Antigen-initiated B lymphocyte differentiation. IX. Characterization of memory AFC progenitors by buoyant density and sedimentation velocity separation.

The characteristics of memory B cell antibody-forming cell (AFC) progenitors from long-term hapten-primed CBA mice were investigated by using sedimentation velocity and buoyant density separation to isolate physically distinct B cell sub-sets. The isolated fractions were assayed by the adoptive immune response to NIP-POL antigen, under conditions where neither T cells nor other accessory cells were limiting the IgM or IgG AFC responses. The results were compared to previous studies on the IgM AFC-progenitors of unprimed adult mice. Splenic IgM and IgG memory AFC-progenitor activity was largely found among the typical B cells of slow to medium sedimentation rate, in contrast to the fastre sedimenting IgM AFC-progenitor activity of unprimed animals. Splenic IgM and IgG memory AFC-progenitor activity was found among the medium to light density cells, and so resembled by this parameter the IgM AFC-progenitor activity in unprimed animals. Thoracic duct lymphocytes from hapten-primed mice also exhibited memory IgM and IgG AFC-progenitor activity in the slow-medium sedimentation range. However, in contrast to spleen, the IgM and IgG memory AFC-progenitor activity in lymph was found among very dense B cells. Two physically distinct sub-populations of memory B cells have thus been identified, namely: i) small, medium-light density, presumably tissue-resident B lymphocytes found in spleen; and ii) small, dense, presumably recirculating B lymphocytes found in lymph. Both physical forms include IgM and IgG progenitors. Both forms are distinct from the larger, medium-light density "virgin" AFC-progenitors in the spleen of unprimed adult mice.     

Separation of mouse testis cells by equilibrium density centrifugation in Renografin gradients

Mouse testis cells have been separated by equilibrium density centrifugation in gradients of Renografin. Intact testis cells were not damaged by the separation procedure provided that, following separation, the osmolarity was reduced gradually. The various cell types were identified microscopically and by ³H-thymidine labelling with similar results. The present technique has demonstrated that significant variations in cell density occur during spermatogenesis. Approximately ten-fold enrichments of nearly all testis cell types were achieved by equilibrium density separation of testis cell suspensions. More homogeneous cell populations were prepared by density gradient centrifugation of cell fractions obtained from velocity sedimentation separations. Overall enrichments of spermatogonia, by 29-fold; pachytene spermatocytes, 45-fold; dividing meiotic cells, 170-fold; round spermatids, 30-fold; step 11–13 elongating spermatids, 12-fold; Leydig cells, 70-fold; and cytoplasmic fragments, 55-fold, were obtained. In this study, a method for preparation of cell suspensions was also developed to produce higher yields of spermatogonia and young primary spermatocytes; however, the density distribution of these cells was altered.     

The generation of memory B-cell subpopulations capable of proliferation and expansion of the pool: effect of time and antigen

The ability of antigen to induce proliferation of memory B lymphocytes, thus perpetuating and expanding the memory cell pool, has been examined using an antibody-forming cell (AFC) assay. This method provided confirmation of previous studies in which serum antibody titer was used as a relative measure of pool size and demonstrated directly that the number of antibody-forming cells is increased. Memory cell subpopulations were prepared by lg velocity sedimentation from recently immunized donors (2 weeks) and tested for their ability to proliferate, thus expanding the memory cell pool. Both large and small immature DNP-specific memory cells displayed antigen-dependent and antigen-independent proliferation while mature cells (8 weeks postpriming) were capable only of antigen-dependent proliferation. Chicken γ-globulin (CGG) specific memory cells were also evaluated in this system and were found to differ from DNP-specific cells in several ways. (A) DNP-specific AFC were found to be concentrated in the spleen while CGG-specific AFC were found predominantly in the bone marrow early after transfer and in the spleen upon delayed challenge. (B) The rate of maturation of CGG-specific memory cells capable of antigen-driven proliferation and pool expansion was delayed in comparison to DNP-specific memory cells. The relationship of these functionally defined subsets to previously described memory cell subpopulations is discussed.     

