Characterization of functionally distinct lymphoid and myeloid cells from human blood and bone marrow. II. Separation by velocity sedimentation

Journal of Immunological Methods, 18 (1977) 79--93 © Elsevier/North-Holland Biomedical Press

79

CHARACTERIZATION OF FUNCTIONALLY DISTINCT LYMPHOID AND MYELOID CELLS FROM HUMAN BLOOD AND BONE MARROW. II. S E P A R A T I O N B Y V E L O C I T Y S E D I M E N T A T I O N *

J.R. WELLS, G. OPELZ and M.J. CLINE Division of Hematology-Oncology, Department of Medicine, and the Department of Surgery, University of California School of Medicine, Los Angeles, CA 90024, U.S.A.

(Received 11 April 1977, accepted 26 May 1977)

Velocity sedimentation in a zonal rotor using gradients of uniform osmolarity was used to separate leukocyte subpopulations from human blood and bone marrow. The separations were performed at high sedimentation rates having the advantage of rapidity over conventional unit gravity separations. Myeloid stem cells (CFU-C) and cells reactive with phytohemagglutinin (PHA), Concanavalin A (Con A), and in mixed leukocyte culture (MLC) were separated and their sedimentation profiles obtained. CFU-C sedimented ahead of lymphoid cells and behind mature myeloid elements. Two distinct marrow subpopulations separated by velocity sedimentation were consistently stimulated by Con A, and variably stimulated by PHA and MLC reactions. Both large cells (predominantly myeloid) and small cells (predominantly lymphoid) from bone marrow were stimulated by Con A in [3H]thymidine incorporation assays. When separated subpopulations showing stimulation by Con A were mixed, inhibition of [3H]thymidine incorporation resulted.

INTRODUCTION Several m e t h o d s are used to s e p a r a t e d i s t i n c t p o p u l a t i o n s f r o m h e t e r o g e n e o u s m i x t u r e s o f cells b a s e d on p h y s i c a l c h a r a c t e r i s t i c s such as size, d e n s i t y , and a d h e r e n c e ( S h o r t m a n , 1 9 7 2 ; P r e t l o w et al., 1975). This r e p o r t describes a r e f i n e m e n t o f the v e l o c i t y s e d i m e n t a t i o n m e t h o d t o s e p a r a t e f u n c t i o n a l l y d i s t i n c t p o p u l a t i o n s o f h u m a n h e m a t o p o i e t i c cells f r o m p e r i p h e r a l b l o o d and b o n e m a r r o w . V e l o c i t y s e d i m e n t a t i o n t e c h n i q u e s s e p a r a t e cells p r i m a r i l y on t h e basis o f size, w i t h large cells s e d i m e n t i n g m o s t r a p i d l y ; cell d e n s i t y has o n l y a m i n o r i n f l u e n c e ( S h o r t m a n , 1972). B o t h b l o o d and b o n e m a r r o w contain a large n u m b e r o f cell t y p e s w i t h c o n s i d e r a b l e h e t e r o g e n e i t y o f size and d e n s i t y . A d i s t i n c t s u b p o p u l a t i o n o f cells, such as m y e l o i d s t e m cells, m a y d e m o n s t r a t e h e t e r o g e n e i t y o f size as cells pass t h r o u g h t h e replicative cycle. C o n s e q u e n t l y , v e l o c i t y s e d i m e n t a t i o n s e p a r a t i o n s o f t h e s e cells yield b r o a d p e a k s o f f u n c t i o n a l l y similar cells. * This study was supported by United States Public Health Service Grants AM 18058 and CA 15688.

