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B Lymphocyte Colony Formation in vitro: Ultrastructural Development of Individual Colonies
Differentiation
Differentiation 17, 77-84 (1980)
0 Springer-Vcrlag I980
B Lymphocyte Colony Formation in vitro: Ultrastructural Development of Individual Colonies POUL BRIX POULSEN and MOGENS H. CLAESSON The Panum Institute, Department of Anatomy A and B, University of Copenhagen, Blegdamsvej 3, DK-2200,Copenhagen N, Denmark
B lymphocyte colony development in agar culture was studied using an electron microscope, and more than 3,000 colony cells were identified and photographed. In early cultures (day 4) lymphoblasts dominated the colonies. From day 5 onwards plasmablasts and small lymphocytes were present in the colonies. From day 6 onward mature plasma cells were observed in increasing numbers. On day 9 of culture the colonies started to degenerate and on day 10 of culture approximately 70% of the colony consisted of pyknotic and degenerating cells. Topographically, the degenerating cells were concentrated in the center of the colony whereas proliferation took place in the periphery. Colony growth occurred in an exponential fashion, the number of viable colony cells being maximal on day 8 of culture (400-600 cells/colony). At this time the frequencies of the four B cell categories were: lymphoblasts 72%, plasmablasts 20%, plasma cells 6%, and small lymphocytes 2%. Recloning experiments showed that dispersed colony cells were capable of forming only small cell clusters. It is concluded that B lymphocyte colony formation reflects a series of B cell developmental stages including the formation of the end cell categories of this lymphocyte lineage.
Introduction B lymphocyte colony formation in vitro is today a well established method for the study of B lymphocyte proliferation capacity [l-41, B lymphocyte differentiation [5-71, and lymphocyte function and regulation [8-lo]. However, very little is known about the proliferation kinetics and the differentiation within the individual B lymphocyte colony (BLC). Although specific antibody production has been shown to occur in individual BLCs [8], it is not known whether the colony cells reach a morphologically recognizable end cell stage, i.e., become plasma cells or small lymphocytes or if they die at some immature stage of development. This lack of knowledge regarding BLC morphology has promted us to investigate the ultrastructure of developing individual colonies. The data presented strongly suggests that the proliferation of colony forming cells and colony cells reflects a whole series
of B lymphocytes developmental stages including B lymphocyte activation, proliferation, and differentiation. Methods Culture of B Lymphocyte Colonies Spleen cell suspensions were prepared from 6-8-week-old BALBlc mice and added to an equal volume mixture of double strength Dulbecco’s modified Eagle’s medium at room temperature and 0.6% Bacto agar (Difco, Detroit, Mich.). The formula for the culture medium has been described elsewhere [l]. Fetal calf serum (FCS) was added in a final concentration of 5%. The cultures were supplemented with 5 X lo-’ M 2-mercdptoethanol and 3% washed sheep red blood cells (SRBC).One ml volumes of agar culture medium containing 15,000 spleen cells were added to 35-mm plastic dishes and allowed to gel. Cultures were incubated for 4- 10 days at 37”C in a fully humidified atmosphere of 10% C02 in air. To visualize the developing colonies 0.5 ml of 0.9% NH&I was added for 5 min at the termination of the culture period. After the SRBC had been lysed the culture was washed twice with 2nd RMPI-1640 medium.
0301-4681/80/0017/0077/$ 01.60
78
P. B. Poulsen and M. H. Claesson: Ultrastructure of B Lymphocyte Colonies
Fixation and Embedding for Electron Microscopy
Colony Recloning Experiments
The largest of the developing cell clones were removed with a Pasteur pipette and fixed in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4"C for 12 h. The clones were then washed for 2 x 30 min in 0.1 M phosphate buffer (pH 7.2) at 4" C. Post-fixation was performed in 2% Os04in 0.1 M phosphate buffer (pH 7.2) for 2 h at 37"C. The clones were then washed in 0.1 M phosphate buffer (pH 7.2) at 4"C for 10 min, and for 30 min at 4" C in distilled water. Subsequently, the clones were placed in 0.5% uranyl acetate in distilled water for 12 h at 4" C, and finally washed for 20 min at 4" C in distilled water. The clones were then dehydrated in increasing concentrations of ethanol and embedded in Epon 812. A series of ultrathin sections of individual clones were cut with a 10 pm distance between each series using an LKB ultratome (see below). The ultrathin sections were stained with uranyl acetate and lead citrate.
In separate experiments colony cells were mass-collected by mechanical disruption of the whole agar in serum free RMPI-1640. The agar was removed by centrifugation of the harvested cells through FCS as described above. The pelleting cells were washed twice and counted for viability before seeding in agar medium for BLC formation.
Results Growth of B Lymphocyte Colonies
B lymphocyte colony forming cells started to divide on days 1-2 of culture. Cultures on day 7 contained approximately 300 colonies per 15,000 cells seeded with more than 50 cells per colony. At day 3 the largest colonies contained 20-30 cells (Table 1). The colonies continued to proliferate in an exponential fashion for the first eight days Of reaching a size of 400-600 cells on day 8 of culture. During this period of growth the number of dead cells with& the colonies was extremely low, but from day 9 of culture colony cells died rapidly and colony proliferation decreased.