Mass isolation of pole cells from Drosophila melanogaster.

Crude cell suspensions were obtained from 3 × 10⁶ pregastrula staged embryos. These cells fractionated into three bands on Renografin density gradients. Using ultrastructural characteristics for identification, pole cells were localized in the two denser bands. EM analyses also revealed a striking difference in the frequency of lipid vacuoles found in the pole-cell cytoplasm versus the cytoplasm of other embryonic cells. This difference has enabled us to identify pole cells by light microscopy using a neutral lipid stain. Through detailed analyses of the Renografin fractions with this stain, we have shown that pole cells resolve into two to three density classes. Evidence is presented which suggests that these density classes reflect different developmental age classes. The size differences between the pole cells and other embryonic cells contained in the enriched pole-cell fractions from the density gradients has enabled us to use sedimentation velocity centrifugation for additional enrichment. A distinct lower band of cells was obtained which consisted of 80% pole cells. Using these procedures, 1–2 × 10⁷ pole cells can be obtained daily.     

Sedimentation behavior of chloroplast ribosomes from Chlamydomonas reinhardtii.

The identity of peaks generated by chloroplast ribosomes of Chlamydomonas reinhardtii were determined by zone velocity sedimentation on sucrose density gradients, and analysis of distribution of ribosomal RNAs in the gradients. The sedimentagion coefficient of the principal peak was 66-70 S (usually 69 S), in good agreement with previously reported values for chloroplast ribosomes of C. reinhardtii, and other organisms. The fast sedimenting side of the 69 S peak contained an excess of chloroplast large subunit. When ribosome dissociation was prevented by sedimentation at low velocity, by aldehyde fixation, or by the presence of nascent polypeptide chains, the principal peak had a sedimentation coefficient of about 75 S. Thus the 69 S peak was an artifact caused by dissociation during centrifugation. Peaks that contained chloroplast ribosomal RNAs were also observed at '60 S' and '45 S' when chloroplast ribosomes were centrifuged unfixed at high velocity. The amounts of '60 S' and '45 S' components were decreased by centrifugation at low speed, or fixation, but sedimentation coefficients remained unchanged. The '60 S', and '45 S' components were identified as large, and small subunits of chloroplast ribosomes, respectively. The artifacts produced by centrifugation of chloroplast ribosomes, are similar to the artifacts produced by centrifuging ribosomes of Escherichia coli. Similar explanations appear to apply to both. We concluded that the 69 S chloroplast ribosome peak occurs because of dissociation of 'tight' couples, and incomplete separation of subunits. Subunit peaks (60 S and 45 S) arise from free subunits, and/or from dissociation of 'loose' couples.     

Isolation of human monocytes according to volume and density

Human peripheral blood monocytes were obtained in very pure preparations by using buoyant density gradient centrifugation followed by velocity sedimentation. We introduced several modifications in the experimental procedure in order to take advantage of the minimal differences in cell density and size between monocytes and lymphocytes. Previous methods used to isolate monocytes based on their physical properties are reviewed, and the theory of velocity sedimentation is discussed with regard to the differences in the methodology used, which account for the different results we obtained.     

SUBCELLULAR ANALYSIS OF THE MOLECULAR FORMS OF ACETYLCHOLINESTERASE IN RAT SKELETAL MUSCLE

Acetylcholinesterase (AChE, EC 3.1.1.7) activity of rat gastrocnemius muscle homogenized in 1 M-NaCl and 0.5% Triton X-100 was separated by velocity sedimentation in sucrose gradients into three molecular forms with sedimentation coefficients of about 4S, 10S and 16S. The distribution of homogenate AChE activity among the three peaks was 53, 34 and 13% respectively. The different molecular forms were found to be heterogeneously distributed in subcellular fractions prepared from sucrose homogenates of muscle, as follows: Subfractions of the crude sarcolemmal fraction were prepared by discontinuous sucrose gradient centrifugation. AChE was recovered in the greatest yield and with the highest specific activity in a light density subfraction (0.6/0.8 M-sucrose interface). The AChE activity in this light density subfraction was mainly (81-88%) the 10S form of the enzyme. The velocity sedimentation profiles of the AChE activity in the more dense subfractions were markedly different in that 16S AChE was a major component.     

