scholarly journals Retrovirus-mediated Gene Transfer into Rat Salivary Gland Cells In Vitro and In Vivo

1997 ◽  
Vol 45 (11) ◽  
pp. 1533-1545 ◽  
Author(s):  
Tibor Barka ◽  
Hendrika M. van der Noen
Keyword(s):  
Enzyme Activity ◽  
Salivary Gland ◽  
Gene Transfer ◽  
Cell Line ◽  
Submandibular Gland ◽  
Acinar Cells ◽  
Retroviral Vector ◽  
Placental Alkaline Phosphatase

A retroviral vector DAP that encodes the human placental alkaline phosphatase (PLAP) and the neomycin-resistant gene was used to transduce the salivary gland-derived cell line A5 in vitro and acinar cells in rat submandibular gland in vivo. Expression of the transduced PLAP gene was established by histochemical staining for heat-resistant AP and by determination of enzyme activity. From the in vitro experiments, we concluded that the salivary gland-derived cell line A5 can be infected by the retroviral vector DAP. In the transduced cells the viral long terminal repeat (LTR) promoter was effective, and the cells expressed heat-stable PLAP which was localized mostly in the plasma membrane and could be released by treatment with bromelain or phosphatidyinositol-specific phospholipase C. A5-DAP cells secreted PLAP into the medium. Clones of A5-DAP cells expressed various levels of the enzyme. The level of enzyme activity in different clones was unrelated to growth rate. Retrograde ductal injection of the viral vector into the duct of the submandibular gland of rats resulted in integration and long-term expression of PLAP gene in acinar cells. Expression of PLAP was seen up to 25 days, the limit of the observation period. To facilitate integration of the viral DNA, cell division of acinar cells was induced by administration of the β-adrenergic agonist isoproterenol before administration of the virus. PLAP was secreted into submandibular saliva. The data support the notion that salivary glands are suitable targets for gene transfer in vivo by a retroviral vector. (J Histochem Cytochem 45:1533–1545, 1997)

scholarly journals Transfer of the ADA gene into human ADA-deficient T lymphocytes reconstitutes specific immune functions

Blood ◽  
1992 ◽  
Vol 80 (5) ◽  
pp. 1120-1124 ◽  
Author(s):  
G Ferrari ◽  
S Rossini ◽  
N Nobili ◽  
D Maggioni ◽  
A Garofalo ◽  
...  
Keyword(s):  
T Cells ◽  
Enzyme Activity ◽  
Gene Transfer ◽  
Peripheral Blood ◽  
Retroviral Vector ◽  
Peripheral Blood Lymphocytes ◽  
Proliferative Potential ◽  
Immune Functions ◽  
Beta Gene

Abstract Peripheral blood lymphocytes obtained from a patient affected by adenosine deaminase (ADA) deficiency and severe combined immunodeficiency were infected with a retroviral vector containing two copies of a human ADA minigene, and injected into bg/nu/xid (BNX) immunodeficient mice. Six to 10 weeks after injection, human T cells were cloned from the spleens of recipient animals and analyzed for proliferative potential, T-cell surface markers, expression of ADA activity, integration of retroviral sequences, T-cell receptor (TCR) beta gene rearrangement, and specificity of antigen recognition. Efficient gene transfer and expression restored proliferative potential in vitro and long-term survival in vivo. All clonable human T lymphocytes obtained from the spleen of recipient animals had high levels of vector-derived ADA enzyme activity and showed predominantly the CD4+ phenotype. Retroviral integrations and TCR-beta gene rearrangements demonstrated the presence of a variety of different clones in the spleens of recipient mice. Furthermore, the combined analyses of vector integration and TCR rearrangement provided evidence that a circulating progenitor cell was transduced by the retroviral vector, giving rise to different and functional TCRs. Evaluation of antigen-specificity demonstrated both alloreactive and foreign antigen specific immune responses. These results suggest that restoration of enzyme activity in human ADA-deficient peripheral blood T cells by retroviral-mediated ADA gene transfer allows in vivo survival and reconstitution of specific immune functions. Therefore, retroviral vector-mediated gene transfer into circulating mononuclear cells could be successful not only in maintaining the metabolic homeostasis, but also for the development of a functional immune repertoire. This is a fundamental prerequisite for the usage of genetically engineered peripheral blood lymphocytes for somatic cell gene therapy of ADA deficiency.