Determination of Colony Cells and Their Position Within the Clones A 1 pm survey section was cut immediately before each series of ultrathin sections, stained with Toluidine blue, and drawn wine. a Reichert Diapan microscope with a side tube projecting the section image onto a piece of drawing paper. All cells on the drawings were identified in the subsequent series of ultrathin sections and photographed using a Philips EM 300 electron microscope. The individual clone was divided arbitrarily into three zones, the central. the intermediate, and the outer zone respectively. The radius of the central zone was set to one third of the clone radius whereas the intermediate zone was situated in the area from one to two thirds of the clone radius. The numbers of lymphoblasts, plasmablasts, small lymphocytes, and plasma cells (see results) were determined by combining the electron micrographs and the drawings of individual survey sections.
Colony Cell Categories
In the present study more than 3,000 individual cells were examined at the ultrastructural level and four colony cell categories were identified by the traditional criteria [111: lymphoblasts, plasmablasts, plasma cells, and small lymphocytes. Figure la-d shows the typical appearance-of these four cell types. In brief, the 'ymphoblast is defined as a large with a euchromatic nucleus with a Small rim Of condensed chromatin at the nucleolemma or no condensed chromatin at all. The cytoplasm contains free ribosomes or polyribosomes and only scanty endoplasmic reticulum. The Goki apparatus is poorly developed (Fig. la).
Colonv Cell Counts Ten to 50 individual colonies were removed from the agar culture at days 3- 10 of culture and transferred to RMpI-1640 medium. The colonies were pooled in groups of 5-10 and dispersed mechanically in 0.5 ml of serum free RMPI-1640 medium using a fine Pasteur pipette. Dispersed cells were layered above 0.5 ml FCS, and spun down through the serum by centrifugation (85OgllO min). The cell pellet was resuspended in 0.2 ml eosin stain and counted, and the number of viable and non-viable cells per colony calculated.
Table 1. Number of cells per B lymphocyte clone on different days of culturea Day of culture Colony cells
3b
4
Viable cells
28 + 7
62
Non-viable cells
0
0
5
+ 18
6
8
7
9
0
0
15 -
30-
221 k 19 392
"Cell number in 10-50 B lymphocyte colonies, pooled in groups of five, were counted Cell numbers were estimated from counts in the culture plate
'
10
103k 35 155 + 3 6 280+73 475 +90 259k 57 1 7 + 33
+ 10
P. B . Poulsen and M. H. Claesson: Ultrastructure of B Lymphocyte Colonies
The plasmablast (Fig. lb) is also a large cell with a euchromatic nucleus, but with condensed chromatin typically in the shape of flakes at the nucleolemma. The cytoplasm contains endoplasmic reticulum sur-
79
rounding the nucleus and the Golgi apparatus is well developed. The chromatin of the plasma cell is condensed forming typical pore complexes at the nucleolemma.
Fig. la-h. B lymphocyte colony cells. a: lymphoblast, b: plasmablast, c: plasma cell, and d: small lymphocyte on day 7 of culture, e-f degenerating colony cells on day 9 of culture, g: late mitotic plasmablast on day 7 of culture, h: broad contract zone between two lymphoblasts on day 6 of culture, a-g: magnification X 8,500, b: magnification X 85,000
80
P. B. Poulsen and M. H. Claesson: Ultrastructure of B Lymphocyte Colonies
The cytoplasm is stuffed with endoplasmic reticulum, which contains a more electron dense material in the cisternae compared to the plasmablast. The Golgi apparatus is well developed in the plasma cell (Fig. lc). Nuclear segmentation is often seen both in the plasmablasts and in the plasma cells. The small lymphocyte (Fig. Id) contains con-
Fig. le-h.
densed chromatin at the nucleolemma as well as small chromatin islands. The cytoplasm is scanty containing only a few mitochondria, free ribosomes, and polyribosomes. Of the viable colony cells the majority (more than 90%) were easily placed in one of these four categories. In the late days of culture, cells in various stages
P. B . Poulsen and M. H. Claesson: Ultrastructure of B Lymphocyte Colonies
of degeneration were observed. Figure le and f shows a pyknotic cell and a cell with severe signs of degeneration with no cytoplasmic organelles left. Many mitotic cells were recognized throughout the culture period, being most frequent among lymphoblasts and plasmablasts including rather differentiated plasmablasts (Fig. lg). Occasionally cell membranes of proliferating cells were observed in close contact with each other for broad zones of 0.1-0.5 pm, the intercellular cleft was less than 100 A and suggested the existence of gap junctions in these areas (Fig. lh). Such cell contacts were most frequently observed in colonies on day 6 of culture. They were not only observed between cells of the same category but were also present between lymphoblasts and plasmablasts.
