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Continuous in vitro culture of human T lymphocytes
Journal of Immunological Methods, 39 (1980) 39--45 Q Elsevier/North-Holland Biomedical Press
39
CONTINUOUS IN VITRO CULTURE OF HUMAN T LYMPHOCYTES
A.W. BOYLSTON and RENE L. ANDERSON Department of Pathology, St. Mary's Hospital Medical School, London W2 1PG, U.K.
(Received 19 March 1980, accepted 9 July 1980)
A method for continuous culture of human T lymphocytes by stimulation with irradiated lymphocytes, lymphoblastoid cells, and phytohaemagglutinin is described. Cultures can be maintained for at least 9 months. Growth appears to be dependent on a soluble factor released by lymphocytes when they are stimulated with lymphoblastoid cells and phytohaemagglutinin.
INTRODUCTION Human T l y m p h o c y t e s can be stimulated into limited growth by mitogens such as phytohaemagglutinin (PHA) and lymphoblastoid cell lines (LCL) (Svedmyr, 1975; Boylston and Anderson, 1979) but continuous growth of human T cells has been difficult to achieve consistently (Morgan et al., 1976; Gillis et al., 1978; Alvarez et al., 1979}. This paper outlines a m e t h o d for growing T cells for periods of at least 6 m o n t h s using a combination of mitogenic agents to generate growth. MATERIALS AND METHODS Cell sources and tissue culture c o n d i t i o n s
Human peripheral blood mononuclear cells (PBL) were prepared from defibrinated venous blood by centrifugation on L y m p h o p r e p (Nyegaard, Oslo, Norway). When required, T lymphocytes and B !ymphocytes were separated by the use of 2-aminoethylisothio-uronium bromide hydrobromide treated sheep red blood cells (AET-SRBC) (Boylston and Anderson, 1979). Lymphoblastoid cell lines were prepared by transforming human PBL depleted of AET-SRBC rosetting cells by t h e m e t h o d of Thorley-Lawson et al. (1977). All cultures were initiated and maintained in RPMI-1640 medium supplemented as described by Click et al. (1972} and Boylston and Anderson (1978) with the addition of 10% foetal bovine serum (FBS). Cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. When irradiated cells were required for a source of growth stimulus, PBL were given 3000 R and LCL were given 6000 R from a Siemens
40 Stabilipan 250 kV X-ray t h e r a p y machine. All cell counts were p e r f o r m e d with a h a e m o c y t o m e t e r , and t rypan blue d y e exclusion was used as a criterion of viability.
Divided culture chambers Tissue culture vessels similar to those described by Feldmann and Basten (1972) were used in some experiments. In these vessels, 20 ml of medium were separated f r om the large o u t e r cham ber by a m e m b r a n e made of Visking dialysis tubing which will allow diffusion of molecules of up to 15,000 d alto n s molecular weight. The inner chamber was covered with a m e m b r a n e with a 0.2 tim pore size ( A c r opor AN-200, Gelman, Ann Arbor, MI, U.S.A.). A p p r o x i m a t e l y 3 ml of medium was present in the out er chamber and 1 ml in the inner chamber. RESULTS These experiments arose out of the observation that human LCLs can stimulate growth of human T l y m p h o c y t e s for 2 or 3 weekly cycles and then the cultures will no longer respond. Since this might occur because a population o f cells required to support proliferation, rather than the cells which actually grow, had died, we investigated w het her adding irradiated PBL as a source o f growth stimulus would allow cont i nued proliferation. In Table 1, the effects of various combinations of mitogenic stimuli (PHA and irradiated LCL) combined with irradiated PBL as a source of growth support are shown. In these experiments the unirradiated growing cells were obtained f r om cultures which had been stimulated with 3 cycles of L CL so th at t h e y had lost their ability to respond to the mitogenic stimuli alone. The results of several experiments are pooled and the data are shown
