Colony growth of T lymphocytes in vitro. Control and regulation of clonal formation.

Human lymphocytes stimulated with PHA in liquid phase (step 1) and then seeded in a two-layer soft agar system (step 2) grew and developed into T cell colonies. Colony formation was enhanced when the agar culture was supplemented with culture fluid obtained from phytohemagglutin-treated lymphocytes (Ly-CF). Untreated lymphocytes plated directly in the soft agar system also developed into colonies if the culture medium contained Ly-CF. Mitogen-sensitized T lymphocytes produced a lymphocyte colony enhancing factor in the culture fluid which stimulated lymphocytes into colony formation. The best plating efficiency (1:250) was attained when blood mononuclear cells were seeded. When spleen cell culture fluid or a highly concentrated blood-adherent cell population was added to the soft agar, colony development was strongly inhibited. Monocytes-macrophages secrete a lymphocyte colony inhibiting factor in the culture medium. These lymphokines exert a competitive influence on T cells and thus control and regulate clonal proliferation.     

Replacement of serum by hemolysate as growth promoter for murine leukemic and normal hemopoietic progenitor cells in culture.

The present study was undertaken to test whether lysates prepared from rat erythrocytes can replace serum as in vitro growth promoters for murine leukemic and normal hemopoietic progenitor cells (CFU-C). Normal bone marrow and three leukemic cell lines (P-1081, L-1210 and P-815) were used in all the experiments. The soft agar technique for cloning hemopoietic cells was used to quantitate cell proliferation. Addition of hemolysate to the agar medium at a final concentration of 4% promoted the growth of a maximal number of leukemic colonies, similar to the number of colonies obtained when 20% serum was added to the soft agar. As for normal CFU-C, addition of 10% hemolysate or 40% horse serum promoted the growth of comparable numbers of colonies. Rat hemolysate can therefore replace horse serum as a growth promoter of leukemic and normal CFU-C present in culture. It was also shown that rat hemolysate cannot substitute for the colony stimulating factor (CSF) needed for the cloning of CFU-C. However, addition of hemolysate, CSF and serum to the soft agar cultures promoted a potentiated rather than an additive growth of CFU-C.     

Serum from patients with non-Hodgkin's lymphoma (NHL) inhibits T-cell colony formation

This study compared T-cell colony formation in soft agar of lymphocytes from the peripheral blood and lymph nodes of patients with non-Hodgkin's lymphoma (NHL) with T-cell colony formation of peripheral blood lymphocytes from normal individuals. Mononuclear cells were separated from other blood and lymph node elements on density gradient columns, phenotyped for T- and B-cell antigens using monoclonal antibodies, and then plated in soft agar cultures. Lymphocytes from peripheral blood and lymph nodes of patients with NHL exhibited less T-cell colony formation (p less than 0.01) than did lymphocytes from normal individuals. This decrease in T-cell colony formation was not the result of the number of T cells or null cells plated, or differences in proportions of T helper and T suppressor cells. However, when sera from patients with NHL were incubated with normal lymphocytes before plating in soft agar, a decrease in number of T-cell colonies was observed (p less than 0.01). We conclude that peripheral blood and lymph node mononuclear cells from patients with NHL have a decreased ability to form T-cell colonies in soft agar cultures and that this decrease is related, at least in part, to the presence of serum factor(s).     

Regulation of the induction of colonies in vitro by normal human lymphocytes.