Ultrastructural Development of B Lymphocyte Colonies Figure 2 diagramatically shows the appearance of the four major cell categories at different periods of culture (day 4-10). On day 4 only lymphoblasts were
81
observed in developing colonies. On day 5 lymphoblasts dominated the developing colonies but plasmablasts and a few small lymphocytes were now present. On day 6 of culture the incidence of plasmablasts and lymphoblasts were identical but from day 7 to day 9 the incidence of lymphoblasts again outnumbered that of plasmablasts. During the late period of culture both small lymphocytes and especially plasma cells appeared in colonies. Figure 2 also illustrates the topographical localization of the four major cell categories within individual colonies. The majority of cells were located in the outer zone of the colony [which made up the major part of the colony (see Methods)]. Except from day 9 plasmablasts and lymphoblasts were found to be evenly distributed throughout the three zones examined whereas small lymphocytes and plasma cells were seen in the intermediate zone and especially in the outer zone of the colony. The majority of mitotic cells were located in the outer colony zone whereas dead cells were first observed in the central part of the colony (data not included in Fig. 2). Figure 3 combines the data from Table 1 and Fig. 2 by showing the fluctuations in absolute num-
Fg.2. The graph illustrates the percentage of the four different cell categories (lymphoblast = LB, plasmablast = PB, small lymphocyte = SL, and plasma cell = PC)in the B cell colonies from day 4 to day 10. The graph also illustrate the distribution on the four different cell categoriesin the central, intermediate, and peripheral zones of the cell colonies, respectively.A total of 15 colonies were studied. Numbers of viable colony cells examined were: day 4 = 51, day 5 = 123, day 6 = 655, day 7 = 203, day 8 = 204, day 9 = 484, and day 10 = 178. Since coloniesfrom the same day of culture did not differ with respect to cell categories or cell position the columns represent the pooled data from 2-3 individual colonies
82
P. B. Poulsen and M. H. Claesson: Ultrastructure of B Lymphocyte Colonies
Fig. 3. The graph illustrates the total number of cells per colony in the four cell categories mentioned in Fig. 2. Data are combined from results present in Table 1 and Fig. 2
Table 2. Cluster formation by mass-collected colony and cluster cellsa
day 9 of culture recloned BLC’s did not form clusters.
Day of recloning
5
6
7
8
9
10
20k5
6 f 3
58f28
17+3
6f4
2 f 2
Cells were mass-harvested from primary 5-10 day BLC cultures and recloned in secondary agar cultures. Data represent cluster formation (cluster = 5-32 cells) expressed as means of triplicate cultures with 10,OOO cells/culture counted on day 6 of culture
a
bers of the four categories of cells as a function of the culture time. Apart from days 6 and 10 the absolute number of lymphoblasts was much higher than that of plasmablasts. It is evident from data in Fig. 3 that the B lymphocyte colony produces a substantial number of both plasma cells and small lymphocytes. Recloning of B Lymphocyte Colony Cells
Table 2 shows the results of the recloning experiments. Colony cells harvested from days 5-6 of culture were only capable of forming a few clusters, whereas day 6 and 7 mass-harvested BLC cultures gave rise to a considerable number of cell clusters and in addition to a few cell colonies containing more than 32 cellskolony (data not included in Table 2). From
Discussion The present ultrastructural study shows that clonal proliferation of B lymphocytes in agar culture results in formation of both of the end cell categories of this lymphocyte lineage, i.e., plasma cells and small lymphocytes. Although the colonies grew exponentially giving rise to 400-600 cells on day 8 of culture, the frequency of end cells produced by the individual B lymphocyte colony was low and the majority of proliferating and differentiating cells died at the blast cell stage of development. The present study also elucidates the topographic localization of colony cells: cells in the center of the colony appeared to degenerate prior to cells in the periphery, whereas blast cells and mitotic cells were located in the peripheral zone of the colony. This pattern of organization within the colony differs markedly from that of proliferating granulocyte-macrophage colonies from the bone marrow where the proliferating cells are located to the center of the colony whereas differentiation takes place in the periphery [12]. From experiments in liquid culture it is well known that polyclonal activators of B lymphocytes lead to the formation of antibody producing cells.
P. B. Poulsen and M. H. Claesson: Ultrastructure of B Lymphocy:e Colonies
These can be assayed as specific plaque-forming cells (PFC) against a variety of different antigens [13, 141. The BLC system also appears to proliferate in response to polyclonal activation induced by 2-ME-activated albumin and SRBC as well as agar mitogen in the culture medium [15, 161. In the liquid culture system mentioned above [14] the frequency of PFCs is high compared to the frequency of mature plasma cells in individual BLCs suggesting either that the colonies are less differentiated than the clones arising in liquid culture or that the cells are capable of secreting antibodies before they reach an end cell stage, i.e., become plasma cells. This last view is supported by the fact that the number of SRBC-PFC obtained from an individual specific SRBC-antibody producing BLC can be as high as 240 [8]. Moreover, the relatively small number of colonies examined in the present study makes it dubious to generalize with regard to the degree of plasma cell development in BLCs. From the present data it is possible to delineate the following order of cell development during BLC formation: (1) small B lymphocyte (BL-CFC) + (2) lymphoblast + (3) lymphoblast/plasmablast + (4) lymphoblast/plasmablast/small lymphocyte (BL-CFC)/plasma cell. The BL-CFC‘s are known to be small lymphocytes themselves, the majority having detectable IgM on their surface [5,7,17]. BL-CFC start to proliferate on day 2 of culture and form only lymphoblasts during the next two days, after which differentiation occurs and both lymphoblasts and plasmablasts are formed in equal numbers. From day 6 onwards lymphoblasts dominate the colonies and small lymphocytes and plasma cells appear in small numbers. From day 8 onwards colony cells start to die. Recloning experiments showed that BL-CFC did not appear in colonies except on day 7 of culture and that this point of time coincided with the appearance of small lymphocytes. This mode of BL-CFC proliferation differ markedly from that of human T lymphocyte colony formation in which the colony forming cell (TL-CFC) exists throughout the ten day culture period [18]. The observation of broad contact zones between blast cells in developing colonies with an intercellular gap of approximately 100 A suggests the presence of the so-called gap junctions in these areas. Such contact structures have recently been described between phytohemagglutinin aggregated rabbit lymphocytes [19], and they might be of importance for synchronization of colony cell proliferation. Freeze-fracture electron microscopic studies are in
83
progress to elucidate the nature of the broad contact zones between colony cells. In conclusion, the present results show that B lymphocyte colony formation reflects a whole series of B lymphocyte developmental stages including formation of all the cell categories belonging to the B lymphocyte lineage. Acknowledgements. Excellent technical work was performed by Mrs. Ursula Rentzmann. This study was supported by grants from “Foreningen ti1 Laeevidenskabens Fremme” and “Kong Christian X’s Foundation”.
