Cancer Immunology, Immunotherapy

, Volume 60, Issue 5, pp 739–749 | Cite as

In vitro generated anti-tumor T lymphocytes exhibit distinct subsets mimicking in vivo antigen-experienced cells

  • Shicheng Yang
  • Gattinoni Luca
  • Fang Liu
  • Yun Ji
  • Zhiya Yu
  • Nicholas P. Restifo
  • Steven A. Rosenberg
  • Richard A. Morgan
Original article

Abstract

The T-lymphocyte pool can be subdivided into naïve (Tn), effector memory (Tem), and central memory (Tcm) T cells. In this study, we characterized in vitro short-term cultured anti-tumor human T lymphocytes generated by lentiviral transduction with an anti-tumor antigen TCR vector. Within 2 weeks of in vitro culture, the cultured T cells showed a Tcm-like phenotype illustrated by a high percentage of CD62L and CD45RO cells. When the cells were sorted into populations that were CD45RO+/CD62L-(Tem), CD45RO+/CD62L+(Tcm), or CD45ROlow/CD62L+(Tn) and co-cultured with antigen-matched tumor lines, the magnitude of cytokine release from these populations for IFNγ (Tn < Tcm < Tem) and IL-2 (Tn > Tcm > Tem) mimicked the types of immune cell responses observed in vivo. In comparing cell-mediated effector function, Tn were found to be deficient (relative to Tcm and Tem) in the ability to form conjugates with tumor cells and subsequent lytic activity. Moreover, analysis of the gene expression profiles of the in vitro cultured and sorted T-cell populations also demonstrated patterns consistent with their in vivo counterparts. When Tcm and Tem were tested for the ability to survive in vivo, Tcm displayed significantly increased engraftment and persistence in NOD/SCID/γc−/− mice. In general, a large percentage of in vitro generated anti-tumor T lymphocytes mimic a Tcm-like phenotype (based on phenotype, effector function, and increased persistence in vivo), which suggests that these Tcm-like cultured T cells may be optimal for adoptive immunotherapy.

Keywords

Gene therapy Lentiviral vector T-cell receptor Central memory cells Effector memory cells Tumor immunity 

Supplementary material

262_2011_977_MOESM1_ESM.eps (722 kb)
Sorting of in vitro cultured anti-tumor T lymphocytes for in vivo engraftment. A. Schematic illustration of in vitro transduced and expanded T lymphocytes. PBMC were activated by anti-CD3/CD28 beads. The next day, cells were transduced with lentiviral vector harboring anti-tumor TCR, and 6 h later, the cells were transferred from 6-well plates to 75-cm2 flasks. The cells were maintained for 14 days before harvesting. B. Calibration for sorting and post-evaluation. Upper panel, the percentage of cells defined by square in Tem and Tcm was sorted; middle panel, post-sort re-evaluation; lower panel, the expression of transduced anti-tumor TCR on sorted Tem and Tcm populations was measured by MART-1 Tetramer staining (EPS 721 kb)
262_2011_977_MOESM2_ESM.eps (431 kb)
Summary for generation of anti-tumor T lymphocytes using anti-CD3/CD28 beads activation and lentiviral vector transduction. Twenty million of PBMC per well of 6-well plates from 6 donors were activated by anti-CD3/CD28 beads on day 0 (beads to cells, 2:1), and on day 1 post-stimulation, the cells were transduced with lentiviral vector harboring MART-1 antigen TCR by spinoculation. Six hours post-transduction, the cells were transferred to 75-cm2 culture dishes with a total volume of 30 ml culture medium in horizontal position, and the cell density was maintained below 1.5 × 106/ml in culture for 14 days. The phenotype of transduced PBMC at day 14 was analyzed using a panel of antibodies as denoted on top of each FACS image. The fold expansion from each donor was shown on last column of the table (EPS 431 kb)
262_2011_977_MOESM3_ESM.xlt (616 kb)
Gene expression profile of sorted Tem, Tcm, and Tn populations analyzed by Microarray. The total RNA extracted from sorted populations was previously described in Fig. 5. One-way hierarchical clustering of sorted T-cell samples was conducted, and 2-fold differentially expressed genes were chosen for analysis. A set of 597 probe IDs was identified as differentially expressed among cultured T-cell subsets and used for clustering (XLT 616 kb)