80 Velocity separation procedures that sediment cells at unit gravity usually require several hours to achieve a separation. We describe separations performed at higher g forces with a reorienting gradient zonal rotor using a shallow Ficoll gradient of uniform osmolarity. These separations were performed in a short time period on a large number of cells, with excellent reproducibility. We examined isolated blood and bone marrow populations in assays for myeloid stem cells, response to PHA and Con A, and as responders and stimulators in MLC. Cell-cell interactions were investigated in these functional tests. Populations of cells were identified whose activity is obscured in heterogeneous, unfractionated blood and bone marrow populations. MATERIALS AND METHODS Bone marrow and blood cell preparation Venous blood (70 ml) and bone marrow (10 ml) were collected from 30 healthy adult volunteers in heparinized syringes. Marrow was sieved through fine screens to produce single cell suspensions. Blood or marrow cells were then sedimented with 3% Dextran in saline, and the supernatants containing most of the nucleated cells were removed and centrifuged at 1 5 0 g for 10 min. The cell pellet was resuspended in 0.75% NH4C1 buffered with 0.003 M Tris pH 7.2 at 37°C for 5 min to lyse erythrocytes and then recentrifuged before a final resuspension in Hanks' balanced salt solution (BSS) at 1 to 1.2 X 107 cells per ml. In all experiments aliquots were removed initially and before loading into the rotor for morphologic and functional analysis. Gradient solutions Gradient solutions were prepared with Ficoll 400 (Pharmacia Fine Chemicals, Piscataway, New Jersey) and Hank's BSS. Light solution: Four per cent Ficoll was prepared by mixing 28 g of Ficoll in 672 ml of Hanks' 1.05X dilution using a blender. The solution was adjusted to pH 7.2 with 7.5% sodium bicarbonate, then filtered through a 0.45-p Nalge filter. Heavy solution: Eight per cent Ficoll in 1.0X Hanks' was prepared by mixing 56 g Ficoll in 644 ml of Hanks' 1.0× dilution before pH adjustment and sterile filtration. Both the heavy and the light gradient solutions were sampled for refractive index and osmolarity determinations. Table 1 shows the values for these parameters. Zonal separations Cell separations were performed with the SZ-14 reorienting gradient zonal rotor (DuPont Instruments, Newtown, CT) (Wells et al., 1972). The use of this rotor for cell separations for yeast has been previously described (Wells

81 TABLE 1 Osmolarity and refractive index of heavy and light solutions used in constructing gradients for velocity sedimentation.

4% Ficoll in 1.05x Hanks' BSS 8% Ficoll in 1.00X Hanks' BSS

Osmolarity (mosm)

Refractive index

286 _+5 286 _+5

1.3415 1.3472

and James, 1972). Modification of this procedure for mammalian cell separations was required. In a typical separation the r o t o r was loaded dynamically with a 60-ml cushion of 50% H ypa que solution (Winthrop-Sterling Laboratories, Menlo Park, CA). The gradient was formed using the GF-2 gradient maker (DuPont Instruments, Newtown, CT) and was dynamically loaded at a rate of 50 to 75 ml per min. Linear gradients of 4 to 8% w/w Ficoll prepared from the light and heavy solutions in a total volume of 1250 ml were employed for most separations. Dynamic loading was carried o u t at 2400 rpm. The r o t o r was then decelerated to 1000 rpm and the t em perat ure allowed to stabilize at 22°C. Centrifuge speed was precisely determined with a strobe light. The cell suspension was loaded into the r o t o r in less than 5 sec using a large h y p o d e r m i c syringe. The cells were allowed to sediment for a predetermined period (usually 10 min at 1000 rpm) and the r o t o r was then slowly decelerated to rest. The gradient was unloaded and collected in 40-ml fractions. Cells were c o u n t e d using a Coulter Counter ZH fitted with a 100-p-aperture tube. Cell volume distributions were measured using a 100-channel Coulter Channelyzer Model C-100 (Coulter Electronics, Inc., Hialeah, FL). H e m o c y t o m e t e r counts were also used to determine nucleated cells per ml. F u n c t i o n a l assays

Cells from gradient fractions were prepared for morphological examination with a Shandon cyt ocent r i f uge (Shandon Scientific, Sewickley, PA) and stained with Wright's stain or for peroxidase or chloroacetate esterase (Willcox et al., 1976). Granuloeyte-macrophage colony-forming cells (CFU-C) were assayed as previously described (Pike and Robinson, 1970; Golde and Cline, 1972). MLC and mitogen response studies to PHA and Con A were perform ed as previously described (Wells et al., 1977). A u t o r a d i o g r a p h y was p e r f o r m e d on bone m arrow and blood cell fractions o f 1 X 106 cells per ml prepared with no additive, added PHA-M (1 : 4 dilution; Difco Laboratories, Detroit, MI), and added in Con A (1 : 64 dilution; Calbiochem, Los Angeles, CA). After incubation at 37°C for 4 days, each sample was labeled with 5 ttCi/ml [ZH]thymidine (1.9 Ci/mM; Schwarz-

82

Mann, Orangeburg, NY). After 24 h, cytocentrifuge slides were prepared and fixed in absolute methanol. Autoradiography was performed using NTB-2 emulsion (Eastman-Kodak, Rochester, NY), exposed for 6 h, developed, and stained with Giemsa for morphological observation. RESULTS

Separation of peripheral blood cells Separated cells were identified by light microscopy. Fig. 1A shows the distribution of blood cells from a single donor following separation at 1020 rpm for 10 min. Two major peaks of cells differing in sedimentation velocity were observed: a large-cell peak consisting of mature granulocytes and

80-

A 60-

40-

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-10

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..-2, U

=4

x ,_1 6u U

I

B

i

4

6

8

10

12 14

16

1 18 20

10

~1

. 22 24

FRACTION NO.

