| Abstract|| |
Background: Gene therapy has been in use to cure hereditary and acquired diseases by incorporating the desired gene into the cells with the help of gammaretroviral vectors. Despite the success of this therapy in X-linked severe combined immunodeficiency syndrome, few patients developed leukemia as a major adverse event due to retroviral insertional mutagenesis within stem cells. In experimental animals also, retroviral-mediated gene transfer technique resulted in the development of leukemia. On the other hand, evidence suggests that mature T cells (TC) are relatively resistant to transformation even after retroviral-mediated transfer of potent oncogenes Tcl1, ΔTrkA and LMO2 with no reported side effects yet. Aims: To further address the safety issue for TC use in gene therapy, this study investigated susceptibility of mature polyclonal TC to malignant transformation by the retroviral-mediated transfer of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) oncogene. Materials and Methods: Wild-type mature TC, isolated from C57BL/6 donor mice (genetic background Ly5.1) were transduced with gamma-retroviral vectors encoding the potent TC oncogene NPM-ALK or the control vector enhanced green fluorescent protein eGFP. The cells were then transplanted into RAG-1 deficient recipient mice (genetic background Ly5.2). Results: Two out of five mice from NPM-ALK oncogene group developed leukemia/lymphoma after latency periods (153 and 250 days, respectively). None of the mice from the control group developed any malignancy throughout the observational period. Conclusion: Mature polyclonal TC are relatively susceptible to malignant transformation after gamma-retroviral mediated transfer of NPM-ALK oncogene; hence safety of TC use in gene therapy should be further investigated to avoid the possible side-effect of development of leukemia/lymphoma.
Keywords: Gamma-retroviral vector, gene therapy, leukemia/lymphoma, mature T cells, stem cells
|How to cite this article:|
Kumar A. Malignant transformation of mature T cells after gammaretrovirus mediated transfer of nucleophosmin-anaplastic lymphoma kinase oncogene. Indian J Pathol Microbiol 2015;58:301-6
|How to cite this URL:|
Kumar A. Malignant transformation of mature T cells after gammaretrovirus mediated transfer of nucleophosmin-anaplastic lymphoma kinase oncogene. Indian J Pathol Microbiol [serial online] 2015 [cited 2020 Jun 6];58:301-6. Available from: http://www.ijpmonline.org/text.asp?2015/58/3/301/162835
| Introduction|| |
Gene therapy is the use of a gene as a pharmaceutical agent to treat a disease. It has been used to cure the hereditary and some acquired diseases caused due to lack of a functional gene. In gene therapy, gene that codes for a therapeutic protein is cloned into a vector, which is used as a vehicle to carry that gene inside the cells within the body. Once inside, the gene is transcribed and translated by the cell machinery to produce RNA and protein, respectively, which in turn treat the patients' disease. Viruses have been successfully used as a vector to carry the desired gene into the cell, amongst which retroviral vectors, derived from retroviruses, are important ones.  They have been used to cure the diseases, such as, adenosine deaminase deficient and X-linked severe combined immunodeficiency (X-SCID). Due to lack of a functional gene, patients suffering from these diseases do not develop mature T and B cells, and, therefore, die due to infections within 1-year of life. In these clinical gene therapies, patients' hematopoietic stem cells were genetically modified with retroviral vectors, which carried the normal copy of the gene, and the patients were cured. , Despite the first successful clinical trial for X-SCID, the danger of retroviral insertional mutagenesis as a major adverse event of gene therapy was revealed.  In experimental animals, retroviral-mediated gene transfer into stem cells and their subsequent transplantation into mice resulted in the development of leukemia. In contrast to stem cells, mature T cells (TC) have been shown to be relatively resistant to transformation even after retroviral-mediated transfer of potent oncogenes Tcl1, ΔTrkA and LMO2.  However, mature TC can self-renew, proliferate and survive for long periods; this renders TC prone to transformation. Therefore, the question of mature TC transformability remains elusive. Here, transformation susceptibility of mature polyclonal TC by retroviral-mediated nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) oncogene transfer was further investigated.