References

  1. 1.
    Dudley ME, Wunderlich JR, Yang JC, Sherry RM, Topalian SL, Restifo NP, Royal RE, Kammula U, White DE, Mavroukakis SA, Rogers LJ, Gracia GJ, Jones SA, Mangiameli DP, Pelletier MM, Gea-Banacloche J, Robinson MR, Berman DM, Filie AC, Abati A, Rosenberg SA (2005) Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J Clin Oncol 23:2346–2357PubMedCrossRefGoogle Scholar
  2. 2.
    Dudley ME, Yang JC, Sherry R, Hughes MS, Royal R, Kammula U, Robbins PF, Huang J, Citrin DE, Leitman SF, Wunderlich J, Restifo NP, Thomasian A, Downey SG, Smith FO, Klapper J, Morton K, Laurencot C, White DE, Rosenberg SA (2008) Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens. J Clin Oncol 26:5233–5239PubMedCrossRefGoogle Scholar
  3. 3.
    Rosenberg SA, Dudley ME (2009) Adoptive cell therapy for the treatment of patients with metastatic melanoma. Curr Opin Immunol 21:233–240PubMedCrossRefGoogle Scholar
  4. 4.
    Morgan RA, Dudley ME, Rosenberg SA (2010) Adoptive cell therapy: genetic modification to redirect effector cell specificity. Cancer J 16:336–341PubMedCrossRefGoogle Scholar
  5. 5.
    Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP, Zheng Z, Nahvi A, de Vries CR, Rogers-Freezer LJ, Mavroukakis SA, Rosenberg SA (2006) Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314:126–129PubMedCrossRefGoogle Scholar
  6. 6.
    Johnson LA, Morgan RA, Dudley ME, Cassard L, Yang JC, Hughes MS, Kammula US, Royal RE, Sherry RM, Wunderlich JR, Lee CC, Restifo NP, Schwarz SL, Cogdill AP, Bishop RJ, Kim H, Brewer CC, Rudy SF, Vanwaes C, Davis JL, Mathur A, Ripley RT, Nathan DA, Laurencot CM, Rosenberg SA (2009) Gene therapy with human and mouse T cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood 9:359–363Google Scholar
  7. 7.
    Berger C, Jensen MC, Lansdorp PM, Gough M, Elliott C, Riddell SR (2008) Adoptive transfer of effector CD8 + T cells derived from central memory cells establishes persistent T cell memory in primates. J Clin Invest 118:294–305PubMedCrossRefGoogle Scholar
  8. 8.
    Gattinoni L, Klebanoff CA, Palmer DC, Wrzesinski C, Kerstann K, Yu Z, Finkelstein SE, Theoret MR, Rosenberg SA, Restifo NP (2005) Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8 + T cells. J Clin Invest 115:1616–1626PubMedCrossRefGoogle Scholar
  9. 9.
    Yang S, Rosenberg SA, Morgan RA (2008) Clinical-scale lentiviral vector transduction of PBL for TCR gene therapy and potential for expression in less-differentiated cells. J Immunother 31:830–839PubMedCrossRefGoogle Scholar
  10. 10.
    Lea NC, Orr SJ, Stoeber K, Williams GH, Lam EW, Ibrahim MA, Mufti GJ, Thomas NS (2003) Commitment point during G0 → G1 that controls entry into the cell cycle. Mol Cell Biol 23:2351–2361PubMedCrossRefGoogle Scholar
  11. 11.
    Suhoski MM, Golovina TN, Aqui NA, Tai VC, Varela-Rohena A, Milone MC, Carroll RG, Riley JL, June CH (2007) Engineering artificial antigen-presenting cells to express a diverse array of co-stimulatory molecules. Mol Ther 15:981–988PubMedCrossRefGoogle Scholar
  12. 12.
    Campbell JJ, Bowman EP, Murphy K, Youngman KR, Siani MA, Thompson DA, Wu L, Zlotnik A, Butcher EC (1998) 6-C-kine (SLC), a lymphocyte adhesion-triggering chemokine expressed by high endothelium, is an agonist for the MIP-3beta receptor CCR7. J Cell Biol 141:1053–1059PubMedCrossRefGoogle Scholar
  13. 13.
    Forster R, Schubel A, Breitfeld D, Kremmer E, Renner-Muller I, Wolf E, Lipp M (1999) CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99:23–33PubMedCrossRefGoogle Scholar
