Human B lymphocyte colony responses. I. General characteristics and modulation by monocytes

The present study investigated the ability of human B cell-enriched subpopulations to focally proliferate and form colonies in semisolid cultures after stimulation with staph protein A (SpA). After 6 days of incubation, cultures of B-enriched populations exhibited distinct colonies, the number being dependent on the concentration of SpA and the cell density. Optimal colony responses were 1.6 x 10(3) per 1 x 10(6) B lymphocytes, and greater than 83% of the colony-forming cells expressed surface immunoglobulin (sIg). The depletion of adherent monocytes from the B cell-enriched preparations decreased the colony responses approximately 3-fold compared with the nondepleted B cell populations. Adding optimal numbers of adherent monocytes to the monocyte-depleted B cells restored the colony responses; however, less augmentation was observed in single-layer co-cultures containing greater than optimal numbers of monocytes. Identical experiments in double-layer semisolid cultures revealed that relatively greater numbers of monocytes were required to enhance B cell colony responses. Thus, progressively higher ratios of monocytes to B cells resulted in increasing numbers of colonies and failed to demonstrate the diminished colony responses observed in the single-layer system. These studies demonstrate that human B cells form distinct colonies when activated by SpA and that normal adherent monocytes modulate the magnitude of colony responses. Although monocytes predominately enhance B cell clonal differentiation, the evidence presented also suggests that, to a lesser extent, soluble inhibitory materials are elaborated.     

In vitro immune response of human peripheral lymphocytes. VI. Distribution and characterization of precursors for PHA- and protein A-induced colony-forming B cells

B cells from peripheral blood or cord blood formed colonies by stimulation with either PHA or protein A. On the other hand, tonsillar B cells did not form protein A-induced colonies, although PHA-induced colony formation was comparable to that observed in peripheral B cells. Lack of protein A-induced colony formation in tonsillar B cells was not due to the defect of helper T cells in preculture or to the presence of suppressor cells but was due to the absence of precursors for colony formation. The result showed that PHA- and protein A-induced colony-forming cells belonged to distinct subsets of B cells. Depletion of mu-bearing cells from peripheral B cells abrogated both PHA- and protein A-induced colony formation. Depletion of delta-bearing cells did not affect PHA- and protein A-induced colony formation and the population enriched with delta-bearing cells also showed colony formation. Depletion of complement receptor (CR)-positive cells removed precursors for both PHA- and protein A-induced colony formation. These results showed that precursor cells for PHA- and protein A-induced colony formation were IgM+, IgD+ and CR+ or IgM+, IgD- and CR+.     

Colony formation in agar by adult bone marrow multipotential hemopoietic cells

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen-stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40–50 per 10⁵ bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony-forming cells. In CBA mice erythroid colony-forming cells were mainly non-cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H-thymidine). Erythroid colony-forming cells sedimented with a peak of 4.5 mm/hr, compared with CFU-S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies. Analysis of the total number of erythroid colony-forming cells and CFU-S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types. In addition to benzidine-positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU-S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony-forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.     

Functional differences between cells from MLR colonies grown in soft agar cultures

This report describes a method of growing soft agar colonies of human T lymphocytes activated in the MLR. Two types of colonies were demonstrated: lower colonies grew within the agar layer, and upper colonies grew on the surface of the agar layer. Three days of priming the lymphocytes in the MLR and the use of supernatants of day-3 MLR cultures to provide T cell colony growth factor were necessary for optimal colony formation. Lymphocytes obtained from colonies were grown in long-term (2 to 4 weeks) cultures to generate sufficient numbers of cells to be tested in different functional assays. Cells from both types of colonies exhibited PLT activity. Upper colony cells showed considerably higher CML activity than lower colony cells (mean percent cytotoxicity 37 +/- 5 vs 6 +/- 3). Cells from both types of colonies contained radiosensitive suppressor cell activity that inhibited the primary MLR. The suppressor cell effect of lower colony cells was specific for the original stimulator, but upper colony cells displayed nonspecific suppressive effects. For both types of colony cells, it appeared that suppressive effects were unrelated to the CML activity of these cells. These data suggest that the soft agar colony assay offers a promising approach to separate subpopulations of lymphocytes activated in the MLR.     

