SCANNING ELECTRON MICROSCOPY OF HUMAN LYMPHOCYTES DURING TRANSFORMATION AND SUBSEQUENT TREATMENT WITH METHOTREXATE

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1 J. Cell Set. a8, (1977) Printed in Great Britain Company of Biologists Limited 1077 SCANNING ELECTRON MICROSCOPY OF HUMAN LYMPHOCYTES DURING TRANSFORMATION AND SUBSEQUENT TREATMENT WITH METHOTREXATE C. CHOO HOFFMANN, KENNETH C. MOORE, CHING-YUAN SHIH AND RAYMOND L. BLAKLEY Tlie Department of Biochemistry, College of Medicine and the Department of Zoology, University of Iowa, Iozoa City, Iowa 52242, U.S.A. SUMMARY Preparations of human peripheral blood lymphocytes containing % T-cells and % B-cells were shown by scanning electron microscopy to consist almost exclusively of cells bearing numerous microvilli, whereas thymocytes were of mixed surface morphology, with both smooth and encrusted forms numerous. T-lymphocytes purified on long nylon columns were all covered with numerous short villi. Stimulation with phytohaemagglutinin for 2 days produced T-lymphoblasts almost exclusively, and as the T-cells enlarged the microvilli lengthened, the increase in length reaching 5-fold by day 3. Addition of sufficient methotrexate on day 3 to arrest proliferation (50 ITM) caused progressive loss of microvilli from the cell surface, with the eventual production of large numbers of smooth cells, the surfaces of which later became pitted, followed by the complete dissolution of the cell. T-lymphocytes were shown to form rosettes with sheep erythrocytes through direct contact of the cell membranes over a significant area, but when, as a result of methotrexate treatment, the lymphocytes had become denuded of microvilli or had reached an advanced state of dissolution, rosettes were no longer formed. INTRODUCTION Studies of the surface morphology of human lymphocytes by scanning electron microscopy (SEM) have been reported from several laboratories (Polliack, Lampen, Clarkson & DeHarven, 1973; Wetzel, Erickson & Levis, 1973; Wetzel et al. 1974; Polliack, Lampen & DeHarven, 1974a; Alexander & Wetzel, 1974; Kay et al. 1974; Polliack & DeHarven, 1975) with considerable disagreement as to the architectural forms most prevalent and the functional identity of cells with each of these forms. The controversy has also extended to the morphology of rosettes formed between lymphocytes and sheep erythrocytes (Kay et al. 1974; Polliack et al. 1974). It was not our major purpose to contribute to this controversy, but rather to use SEM to study the effects of methotrexate, an immunosuppressive drug sometimes used in suppressing graft-versus-host disease, on the surface morphology of transforming T-lymphocytes. However, data obtained in the course of this work are clearly in agreement with reports that both B and T lymphocytes are covered with numerous microvilli so that contrary to the claim of some investigators, surface morphology