  14. 14.
    Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A (1999) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708–712PubMedCrossRefGoogle Scholar
  15. 15.
    Sallusto F, Geginat J, Lanzavecchia A (2004) Central memory and effector memory T cell subsets: function, generation, and maintenance. Annu Rev Immunol 22:745–763PubMedCrossRefGoogle Scholar
  16. 16.
    Willinger T, Freeman T, Hasegawa H, McMichael AJ, Callan MF (2005) Molecular signatures distinguish human central memory from effector memory CD8 T cell subsets. J Immunol 175:5895–5903PubMedGoogle Scholar
  17. 17.
    Gattinoni L, Zhong XS, Palmer DC, Ji Y, Hinrichs CS, Yu Z, Wrzesinski C, Boni A, Cassard L, Garvin LM, Paulos CM, Muranski P, Restifo NP (2009) Wnt signaling arrests effector T cell differentiation and generates CD8 + memory stem cells. Nat Med 15:808–813PubMedCrossRefGoogle Scholar
  18. 18.
    Turtle CJ, Swanson HM, Fujii N, Estey EH, Riddell SR (2009) A distinct subset of self-renewing human memory CD8 + T cells survives cytotoxic chemotherapy. Immunity 31:834–844PubMedCrossRefGoogle Scholar
  19. 19.
    Bouneaud C, Garcia Z, Kourilsky P, Pannetier C (2005) Lineage relationships, homeostasis, and recall capacities of central- and effector-memory CD8 T cells in vivo. J Exp Med 201:579–590PubMedCrossRefGoogle Scholar
  20. 20.
    Klebanoff CA, Gattinoni L, Torabi-Parizi P, Kerstann K, Cardones AR, Finkelstein SE, Palmer DC, Antony PA, Hwang ST, Rosenberg SA, Waldmann TA, Restifo NP (2005) Central memory self/tumor-reactive CD8 + T cells confer superior antitumor immunity compared with effector memory T cells. Proc Natl Acad Sci USA 102:9571–9576PubMedCrossRefGoogle Scholar
  21. 21.
    Levine BL (2008) T lymphocyte engineering ex vivo for cancer and infectious disease. Expert Opin Biol Ther 8:475–489PubMedCrossRefGoogle Scholar
  22. 22.
    Wherry EJ, Teichgraber V, Becker TC, Masopust D, Kaech SM, Antia R, von Andrian UH, Ahmed R (2003) Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat Immunol 4:225–234PubMedCrossRefGoogle Scholar
  23. 23.
    Jones S, Peng PD, Yang S, Hsu C, Cohen CJ, Zhao Y, Abad J, Zheng Z, Rosenberg SA, Morgan RA (2009) Lentiviral vector design for optimal T cell receptor gene expression in the transduction of peripheral blood lymphocytes and tumor-infiltrating lymphocytes. Hum Gene Ther 20:630–640PubMedCrossRefGoogle Scholar
  24. 24.
    Schambach A, Bohne J, Baum C, Hermann FG, Egerer L, von Laer D, Giroglou T (2006) Woodchuck hepatitis virus post-transcriptional regulatory element deleted from X protein and promoter sequences enhances retroviral vector titer and expression. Gene Ther 13:641–645PubMedCrossRefGoogle Scholar
  25. 25.
    Johnson LA, Heemskerk B, Powell DJ Jr, Cohen CJ, Morgan RA, Dudley ME, Robbins PF, Rosenberg SA (2006) Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes. J Immunol 177:6548–6559PubMedGoogle Scholar
  26. 26.
    Yang S, Dudley ME, Rosenberg SA, Morgan RA (2010) A simplified method for the clinical-scale generation of central memory-like CD8 + T cells after transduction with lentiviral vectors encoding antitumor antigen T-cell receptors. J Immunother 33:648–658PubMedCrossRefGoogle Scholar
  27. 27.
    Yang S, Cohen CJ, Peng PD, Zhao Y, Cassard L, Yu Z, Zheng Z, Jones S, Restifo NP, Rosenberg SA, Morgan RA (2008) Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition. Gene Ther 15:1411–1423PubMedCrossRefGoogle Scholar