Fig. 1. Separation o f h u m a n peripheral b l o o d cells by velocity s e d i m e n t a t i o n in a zonal rotor using a s h a l l o w Ficoll gradient. Centrifugation at 1 , 0 2 0 rpm for 10 rain at 22 ° C. 1A: PMN l e u k o c y t e s and b a n d forms (G ©); l y m p h o c y t e s (A A); m o n o c y t e s ( X - - - - x ) ; CFU-C ( o - $). CFU-C profile from b l o o d of a s e c o n d subject.

83 band forms, and a small-cell peak comprised mainly of lymphoid cells. In some experiments an intermediate band of monocytes was seen. The purity of granulocytes in the first five fractions and of lymphocytes in the last five fractions was usually above 95%. Starting concentrations above 1.2 X 107 cells per ml gave unacceptable streaming which was characterized by the presence of l y m p h o c y t e s in the larger cell fractions. Myeloid stem cells (CFU-C) in blood

In all experiments the main peak of CFU-C activity in peripheral blood was 3 to 5 fractions ahead of the lymphoid peak. Figs. 1A and B show the sedimentation profile of CFU-C from two separations of peripheral blood. In all 5 separations where CFU-C was measured, a low peak of larger-sized CFUC was observed 6 to 8 fractions ahead of the main peak. Separation o f bone marrow cells

Fig. 2 shows the distribution of morphologically identifiable bone marrow cells from a single donor following a separation at 1010 rpm for 9.5 min. Rate separations from 22 subjects gave comparable results; however, variations from experiment to experiment were greater with marrow cells than with peripheral blood. Mature granulocytes and lymphoid cells from the bone marrow sedimented as in the peripheral blood, with mature neutrophils and band forms in the rapidly sedimenting fractions and small lymphoid cells in the slowest fractions. The sedimentation profile for lymphocytes was reproducible. The low percentage of lymphocytes (<3%) at the large-cell end of the gradient indicated that few cells were clumping or streaming and sedimenting to the b o t t o m of the gradient as a result of non-ideal sedimentation conditions. Promyelocytes were found throughout the gradient with a consistent peak at the large-cell end. Myelocytes showed the most variation in size distribution, with one or two separate peaks or distributed throughout the gradient in a single broad peak. In several experiments a third population of myelocytes was seen banded against the cushion. Blast cells distributed throughout the gradient but with a shallow peak between the lymphoid and myeloid cells. Nucleated red cell precursors also distributed broadly throughout the gradient, indicating size heterogeneity. Eosinophils were found with the differentiated granulocytes. CFU-C in bone marrow

CFU-C sedimentation profiles were measured in bone marrow samples from 10 subjects. CFU-C sedimented in a broad band with one or two peaks. The sedimentation profiles of CFU-C and myelocytes from four representative bone marrow preparations are illustrated in fig. 3. As shown in this

84

A

60-

50-

40-

30-

V

o

20-

X

-'

10-

0

2

4

6

8

10 12 14

16

8 20 22 24

FRACTION NO. Fig. 2. Separation of h u m a n b o n e marrow by velocity sedimentation. 1A: PMN leukocytes (0 --c;); l y m p h o c y t e s ( A - A); bands forms ( e - - o ) ; myelocytes (~-,4). 1B: Blast forms ( © - - - - © ) ; p r o m y e l o c y t e s (o - - e ) ; and eosinophils (~,-/'~) from the same sample.

figure sedimentation under a variety of g forces resulted in comparable distribution and enrichment of CFU-C. In all separations the profile of CFU-C showed peak activity between the two major peaks of myeloid and lymphoid cells and asymmetrically skewed to the large-cell end of the gradient. The main CFU-C peak was located very near or in the same fraction as the slowest sedimenting myelocyte peak. When a second myelocyte peak was present several fractions ahead of the slower sedimenting one, a second peak of CFU-C activity was also found.