2 152 C. C. Hoffmann, K. C. Moore, C.-Y. Shift and R. L. Blakley cannot form a basis for distinguishing between B and T lymphocytes. We also report on the changes that occur on the surface of T-lymphocytes during transformation in vitro by phytohaemagglutinin (PHA), and during subsequent exposure to methotrexate. This work complements a previous publication (Hoffmann, Ho, Blakley & Thompson, 1976) on the effect of exposure to methotrexate for various periods on blast formation, mitosis, DNA synthesis and blast proliferation. MATERIALS AND METHODS Abbreviations used: HBSS, Hanks' buffered salt solution; MEM, minimum essential medium; PHA, phytohaemagglutinin; and SEM, scanning electron microscopy. Peripheral blood was drawn from healthy donors into heparinized vacutainers and allowed to sediment for 2 h at 37 C. The buffy coat was removed, the leukocytes recovered by centrifugation and resuspended. For studies of transformation the lymphocytes were cultured at an initial density of 10 s per ml in the presence of PHA at a concentration of 8 fig per ml. Other details of the experimental procedure and the purification of T-lymphocytes by passage through a nylon column were as previously described (Hoffmann et al. 1976). When lymphocytes were prepared by separation on a Ficoll-Hypaque gradient the procedure used was that of Boyum (1968). When the effect of methotrexate was studied a sterile solution of the drug was added aseptically to a final concentration of 50 nm on the third day of culture with PHA. For assay of T-cells by spontaneous rosette formation, cultures were pooled, centrifuged at 48 g for 8 min and washed twice with warm (37 C) HBSS containing penicillin (100 units/ml), streptomycin (1 mg/ml) and heat-inactivated human AB serum (1 %, v/v) which had previously been absorbed with an equal volume of packed sheep erythrocytes. The washed cells were resuspended in HBSS to a final concentration of 5 x 10' cells per ml and the rosette assay carried out according to Bentwich, Douglas, Siegel & Kunkel (1973). Sheep erythrocytes were collected under sterile conditions in Alsevers solution, stored at 4 C for up to 14 days and washed 2 or 3 times in HBSS before use. In the assay, a total of cells were counted in a haemocytometer and rosettes were defined by the adherence of one or more erythrocytes. For determination of B-cells, cultures were pooled, centrifuged at 48 g for 8 min and washed twice with 2 % bovine serum albumin in phosphate-buffered saline, ph 7-2, containing 0-02 % sodium azide. Cells were finally resuspended in this buffer to a concentration of x 10 cells per ml and reacted with fluorescein-conjugated rabbit anti-human immunoglobin (Grand Island Biological Co., Grand Island, N.Y.) as described by Dickler & Kunkel (1972). Thymocytes were obtained from a thymus that had been removed from a 16-month female patient in the course of therapeutic cardiac surgery. This material was obtained through the courtesy of Dr John S. Thompson, Department of Internal Medicine. The thymus tissue was gently teased in warm HBSS containing penicillin and streptomycin. The resulting suspension was centrifuged at 50 g for 10 min, the supernatant solution removed, and the pellet washed twice more before resuspending, determining the proportion of T and B lymphocytes and examining by SEM. The ability of cells to exclude dye was measured by the technique of Phillips & Terryberry (1957)- For SEM, cells were washed with warm (37 C) MEM, centrifuged at 48 g for 8 min, resuspended in a minimum volume of MEM, and 2 drops of the suspension gently placed on a glass coverslip. In the case of rosetted lymphocytes, 2 drops of the suspension were placed directly on the coverslip because of the danger of disrupting rosettes during washing. After 20 min at room temperature, the cells were fixed with Karnovsky's reagent (1965) for 1 h and processed according to the method of Wetzel et al. (1973) and Kelley, Dekker & Bluemink ( J 973)- Briefly, the cells on the coverslips were postfixed with 1 % osmium tetroxide in o-i M sodium cacodylate buffer, ph 72, for 1 h, washed thoroughly in the same buffer (5 to 10 times over 20 min) and incubated in excess thiocarbohydrazide for 10 min. After rinsing off the -thiocarbohydrazide, the specimens were fixed again in aqueous 1 % osmium tetroxide for 45 min. The specimens were rinsed thoroughly (3 times) in distilled water and dehydrated through a graded ethanol series and dried by the CO. critical-point procedure (Anderson, 1951)

3 Surface morphology of human lymphocytes 153 in a Sorvall Critical Point Drying System. The dried samples were vacuum coated with carbon and gold palladium and examined in a Cambridge S-4 Stereoscan Electron Microscope at 45 0 tilt. Polaroid type 55 P/N film was used for micrographs. In determining subpopulation proportions at least 1000 cells were counted. RESULTS AND DISCUSSION Relative numbers of T and B lymphocytes in human peripheral blood Table 1 compares literature values for the proportion of T and of B lymphocytes found in preparations of human peripheral blood lymphocytes with the results with lymphocytes from 6 donors in this study. Our results for the proportion of T- lymphocytes are slightly higher than some literature values, and our proportion of B-lymphocytes somewhat lower, but we could see no evidence that this is related to the method of preparation (compare our gravity sedimentation and Ficoll-Hypaque values). The cells identified as T or B lymphocytes in our preparations together accounted for % f tne lymphocyte population. Table 1. Properties of T and B lymphocytes in preparations from peripheral human blood Method of isolation Defibrinate, gelatin sedimentation, CO-Fe powder, Ficoll Isopaque Ficoll-Hypaque Ficoll-Hypaque Ficoll-Hypaque Defibrinate, CO-Fe powder, Ficoll-Isopaque Ficoll-Hypaque Gravity sedimentation Ficoll-Hypaque % of peripheral blood lymphocytes A T B ± S6±8» * 77-8it 27-35t IS 24±7'2f Reference Anderson (1951) Alexander & Wetzel (1974) Sabatini et al. (1963) Levis & Robbins (1970) Lohrmann et al. (1975) Epstein et al. (1974) This study This study A rosette contained 3 or more sheep red blood cells. f By erythrocyte antibody-complement rosettes. In other cases immunofluorescent staining was used. X A rosette contained 1 or more sheep red blood cells. PHA stimulation of lymphocytes When lymphocytes were stimulated with the optimum level of PHA under our experimental conditions, the maximum number of blasts that subsequently accumulated in the culture ranged from 4-4 to 9-2 x io B per ml from an initial culture of io B lymphocytes per ml. No correlation could be found between the proportion of T- lymphocytes in the preparation and the peak number of blasts. This was also illustrated by an experiment in which T-lymphocytes were purified by gravity sedimentation followed by passage through a nylon column. Recovery of T-lymphocytes from the latter was 70-5%. Examination of this preparation after Wright staining showed monocytes to be absent, and immunofluorescent staining showed that B-lymphocytes