Lymphocytes isolated from normal human peripheral blood can be induced to form colonies in vitro by incubation with the appropriate inducer. Phytohemagglutinin can induce colonies with T (thymus-derived) lymphocytes. Optimun colony formation with about two colonies per 10(2) peripheral blood lymphocytes was obtained by adding, in addition to phytohemagglutinin, autologous plasma, autoologous red blood cells, and fresh L-glutamine or L-cystine. In the absence of these fresh amino acids, no colonies were formed at seeding levels below 10(5) cells per 35 mm petri dish. The addition of either of these amino acids gave a 10-fold decrease in the minimum number of cells that had to be seeded for colony formation. Lipopolysaccharide did not induce the formation of colonies, but enhanced the formation of T cell colonies by phytohemagglutinin. The mixing of lymphocytes from persons with and deficient in glucose-6-phosphate-dehydrogenase (EC 1-1-1-49) has shown that phytohemagglutinin-induced colonies can be derived from single cells and are, therefore, clones. No colonies were formed by lethally irradiated cells. Incubation with pokeweed mitogen also induced the formation of colonies. With autologous plasma and autologous red blood cells, pokeweek mitogen induced about one colony per 5 X 10(3) cells seeded and no colonies at seeding levels below 10(5) cells per petri dish. The minimum number of cells needed for colony formation by pokeweed mitogen was not decreased by fresh L-glutamine or L-cystine. The results indicate that normal human lymphocytes can be cloned in vitro and that induction of these lymphocyte colonies can be regulated by lectins and other specific factors.     

T lymphocyte colonies stimulated by different mitogens require diverse culture conditions

Normal human peripheral blood mononuclear cells form colonies of T lymphocytes in a semi-solid agar matrix when stimulated by a variety of mitogens. In this report, we attempt to determine the optimal conditions for the formation of T lymphocyte colonies by cells stimulated with phytohemagglutinin (PHA), pokeweed mitogen (PWM), Concanavalin A (Con A), or Staphylococcal protein A (SPA). We conclude that optimal conditions differ for each mitogen used. Cultures stimulated by PWM or Con A showed a significant requirement for feeder layers composed of human peripheral blood mononuclear cells. Two-mercaptoethanol significantly enhanced the number of T-cell colonies when PWM, Con A, or SPA, but not PHA, were added as mitogens. Fetal calf serum (FCS) was required for optimal conditions when Con A or SPA but not PWM or PHA were used to stimulate mononuclear cells. Cells stimulated by PHA or PWM produced more T-cell colonies in a 2-step assay than a 1-step assay, whereas the reverse was true with Con A or SPA. Optimal cell concentrations, mitogen doses, and culture kinetics also differed for each mitogen used in the T-cell colony assay.     

Human lymphocyte colony formation in agar culture: cell phenotype studies on individual colonies indicate a polyclonal origin of such colonies

Monoclonal origin of human lymphocyte colonies grown in agar culture under mitogenic stimulation is still disputed. To solve this question we used different markers: we failed with the G6PD technic and with the successive staining for X and Y chromosomes on individual colonies. Therefore, individual colonies were investigated for the presence of different cell types using membrane receptor identification and cytochemistry. At different stages of the colony formation, presence of a macrophage surrounded by lymphocytes, of a mixture of T cells and B cells, plasma cells and c.Ig negative cells, in the same colony was demonstrated. The mixture of cells from different lineages in individual colonies indicated a polyclonal origin of such colonies, the capacity for the cells to migrate in a short distance, and the involvement of cell-cell contact throughout the colony formation. Human lymphocyte colony formation appears as a new technic for the study of cellular cooperation.     

Characteristics of rat megakaryocyte colonies and their progenitors in agar culture

The characteristics of megakaryocyte colonies that develop from megakaryocyte progenitors of rat bone marrow stimulated by rat spleen-conditioned medium (SCM) in agar culture were investigated. Colony frequency was optimal on day 7 and increased relative to both the number of cells plated and the concentration of SCM used. Plating efficiencies averaged 72 +/- 16 megakaryocyte colonies/10(5) cells with 0.1 ml SCM/culture. Colonies were categorized as small cell and big cell. Small-cell colonies had a greater proliferative potential, with a mean of 25 cells/colony. Big-cell colonies averaged 15 cells/colony. The ratio of big-cell to small-cell colonies was 0.69 +/- 0.29. Granulocyte-macrophage colonies, which were also stimulated by SCM, accounted for 70% +/- 15% of the total colonies in the cultures. Cytocidal experiments with tritiated thymidine reduced megakaryocyte colony formation by 45% and granulocyte-macrophage colony formation by 21%. The properties of rat, mouse, and human megakaryocyte progenitors as assayed in vitro are compared.     