PHA-, Con A-, and MLC-responsive cells from peripheral blood The response of peripheral blood cells fractionated by velocity sedimentation to PHA, Con A, and in mixed leukocyte culture was measured. As anticipated from previous work, the responsive cells were found in the small-cell

85

A

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._J

,v

D

¢

50-

25-

-100

"50

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1() 12 14 16 118 20 2'2 2~I FRACTION

NO.

Fig. 3. V e l o c i t y s e d i m e n t a t i o n d i s t r i b u t i o n o f CFU-C (v. $) and m y e l o c y t e s (o ©) in b o n e m a r r o w o f f o u r subjects. 3A: 1025 r p m × 8 m i n ; 3B: 1030 r p m × 13 m i n ; 3C: 1020 r p m × 11 m i n ; 3D: 1020 r p m × 11 rain. All s e p a r a t i o n s at 22°C.

end of the gradient where the lymphQid cells were located. The PHA response as measured by [3H]thymidine incorporation was consistently stronger than the Con A or MLC response and appeared in a slightly larger cell fraction (fig. 4). Variation in the magnitude of the response among individuals was considerable, but in all cases the general relationship of the curves was consistent. In 5 of 8 separations the Con A and MLC were bimodal (fig. 4), although these fractions all contained morphologically similar lymphoid cells. The PHA curve was never distinctly bimodal. PHA-, Con A-, and MLC-responsive cells from bone marrow In velocity separations of bone marrow cells the l y m p h o c y t e functional assays yielded more complex profiles than those obtained with peripheral blood. Figs. 5A and B show two studies that gave results similar to those

86

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105. 0. . . . . . . . . . . .

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7 9

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i

1'9 21 2'3 NO.

- Pig. 4. R e s p o n s e s of p e r i p h e r a l b l o o d cells f r a e t i o n a t e d by velocity s e d i m e n t a t i o n to P H A (&), C o n A (&), a n d in MLC (©) reactions. Results are expressed as c p m m i n u s b a c k g r o u n d of [ a l l ] t h y m i d i n e i n c o r p o r a t i o n .

achieved in 11 separate experiments. Mitogen-responsive cells were found both at the small-cell (lymphoid) and at the large-cell {myeloid) ends of the gradient in most of the marrow separations. The PHA response was always found in lymphoid cell fractions and in 7 of 11 experiments was also found in the large-cell fractions. The Con A response was found in both the large- and small-cell ends of the gradient in 9 of 11 experiments, and in only the small cells in two experiments. The Con A response among the large cells was usually stronger than the PHA response. The MLC response was also observed in both the large-cell and the small-cell ends of the gradient in 6 of 11 experiments and was of approximately equal magnitude in both fractions. To determine the character of responding large-cell fractions, they were examined with histochemical techniques (peroxidase and chloroacetate esterase) and [3H]thymidine autoradiography before and after incubation with Con A and PHA. As seen in table 2, few morphologically identifiable lymphoid cells were observed in the large-cell fractions from the gradient before incubation, suggesting t h a t clumping and non-ideal sedimentation of lymphocytes was an unlikely explanation of the mitogen and MLC responses. After incubation with PHA (table 2) or Con A {data not shown) for 4 days the predominant cells were in the granulocytic series as determined by Giemsa-stained differential counts. Increases in blast cells were significant, and thymidine incorporation was predominantly in differentiated granulocytes and blast cells, as confirmed by autoradiography. The data shown in table 2 were obtained from a single bone marrow. Similar results were observed in three studies.

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17 19 21 23

NO.

Fig. 5. Response of bone marrow cells from two subjects (A and B). Cells were separated by velocity sedimentation and tested for reactivity with PHA (A), Con A (A), and in MLC (©). Results are expressed as cpm minus background of [3H]thymidine incorporation. Background radioactivity in each fraction is also shown (× ×).