Growth and characterization of T cell colonies from human thymus

A semisolid microculture system was used to study T cell colonies grown from human thymocytes. Colony growth was absolutely dependent upon media conditioned by peripheral blood leukocytes (PBL) in the presence of phytohemagglutinin. Plating efficiency was further enhanced by the addition of a non-T, adherent, radiation-resistant (7500 rad) PBL subpopulation, but was not enhanced by culture supernatants of these cells. The T colony precursor cell in the thymus occurred with a frequency of 8.0 X 10(-3) and had a surface receptor for the OKT3 monoclonal antibody. Thymocyte colony cells were functionally distinct from PBL and the major thymocyte population. The colony cells proliferated in response to T cell mitogens, but only in the presence of exogenous growth factors. The cells stimulated normal PBL in mixed leukocyte culture (MLC), but did not respond to alloantigens in MLC or in assays of spontaneous cytotoxicity. This culture system should prove helpful in the study of human thymocyte differentiation.     

Human T lymphocyte colonies in agar: a comparison with other T cell assays in healthy subjects and cancer patients.

Colonies of human lymphocytes with T cell characteristics will grow in agar from repeated mitotic divisions with phytohaemagglutinin (PHA) stimulation. The colonies comprise spheres of tightly-packed cells with up to 500-1,000 blast-like cells in each colony. 65% of cells from pooled colonies bound AET-treated sheep red cells. 1,100-2,500 colonies/10(6) peripheral blood lymphocytes developed when cell donors were healthy but lower numbers (350-1,000 colonies/10(6) lymphocytes) were detected in blood from cancer patients. Comparison with other non-specific assays of cell-mediated immunity showed that while 66% of cancer patients were anergic (to five recall antigens) and 78% exhibited depressed mitotic activity in standard cultures with low dose PHA, 100% of these patients revealed T cell colony formation below normal. It is suggested that further studies of T lymphocyte colony-forming cells in healthy people and in a number of disease states may significantly advance our understanding of mechanisms of cell-mediated immunity.     

Spontaneous growth of colonies with mature T lineage phenotype from T-cell-depleted bone marrow precursors in a patient with a lymphoproliferative disorder of the large granular lymphocytes

Bone marrow cells from a patient with pancytopenia and a lymphoproliferative disorder of large granular lymphocytes (LGL) were cultured and tested for their hemopoietic colony-forming potential. Neither erythroid nor granulocyte-macrophage colony formation could be obtained from unfractionated, or LGL-depleted bone marrow cell preparations. However, a spontaneous growth of lymphoid colonies was observed after culturing LGL-depleted (T3-) bone marrow cell suspensions for 25 days. Pooled colonies expanded with recombinant interleukin-2 yielded a population composed predominantly of mature T cells (T3+, Leu 6-). These findings suggest that some (T3-) T cell precursors may mature in the bone marrow and that, in our patient, LGL may have exerted a suppressor effect on this maturational process.     

Mononuclear phagocyte colony formation of human spleen cells in liquid culture

We found that mononuclear phagocytes formed a distinct number of clusters and colonies on the bottom of a culture dish 7 days later but granulocytes did not, when a large number of human spleen cells were cultured in liquid medium. In all gastric cancer bearers and patients with portal hypertension operated on, however, colony formation was restricted to spleen cells from patients with advanced gastric cancer and from a group of patients with portal hypertension. These spleen cells formed mononuclear phagocyte colonies without the help of exogeneous colony stimulating factor (CSF). We further demonstrated that the colony-forming cells were glass non-adherent and nylon wool adherent, and that spontaneous colony formation required cooperation between the colony-forming cells and colony-stimulating cells adherent to a plastic surface.     

Formation in agar of fibroblast-like colonies by cells from the mouse pleural cavity and other sources

Colonies of elongated fibroblast-like cells (stellate colonies) developed in agar cultures of mouse pleural cavity cells mixed with whole blood. Cultures of pleural cells alone developed only abortive clusters of round cells. The frequency of colony-forming cells in the pleural cavity was highest in neonatal mice (200/10⁵ cells) and fell progressively with aging. Stellate colony-forming cells were not in cell cycle but were radiosensitive. In adult mice, only occasional colony-forming cells were detected in peritoneal cavity, thymic, spleen, lymph node or bone marrow cell populations. Stellate colony formation was not stimulated by the granulopoietic regulator, colony stimulating factor. The active component in whole blood required for stellate colony formation was present in plasma but not serum or washed red or white cells.     