TABLE 1 Growth of human T lyrnphocytes by stimulation with mitogens and irradiated peripheral blood lymphocytes. Initial responding inoculum
Irradiated PBL added
2 × 106 2 X 106 2X 106 2X 106 2 X 106 2X 106 2 x 106 None
None None None 2 × 106 2 X 106 2X 106 None 2X 106
Irradiated LCL added 2 X 106 -2X 106 2 × 106 -2 X 106 -2 X 106
PHA (1 pg/ml) added -+ + -+ + -+
Growth index
4 5 2 8 7 16 3 4
Mean
Range
1.4 1.2 1.8 2.6 2.8 8.3
0.5-- 2 0.2--3 0.6--3 0.5--5 0.4--6 4 --20 0.I--0.5 0 --0.I
0.3 0.I
41 TABLE 2 Effect of using autologous or allogeneic PBL and LCL on human T cell growth. n
Stimulating cell source PBL (2 × 106)
LCL (2 × 106)
Autologous Autologous Allogeneic Allogeneic
Autologous Allogeneic Autologous Allogeneic
Growth index
3 5 3 6
Mean
Range
9.1 8.9 8.5 9.3
6--12 7--14 5--10 7--14
as a g r o w t h i n d e x w h i c h was c a l c u l a t e d f r o m t h e f o r m u l a : viable cells p r e s e n t 6 d a y s a f t e r s t i m u l a t i o n / i n i t i a l cells s t i m u l a t e d = g r o w t h index. Cultures w h i c h s h o w n o n e t g r o w t h have a g r o w t h i n d e x o f 1, a n d t h o s e w h i c h die o f f have a g r o w t h i n d e x less t h a n 1 b y this calculation. Six d a y s was c h o s e n as t h e t i m e f o r m e a s u r i n g t h e g r o w t h i n d e x b e c a u s e p i l o t experi m e n t s s h o w e d t h a t t h e cell n u m b e r usually r e a c h e d a m a x i m u m at this t i m e . T h e d a t a in T a b l e 1 s h o w t h a t t h e c o m b i n a t i o n o f irradiated PBL a n d b o t h m i t o g e n s is a m o r e e f f e c t i v e g r o w t h s t i m u l u s t h a n a n y o t h e r c o m b i n a t i o n tested. In t h e e x p e r i m e n t s s h o w n in T a b l e 1, n o a t t e m p t was m a d e to s t u d y t h e e f f e c t o f allogeneic c o m p a r e d w i t h a u t o l o g o u s L C L s and PBLs o n g r o w t h s t i m u l a t i o n . T h e results o f such e x p e r i m e n t s are s h o w n in T a b l e 2 w h e r e it can be seen t h a t an e n t i r e l y allogeneic c o m b i n a t i o n is as e f f e c t i v e as an a u t o l o g o u s c o m b i n a t i o n in p r o m o t i n g cell g r o w t h . T h e e f f e c t o f v a r y i n g n u m b e r s o f L C L and P B L o n g r o w t h is s h o w n in T a b l e 3. While t h e t o t a l n u m b e r o f cells o b t a i n e d is d e p e n d e n t on t h e n u m b e r o f P B L a d d e d , it is i n d e p e n d e n t o f t h e n u m b e r o f LCLs. F o r convenience, a s t a n d a r d p r o t o c o l was a d o p t e d f o r long-terrh cultures: 5 × 106
TABLE3 E f f e c t o f v a r y i n g n u m b e r s o f P B L o r L C L o n mitogenstimulatedTcellgrowth. Initial cell number 5x 5x 5× 5×
106 106 106 106
5× 106 5 × 106 5 × 106
Growth stimulus PBL
LCL
lX 3× 5× 7x
2x 2× 2× 2×
106 106 106 106
3 × 106 3× 106 3 × 106
106 106 106 106
1 × 106 5× 106 7 × 106
Final cell number
Growth index
23x 38× 46× 55×
106 106 106 106
4~6 7.6 9.2 11
33x 106 37× 106 34 × 106
6.6 7.4 6.8
42 TABLE 4 Growth of long-term cultures of human T lymphocytes in vitro. Time interval
1st month 2nd month 3rd month 4th month 5th month 6th month
Number of cultures 8 7 6 6 4 2
Growth index Mean
Range
22.0 8.3 10.0 8.6 12.0 9.2
15.5--30 2.5--13 5.0--18 4.0--14 4.5--20 5.0--12
