Int. J. Cancer: 34, zyxwvutsrqp 471-477 (1984) COLONY FORMATION IN THE ABSENCE OF ADDED GROWTH FACTORS BY PERIPHERAL BLOOD T-CELL COLONY-FORMING CELLS OF PATIENTS WITH T-CELL MALIGNANCIES Vassilis GEORGOULIAS zyxwvutsr ', Aldar BOURINBAIAR, Fraqoise AMESLAND, Chantal CANON, Huguette AUCLAIR and Claude zyxwvutsrqp JASMIN Unitk zyxwvutsrqp d'Oncoginbe Appliqui, INSERM U 268, Hapita1 Paul Brousse, 14-16, Avenue Paul Vaillant Couturier, 94800 VillejuiJ France. zyxwvutsr Clonogenic cells from peripheral blood of I3/ I6 patients with T-cell malignancies generated colonies in methykel- lulose in absence of added growth factors or mitogenic stimulation. Spontaneous colonies were also obtained from purified cell fractions zyxwvutsr (E-OKT3- and/or E+ cells) in 13 zyxwvutsrqp 7 ' and 57 % of the patients, respectively. No spontane- ous colony growth was observedwith mononuclear cells of patients with solid tumors, non-T-cell leukemiasor normal subjects. Colonies consisted of acid-phosphatase-positive, myeloperoxidase- and PAS-negative lymphoblasbbearing T-cell surface markers. Although the phenotype of pooled colony cells from either unfractionated mononuclear cells or E-OKT3-4erived colonies varied from patient to pa- tient, the colonies, like fresh leukemic cells, were mostly composed of relatively immature cells as assessed by the high proportion (>40%) of OKT6+ and OKTIO+ cells and the low proportion (<a%) of OKT3+ and/or E+ cells. Cytogenetic analysis of colony cells revealed either normal metaphases or chromosome anomalies similar to those observed in fresh leukemic cells. Moreover, cells from pri- mary colonies exhibited a capacity for self-renewal in the absence of added growth factors. Normal T-cell colony-forming cells (T-CFC) require stimulation by phytohemagglutinin (PHA) (Rozens- zajn et al., 1975;Claesson et al., 1977; Shen et al., 1977) or antigens (Watson, 1979; Kornbluth et al., 1979) for colony growth in semi-solid media. A T-cell growth factor (TCGF or Interleukin 2) is involved in in vitro proliferation of normal T-CFC in the presence of PHA (Triebel et al., 1981; Moreau and Miller, 1983); addi- tion of TCGF results in continuous growth of normal PHA-stimulated (Ruscetti et al., 1977) and of neoplas- tic human T cells (Mier and Gallo, 1980; Gillis et al., 1981). TCGF is normally synthesized by mitogen-stimu- lated helperlinducer (OKT4 +) and some suppressor/ cytotoxic (OKT8+) T lymphocytes (Meuer et al., 1982; Luger et al., 1982)with the help of Interleukin 1. Sever- al recent studies have reported production of TCGF by fresh leukemic cells (Gillis et al., 1981) and human malignant T-cell lines after activation with either PHA or phorbol diesters (Poiesz et al., 1980; Ruscetti and Gallo, 1981; Solbach et zyxwvutsrqp al., 1981; Friedman et al., 1982). Furthermore, some malignant T-cell lines, ini- tially TCGF-dependent , subsequently become TCGF- independent (Poiesz et al., zyxwvutsrq 1980), as do cultures of umbilical cord human T cells after infection with iso- lates of human T-cell leukemidymphoma virus I (HTLV 1; Popovic et al., 1983). These observations prompted us to study the in vitro proliferation and differentiation of peripheral blood T-CFC from patients with T-cell malignancies and especially their capacity to generate colonies in the absence of added growth factors or mitogenic stimula- tion. We report that T-CFC from such patients spon- taneously form colonies which, as suggested by cy- togenetic studies, are at least partly derived from a malignant clone. These T-CFC are either E+ or imma- ture (E-OKn-Ia-) cells and exhibit a capacity for self- renewal when replated in the absence of added growth factors. PATIENTS AND METHODS Patients Heparinized peripheral blood was obtained from 8 normal subjects, 15 patients with solid tumors, 5 with multiple myeloma, 5 chronic myelogenous (CML), 5 acute non-lymphoid (ANLL), 7 non-T non-B acute lymphoblastic (CALL) leukemias and 16 patients with T-cell malignancies (11 with acute lymphoblastic leukemia and 5 with non-Hodgkin's lymphoma: T- ALL AND T-NHL, respectively). Eight patients were studied prior to any treatment and 8 were in relapse. Monoclonal antibodies The following MAbs were used to characterize either freshly separated patients' mononuclear cells (MNC) or colony cells: those of the OKT series (OKT3, 4, 6, 8, 9 and 10; Ortho, Raritan, NJ); sheep erythrocyte receptor antibody (T11; Coultronics, Margency, France); OKMl antibody detecting monocyteslmacro- phages and relatively differentiated myeloid cells (Or- tho); B-lymphocyte antibody (Bl), anti-CALLA anti- body and anti-HLA-DR (11) antibody (Coultronics). Cell separation Peripheral blood MNC were obtained by Ficoll- Paque density centrifugation (d=1.077 g/cm3) and washed twice with Hanks' balanced salt solution (HBSS) (Institut Pasteur, Paris, France) supplemented with 10 % heat-inactivated fetal calf serum (FCS) Gib- co, Grand Island, NY). Further separation was per- formed by rosette formation with AET- (2-Amino- ethylisothio-uronium hydrobromide; Sigma, St. Louis, M0)-treated sheep red blood cells (AET-SRBC) for 1 h at room temperature (Maosen and Johnson, 1979) followed by a second density centrifugation. Interface cells were incubated with OKT3 [50 pl/l X lo6 cells at a final dilution of 1:400 (vlv)] at 4" C for 45 min and pretested guinea-pig complement [SO-SO pI/l X lo6 cells at a final dilution of 1:5 (vlv); Institut Pasteur] at 37" C for 45 min to further remove contaminating E+ cells. Cells were extensively washed with HBSS and viable cells were counted by trypan blue exclusion. 'To whom reprint requests should be addressed. Received: June 5, 1984 and in revised form July 24, 1984.