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Enhanced E- and EAC-rosette formation by neuraminidase
Journal o f Immunological Me thods, 12 ( 1976) 253--260 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
253
E N H A N C E D E- A N D E A C - R O S E T T E F O R M A T I O N B Y N E U R A M I N I D A S E
TIN HAN and JUN MINOWADA Departments o f Medicine B and Immunology and Immunochemistry Research, Roswell Park Memorial Institute, New York State Department o f Health, Buffalo, New York, 14263
(Received 10 December 1975, accepted 29 March 1976)
Pre-treatment by neuraminidase of lymphocytes obtained from peripheral blood of normal donors significantly enhanced E- and EAC-rosette formation. Of other lymphoid cells only spleen cells showed significant enhancement of E-rosettes. The EAC-rosettes slightly increased when the peripheral blood lymphocytes from patients with acute lymphoblastic leukemia or chronic lymphocytic leukemia and MOLT-4 lymphoid cells were pre-treated with this enzyme. The EAC-rosettes were not increased by neuraminidase treatment of phytohemagglutinin-induced blasts, thymus cells or spleen cells. Pre-treatment of peripheral blood lymphocytes with neuraminidase also increased the proportion of stable E-rosettes resistant to incubation at 37°C and to vigorous shaking. Various concentrations of neuraminidase (1--100 U/ml) produced enhancement of E- and EAC-rosettes with the highest activity at 25 and 50 U/ml. Neuraminidase treatment of sheep red blood cells failed to increase the proportion of E-rosettes of peripheral blood lymphocytes. The increased rosette forming capacity induced by neuraminidase is probably related to changes in lymphocyte surface properties.
INTRODUCTION I t has b e e n k n o w n t h a t V i b r i o c h o l e r a e n e u r a m i n i d a s e ( V C N ) increases the i m m u n o g e n i c i t y a n d / o r a n t i g e n i c i t y o f a v a r i e t y o f t u m o r cells a n d l y m p h o i d cells in a n i m a l s ( S a n f o r d , 1 9 6 7 ; Currie a n d B a g s h a w e , 1 9 6 9 ; Bekesi e t al., 1 9 7 1 ; Schlesinger a n d A m o s , 1 9 7 1 ; S i m m o n s a n d Rios, 1 9 7 1 ; S i m m o n s et al., 1 9 7 1 ) , and the a n t i g e n i c i t y ( H L - A s y s t e m ) o f h u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s ( G r o t h a u s et al., 1 9 7 1 ) . This e n z y m e has also b e e n k n o w n t o increase t h e h u m a n m i x e d l y m p h o c y t e r e a c t i o n ( L u n d g r e n a n d S i m m o n s , 1 9 7 1 ; H a n , 1 9 7 2 ) , l y m p h o c y t e b l a s t o g e n i c r e s p o n s e t o antigens a n d m i t o gens ( H a n , 1 9 7 3 , 1 9 7 5 ) , a n d d e l a y e d skin t e s t r e s p o n s e s (Hart, 1 9 7 4 ) . H u m a n T l y m p h o c y t e s can be i d e n t i f i e d b y t h e i r ability t o f o r m r o s e t t e s w i t h u n s e n s i t i z e d s h e e p red b l o o d cells ( E - r o s e t t e s ) ( J o n d a l e t al., 1 9 7 2 ; Min o w a d a e t al., 1 9 7 2 ) , a n d b y the lack o f r e c e p t o r f o r i m m u n o g l o b u l i n or c o m p l e m e n t . H u m a n B l y m p h o c y t e s , on t h e o t h e r h a n d , can be i d e n t i f i e d b y t h e i r ability t o f o r m r o s e t t e s w i t h sensitized s h e e p red b l o o d cells (EACr o s e t t e s ) ( B i a n c o e t al., 1 9 7 0 ; J o n d a l e t al., 1 9 7 2 ) , a n d b y t h e p r e s e n c e o f r e c e p t o r s f o r a g g r e g a t e d i m m u n o g l o b u l i n ( D i c k l e r and K u n k e l , 1972}.
254 It has recently been reported that the VCN-treatment of either peripheral blood l y m p h o c y t e s or sheep red blood cells increased the proportion of regular E-rosettes formed at 4°C or 20°C and of stable E-rosettes resistant to prolonged incubation at 37°C and to vigorous shaking (Bentwich et al., 1973; Galli and Schlesinger, 1974; Weiner et al., 1973). The present study was designed to investigate the effect of VCN treatment on E- and EAC-rosette formation by various types of human lymphoid cells. MATERIALS AND METHODS Peripheral blood lymphocytes were prepared from heparinized venous blood of healthy donors and patients with acute lymphoblastic leukemia (ALL) or chronic l y m p h o c y t i c leukemia (CLL), by centrifugation over a Ficoll--Hypaque gradient (BCyum, 1968). This preparation usually contains more than 90% lymphocytes, the remaining being m o n o c y t e s and granulocytes. Thymus cells were prepared from a portion of t h y m u s obtained from non-malignant patients at the time of open-heart surgery, as previously described (Hart et al., 