r e s p o n d i n g cells were s t i m u l a t e d w e e k l y with 3 X 106 irradiated PBL, 3 × 106 irradiated L C L and 1 p g / m l PHA. These cultures were initiated in 15 ml c u l t u r e m e d i u m in u p r i g h t 2 5 0 ml plastic tissue culture flasks. An a d d i t i o n a l 10 ml o f culture m e d i u m were a d d e d on d a y 2, and cultures were fed thereafter with sufficient m e d i u m to keep the cell c o n c e n t r a t i o n b e l o w 1 X 104/ ml. Cultures were c o u n t e d o n d a y 6 and an a l i q u o t r e s t i m u l a t e d o n d a y 7. T h e g r o w t h o f several cultures over a 6 m o n t h period is r e c o r d e d in Table 4. A f t e r the first m o n t h , cultures settle into a fairly c o n s i s t e n t g r o w t h pattern. T h e initial very high g r o w t h seen w h e n n o r m a l PBLs are g r o w n is p r o b a b l y d u e to residual g r o w t h p r o m o t i n g cells in t h e i n o c u l u m . These cells p r o b a b l y die over the first m o n t h and t h e n g r o w t h is strictly d e p e n d e n t o n a d d e d PBL. F u r t h e r e x p e r i e n c e with t h e t w o oldest cultures indicates t h a t g r o w t h o f this m a g n i t u d e can be sustained to 9 m o n t h s at least. A t t e m p t s to i d e n t i f y t h e cells w h i c h g r o w in culture b y their surface m e m b r a n e m a r k e r s were m a d e at various times. Table 5 r e c o r d s t h e fact t h a t m o s t o f t h e cells bind A E T - S R B C and are t h e r e f o r e T l y m p h o c y t e s . In these e x p e r i m e n t s 2 0 0 cells were c o u n t e d in each d e t e r m i n a t i o n and n o surface i m m u n o g l o b u l i n positive cells were f o u n d . T h e n a t u r e o f the A E T - S R B C
TABLE 5 AET-SRBC binding cells in continuous human PBL cultures. Culture age (months)
AET-SRBC rosetting cells (%)
Surface Ig positive cells (%)
5 5 4 3 2 1
73 50 81 67 77 66
<0.5 <0.5 ~<0.5 <0.5 <0.5 ~<0.5
43
TABLE 6 R e - e x p r e s s i o n o f A E T - S R B C b i n d i n g b y i n i t i a l l y A E T - S R B C n e g a t i v e cells. Culture age (months)
Initial receptor phenotype
Receptor phenotype after two further growth cycles
5
AET-SRBC positive AET-SRBC negative
78% AET-SRBC positive 75% AET-SRBC positive
< 0 . 5 % Ig p o s i t i v e < 0 . 5 % Ig p o s i t i v e
2
AET-SRBC positive AET-SRBC negative
66% AET-SRBC positive 70% AET-SRBC positive
< 0 . 5 % Ig p o s i t i v e < 0 . 5 % Ig p o s i t i v e
negative cells was examined because of the possibility that there were two distinct populations growing. Cultures were separated into AET-SRBC binding and non-binding populations b y centrifugation on Lymphoprep and then each population was grown for 2 more weekly cycles, and the surface p h e n o t y p e re-examined. The data are shown in Table 6 and suggest that the growing cells are all T l y m p h o c y t e s since initially negative cells have the same percentage of AET-SRBC positive cells as cultures selected for this marker. Presumably the failure of some cells to express this marker is due to their position in the cell cycle. In order to study the mechanisms by which irradiated PBL and LCL stimulate the growth of normal T lymphocytes, cultures were restimulated in vessels constructed so that part or all of the growth inducing inoculum could be separated from the responding population b y a 0.2 pm pore size membrane. If growth is supported by a soluble factor, then growth should occur TABLE 7 Growth of human T lymphocytes Responding cells
Exp. 1 + + + + + -
-
Exp. 2 + + + +
in divided culture chambers.
