scholarly journals 3352 Surgical Adjuvant of Immunomodulatory Gene Circuits for Treatment of Glioblastoma

2019 ◽  
Vol 3 (s1) ◽  
pp. 155-155
Author(s):  
Jordan Matthew Spatz ◽  
Ming Ru Wu ◽  
Karen Weisinger ◽  
Tim Lu ◽  
Manish Aghi
Keyword(s):  
T Cell ◽  
Fluorescent Protein ◽  
Brain Cells ◽  
Tumor Control ◽  
Normal Brain ◽  
Immune Microenvironment ◽  
Gene Circuits ◽  
Immunomodulatory Gene

OBJECTIVES/SPECIFIC AIMS: Glioblastoma (GBM) is a brain cancer with a devastatingly short overall survival of under two years. The poor prognosis of GBM is largely due to cell invasion and maintenance of cancer initiating cells that evade the brain’s innate and adaptive immune responses which enables escape from surgical resection and drives inevitable recurrence. While targeting the brain’s immune microenvironment has long been proposed as a strategy for treating GBM, translational progress has been slow, underscoring the need to investigate the brain’s immune microenvironment for therapeutic avenues. METHODS/STUDY POPULATION: Recent advancements in tunable synthetic immunomodulatory gene circuits targeting metastatic cancers has demonstrated the novel ability to use engineering principles to induce infiltrative cancer cells to express combinatorial immunomodulatory outputs that enable T-cell killing4. Our central hypothesis is: we will be able to significantly improve survival with a lasting immune-mediated control of GBM by using synthetic immunomodulatory gene circuits driving GBM cells to express a local combination of immunomodulatory proteins: human IL15, a surface T-cell engager, PD-L1-CD3 bispecific antibody, and the protein, LIGHT (TNFRSF14). Importantly, the co-expression of LIGHT and anti-PD-L1 therapies was recently shown to rescue PD-L1 checkpoint blockage in the preclinical models of brain tumors and significant enhance survival outcomes highlighting the benefits of novel combinations of immunomodulatory proteins for treatment of GBM. To identify genes whose expression is dramatically upregulated in GBM compared to normal human brain cells, a pooled of six thousand lentiviral oncogene promoters that drive expression of a red-fluorescent protein has been infected into three human GBM cell lines. RESULTS/ANTICIPATED RESULTS: We have successfully infected our GBM cells and are preparing samples for next generation DNA sequencing to determine highly active promoters in GBM that are not expressed in multiple normal brain cells types, astrocytes and neurons. These chosen promoters will then be used to drive an AND gate logic gene circuit immunotherapy outputs which is currently under development for both in-vitro and in-vivo experiments. DISCUSSION/SIGNIFICANCE OF IMPACT: We anticipate that local expression of multiple immune effectors proteins will significantly enhance tumor control and survival in both synergistic murine and human-murine xenograft pre-clinical models of GBM. Ultimately, our goal is to rapidly translate this technology advance into the clinical trial for adult GBM patients.

scholarly journals The GM-CSF–IRF5 signaling axis in eosinophils promotes antitumor immunity through activation of type 1 T cell responses

2020 ◽  
Vol 217 (12) ◽  
Author(s):  
Isabelle C. Arnold ◽  
Mariela Artola-Boran ◽  
Alessandra Gurtner ◽  
Katrin Bertram ◽  
Michael Bauer ◽  
...  
Keyword(s):  
T Cell ◽  
Antitumor Immunity ◽  
Critical Role ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Tumor Control ◽  
Gm Csf ◽  
Cell Responses

The depletion of eosinophils represents an efficient strategy to alleviate allergic asthma, but the consequences of prolonged eosinophil deficiency for human health remain poorly understood. We show here that the ablation of eosinophils severely compromises antitumor immunity in syngeneic and genetic models of colorectal cancer (CRC), which can be attributed to defective Th1 and CD8+ T cell responses. The specific loss of GM-CSF signaling or IRF5 expression in the eosinophil compartment phenocopies the loss of the entire lineage. GM-CSF activates IRF5 in vitro and in vivo and can be administered recombinantly to improve tumor immunity. IL-10 counterregulates IRF5 activation by GM-CSF. CRC patients whose tumors are infiltrated by large numbers of eosinophils also exhibit robust CD8 T cell infiltrates and have a better prognosis than patients with eosinophillow tumors. The combined results demonstrate a critical role of eosinophils in tumor control in CRC and introduce the GM-CSF–IRF5 axis as a critical driver of the antitumor activities of this versatile cell type.

