title: Expansion of Philadelphia chromosome-negative CD3(+)CD56(+) cytotoxic cells from chronic myeloid leukemia patients: in vitro and in vivo efficacy in severe combined immunodeficiency disease mice.
abstract: We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMNCs) by the timed addition of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and the monoclonal antibody (MoAb) OKT3. These cells, termed cytokine-induced killer (CIK) cells, are composed primarily of T cells, and the population of cells with the greatest cytotoxic activity is an otherwise rare population of CD3(+)CD56(+) cells that expand dramatically under these culture conditions. CIK cells were expanded from PBMNCs from 13 patients with chronic myeloid leukemia (CML). These cultures contained a variable number of T cells at the start of the culture (median 44%, range 1% to 64%), yet after 21 to 28 days of culture, virtually all of the cells were CD3(+) T cells (median 97%, range 90% to 99%). The CD3(+)CD56(+) subset of cells expanded significantly (median 25-fold, range 2.2- to 525-fold). CIK cells from all patients showed cytotoxicity against the tumor cell lines OCI-LY8 and K562. In four patients the expanded CIK cells suppressed colony growth of autologous CML blast cells and myeloid progenitor cells. Allogeneic CIK cells from normal donors also suppressed CML colony growth but did not inhibit growth of normal hematopoietic colonies. Twelve of the 13 cultures were exclusively composed of Philadelphia (Ph)-negative cells and one culture had 1 out of 20 Ph-positive metaphases after 4 weeks in culture. Intracellular cytokine production was assayed by fluorescence-activated cell sorter (FACS), and the expanded T-cell cultures produced IL-2, IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha), but not IL-4. Both the CD4(+) and CD8(+) subsets secreted this cytokine profile. To test the in vivo activity of the expanded CIK cells, CML was engrafted into severe combined immunodeficiency disease (SCID) mice using matrigel. After 4 weeks, 4 x 10(7) autologous CIK cells were injected intravenously by tail vein injection into groups of mice, and the animals were sacrificed after a total of 18 weeks. Bcr-abl was detected in the bone marrow or spleen of 5 out of 6 control mice and only 2 out of 13 mice who received the autologous CIK cells (P = .02). In an additional series of animals, the mice did not engraft with CML but instead developed large human Epstein-Barr virus-associated lymphomas by 12 weeks. The mice who received autologous CIK cells at 4 weeks had either no tumor (5) or small tumors (5), whereas all 10 mice that received CIK cells at week 8 developed lymphomas; however, these were not as large as in the 10 control mice who did not receive CIK cells (P = . 03). This study shows that CIK cells, which are Ph chromosome-negative, can be expanded from patients with CML and have potent in vitro and in vivo efficacy against autologous tumor cells.
authors.affiliation: Division of Bone Marrow Transplantation, Department of Medicine; the Department of Pathology, Stanford University Medical School, Stanford, CA, USA.
authors.forename: C
authors.initials: C
authors.lastname: Hoyle
authors.forename: C D
authors.initials: CD
authors.lastname: Bangs
authors.forename: P
authors.initials: P
authors.lastname: Chang
authors.forename: O
authors.initials: O
authors.lastname: Kamel
authors.forename: B
authors.initials: B
authors.lastname: Mehta
authors.forename: R S
authors.initials: RS
authors.lastname: Negrin
mesh_terms: D000818:Animals; D017252:CD3 Complex / Q000032:analysis; D019002:CD56 Antigen / Q000032:analysis; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured / Q000637:transplantation; D003094:Collagen; D004338:Drug Combinations; D020031:Epstein-Barr Virus Infections / Q000635:transmission; D005188:False Negative Reactions; D016044:Fusion Proteins, bcr-abl / Q000032:analysis; D020350:Graft vs Tumor Effect / Q000276:immunology*; D006412:Hematopoietic Stem Cells / Q000187:drug effects; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000009:adverse effects; D007371:Interferon-gamma / Q000096:biosynthesis / Q000494:pharmacology*; D007376:Interleukin-2 / Q000096:biosynthesis / Q000494:pharmacology*; D007621:Karyotyping; D007694:Killer Cells, Natural / Q000276:immunology / Q000637:transplantation* / Q000821:virology; D007797:Laminin; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000276:immunology / Q000473:pathology / Q000628:therapy* / Q000821:virology; D015466:Leukemia, Myeloid, Chronic-Phase / Q000473:pathology; D016393:Lymphoma, B-Cell / Q000209:etiology / Q000821:virology; D051379:Mice; D016513:Mice, SCID; D016853:Muromonab-CD3 / Q000494:pharmacology*; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells / Q000187:drug effects; D010677:Philadelphia Chromosome; D011509:Proteoglycans; D016176:T-Lymphocyte Subsets / Q000187:drug effects / Q000276:immunology* / Q000821:virology; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000276:immunology* / Q000821:virology; D014183:Transplantation, Heterologous / Q000276:immunology; D014184:Transplantation, Homologous / Q000276:immunology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D014410:Tumor Stem Cell Assay
chemical_list: D017252:CD3 Complex; D019002:CD56 Antigen; D004338:Drug Combinations; D007376:Interleukin-2; D007797:Laminin; D016853:Muromonab-CD3; D011509:Proteoglycans; D014409:Tumor Necrosis Factor-alpha; C059485:matrigel; D007371:Interferon-gamma; D003094:Collagen; D016044:Fusion Proteins, bcr-abl