Lower chamber
Upper chamber
PBL
LCL
PBL
LCL
+ -+ -.
+ --+
-+ -+
-+ + --
-
-
.
.
+
+
+ -+ --
+ --+
.
-
-
-
+ -+
-
-
-
+ + --'
Growth index
7.8 8.1 4.2 4.0 0.1 0.I
8.1 9.2 4.6 3.8
44 when the PBL and LCL are separated from the growing cells by such a membrane. In addition, the role of the irradiated LCL can be assessed since if it only provides a non-specific growth supporting factor, t hen this should also diffuse through the m em br ane and n o t require cont act between LCL and PBL. The results of two such experiments are shown in Table 7. Growing cells from 3-month-old cultures were restimulated in vessels with the supporting cells divided in various ways. There is as m uch growth when the PBL and LCL are separated from the responding cells as when all 3 populations are in contact. F u r t h e r m o r e , m a x i m u m growth is obtained when the LCL are in c ont a c t with the PBL rather than separated by a membrane. DISCUSSION These experiments outline a m e t h o d of maintaining normal human T l y m p h o c y t e s in cont i nuous culture. The results of the divided chamber experiments show that growth is stimulated by a soluble factor produced when PBL are stimulated with b o t h PHA and LCL. Therefore, it seems likely that this m e t h o d represents a variation on the soluble growth factor m e th o d s used by others (Morgan et al., 1976; Gillis et al., 1978; Alvarez et al., 1979). However, the growth stimulus produced by used b o t h mitogenic stimuli seems greater than that pr odu ced by PHA alone since our cultures regularly generate 10--20 times as m a n y T cells as supporting PBL, whereas the published figures for soluble T cell growth factor suggest t hat only 2--4 times as m a n y blasts can be grown as cells used to produce growth factor (Gillis et al., 1978; Alvarez et al., 1979; Kurnick et al., 1979). A further difference between our cultures and those report ed is that our cultures do n o t deteriorate at cell densities greater than 0.5 × 106/ml (Kurnick et al., 1979) and when the cells have accidently been allowed to grow w i t h o u t dilution t h e y regularly reach 2 × 106/ml w i t h o u t obvious damage. Furt her information as to w he t he r the growth factor d e m o n s t r a t e d here is the same as th at obtained by others will require comparison of the results from longterm antigen sensitised cultures. So far we have been unable to prepare soluble growth factor which will consistently support continuous growth o f human T cells except in the divided culture vessels discussed above. This may indicate that the growth factor pr oduced by this m e t h o d is less stable than PHA induced growth factor. ACKNOWLEDGEMENTS Dr. A.W. Boylston is a Wellcome Senior Fellow in Clinical Science. This project was supported by grants from the Medical Research Council and t h e Cancer Research Campaign. REFERENCES Alvarez, J.M., A. Silva and M. De Landazuri, 1979, J. Immunol. 123,977. Boylston, A.W. and R.L. Anderson, 1978, Immunology 35,455. Boylston, A.W. and R.L. Anderson, 1979, Scand. J. Immunol. 9, 151.
45 Click, R.E., L. Benck and B.J. Alter, 1972, Cell. Immunol. 3,264. Feldmann, M. and A. Basten, 1972, Eur. J. Immunol. 2,213. Gillis, S., P. Baker, F.W. Ruscetti and K.A. Smith, 1978, J. Exp. Med. 148, 1093. Kurnick, J.T., K.-O. GrSnvik, A.K. Kimura, J.B. Lindblom, J.T. Skoog, O. SjSberg and H. Wigzell, 1979, J. Immunol. 122, 1255. Morgan, D.A., F.W. Ruscetti and R. Gallo, 1976, Science 193, 1007. Svedmyr, E., 1975, Scand. J. Immunol. 4,421. Thorley-Lawson, D.A., L. Chess and J.L. Strominger, 1977, J. Exp. Med. 146,495.
