CELLULAR

IMMUNOLOGY

26, 105-l

13 (1976)

E Rosette Formation at 37°C: A Property of Mitogen-Stimulated Human Peripheral Blood Lymphocytes BARRY M. BERGER, RICHARD AND

Department

M. SCHUMAN,

PETER

of Pathology, School Philadelphia, Received

C.

of Medicine,

Pennsylvania April

RONALD

P. DANIELE,

NOWELL

University 19174

of Pennsylvaka,

5,1976

Peripheral blood lymphocytes from healthy humans formed stable E rosettes with sheep erythrocytes (SRBC) at 37°C after culture with phytohemagglutinin or the divalent cation ionophore A23187. Cells manifesting this phenomenon exhibited “blast” morphology, appeared by 16 hr of culture, increased dramatically in percentage and absolute number by 62 hr, and persisted in large numbers for the duration of culture (182 hr). Unstimulated lymphocytes formed rosettes at 4°C but not at 37°C. Increased “stickiness” due to surface-bound lectin mitogen was not the cause of rosette formation at 37°C. Formation of E rosettes at 37°C has previously been considered a property of lymphocytes less differentiated than the circulating T cell (e.g., thymocytes, leukemic lymphoblasts). The present findings indicate that this property can be “reexpressed” during blastogenesis in culture. This observation also demonstrates technical problems associated with the use of SRBC to qua&ate lymphocytes with complement receptors (B cells) by the EAC rosette assay in culture. False positives resulted from 37°C E rosette formation, but this was overcome by replacing the SRBC with guinea pig erythrocytes in the EAC assay.

INTRODUCTION The formation of rosettes with sheep erythrocytes by human peripheral blood lymphocytes (E rosettes) has been used extensively as a marker for human T lymphocytes (1, ‘2, 8). E rosette formation is temperature dependent and occurs optimally for human lymphocytes between the temperatures of 6 and 24°C during a I-hr incubation (3) or at 4°C for an overnight incubation (4). Human circulating T cells will not form E rosettes if the entire procedure is carried out at 37°C (3, 5) ; however, most lymphocytes isolated from the human thymus will do so (3, 5, 6), as will lymphoblasts from the blood and bone marrow of patients with acute lymphoblastic leukemia of the T cell type (5). We report here that the property of E rosette formation at 37°C is also shared by transformed lymphocytes in cultures of normal human peripheral blood stimulated with either phytohemagglutinin (PHA) or the calcium ionophore, A23187. This observation is of interest in considering normal T cell differentiation; it also exposes technical problems associated with the use of sheep erythrocytes to define surface markers of either T cells or B cells in lymphocyte cultures. 105 Copyright All rights

1976 by AcademicPress,Inc.

o3 reproduction

in any form reserved.

106

HERGER

MATERIALS

ET

AND

AL.