In our body, there are around 10 13 different TC with specific TC receptors (TCRs) to respond to specific antigens. The different TCR expression by TC is caused by the recombination activating genes RAG1 and RAG2. This results in the polyclonality (many clones with specific TCR) of TC. , NPM-ALK is a fusion oncogene, which results due to chromosomal translocation t(2;5)(p23;q35). This translocation fuses the gene encoding ALK, located on chromosome 2p23, with the house-keeping gene NPM on chromosome 5q35. NPM-ALK codes for a constitutively activated enzyme tyrosine kinase, which is oncogenic in nature.  In the presented study, mature TC were used. NPM-ALK was used because of its association with mature TC lymphoma in humans. ,,
The objective of this study was to address the safety issue for TC use in gene therapy. For this purpose, mature polyclonal TC were transduced with retroviral-mediated transfer of NPM-ALK oncogene.
| Materials and Methods|| |
Gamma retroviral vectors and cloning
The control vector MP91-eGFP (a gamma-retroviral vector) has been described. , Human NPM-ALK oncogene (company GeneArt, Germany) was cloned into it. The vector also encodes eGFP, which acts as a marker gene upon expression [Figure 1]. The construct was verified by sequencing.
|Figure 1: Molecular structure of the utilized gamma-retroviral vectors: The upper one is MP91-NPM-ALK (nucleophosmin-anaplastic lymphoma kinase)vector, and the lower one is MP91-eGFP control empty vector. mcs: Multiple cloning site.,|
Click here to view
Mature T lymphocytes were isolated from lymph nodes and spleen of healthy wild-type (WT) donor mice of genetic background C57BL/6J Ly5.1. After transducing with the control vector or NPM-ALK vector, the cells were transplanted into RAG-1 deficient recipient mice of genetic background C57BL/6J Ly5.2 [Figure 2]. RAG-1 deficient mice support long-term engraftment of donor lymphocytes as they do not develop any T or B cells.
|Figure 2: Transplantation principle - C57BL/6 (Ly5.1) wild type mice were used as donors for mature T cells (TC). Isolated mature TC from spleen and lymph nodes were stimulated for 4 days. Subsequently after retroviral transduction, the cells were transplanted into Rag-1 deficient mice (Ly5.2)|
Click here to view
Six to eight weeks old mice C57BL/6J Ly5.1 and Rag-1-deficient C57BL/6J Ly5.2 were obtained from Charles River Laboratories (Sulzfeld, Germany) and The Jackson Laboratory (Bar Harbor, USA). Donor (Ly5.1) hematopoietic cells could be differentiated by the antigen CD45.1 from the Rag-1 deficient recipient (Ly5.2) cells expressing the antigen CD45.2. The mice were maintained and bred according to the guidelines of Federation of European Laboratory Animal Science Associations in the animal husbandry facility of our institute. Healthy (donors) or Symptomatic/leukemic (recipient) mice were sacrificed after anesthesia by breaking the nape and examined for histopathological and flow cytometric analyses. Animal experiments were approved by the Regional Council Darmstadt.