References 1. Metcalf D, Nossal GJV, Warner NL, Miller JFAP, Mandel TE, Layton JE, Gutman GA (1975) Growth of B lymphocyte colonies in vitro. J Exptl Med 142: 1534 2. Claesson MH, Metcalf D (1977) B lymphocyte colony-forming cells in the SJUJ mouse thymus. J Immunol 118: 1208 3. Lala PK, Johnson GR (1978) Monoclonal origin of B lymphocyte colony-forming cells in spleen colonies formed by multipotential hemopoietic stem cells. J Exptl Med 148: 1468 4. Gjedde SB (1980) B lymphocyte colony forming cells in thymus of AKR mice. Exptl Hematol In press 5. Kincade PW, Ralph P (1976) Regulation of clonal B lymphocyte proliferation by anti-immunoglobulin or anti-Ia antibodies. Cold Spring Harbor Symp Quant Biol 41: 245 6. Howard MC, Claesson MH, Johnson GR (1977) Surface immunoglobulin characteristcs of B lymphocyte developmental states and B lymphocyte colony-formingcells. Scand J Immunol 6: 1317 7. Kincade PW, Paige CT, Parkhouse RMR, Lee G (1978) Characterization of murine colony-forming B cells. I. Distribution, resistance to anti-immunoglobulin antibodies, and expression of Ia antigens. J Immunol 120: 1289 8. Claesson MH, Layton JE, Luckenbach GA (1978) Specific antibody-forming B lymphocyte colonies. I. Distribution and nature of SRBC antibody-forming B lymphocyte colonies in mouse lymphomyeloid organs. Immunology 35 : 397 9. Claesson MH (1979) Soluble suppressor activity of concanavalin A-activated spleen cells on B lymphocyte colony formation in vitro. Cell Immunol 42: 344 10. Claesson MH (1979) B lymphocyte colony suppression in vitro: Characteristicsof the suppressor cell. In:Kaplan JG (ed) Proceedings of the 13th International Leucocyte Culture Conference, Ottawa, Canada. ElseviedNorth Holland Biomedical Press, Amsterdam New York Oxford, p 680 11. Mandel TE (1977) The ultrastructure of mammalian lymphocytes and their progeny. In: Marchalonis JJ (ed) The lymphocyte structure and function. New York Basel, p 11 12. Metcalf D (1977) Hemopoietic colonies. In vitro cloning of normal and leukemic cells. Recent Results Cancer Res 61 : 58 13. Fauci AS (1979) Human B cell function in a polyclonally induced plaque forming cell system. Cell triggering and immunoregulation. Immunol Rev 45 : 93 14. Anderson J, Coutinho A, Melchers F (1977) Frequencies of mitogen-reactive B cells in the mouse. 11. Frequencies of B cells producing antibodies which lyse sheep or horse erythrocytes, and trinitrophenylated or nitroiodophenylated sheep erythrocytes. J Exptl Med 145: 1520
84
P. B. Poulsen and M. H. Claesson: Ultrastructure of B Lymphocyte Colonies
15. Claesson MH, Had H-D, Opitz HG (1979) B and T lymphocyte colony formation in agar: 2-Mercaptoethanol-a~tivated albumin can substitute for 2-mercaptoethanol. Cell Immunol 46: 398 16. Kincade PW, Ralph P, Moore MAS (1976) Growth of B lymphocyteclones in semisolidculture is mitogen dependent. J Exptl Med 143: 1265 17. Metcalf D, Wilson JW, Shortman K, Miller JFAP, Stocker J (1976)The nature of cells generating B lymphocyte colonies in vitro. J Cell Physiol 88: 107
18. Smith SD, Sachs L (1979) Difference in the cell proliferation and colony-forming ability of normal human T lymphocytes. Clin Exptl Immunol 37: 348 19. Kapsenberg ML, Leene W (1979) Formation of B type gap junctions between Pha-stimulated rabbit lymphocytes. Exptl Cell Res 120: 211
Received February 1980/Accepted May 1980
Differentiation 17, 77-84 (1980)
0 Springer-Vcrlag I980
B Lymphocyte Colony Formation in vitro: Ultrastructural Development of Individual Colonies POUL BRIX POULSEN and MOGENS H. CLAESSON The Panum Institute, Department of Anatomy A and B, University of Copenhagen, Blegdamsvej 3, DK-2200,Copenhagen N, Denmark
B lymphocyte colony development in agar culture was studied using an electron microscope, and more than 3,000 colony cells were identified and photographed. In early cultures (day 4) lymphoblasts dominated the colonies. From day 5 onwards plasmablasts and small lymphocytes were present in the colonies. From day 6 onward mature plasma cells were observed in increasing numbers. On day 9 of culture the colonies started to degenerate and on day 10 of culture approximately 70% of the colony consisted of pyknotic and degenerating cells. Topographically, the degenerating cells were concentrated in the center of the colony whereas proliferation took place in the periphery. Colony growth occurred in an exponential fashion, the number of viable colony cells being maximal on day 8 of culture (400-600 cells/colony). At this time the frequencies of the four B cell categories were: lymphoblasts 72%, plasmablasts 20%, plasma cells 6%, and small lymphocytes 2%. Recloning experiments showed that dispersed colony cells were capable of forming only small cell clusters. It is concluded that B lymphocyte colony formation reflects a series of B cell developmental stages including the formation of the end cell categories of this lymphocyte lineage.
