In vitro immune response of human peripheral lymphocytes. VII. Effect of anti-mu and anti-delta antibodies on B colony formation and detection of abnormal B cells in patients with juvenile rheumatoid arthritis

Effect of anti-mu and anti-delta antibodies on PHA- and protein A-induced B colony formation was studied. Anti-mu antibody at any concentrations tested did not show inhibitory or enhancing effect on colony formation. On the other hand, anti-delta antibody enhanced both PHA- and protein A-induced colony formation. Optimum concentration of anti-delta antibody for maximum enhancement was 0.1 microgram/ml. and F(ab')2 fragment of anti-delta antibody also showed comparable enhancing effect. Simultaneous addition of IgD with anti-delta antibody abrogated anti-delta-induced enhancement, and anti-delta antibody did not show any facilitation of colony formation in IgM+ IgD- cell population. In marked contrast with normal B cells, anti-mu antibody showed a remarkable enhancing effect on protein A-induced colony formation of B cells from JRA patients. F(ab')2 fragment of anti-mu antibody also showed comparable enhancing effect. Anti-mu antibody did not show any enhancement of colony formation of B cells from several other autoimmune diseases. The result indicated the presence of abnormal B cells in JRA patients.     

Hapten-specific murine colony-forming B cells. II. Delineation of a tolerogen-sensitive subpopulation of colony-forming B cells

B cell subpopulations were studied by using B cell cloning procedures and an in vitro tolerance induction model. Fluorescein- (FL) specific B cells from normal spleens were isolated by using FL gelatin plates and were then cultured in semisolid agar in the presence or absence of tolerogen. Hapten-specific cells grew in soft agar to form discrete colonies. Colony growth is dependent on "mitogens" present in agar, sheep red blood cells (SRBC), and lipopolysaccharide (LPS). For example, SRBC plus LPS potentiate the growth of an increased number of colony-forming B cells (CFU-B) compared to either additive alone. These CFU-B could be triggered by a specific antigen to yield plaque-forming cells (PFC). With tolerogen (FL-sheep gamma-globulin) present in the agar, the number of FL-specific CFU-B was reduced by 25 to 50%. The ability of the remaining colonies to form PFC upon antigenic stimulation was also reduced. This reduction in CFU-B numbers, however, was observed only when the agar contained both SRBC and LPS as mitogenic potentiators of growth; no effect of tolerogen on CFU-B numbers was seen when cells were grown with either additive alone. Interestingly, the effect of tolerogen on CFU-B numbers was abrogated when peritoneal macrophages, in addition to SRBC plus LPS, were present during cloning. It is postulated that unique subpopulations of B cells form colonies under varied cloning conditions and that those CFU-B grown with SRBC plus LPS display an increased sensitivity to growth inhibition by tolerogen.     

Hapten-specific murine colony-forming B cells: in vitro response of colonies to fluoresceinated thymus independent antigens

The ability to clone hapten-specific B cells in agar and to subsequently trigger their clonal progeny to antibody synthesis was investigated. Fluorescein (FL) specific B cells were purified on FL-gelatin dishes and cultured in semisolid agar for 6 to 7 days; individual colonies were then picked for restimulation in microculture. FL-specific B cells could be cloned as efficiently as unpurified splenic B cells. The number of colonies formed depended on the presence of sheep erythrocytes (SRBC) or E. coli lipopolysaccharide (LPS) in the cultures. An additive number of colonies were observed with SRBC + LPS compared to that of SRBC or LPS alone. The colonies obtained from SRBC-containing cultures were stimulatable at high frequency by various FL-conjugated antigens to yield anti-FL PFC. However, colonies grown with LPS as the only additive were not stimulatable by any of the antigens tested. On the other hand, addition of M phi or SRBC as additional "mitogens" along with LPS in the agar resulted in progeny colonies that could respond in vitro. Although M phi did not increase the number of colonies, their presence enhanced the size and in some cases the frequency of stimulatable colonies. These data complement earlier observations in suggesting that different B cell subpopulations may grow under different cloning conditions. Moreover, the ability to stimulate the clonal progeny of single B cells to antibody synthesis should permit further definition of triggering and tolerance events at the single-cell level.     

