Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Technical Report
  • Published:

Amplification of T cells from human cord blood in serum-deprived culture stimulated with stem cell factor, interleukin-7 and interleukin-2

Bone Marrow Transplantation volume 31, pages 713–723 (2003)Cite this article

Summary:

We report the effects exerted by cytokine combinations, including stem cell factor (SCF), interleukin-7, interleukin-4 and interleukin-2, on the amplification of T cells from cord blood (CB) mononuclear cells cultured for 10–11 days under serum-deprived conditions. Of all the combinations investigated, SCF+interleukin-7 sustained the best fold increase (FI) of total nucleated cells (FI=6.4±1.17), amplifying preferentially CD4+ over CD8+ T-cell subsets (FI=4.72±0.79 vs 2.73±1.2, respectively, P<0.05). The addition of interleukin-2 to this combination did not significantly increase the total number of cells generated (FI=7.4±2.27), but allowed preferential amplification of CD8+ over CD4+ T cells (FI=6.04±0.14 vs 1.67±0.6, respectively, P<0.05). Single-strand conformation polymorphism analysis of the T-cell receptor Vβ-chain rearrangements expressed by the expanded T cells indicated that the complexity of the T-cell repertoire had increased after 10 days of culture in the presence of SCF and IL-7. Interestingly, a modest expansion (FI=8.67±1.5) of myeloid progenitor cells was also observed in these cultures. These results indicate that it is possible to expand specific T-cell subsets for adoptive immunotherapy without losing myeloid progenitor cells necessary for neutrophil recovery after CB transplantation, by modulating the cytokines added to the cultures.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Gluckman E . Current status of umbilical cord blood hematopoietic stem cell transplantation. Exp Hematol 2000; 28: 1197–1205.

    Article  CAS  PubMed  Google Scholar 

  2. Rubinstein P, Carrier C, Scaradavou A et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 1998; 339: 1565–1577.

    Article  CAS  PubMed  Google Scholar 

  3. Hassan J, Reen DJ . Cord blood CD4+CD45RA+ T cells achieved a lower magnitude of activation when compared with their adult counterparts. Immunology 1997; 90: 397–401.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Harris DT, Schumacher MJ, Locascio J et al. Phenotypic and functional immaturity of human umbilical cord blood T lymphocytes. Proc Natl Acad Sci USA 1992; 89: 10006–10010.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Risdon G, Gaddy J, Horie et al. Alloantigen priming induces a state of unresponsiveness in human umbilical cord blood T cells. Proc Natl Acad Sci USA 1995; 92: 2413–2417.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kolb HJ, Mittermuller J, Clemm C et al. Donor leukocyte transfusion for treatment of recurrent chronic myelogeneous leukemia in marrow transplant patients. Blood 1990; 76: 2462–2465.

    CAS  PubMed  Google Scholar 

  7. Dazzi F, Szydlo RM, Goldman JM . Donor lymphocyte infusion for relapse of chronic myeloid leukemia after allogeneic stem cell transplant: where we now stand. Exp Hematol 1999; 27: 1477–1486.

    Article  CAS  PubMed  Google Scholar 

  8. Gross S, Pyatt DW, Shpall EJ et al. Ex vivo expansion of CD3- cells from cord blood for use as donor lymphocyte infusions. Exp Hematol 2000; 28(Suppl. 1): 89 (Abstr. 185).

    Article  Google Scholar 

  9. Carlens S, Gilljam M, Remberger M et al. Ex vivo T lymphocyte expansion for retroviral transduction: influence of serum-free media on variations in cell expansion rate and lymphocyte subset distribution. Exp Hematol 2000; 28: 1137–1146.

    Article  CAS  PubMed  Google Scholar 

  10. Skea D, Chang NH, Hedge R et al. Large ex vivo expansion of human umbilical cord blood CD4+ and CD8+ T cells. J Hematother 1999; 8: 129–139.

    Article  CAS  PubMed  Google Scholar 

  11. Azuma H, Yamada Y, Shibuya-Fujiwara N et al. Functional evaluation of ex vivo expanded cord blood lymphocytes: possible use for adoptive cellular immunotherapy. Exp Hematol 2002; 30: 346–351.

    Article  CAS  PubMed  Google Scholar 

  12. Bonini C, Ferrari G, Verzeletti S et al. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science 1997; 276: 1719–1724.

    Article  CAS  PubMed  Google Scholar 

  13. Peschon JJ, Morrissey PJ, Grabstein KH et al. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J Exp Med 1994; 180: 1955–1960.

    Article  CAS  PubMed  Google Scholar 

  14. Hofmeister R, Khaled AR, Benbernou N et al. Interleukin-7: physiological roles and mechanisms of action. Cytokine Growth Factor Rev 1999; 10: 41–60.

