Immortalization of Human and Rhesus Macaque Primary Antigen‐Specific T Cells by Retrovirally Transduced Telomerase Reverse Transcriptase

Eugene V. Barsov1

1 AIDS and Cancer Virus Program, SAIC‐Frederick, Inc., Frederick, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.21B
DOI:  10.1002/0471142735.im0721bs95
Online Posting Date:  November, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Human and rhesus macaque primary antigen‐specific T cells derived from infected or immunized individuals or animals are a valuable material with which to study cellular immune responses against pathogens and tumors. Antigen‐specific T cells can be expanded in vitro but have a finite proliferative life span. After a limited period in culture, primary T cells undergo replicative senescence and stop dividing. This restricts their applicability to short‐term experiments and complicates their use in adoptive immunotherapy. The proliferative life span of primary human and rhesus macaque T cells can be considerably extended by ectopically expressed human telomerase reverse transcriptase (TERT). Antigen‐specific T cells transduced with TERT‐expressing retroviral vectors can proliferate and expand in culture for long periods of time while maintaining their primary T cell characteristics, including antigen‐specific responses. Thus, TERT‐immortalized T cells are an important and valuable resource for studying T cell–mediated immune responses and, potentially, for adoptive immunotherapy. Curr. Protoc. Immunol. 95:7.21B.1‐7.21B.20. © 2011 by John Wiley & Sons, Inc.

Keywords: antigen‐specific T cells; TERT; telomerase; immortalization; retroviral vector; primary T cells; life span; senescence

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Activation of Primary T Cells with Plate‐Bound Anti‐CD3 Antibody
  • Alternate Protocol 1: Activation of Primary T Cells with Soluble Anti‐CD3 Antibody
  • Alternate Protocol 2: Activation of Human Primary T Cells with Concanavalin A
  • Basic Protocol 2: Generation of TERT‐Expressing Retroviral Vector Stocks
  • Alternate Protocol 3: Generation of Stocks from Cloned TERT‐Expressing Retroviral Vector Producer Cell Lines
  • Basic Protocol 3: Transduction of Primary T Cells with TERT Retroviral Vectors
  • Basic Protocol 4: Isolation of TERT‐Transduced T Cells by Immunomagnetic Sorting
  • Basic Protocol 5: Generation of Clonal TERT‐Immortalized T Cell Lines by Limiting Dilution
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Activation of Primary T Cells with Plate‐Bound Anti‐CD3 Antibody

  Materials
  • Mitogenic anti‐CD3 monoclonal antibody: e.g., purified NA/LE mouse anti‐human CD3ɛ antibody (Becton Dickinson, cat. no. 557052; this antibody works well with both human and rhesus T lymphocytes); store at 4°C
  • Dulbecco's phosphate‐buffered saline (D‐PBS; Invitrogen, cat. no. 14190144)
  • Primary T cells: e.g., freshly isolated human or macaque PBMC, antigen‐specific T cell line, or clone
  • T cell medium (see recipe)
  • 24‐, 12‐, or 6‐well polystyrene tissue culture plates (Corning)
  • Centrifuge

Alternate Protocol 1: Activation of Primary T Cells with Soluble Anti‐CD3 Antibody

  Materials
  • Freshly obtained human PBMC (to use as feeder cells; unit 7.1)
  • T cell medium (see recipe)
  • Mitomycin C (Sigma‐Aldrich, cat. no. M4287)
  • Dulbecco's phosphate‐buffered saline (D‐PBS; Invitrogen, cat. no. 14190144)
  • RPMI 1640 medium containing 10% FBS
  • Mitogenic anti‐CD3 monoclonal antibody: e.g., purified NA/LE mouse anti‐human CD3ɛ antibody (Becton Dickinson, cat. no. 557052; this antibody works well with both human and rhesus T lymphocytes); store at 4°C
  • Primary T cells: e.g., freshly isolated human or macaque PBMC, antigen‐specific T cell line or clone
  • γ‐irradiator
  • Centrifuge
  • 25‐cm2 cell culture flasks
  • Additional reagents and equipment for counting cells ( appendix 3A)