scholarly journals The Cysteine-Containing Cell-Penetrating Peptide AP Enables Efficient Macromolecule Delivery to T Cells and Controls Autoimmune Encephalomyelitis

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1134
Author(s):  
Won-Ju Kim ◽  
Gil-Ran Kim ◽  
Hyun-Jung Cho ◽  
Je-Min Choi
Keyword(s):  
Drug Delivery ◽  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Autoimmune Encephalomyelitis ◽  
Optimal Sequence ◽  
T Cell Function

T cells are key immune cells involved in the pathogenesis of several diseases, rendering them important therapeutic targets. Although drug delivery to T cells is the subject of continuous research, it remains challenging to deliver drugs to primary T cells. Here, we used a peptide-based drug delivery system, AP, which was previously developed as a transdermal delivery peptide, to modulate T cell function. We first identified that AP-conjugated enhanced green fluorescent protein (EGFP) was efficiently delivered to non-phagocytic human T cells. We also confirmed that a nine-amino acid sequence with one cysteine residue was the optimal sequence for protein delivery to T cells. Next, we identified the biodistribution of AP-dTomato protein in vivo after systemic administration, and transduced it to various tissues, such as the spleen, liver, intestines, and even to the brain across the blood–brain barrier. Next, to confirm AP-based T cell regulation, we synthesized the AP-conjugated cytoplasmic domain of CTLA-4, AP-ctCTLA-4 peptide. AP-ctCTLA-4 reduced IL-17A expression under Th17 differentiation conditions in vitro and ameliorated experimental autoimmune encephalomyelitis, with decreased numbers of pathogenic IL-17A+GM-CSF+ CD4 T cells. These results collectively suggest the AP peptide can be used for the successful intracellular regulation of T cell function, especially in the CNS.

scholarly journals Immune Escape of Tumors in Vivo by Expression of Cellular Flice-Inhibitory Protein

1999 ◽  
Vol 190 (7) ◽  
pp. 1033-1038 ◽  
Author(s):  
Jan Paul Medema ◽  
Joan de Jong ◽  
Thorbald van Hall ◽  
Cornelis J.M. Melief ◽  
Rienk Offringa
Keyword(s):  
T Cell ◽  
Immune Escape ◽  
Cytotoxic T Cell ◽  
Tumor Control ◽  
Tumor Escape ◽  
Inhibitory Protein ◽  
Cell Immunity ◽  
Induced Apoptosis

The antiapoptotic protein cellular FLICE (Fas-associated death domain–like IL-1β–converting enzyme) inhibitory protein (cFLIP) protects cells from CD95(APO-1/Fas)-induced apoptosis in vitro and was found to be overexpressed in human melanomas. However, cytotoxic T cell–induced apoptosis, which is critically involved in tumor control in vivo, is not inhibited by cFLIP in vitro, as only CD95- and not perforin-dependent lysis is affected. This calls into question whether cFLIP is sufficient to allow escape from T cell–dependent immunity. Using two murine tumors, we directly demonstrate that cFLIP does result in escape from T cell immunity in vivo. Moreover, tumor cells are selected in vivo for elevated cFLIP expression. Therefore, our data indicate that CD95-dependent apoptosis constitutes a more prominent mechanism for tumor clearance than has so far been anticipated and that blockade of this pathway can result in tumor escape even when the perforin pathway is operational.

scholarly journals P09.03 Cathepsin S alterations induce a tumor-promoting immune microenvironment in follicular lymphoma

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A53.1-A53
Author(s):  
JA Hildebrand ◽  
D Bararia ◽  
S Stolz ◽  
S Häbe ◽  
F Osorio-Barrios ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cathepsin S ◽  
Immune Microenvironment ◽  
Cleavage Activity ◽  
Autocatalytic Cleavage ◽  
Substrate Cleavage