Retroviral vector production
Vector supernatants were produced by calcium-phosphate-mediated transient transfection as described. ,
Retroviral transduction and transplantation of mature T cells
After sacrificing mice by breaking the nape, skin was disinfected with 70% ethanol. The mononuclear cells were isolated from mesenteric lymph nodes and spleen of donor mice of Ly5.1. After washing with 2 ml phosphate buffered saline, the cells were stimulated with anti-CD3 (clone 145-2C11) and anti-CD28 (clone 37.51) antibodies (BD PharMingen) coated beads (Invitrogen, Carlsbad, CA) along with 100 U/ml interleukin-2 (IL-2). The stimulated TC enter mitosis, in which nuclear membrane is disintegrated. This facilitates the retroviral vector to integrate directly into the cell genome. Cells with beads were mixed and seeded in six-well culture plates (4 × 10 6 cells/6 ml per well). Cells were cultured under standard culture conditions for 3 days. On 4 th day, cells were collected in a 50 ml Falcon tube. After rigorous pipetting 8-10 times up and down with a 10 ml pipette, cells were debeaded by placing in a magnetic separator. Afterwards, the medium containing debeaded cells was transferred to a new 50 ml Falcon tube. The cells were counted and used for transduction. Stimulated TC were transduced by culturing on virus-preloaded six-well plates. For this purpose, nontissue culture treated six-well plates were coated with 1 ml retronectin solution (fibroncetin protein) overnight at 4°C. The plates were blocked with 3 ml of 2% BSA solution and subsequently washed with 3 ml HBSS. 4 ml virus containing supernatant was added to each well of six-well plate and centrifuged for 30 min at 962 × g at 4°C. This step was repeated twice. Virus in the supernatant adheres unspecifically to the retronectin-coated surface, thus allowing concentration of retroviral particles at the bottom of the plate. 3-4 × 10 6 stimulated TC in mouse special medium were added per well of virus-preloaded plate and incubated overnight. Next day, the cells were transferred onto a second virus-preloaded plate (procedure repeated as above) and cultured for 2 days. 5 × 10 6 transduced TC were injected into tail vein of Rag-1 deficient recipient mice.
Flow cytometry and white blood cell counts of leukaemia/lymphoma samples
The sick mice were sacrificed and their blood and spleen were analyzed by flow cytometry. Blood count was determined using blood counter (Scil animal care, Germany). The samples were stained for the following anti mouse antibodies: CD3 (PE-Cy5), CD19 (PE-Cy5.5), (both from Invitrogen); CD45.1 (PE), CD45.2 (PerCP-Cy5.5), (both from BD PharMingen. Before staining, the samples were incubated with FcR block (Serotec, Duesseldorf, Germany) to prevent non-specific binding to Fc receptors (BD PharMingen). After staining, Cal-lysis solution (Invitrogen) was mixed with the samples to lyse red blood cells. Analysis was performed on FACSCalibur using the Cellquest pro software. 
Sick mice were sacrificed for necropsy and examined for pathological abnormalities. Liver, spleen lymph nodes and bone marrow were fixed in 10% Zinc Formal Fixx (Thermo Shandon, Pittsburgh, USA). Histopathological examination was done by the Department of Pathology of our university.
Ligation-mediated polymerase chain reaction
Ligation-mediated polymerase chain reaction (LM-PCR) was performed according to the protocol. ,, Single cell suspensions of mouse organs were prepared at necropsy. Genomic DNA from organs and blood was isolated using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany).
Retroviral insertion site analysis
Following LM-PCR and sequencing, integration-flanking sequences were BLAST aligned using the NCBI Build 37 mouse genome (accessed August 2009). Genes within 200 kb (up and downstream) of the integrated vector were documented. In addition, the integration junction sequences were aligned to the mouse genome (ensemble, blat) to detect amplified endogenous retrovirus sequences, which were excluded from the statistical analysis.
| Results|| |
Retroviral transduction efficacies for mature T cells
Gamma-retroviral vector encoding NPM-ALK oncogene (MP91/NPM-ALK), or the control vector encoding only eGFP (MP91/eGFP) was transduced ex vivo twice into donor mature T lymphocytes. After 24-48 h of transduction, the cells were observed under fluorescent microscope and also measured by fluorescent activated cell sorting (FACS) for eGFP marker expression. NPM-ALK transduced TC showed 22% and the control vector transduced TC showed 70% transduction efficacies [Figure 3].
|Figure 3: Retroviral transduction efficacies in mature T cells - nucleophosmin-anaplastic lymphoma kinase vector 22%; eGFP control vector 70%|
Click here to view
Two out of five mice were detected to have developed leukemia/lymphoma after latency periods of 153 and 250 days, respectively
After transduction, 1 × 10 7 cells were transplanted into the tail vein of each RAG-1 deficient mouse. Two out of five mice (transplanted with NPM-ALK transduced TC) developed leukemia/lymphoma after latency periods (153 and 250 days, respectively) [Figure 4]. The mice showed protruded abdomen, weight loss and sluggish movements. Before sacrificing, blood was drawn from the tail vein and measured by Scil vet animal hemogram for white blood cell count. The count was under the normal range (<15000/μl). Afterwards, the mice were sacrificed for further analyses, described below. All the mice from the control group did not develop any malignancy and remained healthy.