39
CONTINUOUS IN VITRO CULTURE OF HUMAN T LYMPHOCYTES
A.W. BOYLSTON and RENE L. ANDERSON Department of Pathology, St. Mary's Hospital Medical School, London W2 1PG, U.K.
(Received 19 March 1980, accepted 9 July 1980)
A method for continuous culture of human T lymphocytes by stimulation with irradiated lymphocytes, lymphoblastoid cells, and phytohaemagglutinin is described. Cultures can be maintained for at least 9 months. Growth appears to be dependent on a soluble factor released by lymphocytes when they are stimulated with lymphoblastoid cells and phytohaemagglutinin.
INTRODUCTION Human T l y m p h o c y t e s can be stimulated into limited growth by mitogens such as phytohaemagglutinin (PHA) and lymphoblastoid cell lines (LCL) (Svedmyr, 1975; Boylston and Anderson, 1979) but continuous growth of human T cells has been difficult to achieve consistently (Morgan et al., 1976; Gillis et al., 1978; Alvarez et al., 1979}. This paper outlines a m e t h o d for growing T cells for periods of at least 6 m o n t h s using a combination of mitogenic agents to generate growth. MATERIALS AND METHODS Cell sources and tissue culture c o n d i t i o n s
Human peripheral blood mononuclear cells (PBL) were prepared from defibrinated venous blood by centrifugation on L y m p h o p r e p (Nyegaard, Oslo, Norway). When required, T lymphocytes and B !ymphocytes were separated by the use of 2-aminoethylisothio-uronium bromide hydrobromide treated sheep red blood cells (AET-SRBC) (Boylston and Anderson, 1979). Lymphoblastoid cell lines were prepared by transforming human PBL depleted of AET-SRBC rosetting cells by t h e m e t h o d of Thorley-Lawson et al. (1977). All cultures were initiated and maintained in RPMI-1640 medium supplemented as described by Click et al. (1972} and Boylston and Anderson (1978) with the addition of 10% foetal bovine serum (FBS). Cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. When irradiated cells were required for a source of growth stimulus, PBL were given 3000 R and LCL were given 6000 R from a Siemens
40 Stabilipan 250 kV X-ray t h e r a p y machine. All cell counts were p e r f o r m e d with a h a e m o c y t o m e t e r , and t rypan blue d y e exclusion was used as a criterion of viability.
Divided culture chambers Tissue culture vessels similar to those described by Feldmann and Basten (1972) were used in some experiments. In these vessels, 20 ml of medium were separated f r om the large o u t e r cham ber by a m e m b r a n e made of Visking dialysis tubing which will allow diffusion of molecules of up to 15,000 d alto n s molecular weight. The inner chamber was covered with a m e m b r a n e with a 0.2 tim pore size ( A c r opor AN-200, Gelman, Ann Arbor, MI, U.S.A.). A p p r o x i m a t e l y 3 ml of medium was present in the out er chamber and 1 ml in the inner chamber. RESULTS These experiments arose out of the observation that human LCLs can stimulate growth of human T l y m p h o c y t e s for 2 or 3 weekly cycles and then the cultures will no longer respond. Since this might occur because a population o f cells required to support proliferation, rather than the cells which actually grow, had died, we investigated w het her adding irradiated PBL as a source o f growth stimulus would allow cont i nued proliferation. In Table 1, the effects of various combinations of mitogenic stimuli (PHA and irradiated LCL) combined with irradiated PBL as a source of growth support are shown. In these experiments the unirradiated growing cells were obtained f r om cultures which had been stimulated with 3 cycles of L CL so th at t h e y had lost their ability to respond to the mitogenic stimuli alone. The results of several experiments are pooled and the data are shown