1976). Spleen cells were prepared from a portion of normal spleen obtained from patients with malignant l y m p h o m a (Stage I or II), at the time of laparotomy and splenectomy for staging of the disease. T and B l y m p h o i d cells were obtained from established cell lines (MOLT-4 and B411-4). L y m p h o c y t e suspensions were prepared at a concentration of 5 X 106/ml of phosphate buffered saline (PBS); sheep red blood cells and bovine red blood cells were adjusted to 1% and 2.5% (v/v), respectively. Twenty-five or 50 units of VCN (Calbiochem, Los Angeles, California) were added to lymphocyte suspensions or red blood cell suspensions, which were incubated at 37°C for 30 min. The cells were then washed once with PBS prior to E- or EAC-rosette assay. For E-rosette as a T-cell marker, 0.1 ml of the l y m p h o c y t e suspension at a concentration of 5 X 106/ml was mixed with 0.1 ml of 1% (v/v) sheep red blood cell suspension in a small test tube (7 X 50 mm). Mixed cell suspensions were usually centrifuged at 200 g for 3 min and were left undisturbed at 4°C for 2 h (Standard technique}. In some instances, the mixed cell suspensions were centrifuged at 200 g for 3 min and were left undisturbed at 20°C for 2 h or the mixed cell suspensions were left to settle by gravity, without centrifugation at 20°C for 2 h. A few drops of 0.1% trypan blue solution in PBS were then added to the tube and the cell pellet was gently resuspended, following which, a drop of the cell suspension was examined under a microscope at 500 X magnification. Each sample was tested in duplicate, and at least 200 cells were observed to determine the percentage of Erosettes. A positive E-rosette was defined as a l y m p h o c y t e to which three or more sheep red blood cells were attached. For EAC-rosettes as a B-cell marker, bovine red blood cells were used as the indicator cells since the unsensitized bovine red blood cells do n o t bind
255 w i t h e i t h e r T o r B l y m p h o c y t e s . B o v i n e r e d b l o o d cells w e r e f i r s t s e n s i t i z e d w i t h a p p r o p r i a t e d i l u t i o n o f r a b b i t a n t i b o v i n e e r y t h r o c y t e s e r u m ( a n IgM class a n t i b o d y ) a t 3 7 ° C f o r 3 0 m i n , a n d t h e cells w e r e w a s h e d t h r e e t i m e s w i t h PBS. O n e m l o f 2 , 5 % (v/v) s u s p e n s i o n o f t h e s e n s i t i z e d cells w a s i n c u b a t e d with 1.0 m l of 1 / 1 0 d i l u t i o n of fresh m o u s e s e r u m ( D B A / 2 Ha strain) as a c o m p l e m e n t s o u r c e a t 3 7 ° C f o r 3 0 m i n . T h e cells w e r e t h e n w a s h e d t h r e e t i m e s w i t h P B S a n d w e r e r e s u s p e n d e d a t 1% (v/v) s u s p e n s i o n in PBS f o r E A C - r o s e t t i n g . T h e p r o c e d u r e f o r E A C - r o s e t t i n g was g e n e r a l l y t h e s a m e as t h e s t a n d a r d t e c h n i q u e f o r E - r o s e t t i n g ; h o w e v e r , t h e m i x t u r e o f t e s t l y m p h o c y t e s a n d E A C i n d i c a t o r e r y t h r o c y t e s was l e f t t o s e t t l e b y g r a v i t y , w i t h o u t c e n t r i f u g a t i o n , a t 2 0 ° C f o r 2 h. Statistical analyses were p e r f o r m e d by the S t u d e n t ' s t test. RESULTS E n h a n c e m e n t e f f e c t o f V C N o n E- a n d E A C - r o s e t t e s o f v a r i o u s t y p e s o f l y m p h o i d cells is s h o w n i n t a b l e 1. T h e E- a n d E A C - r o s e t t e s s i g n i f i c a n t l y increased w h e n the peripheral b l o o d l y m p h o c y t e s of n o r m a l d o n o r s were pre-
TABLE 1 Effect of neuraminidase treatment of human lymphocytes on E and EAC-rosettes * Type of lymphoid cells
No. of expts,
Treatment
E-rosette % mean ± S.D.
EAC rosette % mean ± S.D.
Peripheral lymphocytes
25 25
Untreated VCN-treated
62.0 ± 9.6 80.1-+ 9 . 8 " *
22.6 ± 10.9 32.9+- 1 1 . 4 " * *
6 6
Untreated VCN-treated
78.0 ± 9.7 82.6 ± 8.5
2.6 ± 3.8 0.9 ± 1.3
ALL
20 20
Untreated VCN-treated
44.4 ± 28.8 56.3 + 33.3
16.9 -+ 18.2 23.4 ÷ 22.3
CLL
12 12
Untreated VCN-treated
14.2 ± 12.6 27.5 +- 21.2
37.2 ± 29.8 49.0 ± 24.9
Thymus
6 6
Untreated VCN-treated
79.3 -+ 11.2 91.6 ÷ 8.4
5.7 + 4.0 6.7 ± 4.9
Spleen
5 5
Untreated VCN-treated
34.4 ± 14.5 73.7 -+ 8.6 **
56.1 + 11.4 52.6 -+ 8.3
MOLT-4 (T cell line)
3 3
Untreated VCN-treated
67.7 +- 9.6 90.2 -+ 3.3
30.1 ± 29.2 49.0 ± 34.0
B411-4 (B cell line)
1 1
Untreated VCN-treated
PHA-induced blasts
0 0
* Various types of lymphoid cells were treated with VCN, 50 U/ml or without VCN at 37°C for 30 min. E and EAC-rosetting were then performed. **IP < 0.01. *** P < 0.02.