Separation of lymphoid-line cells according to volume and density

Velocity sedimentation and isopycnic centrifugation was used to separate exponentially growing RAJI lymphoid line cells according to size and density. Mixtures of Ficoll, Isopaque and tissue culture medium were used as gradient media. These media had a constant pH, were isotonic, and did not have any significant harmful effect on the cells. The observed variation in cell size paralleled the progression of the cells through the cell cycle, as assessed by thymidine incorporation and impulse cytophotometric determination of DNA contents. Differences in cell density did not reflect the cell cycle phase. No correlation could be established between cell size and density. Velocity sedimentation could be used to obtain cell populations which were relatively pure according to cell cycle phase and growing synchronously for at least 24 h.     

Physical separation of hemopoietic stem cells from cells forming colonies in culture

Mouse bone marrow cells in suspension were separated into a number of fractions on the basis of cell density by equilibrium density gradient centrifugation, or on the basis of cell size by velocity sedimentation. After each type of separation, the cells from the various fractions were assayed for their ability to form macroscopic spleen colonies in irradiated recipient mice, and for their ability to form colonies in a cell culture system. The results from either separation technique demonstrate that cells in some fractions formed more colonies in vivo than in the culture system, while cells in other fractions formed more colonies in culture than in the spleen. The results of control experiments indicate that this separation of the two types of colony-forming cells was not an artifact of the separation procedures. From these experiments it was concluded that the population of cells which form colonies in culture under the conditions used is not identical to the population of cells detected by the spleen colony assay.     

Separation of hepatocytes from suspensions of mouse liver cells using programmed gradient sedimentation in gradients of ficoll in tissue sulture medium

Liver cells were obtained in suspension using a solution of lysozyme in Joklik's modification of minimum essential medium. Hepatocytes were separated in 74.2 ± 12.9% purity from other liver cells having different densities using isopycnic centrifugation, in 97.1 ± 1.9% purity from other liver cells having different diameters using velocity or rate-zonal centrifugation. A previously reported computer integration of the differential sedimentation equation was employed in determining the gradient design and the speed and duration of centrifugation which would permit purification of hepatocytes from other liver cells. More than 98% of the hepatocytes separated by velocity sedimentation excluded trypan blue. Velocity sedimentation is superior to isopycnic centrifugation for the separation of hepatocytes from liver cell suspensions because it gives more highly purified hepatocytes and because it requires lower centrifugal forces for shorter periods of time.     

Antigen-initiated B lymphocyte differentiation. V. Electrophoretic separation of different subpopulations of AFC progenitors for unprimed IgM and memory IgG responses to the NIP determinant.

Spleen cells from CBA mice were separated by continuous, free-buffer film cell electrophoresis, and the capacity of cells in different fractions to mount an adoptive immune response specific for the NIP hapten determined. Experimental conditions were such that AFC progenitor B cells were measured, rather than helper or suppressor T cells. The IgM response of unprimed animals (a virgin or antigen inexperienced population) and the IgG response of long-term hapten-primed animals (a B memory cell population) were compared. The results indicated physical and biological heterogeneity in splenic B cells, with AFC progenitors for unprimed IgM and memory IgG responses being extensively separated.AFC progenitors for a primary IgM response in normal, germ-free and athymic mouse spleen, and bone marrow, separated into three distinct populations. Two of these were of much higher mobility than the typical splenic B cells and separated in the T cell zone. These cells produced a relatively early peak response of AFC after stimulation.AFC progenitors for a secondary IgG response were predominantly typical low-mobility B cells. Three regions of activity were separated, one overlapping part of the IgM progenitors. The slowest migrating activity peaks corresponded to the mobility of some recirculating B cells. These cells produced a more delayed AFC response after stimulation.AFC from the spleens of immunised mice separated as a single, broad, mediummobility peak distinct from most B cells and AFC progenitors. IgM and IgG (memory) AFC had similar electrophoretic characteristics.     