Pluripotential hematopoietic stem cells in post-5-fluorouracil murine bone marrow express the Thy-1 antigen

Expression of the Thy-1 alloantigen by hematopoietic stem and progenitor cells in post-5-fluorouracil (5-FU) murine bone marrow was investigated. FACS analysis of BDF1 bone marrow stained for Thy-1.2 with a triple-layer amplified labeling technique demonstrated that 35% of the total bone marrow population expressed Thy-1.2 (Thy-1.2+). Two distinct size subpopulations were observed in post-5-FU BDF1 marrow. Thy-1.2+ cells were present in both the large and the small subpopulations. FACS-separated bone marrow cells were also plated in methylcellulose cultures. Ninety percent of all colony-forming cells surviving in vivo administration of 5-FU were Thy-1.2+. Replating of primary hemopoietic colonies and morphologic examination of primary and secondary colonies demonstrated that the most primitive stem cells including "stem" (S) cells were Thy-1.2+. These cells (Thy-1.2+) were capable of self-renewal in vitro and exhibited multiple differentiation potentials in comparison to Thy-1.2-cells, which lacked significant self-renewal capability and were mono- or bipotent progenitor cells. Separation of Thy-1.2+ cells into large or small Thy-1.2+ subpopulations showed that only the large Thy-1.2+ colony-forming cells possessed significant self-renewal capacity. Treatment of BDF1 bone marrow with anti-Thy-1.2 plus complement reduced primary colony formation by 67% and eliminated those colony-forming cells which had extensive self-renewal properties. In the presence of PWMSCM, depletion and reconstitution of T lymphocytes had no effect on primary or secondary colony formation. These data demonstrate that Thy-1 is present on primitive hematopoietic stem cells in post-5-FU bone marrow. In addition, they show that the murine S cell is Thy-1+.     

Agar human T cell colony growth promoted by a B + null cell-derived lymphokine distinct from IL 2

Human T cell agar colonies can be grown under PHA stimulation from either mature T cells or their E rosette-negative (E-), OKT3- peripheral blood and bone marrow precursors. Colonies comprise a majority of mature E+, OKT3+ cells and a minor (5 to 10%) population of immature E-, T3-, T8-, T4-, DR+, T10+, RFB1+ cells, which upon replating in subculture, can generate secondary colonies of OKT3+, E+, OKT4+, OKT8+ cells. Secondary colony formation can serve as a test for growth requirement of colony precursors, because it depends on the presence of both PHA and a colony-promoting activity (CPA) recovered in PHA-stimulated B + null or T + adherent cell supernatants. CPA production by B + null cells was not affected by their treatment with OKT3 or D66 (T11-like) monoclonal antibodies (MAB) + complement but was abolished by an anti-HLA-DR MAB + complement. However, B cells sorted by panning with the same anti-HLA-DR MAB did not release CPA, demonstrating the requirement of both B cells and null cells for CPA production. Neither IL 2 nor IL 1 could account for B + null cell-derived CPA.     

Effects of recombinant human interferon alpha on human megakaryocyte and fibroblast colony formation

Effects of recombinant human interferon alpha (HuIFN-alpha) on human megakaryocyte (CFU-MK) and fibroblast (CFU-F) colony-forming cell growth were studied. Concentration-dependent inhibition of both CFU-MK and CFU-F by HuIFN-alpha was demonstrated. Statistically significant suppression of both CFU-MK and CFU-F was seen at a HuIFN-alpha concentration of 1000 U/ml or greater. No significant difference was found between HuIFN-alpha treated cultures and controls for the distribution of CFU-MK types and for the size and cell morphology of CFU-F. When a concentration of 1000 u/ml HuIFN-alpha was added at varying time points during the marrow cultures, decreased numbers of megakaryocyte and fibroblast colonies only appeared at the early days of cultures. When bone marrow cells were incubated with HuIFN-alpha for different periods of time prior to initiation of cultures, a reduction of megakaryocyte colony formation also occurred. These studies demonstrate a suppressive effect of HuIFN-alpha on human CFU-MK and CFU-F growth. This effect seems to occur at the initial stages of CFU-MK and CFU-F development.     

Augmentation of erythroid burst formation by the addition of thymocytes and other myelo-lymphoid cells

Bone marrow from barrier-sustained specific pathogen-free (SPF) CBA and C57BL/6 mice gave relatively low numbers of BFU-E colonies in methylcellulose culture, as compared to conventional mice. Addition of thymocytes to the marrow cultures increased the yield of BFU-E colonies more than fourfold in SPF mice but only 1.5-fold in conventional mice. Colony size was also increased. Increased yield of BFU-E colonies was also obtained by co-culture of bone marrow with lymph node cells or with bone marrow or spleen cells from 900R whole-body-irradiated mice. The effect appeared to be cellular rather than humoral. It was not reproduced by conditioned medium from thymus or pokeweed mitogen stimulated spleen cells. The helper effect of thymus cells was eliminated or reduced by freezing and thawing, or by 48 hours of incubation after irradiation. Treatment of bone marrow cells in vitro with anti-theta serum and complement did not decrease the number of BFU-E colonies. The putative helper cells appear not to be T cells, were non-adherent to the plastic culture dish, and were cortisone resistant and radioresistant. The low BFU-E colony yield from SPF mouse marrow is presumed to be largely the result of deficiency of these non-T helper cells in SPF bone marrow, rather than of BFU-E progenitor cells.     