TABLE 1 Growth of human T lyrnphocytes by stimulation with mitogens and irradiated peripheral blood lymphocytes. Initial responding inoculum
Irradiated PBL added
2 × 106 2 X 106 2X 106 2X 106 2 X 106 2X 106 2 x 106 None
None None None 2 × 106 2 X 106 2X 106 None 2X 106
Irradiated LCL added 2 X 106 -2X 106 2 × 106 -2 X 106 -2 X 106
PHA (1 pg/ml) added -+ + -+ + -+
Growth index
4 5 2 8 7 16 3 4
Mean
Range
1.4 1.2 1.8 2.6 2.8 8.3
0.5-- 2 0.2--3 0.6--3 0.5--5 0.4--6 4 --20 0.I--0.5 0 --0.I
0.3 0.I
41 TABLE 2 Effect of using autologous or allogeneic PBL and LCL on human T cell growth. n
Stimulating cell source PBL (2 × 106)
LCL (2 × 106)
Autologous Autologous Allogeneic Allogeneic
Autologous Allogeneic Autologous Allogeneic
Growth index
3 5 3 6
Mean
Range
9.1 8.9 8.5 9.3
6--12 7--14 5--10 7--14
as a g r o w t h i n d e x w h i c h was c a l c u l a t e d f r o m t h e f o r m u l a : viable cells p r e s e n t 6 d a y s a f t e r s t i m u l a t i o n / i n i t i a l cells s t i m u l a t e d = g r o w t h index. Cultures w h i c h s h o w n o n e t g r o w t h have a g r o w t h i n d e x o f 1, a n d t h o s e w h i c h die o f f have a g r o w t h i n d e x less t h a n 1 b y this calculation. Six d a y s was c h o s e n as t h e t i m e f o r m e a s u r i n g t h e g r o w t h i n d e x b e c a u s e p i l o t experi m e n t s s h o w e d t h a t t h e cell n u m b e r usually r e a c h e d a m a x i m u m at this t i m e . T h e d a t a in T a b l e 1 s h o w t h a t t h e c o m b i n a t i o n o f irradiated PBL a n d b o t h m i t o g e n s is a m o r e e f f e c t i v e g r o w t h s t i m u l u s t h a n a n y o t h e r c o m b i n a t i o n tested. In t h e e x p e r i m e n t s s h o w n in T a b l e 1, n o a t t e m p t was m a d e to s t u d y t h e e f f e c t o f allogeneic c o m p a r e d w i t h a u t o l o g o u s L C L s and PBLs o n g r o w t h s t i m u l a t i o n . T h e results o f such e x p e r i m e n t s are s h o w n in T a b l e 2 w h e r e it can be seen t h a t an e n t i r e l y allogeneic c o m b i n a t i o n is as e f f e c t i v e as an a u t o l o g o u s c o m b i n a t i o n in p r o m o t i n g cell g r o w t h . T h e e f f e c t o f v a r y i n g n u m b e r s o f L C L and P B L o n g r o w t h is s h o w n in T a b l e 3. While t h e t o t a l n u m b e r o f cells o b t a i n e d is d e p e n d e n t on t h e n u m b e r o f P B L a d d e d , it is i n d e p e n d e n t o f t h e n u m b e r o f LCLs. F o r convenience, a s t a n d a r d p r o t o c o l was a d o p t e d f o r long-terrh cultures: 5 × 106
TABLE3 E f f e c t o f v a r y i n g n u m b e r s o f P B L o r L C L o n mitogenstimulatedTcellgrowth. Initial cell number 5x 5x 5× 5×
106 106 106 106
5× 106 5 × 106 5 × 106
Growth stimulus PBL
LCL
lX 3× 5× 7x
2x 2× 2× 2×
106 106 106 106
3 × 106 3× 106 3 × 106
106 106 106 106
1 × 106 5× 106 7 × 106
Final cell number
Growth index
23x 38× 46× 55×
106 106 106 106
4~6 7.6 9.2 11
33x 106 37× 106 34 × 106
6.6 7.4 6.8
42 TABLE 4 Growth of long-term cultures of human T lymphocytes in vitro. Time interval
1st month 2nd month 3rd month 4th month 5th month 6th month
Number of cultures 8 7 6 6 4 2
Growth index Mean
Range
22.0 8.3 10.0 8.6 12.0 9.2
15.5--30 2.5--13 5.0--18 4.0--14 4.5--20 5.0--12