Constancy of cell buoyant density for cultured murine cells

The relationship between cell cycle and cell density was determined for three different lines of mouse cells by equilibrium centrifugation of suspension cultures. The mean cell densities of the three lines differed significantly, with values of 1.0622, 1.0678, 1.0540 gm/ml for 70Z/3, S 107, and ABE 8, respectively. However, the density distributions within each of the three lines were indistinguishable, with an average coefficient of variation about 5% of the mean reduced density (i.e., density minus one). Quantitative DNA analysis of the cells separated by density showed that the proportion of cells in G1, S, and G2 + M phase of the cell cycle changed very little or not at all with cell density. In addition, cells separated by size (and therefore by phase of the cell cycle) using velocity sedimentation had the same means and distributions of densities. These results indicate that there is little or no change in cell density as the cells traverse the life cycle and that buoyant density appears to be a constant property of a cell type.     

Effector mechanisms in allograft rejection. II. Density, electrophoresis, and size fractionation of allograft-infiltrating cells demonstrating several classes of killer cells.

An analysis of killer cells infiltrating “sponge-matrix” allografts during rejection has been performed by preparative fractionation by density centrifugation, velocity sedimentation, and free flow cell electrophoresis and by the use of heterologous anti-T-cell sera. At the peak of rejection, 7 to 8 days after transplantation, the allograft is infiltrated by several classes of killer cells, most notably by non-T lymphocytes, monocytes/macrophages, and T lymphocytes. The predominant cell types capable of performing in vitro lysis of relevant target cells appeared to be monocytes and non-T lymphocytes. T lymphocytes formed only a minority of the killer cells at this stage of the response. In contrast, as also documented earlier, the predominant killer cells in the regional lymph nodes and the spleen of the graft recipient mice were T lymphocytes (blasts).     

Centrifugal elutriation (counterstreaming centrifugation) of cells.

Lindahl first described the separation of cells by velocity sedimentation utilizing a special technique (counterstreaming centrifugation) that was later modified slightly and renamed centrifugal elutriation. Centrifugal elutriation has been applied, with variable degrees of success, to the separation of hemopoietic cells, mouse tumor cells, testicular cells, and a variety of other specialized cells as well as cells in particular phases of the cell cycle. The capacity of the elutriator to separate large numbers of cells is its chief advantage. The purities of the separated cells have not been compared with the purities of cells separated by other methods in most cases; such comparisons would permit more sophisticated comparison of elutriation with other techniques for velocity sedimentation.     

Centrifugal elutriation (counterstreaming centrifugation) of cells

Lindahl first described the separation of cells by velocity sedimentation utilizing a special technique (counterstreaming centrifugation) that was later modified slightly and renamed centrifugal elutriation. Centrifugal elutriation has been applied, with variable degrees of success, to the separation of hemopoietic cells, mouse tumor cells, testicular cells, and a variety of other specialized cells as well as cells in particular phases of the cell cycle. The capacity of the elutriator to separate large numbers of cells is its chief advantage. The purities of the separated cells have not been compared with the purities of cells separated by other methods in most cases; such comparisons would permit more sophisticated comparison of elutriation with other techniques for velocity sedimentation.     

Leukemia in AKR mice: III. Size distribution of suppressor T-cells in AKR leukemia and neonatal mice

Suppression of in vitro antibody forming potential of normal cells by leukemic cells of AKR and normal neonatal mice have many similarities. In both cases the suppression is by cell contact rather than by the elaboration of soluble suppressive factors and the suppression is sensitive to both x-irradiation and mitomycin C treatment. When the size distribution of suppressing cells in thymus and spleen were compared by velocity sedimentation, both leukemic and neonatal suppressing cells had similar size distribution in each organ. Both large and small cells in the thymus suppress but only large cells (sedimentation velocity >3.5 mm/hr) in the spleen are able to suppress. Leukemic cells in lymph node have a splenic size distribution, viz., only large cells suppress. Both large and small cells of a subcutaneously growing long passage AKR lymphoma are able to suppress. While large cells contain the bulk of cells actively incorporating tritiated thymidine and thus probably in cycle, small but significant amounts of incorporation in small suppressing cells is also seen.     