The origin of chicken hematopoietic colonies as assayed in semisolid agar.

This investigation was undertaken to determine whether primitive stem cells and/or fully differentiated macrophages were the source of in vitro colonies derived from hematopoietic tissues. The chicken colony-forming cell (CFC) present in uncultured yolk sac was a nonadherent, presumably undifferentiated cell. The efficiency of colony formation in this case was approximately 0.08%. In contrast to uncultured yolk sac, the CFC present in one-week old yolk sac cultures was evidently a macrophage. Yolk sac cultures, which consisted of greater than 99% macrophages, produced colonies with an efficiency of 1-5% while cultures derived from peritoneal macrophages produced colonies with an efficiency of 10%. Silica selectively destroyed macrophages and reduced the colony forming efficiency of cells derived from yolk sac cultures.     

Hapten-specific murine colony-forming B cells. III. Delineation of functional B cell subpopulations grown under different conditions

The influence of anti-immunoglobulin M (IgM) and anti-IgD on the ability of fluorescein (FL)-specific B cells to proliferate in a colony-forming assay, and of their progeny to further differentiate in response to different FL-antigens was studied. Splenic FL-specific B cells were purified on FL-gelatin plates and were then cultured in semisolid agar in the presence or absence of anti-mu, and anti-delta, or both. Experiments were performed under conditions of either sheep red blood cell (SRBC)-potentiated or SRBC + lipopolysaccharide (LPS)-potentiated colony growth. The resulting colonies were then tested in secondary filler cell-dependent microcultures for the ability to be triggered by different classes of FL-antigens to yield plaque-forming cells (PFC). Anti-delta inhibited 47% of colony growth under both agar culture conditions. Anti-mu inhibited 55% of colony growth in SRBC + LPS-potentiated agar cultures, and inhibited 72% if only SRBC was present. If anti-delta and anti-mu were added together, inhibition was nearly additive. When anti-Ig-treated colonies were tested for PFC responses against FL-polymerized flagellin (POL), both normal and anti-delta resistant colonies, grown under both agar culture conditions, responded well. Anti-mu resistant colonies were refractory to FL-POL challenge. Only normal or anti-delta resistant colonies grown in SRBC + LPS agar cultures were able to respond well to FL-Ficoll, whereas even normal SRBC-potentiated colonies responded poorly. All except SRBC-potentiated, anti-mu treated colonies were able to respond to nonspecific signals present in cultures containing FL-KLH and activated T cell help. These data suggest that addition of specific anti-Ig antibodies, and variation of agar culture conditions, can select for B cell subpopulations responsive only to certain types of antigens.     

Factors affecting the generation of mast cells from multipotential colony-forming cells

The cells responsible for the long-term in vitro generation of murine mast cells have been examined. Sequential analysis of all colony types obtained from cultures of spleen or bone marrow cells showed that only colonies derived from multipotential cells (mixed-erythroid colonies) or mast cell progenitors, contained cells responsible for mast cell generation in liquid cultures. Primary colony growth and subsequent maintenance of mast cells in liquid cultures was dependent upon pokeweed mitogen-stimulated spleen cell-conditioned medium (SCM). Mixed-erythroid colonies from 14-day cultures of spleen cells had the greatest capacity for mast cell generation. Analysis by clone splitting and transfer to high (20%) and low (2.5%) concentrations of SCM showed that the concentration of SCM used in either the primary colony culture or subsequent liquid culture phase altered both the proliferative capacity of the mast cells generated and the frequency of mast cell progenitors within individual mixed-erythroid colonies. Thus, mixed-erythroid colonies stimulated with 2.5% SCM contained the highest proportion of mast cell progenitors (34% of colonies) and when stimulated with 20% SCM, approximately fourfold higher numbers of mast cells were produced at weekly intervals from liquid cultures maintained in 2.5% SCM compared to parallel liquid cultures containing 20% SCM. These studies confirm the hemopoietic origin of mast cells and demonstrate that a factor(s) in SCM is able to modulate their proliferative potential.     