256 TABLE 2 Effect of neuraminidase treatment of human peripheral blood lymphocytes on E and EAC-rosettes * (9 experiments). Type of assay
E-rosette E-rosette E-rosette EAC-rosette
Technique of rosetting
Rosettes % mean (ranges)
Gravity 20 ° C Centrifugation 20°C Centrifugation 4°C (standard) Gravity 20°C (standard)
Untreated lymphocytes
VCN-treated lymphocytes
0.1 (0--0.5) 24.2 (10.0--45.3) 59.6 (36.6--75.8)
59.5 (24.8--83.3)P < 0.001 68.4 (31.6--84.8) P < 0.001 78.8 (61.0--92.7) P < 0.005
20.4 (10.1--40.1)
34.9 (17.6--50.4) P < 0.05
* Peripheral blood lymphocytes were treated with VCN, 50 U]ml or without VCN at 37°C for 30 min. E and EAC-rosetting were then performed according to standard techniques and modified techniques.
t r e a t e d w i t h V C N (P < 0 . 0 l a n d P < 0 . 0 2 , r e s p e c t i v e l y ) . A l t h o u g h t h e E-ros e t t e s i n c r e a s e d f o l l o w i n g t h e V C N t r e a t m e n t o f o t h e r l y m p h o i d cells, t h e d i f f e r e n c e s w e r e n o t s t a t i s t i c a l l y s i g n i f i c a n t e x c e p t f o r t h e s p l e e n cells (P 0 . 0 1 ) , A s l i g h t i n c r e a s e o f E A C - r o s e t t e s was seen w h e n t h e p e r i p h e r a l b l o o d l y m p h o c y t e s f r o m p a t i e n t s w i t h A L L or C L L a n d M O L T - 4 l y m p h o i d cells w e r e p r e - t r e a t e d w i t h VCN. T h e E A C - r o s e t t e s w e r e n o t i n c r e a s e d b y V C N t r e a t m e n t o f P H A - i n d u c e d b l a s t s , t h y m u s cells o r s p l e e n cells. The E-rosettes f o r m e d b y the a t t a c h m e n t of unsensitized sheep red b l o o d cells t o u n t r e a t e d p e r i p h e r a l b l o o d l y m p h o c y t e s w e r e e a s i l y b r o k e n u p b y inc u b a t i o n a t 3 7 ° C f o r 30 m i n o r s h a k i n g . T h u s , n o s t a b l e E - r o s e t t e s o f unt r e a t e d l y m p h o c y t e s w e r e seen. P r e - t r e a t m e n t o f l y m p h o c y t e s w i t h V C N increased the proportion of stable E-rosettes (60--70%) resistant to incubation at 37°C and to vigorous shaking. T h e e f f e c t o f V C N t r e a t m e n t of p e r i p h e r a l b l o o d l y m p h o c y t e s on E-ro-
TABLE 3 Effect of neuraminidase treatment of human thymus cells on E-rosettes * Technique of E-rosetting Gravity, 20 ° C Centrifugation, 20 ° C Centrifugation, 4°C (standard)
E-rosettes % Untreated thymus cells
VCN-treated thymus cells
41.4 68.1 82.5
91.7 96.3 96.3
* Thymus cells were treated with VCN, 50 U/ml or without VCN at 37°C for 30 min. E-rosetting was then performed.
257 TABLE 4 Effect of neuraminidase treatment of red blood cells (RBC) of various species on E-rosettes * of peripheral blood lymphocytes (4 experiments). Source of RBC
Sheep Horse Bovine
E-rosettes % mean (ranges) Untreated RBC
VCN-treated RBC
76.5 (62.6--89.5) 32.2 (27.8--40.3) 0
72.0 (49.5--92.4) 23.6 (16.8--39.4) 0
* Red blood cells were treated with VCN, 50 U/ml or without VCN at 37°C for 30 rain. E-rosetting was then performed according to the standard method.
settes by various techniques and EAC-rosettes by standard technique is presented in table 2. Again, a significant increase in the p r o p o r t i o n o f E- and EAC-rosettes was observed when the peripheral blood l y m p h o c y t e s were pretreated with VCN and rosettings were p e r f o r m e d according to standard tech.niques (P < 0.005 and P < 0.05, respectively). Percentage of E- rosettes of u n t r e a t e d l y m p h o c y t e s decreased by m or e than half when the modified technique with an incubation at 20°C was utilized. Omission of centrifugation followed by incubation at 20°C resulted in virtually no E-rosettes of untreated l y m p h o c y t e s . However, the n u m b e r o f E-rosettes was significantly increased when the l y m p h o c y t e s were treated with VCN prior to either modified technique o f E-rosetting (P < 0.001). The ef f ect of VCN t r e a t m e n t of t h y m u s cells on E-rosettes is shown in table 3. The highest p r o p o r t i o n o f E-rosettes o f u n t r e a t e d t h y m u s cells was seen when the standard technique was used; the lower p r o p o r t i o n s o f E-rosettes o f u n t r e a t e d t h y m u s cells were seen when the modified techniques were used as with peripheral bl ood l y m p h o c y t e s . Again, the percentage of E-rosettes of VCN-treated t h y m u s cells was increased w h e t h e r the standard technique or modified technique was utilized. Various concentrations of VCN (1--100 U/ml) had an enhancing e f f e c t on E- and EAC-rosette f or m a t i on with the highest activity at a c o n c e n t r a t i o n of either 25 or 50 U/ml. Bovine red blood cells did n o t bind t o either u n t r e a t e d or VCN-treated peripheral blood l y m p h o c y t e s or t h y m o c y t e s and as shown in table 4. VCN t r e a t m e n t of sheep red blood cells or horse red blood cells failed t o increase the p r o p o r t i o n of E-rosettes obtained with peripheral blood lymphocytes. DISCUSSION It has r ecen tl y been established t hat E-rosette f o r m a t i o n is a useful tool for the identification of h u m a n T l y m p h o i d cells (Jondal et al., 1972; Mino-
258 wada et al., 1972). The mechanisms of E-rosette formation of lymphocytes with unsensitized sheep red blood cells are n o t fully understood at present. The EAC-rosette formation, on the other hand, has been used as a tool for the identification of human B lymphoid cells with the mechanism involved utilizing the complement receptor property of B cells (Jondal et al., 1972). The present study clearly indicates that the VCN treatment of human lymphoid cells increased the proportion of E- or EAC-rosette formation. In this study, we observed that the E-rosettes formed by binding of sheep red blood cells to untreated peripheral blood at 0--4°C were easily disassociated by incubation at 37°C for 30 min or vigorous shaking; some of the E-rosettes obtained with VCN-treated peripheral blood lymphocytes, on the other hand, were stable after incubation at 37°C for 30 min or vigorous shaking. Similar findings on the effect of VCN on peripheral blood