Antigen modulation of the immune response. VI. Rate of large memory cell appearance in lymph nodes and thoracic duct lymph

We have studied the rate of appearance of memory B-cell subpopulations in the antigen draining lymph nodes and thoracic duct lymph of rats using 1g velocity sedimentation and adoptive transfer. Five days after immunization 100% of the memory response was attributable to large cells. By Days 7, 14, 28, and 77 after priming the large cells contribution to the memory population dropped to 86, 35, 15, and 10% respectively. At the same time the small cell contribution rose from 20% on Day 14 to 46% on Days 28 and 77. The same results were obtained with thoracic duct lymphocytes with the large cells contributing 53% of the response on Day 7 and 20% on Day 150. Appropriate controls were included to show that differential suppression was not responsible for these results. Furthermore, when purified large memory cells were passaged through intermediate hosts for 7 to 11 days, between 76 and 81% of the large cells matured into medium or small lymphocytes. These data show that the earliest memory cells formed after antigen encounter are the blast-like large lymphocytes and that these evolve, through a series of antigen-independent events, first into medium and then small lymphocytes. A model of memory cell development incorporating these results and the results of others is presented.     

Cellular origin of cytotoxic effectors and secondary educated lymphocytes in human mixed leukocyte reaction

Human lymphocytes sensitized in vitro during a mixed leucocyte reaction (MLR) against an allogeneic-stimulating cell respond by blast transformation and generation of specific cytotoxic effector cells. Both proliferation and cytotoxicity are maximum on Days 6 and 7 of culture. On Day 14, no more dividing cells or cytotoxic cells are detected in such primary cultures. Restimulation by the specific priming cell triggers a secondary proliferative response and rapid reappearance of specific cytotoxic effector cells. The velocity sedimentation cell separation method which separates cells according to their size was applied to human lymphocytes sensitized in vitro during an MLR on Day 7 of culture. Blast cells were separated from nondividing small lymphocytes. It was shown that: (1) cytotoxic effectors generated at the peak of a primary response are exclusively present in the isolated blast population; (2) highly cytotoxic secondary effector cells are induced to reappear mainly from the blast-derived population upon restimulation; and (3) secondary educated proliferative cells mainly derive from the blast population. Conversely, the blast-depleted small lymphocyte population is operationally depleted of cells able to respond by proliferation to the priming cell while responding normally against third party control cells. HLA-D region specificity of the secondary proliferative response is suggested.     

Germination of Schizosaccharomyces pombe spores separated by zonal centrifugation

Spores from the fission yeast Schizosaccharomyces pombe (H90 strain) were separated from residual vegetative cells into distinct size classes by zonal density centrifugation. Spores were sized photographically and with a Coulter counter. The kinetics of germination were followed by time-lapse photomicrography. The duration of the pre-germination interval was size dependent. Large spores (˜50 μm³) germinated as early as 5 h after resuspension in nutrient media, smaller ones (˜20 μm³) did so at 11 h. The first cell division occurred 4–5 h later regardless of spore size. The large spores divided more synchronously as shown by the occurrence of peaks in the cell plate index at approximately one doubling time intervals.     

Immunologic specificity of local inhibitory intercellular interactions

The authors previously reported local intercellular interactions suppressing the increase of the antibody-forming cell (AFC) count in the suspension of the spleen cells of nonimmunized mice and sharply elevating with increase in density of the cultivated suspensions. This work showed that preliminary immunization of mice with an antigen eliminated or sharply reduced the accretion inhibition of the AFC cells against the given antigen, but failed to influence the accretion inhibition of cells producing antibodies against another antigen, and the proliferation inhibition of the dividing bulk cells in the culture.