METHODS

Isolation of lymphocytes. Blood was obtained from healthy human donors, nonlaboratory personnel, ranging in age from 20-28 years old. Two hundred milliliters of heparinized blood was placed into a SOO-ml bottle containing 1.0 g of carbonyliron suspended in 40 ml of Ca’+-Mg?+-free Hank’s balanced salt solution (HBSS) and incubated in a 37°C water bath with moderate shaking for 30 min. Two volumes of the same HBSS was then added and the diluted blood dispensed into 40-ml glass conical centrifuge tubes, underlayered with 10 ml of Ficoll-Hypaque, and centrifuged for 40 min at 20°C with 400g at the interface. The lymphocyterich layers were harvested, pooled, and centrifuged at 5OOg for 15 min at 20°C. The lymphocytes were then washed with a hypotonic EDTA solution (7) to lyse residual red cells, and twice with Ca2+-Mg”+-free HBSS for 15 min at 8Og at 20°C. The final pellet was resuspended in Ca”+-Mg’+-free HBSS and adjusted to 5 X 10” cells/ml, the concentration used for cell surface marker assays. The above procedure resulted in a lymphocyte suspension containing less than 3.0% monocytes, no red blood cells, and usually less than one platelet per lymphocyte. Forty to fifty percent of the lymphocytes in the initial blood sample could be recovered without any detectable selective loss of lymphocyte subpopulations. Culture conditions. The lymphocytes were cultured as previously described (8) and stimulated with either 50 pg/ml of PHA-M (Difco) or 2.0 x lo9 mol/ml of A23187 (gift from Eli Lilly Co.). Proliferation was measured at various times (38, 62, 86, 134, 182 hr) by adding 0.25 #Zi of [“Hlthymidine to each of three replicate cultures. These were harvested 16 hr later onto glass fiber filters using a multiple cell harvester (model M 12V, Biomedical Research Institute, Rockville, Md.), washed with distilled water, dried overnight, and counted by standard scintillation techniques. Harvesting cultured lymphocytes. For the cell surface marker assay, 15 cultures with each mitogen were harvested at each time period by adding 0.1 ml of 50 mM EDTA to each culture, resuspending the cells, and pooling the replicates. Cell survival was determined by counting a 0.4-ml sample of each pool in a Coulter particle counter (Coulter Electronics, Hialeah, Fla.). Viability was assayed by tryphan blue exclusion and the number of viable cells per culture was calculated. The pooled cells were then spun down and resuspended at 5 X lo6 cells/ml in HBSS for use in the surface marker assays. Preparation of lymphocyte sw+zce murker indicator cells. Fresh sheep erythrocytes (SRBC) for detecting T cells were prepared from SRBC preserved in Alsever’s solution (received fresh weekly from University of Pennsylvania School of Veterinary Medicine). The SRBC were washed twice with phosphate-buffered saline (pH 7.2) and once with HBSS. A 0.57 o suspension was prepared and used on the same day for the E rosette assays. Erythrocytes coated with antibody and complement (EAC indicator cells), used for detecting cells bearing complement receptors (B cells) were prepared by standard technique (9) either from SRBC or from guinea pig erythrocytes (GPRBC), similarly processed. Polyvalent anti-sheep hemolysin (Gibco) or polyvalent antiGBRBC serum (Cappel Labs, Downingtown, Pa.) was used. Assay of cell surface markers. The standard assay, at 4”C, for E rosettes was carried out using 50 ~1 of lymphocytes at 5 X 10’ cells/ml and 50A of 0.5% E indicator cells as previously described (8) with the following modifications : Ficoll

E ROSETTE

FORMATION

AT

37°C

107

(10) to a final concentration of 1.8 mg/ml was added to each assay instead of human serum and the assays were spun at 80g for 5 min at 10°C incubated for 1 hr at room temperature and then at 4°C overnight. After equilibrating to room temperature and staining with toluidine blue, the rosettes were enumerated on a hemocytometer. Assays were done in triplicate and at least 300 lymphocytes were scored per assay. Lymphocytes binding more than three erythrocytes were counted as rosettes (Fig. 1A). To assay E rosettes at 37°C 50 pl of lymphocytes at 5 X lo6 cells/ml and 50 ~1 of 0.5% E indicator cells were mixed, incubated for 5 min, centrifuged at 1009 for 5 min, and then immediately resuspended, stained, and scored as were the 4°C E rosettes (Fig. 1B). The temperature was maintained at 37°C during the entire procedure. Complement receptor-bearing lymphocytes were assayed using EAC indicator cells as previously reported (Daniele and Rowlands). Fifty microliters of lymphocytes in suspension at 5 x lo6 cells/ml and 20 pl of dithiothreitol 1.0 X 10m3M were mixed and incubated for 5 min followed by the addition of 50 ~1 of EAC indicator cells, preequilibrated to 37”C, for another 5-min incubation. The suspension was centrifuged at 100s for 5 min and then immediately resuspended, stained, and scored as were the E rosettes (Fig. 1C). The temperature was scrupulously maintained at 37°C during the entire procedure. The average percentage of lymphocytes from eight normal donors forming EAC rosettes showed no significant difference between SRBC and GPRBC: EAC-SRBC 18.6%. + 3.0; EAC-GPRBC 17.3% + 3.1. RESULTS Proliferation