BackgroundBy targeted DNA sequencing of 305 diagnostic follicular lymphoma (FL) biopsies, we identified somatic mutations of Cathepsin S (CTSS) in 8% of cases (24/305), mostly clustered at Y132 (19/24) converting Y to D (16/19). Another 13% of FL had CTSS amplifications (37/286), associated with higher CTSS expression (P=0.05). CTSS is a cysteine protease that is highly expressed in endolysosomes of antigen presenting cells and malignant B-cells. CTSS is involved in proteolytical processing of antigenic peptides for presentation on MHC-II to be recognized by antigen specific CD4+ T-cells.1 CTSS is synthesized as an inactive zymogen, which is converted to its active form by autocatalytic cleavage of the autoinhibitory propeptide (pro-CTSS).Materials and MethodsWe used CRISPR/Cas9 to introduce CTSS Y132D into Karpas422, a B-cell lymphoma cell line that harbors the FL hallmark translocation t(14;18). We purified pro-CTSS WT and Y132D and assayed the in vitro autocatalytic cleavage over time. We then tested the impact of CTSS on CD4+ T-cell activation in co-culture assays, in a previously described in vivo model2 which we slightly modified to reflect FL-like conditions, and in primary patient samples.ResultsSingle-cell derived Y132D mutant Karpas422 clones showed >3-fold higher ratios of active CTSS to pro-CTSS (N=4, P=0.0003). Immunoprecipitated CTSS Y132D had >3-fold higher in vitro substrate cleavage activity compared to CTSS wild type (WT) (N=6, P=0.001) which was mediated by an accelerated conversion from pro-CTSS to active CTSS (11 minutes for CTSS Y132D vs 17 minutes for CTSS WT; N=3, P=0.04). Molecular dynamics simulations showed that the Y132D mutation shortens the distances by ~2Å between the catalytic triad of active CTSS (C139, H278, N298) and a stretch of amino acids from the proform (L80, G81, D82, S94), which could facilitate intramolecular cleavage. The higher substrate cleavage activity of CTSS Y132D came along with a high capacity to stimulate antigen specific CD4+ T cell responses in vitro and in vivo. Additionally, CTSS overexpression could phenocopy this high CD4+ T cell activation. Lastly, we aimed to correlate CTSS aberrations with clinical outcome in patients who received standard immunochemotherapy (R-CHOP) for advanced FL (N=51 with available CTSS mutation and gene expression data). Compared to all other patients (N=34), patients with CTSS Y132 mutations or CTSS overexpression (N=17) had longer failure free survival (P=0.012).ConclusionsHere, we provide biochemical, structural, functional and clinical evidence that aberrant CTSS activity induces a supportive immune microenvironment in FL. We propose that aberrant CTSS activity can elicit a CD4+ T-cell driven tumor-promoting immune response, which could be amplified within the microenvironment and substantially impact the biology and clinical course of the disease. Thus, aberrant CTSS activity is a promising biomarker and therapeutic target in FL and potentially also other tumors.ReferencesRiese, R.J., et al., Essential role for cathepsin S in MHC class II-associated invariant chain processing and peptide loading. Immunity 1996; 4(4): p. 357–66.Kim, K.J., et al., Establishment and characterization of BALB/c lymphoma lines with B cell properties. J Immunol 1979; 122(2): p. 549–54.Disclosure InformationJ.A. Hildebrand: None. D. Bararia: None. S. Stolz: None. S. Häbe: None. F. Osorio-Barrios: None. M.D. Bartoschek: None. E. Gaitzsch: None. V. Jurinovic: None. K. Rautter: None. C. Ludwig: None. S. Bultmann: None. H. Leonhardt: None. S. Eustermann: None. K. Hopfner: None. W. Hiddemann: None. M. Bergwelt: None. M. Schmidt-Supprian: None. M.B. Sárosi: None. M. Rudelius: None. V. Passerini: None. J. Mautner: None. O. Weigert: None.

Evaluation of Poly (Glycerol-Adipate) Nanoparticle Uptake in an In Vitro 3-D Brain Tumor Co-Culture Model

2007 ◽  
Vol 232 (8) ◽  
pp. 1100-1108 ◽  
Author(s):  
W. Meng ◽  
P. Kallinteri ◽  
D. A. Walker ◽  
T. L. Parker ◽  
M. C. Garnett
Keyword(s):  
Drug Delivery ◽  
Brain Tumor ◽  
Single Cells ◽  
Brain Slices ◽  
Brain Cells ◽  
Normal Brain ◽  
New Model ◽  
Culture Model