r e s p o n d i n g cells were s t i m u l a t e d w e e k l y with 3 X 106 irradiated PBL, 3 × 106 irradiated L C L and 1 p g / m l PHA. These cultures were initiated in 15 ml c u l t u r e m e d i u m in u p r i g h t 2 5 0 ml plastic tissue culture flasks. An a d d i t i o n a l 10 ml o f culture m e d i u m were a d d e d on d a y 2, and cultures were fed thereafter with sufficient m e d i u m to keep the cell c o n c e n t r a t i o n b e l o w 1 X 104/ ml. Cultures were c o u n t e d o n d a y 6 and an a l i q u o t r e s t i m u l a t e d o n d a y 7. T h e g r o w t h o f several cultures over a 6 m o n t h period is r e c o r d e d in Table 4. A f t e r the first m o n t h , cultures settle into a fairly c o n s i s t e n t g r o w t h pattern. T h e initial very high g r o w t h seen w h e n n o r m a l PBLs are g r o w n is p r o b a b l y d u e to residual g r o w t h p r o m o t i n g cells in t h e i n o c u l u m . These cells p r o b a b l y die over the first m o n t h and t h e n g r o w t h is strictly d e p e n d e n t o n a d d e d PBL. F u r t h e r e x p e r i e n c e with t h e t w o oldest cultures indicates t h a t g r o w t h o f this m a g n i t u d e can be sustained to 9 m o n t h s at least. A t t e m p t s to i d e n t i f y t h e cells w h i c h g r o w in culture b y their surface m e m b r a n e m a r k e r s were m a d e at various times. Table 5 r e c o r d s t h e fact t h a t m o s t o f t h e cells bind A E T - S R B C and are t h e r e f o r e T l y m p h o c y t e s . In these e x p e r i m e n t s 2 0 0 cells were c o u n t e d in each d e t e r m i n a t i o n and n o surface i m m u n o g l o b u l i n positive cells were f o u n d . T h e n a t u r e o f the A E T - S R B C
TABLE 5 AET-SRBC binding cells in continuous human PBL cultures. Culture age (months)
AET-SRBC rosetting cells (%)
Surface Ig positive cells (%)
5 5 4 3 2 1
73 50 81 67 77 66
<0.5 <0.5 ~<0.5 <0.5 <0.5 ~<0.5
43
TABLE 6 R e - e x p r e s s i o n o f A E T - S R B C b i n d i n g b y i n i t i a l l y A E T - S R B C n e g a t i v e cells. Culture age (months)
Initial receptor phenotype
Receptor phenotype after two further growth cycles
5
AET-SRBC positive AET-SRBC negative
78% AET-SRBC positive 75% AET-SRBC positive
< 0 . 5 % Ig p o s i t i v e < 0 . 5 % Ig p o s i t i v e
2
AET-SRBC positive AET-SRBC negative
66% AET-SRBC positive 70% AET-SRBC positive
< 0 . 5 % Ig p o s i t i v e < 0 . 5 % Ig p o s i t i v e
negative cells was examined because of the possibility that there were two distinct populations growing. Cultures were separated into AET-SRBC binding and non-binding populations b y centrifugation on Lymphoprep and then each population was grown for 2 more weekly cycles, and the surface p h e n o t y p e re-examined. The data are shown in Table 6 and suggest that the growing cells are all T l y m p h o c y t e s since initially negative cells have the same percentage of AET-SRBC positive cells as cultures selected for this marker. Presumably the failure of some cells to express this marker is due to their position in the cell cycle. In order to study the mechanisms by which irradiated PBL and LCL stimulate the growth of normal T lymphocytes, cultures were restimulated in vessels constructed so that part or all of the growth inducing inoculum could be separated from the responding population b y a 0.2 pm pore size membrane. If growth is supported by a soluble factor, then growth should occur TABLE 7 Growth of human T lymphocytes Responding cells
Exp. 1 + + + + + -
-
Exp. 2 + + + +
in divided culture chambers.