in Mitogen-Stimulated

Cultures

Proliferation in the PHA cultures characteristically peaked at 72 hr (3O,OOO50,000 dpm) with a rapid decline thereafter. The total number of cells in each culture decreased by 30% in the first 38 hr, increased to 120 hr, with the maximum exceeding the initial innoculum by approximately 40%, and gradually decreased for the remainder of the culture period (182 hr). Proliferation in the A23187-stimulated cultures was similar to that in the PHA cultures except that the peak response in the A23187 cultures was slightly higher (60,000-70,000 dpm) and occurred at 96-120 hr. The total number of cells in the A23187 cultures decreased 35% in the first 38 hr, remained constant up to 134 hr, and declined gradually thereafter. Viability of the intact cells harvested at each point was always greater than 98% in both the PHA and A23187 cultures up to 182 hr of culture. E Rosette Formation

by Mitogen-Stimulated

Lymphocytes

The fraction and absolute number of E rosettes from two typical experiments are illustrated in Table 1. Similar data were obtained from six other experiments which did not include all of the time periods indicated in Table 1. The percentage of lymphocytes forming E rosettes at 4”C, prior to the addition of either PHA or was within the normal range for this laboratory (55-820/a). A23187 (5872%)> The percentage of E rosettes at 4°C after the addition of either PHA or A23187,

108

RER(;ER

ET

AL.

Fro. 1. “Transformed” human lymphocytes cultured with A23187 for 5 days. forming E rosettes with sheep erythrocytes at (a) 4°C and (b) 37°C and (c) with complement-coated sheep erythrocytes (EAC rosettes) at 37°C. Figure la is an autoradiograph, labeled with [‘Hlthymidine, which demonstrates DNA synthesis in one of the rosetted cells. Stained with Diff-Quick, Harleco. X 1900.

. remained relatively constant (mean, 63% ; range, 41-75s) for the duration of culture. The percentage of cells forming E rosettes at 37”C, however, increased from low prkmitogen levels ( < 2%) to an average level of 36% (range 34-3851) in the A23187 cultures and to 4970 (range 46-51%) in the PHA cultures by 62 hr

E ROSETTE

FORMATION

AT

37°C

109

of culture. During this period, the calculated absolute number of cells forming 37°C E rosettes also increased in both the PHA and ionophore cultures. After 6.2 hr, the percentage of 37°C E rosettes remained relatively constant with both mitogens, but the absolute number continued to rise in the PHA cultures until 86-134 hr. In order to further define the time course of this phenomenon, the frequency of 37°C E rosette formation at 3 and 16 hr after exposure to A23187 was assayed in additional experiments. These revealed no increase at 3 hr above the premitogen level (0.5%) ; by 16 hr, however, values had increased to lO-20%. Morphology

and Properties of 37°C E Rosettes

The E rosettes formed at 37°C with cells from mitogen-stimulated cultures were usually large and compact with more than 15 sheep erythrocytes (SRBC) per rosette. In wet preparations, the rosetting cell was usually large (15-20 pm) and in stained smears the morphology was characteristically that of a transformed “blast” (Fig. lb), Some preliminary studies were done to examine certain properties of 37°C E rosettes. It was found that their formation was inhibited by sodium azide and by anti-human lymphocyte globulin, which also inhibit 4°C rosettes formation (11). However, while 37°C rosettes were stable for over 4 hr at 37”C, 4°C rosettes (formed by unstimulated lymphocytes) were not stable for more than 1 hr at 37°C. E Rosettes Forwed by Nonspecific ‘Sticking” Table 1 also indicates that the percentage of 37°C rosettes was consistently higher in the PHA cultures as compared to the ionophore cultures, although the percentTABLE Surface Ept L A23187

Before Mitogen

of Cultured Time After

T = 0 hours

\ rosettedC TOU.I

Markers

mettedd

2

Total rosetred

72 363

Lymphocytes:

