SCANNING ELECTRON MICROSCOPY OF HUMAN LYMPHOCYTES DURING TRANSFORMATION AND SUBSEQUENT ... · 2005. 8. 21. · Alexander & Wetzel (1974) Sabatini et al. (1963) Levis & Robbins (1970)

J. Cell Set. a8, 151-165 (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. BLAKLEYTlie Department of Biochemistry, College of Medicineand the Department of Zoology, University of Iowa,Iozoa City, Iowa 52242, U.S.A.

SUMMARY

Preparations of human peripheral blood lymphocytes containing 72-87 % T-cells and 10—16 % B-cells were shown by scanning electron microscopy to consist almost exclusively of cellsbearing numerous microvilli, whereas thymocytes were of mixed surface morphology, withboth smooth and encrusted forms numerous. T-lymphocytes purified on long nylon columnswere all covered with numerous short villi. Stimulation with phytohaemagglutinin for 2 daysproduced T-lymphoblasts almost exclusively, and as the T-cells enlarged the microvillilengthened, the increase in length reaching 5-fold by day 3. Addition of sufficient methotrexateon day 3 to arrest proliferation (50 ITM) caused progressive loss of microvilli from the cellsurface, with the eventual production of large numbers of smooth cells, the surfaces of whichlater became pitted, followed by the complete dissolution of the cell. T-lymphocytes wereshown to form rosettes with sheep erythrocytes through direct contact of the cell membranesover a significant area, but when, as a result of methotrexate treatment, the lymphocytes hadbecome denuded of microvilli or had reached an advanced state of dissolution, rosettes wereno longer formed.

INTRODUCTION

Studies of the surface morphology of human lymphocytes by scanning electronmicroscopy (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 architecturalforms most prevalent and the functional identity of cells with each of these forms.The controversy has also extended to the morphology of rosettes formed betweenlymphocytes 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 useSEM to study the effects of methotrexate, an immunosuppressive drug sometimesused in suppressing graft-versus-host disease, on the surface morphology of trans-forming T-lymphocytes. However, data obtained in the course of this work are clearlyin agreement with reports that both B and T lymphocytes are covered with numerousmicrovilli so that contrary to the claim of some investigators, surface morphology

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 reporton the changes that occur on the surface of T-lymphocytes during transformationin vitro by phytohaemagglutinin (PHA), and during subsequent exposure to metho-trexate. This work complements a previous publication (Hoffmann, Ho, Blakley &Thompson, 1976) on the effect of exposure to methotrexate for various periods onblast formation, mitosis, DNA synthesis and blast proliferation.

MATERIALS AND METHODS

Abbreviations used: HBSS, Hanks' buffered salt solution; MEM, minimum essentialmedium; PHA, phytohaemagglutinin; and SEM, scanning electron microscopy.

Peripheral blood was drawn from healthy donors into heparinized vacutainers and allowed tosediment for 2 h at 37 °C. The buffy coat was removed, the leukocytes recovered by centri-fugation and resuspended. For studies of transformation the lymphocytes were cultured at aninitial density of 10s per ml in the presence of PHA at a concentration of 8 fig per ml. Otherdetails of the experimental procedure and the purification of T-lymphocytes by passage througha nylon column were as previously described (Hoffmann et al. 1976). When lymphocytes wereprepared 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 addedaseptically 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 at48 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 previouslybeen absorbed with an equal volume of packed sheep erythrocytes. The washed cells wereresuspended in HBSS to a final concentration of 5 x 10' cells per ml and the rosette assaycarried out according to Bentwich, Douglas, Siegel & Kunkel (1973). Sheep erythrocytes werecollected under sterile conditions in Alsevers solution, stored at 4 °C for up to 14 days andwashed 2 or 3 times in HBSS before use. In the assay, a total of 200-300 cells were counted ina 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 washedtwice 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 15-20 x 10°cells per ml and reacted with fluorescein-conjugated rabbit anti-human immunoglobin (GrandIsland 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 femalepatient in the course of therapeutic cardiac surgery. This material was obtained through thecourtesy of Dr John S. Thompson, Department of Internal Medicine. The thymus tissue wasgently teased in warm HBSS containing penicillin and streptomycin. The resulting suspensionwas centrifuged at 50 g for 10 min, the supernatant solution removed, and the pellet washedtwice more before resuspending, determining the proportion of T and B lymphocytes andexamining 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 aglass coverslip. In the case of rosetted lymphocytes, 2 drops of the suspension were placeddirectly on the coverslip because of the danger of disrupting rosettes during washing. After20 min at room temperature, the cells were fixed with Karnovsky's reagent (1965) for 1 h andprocessed according to the method of Wetzel et al. (1973) and Kelley, Dekker & Bluemink(J973)- Briefly, the cells on the coverslips were postfixed with 1 % osmium tetroxide in o-i Msodium cacodylate buffer, pH 72 , for 1 h, washed thoroughly in the same buffer (5 to 10 timesover 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 for45 min. The specimens were rinsed thoroughly (3 times) in distilled water and dehydratedthrough a graded ethanol series and dried by the CO. critical-point procedure (Anderson, 1951)