|Figure 4: Survival curve of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) transplanted mice - 2 out of 5 mice transplanted with NPM-ALK transduced polyclonal T cells (dotted ladder/line) developed leukaemia/lymphoma 153 and 210 days, respectively, after transplantation. None of the mice from control group, transplanted with eGFP control vector developed any malignancy (straight line)|
Click here to view
Histopathological analysis of tumors
Gross features: The autopsy of the mice revealed enlarged liver and spleen. Spleen showed small white mass infiltrations [Figure 5]a. Microscopic features: The histopathological analysis on hematoxylin and eosin staining of the tumors revealed "anaplastic large-cell lymphoma" phenotype. The tumors showed medium-sized lymphoid cells with kidney shaped nucleus and clear cytoplasm [Figure 5]b.
|Figure 5: (a) Autopsy of the mouse — nucleophosmin-anaplastic lymphoma kinase induced tumor development. Gorss enlargements of liver (hepatomegaly) and spleen (splenomegaly) with tumor masses (white patches). (b) Histopathological features - analysis of the tumor mass from spleen, using H and E, which revealed anaplastic large cell lymphoma phenotype (×40)|
Click here to view
Fluorescent activated cell sorting analysis of tumors
The tumor cells were analyzed through FACS to detect the phenotypes of the lymphomas. As frequently observed in human lymphomas, marker expressions were not confined, and typical T-lineage markers (CD3/TCR) were expressed in only 17% of the cells. The other cells did not express any phenotype of T, B or granulocyte markers. To confirm if the tumors developed from the donor cell compartment, the cells were stained with CD45.1 marker, which was positive in these tumors.
Gamma-retroviral integration site analysis
Cancer can be induced in host cells by retroviral-mediated oncogene transfer. Therefore, integration sites of the retroviral vector, encoding NPM-ALK, were analyzed via LM-PCR for all induced tumors to see the transgene-surrounding genes which might have contributed in the development of tumor. The tumors of the both mice showed some interesting genes such as IL-6 and IL-17 within 200 kb upstream and downstream of the transgene integration. These genes are known to play an important role in cell survival and growth. The integration profiles of NPM-ALK were monoclonal to oligoclonal [Figure 6].
|Figure 6: Ligation-mediated polymerase chain reaction of nucleophosmin-anaplastic lymphoma kinase induced tumours - Mono to oligoclonal pattern in both mice tumours (red box), Internal control (blue box). One mouse tumour (A1) shows monoclonal (lane 1), while the other mouse tumour (A2) shows oligoclonal (lane 2) pattern. A: Animal; 100 bp: Marker for base pairs|
Click here to view
| Discussion|| |
To address the safety issue of TC use in gene therapy, the risk of TC transformability by gamma-retroviral mediated transfer of NPM-ALK oncogene was investigated in this study. Previous study suggested, that in comparison to hematopoietic stem cells, mature TC are resistant to transformation by gamma-retroviral mediated transfer of the oncogenes TCL1, ΔTrkA and LMO2.  TC can self-renew, proliferate and survive for long periods in vivo, which make them susceptible to transformation. Therefore, the question of TC transformability needs to be further investigated. For this purpose, mature TC were isolated from WT mice BL6/57J Ly5.1 and transduced with gamma-retroviral vectors encoding NPM-ALK oncogene, or the control gene eGFP. The cells were then transplanted into Rag-1 deficient mice BL6/57J Ly5.2. It was found that two out of five mice, transplanted with NPM-ALK transduced TC, developed leukemia/lymphoma after latency periods (153 and 250 days, respectively). None of the mice from the control group, transplanted with eGFP control vector transduced TC, developed any malignancy and remained healthy throughout the observational period [Figure 4]. The histopathological analysis of the tumors revealed the typical features of anaplastic large-cell lymphoma. NPM-ALK oncogene has already been known to be involved in anaplastic large-cell lymphoma.  FACS analysis of the tumors for CD marker expression also revealed the features of anaplastic lymphoma, which do not express typical TC marker (CD3), as in these tumors, only 17% of the cells expressed CD3 marker.