E Rosettes

Nitogen Addition

38 hours

62

hours

86

hours

52 160

67 207

58 105

67 188

34 113

58

9

292

30

69 233

34 116

71 243

28 93

65 229

219

61 212

51 230

64 288

67 455

7 20

65 194

46 137

68 244

45 231

1

11

2

2

10

2 1

11

72 362

2

58

2

10

292

% wsetted

1

134 hours

63

34

210

96

182 hours

75 212

42 132

55 172

33 109

67 225

38 106

66 184

58 397

50 357

71 507

48 380

59 459

62 317

45 288

41 265

41 189

53 242

PKA

% rosetted Total

roserted

% rasetted

Total rosetted

% rosettes

63

formed entirely

at 37%.

bE rosettes incub’ated at mm temperature incubated at 4OC overnight. %rcenrage

of cells/culture

dTota1 number of cells/culture

for one hour and

forming rosettes. forming iosettes

x 10t3.

110

BERGER

ET

AL.

age of cells rosetting at 4°C was similar with both mitogens. This may have been due in part to surface bound PHA causing nonspecific sticking of SRBC at 37°C and therefore a proportion of “false positive” rosettes. To investigate this possible artifact, lymphocytes were incubated with PHA or A23187, at the concentrations used for culture, for 1 hr at 37°C in 5% COs-humid air followed by two washes with Ca2+-Mg” free HBSS. These cells were resuspended in HBSS and were assayed for 37°C E rosette formation. As a further test for nonspecific stickiness, guinea pig erythrocytes, which do not bind human lymphocytes at any temperature (14)) were also used as 37°C E rosette indicator cells. Lymphocytes incubated with A23187 formed very few rosettes with either type of erythrocyte ( < 2%) ; 3% of lymphocytes incubated with PHA rosetted with guinea pig erythrocytes and 10% rosetted with sheep erythrocytes. Although these ‘data indicate that a minority of the rosettes formed at 37°C with PHAtransformed lymphocytes may reflect artifactual binding of erythrocytes due to the surface-bound PHA, this does not appear to be a significant problem with A23187transfomred lymphocytes. False Positive EAC Rosettes An additional consequence of 37°C E rosette formation by stimulated lymphocytes was interference with the quantitation of cells bearing complement receptors. Lymphocytes bearing complement receptors (B cells) are assayed by their ability to form rosettes with sheep erythrocytes coated with antibody and complement (EAC rosettes) at 37°C (12, 13). The data in Table 2 show an increase in the percentage and number of cells forming EAC rosettes in both experiments 1 and 2 which parallels the increase in cells forming E rosettes at 37°C. By 62 hr, in both the A23187 and PHA cultures, the percentage of EAC rosettes (mean, 4.5% ; range, 2565%) was almost identical to the percentage of cells forming 37°C E rosettes (mean, 42%; range 34-51%), and these levels were maintained throughout the remainder of the culture period. TABLE Surface

Markers

of Cultured

2 Lymphocytes

: EAC

Rosettes

E ROSETTE

FORMATION

AT

111

37°C

500

67 0 x

400

2 3 tJ 2 u

300

cc W a *

200

5 W

U LL 0 100 El m 2 !2

EAC%PRB‘

0

I 24

TIME

w-D-I 48

48

H-4.

iyI 72

-b I 96

96

IN CULTURE

I 120

I 144

120 144

I 168

(hours)