Types of colonies formed by normal human bone marrow, peripheral blood and umbilical cord blood CFUc

Four types of colonies can be formed by progenitor cells (CFUc) in double layer agar cultures: eosinophil, granulocyte, macrophage, and mixed granulocyte-macrophage. We have developed a method for in situ staining of intact agar plates with luxol fast blue and acetoorcein employed to differentiate the type of colonies. Cultures of 10(5) normal human bone marrow cells yielded 89.1 colonies, of which 72% were eosinophil. Cultures of 5 X 10(5) peripheral blood cells, enriched by isopycnic sedimentation, yielded 39.0 colonies, of which 48% were eosinophil. T-cell depleted peripheral blood mononuclear cells yielded 116.9 colonies/5 X 10(5) cells, of which 74% were eosinophil. Umbilical cord blood cells yielded 65.1 colonies/5 X 10(5) cells, of which 23% were eosinophil. Comparing similar numbers of total colonies, there appears to be fewer relative and absolute numbers of eosinophil colonies formed by CFUc recovered from cord blood as opposed to adult peripheral blood and bone marrow. Enumeration of colony numbers without regard to types of colonies is imprecise. The method employed in the present study is easy to perform, reproducible and provides permanent slides.     

Preferential inhibition of murine macrophage colony formation by prostaglandin E.

Murine bone marrow progenitor cells that gave rise to macrophage colonies in semisolid agar were found to be more sensitive to prostaglandins of the E series (PGE) than were precursor cells of granulocytes and megakaryocytes. Macrophage colonies themselves had different sensitivities to the molecule. Precursor cells of macrophages that formed colonies in the presence of a stimulating activity from L cells (L-cell CSA) were inhibited to 50% levels by 3 x 10(-9)-M PGE. Macrophage progenitor cells, which require both L-cell CSA and rat hemolysate for colony growth, were inhibited to the same level by 3 x 10(-7)-M PGE. Other colony types (granulocytes and megakaryocytes) were sensitive to PGE only at concentrations greater than 10(-6) M. Accordingly, addition of different PGE concentrations to the culture assay should allow easy detection of precursor cells with morphologically distinct end cells. The different sensitivities to PGE of two macrophage colony types of different maturation stages indicate that PGE may provide feedback to control macrophage formation by inhibiting proliferation and differentiation of immature monocytoid cells.     

The effect of syngeneic peripheral blood cells on the formation of colonies by normal human bone marrow cells in diffusion chambers in mice.

This paper describes the influence of cells capable of releasing colony stimulating activity (CSA) in vitro on the formation of granulocytic colonies by normal human bone marrow in diffusion chambers in mice. A carbonyl iron method was used to remove phagocytic cells from normal human bone marrow. This treatment prevented spontaneous colony and cluster formation when the cells were cultured in agar in vitro at initial concentrations of 2-5 x 105 cells per ml. However, non-phagocytic bone marrow cells formed granulocytic colonies when inoculated into diffusion chambers at 105 cells per chamber and cultured in 450 R-irradiated or non-irradiated mice. The formation of granulocytic colonies by carbonyl iron treated marrow in diffusion chamber cultures was not consistently enhanced by the admixture of 1.4 x 105 1500 R-irradiated syngeneic light density blood cells (less than 1.077 g/ml) to the chamber inoculum. In contrast, these cells induced cluster or colony formation when added in the same proportion to the marrow cells in agar cultures in vitro. Addition of 1.4 x 105 1500 R-irradiated high density blood cells(greater than 1.077 g/ml) to the inoculum resulted in a slight, non-significant decrease in the number of colonies in diffusion chambers. The stimulating effect of host irradiation on neutrophilic colony formation was independent of the presence of CSA releasing cells in the chamber inoculum.     