Role of T Cells in the Mitogen-Induced Proliferation and Polyclonal Antibody Response of Murine B Cells

The effect of thymus-derived lymphocytes (T cells) on the responsiveness of bone marrow-derived lymphocytes (B cells) to bacterial lipopolysaccharide (LPS) was determined in in vitro experiments. Radiation resistant splenic T cells obtained from euthymic nu/+ mice increased the number of proliferating cells in the cultures of splenic B cells from athymic nu/nu mice even in a nonstimulated state. The radiation resistant T cells augmented significantly the responsiveness of B cells to LPS, as determined by an increase in proliferating cells and polyclonally induced anti-sheep erythrocyte (SRBC) IgM hemolysin plaque-forming cells (PFC). Addition of the T cells to B cell cultures not only augmented the responsiveness of B cells to suboptimal doses of LPS but also enabled B cells to respond to supraoptimal doses of LPS. As is well documented, the radiation resistant T cells were unable to induce the generation of anti-SRBC PFC in B cell cultures, unless the cultures were simultaneously stimulated with SRBC. Colcemid, a specific inhibitor of cell mitosis, blocked almost completely the exponential generation of anti-SRBC PFC in B cell cultures responding to SRBC with the aid of radiation resistant T cells. In contrast, colcemid did not affect the exponential generation of anti-SRBC PFC of a polyclonal nature in B cell cultures responding to LPS, either in the presence or absence of radiation resistant T cells.     

Relation between structure of bacterial lipopolysaccharide and the stimulation of B lymphocytes into colony formation.

Lipopolysaccharide (LPS) was analyzed in order to determine which component, lipid A or polysaccharide (PS), is able to stimulate B lymphocytes from ICR lymph nodes and spleen cells from nude (nu/nu) mice into forming colonies in soft agar culture. Lipid A, obtained by acid hydrolysis of LPS and solubilized by complex-formation with bovine serum albumin, was found to be the active moiety of LPS capable of stimulating colony growth of lymphoid cells in soft agar culture. The PS portion exhibited no significant activity at the concentrations used. Glycolipids from mutant strains of S. minnesota which contain the intact lipid moiety but are deficient in PS content, were as potent as S. abortus equi LPS in stimulating B cells into colony growth. Alkaline hydrolysis of LPS which cleaves ester-linked fatty acids, substantially decreased the number of lymphocyte colonies formed. This indicates that the intact lipid moiety is required for stimulating lymphocytes into colony formation. The synthetic glycolipid, N-palmitoyl-D-glucosamine (NPG), whose structure is similar to some components of lipid A, was also able to induce B lymphocyte colony development. In summary, our data point to lipid A as the active moiety of the endotoxin which induces B lymphocytes to grow and develop into colonies in the 2-layer soft agar culture system.     

Role of phosphatidylinositol 3-kinase in anti-IgM- and anti-IgD-induced apoptosis in B cell lymphomas

Cross-linking of surface Ig receptors with anti IgM (anti-mu heavy chain, anti-mu), but not anti-IgD (anti-delta heavy chain, anti-delta), Abs leads to growth arrest and apoptosis in several extensively characterized B cell lymphomas. By poorly understood mechanisms, both Igs transiently stimulate c-Myc protein expression. However, ultimately, only anti-mu causes a severe loss in c-Myc and a large induction of p27(Kip1) protein expression. Because phosphatidylinositol 3-kinase (PI3K) has been established as a major modulator of cellular growth and survival, we investigated its role in mediating anti-Ig-stimulated outcomes. Herein, we show that PI3K pathways regulate cell cycle progression and apoptosis in the ECH408 B cell lymphoma. Anti-mu and anti-delta driven c-Myc protein changes precisely follow their effects on the PI3K effector, p70(S6K). Upstream of p70(S6K), signaling through both Ig receptors depresses PI3K pathway phospholipids below control with time, which is followed by p27(Kip1) induction. Conversely, anti-delta, but not anti-mu stimulated PI3K-dependent phospholipid return to control levels by 4-8 h. Abrogation of the PI3K pathway with specific inhibitors mimics anti-mu action, potentiates anti-mu-induced cell death and, importantly, converts anti-delta to a death signal. Transfection with active PI3K kinase construct induces anti-mu resistance, whereas transfection with dominant negative PI3K augments anti-mu sensitivity. Our results show that prolonged disengagement of PI3K or down-regulation of its products by anti-mu (and not anti-delta) determines B cell fate.     

