value of the anti-HEL IgG ELISA, data was combined from 3 separate experiments (n=31 mice). An unpaired two-tailed t-test showed a mean of 6.25e-5, SD 5.87e-5 for sickle disease mice, and a mean of 4.14e-5, SD 2.84e-5 for sickle trait mice, p=0.217 (see figure). Baseline co-stimulatory molecule expression of all molecules examined on macrophages and dendritic cells was similar in both sickle cell disease and sickle trait mice. Figure Figure Conclusions: Generation of the HOD mouse, containing a model humoral antigen capable of being presented by the antigen presenting cells in the Berkeley mice, allowed these studies to be performed. These data rule out the hypothesis that Berkeley mice with sickle cell disease are substantially more immunizable to a single transfusion of RBCs containing the HOD foreign antigen than are those with sickle cell trait. However, there is a large standard deviation in alloantibody response between individual mice with sickle cell disease. The varied response may be explained by differing levels of illness in individual mice; ongoing studies are investigating such a potential correlation. Clinically, many patients with sickle cell disease are transfused in times of illness (i.e. acute chest syndrome), and the possibility that illness alters alloantibody response cannot be ruled out by these studies. Furthermore, although the model was designed to be as reductionist as possible, these studies are limited by the highly heterogeneous genetic background of the Berkeley mice (including FVB/N, 129, DBA/2, C57BL/6, and Black Swiss); this diverse background likely influences response to foreign antigen and makes identifying control groups difficult. Finally, although Berkeley mice with sickle cell disease are known to have baseline inflammation associated with their disease, this does not appear to influence co-stimulatory molecule expression on their antigen presenting cells.

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Yamada, Masana, Rika Sasaki, Koki Hirota, and Mitsuaki Yamazaki. "Dementia Enhances Inhibitory Actions of General Anesthetics in Hippocampal Synaptic Transmission." ISRN Anesthesiology 2011 (December 21, 2011): 1–5. http://dx.doi.org/10.5402/2011/837937.

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In order to investigate whether dementia modifies the anesthetic actions in the central nervous systems, we have studied effects of general anesthetics on the hippocampal synaptic transmission using the dementia model mice. Preliminary in vivo experiments revealed that time of loss of righting reflex following sevoflurane inhalation was more shortened in dementia mice than in healthy control mice. Field population spikes of hippocampal CA1 pyramidal neurons were elicited in vitro using orthodromic stimulation of Schaffer collateral commissural fibers (test pulse). The recurrent inhibition was enhanced with the second stimulating electrode placed in alveus hippocampi (prepulse) to activate recurrent inhibition of CA1. The prepulses were applied as train stimuli to activate release and then deplete γ-amino-butyric acid (GABA) at presynaptic terminals of inhibitory interneurons. Sevoflurane and thiopental had greater actions on inhibitory synaptic transmission in dementia model mice than in control mice. The pre-pulse train protocol revealed that the anesthetic-induced GABA discharge was more enhanced in dementia mice than in control mice. Dementia enhances the actions of general anesthetics due to the increase in GABA release from presynaptic terminals.

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Onishi, Toshihiro, Maria Berg, Robert Reger, Leonard Miller, Steve Wolpe, Lynnet Koh, and Richard Childs. "Forced Fucosylation With ASC-101 Enhances The Binding Of Ex Vivo Expanded Human NK Cells To E-Selectin: A Novel Method To Improve The Homing Of Adoptively Transferred NK Cells To The Bone Marrow In Patients With Hematological Malignancies." Blood 122, no. 21 (November 15, 2013): 4499. http://dx.doi.org/10.1182/blood.v122.21.4499.4499.