88 TABLE 2 Differential counts (%) of large-cell fractions from a bone marrow separation before and after incubation with PHA. Cell type

Fraction number 4

6

8

10

12

Before incubation

Segmented PMN Bands Myelocytes Promyelocytes Blasts Monocytes Lymphocytes Eosinophils Other

53 14 25 2 1 0 3 2 0

53 24 19 2 0 0 1 1 0

58 16 21 1 2 0 1 1 0

36 14 39 3 2 0 4 2 0

33 15 42 0 * 3 0 5 0 2

After incubation

Segmented PMN Bands Myelocytes Promyelocytes Blasts Monocytes Lymphocytes Eosinophils Other

7 17 43 21 6 0 0 1 5

11 26 41 4 13 0 3 0 2

7 14 55 9 8 0 2 0 5

8 23 45 9 7 0 2 0 6

9 20 50 10 3 0 1 1 5

* < 0.5%.

M i x t u r e s o f f r a c t i o n a t e d b o n e m a r r o w cells as responders to lectins and in MLC F r o m t h e p r e v i o u s s t u d i e s w i t h d e n s i t y s e p a r a t i o n s o f cells (Wells e t al., 1 9 7 7 ) i t w a s a p p a r e n t t h a t c e r t a i n p h e n o m e n a w e r e d e m o n s t r a b l e in i s o l a t e d b o n e m a r r o w f r a c t i o n s w h i c h w e r e o b s c u r e in u n f r a c t i o n a t e d p o p u l a t i o n s . I n contrast, nucleated cells from peripheral blood behaved similarly whether t e s t s w e r e d o n e o n s t a r t i n g m a t e r i a l o r o n s e p a r a t e d cells. T h e r e f o r e , w e e x a m i n e d c e l l u l a r i n t e r a c t i o n s in m a r r o w b y c o m b i n i n g s e p a r a t e d f r a c t i o n s . Table 3 documents some of these observations. Fractions 6 and 8 (large myel o i d c e l l s ) a n d f r a c t i o n s 2 1 , 2 3 , a n d 2 5 ( s m a l l l y m p h o i d cells) e a c h i n d e p e n d e n t l y d e m o n s t r a t e d e n h a n c e d [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n in r e s p o n s e t o C o n A , P H A , a n d in M L C . W h e n t h e l a r g e - a n d s m a l l - c e l l f r a c t i o n s w e r e c o m b i n e d ( d o u b l i n g t h e n u m b e r o f c e l l s ) , b a c k g r o u n d r a d i o a c t i v i t y in t h e a b s e n c e o f s t i m u l u s w a s a p p r o x i m a t e l y a d d i t i v e , as w a s t h e r e s p o n s e t o P H A . H o w e v e r , the responses to Con A were strikingly reduced on mixing fractions 4 with 2 1 , 6 w i t h 2 3 , a n d 8 w i t h 2 5 . T h e r e s p o n s e in M L C w a s r e d u c e d o n m i x i n g f r a c t i o n s 6 w i t h 2 3 a n d 8 w i t h 2 5 . T h e r e s u l t s s h o w n in t a b l e 3 w e r e

89 TABLE 3 R e s p o n s e o f isolated a n d c o m b i n e d b o n e m a r r o w f r a c t i o n s to lectins and in MLC. Bone marrow fraction

Unstimulated control (cpm)

PHA (cpm)

Con A (cpm)

MLC (cpm)

4 21 4 + 21 e x p e c t e d observed

18418 86 18504 25717

18989 18110 37099 31103

35372 30226 65598 18275

28324 2259 30583 26928

6 23 6 + 23 e x p e c t e d observed

9257 55 9312 13981

16834 20891 37725 39025

27872 17531 45403 14506

21337 7964 29301 5869

8 25 8 + 25 e x p e c t e d observed

9263 88 9351 20431

11244 9664 20908 29286

15069 4143 19212 9719

19950 809 20759 7141

obtained from a single bone marrow preparation. Similar results were observed in three independent experiments. Fractionated peripheral blood cells as stimulators in MLC

Separated peripheral blood cells were used as stimulators of normal allogeneic lymphocytes. Figs. 6A and B show the results of two studies 8"

7-

30'

6-

25,

520,

4'o

3-

~ 10-

1-

5"

0.-

-2

; ; 1~ 1'3 1'5 ;7 19 2'1 2'3 h FRACTION

NO.

FRACTION

NO.

Fig. 6. Peripheral b l o o d cells f r o m t w o s u b j e c t s (A a n d B) as s t i m u l a t o r s o f MLC reactions, t e s t e d against t w o p r e p a r a t i o n s o f allogeneic l y m p h o c y t e s (o • and o ©). Results are e x p r e s s e d as c p m m i n u s b a c k g r o u n d . Negative p o i n t s i n d i c a t e activity b e l o w b a c k g r o u n d .