l y m p h o c y t e s have been reported by other investigators (Bentwich et al., 1973; Galli and Schlesinger, 1974). It has also been reported that the proportion of regular or stable Erosettes was increased b y treating the sheep red blood cells with VCN (Galli and Schlesinger, 1974; Weiner et al., 1973). We, on the other hand, observed that the regular E-rosette formation was n o t enhanced by the VCN treatment of sheep red blood cells or horse red blood cells. Stable E-rosette assays, using untreated l y m p h o c y t e s and VCN-treated sheep red blood cells, were n o t carried out in the present study. It should be pointed out that 30% of untreated MOLT-4 cells (T cells) possessed EAC-rosettes and pre-treatment by neuraminidase of MOLT-4 cells enhanced EAC-rosette formation to 49%. These findings suggest that some of the MOLT-4 cells have receptor for complement. It is evident that normal T l y m p h o c y t e s (thymus cells or PHA-induced lymphoblasts) lack receptor for complement (table 1). It is possible that the MOLT-4 cells represent leukemic T cells having both E- and EAC-receptor expression (West and Herberman, 1974). It should be emphasized again that the MOLT-4 cell line was originally derived from a patient with acute lymphoblastic leukemia (ALL) during relapse. Although human l y m p h o c y t e s have been subclassified into two major subpopulations, namely T-cell and B-cell, there is increasing evidence indicating that each T- or B-cell subpopulation consists of a heterogeneous cell population. At the present time, leukemic l y m p h o c y t e s from cases of ALL frequently demonstrate partial phenotypic expression of both T- and B-cells. It has also been known that a small proportion of the normal peripheral blood l y m p h o c y t e s have such double markers as detected by E-rosette and EAC-rosette or surface immunoglobulin. Despite the presence of EAC in MOLT-4 cells, these cells present most probable evidence suggesting leukemic T cells on the basis of lack of cell surface immunoglobulin and presence of T cell antigens by c y t o t o x i c and immunofluorescent tests (Takada et al., 1974; Seon et al., 1975). The exact mechanisms of increased capacity of VCN-treated lymphocytes to form rosettes with unsensitized sheep red blood cells (E-rosettes) or with sensitized bovine red blood cells (EAC-rosettes) and of increased stability of
259 E-rosettes are not understood at present. The increased capacity of rosette formation is probably related to changes in the cell-surface properties of lymphocytes following the VCN treatment. It is well-known that the VCN removes sialic acid from the cell membrane (Ambrose, 1967). It is possible that some of the receptor sites for unsensitized sheep red blood cells and sensitized bovine red blood cells on the cell membrane of T and B lymphocytes, respectively, are hidden by sialic acid and t h a t the VCN t r e a t m e n t of those lymphocytes unmasks hidden receptors which become available for interaction with sheep or bovine red blood cells. Failure to obtain increased E-rosette formation with VCN-treated sheep red blood cells, observed in the present study, suggests the possibility that the receptor sites for the T lymphocytes on the cell membrane of sheep red blood cells are n o t hidden by sialic acid. An alternative explanation for increased capacity of VCN-treated T lymphocytes to form E-rosettes with sheep red blood cells may be t h a t VCN does not expose hidden receptor sites on the l y m p h o c y t e surface but simply reduces its negative charge, resulting in greater and closer contact between the two cell types. Unsensitized bovine red blood cells do n o t bind with untreated T lymphocytes (personal observation). We observed in the present study t h a t the VCNtreated peripheral blood l y m p h o c y t e s or t h y m o c y t e s failed to form E-rosettes with bovine red blood cells; the VCN-treated bovine red blood cells also failed to bind with untreated peripheral blood lymphocytes. These observations indicate that the T l y m p h o c y t e s truly do n o t possess receptor sites for bovine red blood cells. In the present study, we observed no E-rosette formation of untreated or VCN-treated B l y m p h o i d cells with unsensitized sheep red blood cells. These findings indicate that the B l y m p h o c y t e s really do n o t possess receptor sites for sheep red blood cells. E-rosette assa:~ has been utilized in the past few years to determine the total T l y m p h o c y t e count in patients with various types of neoplasia (Brugarolas et al., 1973; Nemoto et al., 1974). The results on the E-rosettes reported in different studies vary greatly depending on details of the m e t h o d used for the assay. The temperature used for incubation of cells is very critical (Lay et al., 1971; Chapel, 1973). The E-rosette formation is maximal at 4 °C. Centrifugation of cell mixture of lymphocytes and sheep blood cells is also an important step in this assay for optimal rosetting (table 2). In the present study it is clearly demonstrated t h a t the proportion of E-rosettes is further increased by treating the l y m p h o c y t e s with VCN prior to incubation of these cells with unsensitized sheep red blood cells, and that the proportion of EAC-rosettes is also increased by VCN t r e a t m e n t of the l y m p h o c y t e s prior to incubation of these cells with sensitized bovine red blood cells. It is possible that the number of E- or EAC-rosettes observed with untreated lymphocytes falls short of the actual total number of T- or B-cells, respectively. The findings of this study indicate that it is advantageous to treat the peri-
260 pheral b l o o d l y m p h o c y t e s in E- and E A C - r o s e t t e assays t o d e t e r m i n e the t o t a l n u m b e r o f h u m a n T and B l y m p h o c y t e s . ACKNOWLEDGMENTS T h e a u t h o r s t h a n k Mrs. D o r o t h y T a b r o n f o r her t e c h n i c a l assistance. This s t u d y was s u p p o r t e d in p a r t b y U n i t e d States Public Health Service Grants: C A - 1 2 3 1 8 , C A - 1 4 3 2 3 , C A - 1 4 4 1 3 and A I - 0 8 8 9 9 .
