contains CS, the majority of the isolated hybrids is cytolytic and dependent on ..... in CS-free culture medium for 4 wk, we tested whether their phenotype would ...
CORRELATED
EXPRESSION
DEPENDENCE, LECTIN, BETWEEN
OF T CELL GROWTH
SENSITIVITY
AND CYTOLYTIC CYTOLYTIC
TO
VICIA
ACTIVITY
T CELLS
FACTOR
VILLOSA
IN HYBRIDS
AND T LYMPHOMAS*
By ANDREAS CONZELMANN,~: AUGUSTO SILVA,§ MAURIZIO CIANFRIGLIA,I] CHANTAL TOUGNE, RAFICK P. SEKALY, AND MARKUS NABHOLZ From the Genetics Unit, Swiss Institute for Experimental Cancer Research and Ludwig Institute for Cancer Research, Lausanne Branch, 1066 Epalinges, Switzerland
T h e use of conditioned media from leukocyte cultures activated by T cell mitogens (1, 2) has allowed the derivation of cloned cell lines from cytolytic T lymphocytes (CTL), 1 which stably express antigen-specific cytolytic activity (3-5). T h e growthpromoting activity of conditioned media for C T L lines is called T cell growth factor (TCGF) or interleukin 2. It has been purified and consists o f a glycoprotein (gp) with a weight of 15 kilodaltons (kd) in m a n and rat (6, 7) and of 30 kd in the mouse (8). T h e action of T C G F is mediated through specific surface receptors that become expressed on T lymphocytes upon exposure to antigenic stimulator cells or mitogens (9-11). In the experiments to be described in this paper we have used the crude supernatant of concanavalin A (Con A) -stimulated rat spleen cells (CS) as a source of T C G F . Cloned C T L lines can be derived from C T L - c o n t a i n i n g mixed lymphocyte cultures either by immediate cloning in the presence of T C G F and feeder cells (CTLA) or by maintaining cell populations in T C G F for several months before cloning in T C G F without feeder cells (CTL-B) (for discussion see ref. 12). C T L - B lines differ from normal C T L and C T L - A lines in morphology and karyotype (13) and by the fact that their growth is not dependent on feeder cells or on any growth factor besides T C G F . W h e n C T L lines are plated in the absence of T C G F , cell growth stops reversibly in G1 (14; R. Sekaly, unpublished observations). We have reported previously that somatic cell fusion of T C G F - d e p e n d e n t murine C T L - B or C T L - A lines with T C G F - i n d e p e n d e n t murine T l y m p h o m a s yields two types of hybrids (5, 12). W h e n the culture m e d i u m used during the hybrid selection contains CS, the majority of the isolated hybrids is cytolytic and dependent on CS (CS+). Most of these hybrids stably maintain their phenotype. O n the other hand, selection in m e d i u m without CS only yields noncytolytic, CS-independent (CS-) * Supported in part by grants from the Swiss National Foundation and the Swiss Cancer League. :~Present address: Washington University, Division of Hematology-Oncology, 660 S. Euclid, St. Louis, MO 6:t I 10. § Recipient of a fellowship from the Swiss Institute for Experimental Cancer Research. HRecipient of a fellowship from the European Molecular BiologyOrganization. 1Abbreviations used in this paper: Con A, concanavalin A; CS, supernatant from Con A-slimulated rat spleen cells; CS , CS independent; CS+, CS dependent; CTL, cytolytic T lymphocyte; gp, glycoprotein; HAT, hypoxanthine, aminopterin, thymidine; kd, kilodahons; PBS, phosphate-buffered saline; TCGF, T cell growth factor; VV, Vicia villosa lectin. J. ExP. MED.© The Rockefeller University Press • 0022-1007/82/11/1335/17 $1.00 Volume 156 November 1982 1335-1351
1335
1336
T CELL GROWTH FACTOR DEPENDENCE IN T CELL HYBRIDS