Surface morphology of human lymphocytes 153

in a Sorvall Critical Point Drying System. The dried samples were vacuum coated with carbonand gold palladium and examined in a Cambridge S-4 Stereoscan Electron Microscope at 450

tilt. Polaroid type 55 P/N film was used for micrographs. In determining subpopulation pro-portions 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 lymphocytesfound in preparations of human peripheral blood lymphocytes with the results withlymphocytes from 6 donors in this study. Our results for the proportion of T-lymphocytes are slightly higher than some literature values, and our proportion ofB-lymphocytes somewhat lower, but we could see no evidence that this is related tothe method of preparation (compare our gravity sedimentation and Ficoll-Hypaquevalues). The cells identified as T or B lymphocytes in our preparations togetheraccounted for 86-99 % °f t n e 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-HypaqueFicoll-HypaqueFicoll-HypaqueDefibrinate, CO-Fe powder,Ficoll-Isopaque

Ficoll-HypaqueGravity sedimentationFicoll-Hypaque

% of peripheralblood lymphocytes

A

T

70

648 ±3769-8270-80

S6±8»

52-8172-87*77-8it

B

27-35t

20-3020-30IS

24±7'2f

20-4410-5-15-812-0-17-9

Reference

Anderson (1951)

Alexander & Wetzel (1974)Sabatini et al. (1963)Levis & Robbins (1970)Lohrmann et al. (1975)

Epstein et al. (1974)This studyThis 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 ourexperimental conditions, the maximum number of blasts that subsequently accumu-lated in the culture ranged from 4-4 to 9-2 x ioB per ml from an initial culture of ioB

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 illustratedby an experiment in which T-lymphocytes were purified by gravity sedimentationfollowed by passage through a nylon column. Recovery of T-lymphocytes from thelatter was 70-5%. Examination of this preparation after Wright staining showedmonocytes to be absent, and immunofluorescent staining showed that B-lymphocytes

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 typicallymphocytes in a gravity-sedimentated preparation with some platelets in the back-ground ; B, monocyte present in the preparation; C, lymphocytes typical of the majorpopulation with short microvilli (upper left) and of the minor subpopulation with longvilli (lower left), x 8000.

were absent, but 9% of the lymphocytes did not form spontaneous rosettes withsheep red blood cells. Although this preparation therefore contained a higher pro-portion of rosetting T-lymphocytes (91 ±2*6%), the peak blast count was only3-5 x io5, below the usual range. This suggests a role of other (adhesive) cells in stimu-lating the response of T-lymphocytes to PHA, in agreement with the finding of others

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 unfractionatedlymphocytes were found to be almost exclusively (95 %) T-lymphoblasts, as indicatedby the formation of spontaneous rosettes with sheep red blood cells, and by the smallproportion (~ 3 %) of cells labelled by immunofluorescence staining, most or all ofwhich were the B-cells originally present in the culture. This is in good agreementwith 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-cellorigin, while 99 % of the blasts were T-lymphoblasts in PHA-stimulated cultures ofpurified T-lymphocytes.

Table 2. Characteristics of lymphocyte populations prepared from humanperipliera. bloodby various procedures*

Characteristics

Subpopulation, %Short stubbyLong-villousRuffledSmooth

Cell diameter, /*mMean ± S.E.Range

No. of villi per cellMean ± S.E.Range

Length of villi, fim

Gravitysedimentation

9 0 3

6 72 3°'5

4'55±°-34(4-15-5-29)

369±i45(280-536)

0-2-0-66

* Each measurement was performed

Method of preparation

Ficoll-Hypaque

9464-41 0

0

463 ±0-45(3-85-5-23)

398 ±90(312-448)0-19-0-67

on samples of at least 900

Nylon column

~ 100

0

0

0

4-97 ± 0-49(3-76-5-65)

482±100(380-580)o-i6-O'57

cells.

