Search tips
Search criteria 


Logo of jexpmedHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Exp Med. 1990 July 1; 172(1): 303–313.
PMCID: PMC2188143

The generation of natural killer (NK) cells from NK precursor cells in rat long-term bone marrow cultures


In this report, we describe a novel long-term bone marrow culture (LTBMC) system to study the origin and generation of natural killer (NK) cells from NK precursors. Rat bone marrow was cultured for 4 wk in RPMI 1640 with 5% fetal calf serum and 2-mercaptoethanol to allow the formation of an adherent stromal cell layer containing NK precursor cells. After addition of interleukin 2 (IL-2), the LTBMC generated high numbers (up to 100-fold expansion in 7 d) of pure 3.2.3+ large granular lymphocytes with lytic activity against NK-sensitive and -resistant tumor targets, as well as antibody-dependent cellular cytotoxicity. NK activity in LTBMC could be detected 3 d after addition of as little as 1 U/ml rIL-2, whereas lymphokine-activated killer activity was found 5 d after addition of at least 10 U/ml rIL-2. In vivo depletion and in vitro complement lysis studies showed that the NK precursor cells in LTBMC did not express the NK-associated surface markers asialo GM1 or 3.2.3. We also found that LTBMC cells did not exhibit colony growth in granulocyte/macrophage or spleen colony-forming unit assays. The generation of NK cells from NK precursors required, in addition to IL- 2, a second growth/maturation factor(s), which was present in the conditioned medium of the LTBMC. This LTBMC system provides a unique in vitro model to study the development of NK cells from precursor cells, the role of the bone marrow stromal microenvironment in this development, and the lineage relationship of NK cells to other hematopoietic cells.