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253
E N H A N C E D E- A N D E A C - R O S E T T E F O R M A T I O N B Y N E U R A M I N I D A S E
TIN HAN and JUN MINOWADA Departments o f Medicine B and Immunology and Immunochemistry Research, Roswell Park Memorial Institute, New York State Department o f Health, Buffalo, New York, 14263
(Received 10 December 1975, accepted 29 March 1976)
Pre-treatment by neuraminidase of lymphocytes obtained from peripheral blood of normal donors significantly enhanced E- and EAC-rosette formation. Of other lymphoid cells only spleen cells showed significant enhancement of E-rosettes. The EAC-rosettes slightly increased when the peripheral blood lymphocytes from patients with acute lymphoblastic leukemia or chronic lymphocytic leukemia and MOLT-4 lymphoid cells were pre-treated with this enzyme. The EAC-rosettes were not increased by neuraminidase treatment of phytohemagglutinin-induced blasts, thymus cells or spleen cells. Pre-treatment of peripheral blood lymphocytes with neuraminidase also increased the proportion of stable E-rosettes resistant to incubation at 37°C and to vigorous shaking. Various concentrations of neuraminidase (1--100 U/ml) produced enhancement of E- and EAC-rosettes with the highest activity at 25 and 50 U/ml. Neuraminidase treatment of sheep red blood cells failed to increase the proportion of E-rosettes of peripheral blood lymphocytes. The increased rosette forming capacity induced by neuraminidase is probably related to changes in lymphocyte surface properties.
INTRODUCTION I t has b e e n k n o w n t h a t V i b r i o c h o l e r a e n e u r a m i n i d a s e ( V C N ) increases the i m m u n o g e n i c i t y a n d / o r a n t i g e n i c i t y o f a v a r i e t y o f t u m o r cells a n d l y m p h o i d cells in a n i m a l s ( S a n f o r d , 1 9 6 7 ; Currie a n d B a g s h a w e , 1 9 6 9 ; Bekesi e t al., 1 9 7 1 ; Schlesinger a n d A m o s , 1 9 7 1 ; S i m m o n s a n d Rios, 1 9 7 1 ; S i m m o n s et al., 1 9 7 1 ) , and the a n t i g e n i c i t y ( H L - A s y s t e m ) o f h u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s ( G r o t h a u s et al., 1 9 7 1 ) . This e n z y m e has also b e e n k n o w n t o increase t h e h u m a n m i x e d l y m p h o c y t e r e a c t i o n ( L u n d g r e n a n d S i m m o n s , 1 9 7 1 ; H a n , 1 9 7 2 ) , l y m p h o c y t e b l a s t o g e n i c r e s p o n s e t o antigens a n d m i t o gens ( H a n , 1 9 7 3 , 1 9 7 5 ) , a n d d e l a y e d skin t e s t r e s p o n s e s (Hart, 1 9 7 4 ) . H u m a n T l y m p h o c y t e s can be i d e n t i f i e d b y t h e i r ability t o f o r m r o s e t t e s w i t h u n s e n s i t i z e d s h e e p red b l o o d cells ( E - r o s e t t e s ) ( J o n d a l e t al., 1 9 7 2 ; Min o w a d a e t al., 1 9 7 2 ) , a n d b y the lack o f r e c e p t o r f o r i m m u n o g l o b u l i n or c o m p l e m e n t . H u m a n B l y m p h o c y t e s , on t h e o t h e r h a n d , can be i d e n t i f i e d b y t h e i r ability t o f o r m r o s e t t e s w i t h sensitized s h e e p red b l o o d cells (EACr o s e t t e s ) ( B i a n c o e t al., 1 9 7 0 ; J o n d a l e t al., 1 9 7 2 ) , a n d b y t h e p r e s e n c e o f r e c e p t o r s f o r a g g r e g a t e d i m m u n o g l o b u l i n ( D i c k l e r and K u n k e l , 1972}.
254 It has recently been reported that the VCN-treatment of either peripheral blood l y m p h o c y t e s or sheep red blood cells increased the proportion of regular E-rosettes formed at 4°C or 20°C and of stable E-rosettes resistant to prolonged incubation at 37°C and to vigorous shaking (Bentwich et al., 1973; Galli and Schlesinger, 1974; Weiner et al., 1973). The present study was designed to investigate the effect of VCN treatment on E- and EAC-rosette formation by various types of human lymphoid cells. MATERIALS AND METHODS Peripheral blood lymphocytes were prepared from heparinized venous blood of healthy donors and patients with acute lymphoblastic leukemia (ALL) or chronic l y m p h o c y t i c leukemia (CLL), by centrifugation over a Ficoll--Hypaque gradient (BCyum, 1968). This preparation usually contains more than 90% lymphocytes, the remaining being m o n o c y t e s and granulocytes. Thymus cells were prepared from a portion of t h y m u s obtained from non-malignant patients at the time of open-heart surgery, as previously described (Hart et al., 1976). Spleen cells were prepared from a portion of normal spleen obtained from patients with malignant l y m p h o m a (Stage I or II), at the time of laparotomy and splenectomy for staging of the disease. T and B l y m p h o i d cells were obtained from established cell lines (MOLT-4 and B411-4). L y m p h o c y t e suspensions were prepared at a concentration of 5 X 106/ml of phosphate buffered saline (PBS); sheep red blood cells and bovine red blood