Functional Cellular Maturation in Cultures of Human Haematopoietic Cells

S ummary . The capacity of cells grown in colonies derived from human haematopoietic cells to phagocytize bacteria and to reduce nitroblue tetrazolium (NBT) was studied as a measurement of functional maturation. Marrow cells from 12 non-leukaemic patients, fivc patients with acute non-lymphocytic leukacmia, and cells taken from a myeloblastic cell line established from a patient with acute myelo-genous leukaemia (AML) were studied. Cells were cultured in soft agar with normal human leucocyte feeder layers as the source of stimulating factor. While the leukaemic marrows gave rise to fewer colonies than the non-leukaemic marrows, colony formation by the AML cell line was extensive. In all instances colony formation was completely dependent upon the presence of the leucocyte feeder layers. Suspensions of pooled colonies contained 42% mature granulocytes in non-leukaemic cultures and 41% in the leukaemic cultures. Granulocytes from these cultures showed active phagocytosis of Staphylococcus aureus and reduction of NBT. In cells taken directly from the liquid suspension culture of the AML cell line, no mature granulocytes nor phagocytosis were noted, whereas 35% and 26% of the cells removed from the colonies formed by these cells were classified as mature granulocytes, or demonstrated phagocytosis and NBT reduction respectively. These observations indicate that granulocytes in soft agar colonies derived from human haematopoietic cells are functionally mature, and that cells derivcd from a human AML cell line have the capacity to form colonies with normal maturation, if they are provided with an appropriate stimulating factor.     

The role of the cellular sodium pump in human granulopoiesis in vitro

The specific sodium pump antagonist ouabain was used to study the effect of sodium pump inhibition on granulopoietic colony formation by normal human bone marrow cells cultured in soft agar for 7 d. Suppression of colony formation was dose-dependent and occurred at a low and reproducible concentration. The use of an extended range of concentrations (1 X 10(-19) M to 1 X 10(-4) M) revealed no additional effect. Inhibition occurred when colony formation was stimulated by either normal human white blood cells in a soft agar underlayer, or the inclusion of pre-formed colony-stimulating activity. Sensitivity to inhibition was similar under either set of conditions, the 50% inhibitory concentrations being 2.70 +/- 0.43 (SE) X 10(-8) M and 2.83 +/- 0.21 (SE) X 10(-8) M respectively. This is interpreted as showing an effect primarily on the colony-forming cells rather than on the cellular production of colony-stimulating activity. Inhibition of colony formation by ouabain was opposed by the addition of extra potassium to the culture medium, confirming that the inhibition was mediated via a perturbation of monovalent cation exchange. Inhibition by 2 X 10(-7) M ouabain appeared to be reversible by simple washing after exposure for up to 24 h, suggesting that the inhibitory effect of ouabain is not primarily cytotoxic.     

Growth of canine T-lymphocyte colonies in vitro.

Canine lymphocytes from peripheral blood, lymph nodes, thymus and bone marrow were stimulated with phytohemagglutinin-P (PHA) or concanavalin-A (CON-A) to form colonies in methylcellulose. Lymphocytes exposed to mitogens in liquid phase formed clumps the size of colonies. Lymphocyte clumping was eliminated by plating cells directly into methylcellulose, but high concentrations of mitogens (CON-A or PHA is greater than 10 mg/10(6) lymphocytes) were required in order to get subsequent colony formation. Thus, in contrast to published reports, exposure of lymphocytes to mitogen prior to plating was not required for cloning of canine peripheral blood lymphocytes. Colonies from thymus, lymph node, or peripheral blood consisted predominantly of T lymphocytes, whereas cultures from bone marrow also produced colonies with macrophage morphology and surface-adherent colonies with mesenchymal morphology.     