  28. 28.
    Voss CY, Deola S, Fleisher TA, Marincola FM (2009) Increased effector-target cell conjugate formation due to HLA restricted specific antigen recognition. Immunol Res 45:13–24PubMedCrossRefGoogle Scholar
  29. 29.
    Topalian SL, Solomon D, Rosenberg SA (1989) Tumor-specific cytolysis by lymphocytes infiltrating human melanomas. J Immunol 142:3714–3725PubMedGoogle Scholar
  30. 30.
    Hinrichs CS, Borman ZA, Cassard L, Gattinoni L, Spolski R, Yu Z, Sanchez-Perez L, Muranski P, Kern SJ, Logun C, Palmer DC, Ji Y, Reger RN, Leonard WJ, Danner RL, Rosenberg SA, Restifo NP (2009) Adoptively transferred effector cells derived from naive rather than central memory CD8 + T cells mediate superior antitumor immunity. Proc Natl Acad Sci USA 106:17469–17474PubMedCrossRefGoogle Scholar
  31. 31.
    Campbell JJ, Murphy KE, Kunkel EJ, Brightling CE, Soler D, Shen Z, Boisvert J, Greenberg HB, Vierra MA, Goodman SB, Genovese MC, Wardlaw AJ, Butcher EC, Wu L (2001) CCR7 expression and memory T cell diversity in humans. J Immunol 166:877–884PubMedGoogle Scholar
  32. 32.
    Wargo JA, Robbins PF, Li Y, Zhao Y, El-Gamil M, Caragacianu D, Zheng Z, Hong JA, Downey S, Schrump DS, Rosenberg SA, Morgan RA (2009) Recognition of NY-ESO-1 + tumor cells by engineered lymphocytes is enhanced by improved vector design and epigenetic modulation of tumor antigen expression. Cancer Immunol Immunother 58:383–394PubMedCrossRefGoogle Scholar
  33. 33.
    Parkhurst MR, Yang JC, Langan RC, Dudley ME, Nathan DA, Feldman SA, Davis JL, Morgan RA, Merino MJ, Sherry RM, Hughes MS, Kammula US, Phan GQ, Lim RM, Wank SA, Restifo NP, Robbins PF, Laurencot CM, Rosenberg SA (2010) T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis. Mol Ther [Epub ahead of print]Google Scholar
  34. 34.
    Neeson P, Shin A, Tainton KM, Guru P, Prince HM, Harrison SJ, Peinert S, Smyth MJ, Trapani JA, Kershaw MH, Darcy PK, Ritchie DS (2010) Ex vivo culture of chimeric antigen receptor T cells generates functional CD8 + T cells with effector and central memory-like phenotype. Gene Ther 17:1105–1116PubMedCrossRefGoogle Scholar
  35. 35.
    Yi CH, Terrett JA, Li QY, Ellington K, Packham EA, Armstrong-Buisseret L, McClure P, Slingsby T, Brook JD (1999) Identification, mapping, and phylogenomic analysis of four new human members of the T-box gene family: EOMES, TBX6, TBX18, and TBX19. Genomics 55:10–20PubMedCrossRefGoogle Scholar
  36. 36.
    Pennica D, Nedwin GE, Hayflick JS, Seeburg PH, Derynck R, Palladino MA, Kohr WJ, Aggarwal BB, Goeddel DV (1984) Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin. Nature 312:724–729PubMedCrossRefGoogle Scholar
  37. 37.
    Lasorella A, Boldrini R, Dominici C, Donfrancesco A, Yokota Y, Inserra A, Iavarone A (2002) Id2 is critical for cellular proliferation and is the oncogenic effector of N-myc in human neuroblastoma. Cancer Res 62:301–306PubMedGoogle Scholar
  38. 38.
    Mattarollo SR, Rahimpour A, Choyce A, Godfrey DI, Leggatt GR, Frazer IH (2010) Invariant NKT cells in hyperplastic skin induce a local immune suppressive environment by IFN-gamma production. J Immunol 184:1242–1250PubMedCrossRefGoogle Scholar
  39. 39.
    International Human Genome Sequencing Consortium (2004) Finishing the euchromatic sequence of the human genome. Nature 431:931–945Google Scholar
  40. 40.