Alternate Protocol 2: Activation of Human Primary T Cells with Concanavalin A

  Materials
  • Concanavalin A (Sigma‐Aldrich, cat. no. C0412); dissolve in Dulbecco's phosphate‐buffered saline (D‐PBS; Invitrogen, cat. no. 14190144) at a final concentration of 1 mg/ml; aliquot and store frozen at −20°C
  • T cell medium (see recipe)
  • Primary T cells: e.g., freshly isolated human or macaque PBMC, antigen‐specific T cell line or clone
  • Polystyrene 24‐, 12‐, or 6‐well tissue culture plates (Corning)

Basic Protocol 2: Generation of TERT‐Expressing Retroviral Vector Stocks

  Materials
  • GP2‐293 packaging cell line (Invitrogen), maintained in packaging cell medium with penicillin‐streptomycin as subconfluent monolayer
  • Packaging cell medium with penicillin‐streptomycin (see recipe)
  • Dulbecco's phosphate‐buffered saline (D‐PBS; Invitrogen, cat. no. 14190144)
  • 0.05% trypsin‐EDTA (Invitrogen, cat. no. 2530054); store at 4°C.
  • Packaging cell medium without antibiotics (see recipe)
  • OptiMEM reduced serum medium (Invitrogen, cat. no. 31985062); store at 4°C
  • hTERT‐expressing retroviral vector construct—two constructs expressing either ΔNGFR (Andersen et al., ) or GFP cDNA as marker genes are available; store frozen at −20°C (hTERT‐expressing retroviral constructs and producer cell lines can be obtained from Dr. Eugene Barsov, ; they are not available commercially)
  • Retroviral envelope expression constructs (GaLV, or RD114); store frozen at −20°C
  • Lipofectamine 2000 transfection reagent (Invitrogen, cat. no. 11668019); store at 4°C
  • 1 M HEPES buffer pH 7.5 (Invitrogen, cat. no. 15630080)
  • Sterile polystyrene round‐bottom tubes with snap‐top (12 × 75 mm, BD Falcon, cat. no. 352058)
  • 10‐cm poly‐D‐lysine coated cell culture plates (Becton Dickinson, cat. no. 354469)
  • Centrifuge
  • 15‐ml polypropylene tubes (e.g., BD Falcon)
  • Additional reagents and equipment for counting viable cells ( appendix 3A)

Alternate Protocol 3: Generation of Stocks from Cloned TERT‐Expressing Retroviral Vector Producer Cell Lines

  • GP2xTERT11 or GP2xTERT‐G22 vector producer cell lines (hTERT‐expressing retroviral constructs and producer cell lines can be obtained from Dr. Eugene Barsov, ; they are not available commercially)

Basic Protocol 3: Transduction of Primary T Cells with TERT Retroviral Vectors

  Materials
  • RetroNectin working solution (see recipe)
  • Blocking solution (see recipe)
  • Hanks balanced salt solution (HBSS; Invitrogen, cat. no. 14170112); store at 4°C
  • TERT retroviral vector stock, freshly prepared as described above in protocol 4 or protocol 3 (avoid storing the preparation); to minimize retroviral particle inactivation and prevent vector titer loss, harvest retroviral vector‐containing cell culture medium and prepare the stock at the time when RetroNectin‐coated plates are being blocked by the blocking solution (see step 4, below)
  • Target T cells that were activated 72 hr before transduction as described above in protocol 1 or Alternate Protocols protocol 21 and protocol 32; the cells should be robustly proliferating
  • T cell medium (see recipe), freshly prepared
  • Anti‐human NGFR (CD271) monoclonal antibody conjugated with PE (Becton‐Dickinson, cat. no. 557196)
  • Non‐tissue‐culture‐treated multi‐well plates (BD Falcon: 12‐well plates, cat. no. 351143; 24‐well plates, cat. no. 351147)
  • Refrigerated centrifuge.
  • Centrifuge with multi‐well‐plate holders
  • Additional reagents and equipment for counting viable cells ( appendix 3A)