Anchorage-independent colony formation of SV40 transformed BALB/c-3T3 cells in serum-free medium: role of cell- and serum-derived factors

The colony-forming response of SV40 transformed BALB/c-3T3 cells in agarose suspension culture was studied in a serum-free medium (with insulin, transferrin and serum albumin as the only macromolecular supplements) that was optimized for colony formation of fibronectin-attached monolayer cultures. In this serum-free medium, the SV3T3 cells fail to form colonies in agarose suspension. However, they can be induced to anchorage-independent colony formation by the growth factors that are additionally required by their untransformed counterparts for proliferation in monolayer culture. The SV3T3 cells are also rendered anchorage-independent for colony formation in serum-free medium by conditioned medium from dense monolayer serum-free SV3T3 cultures. These experiments suggest that it is the cell-substrate interaction that is responsible for the growth factor autonomy of fibronectin-attached transformed cells.     

Colony formation in agar by multipotential hemopoietic cells.

Agar cultures of CBA fetal liver, peripheral blood, yolk sac and adult marrow cells were stimulated by pokeweed mitogen-stimulated spleen conditioned medium. Two to ten percent of the colonies developing were mixed colonies, documented by light or electron microscopy to contain erythroid, neutrophil, macrophage, eosinophil and megakaryocytic cells. No lymphoid cells were detected. Mean size for 7-day mixed colonies was 1,800-7,300 cells. When 7-day mixed colonies were recloned in agar, low levels of colony-forming cells were detected in 10% of the colonies but most daughter colonies formed were small neutrophil and/or macrophage colonies. Injection of pooled 7-day mixed colony cells to irradiated CBA mice produced low numbers of spleen colonies, mainly erythroid in composition. Karyotypic analysis using the T6T6 marker chromosome showed that some of these colonies were of donor origin. With an assumed f factor of 0.2, the mean content of spleen colony-forming cells per 7-day mixed colony was calculated to vary from 0.09 to 0.76 according to the type of mixed colony assayed. The fetal and adult multipotential hemopoietic cells forming mixed colonies in agar may be hemopoietic stem cells perhaps of a special or fetal type.     

Overexpression of insulin receptors in fibroblast and ovary cells induces a ligand-mediated transformed phenotype.

To investigate whether overexpression of the insulin receptor results in altered cell growth we used NIH 3T3 cells transfected with a bovine papilloma virus/insulin receptor cDNA construct (3T3/HIR). These cells expressed high numbers of insulin receptors (mean +/- sd, 631.0 +/- 16.7 ng receptors/10(6) cells). Insulin significantly stimulated the growth of 3T3/HIR cells maintained in serum-free medium. Moreover, in these cells, insulin induced marked phenotypic changes, including alterations in cell shape, loss of contact inhibition, and focal growth. In contrast to 3T3/HIR cells, insulin was without effect in either wild-type 3T3 cells (3T3/wt), 3T3 cells transfected with the neomycin resistance gene (3T3/NEO), or the bovine papilloma virus (3T3/BPV). To assess the presence of anchorage-independent growth, cells were seeded in soft agar and inspected for colony formation. 3T3/HIR cells showed absent or minimal colony growth in the absence of insulin. However, there was a dose-dependent insulin-stimulated increase in both colony size and number. Insulin-stimulated colony formation was specifically inhibited by an insulin antagonist, monoclonal antibody MA-10. In the presence of 100 nM insulin, about 3% of cells formed large colonies. Insulin neither stimulated growth nor induced colony formation in 3T3/wt cells or 3T3/NEO cells. Insulin also stimulated colony formation in CHO cells transfected with an insulin receptor cDNA construct. In conclusion, overexpression of normal insulin receptors induces a ligand-dependent transformed phenotype. This phenomenon may have clinical relevance by conferring a selective growth advantage to tumor cells with high numbers of insulin receptors.     

Macrophage requirements of CR- and CR+ B lymphocytes for antibody production in vitro.

A Sephadex G-10 column coated with antigen-antibody complexes and complement retains complement receptor-bearing (CR+) mouse spleen cells. The effluent is rich in thymus-derived cells (T cells), and contains bone marrow-derived cells (B cells) which carry surface immunoglobulin (Ig), Ir-associated antigen (Ia), and Fc receptors, but no complement receptors (CR-). Although both unfractionated and CR- B cell populations are capable of producing antibody to red cell antigens, they differ in their requirements for the initiation of the response. Unfractionated B cells cooperate with primed as well as unprimed helper T cells; macrophages are required for this cooperation but can be replaced by 2-mercaptoethanol. CR- B cells cooperate with primed but not with unprimed T cells provided macrophages are added to cultures. After addition of culture supernatant from BCG-activated macrophages CR- B cells cooperate with both unprimed and primed T helper cells.