Differentiation and Characterization of T Cells

Paul Robertson1, David T. Scadden1

1 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 22F.8
DOI:  10.1002/0471142735.im22f08s69
Online Posting Date:  November, 2005
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Abstract

This paper outlines current standard methods of inducing T lymphopoiesis in vitro and in vivo. Reference is made to both murine and human systems. In addition to differentiation assays, methods to optimally characterize output cells are discussed. In bringing together a number of existing protocols, many techniques important in investigating T cell development can be reviewed in one place.

Keywords: T cell development; Fetal thymic organ culture (FTOC); Intrathymic injection; T cell characterization

     
 
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Table of Contents

  • Basic Protocol 1: T Cell Differentiation of Human CD34 Cells using Nod‐Scid Fetal Thymic Organ Culture
  • Basic Protocol 2: T Cell Differentiation of CD34 Cells using Intrathymic Injection of Nod‐Scid Thymus
  • Basic Protocol 3: Characterization of T Cells
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: T Cell Differentiation of Human CD34 Cells using Nod‐Scid Fetal Thymic Organ Culture

  Materials
  • Day 14 to 15 fetal NOD‐LtSz‐scid/scid (NOD‐SCID) mice from timed‐pregnant females (e.g., The Jackson Laboratory; see unit 1.1 for precautions when housing immunocompromised mice)
  • Complete IMDM‐10 medium ( appendix 2A)
  • Human cord blood or bone marrow
  • Terasaki plates (e.g., Nunc; see unit 3.18)
  • Additional reagents and equipment isolation of thymic lobes from mice (unit 3.18, Basic Protocol), human CD34 hematopoietic progenitor cell isolation (unit 22.1), and reconsititution of thymic lobes with stem cells (unit 3.18, Alternate Protocol for hematopoietic depletion and reconstruction of fetal thymic lobes, steps 6 to 8)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 2: T Cell Differentiation of CD34 Cells using Intrathymic Injection of Nod‐Scid Thymus

  Materials
  • C57BL/6 mice (e.g., The Jackson Laboratory)
  • Ketamine⋅HCl (e.g., Fort Dodge Animal Health)
  • Xylazine (e.g., Fort Dodge Animal Health)
  • Suspension of stem or progenitor cells (unit 22.1)
  • Betadine (10% povidone‐iodine; Purdue Frederick) or 70% ethanol
  • Cesium irradiator
  • Sterile 1‐cc syringe with 25‐G needle
  • Dissecting board
  • Paper towels, sterile
  • Gauze, sterile
  • Elastic bands
  • Directional light source
  • Surgical instruments (sterile) including:
    • Fine‐point scissors, (10‐cm, straight, blunt‐ended)
    • 2 pairs fine‐point forceps (10‐cm, half‐curved)
    • Autoclip wound applier for 9‐mm clips
  • 9‐mm stainless steel wound clips
  • Additional reagents and equipment for mouse anesthesia using ketamine and xylazine (unit 1.4), intraperitoneal injection (unit 1.6), thymectomy (unit 1.10), microisolation housing (unit 1.2), euthanasia (unit 1.8), and preparing single‐cell suspension (unit 3.1)
NOTE: The technique described below depends on survival surgery, and sterility is vital for success. It is assumed proper sterile technique is followed throughout. Also see unit 1.10.
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Figures

Videos

Literature Cited

Literature Cited
   Allman, D., Sambandam, A., Kim S., Miller, J.P., Pagan, A., Well, D., Meraz, A., and Bhandoola, A. 2003. Thymopoietic independent of common lymphoid progenitors. Nat. Immunol. 4:168‐174.
   Anderson, G., Owen, J.J., Moore, N.C., and Jenkinson, E.J. 1994. Characteristics of an in vitro system of thymocyte positive selection. J. Immunol. 153:1915‐1920.
   Douek, D.C., McFarland, R.D., Keiser, P.H., Gage, E.A., Massey, J.M., Haynes, B.F., Polis, M.A., Haase, A.T., Feinberg, M.B., Sullivan, J.L., Jamieson, B.D., Zack, J.A., Picker, L.J., and Koup, R.A. 1998. Changes in thymic function with age and during treatment of HIV infection. Nature 396:690‐695.
   Freedman, A.R., Zhu, H., Levine, J.D., Kalams, S., and Scadden, D.T. 1996. Generation of human T lymphocytes from bone marrow CD34+ cells in vitro. Nat Med. 2:46‐51.
   Gorski, J., Yassai, M., Zhu, X., Kissela, B., Keever, C., and Flomberg, N. 1994. Circulating T cell repertoire complexity in normal individuals and bone marrow recipients analyzed by CDR3 size spectratyping: Correlation with immune status. J. Immunol. 152:5109‐5119.
   Hazenberg, M.D., Otto, S.A., Cohen Stuart, J.W., Verschuren, M.C., Borleffs, J.C., Boucher, C.A., Coutinho, R.A., Lange, J.M., Rinke de Wit, T.F., Tsegaye, A., van Dongen, J.J., Hamann, D., de Boer, R.J., and Miedema, F. 2000. Increased cell division but not thymic dysfunction rapidly affects the T cell receptor excision circle content of the native population in HIV‐1 infection. Nat. Med. 396:690‐695.
   Kerre, T.C., De Smedt, G., De Smedt, M., Zippelius, A., Pittet, M.J., Langerak, A.W., De Bosscher, J., Offner, F., Vandekerckhove, B., and Plum, J. 2002. Adapted NOD/SCID model supports development of phenotypically and functionally mature T cells from human umbilical cord blood CD34(+) cells. Blood 99:1620‐1626.
   Kollet, O., Peled, A., Byk, T., Ben‐Hur, H., Greiner, D., Shultz, L., and Lapidot, T. 2000. β2 microglobulin‐deficient (β2m(null)) NOD/SCID mice are excellent recipients for studying human stem cell function. Blood 95:3102‐3105.
   Kong, F., Chen, C.H., and Cooper, M.D. 1998. Thymic function can be accurately monitored by the level of recent T‐cell emigrant in the circulation. Immunity 8:97‐104.
   Poznansky, M.C., Evans, R.H., Foxall, R.B., Olszak, I.T., Piascik, A.H., Hartman, K.E., Brander, C., Meyer, T.H., Pykett, J.J., Chabner, K.T., Kalams, S.A., Rosenzweig, M., and Scadden, D.T. 2000. Efficient generation of human T cells from a tissue engineered thymic organoid. Nature Biotechnol. 18:729‐734.
   Sempowski, G.D. and Haynes, B.F. 2002. Immune reconstitution in patients with HIV infection. Annu. Rev. Med. 53:269‐284.
   Spits, H. 2002 Development of αβ T cells in the human thymus. Nat. Immunol. Rev. 2:760‐772.
   Suniara, R.K., Jenkinson, E.J., and Owen, J.J.T. 1999. Studies on the phenotype of migrant thymic stem cells. Eur. J. Immunol. 29:75‐80.
   Ye, P. and Kirschner, D.E. 2002. Reevaluation of T cell receptor circles as a measure of recent thymic emigrants. J. Immunol. 169:4968‐4979.
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