hybrids. These h y b r i d s also m a i n t a i n their p h e n o t y p e in that they do not b e c o m e cytolytic when grown in presence of CS. Thus, these characteristics are fixed a n d inherited over m a n y cell generations. W e decided to test the hypothesis that one t y p e of h y b r i d is derived by c h r o m o s o m a l segregation from the o t h e r type. U n d e r this assumption, it a p p e a r e d more p r o b a b l e that C S - hybrids are derived from CS + t h a n vice versa, because (a) we consistently found significantly m o r e h y b r i d s when selecting in CS t h a n without CS, regardless of w h e t h e r C T L lines were fused with a d i p l o i d or t e t r a p l o i d T l y m p h o m a line, a n d (b) we d i d not o b t a i n C S - h y b r i d s in a cross involving a t e t r a p l o i d C T L line a n d a d i p l o i d T l y m p h o m a line (12). Therefore, we hypothesized that CS ÷ h y b r i d s c o n t a i n a C T L line c h r o m o s o m e c a r r y i n g one or several genes that r e n d e r a cell CS + a n d that b o t h copies of this c h r o m o s o m e are lost in the C S - hybrids. H e r e we report on the isolation a n d c h a r a c t e r i z a t i o n o f C S variants derived from cloned CS ÷ hybrids. T h e striking correlation between the expression of CS d e p e n d e n c e a n d cytolytic activity i n d u c e d us to e x a m i n e w h e t h e r o t h e r markers typical o f C T L lines c o n t i n u e to be expressed in the cytolytic hybrids a n d are lost in C S - variants. T130 a n d T145 have been described as surface gp specific for resting a n d m a t u r e c y t o l y t i c a l l y active T lymphocytes in the mouse, respectively (15, 16). W e have previously r e p o r t e d (17) that m u r i n e C T L lines express T145 b u t not T130, whereas m u r i n e T l y m p h o m a s contain T130 b u t not T145. It is possible t h a t T145 a n d T130 represent differently glycosylated forms o f the same protein, b u t it is not clear w h e t h e r the expression of T145 is i m p o r t a n t for the cytolytic function (17). T h e search for a specific reagent for T145 led to the discovery o f a lectin from Vicia villosa (VV), which has high affinity for t e r m i n a l N-acetyl-D-galactosamine (18) a n d binds T145 b u t not T130 of the l y m p h o b l a s t s c o n t a i n e d in m i x e d leukocyte cultures (19). All C T L lines tested so far b i n d high a m o u n t s o f V V a n d are very susceptible to its cytotoxic effect, whereas T l y m p h o m a s b i n d 500-1,000-fold less V V a n d are highly resistant (17). Therefore, we have c o m p a r e d the sensitivity to V V o f CS + h y b r i d s a n d C S - variants. Materials and Methods The culture medium used for all cell types was Dulbecco's modified Eagle's medium supplemented as previously described (20), containing 5% fetal calf serum. CS was produced by culturing rat spleen cells from outbred OFA rats in this medium with 5 /~g/ml of Con A for 48 h. For the growth of TCGF-dependent cell lines, a final concentration of 25% of this crude supernatant was added to the culture medium. All cell lines and hybrids were grown in 60-mm plastic tissue culture dishes and subcultured twice weekly by splitting at ratios of 1:10-1:50. Hybrids, clones, and hybrid variants isolated in 96-well microtiter plates in the presence of irradiated macrophages were grown in 24-well plates (Costar, Data Packaging, Cambridge, MA) for 1 wk before being transferred into petri dishes. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. B~i.I.SF.1 is a cloned murine CTL line derived from a female C57BL/6 mouse (21). At the time of fusion it had been in culture for >2 yr and had strong cytolytic activity against the BALB/c myeloma S194. BW5147 is a spontaneous T lymphoma from an AKR mouse. A thioguanine- and ouabain-resistant double mutant from the BW5147 T cell lymphoma was obtained from B. Hyman, The Salk Institute, San Diego, CA. B6.1.SF.1 and the TCGFdependent hybrids are slightly adherent and were removed by a 5-min incubation in EDTA (0.2 g/l) in phosphate-buffered saline (PBS). Cells were checked monthly for mycoplasma contamination by the method of Fogh (22) as modified by J. W. Stocker (personal communication) and were always found to be mycoplasma free. Production ofttybrids. Before fusion, the BW5147 line was grown for two passages in medium Cell Culture and Cell Lines.
CONZELMANN ET AL.