Full Text

The Full Text of this article is available as a PDF (1.0M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bukowski JF, Warner JF, Dennert G, Welsh RM. Adoptive transfer studies demonstrating the antiviral effect of natural killer cells in vivo. J Exp Med. 1985 Jan 1;161(1):40–52. [PMC free article] [PubMed]
  • Trinchieri G. Biology of natural killer cells. Adv Immunol. 1989;47:187–376. [PubMed]
  • Herberman RB, Nunn ME, Lavrin DH. Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic acid allogeneic tumors. I. Distribution of reactivity and specificity. Int J Cancer. 1975 Aug 15;16(2):216–229. [PubMed]
  • Herberman RB. Cell-mediated immunity to tumor cells. Adv Cancer Res. 1974;19(0):207–263. [PubMed]
  • Ortaldo JR, Herberman RB. Heterogeneity of natural killer cells. Annu Rev Immunol. 1984;2:359–394. [PubMed]
  • Reynolds CW, Bonyhadi M, Herberman RB, Young HA, Hedrick SM. Lack of gene rearrangement and mRNA expression of the beta chain of the T cell receptor in spontaneous rat large granular lymphocyte leukemia lines. J Exp Med. 1985 May 1;161(5):1249–1254. [PMC free article] [PubMed]
  • Lanier LL, Cwirla S, Federspiel N, Phillips JH. Human natural killer cells isolated from peripheral blood do not rearrange T cell antigen receptor beta chain genes. J Exp Med. 1986 Jan 1;163(1):209–214. [PMC free article] [PubMed]
  • Biron CA, van den Elsen P, Tutt MM, Medveczky P, Kumar V, Terhorst C. Murine natural killer cells stimulated in vivo do not express the T cell receptor alpha, beta, gamma, T3 delta, or T3 epsilon genes. J Immunol. 1987 Sep 1;139(5):1704–1710. [PubMed]
  • Ortaldo JR, Sharrow SO, Timonen T, Herberman RB. Determination of surface antigens on highly purified human NK cells by flow cytometry with monoclonal antibodies. J Immunol. 1981 Dec;127(6):2401–2409. [PubMed]
  • Perussia B, Trinchieri G. Antibody 3G8, specific for the human neutrophil Fc receptor, reacts with natural killer cells. J Immunol. 1984 Mar;132(3):1410–1415. [PubMed]
  • Phillips JH, Babcock GF. NKP-15: a monoclonal antibody reactive against purified human natural killer cells and granulocytes. Immunol Lett. 1983 Mar;6(3):143–149. [PubMed]
  • Hercend T, Griffin JD, Bensussan A, Schmidt RE, Edson MA, Brennan A, Murray C, Daley JF, Schlossman SF, Ritz J. Generation of monoclonal antibodies to a human natural killer clone. Characterization of two natural killer-associated antigens, NKH1A and NKH2, expressed on subsets of large granular lymphocytes. J Clin Invest. 1985 Mar;75(3):932–943. [PMC free article] [PubMed]
  • Glimcher L, Shen FW, Cantor H. Identification of a cell-surface antigen selectively expressed on the natural killer cell. J Exp Med. 1977 Jan 1;145(1):1–9. [PMC free article] [PubMed]
  • Young WW, Jr, Hakomori SI, Durdik JM, Henney CS. Identification of ganglio-N-tetraosylceramide as a new cell surface marker for murine natural killer (NK) cells. J Immunol. 1980 Jan;124(1):199–201. [PubMed]
  • Chambers WH, Vujanovic NL, DeLeo AB, Olszowy MW, Herberman RB, Hiserodt JC. Monoclonal antibody to a triggering structure expressed on rat natural killer cells and adherent lymphokine-activated killer cells. J Exp Med. 1989 Apr 1;169(4):1373–1389. [PMC free article] [PubMed]
  • Cantrell DA, Robins RA, Brooks CG, Baldwin RW. Phenotype of rat natural killer cells defined by monoclonal antibodies marking rat lymphocyte subsets. Immunology. 1982 Jan;45(1):97–103. [PubMed]
  • Reynolds CW, Sharrow SO, Ortaldo JR, Herberman RB. Natural killer activity in the rat. II. Analysis of surface antigens on LGL by flow cytometry. J Immunol. 1981 Dec;127(6):2204–2208. [PubMed]
  • Haller O, Wigzell H. Suppression of natural killer cell activity with radioactive strontium: effector cells are marrow dependent. J Immunol. 1977 Apr;118(4):1503–1506. [PubMed]
  • Seaman WE, Gindhart TD, Greenspan JS, Blackman MA, Talal N. Natural killer cells, bone, and the bone marrow: studies in estrogen-treated mice and in congenitally osteopetrotic (mi/mi) mice. J Immunol. 1979 Jun;122(6):2541–2547. [PubMed]
  • Kumar V, Ben-Ezra J, Bennett M, Sonnenfeld G. Natural killer cells in mice treated with 89strontium: normal target-binding cell numbers but inability to kill even after interferon administration. J Immunol. 1979 Oct;123(4):1832–1838. [PubMed]
  • Hackett J, Jr, Tutt M, Lipscomb M, Bennett M, Koo G, Kumar V. Origin and differentiation of natural killer cells. II. Functional and morphologic studies of purified NK-1.1+ cells. J Immunol. 1986 Apr 15;136(8):3124–3131. [PubMed]
  • Ortaldo JR, Sharrow SO, Timonen T, Herberman RB. Determination of surface antigens on highly purified human NK cells by flow cytometry with monoclonal antibodies. J Immunol. 1981 Dec;127(6):2401–2409. [PubMed]