Introduction B lymphocyte colony formation in vitro is today a well established method for the study of B lymphocyte proliferation capacity [l-41, B lymphocyte differentiation [5-71, and lymphocyte function and regulation [8-lo]. However, very little is known about the proliferation kinetics and the differentiation within the individual B lymphocyte colony (BLC). Although specific antibody production has been shown to occur in individual BLCs [8], it is not known whether the colony cells reach a morphologically recognizable end cell stage, i.e., become plasma cells or small lymphocytes or if they die at some immature stage of development. This lack of knowledge regarding BLC morphology has promted us to investigate the ultrastructure of developing individual colonies. The data presented strongly suggests that the proliferation of colony forming cells and colony cells reflects a whole series
of B lymphocytes developmental stages including B lymphocyte activation, proliferation, and differentiation. Methods Culture of B Lymphocyte Colonies Spleen cell suspensions were prepared from 6-8-week-old BALBlc mice and added to an equal volume mixture of double strength Dulbecco’s modified Eagle’s medium at room temperature and 0.6% Bacto agar (Difco, Detroit, Mich.). The formula for the culture medium has been described elsewhere [l]. Fetal calf serum (FCS) was added in a final concentration of 5%. The cultures were supplemented with 5 X lo-’ M 2-mercdptoethanol and 3% washed sheep red blood cells (SRBC).One ml volumes of agar culture medium containing 15,000 spleen cells were added to 35-mm plastic dishes and allowed to gel. Cultures were incubated for 4- 10 days at 37”C in a fully humidified atmosphere of 10% C02 in air. To visualize the developing colonies 0.5 ml of 0.9% NH&I was added for 5 min at the termination of the culture period. After the SRBC had been lysed the culture was washed twice with 2nd RMPI-1640 medium.
0301-4681/80/0017/0077/$ 01.60
78
P. B. Poulsen and M. H. Claesson: Ultrastructure of B Lymphocyte Colonies
Fixation and Embedding for Electron Microscopy
Colony Recloning Experiments
The largest of the developing cell clones were removed with a Pasteur pipette and fixed in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4"C for 12 h. The clones were then washed for 2 x 30 min in 0.1 M phosphate buffer (pH 7.2) at 4" C. Post-fixation was performed in 2% Os04in 0.1 M phosphate buffer (pH 7.2) for 2 h at 37"C. The clones were then washed in 0.1 M phosphate buffer (pH 7.2) at 4"C for 10 min, and for 30 min at 4" C in distilled water. Subsequently, the clones were placed in 0.5% uranyl acetate in distilled water for 12 h at 4" C, and finally washed for 20 min at 4" C in distilled water. The clones were then dehydrated in increasing concentrations of ethanol and embedded in Epon 812. A series of ultrathin sections of individual clones were cut with a 10 pm distance between each series using an LKB ultratome (see below). The ultrathin sections were stained with uranyl acetate and lead citrate.
In separate experiments colony cells were mass-collected by mechanical disruption of the whole agar in serum free RMPI-1640. The agar was removed by centrifugation of the harvested cells through FCS as described above. The pelleting cells were washed twice and counted for viability before seeding in agar medium for BLC formation.
Results Growth of B Lymphocyte Colonies
B lymphocyte colony forming cells started to divide on days 1-2 of culture. Cultures on day 7 contained approximately 300 colonies per 15,000 cells seeded with more than 50 cells per colony. At day 3 the largest colonies contained 20-30 cells (Table 1). The colonies continued to proliferate in an exponential fashion for the first eight days Of reaching a size of 400-600 cells on day 8 of culture. During this period of growth the number of dead cells with& the colonies was extremely low, but from day 9 of culture colony cells died rapidly and colony proliferation decreased.