FIG. 2. Summary of the interrelationships among proliferation, cell survival, and cell surface markers as functions of time for experiment 2 in Tables 1 and 2, with A23187 as the mitogen. The peak of proliferation (DPM) is unusually late, but otherwise the experiment is typical. The total cells per culture fell moderately over the first 38 hr and remained relatively constant thereafter. The cells forming E rosettes at 4°C (E-4°C) followed a similar time course. In contrast, the cells forming E rosettes at 37°C (E-37°C) or EAC rosettes with sheep erythrocytes (EAC-SRBC) nearly tripled, from low initial levels, during the first 62 hr and then remained constant. Cells forming EAC rosettes with guinea pig erythrocytes (EAC-GPRBC) , which initially equaled the EAC-SRBC, decreased to almost zero by 38 hr and remained very low thereafter. These presumably represent the “true” EAC rosette values. To further define the interaction between the mitogen-stimulated lymphocytes and the EAC indicator cells, sheep erythrocytes were coated with antibody alone (EA cells). It was found that the percentage of cells forming rosettes with EA cells corresponded closely to the percentage forming both EAC rosettes and 37°C E rosettes. In order to detect the true magnitude of the complement receptor-bearing population, guinea pig erythrocytes (GPRBC), which will not form E rosettes with stimulated (unpublished observation) or unstimulated human lymphocytes at any temperature, were coated with antibody and complement for use as EAC indicator cells. These were equally as sensitive as sheep erythrocytes in detecting complement receptor-bearing lymphocytes before culture (see Materials and Methods). The data in Table 2 show that the percentage of cells with complement receptors, as detected by the guinea pig erythrocytes, decreased in both experiments from lO11% before mitogen addition, to l.O-2.0% after 38 hr of culture with either A23187

112

RERGER

ET

AL.

or PHA, and was maintained at a low level (0.3-4s j thereafter. These data contrasted sharply with the markedly increased levels of rosettes formed by coated sheep erythrocytes and presumably represent a measure of the true frequency of complement receptor-bearing cells (B cells) in culture. Figure 2 summarizes, for a typical experiment, the interrelationships existing among proliferation, number of cells per culture, and cell surface markers, as functions of time in culture with A23187 as the mitogen. The data in Fig. 2 were taken from experiment 2 in Tables 1 and 2. DISCUSSION The present data indicate that human circulating lymphocytes, after being cultured in the presence of PHA or a divalent cation ionophore, A23187, will form E rosettes at 37°C a property not manifested by unstimulated cells, although the latter do rosette at 4°C. Morphologically, the cells forming E rosettes at 37°C are “transformed” lymphocytes. In culture, the time of appearance of cells rosetting at 37°C was similar for both mitogens, but varied somewhat with the lymphocyte donor. Studies are under way to determine more precisely the relationship between the time course of this phenomenon and other early indicators of blastogenesis such as altered movement of cations across the plasma membrane ( 15)) enhanced phospholipid turnover ( 16)) and increased RNA synthesis ( 17). The exact structure of the sheep erythrocyte “receptor” in the formation of E rosettes, at either 4 or 37”C, is presently unknown. Consequently, it is difficult to speculate whether a different receptor is being “unmasked” in the transformed lymphocytes or whether the receptor normally involved in forming rosettes at 4°C is increased in quantity (21) or density or modified in structure. It has been suggested that E rosette formation at 37°C is a property of a T cell which is less differentiated than the small circulating T lymphocyte (5, 18). This view is based on the observation that E rosette formation at 37°C occurs with a majority of lymphocytes from the thymuses of children (3, 5, 6) and also with lymphoblasts from the peripheral blood and bone marrow of some patients with acute lymphocytic leukemia (5). The present data indicate that if this property does relate to T cell differentiation, it is not irreversibly lost in the “mature” circulating T cell, but can be reexpressed in association with mitogen-induced blast transformation. It is not known if daughter cells, “redifferentiating” in culture, again lose the ability to form E rosettes at 37°C. Along with providing some insight into human T cell differentiation, the present observation demonstrates significant technical problems for investigators studying changes in cell surface markers of lymphocyte populations in mitogen-stimulated cultures and perhaps in antigen-stimulated cultures as well. The data indicate that the sheep erythrocyte (SRBC) should not be used in such cultures as a complement carrier in the EAC rosette assay to quantitate complement receptor-bearing cells (B cells). In addition to true EAC rosettes, false positive “EAC” rosettes will be formed by transformed T cells binding directly to the SRBC under the 37°C assay conditions. Accurate quantitation of B cells thus becomes impossible as the true and false EAC rosettes are indistinguishable with the light microscope. Fortunately, the problem can be overcome by using guinea pig erythrocytes (GPRBC) as the complement carrier instead of SRBC. Guinea pig erythrocytes