4 C. C. Hoffmann, K. C. Moore, C.-Y. Shih and R. L. Blakley Fig. i. Surface morphology of lymphocytes from peripheral blood, A, cluster of typical lymphocytes in a gravity-sedimentated preparation with some platelets in the background ; B, monocyte present in the preparation; C, lymphocytes typical of the major population with short microvilli (upper left) and of the minor subpopulation with long villi (lower left), x were absent, but 9% of the lymphocytes did not form spontaneous rosettes with sheep red blood cells. Although this preparation therefore contained a higher proportion of rosetting T-lymphocytes (91 ±2*6%), the peak blast count was only 3-5 x io 5, below the usual range. This suggests a role of other (adhesive) cells in stimulating the response of T-lymphocytes to PHA, in agreement with the finding of others

5 Surface morphology of human lymphocytes 155 that macrophages and monocytes have such an effect (Levis & Robbins, 1970; Lohrmann, Novikofs & Graw, 1975; Epstein, Kreth & Herzenberg, 1974). The blasts formed during the proliferative response to PHA of unfractionated lymphocytes were found to be almost exclusively (95 %) T-lymphoblasts, as indicated by the formation of spontaneous rosettes with sheep red blood cells, and by the small proportion (~ 3 %) of cells labelled by immunofluorescence staining, most or all of which were the B-cells originally present in the culture. This is in good agreement with the results of Greaves, Janossy & Doenhoff (1974) who reported that 92-96% of the blastoid cells in cultures of PHA-stimulated tonsil lymphocytes were of T-cell origin, while 99 % of the blasts were T-lymphoblasts in PHA-stimulated cultures of purified T-lymphocytes. Table 2. Characteristics of lymphocyte populations prepared from humanperipliera. blood by various procedures* Characteristics Subpopulation, % Short stubby Long-villous Ruffled Smooth Cell diameter, /*m Mean ± S.E. Range No. of villi per cell Mean ± S.E. Range Length of villi, fim Gravity sedimentation '5 4'55± -34 ( ) 369±i45 ( ) Method of preparation Ficoll-Hypaque * Each measurement was performed on samples of at least 900 cells ±0-45 ( ) 398 ±90 ( ) Nylon column ~ ± 0-49 ( ) 482±100 ( ) o-i6-o'57 SEM of peripheral blood lymphocytes Populations of unstimulated peripheral lymphocytes were shown by SEM to consist of villous cells (Fig. 1 A), on most of which the villi were short or stubby (Fig. ic). A subpopulation (Fig. ic) had appreciably longer villi, with mean length o-6i /jm compared with a mean length of o-i /tm for the majority. Another group had ridges or ruffles (Fig. IB) which have been shown by Alexander & Wetzel (1974) to be characteristic of monocytes. Cells prepared with Ficoll-Hypaque did not differ significantly in surface morphology from lymphocytes prepared by gravity sedimentation (Table 2). Wright stain showed that lymphocytes prepared by the latter method contained % lymphocytes, % polymorphs and 9-1 ±1-3% monocytes, but the results of Wetzel et al. (1973, 1974; Alexander & Wetzel, 1974) indicate that polymorphs and at least some monocytes are not readily