Lower chamber
Upper chamber
PBL
LCL
PBL
LCL
+ -+ -.
+ --+
-+ -+
-+ + --
-
-
.
.
+
+
+ -+ --
+ --+
.
-
-
-
+ -+
-
-
-
+ + --'
Growth index
7.8 8.1 4.2 4.0 0.1 0.I
8.1 9.2 4.6 3.8
44 when the PBL and LCL are separated from the growing cells by such a membrane. In addition, the role of the irradiated LCL can be assessed since if it only provides a non-specific growth supporting factor, t hen this should also diffuse through the m em br ane and n o t require cont act between LCL and PBL. The results of two such experiments are shown in Table 7. Growing cells from 3-month-old cultures were restimulated in vessels with the supporting cells divided in various ways. There is as m uch growth when the PBL and LCL are separated from the responding cells as when all 3 populations are in contact. F u r t h e r m o r e , m a x i m u m growth is obtained when the LCL are in c ont a c t with the PBL rather than separated by a membrane. DISCUSSION These experiments outline a m e t h o d of maintaining normal human T l y m p h o c y t e s in cont i nuous culture. The results of the divided chamber experiments show that growth is stimulated by a soluble factor produced when PBL are stimulated with b o t h PHA and LCL. Therefore, it seems likely that this m e t h o d represents a variation on the soluble growth factor m e th o d s used by others (Morgan et al., 1976; Gillis et al., 1978; Alvarez et al., 1979). However, the growth stimulus produced by used b o t h mitogenic stimuli seems greater than that pr odu ced by PHA alone since our cultures regularly generate 10--20 times as m a n y T cells as supporting PBL, whereas the published figures for soluble T cell growth factor suggest t hat only 2--4 times as m a n y blasts can be grown as cells used to produce growth factor (Gillis et al., 1978; Alvarez et al., 1979; Kurnick et al., 1979). A further difference between our cultures and those report ed is that our cultures do n o t deteriorate at cell densities greater than 0.5 × 106/ml (Kurnick et al., 1979) and when the cells have accidently been allowed to grow w i t h o u t dilution t h e y regularly reach 2 × 106/ml w i t h o u t obvious damage. Furt her information as to w he t he r the growth factor d e m o n s t r a t e d here is the same as th at obtained by others will require comparison of the results from longterm antigen sensitised cultures. So far we have been unable to prepare soluble growth factor which will consistently support continuous growth o f human T cells except in the divided culture vessels discussed above. This may indicate that the growth factor pr oduced by this m e t h o d is less stable than PHA induced growth factor. ACKNOWLEDGEMENTS Dr. A.W. Boylston is a Wellcome Senior Fellow in Clinical Science. This project was supported by grants from the Medical Research Council and t h e Cancer Research Campaign. REFERENCES Alvarez, J.M., A. Silva and M. De Landazuri, 1979, J. Immunol. 123,977. Boylston, A.W. and R.L. Anderson, 1978, Immunology 35,455. Boylston, A.W. and R.L. Anderson, 1979, Scand. J. Immunol. 9, 151.
45 Click, R.E., L. Benck and B.J. Alter, 1972, Cell. Immunol. 3,264. Feldmann, M. and A. Basten, 1972, Eur. J. Immunol. 2,213. Gillis, S., P. Baker, F.W. Ruscetti and K.A. Smith, 1978, J. Exp. Med. 148, 1093. Kurnick, J.T., K.-O. GrSnvik, A.K. Kimura, J.B. Lindblom, J.T. Skoog, O. SjSberg and H. Wigzell, 1979, J. Immunol. 122, 1255. Morgan, D.A., F.W. Ruscetti and R. Gallo, 1976, Science 193, 1007. Svedmyr, E., 1975, Scand. J. Immunol. 4,421. Thorley-Lawson, D.A., L. Chess and J.L. Strominger, 1977, J. Exp. Med. 146,495.