E

ROSETTE

FORMATION

AT

37°C

113

will not bind directly to either unstimulated or transformed human lymphocytes and, when coated with complement, are equally as sensitive as SRBC in detecting cells bearing a complement receptor. A second technical problem involves rosette formation due to nonspecific lymphocyte “stickiness” caused by lectin mitogens bound to the cell membrane. This was a minor source of false positive rosettes in PHA cultures during the first 24 hr, but did not occur after exposure to A23187. After recognition of these various technical problems, the changes in the size of the T and B populations, as demonstrated by rosette formation, in cultures stimulated with A23187 suggest that for human peripheral blood lymphocytes, this ionophore is primarily mitogenic for T cells. This view is also supported by preliminary studies in our laboratory of purified human T cell populations and of lymphocytes from both B cell and T cell chronic lymphocytic leukemia in which the responses to PHA and to A23187 have been consistently parallel. This work is preliminary, however, and other reports with human cells as well as other species (19, 20) have been conflicting as to whether the calcium ionophore may effectively stimulate B cells also. Resolution of this question with respect to man is of particular interest in view of the present lack of a potent mitogen for human B cells. ACKNOWLEDGMENTS This investigation was supported by USPHS Grants No. CA-12779, No. CA-15822, and No. CA-05022. Dr. Daniele is the recipient of a Pulmonary Young Investigator Award from the National Heart and Lung Institute (HL 17221).

REFERENCES 1. Wybran, J., Carr, C., and Fudenberg, H. H., J. Clzlz. Znzx~t. 51, 2537, 1972. 2. Jondal, M., Helm, G., and Wigzell, H., J. Exp. Med. 136, 207, 1972. 3. Mendes, M. F., Tolnai, M. E. A., Silveira, N. P. A., Gilbertsen, R. B., and Metzgar, R. S., J. Zmmunol. 111, 860, 1973 4. Mahowald, M. L., Peters, T., Handwerger, B., Caperton, E. M., Jr., and Douglas, S. D., Cl&z. Res. 23, 493A, October 1975. 5. Borella, L., and Sen, L., J. Zmmunol. 114, 187, 1975. 6. Galili, U., and Schlesinger, M., J. Zmmunol. 115, 827, 1975. 7. Severson, C. D., Greazel, N. A., and Thompson, J. S., J. Zmmunol. Metlz. 4, 369, 1974. 8. Nowell, P. C., Daniele, R P., and Winger, L. A., J. Reticzlloendo. Sot. 17, 47, 1975. 9. Daniele, R. P., and Rowlands, D. T., Jr., Transplantation 17, 126, 1974. 10. Pattengale, P. K., and Reichelderfer, P. S., Imm.unoZ. Commun. 4, 179, 1975. 11. Bentwich, Z., Douglas, S. D., Siegal, F. P., and Kunkel, H. G., Clin. Zmmunol. Immunopathol. 1, 511, 1973. 12. Nussensweig, V., Bianco, C., Dukor, P., and Eden, A., In “Progress in Immunology” (B. Amos, Ed.), p. 73. Academic Press, New York, 1971. 13. Silveira, N. P. A., Mendes, N. F., and Tolnai, M. E. A., J. Zmmunol. 108, 1456, 1972. 14. Lay, W. H., Mendes, N. F., Bianco, C., and Nussenzweig, V., Nature (London) 230, 531, 1971. 15. Greaves, M., and Janossy, G., Transplant. Rev. 11, 87, 1972. 16. Fisher, D. B., and Mueller, G. C., Biochim. Biophys. Acfa 248, 434, 1971. 17. Cooper, H. L., Transplant. Rev. 11, 1, 1972. 18. Gelfand, E. W., and Chechnik, B. E., N. Engl. J. Med. 294, 275, 1976. 19. Maino, V. C., Green, N. M., and Crumpton, M. J., Nature (London) 251, 324, 1974. 20. Hovi, T., Allison, A. C., and Williams, S. C., Exp. Cell Res. 96, 92, 1976. 21. Owen, F. L., and Fanger, M. W., J. Immunol. 115, 765, 1975.