T-lymphocyte colonies in human cord blood

The capacity of human cord blood (CB) lymphocytes to form T-colonies was studied with a double layer technique. The mean number of colonies in CB was 91 +/- 70.5 SD (X 10(5) cells), significantly lower than in adult blood, mean 182 +/- 58.0 (X 10(5). In 28 of the 50 CB samples tested the colony numbers were below the normal range for adult lymphocytes. There was no direct correlation between number of colonies and percentage of E-rosette-forming cells in CB. Some CB samples with a high proportion of E-rosettes formed few T-colonies, suggesting that not all E-rosette positive cells are capable of producing T-colonies. On the other hand, some CB samples with a low proportion of E-rosettes formed normal numbers of T-colonies. Purification of two populations of T-cell enriched and T-cell depleted lymphocytes confined the T-colony growth in CB, as in adult blood, to the former fraction, excluding that T-colonies could be obtained from E-rosette negative lymphocytes. This indicates that, from birth onwards, T-lymphocyte colonies originate from E-rosette positive cells. Whether the low growth observed in CB results from lack of maturation of T-lymphocytes or from the presence of specific subsets of T-lymphocytes is not clear at the present time.     

Granulocyte-macrophage colonies in cultures of human fetal liver cells: morphologic and ultrastructural analysis of proliferation and differentiation

Fetal liver cells from 6-12-week-old human fetuses were cultured in soft agar to study growth patterns of the granulocyte-macrophage colony forming cells (CFU(c)) and to characterize the cellular components of these colonies by morphologic, cytochemical and ultrastructural methods. Liver cell suspensions prepared from 31 fetuses obtained by vaginal interruptions of pregnancies, were seeded in soft agar over feeder layers of normal human leukocytes. At all gestational ages examined, agar colony numbers ranged from 44 +/- 15 to 89 +/- 44/2 x 10(5) cells seeded. Colony frequencies, size and gross morphology closely resembled those derived from adult human marrow. Morphologic, cytochemical and ultrastructural examinations showed that 92% of the colonies were granulocytic with incomplete maturation, as found in adult human marrow colonies. Density fractionation of the cells produced a low density cellular fraction which gave a 3- to 5-fold improved cloning efficiency. This study shows that human fetal livers of 6-12 weeks gestational age contain CFU(c) comparable to that found in adult marrow in their frequency, size, density and dependence on colony stimulating factor, and which differentiate mainly into mature or immature granulocytes. It is suggested that the lack of granulopoiesis in vivo in the early human fetal liver is probably not related to CFU(c) deficiency or defective differentiation. An alternative explanation involving impaired regulatory mechanism(s) should be sought.     

Human mixed cell colonies: unicellular or multicellular origin–analysis by G-6-PD

Marrow and peripheral blood cells from normal women heterozygous (GdB/GdA) at the X-chromosome-linked glucose-6-phosphate dehydrogenase (G-6-PD) locus were cultured at cell concentrations ranging from 2 X 10(4)/ml to 4 X 10(5)/ml to test formally the plating conditions necessary for reliable enumeration of multipotent stem cells (CFU-mix). The culture system was rigorously tested by plating cells obtained after velocity sedimentation and the G-6-PD enzyme type of individual colonies was determined. At cell concentrations less than or equal to 7.5 X 10(4)/ml for marrow and less than or equal to 1 X 25 X 10(5)/ml for peripheral blood, mixed-cell colonies had either type A or type B enzyme, but not both. At higher cell concentrations, significant numbers of colonies showed both enzyme types and therefore arose from more than one cell. These studies demonstrate that enumeration of CFU-mix by in vitro colony assay is accurate only at low cell concentrations. Studies of haematopoietic differentiation relying on in vitro colony assays of multipotent stem cells must be carefully analysed in light of these data.     

Induction of erythropoietic colonies in a human chronic myelogenous leukemia cell line.

The ability of cells derived from the K562 cell line to generate erythropoietic colonies was studied. The K562 cell line was derived from a patient with chronic myelogenous leukemia 8 yr ago by Lozzio and Lozzio. Rare benzidine-positive colonies formed when these cells were cloned in plasma clots (3 +/- 1/10(4) cells), and their number was not substantially increased by the addition of erythropoietin (9.5 +/- 1/10(4) cells). Sodium butyrate was capable of markedly enhancing the number of benzidine-positive colonies (19.5 +/- 1/10(4) cells) formed, while the combination of sodium butyrate plus erythropoietin exerted a synergistic effect on erythropoietic colony formation (57 +/- 4/10(4) cells). The K562 cell line is a long-term culture system that contains human erythropoietic stem cells. This cell line should be useful in future studies on the cellular and molecular events associated with human erythroid cell differentiation.     