6 156 C. C. Hoffmann, K. C. Moore, C.-Y. Shih and R. L. Blakley distinguishable from lymphocytes by SEM. T-lymphocytes prepared by passage through a nylon column all appeared as cells with many short microvilli. Our preparations of lymphocytes contained few or no smooth cells (Table 2). Our results are thus in agreement with those of Alexander & Wetzel (1974) and Kay et al. (1974) who also found that all human peripheral lymphocytes are villous, and with those of Baur, Thurman & Goldstein (1975), Criswell, Rich, Dardano & Kimzey (1975) and Van Ewijk, Brons & Rozing (1971), who found that in the mouse both B- lymphocytes and T-lymphocytes are villous, at least in some environments, including the peripheral blood. These results are in contrast to those of Polliack et al. (1973, 1974a, b) who have stated that 'in many cases B and T lymphocytes can be distinguished by their surface architecture as seen under the SEM', 'relatively smooth' cells being identified with T-lymphocytes and 'villous' cells with B-lymphocytes. Although Polliack & DeHarven (1975) have later modified this statement to the extent that they allow that accurate identification of lymphocyte population is difficult by SEM alone without parallel immunologic identification, they still show micrographs with considerable numbers of smooth cells as well as many cells with relatively few microvilli. Alexander & Wetzel (1974) have suggested that the different results of Polliack et al. are due to their method of sample preparation, that is, collection on silver membranes for variable time periods before fixation in glutaraldehyde. Alexander & Wetzel (1974) consider that this procedure involves large and possibly selective losses of cells and has a nonspecific smoothing effect on the surface of cells. Lin, Wallach & Tsai (1973) have shown that an established line of bovine lymphocytes exhibits fewer microvilli at lower temperatures and our results with methotrexatetreated cells, reported below, indicate that dead or dying cells lose their microvilli and eventually become quite smooth before dissolution. We also show that the microvilli increase in length with the increased metabolic activity associated with transformation. It therefore seems probable that any preparation of peripheral blood monocytes containing a significant number of non-villous cells, or cells with few microvilli, gives a false representation of the surface morphology of normal, live lymphocytes. Effect of transformation by PHA on lymphocyte surface morphology Although the surface morphology did not differ significantly with the method of lymphocyte isolation, the appearance of lymphocytes undergoing transformation with PHA showed significant changes during the culture period (Table 3). The data, which were obtained with T-lymphocytes purified on a nylon column, show that although there was no increase in cell diameter after 1 day of incubation with PHA, by this time the subpopulation having smaller villi had almost disappeared. After 2 days of incubation with PHA the majority of cells were now blastoid with diameters about twice those of the unstimulated cells (Fig. 2 A) and had microvilli with a mean length 4 times that of the microvilli on unstimulated cells. The mean villous length was still greater on day 3, some villi reaching a length of over 3 /im, but by day 5 when the peak of blast proliferation was over (Hoffmann et al. 1976) the mean length of the microvilli had started to decrease. These changes are illustrated in Fig. 2, and

7 Table - 3. Changes in morphology of purified T-lymphocytes during incubation with PHA - Period of incubation, days 2 I E A I \\ Characteristics -PHA + PHA - PHA + PHA - PHA + PHA - PHA +PEL4 $ k (a) Short, stubby villi 90.2 I ' ' I (6) Long villi '0 o o 23.7 (c) Ruffled o o 6.2 o 3 '0 o 1'5 o % b Subpopulations, % & Cell diameter, prn 5.0 f 0.5 5'5 f 0.9 4'3 f 0'5 8.2 f f 0.4 7'9 f 1'5 4.8 f k 1'5 Villi per cell 48zf1oo 37of f f f f f f 139 a Mean length of villi, pm q Subpopulation (a) 0.35 f f ko fo fo.16 1.ogfo.32 3 Subpopulation (b) 0.61 f f f f ko.31 % Range of villi length, pm Subpopulation (a) ' ' Subpopulation (b) 0' ' I '54-1'19-3' L

8 C. C. Hoffmann, K. C. Moore, C.-Y. Shih and R. L. Blakley Fig. 2. Changes in surface morphology of lymphocytes undergoing transformation by PHA. Peripheral blood T-lymphocytes obtained by purification on a nylon column, were incubated with PHA for various periods. A, unstimulated T-lymphocyte; B, typical cluster of stimulated cells on the third day of incubation; c, lymphocyte on second day of stimulation with particularly long villi; and D, 5th day of stimulation. x8ooo.