cells were adjusted to 1% and 2.5% (v/v), respectively. Twenty-five or 50 units of VCN (Calbiochem, Los Angeles, California) were added to lymphocyte suspensions or red blood cell suspensions, which were incubated at 37°C for 30 min. The cells were then washed once with PBS prior to E- or EAC-rosette assay. For E-rosette as a T-cell marker, 0.1 ml of the l y m p h o c y t e suspension at a concentration of 5 X 106/ml was mixed with 0.1 ml of 1% (v/v) sheep red blood cell suspension in a small test tube (7 X 50 mm). Mixed cell suspensions were usually centrifuged at 200 g for 3 min and were left undisturbed at 4°C for 2 h (Standard technique}. In some instances, the mixed cell suspensions were centrifuged at 200 g for 3 min and were left undisturbed at 20°C for 2 h or the mixed cell suspensions were left to settle by gravity, without centrifugation at 20°C for 2 h. A few drops of 0.1% trypan blue solution in PBS were then added to the tube and the cell pellet was gently resuspended, following which, a drop of the cell suspension was examined under a microscope at 500 X magnification. Each sample was tested in duplicate, and at least 200 cells were observed to determine the percentage of Erosettes. A positive E-rosette was defined as a l y m p h o c y t e to which three or more sheep red blood cells were attached. For EAC-rosettes as a B-cell marker, bovine red blood cells were used as the indicator cells since the unsensitized bovine red blood cells do n o t bind
255 w i t h e i t h e r T o r B l y m p h o c y t e s . B o v i n e r e d b l o o d cells w e r e f i r s t s e n s i t i z e d w i t h a p p r o p r i a t e d i l u t i o n o f r a b b i t a n t i b o v i n e e r y t h r o c y t e s e r u m ( a n IgM class a n t i b o d y ) a t 3 7 ° C f o r 3 0 m i n , a n d t h e cells w e r e w a s h e d t h r e e t i m e s w i t h PBS. O n e m l o f 2 , 5 % (v/v) s u s p e n s i o n o f t h e s e n s i t i z e d cells w a s i n c u b a t e d with 1.0 m l of 1 / 1 0 d i l u t i o n of fresh m o u s e s e r u m ( D B A / 2 Ha strain) as a c o m p l e m e n t s o u r c e a t 3 7 ° C f o r 3 0 m i n . T h e cells w e r e t h e n w a s h e d t h r e e t i m e s w i t h P B S a n d w e r e r e s u s p e n d e d a t 1% (v/v) s u s p e n s i o n in PBS f o r E A C - r o s e t t i n g . T h e p r o c e d u r e f o r E A C - r o s e t t i n g was g e n e r a l l y t h e s a m e as t h e s t a n d a r d t e c h n i q u e f o r E - r o s e t t i n g ; h o w e v e r , t h e m i x t u r e o f t e s t l y m p h o c y t e s a n d E A C i n d i c a t o r e r y t h r o c y t e s was l e f t t o s e t t l e b y g r a v i t y , w i t h o u t c e n t r i f u g a t i o n , a t 2 0 ° C f o r 2 h. Statistical analyses were p e r f o r m e d by the S t u d e n t ' s t test. RESULTS E n h a n c e m e n t e f f e c t o f V C N o n E- a n d E A C - r o s e t t e s o f v a r i o u s t y p e s o f l y m p h o i d cells is s h o w n i n t a b l e 1. T h e E- a n d E A C - r o s e t t e s s i g n i f i c a n t l y increased w h e n the peripheral b l o o d l y m p h o c y t e s of n o r m a l d o n o r s were pre-
TABLE 1 Effect of neuraminidase treatment of human lymphocytes on E and EAC-rosettes * Type of lymphoid cells
No. of expts,
Treatment
E-rosette % mean ± S.D.
EAC rosette % mean ± S.D.
Peripheral lymphocytes
25 25
Untreated VCN-treated
62.0 ± 9.6 80.1-+ 9 . 8 " *
22.6 ± 10.9 32.9+- 1 1 . 4 " * *
6 6
Untreated VCN-treated
78.0 ± 9.7 82.6 ± 8.5
2.6 ± 3.8 0.9 ± 1.3
ALL
20 20
Untreated VCN-treated
44.4 ± 28.8 56.3 + 33.3
16.9 -+ 18.2 23.4 ÷ 22.3
CLL
12 12
Untreated VCN-treated
14.2 ± 12.6 27.5 +- 21.2
37.2 ± 29.8 49.0 ± 24.9
Thymus
6 6
Untreated VCN-treated
79.3 -+ 11.2 91.6 ÷ 8.4
5.7 + 4.0 6.7 ± 4.9
Spleen
5 5
Untreated VCN-treated
34.4 ± 14.5 73.7 -+ 8.6 **
56.1 + 11.4 52.6 -+ 8.3
MOLT-4 (T cell line)
3 3
Untreated VCN-treated
67.7 +- 9.6 90.2 -+ 3.3
30.1 ± 29.2 49.0 ± 34.0
B411-4 (B cell line)
1 1
Untreated VCN-treated
PHA-induced blasts
0 0
* Various types of lymphoid cells were treated with VCN, 50 U/ml or without VCN at 37°C for 30 min. E and EAC-rosetting were then performed. **IP < 0.01. *** P < 0.02.
256 TABLE 2 Effect of neuraminidase treatment of human peripheral blood lymphocytes on E and EAC-rosettes * (9 experiments). Type of assay
E-rosette E-rosette E-rosette EAC-rosette
Technique of rosetting
Rosettes % mean (ranges)
Gravity 20 ° C Centrifugation 20°C Centrifugation 4°C (standard) Gravity 20°C (standard)
Untreated lymphocytes
VCN-treated lymphocytes
0.1 (0--0.5) 24.2 (10.0--45.3) 59.6 (36.6--75.8)
59.5 (24.8--83.3)P < 0.001 68.4 (31.6--84.8) P < 0.001 78.8 (61.0--92.7) P < 0.005
20.4 (10.1--40.1)
34.9 (17.6--50.4) P < 0.05
* Peripheral blood lymphocytes were treated with VCN, 50 U]ml or without VCN at 37°C for 30 min. E and EAC-rosetting were then performed according to standard techniques and modified techniques.