Biologic effects of in vitro X-irradiation of murine long-term bone marrow cultures on the production of granulocyte-macrophage colony-stimulating factors

The production of colony-stimulating factor (CSF) by murine long-term bone marrow culture (LTBMC) was studied by a technique involving measurement of colony formation in agar overlay by fresh marrow target cells. Colonies were removed and microscopically examined for morphology and histochemistry. LTBMCs were exposed to x-irradiation at 200 rad/min prior to the overlay. Nonirradiated control LTBMCs induced 51.5 +/- 11 granulocyte-macrophage colonies per 2 X 10(5) target cells. Irradiation of LTBMCs to 6000 rad revealed a six-fold plateau-maximum increase in the number of colonies. There was occasional appearance of macroscopic mixed colonies containing granulocytes, macrophages, and megakaryocytes over irradiated but not control LTBMCs. Irradiated cells in the adherent stromal layer of LTBMCs continuously produced CSF that was detectable in the cell-free supernatants for up to seven weeks after irradiation and after doses as high as 10,000 rad. Shielding of the x-ray beam over half of the culture surface by a 10-half-value-layer lead block produced increased colony formation by target cells near the exposed surface area. The data indicate that CSF production by adherent cells within LTBMC persists after supralethal doses of x-irradiation. The mechanism of the increased colony formation by target cells overlaid on irradiated stromal cells involves factors relative to the local microenvironment.     

Staining of granulopoietic agar colonies with a modified Papanicolaou technique.

The aceto-orcein method of staining agar cultured granulopoietic marrow cells has not been entirely satisfactory. In staining whole colonies the original colony structure is lost and the colony cells have blurred cytoplasm. A slight modification of the Papanicolaou technique used for staining vaginal smears improved the quality of stained granulopoietic agar preparations. Smeared Papanicolaou stained colony cells could be compared with smeared ordinary Giemsa stained blood and marrow cells. The Papanicolaou method enabled staining of agar colonies without manipulation of the original colony structure. The preparations of transferred intact colonies had cells with distinct cytoplasm and clear cellular outlines and a background without disturbing masses of stained agar.     

Comparisons of 12-tetradecanoylphorbol-13-acetate (TPA) and PHA as mitogens in the T-lymphocyte colony assay

12-O-tetradecanoylphorbol-13-acetate (TPA) has multiple effects on the capacity of human T-lymphocytes to form colonies in soft agar. This compound is directly mitogenic for T-lymphocytes; the optimal concentration (100 ng/ml) stimulates an average of 2862 +/- 583 colonies/7.5 X 10(5) cells plated. Furthermore, TPA can act synergistically with PHA to induce a greater number of colonies than can either mitogen alone. Sephadex G10 nonadherent (NA) cells can be directly stimulated by TPA; by contrast, these isolated T cells do not respond to PHA alone. These data indicate that the phorbol ester is able to provide an inductive signal for T cells, allowing them to respond to the plant lectin. Using T-cell subsets isolated by monoclonal antibodies and complement cytotoxicity, PHA is able to induce colony growth of T4 cells; T8 cells fail to respond unless cocultured with exogenous IL-2. TPA can directly stimulate colony formation by both subsets. In cultures stimulated with either TPA or PHA, approximately equal numbers of colonies are generated in the presence of IL-2, suggesting that T4 and T8 cells have similar proliferative capabilities. Phenotypic studies of cells contained in colonies showed differences between the two mitogens. With PHA, more than 98% are both T11 and T3 positive; by contrast, approximately one-third of the cells stimulated by TPA are T11 +, T3-.