Neonatal or hepatocyte growth factor–potentiated adult gene therapy with a retroviral vector results in therapeutic levels of canine factor IX for hemophilia B

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 3924-3932 ◽  
Author(s):  
Lingfei Xu ◽  
Cuihua Gao ◽  
Mark S. Sands ◽  
Shi-Rong Cai ◽  
Timothy C. Nichols ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Growth Factor ◽  
Gene Transfer ◽  
Hepatocyte Growth Factor ◽  
Retroviral Vector ◽  
Factor Ix ◽  
Hemophilia B ◽  
Hepatocyte Growth

AbstractHemophilia B is a bleeding disorder resulting from factor IX (FIX) deficiency that might be treated with gene therapy. Neonatal delivery would correct the disease sooner than would transfer into adults, and could reduce immunological responses. Neonatal mice were injected intravenously with a Moloney murine leukemia virus–based retroviral vector (RV) expressing canine FIX (cFIX). They achieved 150% to 280% of normal cFIX antigen levels in plasma (100% is 5 μg/mL), which was functional in vitro and in vivo. Three newborn hemophilia B dogs that were injected intravenously with RV achieved 12% to 36% of normal cFIX antigen levels, which improved coagulation tests. Only one mild bleed has occurred during 14 total months of evaluation. This is the first demonstration of prolonged expression after neonatal gene therapy for hemophilia B in mice or dogs. Most animals failed to make antibodies to cFIX, demonstrating that neonatal gene transfer may induce tolerance. Although hepatocytes from newborns replicate, those from adults do not. Adult mice therefore received hepatocyte growth factor to induce hepatocyte replication prior to intravenous injection of RV. This resulted in expression of 35% of normal cFIX antigen levels for 11 months, although all mice produced anti-cFIX antibodies. This is the first demonstration that high levels of FIX activity can be achieved with an RV in adults without a partial hepatectomy to induce hepatocyte replication. We conclude that RV-mediated hepatic gene therapy is effective for treating hemophilia B in mice and dogs, although the immune system may complicate gene transfer in adults.

scholarly journals An In Vitro Barrier Model of the Human Submandibular Salivary Gland Epithelium Based on a Single Cell Clone of Cell Line HTB-41: Establishment and Application for Biomarker Transport Studies

Biomedicines ◽  
2020 ◽  
Vol 8 (9) ◽  
pp. 302
Author(s):  
Grace C. Lin ◽  
Merima Smajlhodzic ◽  
Anna-Maria Bandian ◽  
Heinz-Peter Friedl ◽  
Tamara Leitgeb ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Cell Line ◽  
Submandibular Gland ◽  
Immunofluorescent Staining ◽  
Epithelial Barrier ◽  
Submandibular Salivary Gland ◽  
Iron Storage ◽  
Model Based ◽  
Transport Studies

The blood–saliva barrier (BSB) consists of the sum of the epithelial cell layers of the oral mucosa and salivary glands. In vitro models of the BSB are inevitable to investigate and understand the transport of salivary biomarkers from blood to saliva. Up to now, standardized, cell line-based models of the epithelium of the submandibular salivary gland are still missing for this purpose. Therefore, we established epithelial barrier models of the submandibular gland derived from human cell line HTB-41 (A-253). Single clone isolation resulted in five different clones (B2, B4, B9, D3, and F11). Clones were compared to the parental cell line HTB-41 using measurements of the transepithelial electrical resistance (TEER), paracellular marker permeability assays and analysis of marker expression for acinar, ductal, and myoepithelial cells. Two clones (B9, D3) were characterized to be of acinar origin, one clone (F11) to be of myoepithelial origin and one isolation (B4) derived from two cells, to be presumably a mixture of acinar and ductal origin. Clone B2, presumably of ductal origin, showed a significantly higher paracellular barrier compared to other clones and parental HTB-41. The distinct molecular identity of clone B2 was confirmed by immunofluorescent staining, qPCR, and flow cytometry. Experiments with ferritin, a biomarker for iron storage, demonstrated the applicability of the selected model based on clone B2 for transport studies. In conclusion, five different clones originating from the submandibular gland cell line HTB-41 were successfully characterized and established as epithelial barrier models. Studies with the model based on the tightest clone B2 confirmed its suitability for transport studies in biomarker research.