Basic Protocol 4: Isolation of TERT‐Transduced T Cells by Immunomagnetic Sorting

  Materials
  • TERT‐transduced primary T cell culture (see protocols above)
  • Magnetic sorting buffer (see recipe); keep on ice during use
  • Anti‐human NGFR (CD271) monoclonal antibody conjugated to PE (Becton Dickinson, cat. no. 557196)
  • Anti‐PE paramagnetic microbeads (Miltenyi Biotec, cat. no. 130‐048‐801); store at 4°C
  • T cell medium (see recipe)
  • 15‐ml polypropylene centrifuge tubes (Corning, cat. no. 430052)
  • Refrigerated centrifuge
  • LS MACS separation columns (Miltenyi Biotec, cat. no. 130‐042‐401); store at room temperature
  • MidiMACS Separation Unit (Miltenyi Biotec, cat. no. 130‐042‐302); prechill at 4°C before cell separation
  • MACS MultiStand (Miltenyi Biotec, cat. no. 130‐042‐303); prechill at 4°C before cell separation
  • 25‐cm2 cell culture flask
  • Microscope
  • Additional reagents and equipment for counting viable cells ( appendix 3A) and preparing feeder cells ( protocol 2)

Basic Protocol 5: Generation of Clonal TERT‐Immortalized T Cell Lines by Limiting Dilution