Despite the inherent problems associated with in vivo animal models of tumor growth and metastases, many of the current in vitro brain tumor models also do not accurately mimic tumor-host brain interactions. Therefore, there is a need to develop such co-culture models to study tumor biology and, importantly, the efficacy of drug delivery systems targeting the brain. So far, few investigations of this nature have been published. In this paper we describe the development of a new model system and its application to drug delivery assessment. For our new model, a co-culture of DAOY cell brain tumor aggregates and organo-typic brain slices was developed. Initially, the DAOY aggregates attached to cerebellum slices and invaded as a unit. Single cells in the periphery of the aggregate detached from the DAOY aggregates and gradually replaced normal brain cells. This invasive behavior of DAOY cells toward organotypic cerebellum slices shows a similar pattern to that seen in vivo. After validation of the co-culture model using transmission electron microscopy, nanoparticle (NP) uptake was then evaluated. Confocal micrographs illustrated that DAOY cells in this co-culture model took up most of the NPs, but few NPs were distributed into brain cells. This finding corresponded with results of NP uptake in DAOY and brain aggregates reported elsewhere.

scholarly journals Engineered red blood cells as an off-the-shelf allogeneic anti-tumor therapeutic

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xuqing Zhang ◽  
Mengyao Luo ◽  
Shamael R. Dastagir ◽  
Mellissa Nixon ◽  
Annie Khamhoung ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Checkpoint Inhibitors ◽  
Critical Role ◽  
Cellular Immunotherapy ◽  
Tumor Control

AbstractCheckpoint inhibitors and T-cell therapies have highlighted the critical role of T cells in anti-cancer immunity. However, limitations associated with these treatments drive the need for alternative approaches. Here, we engineer red blood cells into artificial antigen-presenting cells (aAPCs) presenting a peptide bound to the major histocompatibility complex I, the costimulatory ligand 4-1BBL, and interleukin (IL)-12. This leads to robust, antigen-specific T-cell expansion, memory formation, additional immune activation, tumor control, and antigen spreading in tumor models in vivo. The presence of 4-1BBL and IL-12 induces minimal toxicities due to restriction to the vasculature and spleen. The allogeneic aAPC, RTX-321, comprised of human leukocyte antigen-A*02:01 presenting the human papilloma virus (HPV) peptide HPV16 E711-19, 4-1BBL, and IL-12 on the surface, activates HPV-specific T cells and promotes effector function in vitro. Thus, RTX-321 is a potential ‘off-the-shelf’ in vivo cellular immunotherapy for treating HPV + cancers, including cervical and head/neck cancers.

scholarly journals GD2 or HER2 targeting T cell engaging bispecific antibodies to treat osteosarcoma

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Jeong A. Park ◽  
Nai-Kong V. Cheung
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitors ◽  
Checkpoint Inhibitors ◽  
Tumor Control ◽  
Osteosarcoma Cell

Abstract Background The cure rate for metastatic osteosarcoma has not substantially improved over the past decades. Clinical trials of anti-HER2 trastuzumab or anti-GD2 dinutuximab for metastatic or refractory osteosarcoma were not successful, and neither was immune checkpoint inhibitors (ICIs). Methods We tested various target antigen expressions on osteosarcoma cell lines using flow cytometry and analyzed in vitro T cell engaging BsAb (T-BsAb)-dependent T cell-mediated cytotoxicity using 4-h 51Cr release assay. We tested in vivo anti-tumor activities of T-BsAb targeting GD2 or HER2 in established osteosarcoma cell line or patient-derived xenograft (PDX) mouse models carried out in BALB-Rag2−/−IL-2R-γc-KO (BRG) mice. We also generated ex vivo BsAb-armed T cells (EATs) and studied their tumor-suppressive effect against osteosarcoma xenografts. In order to improve the anti-tumor response, ICIs, anti-human PD-1 (pembrolizumab) or anti-human PD-L1 (atezolizumab) antibodies were tested their synergy with GD2- or HER2-BsAb against osteosarcoma. Results GD2 and HER2 were chosen from a panel of surface markers on osteosarcoma cell lines and PDXs. Anti-GD2 BsAb or anti-HER2 BsAb exerted potent anti-tumor effect against osteosarcoma tumors in vitro and in vivo. T cells armed with anti-GD2-BsAb (GD2-EATs) or anti-HER2-BsAb (HER2-EATs) showed significant anti-tumor activities as well. Anti-PD-L1 combination treatment enhanced BsAb-armed T cell function in vivo and improved tumor control and survival of the mice, when given sequentially and continuously. Conclusion Anti-GD2 and anti-HER2 BsAbs were effective in controlling osteosarcoma. These data support the clinical investigation of GD2 and HER2 targeted T-BsAb treatment in combination with immune checkpoint inhibitors, particularly anti-PD-L1, in patients with osteosarcoma to improve their treatment outcome.