Role of laminin-1 and TGF-beta 3 in acinar differentiation of a human submandibular gland cell line (HSG)

1996 ◽  
Vol 109 (8) ◽  
pp. 2013-2021 ◽  
Author(s):  
M.P. Hoffman ◽  
M.C. Kibbey ◽  
J.J. Letterio ◽  
H.K. Kleinman
Keyword(s):  
Salivary Gland ◽  
Cell Line ◽  
Submandibular Gland ◽  
Neutralizing Antibodies ◽  
Gland Cell ◽  
Specific Protein ◽  
Model System ◽  
Tgf Beta ◽  
Laminin 1

Previous studies show that culturing an immortalized human submandibular gland cell line (HSG) on Matrigel, a basement membrane extract, induces cytodifferentiation. We have further defined this model system and identified factors involved in HSG cell acinar development and cyto-differentiation. Acinar development is marked by cell migration into multi-cellular spherical structures, cell proliferation and apoptosis of the centrally localized cells. In addition, functional differentiation was determined by indirect immunofluorescence and immunoblot analysis for cystatin, a salivary gland acinar cell-specific protein found to be produced by differentiated HSG cells. Matrigel contains multiple extracellular matrix proteins, however, laminin-1 was identified as the major matrix component that induced HSG cell acinar development and cytodifferentiation. Antibodies against specific components of Matrigel and against cell surface adhesion molecules were added to cells in culture to identify components important for HSG cell acinar differentiation. Immunostaining of HSG cell acini identified TGF-beta 2 and beta 3 as the predominant isoforms within the cells. Neutralizing antibodies directed against TGF-beta 3 significantly decreased (P < or = 0.0002) the size of acini formed. These results indicate that multiple components, including laminin-1 and TGF-beta 3, contribute to HSG cell acinar development. This model system will be useful to study acinar differentiation and salivary gland-specific protein expression in vitro.

Lateral Gene Transfer Contributing to Leukemogenesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1235-1235
Author(s):  
Joo Hyun Lee ◽  
Cynthia R. Giver ◽  
Sravanti Rangaraju ◽  
Edmund K Waller
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Tumor Cell ◽  
Cell Line ◽  
Tumor Cell Line ◽  
Hematopoietic Stem ◽  
Precursor Frequency ◽  
Marrow Cells