  Materials
  • TERT‐transduced T cell line prepared by immunomagnetic (see protocol 7) or FACS sorting
  • T cell cloning medium (see recipe), freshly prepared
  • T cell medium (see recipe but use 20% FBS) with double concentration (200 IU/ml) of IL‐2
  • T cell medium (see recipe; use 10% FBS) containing 100 IU/ml (normal concentration) of IL‐2
  • 50‐ml polypropylene centrifuge tubes (Corning, cat. no. 430290)
  • Multichannel pipettor and sterile plastic 50‐ml reagent reservoirs (Corning, cat. no. 4870)
  • Polystyrene round‐bottom 96‐well tissue‐culture plates (Costar, cat. no. 3799)
  • Additional reagents and equipment for counting viable cells ( appendix 3A) and preparation of feeder cells ( protocol 2)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Akari, H., Mori, K., Terao, K., Otani, I., Fukasawa, M., Mukai, R., and Yoshikawa, Y. 1996. In vitro immortalization of Old World monkey T lymphocytes with Herpesvirus saimiri: Its susceptibility to infection with simian immunodeficiency viruses. Virology 218:382‐388.
   Andersen, H., Barsov, E.V., Trivett, M.T., Trubey, C.M., Giavedoni, L.D., Lifson, J.D., Ott, D.E., and Ohlen, C. 2007. Transduction with human telomerase reverse transcriptase immortalizes a rhesus macaque CD8+ T cell clone with maintenance of surface marker phenotype and function. AIDS Res. Hum. Retroviruses 23:456‐465.
   Barsov, E.V. 2009. Selective immortalization of tumor‐specific T cells to establish long‐term T‐cell lines maintaining primary cell characteristics. Methods Mol. Biol. 511:143‐158.
   Barsov, E.V., Andersen, H., Coalter, V.J., Carrington, M., Lifson, J.D., and Ott, D.E. 2006. Capture of antigen‐specific T lymphocytes from human blood by selective immortalization to establish long‐term T‐cell lines maintaining primary cell characteristics. Immunol. Lett. 105:26‐37.
   Brey, E.M. and Greisler, H.P. 2005. Telomerase expression in somatic cells. Lancet 365:2068‐2069.
   Chebel, A., Rouault, J.P., Urbanowicz, I., Baseggio, L., Chien, W.W., Salles, G., and Ffrench, M. 2009. Transcriptional activation of hTERT, the human telomerase reverse transcriptase, by nuclear factor of activated T cells. J. Biol. Chem. 284:35725‐35734.
   Coussens, M., Yamazaki, Y., Moisyadi, S., Suganuma, R., Yanagimachi, R., and Allsopp, R. 2006. Regulation and effects of modulation of telomerase reverse transcriptase expression in primordial germ cells during development. Biol. Reprod. 75:785‐791.
   Dagarag, M., Evazyan, T., Rao, N., and Effros, R.B. 2004. Genetic manipulation of telomerase in HIV‐specific CD8+ T cells: Enhanced antiviral functions accompany the increased proliferative potential and telomere length stabilization. J. Immunol. 173:6303‐6311.
   Dolci, S., Levati, L., Pellegrini, M., Faraoni, I., Graziani, G., Di Carlo, A., and Geremia, R. 2002. Stem cell factor activates telomerase in mouse mitotic spermatogonia and in primordial germ cells. J. Cell Sci. 115:1643‐1649.
   Duboise, S.M., Guo, J., Czajak, S., Desrosiers, R.C., and Jung, J.U. 1998. STP and Tip are essential for herpesvirus saimiri oncogenicity. J. Virol. 72:1308‐1313.
   Effros, R.B. 2007. Telomerase induction in T cells: A cure for aging and disease? Exp. Gerontol. 42:416‐420.
   Greider, C.W. 1994. Mammalian telomere dynamics: Healing, fragmentation shortening and stabilization. Curr. Opin. Genet. Dev. 4:203‐211.
   Guo, J., Williams, K., Duboise, S.M., Alexander, L., Veazey, R., and Jung, J.U. 1998. Substitution of ras for the herpesvirus saimiri STP oncogene in lymphocyte transformation. J. Virol. 72:3698‐3704.
   Haruta, Y., Hiyama, K., Ishioka, S., Hozawa, S., Maeda, H., and Yamakido, M. 1999. Activation of telomerase is induced by a natural antigen in allergen‐specific memory T lymphocytes in bronchial asthma. Biochem. Biophys. Res. Commun. 259:617‐623.
   Hemann, M.T., Hackett, I.J.A., and Greider, C.W. 2000. Telomere length, telomere‐binding proteins, and DNA damage signaling. Cold Spring Harb. Symp. Quant. Biol. 65:275‐279.
   Hiyama, E. and Hiyama, K. 2007. Telomere and telomerase in stem cells. Br. J. Cancer 96:1020‐1024.
   Hiyama, K., Hirai, Y., Kyoizumi, S., Akiyama, M., Hiyama, E., Piatyszek, M.A., Shay, J.W., Ishioka, S., and Yamakido, M. 1995. Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J. Immunol. 155:3711‐3715.
   Hooijberg, E., Ruizendaal, J.J., Snijders, P.J., Kueter, E.W., Walboomers, J.M., and Spits, H. 2000. Immortalization of human CD8+ T cell clones by ectopic expression of telomerase reverse transcriptase. J. Immunol. 165:4239‐4245.