scholarly journals VEGFR-2 redirected CAR-T cells are functionally impaired by soluble VEGF-A competition for receptor binding

2021 ◽  
Vol 9 (8) ◽  
pp. e002151
Author(s):  
Evripidis Lanitis ◽  
Paris Kosti ◽  
Catherine Ronet ◽  
Elisabetta Cribioli ◽  
Giorgia Rota ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Vasculature ◽  
Tumor Control ◽  
Car T Cells ◽  
Car T Cell ◽  
Anti Vegf ◽  
Car T

BackgroundThe adoptive transfer of chimeric antigen receptor (CAR)-T cells has emerged as a potent immunotherapy against some hematological malignancies but not yet for epithelial-derived solid tumors. One critical issue is the paucity of broadly expressed solid tumor antigens (TAs), and another is the presence of suppressive mechanisms in the tumor microenvironment (TME) that can impair CAR-T cell homing, extravasation and effector functions. TAs expressed by endothelial cells of the tumor vasculature are of clinical interest for CAR therapy because of their genomic stability and accessibility to circulating T cells, as well as their expression across multiple tumor types. In this study, we sought to explore limitations to the efficacy of second-generation (2G) murine CAR-T cells redirected against the vascular endothelial growth factor receptor-2 (VEGFR-2) with the well-characterized single-chain variable fragment DC101.MethodsPrimary murine T cells were retrovirally transduced to express a 2G anti-VEGFR-2-CAR, and the in vitro binding to VEGFR-2, as well as reactivity against TA-expressing cells, was evaluated in the absence versus presence of exogenous VEGF-A. The CAR-T cells were further tested in vivo for tumor control alone and in combination with anti-VEGF-A antibody. Finally, we performed ex vivo phenotypic analyses of tumor-infiltrating CAR-T cells for the two treatment groups.ResultsIn line with previous reports, we observed poor control of B16 melanoma by the 2G anti-VEGFR-2 CAR-T cells as a monotherapy. We further showed that VEGFR-2 is not downregulated by B16 melanoma tumors post treatment, but that its soluble ligand VEGF-A is upregulated and furthermore competes in vitro with the CAR-T cells for binding to VEGFR-2. This competition resulted in impaired CAR-T cell adhesion and effector function in vitro that could be restored in the presence of anti-VEGF-A antibody. Finally, we demonstrated that coadministration of anti-VEGF-A antibody in vivo promoted CAR-T cell persistence and tumor control and was associated with reduced frequencies of PD-1+ Ki67- and LAG-3+ Ki67- CAR-T cells in the TME.ConclusionsThis study represents the first example of impaired function of a vasculature-targeted CAR by an angiogenic ligand and rationalizes the use of combinatorial therapies that target the tumor vasculature and augment CAR-T cell effector function.

Inhibition of T Cell Alloreactivity by Gr-1+Mac-1+ Myeloid Suppressor Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5177-5177
Author(s):  
Xiao Chen ◽  
Jon Anderson ◽  
William R. Drobyski
Keyword(s):  
T Cell ◽  
Adoptive Transfer ◽  
Fluorescent Protein ◽  
Suppressor Cells ◽  
Major Complication ◽  
Spleen Cells ◽  
Regulatory Cell ◽  
Myeloid Suppressor Cells