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Introduction The ability of adoptively infused NK cells to home and traffic to the microenvironment where the tumor resides may be a critical determinant of their ability to mediate clinically meaningful anti-tumor effects. The initial step in leukocyte emigration from post-capillary venules, referred to as “tethering”, is a low-affinity interaction between leukocyte ligands with selectins expressed on endothelial cells. Since E-selectin is constitutively expressed on endothelium of skin and bone marrow in humans, leukocyte recruitment to bone marrow is thought to be largely dependent on E-selectin-binding. Among E-selectin ligands, only ligands bearing sialyl Lewis X with a terminal fucose (fucosylated) are functional forms that actively bind to E-selectin. One of the pitfalls of ex vivo NK cell expansion for adoptive infusion in humans is that expanded NK cells express predominantly non-fucosylated E-selectin ligands. We hypothesized that ex vivo fucosylation could enhance the binding capacity of E-selectin ligands on NK cells improving their homing into bone marrow where hematological malignancies reside. Methods CD56+/CD3- NK cells were isolated from normal human subjects by immuno-magnetic bead selection and were expanded ex vivo over 7-21 days by co-culturing with irradiated EBV-LCL feeder cells in IL-2 containing medium. Expanded NK cells were incubated for 30 minutes at room temperature with GDP-fucose and alpha1,3 fucosyltransferase-VI (ASC-101). The levels of fucosylation were determined by CLA surface expression measured by flow cytometry using the antibody HECA-452. After fucosylation, NK cells were analyzed by flow cytometry to assess for phenotype changes, viability and stability of fucosylation. Chromium release assays were performed to assess NK cell cytotoxicity against tumor cells. To determine whether fucosylated NK cells had enhanced binding to E-selectin, the binding capacity of NK cells to human recombinant E-selectin/Fc Chimera protein was evaluated by flow cytometry. Results Expanded human NK cells had low levels of baseline fucosylation, ranging from only 10-25%. Expanded NK cells were successfully fucosylated with ASC-101 in a dose-dependent manner (figure); the MFI of CLA on NK cells peaked at 25 ug/ml of ASC-101, with nearly 100% of NK cells being fucosylated (CLA positive). Fucosylation did not affect NK cell viability nor was it associated with changes in NK cell phenotype including surface expression of CD16, CD56, KIR2DL1, KIR2DL2/3, KIR3DL1, NKG2A, NKG2D, TRAIL, perforin, or granzymes A/B. Ex vivo cell culture showed fucosylation was sustained at nearly 100% for 48 hours, but then rapidly declined returning to baseline levels by 96 hours. NK cell cytotoxicity against tumor targets including K562 cells and myeloma cells was preserved and unaffected by fucosylation. Fucosylation significantly enhanced the binding capacity of NK cells to human E-selectin. Further, NK cell binding to recombinant human E-selectin/Fc chimera protein directly correlated with the degree of NK cell fucosylation (figure), which was dose-dependent on the ASC-101 concentration. The effects of forced fucosylation on the ability of human NK cells to home to the bone marrow following adoptive transfer into immuno-deficient mice is currently being explored. Conclusion Expanded NK cells primarily express non-glycosylated ligands for E-selectin, potentially limiting their ability to home to the bone marrow following adoptive transfer in humans with hematological malignancies. Ligands for E-selectin on the surface of expanded NK cells can be glycosylated ex vivo rapidly and to high degrees using ASC-101, significantly enhancing their ability to bind E-selectin. These data suggest forced fucosylation of NK cells could be used as a novel approach to improve the antitumor effects of adoptive NK cell infusions in patients with hematological malignancies. Disclosures: Miller: America Stem Cell Inc: Employment. Wolpe:American Stem Cell, Inc: Employment. Koh:America Stem Cell Inc: Employment.

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Mullins, Eric S., Kathryn E. Talmage, Matthew Flick, Keith W. Kombrinck, Richard T. Strait, and Jay L. Degen. "Transfusion Related Acute Lung Injury (TRALI) Is Ameliorated by Genetic Elimination of Fibrin(ogen) in Mice." Blood 114, no. 22 (November 20, 2009): 3189. http://dx.doi.org/10.1182/blood.v114.22.3189.3189.