1337
containing thioguanine and ouabain to eliminate any revertants. Exponentially growing B6.1.SF.1 (8 × 106) and BW5147 (10 7) cells were fused using 45% polyethylene glycol in a procedure essentially identical to the one described by Galfr~ et al. (23). Hybrids were selected in culture medium containing 5 × 10-4 M hypoxanthine, 5 × 10-v M aminopterin, 1.6 × 10-5 M thymidine (HAT) and 10-3 ouabain containing 25% of CS. Aliquots of 2 × 104 cells (based on the initial concentration of the fusion mixture) were dispensed into flat-bottomed wells of 96-well microtiter plates (Costar, Data Packaging)) containing 104 irradiated (2,000 rad) C57BL/6 mouse peritoneal macrophages (3), in a total volume of 200 pl of selective medium. Cells were fed weekly by replacing -2/3 of the selective medium. After 25 d, growing hybrids from 10 different wells were detached by vigorous pipetting with a micropipette and half of the cells were used to measure the cytolytic activity of the hybrids (see below). Assay of Cytolytic Activity. Culture medium was used throughout the test. Cells were harvested, washed twice, counted, and placed into round-bottomed 96-well microtiter plates. After serial dilution of the cells, 104 ~lCr-labeled S194 target cells/well and Con A (10 # g / m l final concentration) were added. Plates were centrifuged for 1 min at 1,000 rpm. and incubated for 4 h at 37°C. Specific isotope release into the medium was determined according to Brunner et al. (24). The same procedure was used for testing cells from cloning plates, except that these were not counted or serially diluted. Cloning of Cytolytic Hybrids. Seven independent hybrids were cloned by placing an average of one cell/well/200 pl of culture medium supplemented with 25% CS into flat-bottomed 96well plates, which, in addition, contained 104 irradiated (2,000 rad) murine macrophages from a peritoneal wash. The cloning efficiency in this first cloning ranged from 25 to 100%. Growing clones were detached and tested for cytolytic activity in the same way as the original hybrids. Cytolytically active clones were obtained from six of the seven hybrids and all but one cloned hybrid (15.4) were recloned once more. In this recloning step, the cloning efficiency was between 80 and 100%. One clone, 9.4, was also recloned by micromanipulation of single cells into cloning wells. Selection of CS-independent Variants. Variants were selected in flat-bottomed 96-well microtiter plates containing 104 irradiated (2,000 rad) murine peritoneal macrophages, which effected the removal of dead cells by phagocytosis. TCGF-independent, cloned hybrids were washed, resuspended in normal culture medium, and added to the plates at 2 × 104-2 × 10' cells/200 /×l/well. Cells were fed weekly, and after a culture period of 14-18 d, wells containing growing colonies or surviving cells were harvested and assayed for cytolytic activity. From each hybrid some C S - variants were isolated and maintained in normal culture medium in petri dishes. Determination of VV Resistance, TCGF Dependence, and Titration of CS on Different Cell Lines. For these assays, 104 cells/well/ml were placed in 16-mm, 24-well plates and cultured for 4 d. Cells from individual wells were then counted with a coulter counter. To determine their V V resistance, the cells were grown in serial dilutions of purified V V lectin (obtained from J. P. Mach, prepared as described [17]) and 25% of CS. T C G F dependence was assessed by washing the cells and culturing either in culture medium or in medium supplemented with 25% of CS. The relative dependence of cell lines was determined by culturing the washed cells in serial dilutions of CS. Determination of TCGF Receptors. The capacity of different cell lines to remove T C G F from CS was measured by absorption. The relative amount of T C G F present in CS was measured by its capacity to stimulate growth of the TCGF-dependent C T L line BD.2.118, a line developed in our laboratory that grows in suspension. Aliquots containing 4 × 103 cells/100 #1 were added to serial dilutions of a test sample in 96-well round-bottomed microtiter plates. After 48 h of culture and a final [3H]thymidine pulse of 6 h (1/~Ci/well), cells were transferred and washed on glass filters with an automatic cell harvester, and [3H]thymidine incorporation