As in the presented study, we used gamma-retroviral vector with a strong long terminal repeat-enhancer, retroviral integration sites were analyzed, which might have caused the insertional mutagenesis to aid in the development of lymphoma. For this purpose, LM-PCR was performed, which revealed some interesting genes, such as IL-6 and IL-17, near the transgene integration sites. IL-6 is involved in cell proliferation and survival.  IL-17 promotes the formation and growth of prostate adenocarcinoma.  As these genes promote proliferation and growth of the cells, these genes might have contributed in the development of leukemia/lymphoma. It has already been shown that retroviral-mediated gene transfer into host cells causes dysregulation of oncogenes and tumor suppressor genes near its vicinity of integration, resulting in uncontrolled proliferation of cells. ,,
As two out of five mice transplanted with NPM-ALK transduced TC developed tumors after longer latency periods (153 and 250 days, respectively), an interesting potential mechanism in delayed or controlled leukemogenesis in these cells could be competition between different clones of TC for stimulatory niches from major histocompatibility complex (MHC)/self-peptide complex on the antigen presenting cells (APC). Peripheral T lymphocytes get their survival signal by interacting through their TCR with the specific MHC/self-peptide complex, presented by APC. Homeostatic mechanisms conserve the polyclonality of the TC population.  and regulate the proliferation of peripheral T lymphocytes.  In this way, TC-clone size is restricted by competition between different clones of TC.  Thus it might be possible that the nontransformed TC competed the transformed TC clone for the stimulatory niche and caused delay in the development of leukemia/lymphoma in two mice and hindered the development of leukemia/lymphoma in remaining three mice transplanted with NPM-ALK transduced TC. However in contrast to that, it was described that activated/memory TC do not depend upon MHC-TCR interaction for survival signals, and their proliferation is essentially controlled by cytokines. ,, In the present study, only memory TC were used, since the ex vivo stimulation with anti CD3/anti CD28 antibodies converted the naive mature TC to memory TC.  On the other hand, it could be shown that after the ablation of TCR, naive and memory TC disappeared due to deprivation of MHC-TCR signal. ,
The development of leukemia/lymphoma in two mice could also be due to NPM-ALK oncogenecity itself, as it is a more potent oncogene and, therefore, could transform mature polyclonal TC. Its association with mature T-cell lymphoma in humans is supported by many studies. ,, Our finding of relative susceptibility of mature polyclonal TC to transformation alarms that gene transfer via gamma-retroviral vector into these cells in gene therapy needs to be further investigated for safety issue. This could be achieved by increasing the number of mice and using more oncogenes, using retroviral vectors as a vehicle for gene transfer.
| Conclusion|| |
Mature polyclonal T lymphocytes were found to be relatively susceptible to transformation after gamma-retroviral mediated transfer of NPM-ALK oncogene.
| References|| |
Dunbar CE. Gene transfer to hematopoietic stem cells: Implications for gene therapy of human disease. Annu Rev Med 1996;47:11-20.
Hacein-Bey-Abina S, Le Deist F, Carlier F, Bouneaud C, Hue C, De Villartay JP, et al.
Sustained correction of X-linked severe combined immunodeficiency by ex vivo
gene therapy. N Engl J Med 2002;346: 1185-93.
Aiuti A, Slavin S, Aker M, Ficara F, Deola S, Mortellaro A, et al.
Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 2002;296:2410-3.
Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E, et al.
A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003;348:255-6.
Newrzela S, Cornils K, Li Z, Baum C, Brugman MH, Hartmann M, et al.
Resistance of mature T cells to oncogene transformation. Blood 2008;112:2278-86.
Davis MM, Bjorkman PJ. T-cell antigen receptor genes and T-cell recognition. Nature 1988;334:395-402.
Nikolich-Zugich J, Slifka MK, Messaoudi I. The many important facets of T-cell repertoire diversity. Nat Rev Immunol 2004;4:123-32.