Activation of rat B lymphocytes. II. Functional and structural changes in "aged" rat B lymphocytes

Anti-mu, anti-gamma, and anti-delta antibodies induce proliferation of splenic B lymphocytes from young Lewis rats, measured by 3H-TdR uptake. In contrast, splenic B cells of aged Lewis rats respond poorly or not at all to these reagents. T lymphocytes or interleukin 2 (IL-2) of young or aged rats augment the uptake of 3H-TdR in cultures of "young" B cells responding to anti-Ig reagents or LPS and DxS, but have no significant effect on the responses of "old" B cells. Analysis of spleen cells of young and aged rats in a fluorescence-activated cell sorter indicates the density of mu, gamma, and delta isotypes is reduced in "old" B cells, and that B cells of aged rats are significantly larger than those of young rats. These results delineate anatomic and structural changes in B lymphocytes of aged rats.     

Lymphoma models for B cell activation and tolerance. VIII. Cross-desensitization by sIgM and sIgD and its effects on growth regulation by anti-isotype antibodies

ECH408-1 is a murine B cell lymphoma expressing idiotypically and allotypically distinguishable transfected and endogenous IgD. Previously, we demonstrated that this cell line was not growth inhibited by antibodies directed at membrane IgD, but could be inhibited by antibodies which crosslink membrane IgM. Herein, we demonstrate that both anti-mu and anti-delta will cause calcium mobilization in this transfected cell line; this is followed by a period during which antibodies against the alternative isotype are unable to induce significant increases in intracellular calcium concentrations. This phenomenon, called "desensitization," is short-lived, lasting 20 min. We further demonstrate that acute desensitization of these cells by anti-delta has no effect on immediate growth inhibition which is elicited by anti-mu. These data confirm our earlier proposal that the rapid, initial calcium response seen in these lymphomas is not required for the negative signal for growth. Moreover, we also demonstrate that pretreatment of these lymphoma cells with phorbol myristate acetate (PMA) also renders these lymphoma cells temporarily incapable of manifesting a significant calcium signal. Nonetheless, PMA-pretreated B lymphoma cells are not altered in their subsequent sensitivity to anti-mu growth inhibition, nor are they affected in their resistance to inhibition by anti-delta. Our data confirm the proposal that neither the calcium signal nor protein kinase-C activation is involved in the modulation of B lymphoma growth.     

Differential expression of a surface antigen recognized by a monoclonal antibody, J11d, on unprimed and primed B cells

Functional studies of both polyclonal and antigen-specific responses have suggested that murine B cells differ in the expression of an antigen recognized by a rat anti-mouse monoclonal antibody, called J11d. Using both positive and negative selection, we now demonstrate that the J11d marker is differentially displayed on B lymphocytes responding to LPS vs anti-mu, as well as on unprimed vs specific antigen-primed B cells. Thus, cytotoxic elimination of cells expressing high levels of J11d (J11d-hi) reduced LPS-driven B cell proliferation by 60 to 80% but had no effect on anti-mu stimulated B cell growth. Interestingly, equal numbers of positively selected J11d-hi B cells responded similarly to LPS and anti-mu plus B cell growth factors, a result that suggests that the response to anti-mu of the J11d-lo B cells is normally masked by the majority J11-d-hi cells. In further studies, the primary PFC response of normal murine spleen cells to fluorescein (FL)-coupled TI antigens or to LPS in vitro was reduced dramatically by cytotoxic J11d antibody treatment. In contrast, the anti-FL PFC response of spleen cells from mice primed 1 wk previously with FL-Ficoll was not affected by J11d antibody treatment, whereas the response of these FL-primed B cells to TNP (to which the mice were not primed) was greatly reduced by J11d + complement treatment. Our data indicate that antigen-experienced (activated) B cells are primarily found in the J11d-lo B cell subset and that unprimed (resting) B cells are found in the J11d-hi population, although both populations of murine B cells can respond to anti-mu. These studies also provide further evidence for B cell heterogeneity.     