was determined in a/~-counter. Plateau levels of incorporation reached 5-10 × 104 cpm, whereas control values in the absence of CS were 200-300 cpm. A 1:8 dilution of CS in culture medium was used in absorption experiments, as this was the lowest concentration giving plateau values of incorporation. To remove T C G F from cells grown in CS before the absorption procedure, exponentially growing cells were harvested, washed three times, resuspended, cultured for 6 h in normal culture medium, and then washed once more. Cells grown without CS were simply washed. Sometimes, washed cells were fixed in 2% glutaraldehyde for 5 rain at room tempera-
1338
T CEI,I, GROWTH FACTOR DEPENDENCE IN T CEI,L ttYBRIDS
ture, washed, and frozen. For absorption, serial dilutions of washed cells in culture medium were added to round-bottomed 96-well microtiter wells and cooled to 4°C. Ice-cold CS was added at a final concentration of 1:8 and absorption was allowed to proceed for 60 rain at 4°C with occasional shaking. (Previous absorption studies showed that half-maximal absorption was reached within 5 min.) After centrifugation, the supernatants were removed, irradiated (2,000 tad), and frozen, and the remaining TCGF activity was measured as described above. Duplicate samples were tested in both the absorption and TCGF assays, and the percent unabsorbed TCGF was calculated by comparison with the CS titration curve, which had been linearized by probit plot as described by Smith (25). Cell Surface Labehng with Nat~H4. Cells were labeled using a procedure essentially identical to the one described by Gahmberg et al. (26). All cells to be labeled were grown for 3 d in culture medium supplemented with 25% CS, regardless of their dependence on CS. Exponentially growing cells were washed, incubated at room temperature for 10 rain in 50 mM c~methylmannoside in PBS to remove residual Con A, and then washed three times, resuspended tO 10 7 cells/ml in Dulbecco's PBS, and incubated with galactoseoxidase (5 U/ml) and neuraminidase (0.025 U/ml) for 60 rain at 37°C. After an additional three washes, the cells were resuspended to 107/ml and reduced with 1 mCi/ml of NaBaH4 for 40 min. They were then washed twice more and lysed in 0.5% NP-40 plus 1 mM PMSF for 30 rain on ice and frozen at -70°C. Alternatively, cells were oxidized with sodium metaperiodate according to Gahmberg and Andersson (27). In this case, washed cells were resuspended to a concentration of 10 7 cells/ml in PBS, treated with periodate at a final concentration of 1 mM on ice for 5 min, and then washed and reduced as cells oxidized with galactoseoxidase. After addition of sodium dodecyl sulfate, the lysates were boiled for 3 rain as described previously (17), and gel electrophoresis was performed in 8% polyacrylamide slab gels according to Laemmli (28). Gels were processed by fluorography as described by Bonner and Laskey (29). Determination of the Cellular DNA Content. Exponentially growing cells or freshly isolated mouse thymocytes were washed, and chicken erythrocytes were added to each sample as an internal control. The mixture was then treated with RNase (80 Kunitz U/ml) for 1 min and stained with the DNA-binding dye propidium iodide (50/*g/ml) in the presence of 0.05(~ NP40 as described by Taylor and Milthorpe (30). The cells were then passed on a FACS-II flow cytometer (B-D FACS Systems, Becton, Dickinson & Co., Sunnyvale, CA) using 488-nm excitation wavelength. Materials. Plastics were obtained from Nunc (Bio AG/Gibco Europe, Basel, Switzerland); fetal calf serum from Seromed GmbH (Munich, FRG); Con A from Pharmacia Fine Chemicals AB (Uppsala, Sweden); thioguanine, ~-methylmannoside, phenylmethanesulfonyl fluoride, RNase, and propidium iodide from Sigma Chemical Co. (St. Louis, MO); hypoxanthine, EDTA, and 2'deoxythymidine from Merck & Co. (Rahway, NJ); polyethylene glycol, ouabain, amethopterine, and sodium dodecyl sulfate from Serva Feinbiochemica GmbH (Heidelberg, FRG); galactose-oxidase from Kabi AG (Stockholm, Sweden), Nonidet P-40 from Dr. W. Kolb AG (Hedingen, Switzerland); '~XCrfrom Institut National des Radioelements (Fleurus, Belgium) Belgium; and all other radiochemicals, including a 14C-methylated protein mixture, from Amersham International plc (Amersham, England). Results
Description of Hybrids.