Abstract The uncontrolled proliferation of genetically mutated cells is the commonly understood mechanism for cancer growth and invasion, with accumulation of new mutations in daughter cells leading to clonal diversity of cancer derived from a single founding event. The genetic alterations are passed to new generations by cell division and vertical gene transfer. Viral transmission of oncogenes represents a known mechanism of lateral gene transfer in cancer initiation. Some experimental systems have also suggested that circulating DNA or micro-vesicles may contribute to lateral oncogene transfer in tumorigenesis. We hypothesized that interactions between leukemic cells and adjacent normal hematopoietic stem or progenitor cells may provide an alternative mechanism for the accumulation of mutated genes and the multiplicity of distinct clones in leukemia. To test this hypothesis, we performed experiments to determine whether tumorigenic properties could be transferred from a tumor cell line to normal mouse bone marrow cells using both in vivo and in vitro and systems. B6-GFP+ mice were injected i.v. with 200,000 C1498-Luc cells (a B6-derived NKT-cell-like mouse tumor cell line expressing luciferase and DSRed). Bioluminescent imaging was used to monitor the progression of tumor cell growth in recipients. At 1 month after tumor-cell inoculation, marrow from these mice was harvested and FACS-sorted for GFP+ cells (to eliminate C1498 cells), and then cultured on irradiated stromal cell layers in 96-well plates in a limiting dilution analysis for Poisson analysis of GFP+ clonogenic precursor frequency on day 9. On day 10, cells were harvested from culture and GFP+ cells resorted onto fresh stromal layers for second and third determinations of GFP+ clonogenic precursor frequency on days 15 and 18. As shown in Figure 1, the frequency of clonogenic precursors increased with each successive determination for marrow from C1498-injected mice, while control cultures from non-injected mice showed no increase in precursor frequency, suggesting that exposure to C1498 cells conferred a growth advantage to the marrow cells in the tumor-cell injected mice. Similar results were obtained using an in vitro system of co-culture using C1498 cells and GFP+ bone marrow cells, followed by serial rounds of GFP+ sorting and Poisson analysis, showing increases in clonogenic frequency over 5 successive sorts and re-cultures over a 2-month period, while control cultures showed decreased clonogenic frequencies over the course of the experiment. To confirm these observations in vivo, B6-GFP mice were injected with C1498-Luc and marrow was harvested after a month and sorted for GFP+ cells. The sorted marrow was transplanted into 11Gy-irradiated (FVB x B6albino)F1 recipients (5 x 106 cells per recipient, n=5). Control recipients were irradiated and transplanted with GFP+ marrow from non-injected donors. All recipients developed full hematopoietic engraftment with GFP+ cells. At 6 months post-transplant, a tumor was observed near the left shoulder of one of the recipients of C1498-exposed GFP+ marrow. Figure 2 shows IVIS GFP imaging of this mouse with the GFP+ tumor along with control animals. The tumor was not positive for luciferase expression. The mouse was sacrificed and the tumor excised and a portion was dissociated for flow cytometric analysis and culturing (with other segments reserved for subsequent histological and genetic analysis). Both GFP+ and non-GFP cells were found in the dissociated tumor cell suspension. The GFP+ cells were hematopoietic in origin (CD45+) and exhibited a mixed phenotype containing markers expressed on C1498 (DX5+) and myeloid lineage cells (CD11b+) as well as Sca-1, a stem cell marker. Cultures of the GFP+ tumor yielded a population of GFP+ mononuclear cells. These data are consistent with a model in which growth-promoting or transforming genes from cancer cells become incorporated within a healthy hematopoietic stem or progenitor cell, which contributes to the genetic diversity of the cancer through the initiation a new transformed clone. Genetic analysis with deep sequencing will compare the DNA sequences between the parental C1498 cell line, sorted populations of clonogenic GFP+ cells obtained from the in vitro and in vivo experiments, and the GFP+ tumor cells to confirm the transformation of healthy bone marrow hematopoietic stem cells with genetic sequences derived from the C1498 cells. Disclosures No relevant conflicts of interest to declare.

Origin of Cyclic Adenosine Monophosphate in Saliva

1975 ◽  
Vol 54 (3) ◽  
pp. 535-539 ◽  
Author(s):  
Takao Kanamori ◽  
Toshiharu Nagatsu ◽  
Shosei Matsumoto
Keyword(s):  
Salivary Gland ◽  
Cyclic Amp ◽  
Submandibular Gland ◽  
Submaxillary Gland ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate

The level of cyclic adenosine monophosphate (AMP) in duct saliva from the dog submandibular gland was increased after cyclic AMP was administered intravenously in vivo. Isoproterenol increased the level of cyclic AMP in plasma and saliva in vivo and in salivary gland slices in vitro, but increased the level only slightly in saliva in a perfused dog submaxillary gland.

scholarly journals Transfer of the ADA gene into human ADA-deficient T lymphocytes reconstitutes specific immune functions