SEM of peripheral blood lymphocytes

Populations of unstimulated peripheral lymphocytes were shown by SEM toconsist 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 lengtho-6i + 0-18 /jm compared with a mean length of 0-35 + o-i /tm for the majority. An-other group had ridges or ruffles (Fig. IB) which have been shown by Alexander &Wetzel (1974) to be characteristic of monocytes. Cells prepared with Ficoll-Hypaquedid not differ significantly in surface morphology from lymphocytes prepared bygravity sedimentation (Table 2). Wright stain showed that lymphocytes prepared bythe latter method contained 88-416-4% lymphocytes, 5-9 + 0-8% polymorphs and9-1 ±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

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

distinguishable from lymphocytes by SEM. T-lymphocytes prepared by passagethrough a nylon column all appeared as cells with many short microvilli.

Our preparations of lymphocytes contained few or no smooth cells (Table 2). Ourresults 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 withthose 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, includingthe 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 distin-guished 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 extentthat they allow that accurate identification of lymphocyte population is difficult bySEM alone without parallel immunologic identification, they still show micrographswith considerable numbers of smooth cells as well as many cells with relatively fewmicrovilli. Alexander & Wetzel (1974) have suggested that the different results ofPolliack et al. are due to their method of sample preparation, that is, collection onsilver membranes for variable time periods before fixation in glutaraldehyde. Alexander& Wetzel (1974) consider that this procedure involves large and possibly selectivelosses 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 lymphocytesexhibits fewer microvilli at lower temperatures and our results with methotrexate-treated cells, reported below, indicate that dead or dying cells lose their microvilli andeventually become quite smooth before dissolution. We also show that the microvilliincrease in length with the increased metabolic activity associated with transformation.It therefore seems probable that any preparation of peripheral blood monocytescontaining a significant number of non-villous cells, or cells with few microvilli, givesa 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 oflymphocyte isolation, the appearance of lymphocytes undergoing transformation withPHA showed significant changes during the culture period (Table 3). The data,which were obtained with T-lymphocytes purified on a nylon column, show thatalthough 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. After2 days of incubation with PHA the majority of cells were now blastoid with diametersabout twice those of the unstimulated cells (Fig. 2 A) and had microvilli with a meanlength 4 times that of the microvilli on unstimulated cells. The mean villous lengthwas still greater on day 3, some villi reaching a length of over 3 /im, but by day 5when the peak of blast proliferation was over (Hoffmann et al. 1976) the mean lengthof the microvilli had started to decrease. These changes are illustrated in Fig. 2, and

Tab

le 3

. Cha

nges

in m

orph

olog

y of

pur

ified

T-l

ymph

ocyt

es d

urin

g in

cuba

tion

wit

h P

HA

Per

iod

of i

ncub

atio

n, d

ays

2

I 2

3 4

E A

-

I \ \ -

Cha

ract

eris

tics

-P

HA

+ P

HA

- P

HA

+ P

HA

- P

HA

+ P

HA

- P

HA

+

PE

L4

$ S

ubpo

pula

tion

s, %

k

(a)

Sho

rt,

stu

bb

y v

illi

90

.2

I '0

93

.8

3'1

96

.4

I .8

98

.5

76

-3

& (6

) L

on

g v

illi

9

.8

99'0

o

96.9

0

.6

98.2

o

23.7

(c

) R

uffl

ed

o o

6.2

o 3 '

0

o

1'5

o

%

b

Cel

l di

amet

er, p

rn

5.0

f 0.

5 5'

5 f 0

.9

4'3

f 0

'5

8.2 f 0

.6

5.0

f 0.

4 7

'9 f 1

'5

4.8

f 0.

5 7.

2 k

1'5

Vil

li p

er c

ell

48

zf1

oo

3

7o

f14

28

4 f

54

614 f 3

8 4

13

f 39

37

2 f

201

386

f 66

5

40

f 13

9 a

Mea

n l

engt

h of

vil

li,

pm

q

S

ubpo

pula

tion

(a)

0.

35 f

0.1

0

-

0.31

f 0

.09

-

0.3

8k

o.1

5

0.8

5fo

.26

0

.30

fo.1

6

1.o

gfo

.32

3

Sub

popu

lati

on (

b)

0.61

f 0

.18

0.58

f 0

.18

-

1.24

f 0

.42

-

1.94

f 0

.60

-

1.3

4k

o.3

1

%

Ran

ge o

f vi

lli l

engt

h, p

m

8 L

Sub

popu

lati

on (

a)

0.14

-0'7

5 -

0.15

-0'5

7 -

0.15

-0.8

5 0.

