T Lymphocytes Are Not Immune
Authors: Gianpietro Dotti
Journal: Molecular Therapy (2013)

Administration of genetically modified T lymphocytes to cancer patients has increased exponentially over the past 15 years, and these cells have been given to many hundreds of patients. These studies have often used retroviral vectors, and, in sharp contrast to clinical trials using hematopoietic stem cells as targets, gene-modified T cells have not subsequently undergone malignant transformation. This has led to the impression that the more differentiated state of T lymphocytes may offer protection from retroviral mutagenesis. In this issue of Molecular Therapy, however, Heinrich and colleagues document the occurrence of T-cell lymphoma in RAG−/− mice transplanted with OT-1 transgenic T cells genetically modified with a gammaretroviral vector encoding the green fluorescent protein, suggesting that, although T cells may be relatively insensitive to malignant transformation by gammaretroviruses, they are not immune.1Heinrich T Rengstl B Muik A Petkova M Schmid F Wistinghausen R et al.Mature T-cell lymphomagenesis induced by retroviral insertional activation of Janus kinase 1.Mol Ther. 2013; 21: 1160-1168Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Early gene transfer studies focused primarily on detecting T-cell persistence and trafficking to the tumor site by the expression of nonfunctional genes.2Rosenberg SA Aebersold P Cornetta K Kasid A Morgan RA Moen R et al.Gene transfer into humans—immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction.N Engl J Med. 1990; 323: 570-578Crossref PubMed Scopus (1081) Google Scholar,3Heslop HE Ng CY Li C Smith CA Loftin SK Krance RA et al.Long-term restoration of immunity against Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes.Nat Med. 1996; 2: 551-555Crossref PubMed Scopus (707) Google Scholar The herpes simplex thymidine kinase is one of the earliest examples of a therapeutic gene expressed in T lymphocytes to pharmacologically induce their death in case of graft-versus-host disease in patients who received an allogeneic hematopoietic stem cell transplant.4Bonini C Ferrari G Verzeletti S Servida P Zappone E Ruggieri L et al.HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia.Science. 1997; 276: 1719-1724Crossref PubMed Scopus (1041) Google Scholar More recently, the expression of T-cell receptors and chimeric antigen receptors to redirect the antigen specificity of polyclonal T lymphocytes set off a new wave of enthusiasm for adoptive immunotherapy in cancer patients.5Morgan RA Dudley ME Wunderlich JR Hughes MS Yang JC Sherry RM et al.Cancer regression in patients after transfer of genetically engineered lymphocytes.Science. 2006; 314: 126-129Crossref PubMed Scopus (2114) Google Scholar,6Pule MA Savoldo B Myers GD Rossig C Russell HV Dotti G et al.Virus-specific T cells engineered to coexpress tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma.Nat Med. 2008; 14: 1264-1270Crossref PubMed Scopus (923) Google Scholar,7Kalos M Levine BL Porter DL Katz S Grupp SA Bagg A et al.T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.Sci Transl Med. 2011; 3: 95ra73Crossref PubMed Scopus (1801) Google Scholar Because T lymphocytes are rapidly dividing cells, all these studies have used vectors that produce genomic integration of the transgene of interest, which is critical to ensure its long-term functional expression in progeny cells. Most of these studies over the past 20 years have used gammaretroviral vectors for this purpose—in particular, vectors derived from the Moloney murine leukemia retrovirus.8Bear AS Morgan RA Cornetta K June CH Binder-Scholl G Dudley ME et al.Replication-competent retroviruses in gene-modified T cells used in clinical trials: is it time to revise the testing requirements?.Mol Ther. 2012; 20: 246-249Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar A drawback of this powerful system, however, is the potential to induce genotoxic events by deregulating the expression of oncogenes, ultimately leading to neoplastic transformation of the transduced cells. Although this phenomenon was well recognized in murine models, genotoxicity related to insertion of gammaretroviral vectors in human subjects remained a theoretical risk until 2003, when integration of a retroviral vector encoding the common γ-chain of the interleukin-2 receptor caused deregulated expression of the LMO2 gene in hematopoietic stem cells and resulted in T-cell leukemia in patients with X-linked severe combined immunodeficiency who had been cured of their disease by gene transfer.9Hacein–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: 255-256Crossref PubMed Scopus (1612) Google Scholar These malignant T cells were derived from the transduced stem cell population. Subsequently, neoplastic transformation was also reported in patients treated for chronic granulomatous disease.10Stein S Ott MG Schultze-Strasser S Jauch A Burwinkel B Kinner A et al.Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease.Nat Med. 2010; 16: 198-204Crossref PubMed Scopus (632) Google Scholar Although similar retroviral vectors have been used to modify mature human T lymphocytes, genotoxicity has not been evident in patients who have received these cells. Human T lymphocytes, like hematopoietic stem cells, are susceptible to insertional mutagenesis, but these events do not necessarily evolve to a malignant phenotype in this cell subset. T lymphocytes are generally stimulated by crosslinking of the T-cell receptor and cytokines to allow retroviral integration, and elegant analyses have shown that integrated proviruses are located predominantly around promoters of genes that are active in T cells at the time of transduction, probably because the chromatin configuration of these active genes makes them more accessible.11Recchia A Bonini C Magnani Z Urbinati F Sartori D Muraro S et al.Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells.Proc Natl Acad Sci USA. 2006; 103: 1457-1462Crossref PubMed Scopus (167) Google Scholar The insertional gene activation in T cells is determined predominantly by the characteristics of the transcriptional regulatory elements carried by the vector rather than by the vector type or design.12Maruggi G Porcellini S Facchini G Perna SK Cattoglio C Sartori D et al.Transcriptional enhancers induce insertional gene deregulation independently from the vector type and design.Mol Ther. 2009; 17: 851-856Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar Vector integration can cause deregulated expression of the nearby genes in T lymphocytes,11Recchia A Bonini C Magnani Z Urbinati F Sartori D Muraro S et al.Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells.Proc Natl Acad Sci USA. 2006; 103: 1457-1462Crossref PubMed Scopus (167) Google Scholar but, in sharp contrast to hematopoietic stem cells, T lymphocytes themselves appear “resistant” to malignant transformation. Indeed, even constitutive expression of oncogenes, such as LMO2 and TCL1, obtained by gammaretroviral gene transfer in mature mouse T lymphocytes, did not cause leukemia/lymphoma in RAG−/− mice.13Newrzela S Cornils K Li Z Baum C Brugman MH Hartmann M et al.Resistance of mature T cells to oncogene transformation.Blood. 2008; 112: 2278-2286Crossref PubMed Scopus (170) Google Scholar Published clinical trials confirm the general assumption that these vectors are safe in T lymphocytes; neither clonal expansion nor malignant transformation has been reported in patients followed for more than 10 years.14Ciceri F Bonini C Stanghellini MT Bondanza A Traversari C Salomoni M et al.Infusion of suicide-gene-engineered donor lymphocytes after family haploidentical haemopoietic stem-cell transplantation for leukaemia (the TK007 trial): a non-randomised phase I–II study.Lancet Oncol. 2009; 10: 489-500Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar,15Heslop HE Slobod KS Pule MA Hale GA Rousseau A Smith CA et al.Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients.Blood. 2010; 115: 925-935Crossref PubMed Scopus (632) Google Scholar As reported in this issue of Molecular Therapy, however, it is clear that concerns about malignant transformation of human T lymphocytes cannot be fully laid to rest. Continuing their exploratory work on insertional mutagenesis in T lymphocytes, Heinrich and colleagues documented T-cell lymphomas in RAG−/− mice transplanted with OT-1 transgenic T cells genetically modified with a gammaretroviral vector encoding green fluorescent protein.1Heinrich T Rengstl B Muik A Petkova M Schmid F Wistinghausen R et al.Mature T-cell lymphomagenesis induced by retroviral insertional activation of Janus kinase 1.Mol Ther. 2013; 21: 1160-1168Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Lymphomagenesis occurred with a long latency and was associated with deregulation of the Jak1/STAT3 pathway secondary to the forward-oriented provirus integration between exons 1 and 2 of the Jak1 gene. Although this observation is troubling, some striking differences between human studies and the mouse model should be taken into account. The genotoxic event was obtained in very “stringent experimental