All results reported here were o b t a i n e d with hybrids from a fusion called T2. In this cross, a t h i o g u a n i n - an d ouabain-resistant m u t a n t of the m u r i n e T l y m p h o m a BW5147 was crossed with the m u r i n e C T L - B line B6.1.SF. 1 (derived by yon B o e h m e r et al. [21]) an d hybrids were selected in H A T an d ouabain. Selecting in CS, we observed colonies in 36 o f 48 wells, w i t h o u t CS in 2 of 48 wells. No colonies were observed in a mock fusion w i t h o u t p o l y e t h y l e n e glycol. T o confirm the hybrid n a t u r e o f the isolates, we exploited the fact that the two p a r e n t a l lines carry different Thy-1 alleles. U s i n g m o n o c l o n a l antibodies against Thy-1.1 an d T h y 1.2, we found that all isolates expressed b o t h of these d et er m i n an t s. S o m e hybrids were screened for cytolytic activity, an d 7 out of 10 selected in CS had strong activity.
CONZELMANN ET AL.
1339
T h e active h y b r i d s were cloned. H y b r i d s are n a m e d b y a series of n u m b e r s , the first of which indicates the original well from which the h y b r i d was isolated, the second a n d further n u m b e r s used for clones a n d subclones o b t a i n e d from the original isolate. Thus, h y b r i d s with different first n u m b e r s are derived from i n d e p e n d e n t fusion events. Clones derived b y m i c r o m a n i p u l a t i o n are i n d i c a t e d b y the letter M. T h e p h e n o t y p e o f the h y b r i d clones s t u d i e d further was tested on d a y 88 after fusion a n d is described in T a b l e I. All o f t h e m d i s p l a y cytolytic activity, are sensitive to V V , a n d d e p e n d on CS for growth. Selection and Properties of CS- Variants. 91-96 d after fusion, i.e., 26-30 days after cloning, cells of the various h y b r i d s were transferred into m e d i u m w i t h o u t CS a n d p l a t e d at different cell c o n c e n t r a t i o n s (2 X 104 to 20/well) in m i c r o t i t e r plates. After 10-14 d, colonies o f C S - variants from four h y b r i d s b e c a m e visible to the n a k e d eye. A s s u m i n g 100% cloning efficiency, we c a l c u l a t e d the p r o p o r t i o n s of C S - variants (V) present at the b e g i n n i n g o f the selection using Poisson statistics, a n d from this figure a n d the e s t i m a t e d n u m b e r of generations (n) elapsed b e t w e e n cloning a n d the b e g i n n i n g of v a r i a n t selection, we d e t e r m i n e d the rate of v a r i a n t p r g d u c t i o n (f) b y the e q u a t i o n f - - V / n (31). V a l u e s for f are 3 × 10 -~ (clone 9.4.7), 6 X 10 -~ (clone 15.4), 7 X 10 -6 (clone 16.8.15), a n d 1.3 X 10 -4 (clone 25.2.16). Clone 23.6.9 d i d not yield C S - variants in two experiments: in this h y b r i d , f was therefore < 4 X 10 -s. These calculations neglect the fact t h a t C S - variants a n d C S + h y b r i d s have not necessarily the s a m e growth rates a n d C S - variants m a y be selected for i n a d v e r t e n t l y , as T C G F m i g h t b e c o m e l i m i t i n g occasionally before subculture. T h e y also do not take into account fluctuation of the n u m b e r of variants due to the r a n d o m s a m p l i n g when cultures are split for r o u t i n e transfer. H y b r i d s 9.4.M3 a n d 33.4.14 d i d not give rise to distinct C S - colonies, b u t the p l a t e d cells seemed to r e m a i n alive a n d , after s u b c u l t u r i n g the content o f several wells with the highest cell density (2 × 104), we o b t a i n e d cell p o p u l a t i o n s that grew in the absence of CS from b o t h o f these hybrids. C S - variants were designated b y the a d d i t i o n of the n u m b e r of the isolate p r e c e e d e d b y the s y m b o l CS to the n u m b e r o f the p a r e n t a l hybrid. T h e variants were not cloned a n d different variants isolated from the same h y b r i d are not necessarily i n d e p e n d e n t . TABLE I
Characteristics of Cloned Hybrids Cytolytic* activity
VV resistance:~
CS dependence§
Cell line B6.1.SF.I BW5147 9.4.M3 9.4.7 15.4 23.6.9 25.2.16 33.4.14
1:1
0.3:1
80 -44 45 39 24 19 22
60 -30 33 24 12 12 12
50%
90%
1.1 >154 1.9 1.9 2.1 1.9 2.5 1.9
3.2 >154 5.8 6.3 9.2 5.8 6.5 6.5
+CS
66 77 50 42 41 44 66 39.5
-CS
0.5 138 2.3 1.2 1.6 0.9 1.5 0.4
* Percent specific 51CFrelease at 1:1 and 0.3:1 CTL/target cell ratios. :~Concentration of VV (#g/ml) that reduces cell recovery by 50 and 90%. § Ratio of the number of cells recovered after 90 h ofcuhure to the number of cells put into culture at time 0 either with (+) or without (-) CS.