Determination of Colony Cells and Their Position Within the Clones A 1 pm survey section was cut immediately before each series of ultrathin sections, stained with Toluidine blue, and drawn wine. a Reichert Diapan microscope with a side tube projecting the section image onto a piece of drawing paper. All cells on the drawings were identified in the subsequent series of ultrathin sections and photographed using a Philips EM 300 electron microscope. The individual clone was divided arbitrarily into three zones, the central. the intermediate, and the outer zone respectively. The radius of the central zone was set to one third of the clone radius whereas the intermediate zone was situated in the area from one to two thirds of the clone radius. The numbers of lymphoblasts, plasmablasts, small lymphocytes, and plasma cells (see results) were determined by combining the electron micrographs and the drawings of individual survey sections.
Colony Cell Categories
In the present study more than 3,000 individual cells were examined at the ultrastructural level and four colony cell categories were identified by the traditional criteria [111: lymphoblasts, plasmablasts, plasma cells, and small lymphocytes. Figure la-d shows the typical appearance-of these four cell types. In brief, the 'ymphoblast is defined as a large with a euchromatic nucleus with a Small rim Of condensed chromatin at the nucleolemma or no condensed chromatin at all. The cytoplasm contains free ribosomes or polyribosomes and only scanty endoplasmic reticulum. The Goki apparatus is poorly developed (Fig. la).
Colonv Cell Counts Ten to 50 individual colonies were removed from the agar culture at days 3- 10 of culture and transferred to RMpI-1640 medium. The colonies were pooled in groups of 5-10 and dispersed mechanically in 0.5 ml of serum free RMPI-1640 medium using a fine Pasteur pipette. Dispersed cells were layered above 0.5 ml FCS, and spun down through the serum by centrifugation (85OgllO min). The cell pellet was resuspended in 0.2 ml eosin stain and counted, and the number of viable and non-viable cells per colony calculated.
Table 1. Number of cells per B lymphocyte clone on different days of culturea Day of culture Colony cells
3b
4
Viable cells
28 + 7
62
Non-viable cells
0
0
5
+ 18
6
8
7
9
0
0
15 -
30-
221 k 19 392
"Cell number in 10-50 B lymphocyte colonies, pooled in groups of five, were counted Cell numbers were estimated from counts in the culture plate
'
10
103k 35 155 + 3 6 280+73 475 +90 259k 57 1 7 + 33
+ 10
P. B . Poulsen and M. H. Claesson: Ultrastructure of B Lymphocyte Colonies
The plasmablast (Fig. lb) is also a large cell with a euchromatic nucleus, but with condensed chromatin typically in the shape of flakes at the nucleolemma. The cytoplasm contains endoplasmic reticulum sur-
79
rounding the nucleus and the Golgi apparatus is well developed. The chromatin of the plasma cell is condensed forming typical pore complexes at the nucleolemma.
Fig. la-h. B lymphocyte colony cells. a: lymphoblast, b: plasmablast, c: plasma cell, and d: small lymphocyte on day 7 of culture, e-f degenerating colony cells on day 9 of culture, g: late mitotic plasmablast on day 7 of culture, h: broad contract zone between two lymphoblasts on day 6 of culture, a-g: magnification X 8,500, b: magnification X 85,000
80
P. B. Poulsen and M. H. Claesson: Ultrastructure of B Lymphocyte Colonies
The cytoplasm is stuffed with endoplasmic reticulum, which contains a more electron dense material in the cisternae compared to the plasmablast. The Golgi apparatus is well developed in the plasma cell (Fig. lc). Nuclear segmentation is often seen both in the plasmablasts and in the plasma cells. The small lymphocyte (Fig. Id) contains con-
Fig. le-h.
densed chromatin at the nucleolemma as well as small chromatin islands. The cytoplasm is scanty containing only a few mitochondria, free ribosomes, and polyribosomes. Of the viable colony cells the majority (more than 90%) were easily placed in one of these four categories. In the late days of culture, cells in various stages
P. B . Poulsen and M. H. Claesson: Ultrastructure of B Lymphocyte Colonies
of degeneration were observed. Figure le and f shows a pyknotic cell and a cell with severe signs of degeneration with no cytoplasmic organelles left. Many mitotic cells were recognized throughout the culture period, being most frequent among lymphoblasts and plasmablasts including rather differentiated plasmablasts (Fig. lg). Occasionally cell membranes of proliferating cells were observed in close contact with each other for broad zones of 0.1-0.5 pm, the intercellular cleft was less than 100 A and suggested the existence of gap junctions in these areas (Fig. lh). Such cell contacts were most frequently observed in colonies on day 6 of culture. They were not only observed between cells of the same category but were also present between lymphoblasts and plasmablasts.