Abstract Graft-versus-host-disease (GVHD) remains the major complication of allogeneic stem cell transplantation (SCT). While pharmacological approaches have long been the mainstay of GVHD prevention, recent strategies have focused on cellular approaches, specifically the use of regulatory cell populations to prevent or mitigate the severity of GVHD. Gr-1+Mac-1+ myeloid-derived cells, termed myeloid suppressor cells (MSCs), are a regulatory cell population that has been shown to play an important role in the suppression of tumor and anti-infectious immunity. In these studies, we examined whether these cells were detectable in GVHD recipients and played any role in the suppression of alloreactive T cell responses in vitro and in vivo. Lethally irradiated Balb/c (H-2d) mice were transplanted with MHC-incompatible C57BL/6 (H-2b) bone marrow and spleen cells to induce GVHD. Immature myeloid cells with a Gr-1+Mac-1+ phenotype were found to constitute 60% of all splenocytes at day 10 post-SCT thereafter declining to 30% by day 21. MSCs isolated from the spleens of mice with GVHD expressed high levels of class I and II, but low levels of the costimulatory molecules CD80, CD86, CD40 and were negative for CD11c. Highly purified Gr-1+ Mac-1+ cells obtained from the spleens of GVHD mice 10 or 21 days post-SCT potently suppressed naive T cell proliferation in a standard MLC by 70% at a responder to suppressor ratio of 1:1. Notably, MSCs could also be generated in vitro from normal B6 BM after 6 days in culture with G-CSF and were equally potent at suppressing T cell alloreactivity. Suppression was partially mediated by nitric oxide (NO) as addition of the NO inhibitor L-NMA reversed 50% of the inhibitory effect. To determine whether MSCs exerted a suppressive effect in vivo, lethally irradiated Balb mice were transplanted with B6 BM plus naïve spleen cells with or without MSCs obtained from the spleens of GVHD animals 10 or 21 days post-BMT. The adoptive transfer of MSCs failed to protect mice from GVHD when assessed by overall survival, serial weight measurements or histological analysis. As an alternative approach to augment GVHD protection, G-CSF was administered for 14 days beginning at the time of BMT to enhance the in vivo survival of MSCs. G-CSF administration in both irradiated and non-irradiated MHC-incompatible GVHD mouse models, however, also had no protective effect. We reasoned that a possible explanation for the lack of an effect in vivo was that MSCs did not appropriately localize to nodal sites where GVHD is initiated. To examine this question, B6-green fluorescent protein (GFP) mice were used as donor animals to permit the detection and trafficking of MSCs in vivo. Adoptive transfer of B6 GFP-MSCs from the spleens of GVHD mice 10 days after SCT demonstrated that these cells could be detected in the secondary lymphoid organs of recipient mice 20 hours after BMT, but were completely absent by day 3, supporting the premise that the lack of protection was attributable, at least in part, to the inability of these cells to persist at nodal sites where T cell priming against host alloantigens was occurring. These results indicate that MSCs, which are present in the spleen of GVHD recipients, show potent T cell suppressive capacity in vitro, but fail to mediate a similar effect in vivo. Lack of protection appears to be due to the inability of these cells to localize to all critical secondary lymphoid sites underscoring the importance that the capability of regulatory cells to migrate to and persist at appropriate tissue sites plays in the design of cellular strategies to prevent GVHD.

scholarly journals Construction and Characterization of a Full-Length Infectious Simian T-Cell Lymphotropic Virus Type 3 Molecular Clone

2007 ◽  
Vol 81 (12) ◽  
pp. 6276-6285 ◽  
Author(s):  
Sébastien Alain Chevalier ◽  
Marine Walic ◽  
Sara Calattini ◽  
Adeline Mallet ◽  
Marie-Christine Prévost ◽  
...  
Keyword(s):  
T Cell ◽  
Virus Type ◽  
Fluorescent Protein ◽  
Experimental Studies ◽  
Full Length ◽  
Cell Culture Supernatant ◽  
Molecular Clone ◽  
Human T Cell

ABSTRACT Together with their simian T-cell lymphotropic virus (STLV) equivalent, human T-cell lymphotropic virus type 1 (HTLV-1), HTLV-2, and HTLV-3 form the primate T-cell lymphotropic virus (PTLV) group. Over the years, understanding the biology and pathogenesis of HTLV-1 and HTLV-2 has been widely improved by the creation of molecular clones. In contrast, so far, PTLV-3 experimental studies have been restricted to the overexpression of the tax gene using reporter assays. We have therefore decided to construct an STLV-3 molecular clone. We generated a full-length STLV-3 proviral clone (8,891 bp) by PCR amplification of overlapping fragments. This STLV-3 molecular clone was then transfected into 293T cells. Reverse transcriptase PCR experiments followed by sequence analysis of the amplified products allowed us to establish that both gag and tax/rex mRNAs were transcribed. Western blotting further demonstrated the presence of the STLV-3 p24 gag protein in the cell culture supernatant from transfected cells. Transient transfection of 293T cells and of 293T-long terminal repeat-green fluorescent protein cells with the STLV-3 clone promoted syncytium formation, a hallmark of PTLV Env expression, as well as the appearance of fluorescent cells, also demonstrating that the Tax3 protein was expressed. Virus particles were visible by electron microscopy. These particles are infectious, as demonstrated by our cell-free-infection experiments with purified virions. All together, our data demonstrate that the STLV-3 molecular clone is functional and infectious. This clone will give us a unique opportunity to study in vitro the different pX transcripts and the putative presence of antisense transcripts and to evaluate the PTLV-3 pathogenicity in vivo.

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