1340
T CELL GROWTH
FACTOR
DEPENDENCE
PERCENT 20 I
SPECIFIC
SlCr R E L E A S E
40 I
60 I
9.4.M3 9.4.7
80 I
U
•
)OO
IN T C E L L I I Y B R I D S
¢bo#o
•
•
100 [
ooo
•
15.4
008800 oqpoo°o • •
16.8.15
•
25.2.16
•
~
FIG. 1. Cytolytic activity of CS + clones (O) or CS variants (C)). C o n t e n t s of wells c o n t a i n i n g visible colonies were harvested at 14-18 d after p l a t i n g in microtiter plates. Cells were assayed without c o u n t i n g on S194 target cells in the presence of Con A. T A B L E II Cytolytic Activity of CS- Variants*
Cell line
Parental hybrid
9.4.M3 9.4.7 15.4 16.8.15 25.2.16 33.4.14
60 61 51 39 33 45
CS variants 0, 0, 0, 0, 0, 0
0, 0, 0, 0, 0,
1 0, 0, 0, 0,
6 0 0 0
CS+ subclones 41, 37 65, 66 37, 35 52
* N u m b e r s indicate percent specific ~lCr release at a 10:1 c y t o l y t i c / t a r g e t cell ratio.
14 and 18 d after initiation of the selection, growing colonies or surviving cells (9.4.M3) were collected from the selection plates and their cytolytic activity was measured in a semiquantitative assay. As shown in Fig. 1, C S - variants had relatively low cytolytic activity in comparison with subclones isolated simultaneously in CSsupplemented medium. T h e remaining cytolytic activity of C S - variants might be due to the persistence of CS ÷ cytolytic hybrids in the selection plates, because 1 wk later, after having been transferred to 60-mm culture dishes, none of the C S - variants had any detectable cytolytic activity, whereas the parental hybrid clones, which had been grown in CS during the whole duration of selection, maintained their lytic capacity (Table II). 28 or 61 d after the beginning of the selection of C S - variants, the hybrids and variants were tested for dependence on CS and V V resistance (Table III). C S - variants were CS independent, whereas all parental hybrids still depended on CS, except 33.4.14. In this hybrid, a high proportion of C S - variants appears to have accumulated at the time of testing (day 61 after the beginning of selection). Fig. 2 shows the growth inhibition of two hybrids and its variants by different doses of VV. Similar titration curves served as basis for calculations of the sensitivity to V V of the other hybrids (Table III). All C S - variants are highly resistant to V V with the exception of 9.4.M3.CS69, in which, as discussed later on, CS ÷ cells probably were not completely eliminated by the selection procedure. Stability of the Phenotype of CS- Variants. After the C S - variants had been maintained in CS-free culture medium for 4 wk, we tested whether their phenotype would revert
CONZELMANN ET AL.