Morris SW, Kirstein MN, Valentine MB, Dittmer K, Shapiro DN, Look AT, et al.
Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin′s lymphoma. Science 1995;267:316-7.
Chiarle R, Gong JZ, Guasparri I, Pesci A, Cai J, Liu J, et al.
NPM-ALK transgenic mice spontaneously develop T-cell lymphomas and plasma cell tumors. Blood 2003;101:1919-27.
Kuefer MU, Look AT, Pulford K, Behm FG, Pattengale PK, Mason DY, et al.
Retrovirus-mediated gene transfer of NPM-ALK causes lymphoid malignancy in mice. Blood 1997;90:2901-10.
Meyer J, Rhein M, Schiedlmeier B, Kustikova O, Rudolph C, Kamino K, et al.
Remarkable leukemogenic potency and quality of a constitutively active neurotrophin receptor, deltaTrkA. Leukemia 2007;21:2171-80.
Newrzela S, Al-Ghaili N, Heinrich T, Petkova M, Hartmann S, Rengstl B, et al.
T-cell receptor diversity prevents T-cell lymphoma development. Leukemia 2012;26:2499-507.
Zhang Q, Liu S, Ge D, Zhang Q, Xue Y, Xiong Z, et al.
Interleukin-17 promotes formation and growth of prostate adenocarcinoma in mouse models. Cancer Res 2012;72:2589-99.
Kustikova O, Fehse B, Modlich U, Yang M, Düllmann J, Kamino K, et al.
Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking. Science 2005;308:1171-4.
Modlich U, Kustikova OS, Schmidt M, Rudolph C, Meyer J, Li Z, et al
. Leukaemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis. Blood 2005;105:4235-46.
Schmidt M, Hoffmann G, Wissler M, Lemke N, Müssig A, Glimm H, et al.
Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples. Hum Gene Ther 2001;12:743-9.
Grivennikov S, Karin E, Terzic J, Mucida D, Yu GY, Vallabhapurapu S, et al.
IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer. Cancer Cell 2009;15:103-13.
Schwarzwaelder K, Howe SJ, Schmidt M, Brugman MH, Deichmann A, Glimm H, et al.
Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo
. J Clin Invest 2007;117:2241-9.
Li Z, Düllmann J, Schiedlmeier B, Schmidt M, von Kalle C, Meyer J, et al.
Murine leukemia induced by retroviral gene marking. Science 2002;296:497.
Min B, Paul WE. Endogenous proliferation: Burst-like CD4 T cell proliferation in lymphopenic settings. Semin Immunol 2005;17:201-7.
Troy AE, Shen H. Cutting edge: Homeostatic proliferation of peripheral T lymphocytes is regulated by clonal competition. J Immunol 2003;170:672-6.
Jameson SC. Maintaining the norm: T-cell homeostasis. Nat Rev Immunol 2002;2:547-56.
Seddon B, Tomlinson P, Zamoyska R. Interleukin 7 and T cell receptor signals regulate homeostasis of CD4 memory cells. Nat Immunol 2003;4:680-6.
Schluns KS, Lefrançois L. Cytokine control of memory T-cell development and survival. Nat Rev Immunol 2003;3:269-79.
Marktel S, Magnani Z, Ciceri F, Cazzaniga S, Riddell SR, Traversari C, et al.
Immunologic potential of donor lymphocytes expressing a suicide gene for early immune reconstitution after hematopoietic T-cell-depleted stem cell transplantation. Blood 2003;101:1290-8.
Labrecque N, Whitfield LS, Obst R, Waltzinger C, Benoist C, Mathis D. How much TCR does a T cell need? Immunity 2001;15: 71-82.
Polic B, Kunkel D, Scheffold A, Rajewsky K. How alpha beta T cells deal with induced TCR alpha ablation. Proc Natl Acad Sci U S A 2001;98:8744-9.
Dr. Ashok Kumar
King Faisal University, AL Ahsa
Source of Support: The work was supported by Deutsche
Forschungsgemeinschaft, the German Research Foundation., Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]