9 Surface morphology of human Lymphocytes clearly show the correlation between the degree of metabolic activity of the lymphocyte and its surface morphology. Surface morphology of human thymocytes The preceding suggested that the surface morphology of human thymocytes should be reexamined. Polliack et al. (1973) found in a thymocyte population under SEM a large proportion (70%) of smooth cells, some of which had a small number of short Fig. 3. Typical cluster of human thymocytes, prepared as described in the Methods section, x The group contains representatives of 3 major types: cells with almost completely smooth surfaces (a), with small numbers of stubby microvilli (6), and with a crust-like structure covering part (c) or most (d) of the cell surface. stub-like projections (up to 10 visible). Most of the remainder had projections on the exposed cell surface and cells with densely villous surfaces were rare. Our results are in general similar, except that there were few cells with small numbers of stubby villi and a large number of cells exhibiting a crust-like structure which overlays the cell surface (Fig. 3). Although the interpretation of this structure is obviously hazardous, its appearance suggests that it is an early stage in the development of the dense covering of microvilli present on T-lymphocytes that have been released into Ctrl?

10 i6o C. C. Hoffmann, K. C. Moore, C.-Y. Shih and R. L. Blakley

11 Surf ace morphology of human lymphocytes 161 the lymph and blood. On this hypothesis the surface of thymocytes would change as they mature from smooth, to smooth with a few projections, then to encrusted, and finally to fully villous. When this thymocyte preparation was examined for formation of spontaneous rosettes with sheep red blood cells, % of the population formed rosettes. On the other hand only 1 5 ± o- 5 % of the population stained positively with fluoresceinconjugated anti-ig. Effect of methotrexate on surface morphology of transforming lymphocytes We have previously reported results showing that when 50 nm methotrexate was added to peripheral blood lymphocytes on the third day of culture with PHA, the proliferation of T-lymphoblasts was arrested. When cells were removed from such Table 4. Percentage of lymphocytes of different surf ace morphology under scanning electron microscopy after methotrexate treatment Subpopulation type 1 \ Period of exposure Half Dyeto methotrexate, h Villous, % villous, % Smooth, % Pitted, % absorbing, % Control n Lymphocytes were prepared by gravity sedimentation. Methotrexate (50 nm) added on day 3 of incubation with PHA. a culture on successive days and examined by SEM for changes in surface morphology, it became apparent that exposure to methotrexate rapidly initiated the progressive loss of microvilli from the surface of many cells. As early as 6 h after the addition of methotrexate to the cultures, cells were present on which the microvilli had disappeared over portions of their surfaces (Fig. 4). By 24 h after methotrexate addition, cells could be observed on which no microvilli were present, and with further incubation these non-villous cells became 'pitted' and showed more and more evidence of dissolution (Fig. 4, Table 4). As might be expected from the close relationship between cell viability and the ability to exclude dyes, the proportion of pitted cells corresponds closely with the proportion of cells unable to exclude dyes (Table 4). Fig. 4. Effect of methotrexate on the surface morphology of PHA-transformed lymphocytes. Methotrexate (50 nm) was added to a PHA-stimulated culture on day 3 of incubation. After various periods of exposure of the cells to methotrexate samples were removed for SEM. A, villous cell before methotrexate treatment; B, partly denuded cell (12 h); c, completely denuded cell (24 h); D, denuded cell with pitted surface (48 h); E, F, cells undergoing dissolution at 72 and 96 h, respectively, x 6700.