Colony formation by subpopulations of human T lymphocytes. VI. Further studies on colony phenotype, function, and cloning efficiency

Phytohemagglutinin (PHA)-induced colony formation in semisolid agar medium by human peripheral blood T lymphocytes showed an increasing cloning efficiency with decreasing numbers of cultured cells. Ninety percent of CD4+ cells (inducer/helper phenotype) and 20% of CD8+ cells (cytotoxic/suppressor phenotype) formed colonies when cultured at 10-200 cells/ml culture in the presence of sheep red blood cells (SRBC) and a source of interleukin-2 (IL-2). Probably all T-colony-forming cells, but none of the subsequent colony cells, expressed the Leu-8 antigen. The cloning efficiencies of FACS-sorted cells expressing the natural killer antigenic phenotypes Leu-7+ and CD16+ were found to be less than 1%. The costimulatory effect of red blood cells for colony formation was specific for SRBC and not observed in the presence of red cells obtained from seven other species including man. All T-lymphocyte colonies obtained from unseparated peripheral blood mononuclear cells expressed the CD25 antigen (IL-2 receptor) and colonies were always composed of either CD4+ or CD8+ cells. None of the colony cells expressed the Leu-8 or the CD16 antigens. By their specific morphology in agar culture the majority of colonies composed of CD4+ cells were easily recognized, but but approximately one-third of the CD4+ colonies could not be distinguished from colonies composed of CD8+ cells. On expansion of individual colonies in liquid subculture in the presence of interleukin-2, approximately 15% of the colonies developed natural killer (NK)-like cytotoxic activity, being capable of direct killing of K562 tumor cells. It is concluded that the present method for growing human T colonies exhibits the same cloning efficiency as the most efficient liquid culture systems. Individual T colonies are composed exclusively of T inducer/helper or T cytotoxic/suppressor cells, they are never of mixed phenotype, and they do not contain cells of natural killer phenotype. Regulatory mechanisms influencing colony formation are operating between and within the various subsets of T lymphocytes.     

Analysis of the effects of erythrocytes on mitogen-dependent clonal proliferation of murine B lymphocytes

Addition of intact erythrocytes to semisolid agar cultures of murine B cells dramatically improves cloning efficiency and affects colony morphology. In this study, we investigated possible mechanisms through which this might occur. Specific modification of sheep erythrocyte (SRBC) membranes by treatment with trypsin but not other enzymes improved colony potentiation and erythrocytes from rats, mice, and humans were also effective after trypsin treatment. In addition, autoantibody-coated murine erythrocytes were superior to normal cells in this regard. These observations suggest that erythrocytes enhance lymphocyte survival and/or proliferation by means of particular membrane-mediated processes. The possible importance of erythrocytes as scavengers of toxic hydroxyl radicals was also investigated. Deliberately generated radicals formed by addition of dihydroxyfumaric acid and iron were effectively countered by addition of SRBC. More detailed analyses revealed that of several endogenously produced toxic species, hydrogen peroxide may be the most important under ordinary culture conditions. That is, addition of catalase but not Superoxide dismutase or mannitol improved cloning efficiency in cultures lacking SRBC. These studies suggest that erythrocytes have a beneficial effect on lymphocyte survival and function in culture through at least two mechanisms.     

Immune reactivity of frozen-thawed murine spleen cells.

Spleen cells from mice immunized with SRBC were subjected to controlled rate freezing to ?100 °C. Complete recovery of PFC was obtained with DMSO used as the cryopreservative. Simple dilution of spleen cells in DMSO, or a single cycle of freezing and thawing in DMSO prior to short-term culture, resulted in early loss of recoverable cells. A single cycle of freezing and thawing inhibited the in vitro immune response to SRBC while having little effect on the response to TNP-T4. The in vitro blastogenic responses to LPS and PHA-P were severely reduced in cultures of frozen and thawed cells.     

In vitro development of a primary antibody response with lymph node cells.