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Abstract Abstract 3189 Poster Board III-126 Transfusion related acute lung injury (TRALI) is the leading cause of transfusion related deaths in the United States. The incidence of TRALI is estimated at 1 in 5000 transfusions and can occur with all blood products, however it is most common with transfused platelets. TRALI is associated with rapid onset of non-cardiogenic pulmonary edema, generally within a few hours of a transfusion. The resulting respiratory distress is often severe enough to require mechanical ventilation, up to 70% of patients in some case studies, and mortality rates associated with TRALI are high. While the etiology of TRALI is still not fully defined, donor anti-HLA class I and II antibodies have been reported in transfused blood products responsible for episodes of TRALI. An experimental system for inducing TRALI in mice has recently been described that depends on a single intravenous injection of a monoclonal antibody against MHC-I (mAB 34-1-2S against H2Kd). Here, experimental animals develop tachypnea, pulmonary edema, and capillary leak within minutes of the injection. Lungs, harvested from animals at two hours following challenge, were found to exhibit profuse fibrin deposition within the lungs which was prominent both in perivascular regions and along alveolar walls. Given the copious fibrin deposition associated with the challenge, we hypothesized that thrombin activation and intra- and extra-vascular fibrin deposition exacerbates disease pathologies associated with TRALI. To test this concept, we first challenged animals genetically altered to carry low levels of prothrombin (∼10% of normal) with this model of TRALI. Low prothrombin mice were consistently more tolerant than wildtype mice of the challenge with mAB 34-1-2S based both on overall appearance and activity after initial injection and low prothrombin mice rarely progressed to moribundity. Complementary studies of fibrinogen-deficient and -sufficient animals suggest that mice lacking coagulation function were also tolerant of the TRALI challenge, suggesting that fibrin deposition aggravates pathology. Lungs harvested from cohorts of control and fibrinogen-null mice at either two or four hours after antibody injection revealed significantly higher pulmonary edema in fibrinogen-sufficient relative to fibrinogen-null animals based on wet-to-dry ratios. To further elucidate the mechanisms coupling fibrin(ogen) to TRALI pathobiology, comparative studies were done of mice expressing a mutant form of fibrinogen (Fibγ390-396A) that retains full clotting function, but lacks the binding motif for the leukocyte integrin receptor, Mac-1. These mice also exhibited diminished pulmonary edema in comparison to controls, suggesting that fibrin-coupled inflammatory processes may drive lung disease. Taken together, these studies suggest that strategies to limit the hemostatic pathways or uncouple fibrin from secondary inflammatory events might be advantageous in the clinical management of TRALI. Disclosures No relevant conflicts of interest to declare.

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Byrd, Wyatt, and Frederick J. Cassels. "The encapsulation of enterotoxigenic Escherichia coli colonization factor CS3 in biodegradable microspheres enhances the murine antibody response following intranasal administration." Microbiology 152, no. 3 (March 1, 2006): 779–86. http://dx.doi.org/10.1099/mic.0.28667-0.

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The aim of this study was to measure serum and mucosal antibody responses following intranasal administration of biodegradable poly(dl-lactide-co-glycolide) (PLGA) microspheres loaded with the CS3 colonization factor isolated from enterotoxigenic Escherichia coli (ETEC). The response was compared against that measured in mice similarly administered the native CS3 antigen and in mice co-administered, along with the CS3 antigen, a known mucosal adjuvant, the R192G mutant heat-labile enterotoxin (mLT). The integrity of the CS3 antigen released from the microspheres was maintained as determined by SDS-PAGE and immunoblotting. Native CS3 induced serum and mucosal (bronchoalveolar, small intestinal and faecal) IgG and IgA responses. The co-administration of the mLT mucosal adjuvant significantly enhanced (P<0·001) serum and mucosal antibody responses to the CS3 protein. Likewise, the CS3-loaded PLGA microspheres induced significantly greater (P<0·001) serum and mucosal antibody responses than native CS3, as well as inducing antibody responses superior to those of the CS3 plus mLT formulation. Following administration of CS3 plus mLT, the mice became distressed (loss of activity, increased huddling, ruffled fur), a situation not seen following administration of the CS3-loaded PLGA microspheres. The results in this trial show that the CS3-loaded PLGA microspheres when administered intranasally to mice caused no observable distress to the mice and significantly (P<0·001) enhanced the immunogenicity of the CS3 protein.

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Journal articles on the topic 'Mice trail following'

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