   Hsu, C., Jones, S.A., Cohen, C.J., Zheng, Z., Kerstann, K., Zhou, J., Robbins, P.F., Peng, P.D., Shen, X., Gomes, T.J., Dunbar, C.E., Munroe, D.J., Stewart, C., Cornetta, K., Wangsa, D., Ried, T., Rosenberg, S.A., and Morgan, R.A. 2007. Cytokine‐independent growth and clonal expansion of a primary human CD8+ T‐cell clone following retroviral transduction with the IL‐15 gene. Blood 109:5168‐5177.
   Jung, J.U. and Desrosiers, R.C. 1991. Identification and characterization of the herpesvirus saimiri oncoprotein STP‐C488. J. Virol. 65:6953‐6960.
   Jung, J.U. and Desrosiers, R.C. 1992. Herpesvirus saimiri oncogene STP‐C488 encodes a phosphoprotein. J. Virol. 66:1777‐1780.
   Jung, J.U. and Desrosiers, R.C. 1995. Association of the viral oncoprotein STP‐C488 with cellular ras. Mol. Cell Biol. 15:6506‐6512.
   Lansdorp, P.M. 2005. Role of telomerase in hematopoietic stem cells. Ann. N.Y. Acad. Sci. 1044:220‐227.
   Lewis, P.F. and Emerman, M. 1994. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J. Virol. 68:510‐516.
   Li, Y., Zhi, W., Wareski, P., and Weng, N.P. 2005. IL‐15 activates telomerase and minimizes telomere loss and may preserve the replicative life span of memory CD8+ T cells in vitro. J. Immunol. 174:4019‐4024.
   Loffredo, J.T., Rakasz, E.G., Giraldo, J.P., Spencer, S.P., Grafton, K.K., Martin, S.R., Napoe, G., Yant, L.J., Wilson, N.A., and Watkins, D.I. 2005. Tat(28‐35)SL8‐specific CD8+ T lymphocytes are more effective than Gag(181‐189)CM9‐specific CD8+ T lymphocytes at suppressing simian immunodeficiency virus replication in a functional in vitro assay. J. Virol. 79:14986‐14991.
   Luiten, R.M., Pene, J., Yssel, H., and Spits, H. 2003. Ectopic hTERT expression extends the life span of human CD4+ helper and regulatory T‐cell clones and confers resistance to oxidative stress‐induced apoptosis. Blood 101:4512‐4519.
   Minang, J.T., Barsov, E.V., Yuan, F., Trivett, M.T., Piatak, M. Jr., Lifson, J.D., Ott, D.E., and Ohlen, C. 2008. Efficient inhibition of SIV replication in rhesus CD4+ T‐cell clones by autologous immortalized SIV‐specific CD8+ T‐cell clones. Virology 372:430‐441.
   Roe, T., Reynolds, T.C., Yu, G., and Brown, P.O. 1993. Integration of murine leukemia virus DNA depends on mitosis. Embo J. 12:2099‐2108.
   Roth, A., Yssel, H., Pene, J., Chavez, E.A., Schertzer, M., Lansdorp, P.M., Spits, H., and Luiten, R.M. 2003. Telomerase levels control the lifespan of human T lymphocytes. Blood 102:849‐857.
   Rufer, N., Migliaccio, M., Antonchuk, J., Humphries, R.K., Roosnek, E., and Lansdorp, P.M. 2001. Transfer of the human telomerase reverse transcriptase (TERT) gene into T lymphocytes results in extension of replicative potential. Blood 98:597‐603.
   Slijepcevic, P. and Al‐Wahiby, S. 2005. Telomere biology: Integrating chromosomal end protection with DNA damage response. Chromosoma 114:275‐285.
   Vaziri, H. and Benchimol, S. 1996. From telomere loss to p53 induction and activation of a DNA‐damage pathway at senescence: The telomere loss/DNA damage model of cell aging. Exp. Gerontol. 31:295‐301.
   Verra, N.C., Jorritsma, A., Weijer, K., Ruizendaal, J.J., Voordouw, A., Weder, P., Hooijberg, E., Schumacher, T.N., Haanen, J.B., Spits, H., and Luiten, R.M. 2004. Human telomerase reverse transcriptase‐transduced human cytotoxic T cells suppress the growth of human melanoma in immunodeficient mice. Cancer Res. 64:2153‐2161.
   Wallace, D.L., Berard, M., Soares, M.V., Oldham, J., Cook, J.E., Akbar, A.N., Tough, D.F., and Beverley, P.C. 2006. Prolonged exposure of naive CD8+ T cells to interleukin‐7 or interleukin‐15 stimulates proliferation without differentiation or loss of telomere length. Immunology 119:243‐253.
   Weng, N.P., Levine, B.L., June, C.H., and Hodes, R.J. 1996. Regulated expression of telomerase activity in human T lymphocyte development and activation. J. Exp. Med. 183:2471‐2479.
   Weng, N., Levine, B.L., June, C.H., and Hodes, R.J. 1997a. Regulation of telomerase RNA template expression in human T lymphocyte development and activation. J. Immunol. 158:3215‐3220.
   Weng, N.P., Palmer, L.D., Levine, B.L., Lane, H.C., June, C.H., and Hodes, R.J. 1997b. Tales of tails: Regulation of telomere length and telomerase activity during lymphocyte development, differentiation, activation, and aging. Immunol. Rev. 160:43‐54.
   Weng, N.P., Hathcock, K.S., and Hodes, R.J. 1998. Regulation of telomere length and telomerase in T and B cells: A mechanism for maintaining replicative potential. Immunity 9:151‐157.
   Yang, Y., An, J., and Weng, N.P. 2008. Telomerase is involved in IL‐7‐mediated differential survival of naive and memory CD4+ T cells. J. Immunol. 180:3775‐3781.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library