Utilizing a variety of lymphoid tissues from three common laboratory species, comparative studies were performed to investigate the competence of the dissociated cells to respond to a heterologous erythrocyte with the development of specific plaque-forming cells. Dissociated spleen cells harvested from BDF1 mice consistently developed specific plaque-forming cells (PFC) to sheep red blood cells (SRBC), while hamster spleen cells inconsistently developed specific antibody-forming cells to SRBC. Under identical conditions, guinea pig spleen cells did not develop significant numbers of PFC to SRBC. However, lymph node cell cultures of all three species tested yielded specific PFC. In the mouse and hamster lymph node cell cultures, the yield of PFC per culture or per 10⁶ recovered viable cells was always greater than the yield from companion spleen cell cultures. Guinea pig mesenteric lymph node cell cultures developed the major PFC response to SRBC, while both mesenteric and peripheral lymph node cell cultures from hamsters were equivalent in their response to SRBC. The data demonstrate that it is possible to develop a primary antibody response to SRBC in vitro utilizing normal endogenous hamster or guinea pig lymphoid cells, if lymph nodes are the source of cells.     

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.     

Structural and serological studies of lipopolysaccharides of Citrobacter O35 and O38 antigenically related to Salmonella

Abstract Lipopolysaccharide (LPS) was administered into sheep red blood cells (SRBC)-primed mice, and the effect of LPS on SRBC-specific memory cells was investigated. Spleen cells from SRBC-primed mice which were injected with LPS exhibited much lower in vitro secondary plaque-forming cells (PFC) responses to SRBC than those from untreated SRBC-primed mice. The in vitro anti-SRBC response of the spleen cells to LPS was also reduced. The combination experiments of B cells and T cells from SRBC-primed mice which were injected with or without LPS demonstrated that the reduction of immune responses to SRBC after administration of LPS was caused by the defect of SRBC-specific B memory cells, but not T memory cells. B cell type rosette-forming cells (RFC) for SRBC markedly decreased after injection of LPS, while PFC as antibody-forming cells did not increase subsequently. Therefore, the reduction of RFC was not due to their differentiation into PFC. The lymphoid follicles in the spleens from mice injected with LPS were stained positively by in situ nick end labeling specific for fragmented DNA. A large percentage of Ig⁺ spleen cells from SRBC-primed mice which were injected with LPS was also stained positively. The injection of glucocorticoids into SRBC-primed mice induced similar reduction of B memory cells. It was suggested that LPS might induce apoptosis of B memory cells and regulate B cell memory in antigen-nonspecific manner.     

Role of mercaptoethanol and endotoxin in stimulating B lymphocyte colony formation in vitro.

Mercaptoethanol is necessary to permit B lymphocyte colony formation in semi-solid agar cultures of cells from normal mouse lymphoid organs. Transfer studies on developing colonies showed that, in part, this was a direct action on B lymphocyte colony cells but evidence was produced that in the presence of mercaptoethanol lymphoid organ cells release a factor promoting colony growth. Endotoxin strongly potentiated B lymphocyte colony formation in vitro by a direct action on colony cells but in the absence of mercaptoethanol did not allow cell survival or proliferation.     

Formation of heterophile antibodies by human tonsilar lymphocytes: II. In vitro stimulation with allogeneic cells

Short-term cultures of human tonsilar lymphocytes (HTL), 5 × 10⁶ cells/culture, in medium RPMI 1640 supplemented with human group AB serum were studied for the production of plaque-forming cells (PFC) against sheep (SRBC) and bovine (BRBC) red blood cells following in vitro stimulation by various allogeneic lymphoid cells. Of 55 HTL specimens examined, 48 produced a significant number (50–300/culture) of PFC against SRBC and/or BRBC following the in vitro stimulation. The optimal doses of the stimulator HTL and peripheral blood lymphocytes (PBL) were 10⁷ and 5 × 10⁶/culture, respectively. After the stimulation, PFC appeared in significant numbers on the third day, reached the peak number on the sixth day, and decreased sharply in number thereafter. Removal of E-rosetting cells from both stimulator and responder populations abolished the PFC formation. PFC formation against SRBC was inhibited by solubilized Forssman antigen, while PFC formation against BRBC was inhibited strongly by Hanganutziu-Deicher antigen, hardly by Paul-Bunnell antigen and not at all by Forssman antigen. Supernatants of mixed lymphocyte culture of PBL were shown to enhance PFC formation of HTL cultures stimulated by allogeneic lymphocytes. The results of this study indicated that in vivo primed B cells of the HTL were triggered in vitro by allogeneic stimulation for the heterophile antibody formation. Since these antibodies are apparently directed against Forssman and Hanganutziu-Deicher antigens, the “allo” nature of these antigens as well as their relationship to the previously described heterophile transplantation antigens have to be clarified.     