Blood ◽  
1992 ◽  
Vol 80 (5) ◽  
pp. 1120-1124
Author(s):  
G Ferrari ◽  
S Rossini ◽  
N Nobili ◽  
D Maggioni ◽  
A Garofalo ◽  
...  
Keyword(s):  
T Cells ◽  
Enzyme Activity ◽  
Gene Transfer ◽  
Peripheral Blood ◽  
Retroviral Vector ◽  
Peripheral Blood Lymphocytes ◽  
Proliferative Potential ◽  
Immune Functions ◽  
Beta Gene

Peripheral blood lymphocytes obtained from a patient affected by adenosine deaminase (ADA) deficiency and severe combined immunodeficiency were infected with a retroviral vector containing two copies of a human ADA minigene, and injected into bg/nu/xid (BNX) immunodeficient mice. Six to 10 weeks after injection, human T cells were cloned from the spleens of recipient animals and analyzed for proliferative potential, T-cell surface markers, expression of ADA activity, integration of retroviral sequences, T-cell receptor (TCR) beta gene rearrangement, and specificity of antigen recognition. Efficient gene transfer and expression restored proliferative potential in vitro and long-term survival in vivo. All clonable human T lymphocytes obtained from the spleen of recipient animals had high levels of vector-derived ADA enzyme activity and showed predominantly the CD4+ phenotype. Retroviral integrations and TCR-beta gene rearrangements demonstrated the presence of a variety of different clones in the spleens of recipient mice. Furthermore, the combined analyses of vector integration and TCR rearrangement provided evidence that a circulating progenitor cell was transduced by the retroviral vector, giving rise to different and functional TCRs. Evaluation of antigen-specificity demonstrated both alloreactive and foreign antigen specific immune responses. These results suggest that restoration of enzyme activity in human ADA-deficient peripheral blood T cells by retroviral-mediated ADA gene transfer allows in vivo survival and reconstitution of specific immune functions. Therefore, retroviral vector-mediated gene transfer into circulating mononuclear cells could be successful not only in maintaining the metabolic homeostasis, but also for the development of a functional immune repertoire. This is a fundamental prerequisite for the usage of genetically engineered peripheral blood lymphocytes for somatic cell gene therapy of ADA deficiency.

scholarly journals The murine cytomegalovirus MCK-2 facilitates in vivo infection transfer from dendritic cells to salivary gland acinar cells.

2021 ◽  
Author(s):  
Jiawei Ma ◽  
Kimberley Bruce ◽  
Philip G. Stevenson ◽  
Helen E. Farrell
Keyword(s):  
Dendritic Cells ◽  
Salivary Gland ◽  
Epithelial Cells ◽  
Salivary Glands ◽  
Immune Evasion ◽  
Acinar Cells ◽  
Lung Infection ◽  
Human Cmv

The cytomegaloviruses (CMVs) spread systemically via myeloid cells and demonstrate a broad tissue tropism. Human CMV (HCMV) UL128 encodes a component of the virion pentameric complex (PC) that is important for entry into epithelial cells and cell-cell spread in vitro . It possesses N-terminal amino acid sequences similar to C-C chemokines. While the species-specificity of HCMV precludes confirmation of UL128 function in vivo , UL128-like counterparts in experimental animals have demonstrated a role for salivary gland infection. How they achieve this has not been defined, although effects on monocyte tropism and immune evasion have been proposed. By tracking infected cells following lung infection, we show that although the UL128-like protein in mouse CMV (MCMV; designated MCK-2), facilitated entry into lung macrophages, it was dispensable for subsequent viremia mediated by CD11c + dendritic cells (DC) and extravasation to the salivary glands. Notably, MCK-2 was important for transfer of MCMV infection from DC to salivary gland acinar epithelial cells. Acinar cell infection of MCMVs deleted of MCK-2 was not rescued by T-cell depletion, arguing against an immune evasion mechanism for MCK-2 in the salivary glands. In contrast to lung infection, peritoneal MCMV inoculation yields a mixed monocyte/DC viremia. In this setting, MCK-2 again promoted DC-dependent infection of salivary gland acinar cells, but it was not required for monocyte-dependent spread to the lung. Thus, the action of MCK-2 in MCMV spread was specific to DC-acinar cell interaction. IMPORTANCE Cytomegaloviruses (CMVs) establish myeloid cell-associated viremias and persistent shedding from the salivary glands. In vitro studies with human CMV (HCMV) have implicated HCMV UL128 in epithelial tropism, but its role in vivo is unknown. Here we analysed how a murine CMV (CMV) protein with similar physical properties - designated MCK-2 - contributes to host colonization. We demonstrate that MCK-2 is dispensable for the initial systemic spread from primary infection sites, but within the salivary gland facilitates transfer of infection from dendritic cells (DC) to epithelial acinar cells. Virus transfer from extravasated monocytes to the lungs did not require MCK-2, indicating a tissue-specific effect. These results provide new information about how persistent viral tropism determinants operate in vivo .