    Article  CAS  PubMed  Google Scholar 

  15. Sadlack B, Kuhn R, Schorle H et al. Development and proliferation in lymphocytes in mice deficient for both interleukins-2 and -4. Eur J Immunol 1994; 24: 281–284.

    Article  CAS  PubMed  Google Scholar 

  16. Kuhn R, Rajevsky K, Muller W . Generation and analysis of interleukin-4 deficient mice. Science 1991; 254: 707–710.

    Article  CAS  PubMed  Google Scholar 

  17. Gluckman E, Rocha V, Boyer-Chammard A et al. Outcome of cord-blood transplantation from related and unrelated donors. N Engl J Med 1997; 337: 373–381.

    Article  CAS  PubMed  Google Scholar 

  18. Forte L, Migliaccio G, Sanchez M, Migliaccio AR et al. Effects of cell banking manipulations on ex vivo amplification of umbilical cord blood. Ann Inst Super Sanità 2000; 36: 333–342.

    CAS  Google Scholar 

  19. Sanchez M, Alfani E, Visconti G et al. Thymus-independent T-cell differentiation in vitro. Br J Haematol 1998; 103: 1198–1205.

    Article  CAS  PubMed  Google Scholar 

  20. Migliaccio G, Migliaccio AR, Adamson JW . In vitro differentiation of human granulocyte/macrophage and erythroid progenitors: comparative analysis of the influence of recombinant human erythropoietin, G-CSF, GM-CSF, and IL-3 in serum-supplemented and serum-deprived cultures. Blood 1988; 72: 248–256.

    CAS  PubMed  Google Scholar 

  21. Roncarolo MG, Bigler M, Ciuti E et al. Immune responses by cord blood cells. Blood Cells 1994; 20: 573–586.

    CAS  PubMed  Google Scholar 

  22. Choi Y, Kotzin B, Herron L et al. Interaction of Staphylococcus aureus toxin ‘superantigens’ with human T-cells. Proc Natl Acad Sci USA 1989; 86: 8941–8945.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Yamamoto K, Masuko-Hongo K, Tanaka A et al. Establishment and application of a novel T-cell clonality analysis using single-strand conformation polymorphism of T-cell receptor messenger signals. H Immunol 1996; 48: 23–31.

    Article  CAS  Google Scholar 

  24. Maniatis T, Fritsch EF and Sambrook J . Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Lab. Press: NY, 1989.

    Google Scholar 

  25. Migliaccio G, Migliaccio AR, Druzin ML et al. Long-term generation of colony-forming cells in liquid culture of CD34+ cord blood cells in the presence of recombinant human stem cell factor. Blood 1992; 79: 2620–2627.

    CAS  PubMed  Google Scholar 

  26. Migliaccio AR, Migliaccio G, Adamson JW . Expansion of human neonatal progenitor cells in vitro under serum-deprived conditions. Blood Cells 1994; 20: 424–428.

    CAS  PubMed  Google Scholar 

  27. Rossmanith T, Schroder B, Bug G et al. Interleukin 3 improves the ex vivo expansion of primitive human cord blood progenitor cells and maintains the engraftment potential of scid repopulating cells. Stem Cells 2001; 19: 313–320.

    Article  CAS  PubMed  Google Scholar 

  28. Freedman AR, Zhu H, Levine JD et al. Generation of human lymphocytes from bone marrow CD34+ cells in vitro. Nat Med 1996; 2: 46–51.

    Article  CAS  PubMed  Google Scholar 

  29. Plum J, De Smedt M, Defresne M-P et al. Interleukin-7 is a critical growth factor in early human T-cell development. Blood 1996; 88: 4239–4245.

    CAS  PubMed  Google Scholar 

  30. Hirayama F, Aiba Y, Ikebuchi K et al. Differentiation in culture of murine primitive lymphohematopoietic progenitors toward T-cell lineage. Blood 1999; 93: 4187–4195.

    CAS  PubMed  Google Scholar 

  31. Lima M, Almeida J, dos Ajos Teixeira M et al. The ‘ex vivo’ pattern of CD2/CD7, CD57/CD11c, CD387CD11b, CD45RA/CD45RO, and CD11a/HLA-DR expression identify acute/early and chronic/late NK-cell activation state. Blood Cells Mol Dis 2002; 28: 181–190.

    Article  PubMed  Google Scholar 

  32. Alfani E, Migliaccio AR, Sanchez M et al. Characterization of the T-cell receptor repertoire of neonatal T cells by RT–PCR and single strand conformation polymorphism analysis. Bone Marrow Transplant 2000; 26: 83–89.

    Article  CAS  PubMed  Google Scholar 

  33. Brandt J, Briddell RA, Srour EF et al. Role of c-kit ligand in the expansion of human hematopoietic progenitor cells. Blood 1992; 79: 634–641.