Ultrastructural Development of B Lymphocyte Colonies Figure 2 diagramatically shows the appearance of the four major cell categories at different periods of culture (day 4-10). On day 4 only lymphoblasts were
81
observed in developing colonies. On day 5 lymphoblasts dominated the developing colonies but plasmablasts and a few small lymphocytes were now present. On day 6 of culture the incidence of plasmablasts and lymphoblasts were identical but from day 7 to day 9 the incidence of lymphoblasts again outnumbered that of plasmablasts. During the late period of culture both small lymphocytes and especially plasma cells appeared in colonies. Figure 2 also illustrates the topographical localization of the four major cell categories within individual colonies. The majority of cells were located in the outer zone of the colony [which made up the major part of the colony (see Methods)]. Except from day 9 plasmablasts and lymphoblasts were found to be evenly distributed throughout the three zones examined whereas small lymphocytes and plasma cells were seen in the intermediate zone and especially in the outer zone of the colony. The majority of mitotic cells were located in the outer colony zone whereas dead cells were first observed in the central part of the colony (data not included in Fig. 2). Figure 3 combines the data from Table 1 and Fig. 2 by showing the fluctuations in absolute num-
Fg.2. The graph illustrates the percentage of the four different cell categories (lymphoblast = LB, plasmablast = PB, small lymphocyte = SL, and plasma cell = PC)in the B cell colonies from day 4 to day 10. The graph also illustrate the distribution on the four different cell categoriesin the central, intermediate, and peripheral zones of the cell colonies, respectively.A total of 15 colonies were studied. Numbers of viable colony cells examined were: day 4 = 51, day 5 = 123, day 6 = 655, day 7 = 203, day 8 = 204, day 9 = 484, and day 10 = 178. Since coloniesfrom the same day of culture did not differ with respect to cell categories or cell position the columns represent the pooled data from 2-3 individual colonies
82
P. B. Poulsen and M. H. Claesson: Ultrastructure of B Lymphocyte Colonies
Fig. 3. The graph illustrates the total number of cells per colony in the four cell categories mentioned in Fig. 2. Data are combined from results present in Table 1 and Fig. 2
Table 2. Cluster formation by mass-collected colony and cluster cellsa
day 9 of culture recloned BLC’s did not form clusters.
Day of recloning
5
6
7
8
9
10
20k5
6 f 3
58f28
17+3
6f4
2 f 2
Cells were mass-harvested from primary 5-10 day BLC cultures and recloned in secondary agar cultures. Data represent cluster formation (cluster = 5-32 cells) expressed as means of triplicate cultures with 10,OOO cells/culture counted on day 6 of culture
a
bers of the four categories of cells as a function of the culture time. Apart from days 6 and 10 the absolute number of lymphoblasts was much higher than that of plasmablasts. It is evident from data in Fig. 3 that the B lymphocyte colony produces a substantial number of both plasma cells and small lymphocytes. Recloning of B Lymphocyte Colony Cells
Table 2 shows the results of the recloning experiments. Colony cells harvested from days 5-6 of culture were only capable of forming a few clusters, whereas day 6 and 7 mass-harvested BLC cultures gave rise to a considerable number of cell clusters and in addition to a few cell colonies containing more than 32 cellskolony (data not included in Table 2). From
Discussion The present ultrastructural study shows that clonal proliferation of B lymphocytes in agar culture results in formation of both of the end cell categories of this lymphocyte lineage, i.e., plasma cells and small lymphocytes. Although the colonies grew exponentially giving rise to 400-600 cells on day 8 of culture, the frequency of end cells produced by the individual B lymphocyte colony was low and the majority of proliferating and differentiating cells died at the blast cell stage of development. The present study also elucidates the topographic localization of colony cells: cells in the center of the colony appeared to degenerate prior to cells in the periphery, whereas blast cells and mitotic cells were located in the peripheral zone of the colony. This pattern of organization within the colony differs markedly from that of proliferating granulocyte-macrophage colonies from the bone marrow where the proliferating cells are located to the center of the colony whereas differentiation takes place in the periphery [12]. From experiments in liquid culture it is well known that polyclonal activators of B lymphocytes lead to the formation of antibody producing cells.
P. B. Poulsen and M. H. Claesson: Ultrastructure of B Lymphocy:e Colonies
These can be assayed as specific plaque-forming cells (PFC) against a variety of different antigens [13, 141. The BLC system also appears to proliferate in response to polyclonal activation induced by 2-ME-activated albumin and SRBC as well as agar mitogen in the culture medium [15, 161. In the liquid culture system mentioned above [14] the frequency of PFCs is high compared to the frequency of mature plasma cells in individual BLCs suggesting either that the colonies are less differentiated than the clones arising in liquid culture or that the cells are capable of secreting antibodies before they reach an end cell stage, i.e., become plasma cells. This last view is supported by the fact that the number of SRBC-PFC obtained from an individual specific SRBC-antibody producing BLC can be as high as 240 [8]. Moreover, the relatively small number of colonies examined in the present study makes it dubious to generalize with regard to the degree of plasma cell development in BLCs. From the present data it is possible to delineate the following order of cell development during BLC formation: (1) small B lymphocyte (BL-CFC) + (2) lymphoblast + (3) lymphoblast/plasmablast + (4) lymphoblast/plasmablast/small lymphocyte (BL-CFC)/plasma cell. The BL-CFC‘s are known to be small lymphocytes themselves, the majority having detectable IgM on their surface [5,7,17]. BL-CFC start to proliferate on day 2 of culture and form only lymphoblasts during the next two days, after which differentiation occurs and both lymphoblasts and plasmablasts are formed in equal numbers. From day 6 onwards lymphoblasts dominate the colonies and small lymphocytes and plasma cells appear in small numbers. From day 8 onwards colony cells start to die. Recloning experiments showed that BL-CFC did not appear in colonies except on day 7 of culture and that this point of time coincided with the appearance of small lymphocytes. This mode of BL-CFC proliferation differ markedly from that of human T lymphocyte colony formation in which the colony forming cell (TL-CFC) exists throughout the ten day culture period [18]. The observation of broad contact zones between blast cells in developing colonies with an intercellular gap of approximately 100 A suggests the presence of the so-called gap junctions in these areas. Such contact structures have recently been described between phytohemagglutinin aggregated rabbit lymphocytes [19], and they might be of importance for synchronization of colony cell proliferation. Freeze-fracture electron microscopic studies are in
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progress to elucidate the nature of the broad contact zones between colony cells. In conclusion, the present results show that B lymphocyte colony formation reflects a whole series of B lymphocyte developmental stages including formation of all the cell categories belonging to the B lymphocyte lineage. Acknowledgements. Excellent technical work was performed by Mrs. Ursula Rentzmann. This study was supported by grants from “Foreningen ti1 Laeevidenskabens Fremme” and “Kong Christian X’s Foundation”.