A Gene Transfer Approach towards Hemophilia A Correction Using the Piggybac Transposon Vector.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1477-1477 ◽  
Author(s):  
Janice M. Staber ◽  
Erin R Burnight ◽  
Pavel Korsakov ◽  
Joseph Kaminski ◽  
Nancy L Craig ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Factor Viii ◽  
Cell Line ◽  
Hemophilia A ◽  
Vector System ◽  
Dna Repeats ◽  
Human Factor Viii ◽  
The Impact

Abstract Abstract 1477 Human Factor VIII (hFVIII) deficiency offers advantages as a disease target for gene therapy as small increases in factor VIII levels will alter the bleeding phenotype. In addition, both mouse and dog models of the disease are available for preclinical studies. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Wu et al and others reported that piggyBac transposase is highly efficient at catalyzing transposition in mammalian cells in vitro (PNAS 103: 15008–15013, 2006). Advantages of this novel nonviral vector system include a large transgene cassette capacity and ease of production and purification. We hypothesize that a PB transposon vector carrying a reporter gene cassette or the human FVIII cDNA along with a codon-optimized (co-) or hyperactive (hyp-) transposase will confer persistent gene expression and correction of the hemophilia A bleeding phenotype with the FVIII cDNA. PB transposons were engineered to carry a puromycin resistance gene (PB puro), a human alpha1 antitrypsin reporter (PB hAAT), or hFVIII gene (B domain deleted or a partial B domain-226 amino acids/N6). We evaluated co- and hyp-transposase-mediated transposition in the Huh-7 human hepatoma cell line to verify function in hepatocytes. Using the PB puro vector, we demonstrated that the hyp-transposase generated a 2 fold higher transposition efficiency than the co-transposase in hepatocytes. We investigated the impact of varying the ratio of transposon to transposase; we screened ratios of 5:1, 2:1, 1:1, 1:2, and 1:5 in the Huh-7 cell line. Overall, the 1:2 and 1:1 ratios gave the greatest transposition efficiency in vitro. We evaluated the in vivo gene transfer efficiency in mice by hydrodynamic tail-vein injection using PB hAAT driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Either a low (5 micrograms transposon) or high (25 micrograms transposon) dose was given with varying amounts of hyp-transposase to generate an in vivo dose response curve. Serum hAAT levels were measured prior to injection and then monthly for 3 months. Results revealed the 1:1 ratio at the high transposon dose generated higher level of expression compared to all other doses with expression stable in all groups for 3 months. PB vectors encoding hFVIII have been prepared, and our studies with these vectors are ongoing. These data show that the PB vector can be used to deliver transgene expression to the liver and achieve long term expression of a secreted protein. Disclosures: Staber: Bayer Healthcare: Research Funding.