1341
TABLE III Phenotype of CS- Variants * CS dependence:~
VV resistance§
Cell line -CS
50%
90%
9.4.M3 9.4.M3.CS52 9.4.M3.CS69
+CS 40 16 75
0.7 15 38
1.8
5.5 >154 2.3
9.4.7 9.4.7.CS 113
62 l1
1.2 10
4.8
7.8 >154
15.4
63 52 53 39
2.2
1.8
> 154
15.4.CS73 15.4.CS74 15.4.CS146
57 62 52
16.8.15 16.8.CS116 16.8.CS135
52 29 48
0.6 36 52
2.4
2.5 >154 >154
25.2.16 25.2.16.CS77 25.2.16.CS142 25.2.16.CS 143
60 57 36 46
0.7 70 30 80
1.4
2.7 >154 >154 >154
33.4.14" 33.4.14.CS82"
98 73
19 79
2.1
22.3 > 154
B6. I.SF.1 BW5147
82 36
0.7 124
1.1
1.8 >154
1.2
>154 >154 >154
* Experiments were done 24-28 d after the beginning of the selection except for 33.4.14 and 33.4.14.CS82, which were tested 61 d after the beginning of selection. :~ Ratio of the number cells recovered after 90 h of culture to the number of cells put into culture at time 0 either with (+) or without (-) CS. § Concentration of VV ~g/ml) that reduces cell recovery by 50 and 90%. by c u l t u r i n g t h e m a g a i n in C S for a f u r t h e r 6 wk. C o n c o m i t a n t r e a p p e a r a n c e o f C S d e p e n d e n c e , h i g h c y t o l y t i c a c t i v i t y , a n d V V sensitivity was o b s e r v e d o n l y in t h e case o f 9 . 4 . M 3 . C S 6 9 , b u t not in 9 . 4 . M 3 . C S 5 2 a n d t w o o t h e r C S - v a r i a n t s . T h e b e h a v i o r o f h y b r i d 9 . 4 . M 3 d u r i n g t h e selection o f C S - v a r i a n t s suggests t h a t t h e r e a p p e a r a n c e o f t h e p a r e n t a l p h e n o t y p e in 9 . 4 . M 3 . C S 6 9 r e f l e c t e d a r e l a t i v e i n c r e a s e o f C S + cells t h a t h a d e s c a p e d selection r a t h e r t h a n a c h a n g e in p h e n o t y p e o f C S - cells. 9 . 4 . M 3 . C S 6 9 cells m a i n t a i n e d for 6 w k e i t h e r w i t h or w i t h o u t C S w e r e c l o n e d b y m i c r o m a n i p u l a t i o n . All t h e clones w e o b t a i n e d w e r e e i t h e r c y t o l y t i c , C S +, a n d V V sensitive or n o n c y t o l y t i c , C S - , a n d V V resistant. T h e l a t t e r d i d n o t r e v e r t to t h e p a r e n t a l C S + p h e n o t y p e d u r i n g c u l t u r e in CS. W e t h e r e f o r e p r e s u m e t h a t t h e 9 . 4 . M 3 . C S 6 9 p o p u l a t i o n still c o n t a i n e d C S + cells. T h i s is s u p p o r t e d b y t h e p a r t i a l C S d e p e n d e n c e a n d V V sensitivity o b s e r v e d at 28 d a f t e r t h e b e g i n n i n g o f selection ( T a b l e III). H o w e v e r , t h e persistence o f C S + h y b r i d s d u r i n g > 3 0 p o p u l a t i o n d o u b l i n g s in t h e a b s e n c e o f C S is p u z z l i n g . W e s e a r c h e d in v a i n for T C G F a c t i v i t y in t h e c u l t u r e s u p e r n a t a n t s o f 9 . 4 . M 3 . C S 6 9 , b u t it is possible t h a t these cells p r o d u c e a n a m o u n t too
1342
T CELL GROWTH FACTOR DEPENDENCE IN T CELl. ttYBRII)S
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Fro. 3. Absorption of TCGF by various cell concentrations. BW5147 (O); B6. t.SF. 1 (0); 25.2.16 (&); 25.2.16.CS142 (A). low to b e d e t e c t e d b y t h e c u r r e n t m e t h o d s b u t s u f f i c i e n t to a l l o w s u r v i v a l a n d slow g r o w t h o f C S ÷ cells. W e c a n n o t e x c l u d e t h e p o s s i b i l i t y t h a t t h e 9 . 4 . M 3 p o p u l a t i o n c o n t a i n e d a class o f v a r i a n t s t h a t is e i t h e r n o t c o m p l e t e l y o r n o t i r r e v e r s i b l y C S - . T h e o c c u r r e n c e o f s u c h v a r i a n t s m i g h t b e r e l a t e d to t h e fact t h a t n o d i s t i n c t C S - c o l o n i e s w e r e o b s e r v e d in t h e s e l e c t i o n p l a t e o f 9 . 4 . M 3 , w h i c h i n d i c a t e s t h a t n o C S v a r i a n t s p r e - e x i s t e d in this c l o n e at t h e b e g i n n i n g o f t h e s e l e c t i o n .
CONZELMANN ET AL.