38-1

.33

0.1

1-0

.65

0.24

-1.8

0 S

ubpo

pula

tion

(b)

0'

41-0

.92

0'27

-0.9

I

-

0.52

-2'5

4 -

1'19

-3'3

9 -

0.83

-2.0

8

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

Fig. 2. Changes in surface morphology of lymphocytes undergoing transformation byPHA. 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 onsecond day of stimulation with particularly long villi; and D, 5th day of stimulation.x8ooo.

Surface morphology of human Lymphocytes

clearly show the correlation between the degree of metabolic activity of the lymphocyteand its surface morphology.

Surface morphology of human thymocytes

The preceding suggested that the surface morphology of human thymocytes shouldbe reexamined. Polliack et al. (1973) found in a thymocyte population under SEM alarge 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 Methodssection, x 8060. The group contains representatives of 3 major types: cells with almostcompletely smooth surfaces (a), with small numbers of stubby microvilli (6), and witha 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 10-40 projectionson the exposed cell surface and cells with densely villous surfaces were rare. Ourresults are in general similar, except that there were few cells with small numbers ofstubby villi and a large number of cells exhibiting a crust-like structure which overlaysthe cell surface (Fig. 3). Although the interpretation of this structure is obviouslyhazardous, its appearance suggests that it is an early stage in the development of thedense covering of microvilli present on T-lymphocytes that have been released into

Ctrl?

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

Surf ace morphology of human lymphocytes 161

the lymph and blood. On this hypothesis the surface of thymocytes would change asthey mature from smooth, to smooth with a few projections, then to encrusted, andfinally to fully villous.

When this thymocyte preparation was examined for formation of spontaneousrosettes with sheep red blood cells, 98-912-0% of the population formed rosettes.On the other hand only 1 • 5 ± o- 5 % of the population stained positively with fluorescein-conjugated anti-Ig.

Effect of methotrexate on surface morphology of transforming lymphocytes

We have previously reported results showing that when 50 nM methotrexate wasadded to peripheral blood lymphocytes on the third day of culture with PHA, theproliferation of T-lymphoblasts was arrested. When cells were removed from such

Table 4. Percentage of lymphocytes of different surf ace morphology under scanning electronmicroscopy after methotrexate treatment

Subpopulation type1 \

Period of exposure Half Dye-to methotrexate, h Villous, % villous, % Smooth, % Pitted, % absorbing, %

Control 88-5 22 9-1 — 36 692 215 93 — 4

12 63-9 17-7 85 — 224 64-8 47 28-5 — 848 604 33 18-1 18-2 2572 46-3 51 14-9 338 4496 412 19 n-6 453 46

Lymphocytes were prepared by gravity sedimentation. Methotrexate (50 nM) added on day 3of 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 progressiveloss of microvilli from the surface of many cells. As early as 6 h after the addition ofmethotrexate to the cultures, cells were present on which the microvilli had dis-appeared 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 in-cubation these non-villous cells became 'pitted' and showed more and more evidenceof dissolution (Fig. 4, Table 4). As might be expected from the close relationshipbetween cell viability and the ability to exclude dyes, the proportion of pitted cellscorresponds closely with the proportion of cells unable to exclude dyes (Table 4).

Fig. 4. Effect of methotrexate on the surface morphology of PHA-transformed lympho-cytes. Methotrexate (50 nM) was added to a PHA-stimulated culture on day 3 ofincubation. After various periods of exposure of the cells to methotrexate samples wereremoved for SEM. A, villous cell before methotrexate treatment; B, partly denudedcell (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.