12 162 C. C. Hoffmann, K. C. Moore, C.-Y. Shih and R. L. Blakley Surprisingly, it was found that even after prolonged exposure to methotiexate, some cells retained normal morphology despite the fact that others were in an advanced stage of dissolution (Table 4). The explanation of this observation is uncertain. It is conceivable that the cells which survive with normal morphology, even after 4 days of exposure to 50 nm methotrexate (a concentration sufficient to arrest proliferation completely (Hoffman et al. 1976)), may represent a subpopulation of T-lymphocytes different in both function and metabolism from those that undergo dissolution. There is, of course, much evidence in the literature to indicate the existence of subpopulations of T-lymphocytes with different functions. Presumably a particular subpopulation would survive because its cell kinetics are such that the blockade of thymidylate synthesis does not result in the thymineless death that overtakes the majority. Alternatively, it is possible that the population is effectively homogeneous and that destruction or survival is determined simply by the stage of the cell cycle at which a cell happens to be at the time of methotrexate addition. Since the main metabolic effect of methotrexate is to block thymidylate synthesis (Hoffmann et al. 1976), the drug will produce a critical effect only during S-phase. Cells that enter S-phase at the time of methotrexate addition or soon thereafter may therefore be the ones that exhibit severe morphological changes, whereas those which had completed 5-phase shortly before methotrexate addition may be the ones which survive unchanged morphologically. Since the number of cells passing through mitosis declines rapidly from the fourth day after PHA-stimulation (Hoffman et al. 1976), i.e. the day after methotrexate was added, only a small proportion of cells enter 5-phase and become subject to the severe morphological changes during the third and fourth days of exposure to methotrexate. This could explain why the decline in the number of cells with normal morphology is much less on the fourth day of exposure to methotrexate than on the first day. Rosetting of T-lymphocytes and effect of methotrexate SEM has previously been used to study the formation of rosettes between lymphocytes and sheep red blood cells by Polliack et al. (19746) and by Kay et al. (1974). The latter group reported that microvilli form the attachment between lymphoblasts and sheep red blood cells and speculated that the receptor sites for the interaction are on the microvilli. However, Polliack et al. observed attachment of the red cells directly to the surface of the lymphocytes and considered that the area of contact was much smaller for T-lymphocytes than for B-lymphocytes. In our experiments lymphocytes were stimulated with PHA to give T-lymphoblasts as described above. After 3 days of incubation the latter were treated with sheep red blood cells and the rosettes examined by SEM. We found no evidence (Fig. 5) that the erythrocytes were attached to the lymphoblasts by microvilli as reported by Kay et al. (1974) or that the rosetted lymphoblasts were more villous than lymphoblasts untreated with red cells as stated by Polliack et al. (1974) for peripheral blood lymphocytes and by Lin & Wallach (1974) for an established lymphocyte line. Moreover the area of surface contact between red cell and lymphoblast was not so small as to be accurately described as a ' point attachment' as in the report of Polliack et al. (Fig. 5B). This is in agreement with TEM

13 Surface morphology of human lymphocytes 163 Fig. 5. Interaction between human lymphocytes and sheep red blood cells, A, rosette, x 8000; B, detail of interaction between the erythrocyte and the lymphocyte membrane, x

14 164 C. C. Hoffmann, K. C. Moore, C.-Y. Shift and R. L. Blakley results (Kataoka, Minowada & Pressman, 1975) which show that the erythrocyte and lymphocyte membranes have an extensive area of contact. When PHA-stimulated lymphocytes exposed to methotrexate were treated with red cells it became apparent that cells with membrane showing obvious deterioration were unable to form rosettes. This observation is in agreement with previous reports (Sabatini, Bensch & Barrnett, 1963; Alexander & Wetzel, 1974) that the capacity to form rosettes is dependent on the presence of an intact cell membrane, metabolic activity and structural integrity. This research was supported by Research Grant CA14230 from the National Cancer Institute of the United States National Institutes of Health. REFERENCES ALEXANDER, E. L. & WETZEL, B. (1974). Human lymphocytes. Similarity of B and T-cell surface morphology. Science, N. Y. 188, ANDERSON, T. F. (1951). Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans N.Y. Acad. Set. 13, BAUR, P. S., THURMAN, G. B. & GoLDSTErN, A. L. (1975). Reappraisal of lymphocyte classification by means of surface morphology, J. Immun. 115, BENTWICH, Z., DOUGLAS, S. D., SIEGEL, F. P. & KUNKEL, H. G. (1973). Human lymphocytesheep erythrocyte rosette formation: some characteristics of the interaction. Clin. Immun. Immunopath. 1, BOYUM, A. (1968). Separation of leucocytes from blood and bone marrow. Scand. J. clin. Lab. Invest, ai, Suppl. 97, CRISWELL, B. S., RICH, R. R., DARDANO, J. & KJMZBY, S. L. (1975). Scanning electron microscopy of normal and mitogen-stimulated mouse lymphoid cells. Cell. Immun. 19, DICKLER, H. B. & KUNKEL, H. (1972). Interaction of aggregated y-globulin with B lymphocytes. J. exp. Med. 136, EPSTEIN, L. B., KRETH, H. W. & HERZENBERC, L. A. (1974). Fluorescence-activated cell sorting of human B and T lymphocytes. II. Identification of the cell type responsible for interferon production and cell proliferation in response to mitogens. Cell. Immun. 12, GREAVES, M., JANOSSY, G. & DOENHOFF, M. (1974). Selective triggering of human B and T lymphocytes in vitro by polyclonal mitogens. J. exp. Med. 140, HOFFMAN, C. C, HO, Y. K., BLAKLEY, R. L. & THOMPSON, J. S. (1976). Comparative effects of selected antifolates on transforming human lymphocytes and on established human lymphoblastic cell lines. Biochem. Pharmac. 25, KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137A-138A. KATAOKA, K., MINOWADA, J. & PRESSMAN, D. (1975). Electron microscope study on human lymphocyte-sheep erythrocyte rosettes. J. natn. Cancer Inst. 55, KAY, M. M., BELOHRADSKY, B., YEE, K., VOGEL, J., BUTCHER, D., WYBRAN, J. & FUDENBERG, H. H. (1974). Cellular interactions: scanning electron microscopy of human thymus-derived rosette-forming lymphocytes. Clin. Immun. Immunopatli. 2, KELLEY, R. O., DEKKER, R. A. F. & BLUEMINK, J. G. (1973). Ligand-mediated osmium binding: its application in coating biological specimens for scanning electron microscopy (SEM). J. Cell Biol. 59, 165 a. LEVIS, W. R. & ROBBINS, J. H. (1970). Effect of glass-adherent cells on the blastogenic response of 'purified' lymphocytes to phytohemagglutinin. Expl Cell Res. 61, LIN, P. S. & WALLACH, D. F. H. (1974). Surface modification of T-lymphocytes observed during resetting. Science, N.Y. 184, LIN, P. S., WALLACH, D. F. H. & TSAI, S. (1973). Temperature-induced variations in the surface topology of cultured lymphocytes are revealed by scanning electron microscopy. Proc. natn. Acad. Sci. U.S.A. 70,