conditions,”1Heinrich T Rengstl B Muik A Petkova M Schmid F Wistinghausen R et al.Mature T-cell lymphomagenesis induced by retroviral insertional activation of Janus kinase 1.Mol Ther. 2013; 21: 1160-1168Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar using T cells with a highly restricted T-cell receptor repertoire. By contrast, T-cell therapy approaches use polyclonal populations of T cells. Moreover, in contrast to the murine study, human T cells harboring forward-oriented proviruses within introns or exons frequently cause loss of gene function, leading to a survival disadvantage in vivo transfer rather than to aberrant expansion.16Cattoglio C Maruggi G Bartholomae C Malani N Pellin D Cocchiarella F et al.High-definition mapping of retroviral integration sites defines the fate of allogeneic T cells after donor lymphocyte infusion.PLoS One. 2010; 5: e15688Crossref PubMed Scopus (39) Google Scholar The intrinsic characteristics of mature T lymphocytes, such as antigen dependency, epigenetic regulation, and the negative-feedback check points that contract the immune responses upon elimination of the antigen, actively control the size of the T-lymphocyte population. These control mechanisms certainly help explain the lack of leukemia/lymphoma transformation in clinical trials, despite the molecular evidence of potentially dangerous insertional mutagenesis following gammaretroviral transduction of T lymphocytes. Although the above observations should reassure us that gammaretroviral vectors are relatively safe when used with T lymphocytes, we should remember that the longest observational period relates to studies in which marking and suicide genes were used. The field of cancer immunotherapy is now rapidly moving to use T cells modified with genes that directly affect their intrinsic biological properties. For example, the constitutive expression of potent costimulatory endodomains included within chimeric antigen receptors, such as 4–1BB, alters the physiological kinetics of these molecules in immune responses.7Kalos M Levine BL Porter DL Katz S Grupp SA Bagg A et al.T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.Sci Transl Med. 2011; 3: 95ra73Crossref PubMed Scopus (1801) Google Scholar In addition, subsets of T lymphocytes with “stemness” properties have been recently identified17Gattinoni L Lugli E Ji Y Pos Z Paulos CM Quigley MF et al.A human memory T cell subset with stem cell-like properties.Nat Med. 2011; 17: 1290-1297Crossref PubMed Scopus (1240) Google Scholar that may become appealing targets for gene delivery to ensure longevity. Such applications may generate “stringent experimental conditions” in human subjects and, by creating a selective advantage for these specific T-cell subsets, may allow insertional mutagenesis in T lymphocytes to have less benign consequences. How should these results affect our plans and procedures? Whereas long-term observation of patients enrolled in clinical trials showed that the risk of infusing replication-competent retrovirus into patients is abrogated when manufactured vectors are carefully screened before release,8Bear AS Morgan RA Cornetta K June CH Binder-Scholl G Dudley ME et al.Replication-competent retroviruses in gene-modified T cells used in clinical trials: is it time to revise the testing requirements?.Mol Ther. 2012; 20: 246-249Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar the study by Heinrich et al.1Heinrich T Rengstl B Muik A Petkova M Schmid F Wistinghausen R et al.Mature T-cell lymphomagenesis induced by retroviral insertional activation of Janus kinase 1.Mol Ther. 2013; 21: 1160-1168Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar alerts the gene therapy community to the need for continued long-term monitoring of patients for molecular events related to insertional mutagenesis. Fortunately, high-definition mapping of retroviral integrants is now much faster and more affordable, facilitating these studies. There also continues to be a need to develop vectors with greater intrinsic safety that nonetheless will guarantee the same scalability of robust gene transfer that exists with current retroviral vectors, allowing successful approaches to be broadly applied. Finally, the development of efficient suicide genes that can eliminate transformed cells may make T cells safer if integration should ever become a clinical issue.18Di Stasi A Tey SK Dotti G Fujita Y Kennedy-Nasser A Martinez C et al.Inducible apoptosis as a safety switch for adoptive cell therapy.N Engl J Med. 2011; 365: 1673-1683Crossref PubMed Scopus (1045) Google Scholar