Effects of inositol, LiCl, and heparin on the antibody responses to SRBC by normal and immunodeficient XID mice

Spleen cells from na?ve adult immunocompetent and immunodeficient XID mice were cultured on agar containing sheep red blood cells (SRBC) with and without myo-inositol, scyllo-inositol, lithium chloride, or heparin, and after 1 or 2 days the number of colonies of antiSRBC antibody-forming cells (PFC) were determined. It was found that myo-inositol and scyllo-inositol at one-tenth the concentration were equally effective in increasing the number of specific PFC. Myo-inositol, scyllo-inositol, and lithium chloride accelerated the appearance of direct foci in cultures of spleen cells from normal and XID mice. When heparin was added to cultures of XID spleen cells, PFC were found to be increased on Day 1; however, PFC and foci were not increased in cultures of spleen cells from competent mice until 1 day later. The addition of combinations of these agents to cultures of spleen cells had no positive or negative effect on the generation of foci or PFC. Normal mice given heparin intraperitoneally with SRBC had increased splenic PFC on Days 3 and 4 but not on Day 7. The results suggest that these agents modulate B-cell responses by increasing the rate of proliferation and/or secretion through a signaling pathway(s) distal to, or more likely, independent of Bruton's tyrosine Kinase (BTK). It is not clear that the mechanism is the same with each agent.     

The chicken's femoral-lymph nodules: T and B cells and the immune response.

Femoral lymph nodules (FLN), which are barely perceptible in normal birds, after a footpad injection of sheep red blood cells (SRBC) may either significantly enlarge (responder) or remain reduced in size (nonresponder). There were approximately 38% T cells and 53% B cells in the FLN of responder chickens. Significantly more plaque-forming cells (PFC) developed in the FLN than in the spleen after a footpad injection of SRBC. Total antibody, mercaptoethanol- (ME) resistant, and ME-sensitive fractions were significantly higher in birds given i.v. than in those given footpad injections. There were no differences in PFC and agglutinin titers between FLN-responders and nonresponders. The number of PFC in FLN exceeded the number of splenic PFC previously reported. The high PFC response of the FLN may reflect the large percentage of B cells in this lymphomyeloid tissue or the presence of antigen-experienced B cells in the FLN. Although FLN may influence a systemic immune response its major role appears to be restricted to a local response.     

Mononuclear macrophage (M-CFC) colony formation in semisolid media in the absence of exogenous GM-CSF

Human fetal bone marrow (FBM) cells were examined for the ability to form colonies in the absence of exogenous colony-stimulating factor (CSF) in double layer agar, methylcellulose (MC), and in agar-MC (agar underlayer, MC overlayer) culture systems. Without exogenous CSF, macrophage colonies (M-CFC) were formed in a combined culture of agar and MC. Aggregates of 5-40 cells were observed on day 7. Gradually, large compact colonies which survived for 10-12 weeks of cultivation, were formed. They were composed of mononuclear monocytes and multinucleated cells. M-CFC progenitors were nonadherent, but their progeny became adherent during differentiation within the colony. Colony formation was cell-dose-dependent. Depletion of monocytes increased the number of colonies in agar-MC cultures and stimulated the development of some macrophage colonies in MC. Survival of monocyte progenitors was not dependent on CSF. Neither was their proliferation nor partial differentiation in agar-MC cultures. CSF increased M-CFC colony efficiency, however, if it was present when cultures were initiated. Addition of CSF to M-CFC growing for 2-5 weeks in CSF-deprived medium stimulated monocytes proliferation and transformation into macrophages. Epithelioid cells, an increase in the number of giant multinucleated cells, and granulocyte multiplication were also observed. The absolute dependence of macrophage colony formation on CSF described by others might be a result of inadequate culture conditions due to agar rather than an intrinsic physiological requirement.