t r e a t e d w i t h V C N (P < 0 . 0 l a n d P < 0 . 0 2 , r e s p e c t i v e l y ) . A l t h o u g h t h e E-ros e t t e s i n c r e a s e d f o l l o w i n g t h e V C N t r e a t m e n t o f o t h e r l y m p h o i d cells, t h e d i f f e r e n c e s w e r e n o t s t a t i s t i c a l l y s i g n i f i c a n t e x c e p t f o r t h e s p l e e n cells (P 0 . 0 1 ) , A s l i g h t i n c r e a s e o f E A C - r o s e t t e s was seen w h e n t h e p e r i p h e r a l b l o o d l y m p h o c y t e s f r o m p a t i e n t s w i t h A L L or C L L a n d M O L T - 4 l y m p h o i d cells w e r e p r e - t r e a t e d w i t h VCN. T h e E A C - r o s e t t e s w e r e n o t i n c r e a s e d b y V C N t r e a t m e n t o f P H A - i n d u c e d b l a s t s , t h y m u s cells o r s p l e e n cells. The E-rosettes f o r m e d b y the a t t a c h m e n t of unsensitized sheep red b l o o d cells t o u n t r e a t e d p e r i p h e r a l b l o o d l y m p h o c y t e s w e r e e a s i l y b r o k e n u p b y inc u b a t i o n a t 3 7 ° C f o r 30 m i n o r s h a k i n g . T h u s , n o s t a b l e E - r o s e t t e s o f unt r e a t e d l y m p h o c y t e s w e r e seen. P r e - t r e a t m e n t o f l y m p h o c y t e s w i t h V C N increased the proportion of stable E-rosettes (60--70%) resistant to incubation at 37°C and to vigorous shaking. T h e e f f e c t o f V C N t r e a t m e n t of p e r i p h e r a l b l o o d l y m p h o c y t e s on E-ro-
TABLE 3 Effect of neuraminidase treatment of human thymus cells on E-rosettes * Technique of E-rosetting Gravity, 20 ° C Centrifugation, 20 ° C Centrifugation, 4°C (standard)
E-rosettes % Untreated thymus cells
VCN-treated thymus cells
41.4 68.1 82.5
91.7 96.3 96.3
* Thymus cells were treated with VCN, 50 U/ml or without VCN at 37°C for 30 min. E-rosetting was then performed.
257 TABLE 4 Effect of neuraminidase treatment of red blood cells (RBC) of various species on E-rosettes * of peripheral blood lymphocytes (4 experiments). Source of RBC
Sheep Horse Bovine
E-rosettes % mean (ranges) Untreated RBC
VCN-treated RBC
76.5 (62.6--89.5) 32.2 (27.8--40.3) 0
72.0 (49.5--92.4) 23.6 (16.8--39.4) 0
* Red blood cells were treated with VCN, 50 U/ml or without VCN at 37°C for 30 rain. E-rosetting was then performed according to the standard method.
settes by various techniques and EAC-rosettes by standard technique is presented in table 2. Again, a significant increase in the p r o p o r t i o n o f E- and EAC-rosettes was observed when the peripheral blood l y m p h o c y t e s were pretreated with VCN and rosettings were p e r f o r m e d according to standard tech.niques (P < 0.005 and P < 0.05, respectively). Percentage of E- rosettes of u n t r e a t e d l y m p h o c y t e s decreased by m or e than half when the modified technique with an incubation at 20°C was utilized. Omission of centrifugation followed by incubation at 20°C resulted in virtually no E-rosettes of untreated l y m p h o c y t e s . However, the n u m b e r o f E-rosettes was significantly increased when the l y m p h o c y t e s were treated with VCN prior to either modified technique o f E-rosetting (P < 0.001). The ef f ect of VCN t r e a t m e n t of t h y m u s cells on E-rosettes is shown in table 3. The highest p r o p o r t i o n o f E-rosettes o f u n t r e a t e d t h y m u s cells was seen when the standard technique was used; the lower p r o p o r t i o n s o f E-rosettes o f u n t r e a t e d t h y m u s cells were seen when the modified techniques were used as with peripheral bl ood l y m p h o c y t e s . Again, the percentage of E-rosettes of VCN-treated t h y m u s cells was increased w h e t h e r the standard technique or modified technique was utilized. Various concentrations of VCN (1--100 U/ml) had an enhancing e f f e c t on E- and EAC-rosette f or m a t i on with the highest activity at a c o n c e n t r a t i o n of either 25 or 50 U/ml. Bovine red blood cells did n o t bind t o either u n t r e a t e d or VCN-treated peripheral blood l y m p h o c y t e s or t h y m o c y t e s and as shown in table 4. VCN t r e a t m e n t of sheep red blood cells or horse red blood cells failed t o increase the p r o p o r t i o n of E-rosettes obtained with peripheral blood lymphocytes. DISCUSSION It has r ecen tl y been established t hat E-rosette f o r m a t i o n is a useful tool for the identification of h u m a n T l y m p h o i d cells (Jondal et al., 1972; Mino-
258 wada et al., 1972). The mechanisms of E-rosette formation of lymphocytes with unsensitized sheep red blood cells are n o t fully understood at present. The EAC-rosette formation, on the other hand, has been used as a tool for the identification of human B lymphoid cells with the mechanism involved utilizing the complement receptor property of B cells (Jondal et al., 1972). The present study clearly indicates that the VCN treatment of human lymphoid cells increased the proportion of E- or EAC-rosette formation. In this study, we observed that the E-rosettes formed by binding of sheep red blood cells to untreated peripheral blood at 0--4°C were easily disassociated by incubation at 37°C for 30 min or vigorous shaking; some of the E-rosettes obtained with VCN-treated peripheral blood lymphocytes, on the other hand, were stable after incubation at 37°C for 30 min or vigorous shaking. Similar findings on the effect of VCN on peripheral blood l y m p h o c y t e s have been reported by other investigators (Bentwich et al., 1973; Galli and Schlesinger, 1974). It has also been reported that the proportion of regular or stable Erosettes was increased b y treating the sheep red blood cells with VCN (Galli and Schlesinger, 1974; Weiner et al., 1973). We, on the other hand, observed that the regular E-rosette formation was n o t enhanced