15 Surface morphology of human lymphocytes 165 LOHRMANN, H.-P., NOVIKOFS, L. & GRAW, R. G., JR. (1975). Cellular interactions in the proliferative response of human T and B lymphocytes to phytomitogens and allogeneic lymphocytes. J. exp. Med. 139, PHILLIPS, H. J. & TERRYBERRY, H. (1957). Counting actively metabolizing tissue cultured cells. Expl Cell Res. 13, POLLIACK, A. & DEHARVEN, E. (1975). An interpretative review. Surface features of normal and leukemic lymphocytes as seen by scanning electron microscopy. Clin. Imrmm. Imrnunopatti. 33, POLLIACK, A., Fu, S. M., DOUGLAS, S. D., BENTWICH, Z., LAMPEN, N. & DEHARVEN, E. (1974). Scanning electron microscopy of human lymphocyte-sheep erythrocyte rosettes. J. exp. Med. 140, POLLIACK, A., LAMPEN, N., CLARKSON, B. D. & DEHARVEN, E. (1973). Identification of human B and T lymphocytes by scanning electron microscopy. J. exp. Med. 138, POLLIACK, A., LAMPEN, N. & DEHARVEN, E. (1974a). Scanning electron microscopy of lymphocytes of known B and T derivation. In Scanning Electron Microscopy 1974 (ed. O. Johari & I. Corvin), pp Chicago: Illinois Institute of Technology Research Institute. SABATINI, D. D., BENSCH, K. & BARRNETT, J. J. (1963). Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, VAN EWIJK, W., BRONS, N. H. C. & ROZING, J. (1971). Scanning electron microscopy of homing and recirculating lymphocyte populations. Cell. Immun. 19, WETZEL, B., CANNON, G. B., ALEXANDER, E. L., ERICKSON, B. W., JR. & WESTBROOK, E. W. (1974). A critical approach to the scanning electron microscopy of cells in suspension. In Scanning Electron Microscopy 1974 (ed. O. Johari & I. Corvin), pp Chicago: Illinois Institute of Technology Research Institute. WETZEL, B., ERICKSON, B. W., JR. & LEVIS, W. R. (1973). The need for positive identification of leucocytes examined by scanning electron microscopy. In Scanning Electron Microscopy! 1973 (ed. O. Johari & J. Corvin), pp Chicago: Illinois Institute of Technology Research Institute. {Received 12 April 1977)