90

4035" 30' 25. x

20. 15,

u

10.

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5. 0.

.

.

.

.

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~ 1 ; 1'1 1'3 I'5 1"7 119 211 ~3 FRACTION

NO.

Fig. 7. F r a c t i o n a t e d i r r a d i a t e d b o n e m a r r o w cells f r o m a single s u b j e c t as s t i m u l a t o r s of MLC r e a c t i o n s , tested against t w o p r e p a r a t i o n s of allogeneic l y m p h o c y t e s (e -e and ©-©).

giving results similar to those observed in 5 separate experiments. As shown in fig. 6B the MLC response to the separated small mononuclear cells was bimodal when tested against two allogeneic lymphoid responder populations. In fig. 6A the distribution of stimulator lymphocytes was bimodal when tested against one allogeneic population and had a single peak when tested against another. F r a c t i o n a t e d b o n e m a r r o w cells as s t i m u l a t o r s in M L C

Cells acting as stimulators in MLC reaction against allogeneic lymphoid cells were found throughout a broad portion of the gradient in bone marrow separations. Fig. 7 shows a profile from a representative separation from a single subject. The predominant activity was found in more slowly sedimenting cells (fractions 10 to 23). DISCUSSION

We have used the technique of velocity sedimentation in a zonal rotor to fractionate human peripheral blood and bone marrow into subpopulations that are functionally distinct. The design of the gradient gives uniform osmolarity with a sufficient slope of decreasing density to allow reorientation without resultant mixing but is sufficiently shallow t h a t the more rapidly moving cells are not seriously retarded as they move through the gradient. Relatively concentrated cell suspensions can be dynamically layered onto this gradient using the reorienting gradient zonal rotor without cell clumping and with an acceptable a m o u n t of starting-zone widening.

91 The usefulness of any cell separation technique is dependent upon its reproducibility and ease of application. Conventional cell separations in a high g field are difficult due to the high sedimentation coefficient of whole cells and interaction of cells with the walls of the centrifuge tube. In the described system, the sample is loaded while the rotor is running at an accurately measured and stable speed; and since centrifugation time can be carefully monitored, the exact force × time (w2t) values can be reproduced. Due to the design of the rotor, wall effects are eliminated. The principal observations derived from the initial application of this technique were these: 1) Human myeloid stem cells (CFU-C) in both blood and bone marrow are heterogeneous with respect to sedimentation velocity. 2) Small lymphoid cells responding to PHA, Con A, and in an MLC reaction show a large range of sedimentation rates and are congruent. 3) Cells with widely differing sedimentation velocities are capable of acting as stimulators in MLC reactions. 4) A heterogeneous fraction of rapidly sedimenting nonlymphoid cells in the bone marrow responds to Con A by increased [3H]thymidine incorporation; the same cell population responds to a lesser extent in MLC reactions and variably to PHA. 5) Marrow cell populations of large (myeloid) and small (lymphoid) cells which individually manifest a proliferative response to Con A and in MLC show a markedly reduced response when added together. The observed profiles of human CFU-C in blood and marrow are consistent with the reported descriptions of heterogeneous populations of CFU-C in the mouse and m o n k e y (Worton et al., 1969; Williams and Moore, 1973; Metcalf and MacDonald, 1975). This heterogeneity may reflect cells at different stages of the replicative cycle (Van den Engh et al., 1976) or the resolution of cell populations with differing degrees of maturation, as suggested in previous work (Metcalf et al., 1974; Metcalf and MacDonald, 1975). Conceivably, the heterogeneity might also reflect the interactions of potentiating cells such as macrophages of myelocytes, or suppressive cells such as mature granulocytes. Our profiles, which show a second peak o f CFU-C activity about 6 to 8 fractions ahead of the main CFU-C peak, support the idea of cells in different stages of the cell cycle, since this degree of separation indicates a twofold range of cell sizes (sedimentation coefficients of I : 1.6) (MacDonald and Miller, 1970). The observation of proliferative response of large, predominantly myeloid cells to plant lectins and in mixed leukocyte culture was unexpected. Several explanations were considered: 1) The response is due to lymphoid cells that had aggregated into large clumps that sedimented with the larger cells; 2) the response is due to stimulation by lectin and allogeneic l y m p h o c y t e s of a population of cells producing colony-stimulating factors with resultant proliferation of myeloid progenitors; 3) the response is a direct effect of lectins and allogeneic l y m p h o c y t e s on large myeloid precursor cells. Few lymphoid cells were observed at the large-cell end of the gradient, either initially or after four days of incubation with PHA or Con A. After