by the VCN treatment of sheep red blood cells or horse red blood cells. Stable E-rosette assays, using untreated l y m p h o c y t e s and VCN-treated sheep red blood cells, were n o t carried out in the present study. It should be pointed out that 30% of untreated MOLT-4 cells (T cells) possessed EAC-rosettes and pre-treatment by neuraminidase of MOLT-4 cells enhanced EAC-rosette formation to 49%. These findings suggest that some of the MOLT-4 cells have receptor for complement. It is evident that normal T l y m p h o c y t e s (thymus cells or PHA-induced lymphoblasts) lack receptor for complement (table 1). It is possible that the MOLT-4 cells represent leukemic T cells having both E- and EAC-receptor expression (West and Herberman, 1974). It should be emphasized again that the MOLT-4 cell line was originally derived from a patient with acute lymphoblastic leukemia (ALL) during relapse. Although human l y m p h o c y t e s have been subclassified into two major subpopulations, namely T-cell and B-cell, there is increasing evidence indicating that each T- or B-cell subpopulation consists of a heterogeneous cell population. At the present time, leukemic l y m p h o c y t e s from cases of ALL frequently demonstrate partial phenotypic expression of both T- and B-cells. It has also been known that a small proportion of the normal peripheral blood l y m p h o c y t e s have such double markers as detected by E-rosette and EAC-rosette or surface immunoglobulin. Despite the presence of EAC in MOLT-4 cells, these cells present most probable evidence suggesting leukemic T cells on the basis of lack of cell surface immunoglobulin and presence of T cell antigens by c y t o t o x i c and immunofluorescent tests (Takada et al., 1974; Seon et al., 1975). The exact mechanisms of increased capacity of VCN-treated lymphocytes to form rosettes with unsensitized sheep red blood cells (E-rosettes) or with sensitized bovine red blood cells (EAC-rosettes) and of increased stability of
259 E-rosettes are not understood at present. The increased capacity of rosette formation is probably related to changes in the cell-surface properties of lymphocytes following the VCN treatment. It is well-known that the VCN removes sialic acid from the cell membrane (Ambrose, 1967). It is possible that some of the receptor sites for unsensitized sheep red blood cells and sensitized bovine red blood cells on the cell membrane of T and B lymphocytes, respectively, are hidden by sialic acid and t h a t the VCN t r e a t m e n t of those lymphocytes unmasks hidden receptors which become available for interaction with sheep or bovine red blood cells. Failure to obtain increased E-rosette formation with VCN-treated sheep red blood cells, observed in the present study, suggests the possibility that the receptor sites for the T lymphocytes on the cell membrane of sheep red blood cells are n o t hidden by sialic acid. An alternative explanation for increased capacity of VCN-treated T lymphocytes to form E-rosettes with sheep red blood cells may be t h a t VCN does not expose hidden receptor sites on the l y m p h o c y t e surface but simply reduces its negative charge, resulting in greater and closer contact between the two cell types. Unsensitized bovine red blood cells do n o t bind with untreated T lymphocytes (personal observation). We observed in the present study t h a t the VCNtreated peripheral blood l y m p h o c y t e s or t h y m o c y t e s failed to form E-rosettes with bovine red blood cells; the VCN-treated bovine red blood cells also failed to bind with untreated peripheral blood lymphocytes. These observations indicate that the T l y m p h o c y t e s truly do n o t possess receptor sites for bovine red blood cells. In the present study, we observed no E-rosette formation of untreated or VCN-treated B l y m p h o i d cells with unsensitized sheep red blood cells. These findings indicate that the B l y m p h o c y t e s really do n o t possess receptor sites for sheep red blood cells. E-rosette assa:~ has been utilized in the past few years to determine the total T l y m p h o c y t e count in patients with various types of neoplasia (Brugarolas et al., 1973; Nemoto et al., 1974). The results on the E-rosettes reported in different studies vary greatly depending on details of the m e t h o d used for the assay. The temperature used for incubation of cells is very critical (Lay et al., 1971; Chapel, 1973). The E-rosette formation is maximal at 4 °C. Centrifugation of cell mixture of lymphocytes and sheep blood cells is also an important step in this assay for optimal rosetting (table 2). In the present study it is clearly demonstrated t h a t the proportion of E-rosettes is further increased by treating the l y m p h o c y t e s with VCN prior to incubation of these cells with unsensitized sheep red blood cells, and that the proportion of EAC-rosettes is also increased by VCN t r e a t m e n t of the l y m p h o c y t e s prior to incubation of these cells with sensitized bovine red blood cells. It is possible that the number of E- or EAC-rosettes observed with untreated lymphocytes falls short of the actual total number of T- or B-cells, respectively. The findings of this study indicate that it is advantageous to treat the peri-
260 pheral b l o o d l y m p h o c y t e s in E- and E A C - r o s e t t e assays t o d e t e r m i n e the t o t a l n u m b e r o f h u m a n T and B l y m p h o c y t e s . ACKNOWLEDGMENTS T h e a u t h o r s t h a n k Mrs. D o r o t h y T a b r o n f o r her t e c h n i c a l assistance. This s t u d y was s u p p o r t e d in p a r t b y U n i t e d States Public Health Service Grants: C A - 1 2 3 1 8 , C A - 1 4 3 2 3 , C A - 1 4 4 1 3 and A I - 0 8 8 9 9 .
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