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 advancedstage of dissolution (Table 4). The explanation of this observation is uncertain. It isconceivable that the cells which survive with normal morphology, even after 4 days ofexposure to 50 nM methotrexate (a concentration sufficient to arrest proliferationcompletely (Hoffman et al. 1976)), may represent a subpopulation of T-lymphocytesdifferent in both function and metabolism from those that undergo dissolution. Thereis, of course, much evidence in the literature to indicate the existence of subpopu-lations of T-lymphocytes with different functions. Presumably a particular subpopu-lation would survive because its cell kinetics are such that the blockade of thymidylatesynthesis does not result in the thymineless death that overtakes the majority. Alter-natively, it is possible that the population is effectively homogeneous and that des-truction or survival is determined simply by the stage of the cell cycle at which a cellhappens to be at the time of methotrexate addition. Since the main metabolic effectof methotrexate is to block thymidylate synthesis (Hoffmann et al. 1976), the drug willproduce a critical effect only during S-phase. Cells that enter S-phase at the time ofmethotrexate addition or soon thereafter may therefore be the ones that exhibit severemorphological changes, whereas those which had completed 5-phase shortly beforemethotrexate addition may be the ones which survive unchanged morphologically.Since the number of cells passing through mitosis declines rapidly from the fourthday after PHA-stimulation (Hoffman et al. 1976), i.e. the day after methotrexate wasadded, only a small proportion of cells enter 5-phase and become subject to the severemorphological changes during the third and fourth days of exposure to methotrexate.This could explain why the decline in the number of cells with normal morphologyis 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 lympho-cytes and sheep red blood cells by Polliack et al. (19746) and by Kay et al. (1974). Thelatter group reported that microvilli form the attachment between lymphoblasts andsheep red blood cells and speculated that the receptor sites for the interaction are onthe microvilli. However, Polliack et al. observed attachment of the red cells directlyto the surface of the lymphocytes and considered that the area of contact was muchsmaller for T-lymphocytes than for B-lymphocytes. In our experiments lymphocyteswere stimulated with PHA to give T-lymphoblasts as described above. After 3 daysof incubation the latter were treated with sheep red blood cells and the rosettesexamined by SEM. We found no evidence (Fig. 5) that the erythrocytes were attachedto the lymphoblasts by microvilli as reported by Kay et al. (1974) or that the rosettedlymphoblasts were more villous than lymphoblasts untreated with red cells as statedby 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 redcell and lymphoblast was not so small as to be accurately described as a ' point attach-ment' as in the report of Polliack et al. (Fig. 5B). This is in agreement with TEM

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 20000.

164 C. C. Hoffmann, K. C. Moore, C.-Y. Shift and R. L. Blakley

results (Kataoka, Minowada & Pressman, 1975) which show that the erythrocyte andlymphocyte membranes have an extensive area of contact.

When PHA-stimulated lymphocytes exposed to methotrexate were treated withred cells it became apparent that cells with membrane showing obvious deteriorationwere unable to form rosettes. This observation is in agreement with previous reports(Sabatini, Bensch & Barrnett, 1963; Alexander & Wetzel, 1974) that the capacity toform rosettes is dependent on the presence of an intact cell membrane, metabolicactivity and structural integrity.

This research was supported by Research Grant CA14230 from the National CancerInstitute of the United States National Institutes of Health.

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ANDERSON, T. F. (1951). Techniques for the preservation of three-dimensional structure inpreparing specimens for the electron microscope. Trans N.Y. Acad. Set. 13, 130-134.

BAUR, P. S., THURMAN, G. B. & GoLDSTErN, A. L. (1975). Reappraisal of lymphocyte classifi-cation by means of surface morphology, J. Immun. 115, 1375-1380.

BENTWICH, Z., DOUGLAS, S. D., SIEGEL, F. P. & KUNKEL, H. G. (1973). Human lymphocyte-sheep erythrocyte rosette formation: some characteristics of the interaction. Clin. Immun.Immunopath. 1, 511-522.

BOYUM, A. (1968). Separation of leucocytes from blood and bone marrow. Scand. J. clin. Lab.Invest, ai, Suppl. 97, 9-109.

CRISWELL, B. S., RICH, R. R., DARDANO, J. & KJMZBY, S. L. (1975). Scanning electron micro-scopy of normal and mitogen-stimulated mouse lymphoid cells. Cell. Immun. 19, 336—348.

DICKLER, H. B. & KUNKEL, H. (1972). Interaction of aggregated y-globulin with B lymphocytes.J. exp. Med. 136, 191-196.

EPSTEIN, L. B., KRETH, H. W. & HERZENBERC, L. A. (1974). Fluorescence-activated cell sortingof human B and T lymphocytes. II. Identification of the cell type responsible for interferonproduction and cell proliferation in response to mitogens. Cell. Immun. 12, 407-421.

GREAVES, M., JANOSSY, G. & DOENHOFF, M. (1974). Selective triggering of human B and Tlymphocytes in vitro by polyclonal mitogens. J. exp. Med. 140, 1-18.

HOFFMAN, C. C, HO, Y. K., BLAKLEY, R. L. & THOMPSON, J. S. (1976). Comparative effectsof selected antifolates on transforming human lymphocytes and on established humanlymphoblastic cell lines. Biochem. Pharmac. 25, 1947-1954.

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{Received 12 April 1977)