92 such incubations the predominant cells were myelocytes and mature granulocytes. Increases in blast cells were also significant. These observations suggest that myeloid rather than lymphoid cells account for the observed enhanced responses. The possibility that the proliferative response to lectins and in MLC of large myeloid cells is due to production of colony-stimulating activity is currently under investigation. Lectins have been generally utilized as mitogenic stimulants of lymphoid cell subpopulations, and only small amounts of published data suggest a greater spectrum of activity. Dicke and his colleagues (1976) recently reported that PHA stimulates colony formation by marrow cells from patients with acute myeloblastic leukemia. This observation suggests a PHA effect on primitive myeloid cells. Our data also suggest that certain plant lectins and irradiated allogeneic l y m p h o c y t e s may stimulate the proliferation of nonlymphoid hematopoietic cells. Both unfractionated and fractionated peripheral blood samples responded predictably to PHA, Con A and in MLC with increased thymidine uptake. In contrast, our previous studies demonstrated that incubation of unfractionated marrow cells with Con A resulted in isotope incorporation below that of controls and demonstrated a variable reduction with PHA and in MLC (Wells et al., 1977). When marrow samples were separated by velocity sedimentation, two subpopulations of cells showed enhanced [3H]thymidine incorporation in response to Con A and in MLC. These fractions were found at both the large- and the small-cell ends of the gradient. Combination of these separated cell fractions gave reduced responses similar to the starting material and to fractions of cells separated b y density (Wells et al., 1977). We presume that the diminished isotope incorporation reflects decreased DNA synthesis, although alternative mechanisms have not been excluded. In our previous report we have discussed the possibility that reduction in isotope incorporation below background might be due to triggering of cytotoxic bone marrow cells b y Con A (Wells et al., 1977). Shou et al. (1976) have described a p h e n o m e n o n whereby Con A-treated human peripheral blood mononuclear cells inhibit thymidine incorporation b y either autologous or allogeneic lymphoid cells stimulated by a variety of mitogens, including Con A. Our observations on cells separated b y size suggest that a large-cell subpopulation and a small-cell subpopulation must be present before inhibition occurs. ACKNOWLEDGEMENTS The authors are grateful for the skillful technical assistance of Ann Sullivan and Mary Willcox-Langley. We also wish to thank Judy Lesch, Mark Hermes, Madeleine Winberg, and Larry Cardman for their excellent technical assistance in performing in vitro transformation studies. REFERENCES Dicke, K.A., G. Spitzer and M.J. Ahearn, 1976, Nature 259,129. Golde, D.W. and M.J. Cline, 1972, J. Clin. Invest. 51, 2981.

93 MacDonald, H.R. and R.G. Miller, 1970, Biophysics 10,834. Metcalf, D. and H.R. MacDonald, 1975, J. Cell. Physiol. 85,643. Metcalf, D., J. Parker, H.M. Chester and P.W. Kincade, 1974, J. Cell. Physiol. 84, 275. Pike, B.L., and W.A. Robinson, 1970, J. Cell. Physiol. 76, 77. Pretlow, T.G., II, E.E. Weir and J.G. Zettergren, 1975, Int. Rev. Exp. Pathol. 14, 91. Shortman, K., 1972, Ann. Rev. Biophys. Bioeng. 1, 93. Shou, L., S.A. Schwartz and R.A. Good, 1976, J. Exp. Med. 143, 1100. Van den Engh, G.J., A.H. Mulder and N.T. Williams, 1976, Abstract, 5th Annual Meeting of the Society of Experimental Hematology, August, 1976, p. 136. Wells, J.R. and T.W. James, 1972, Exp. Cell. Res. 75,465. Wells, J.R., P. Sheeler and D.M. Gross, 1972, Anal. Biochem. 46, 7. Wells, J.R., G. Opelz and M.J. Cline, 1977. J. Immunol. Methods 17, xx--xxx. Willcox, M.B., D.W. Golde and M.J. Cline, 1976, J. Histochem. Cytochem. 21, 979. Williams, N. and M. Moore, 1973, J. Cell. Physiol. 82, 81. Worton, R.G., E.A. McCulloch and J.E. Till, 1969, J. Exp. Med. 130, 91.