    Meier M, Kwong PC, Fregeau CJ, Atkinson EA, Burrington M, Ehrman N, Sorensen O, Lin CC, Wilkins J, Bleackley RC (1990) Cloning of a gene that encodes a new member of the human cytotoxic cell protease family. Biochemistry 29:4042–4049PubMedCrossRefGoogle Scholar
  41. 41.
    Yabe T, McSherry C, Bach FH, Houchins JP (1990) A cDNA clone expressed in natural killer and T cells that likely encodes a secreted protein. J Exp Med 172:1159–1163PubMedCrossRefGoogle Scholar
  42. 42.
    Davila S, Froeling FE, Tan A, Bonnard C, Boland GJ, Snippe H, Hibberd ML, Seielstad M (2010) New genetic associations detected in a host response study to hepatitis B vaccine. Genes Immun 11:232–238PubMedCrossRefGoogle Scholar
  43. 43.
    Kreil S, Waghorn K, Ernst T, Chase A, White H, Hehlmann R, Reiter A, Hochhaus A, Cross NC (2010) A polymorphism associated with STAT3 expression and response of chronic myeloid leukemia to interferon alpha. Haematologica 95:148–152PubMedCrossRefGoogle Scholar
  44. 44.
    Jesse S, Koenig A, Ellenrieder V, Menke A (2010) Lef-1 isoforms regulate different target genes and reduce cellular adhesion. Int J Cancer 126:1109–1120PubMedGoogle Scholar
  45. 45.
    Davila S, Froeling FE, Tan A, Bonnard C, Boland GJ, Snippe H, Hibberd ML, Seielstad M (2010) New genetic associations detected in a host response study to hepatitis B vaccine. Genes Immun 11:(3)232–238Google Scholar
  46. 46.
    Jorgensen JS, Gao L (2005) Irx3 is differentially up-regulated in female gonads during sex determination. Gene Expr Patterns 5:756–762PubMedCrossRefGoogle Scholar
  47. 47.
    Joulin V, Bories D, Eleouet JF, Labastie MC, Chretien S, Mattei MG, Romeo PH (1991) A T-cell specific TCR delta DNA binding protein is a member of the human GATA family. EMBO J 10:1809–1816PubMedGoogle Scholar
  48. 48.
    Sasaki T, Brakebusch C, Engel J, Timpl R (1998) Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin. EMBO J 17:1606–1613PubMedCrossRefGoogle Scholar
  49. 49.
    Latza U, Durkop H, Schnittger S, Ringeling J, Eitelbach F, Hummel M, Fonatsch C, Stein H (1994) The human OX40 homolog: cDNA structure, expression and chromosomal assignment of the ACT35 antigen. Eur J Immunol 24:677–683PubMedCrossRefGoogle Scholar
  50. 50.
    Arm JP, Nwankwo C, Austen KF (1997) Molecular identification of a novel family of human Ig superfamily members that possess immunoreceptor tyrosine-based inhibition motifs and homology to the mouse gp49B1 inhibitory receptor. J Immunol 159:2342–2349PubMedGoogle Scholar
  51. 51.
    King M, Pearson T, Shultz LD, Leif J, Bottino R, Trucco M, Atkinson MA, Wasserfall C, Herold KC, Woodland RT, Schmidt MR, Woda BA, Thompson MJ, Rossini AA, Greiner DL (2008) A new Hu-PBL model for the study of human islet alloreactivity based on NOD-scid mice bearing a targeted mutation in the IL-2 receptor gamma chain gene. Clin Immunol 126:303–314PubMedCrossRefGoogle Scholar
  52. 52.
    Shultz LD, Ishikawa F, Greiner DL (2007) Humanized mice in translational biomedical research. Nat Rev Immunol 7:118–130PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag (outside the USA) 2011

Authors and Affiliations

  • Shicheng Yang
    • 1
  • Gattinoni Luca
    • 1
  • Fang Liu
    • 1
  • Yun Ji
    • 1
  • Zhiya Yu
    • 1
  • Nicholas P. Restifo
    • 1
  • Steven A. Rosenberg
    • 1
  • Richard A. Morgan
    • 1
  1. 1.Surgery Branch, Center for Cancer Research, National Cancer InstituteNational Institutes of HealthBethesdaUSA

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