  • Lotzová E, Savary CA, Gray KN, Raulston GL, Jardine JH. Natural killer cell profile of two random-bred strains of athymic rats. Exp Hematol. 1984 Sep;12(8):633–640. [PubMed]
  • Herberman RB, Nunn ME, Holden HT, Lavrin DH. Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. II. Characterization of effector cells. Int J Cancer. 1975 Aug 15;16(2):230–239. [PubMed]
  • Dorshkind K, Pollack SB, Bosma MJ, Phillips RA. Natural killer (NK) cells are present in mice with severe combined immunodeficiency (scid). J Immunol. 1985 Jun;134(6):3798–3801. [PubMed]
  • Hackett J, Jr, Bosma GC, Bosma MJ, Bennett M, Kumar V. Transplantable progenitors of natural killer cells are distinct from those of T and B lymphocytes. Proc Natl Acad Sci U S A. 1986 May;83(10):3427–3431. [PubMed]
  • Lohmann-Matthes ML, Domzig W, Roder J. Promonocytes have the functional characteristics of natural killer cells. J Immunol. 1979 Oct;123(4):1883–1886. [PubMed]
  • Sjögren HO, Hellström I. Induction of polyoma specific transplantation antigenicity in Moloney leukemia cells. Exp Cell Res. 1965 Oct;40(1):208–212. [PubMed]
  • DUNN TB, POTTER M. A transplantable mast-cell neoplasm in the mouse. J Natl Cancer Inst. 1957 Apr;18(4):587–601. [PubMed]
  • Hünig T, Wallny HJ, Hartley JK, Lawetzky A, Tiefenthaler G. A monoclonal antibody to a constant determinant of the rat T cell antigen receptor that induces T cell activation. Differential reactivity with subsets of immature and mature T lymphocytes. J Exp Med. 1989 Jan 1;169(1):73–86. [PMC free article] [PubMed]
  • Tanaka T, Masuko T, Yagita H, Tamura T, Hashimoto Y. Characterization of a CD3-like rat T cell surface antigen recognized by a monoclonal antibody. J Immunol. 1989 Apr 15;142(8):2791–2795. [PubMed]
  • Barlozzari T, Reynolds CW, Herberman RB. In vivo role of natural killer cells: involvement of large granular lymphocytes in the clearance of tumor cells in anti-asialo GM1-treated rats. J Immunol. 1983 Aug;131(2):1024–1027. [PubMed]
  • van den Brink MR, Hunt LE, Hiserodt JC. In vivo treatment with monoclonal antibody 3.2.3 selectively eliminates natural killer cells in rats. J Exp Med. 1990 Jan 1;171(1):197–210. [PMC free article] [PubMed]
  • Barlozzari T, Leonhardt J, Wiltrout RH, Herberman RB, Reynolds CW. Direct evidence for the role of LGL in the inhibition of experimental tumor metastases. J Immunol. 1985 Apr;134(4):2783–2789. [PubMed]
  • Shadduck RK. Leukocyte colony-stimulating factor and inhibitor activity. J Lab Clin Med. 1976 Jun;87(6):1041–1049. [PubMed]
  • Koo GC, Manyak CL. Generation of cytotoxic cells from murine bone marrow by human recombinant IL 2. J Immunol. 1986 Sep 15;137(6):1751–1756. [PubMed]
  • Migliorati G, Cannarile L, Herberman RB, Bartocci A, Stanley ER, Riccardi C. Role of interleukin 2 (IL 2) and hemopoietin-1 (H-1) in the generation of mouse natural killer (NK) cells from primitive bone marrow precursors. J Immunol. 1987 Jun 1;138(11):3618–3625. [PubMed]
  • Sarneva M, Vujanovic NL, Van den Brink MR, Herberman RB, Hiserodt JC. Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells. Cell Immunol. 1989 Feb;118(2):448–457. [PubMed]
  • Kasai M, Yoneda T, Habu S, Maruyama Y, Okumura K, Tokunaga T. In vivo effect of anti-asialo GM1 antibody on natural killer activity. Nature. 1981 May 28;291(5813):334–335. [PubMed]
  • Whitlock CA, Witte ON. Long-term culture of B lymphocytes and their precursors from murine bone marrow. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3608–3612. [PubMed]
  • Hayashi J, Medlock ES, Goldschneider I. A selective culture system for generating terminal deoxynucleotidyl transferase-positive (TdT+) lymphoid precursor cells in vitro. I. Description of the culture system. J Exp Med. 1984 Dec 1;160(6):1622–1639. [PMC free article] [PubMed]
  • Dexter TM, Allen TD, Lajtha LG. Conditions controlling the proliferation of haemopoietic stem cells in vitro. J Cell Physiol. 1977 Jun;91(3):335–344. [PubMed]
  • Cell JW. Anglo-Indian medical theory and the origins of segregation in West Africa. Am Hist Rev. 1986 Apr;91(2):307–335. [PubMed]
  • Seaman WE, Merigan TC, Talal N. Natural killing in estrogen-treated mice responds poorly to poly I.C despite normal stimulation of circulating interferon. J Immunol. 1979 Dec;123(6):2903–2905. [PubMed]
  • Levy EM, Kumar V, Bennett M. Natural killer activity and suppressor cells in irradiated mice repopulated with a mixture of cells from normal and 89Sr-treated donors. J Immunol. 1981 Oct;127(4):1428–1432. [PubMed]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press