Forced Expression of CC Chemokine Receptor 2 Enhances Anti-Cancer Reactivity Mediated by T Lymphocytes Beforehand Redirected Toward WT1 Inside the Tumor Microenvironment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2059-2059
Author(s):  
Hiroaki Asai ◽  
Hiroshi Fujiwara ◽  
Toshiki Ochi ◽  
Jun An ◽  
Toshiaki Shirakata ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Gene Transfer ◽  
Cell Line ◽  
Cd8 T Cells ◽  
Tcr Signaling ◽  
Ccr2 Gene ◽  
Anti Cancer

Abstract Abstract 2059 Background & Purpose: Redirected T-cell based adoptive therapy using cancer antigen-specific T-cell receptor (TCR) gene transfer has proven promise, however its clinical efficacy still remains unsatisfactory. The less accumulation in number of infused redirected T cells at the local tumor site is one of the causes. In order to accumulate those tumor-responsive T cells inside tumor microenvironment, chemokine produced by tumor cells and/or tumor associated cells is an attractive target. In this study, we examined the feasibility of CC chemokine receptor 2 (CCR2) gene transfer into T cells beforehand redirected toward WT1 in order to enhance the anti-cancer reactivity, both in vitro and in vivo. Methods: HLA-A*24:02-restricted and WT1235–243-specific TCR-a/b genes were introduced into normal CD8+ T cells using our novel retroviral TCR-gene expression vector encompassing silencers for endogenous TCRs (WT1-si-TCR vector). mRNA expression of total 11 chemokines expressed by 10 human lung cancer cell lines was examined using QRT-PCR, then CCL2 (variably produced in 7 out of 10 examined cell lines) and the small lung cancer cell line, LK79 which abundantly produced CCL2 was chosen for the proof of concept. Cloned CCR2 gene was retrovirally introduced into Jurkat cells, Jurkat/MA cells and normal CD8+ T cells similarly redirected beforehand using WT1-siTCR vector. Introduced CCR2 was validated using flow cytometer and transwell experiments. Cytotoxicity was examined using standard 51chromium release assay. Cooperative functionality composed of CCL2-directinality and WT1-specific antitumor cytotoxicity mediated by those double gene transfectants was examined in vitro; values of LDH released from destroyed LK79 cells in the bottom well by migrated double gene transduced CD8+ T cells from the upper well were measured. Antitumor reactivity in vivo was assessed using xenograft mouse model using luciferase-transduced LK79 cells (LK79-luc). Direct effect of CCL2 ligation on WT1-TCR signaling in double gene transfectant was assessed using luciferase assay with double gene transduced TCR− Jurkat/MA cell line, which stably expresses hCD8a and NFAT-luciferase reporter genes (Jurkat/MA/CD8a/luc; kindly provided by Prof. Erik Hooijberg, Netherlands). Results: CCL2 sensitivity was successfully introduced by CCR2 gene transfer; CCR2 transduced Jurkat successfully directed toward CCL2 producing cell line, LK79 cells. CCR2 gene transduction did not impede WT1-specific cytotoxicity mediated by CD8+ T cells beforehand redirected using WT1-siTCR gene transfer, rather double gene transduction cooperatively endowed those transfectants with CCL2 sensitivity and WT1-specific cytotoxicity against LK79 cells. Furthermore, in vivo assay using xenograft mouse model, growth of subcutaneously inoculated LK79-luc cells was more efficiently suppressed by those double gene transduced CD8+ T cells than WT1-siTCR single gene trnasfectants, in particular, immediately after adoptive transfer. Finally, CCL2 synergistically enhanced the magnitude of cognate peptide evoked WT1-specific TCR signaling in a dose dependent manner. Even without WT1 peptide ligation, such TCR signaling was similarly evoked by CCL2 to some extent. Conclusion: In this study, our results demonstrate that forced expression of CCR2 on CTL beforehand redirected toward WT1 enhanced its anti-cancer reactivity both in vitro and in vivo. This in vivo enhancement of antitumor reactivity may be caused by increased number of effector cells and enhanced WT1-TCR signaling generated both by CCL2 in the local tumor microenvironment. Although further studies are warranted, CCR2 gene transfer into redirected WT1-specific tumor-reactive CTL may be feasible for the treatment of human cancers. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document