References 1. Metcalf D, Nossal GJV, Warner NL, Miller JFAP, Mandel TE, Layton JE, Gutman GA (1975) Growth of B lymphocyte colonies in vitro. J Exptl Med 142: 1534 2. Claesson MH, Metcalf D (1977) B lymphocyte colony-forming cells in the SJUJ mouse thymus. J Immunol 118: 1208 3. Lala PK, Johnson GR (1978) Monoclonal origin of B lymphocyte colony-forming cells in spleen colonies formed by multipotential hemopoietic stem cells. J Exptl Med 148: 1468 4. Gjedde SB (1980) B lymphocyte colony forming cells in thymus of AKR mice. Exptl Hematol In press 5. Kincade PW, Ralph P (1976) Regulation of clonal B lymphocyte proliferation by anti-immunoglobulin or anti-Ia antibodies. Cold Spring Harbor Symp Quant Biol 41: 245 6. Howard MC, Claesson MH, Johnson GR (1977) Surface immunoglobulin characteristcs of B lymphocyte developmental states and B lymphocyte colony-formingcells. Scand J Immunol 6: 1317 7. Kincade PW, Paige CT, Parkhouse RMR, Lee G (1978) Characterization of murine colony-forming B cells. I. Distribution, resistance to anti-immunoglobulin antibodies, and expression of Ia antigens. J Immunol 120: 1289 8. Claesson MH, Layton JE, Luckenbach GA (1978) Specific antibody-forming B lymphocyte colonies. I. Distribution and nature of SRBC antibody-forming B lymphocyte colonies in mouse lymphomyeloid organs. Immunology 35 : 397 9. Claesson MH (1979) Soluble suppressor activity of concanavalin A-activated spleen cells on B lymphocyte colony formation in vitro. Cell Immunol 42: 344 10. Claesson MH (1979) B lymphocyte colony suppression in vitro: Characteristicsof the suppressor cell. In:Kaplan JG (ed) Proceedings of the 13th International Leucocyte Culture Conference, Ottawa, Canada. ElseviedNorth Holland Biomedical Press, Amsterdam New York Oxford, p 680 11. Mandel TE (1977) The ultrastructure of mammalian lymphocytes and their progeny. In: Marchalonis JJ (ed) The lymphocyte structure and function. New York Basel, p 11 12. Metcalf D (1977) Hemopoietic colonies. In vitro cloning of normal and leukemic cells. Recent Results Cancer Res 61 : 58 13. Fauci AS (1979) Human B cell function in a polyclonally induced plaque forming cell system. Cell triggering and immunoregulation. Immunol Rev 45 : 93 14. Anderson J, Coutinho A, Melchers F (1977) Frequencies of mitogen-reactive B cells in the mouse. 11. Frequencies of B cells producing antibodies which lyse sheep or horse erythrocytes, and trinitrophenylated or nitroiodophenylated sheep erythrocytes. J Exptl Med 145: 1520
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P. B. Poulsen and M. H. Claesson: Ultrastructure of B Lymphocyte Colonies
15. Claesson MH, Had H-D, Opitz HG (1979) B and T lymphocyte colony formation in agar: 2-Mercaptoethanol-a~tivated albumin can substitute for 2-mercaptoethanol. Cell Immunol 46: 398 16. Kincade PW, Ralph P, Moore MAS (1976) Growth of B lymphocyteclones in semisolidculture is mitogen dependent. J Exptl Med 143: 1265 17. Metcalf D, Wilson JW, Shortman K, Miller JFAP, Stocker J (1976)The nature of cells generating B lymphocyte colonies in vitro. J Cell Physiol 88: 107
18. Smith SD, Sachs L (1979) Difference in the cell proliferation and colony-forming ability of normal human T lymphocytes. Clin Exptl Immunol 37: 348 19. Kapsenberg ML, Leene W (1979) Formation of B type gap junctions between Pha-stimulated rabbit lymphocytes. Exptl Cell Res 120: 211
Received February 1980/Accepted May 1980