    CAS  PubMed  Google Scholar 

  34. Parkin J, Cohen B . An overview of the immune system. Lancet 2001; 357: 1777–1789.

    Article  CAS  PubMed  Google Scholar 

  35. Von Freeden-Jeffry U, Vieira P, Lucian LA et al. Lymphopenia in interleukin (IL)-7 gene-delete mice identifies IL-7 as a non-redundant cytokine. J Exp Med 1995; 181: 1519–1526.

    Article  CAS  PubMed  Google Scholar 

  36. Wiles MV, Ruiz P, Imhof BA . Interleukin-7 expression during mouse thymus development. Eur J Immunol 1992; 22: 1037–1042.

    Article  CAS  PubMed  Google Scholar 

  37. Swain SL, Bradley LM, Croft M et al. Helper T-cell subsets: phenotype, function and the role of lymphokines in their development. Immunol Rev 1991; 123: 115–144.

    Article  CAS  PubMed  Google Scholar 

  38. Spellberg B, Edwards Jr JE . Type 1/type 2 immunity in infectious diseases. Clin Infect Dis 2001; 32: 76–102.

    Article  CAS  PubMed  Google Scholar 

  39. Chervenak R, Dempsey D, Soloff RS, Smithson G . In vitro growth of bone marrow resident T-cell precursors supported by mast-cell growth factor and IL-3. J Immunol 1992; 149: 2851–2856.

    CAS  PubMed  Google Scholar 

  40. Yeoman H, Clark DR, De Luca D . Development of CD4 and CD8 single positive T cells in human thymus organ culture: IL-7 promotes human T-cell production by supporting immature T cells. Dev Comp Immunol 1996; 20: 241–263.

    Article  CAS  PubMed  Google Scholar 

  41. Tagoh H, Kishi H, Okumura A et al. Induction of recombination activating gene expression in a human lymphoid progenitor cell line: requirement of two separate signals from stromal cells and cytokines. Blood 1996; 88: 4463–4473.

    CAS  PubMed  Google Scholar 

  42. Garcia-Ojeda ME, Dejbakhsh-Jones S, Weissman IL, Strober S . An alternate pathway for T-cell development supported by the bone marrow microenvironment: recapitulation of thymic maturation. J Exp Med 1998; 187: 1813–1823.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Takeshita T, Asao H, Ohtani K et al. Cloning of the gamma chain of the human IL-2 receptor. Science 1992; 257: 379–382.

    Article  CAS  PubMed  Google Scholar 

  44. Noguchi M, Nakamura Y, Russell SM et al. Interleukin-2 receptor gamma chain: a functional component of the interleukin-7 receptor. Science 1993; 262: 1877–1880.

    Article  CAS  PubMed  Google Scholar 

  45. Kondo M, Takeshita T, Ishii N et al. Sharing of the interleukin-2 (IL-2) receptor gamma chain between receptor for IL-2 and IL-4. Science 1993; 262: 1874–1877.

    Article  CAS  PubMed  Google Scholar 

  46. Russell SM, Keegan AD, Harada N et al. Interleukin-2 receptor gamma chain: a functional component of the interleukin-4 receptor. Science 1993; 262: 1880–1883.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr M Marceca (II Clinica Ostetrica, Università agli Studi ‘La Sapienza’, Rome, Italy) for providing CB samples. This work was supported by institutional funds of Istituto Superiore di Sanita', Progetto Finalizzato 1%, Ricerca Corrente and Progetti di Ricerca di Interesse Nazionale 2000, from the Ministry of Health; Progetto Strategico Oncologia CNR-MIUR legge 449/99 and Grant no. E1172 from the Telethon Foundation.

Author information

Authors and Affiliations

  1. Laboratory of Cell Biology, Istituto Superiore di Sanita', Rome, Italy

    M Sanchez, E Alfani & G Migliaccio

  2. Clinical Biochemistry, Istituto Superiore di Sanita', Rome, Italy

    A R Migliaccio

  3. Universita' ‘G. D’Annunzio', Chieti, Italy

    A R Migliaccio & G Migliaccio

  4. Ospedale Spirito Santo, Pescara, Italy

    T Bonfini

Authors
  1. M Sanchez
    View author publications

    Search author on:PubMed Google Scholar

  2. E Alfani
    View author publications

    Search author on:PubMed Google Scholar

  3. A R Migliaccio
    View author publications

    Search author on:PubMed Google Scholar

  4. T Bonfini
    View author publications

    Search author on:PubMed Google Scholar

  5. G Migliaccio
    View author publications

    Search author on:PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sanchez, M., Alfani, E., Migliaccio, A. et al. Amplification of T cells from human cord blood in serum-deprived culture stimulated with stem cell factor, interleukin-7 and interleukin-2. Bone Marrow Transplant 31, 713–723 (2003). https://doi.org/10.1038/sj.bmt.1703904

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/sj.bmt.1703904

Keywords

Search

Advanced search

Quick links