1343
Fic. 4. Photomicrographs of 25.12.15 (A) and 25.2.16.CS142 (B).
Fxc. 5. Surface glycoproteins of CS + hybrids and CS- variants separated by gel electrophoresis. Cells were labeled by oxidation with galactose-oxidase/neuraminidase and subsequently reduced with NaB3H4 at the beginning of CS- variant selection (day 100, F, I) or at day 145 (C, D, E, G, H, K). A, B6.1.SF. 1; B, BW5147; C, 9.4.M3; D, 9.4.M3.CS52; E, 9.4.M3.CS69; F, 16.8.15; G, 16.8.15.CS135; H, I, 25.2.16; K, 25.2.16.CS142. 50,000 cpm were loaded in each slot except for B, where 100,000 cpm were loaded. Weight standards indicated at the left side of gel I were ~4Cmethylated derivatives of myosin (200 kd), phosphorylase b (92.5 kd), bovine serum albumin (69 kd), and ovalbumin (46 kd). The T130 and T145 gps of BW5147 and B6. I.SF. 1, respectively, are indicated by an asterisk. Bands, or groups of bands, of the parental hybrids are designated by letters a-f.
Expression of TCGF Receptors in the CS- Variants. W e m e a s u r e d t h e c a p a c i t y o f h y b r i d s 25.2.16 a n d 25.2.16.CS142 to r e m o v e T C G F f r o m CS. R e s u l t s o b t a i n e d w i t h this a b s o r p t i o n assay (25) are in a c c o r d a n c e w i t h m o r e direct q u a n t i t a t i o n s o f surface r e c e p t o r s u s i n g p u r i f i e d , r a d i o l a b e l e d T C G F (6). As c o m p a r e d w i t h B6.1.SF. 1, t h e h y b r i d a n d its C S - v a r i a n t h a v e ~ 2 - f o l d l o w e r c a p a c i t y to a b s o r b , w h e r e a s t h a t o f B W 5 1 4 7 is at least 15-fold l o w e r (Fig. 3). E s s e n t i a l l y i d e n t i c a l results h a v e b e e n o b t a i n e d w i t h g l u t a r a l d e h y d e - f i x e d cells ( d a t a n o t shown). T h u s , a l t h o u g h i n d e p e n d -
1344
T CELL GROWTH FACTOR DEPENDENCE IN T CELL HYBRIDS RATIO G 1-PEAK'HEN ERYTHROCYTES 5
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Fic. 6. DNA content of hybrids was measured at day 96, i.e., before the selection of CS- variants was begun (11), or 28 d later (0, (3). Results are given as the ratio of the channel number containing the G]-peak of the test cells to that containing the peak of chicken erythrocytes. Each value is based on the measurement of 3,000 cells. Bars indicate 1 SD from the mean. Open symbols, CS- cells; closed symbols, CS + cells. Sum of the DNA of the two parental cells ( - -).
e n t o f T C G F , 2 5 . 2 . 1 6 . C S 142 still a b s o r b s T C G F as e f f i c i e n t l y as t h e p a r e n t a l h y b r i d . Morphology and Adherence to Plastic of CS + and CS- Hybrids. B 6 . 1 . S F . 1 cells a d h e r e to tissue c u l t u r e p l a s t i c as d o m a n y o t h e r C T L lines i n c o n t r a s t to B W 5 1 4 7 a n d o t h e r T l y m p h o m a s . T o c o m p a r e h y b r i d s w i t h r e g a r d to t h i s p r o p e r t y , C S + a n d C S - cells w e r e g r o w n i n tissue c u l t u r e d i s h e s for 3 d in t h e p r e s e n c e o f C o n A - f r e e CS. T h e r e a f t e r , t h e p e r c e n t a g e o f cells t h a t c o u l d b e r e m o v e d b y g e n t l e w a s h e s w a s d e t e r m i n e d . F o r C S + h y b r i d s , t h e v a l u e s r a n g e d f r o m 12 to 32%, w h e r e a s t h e y w e r e b e t w e e n 85 a n d 95% for t h e C S - v a r i a n t s . T h u s , t h e C S + a n d C S - h y b r i d s r e s e m b l e t h e C T L a n d t h y m o m a p a r e n t a l cells lines, r e s p e c t i v e l y . M i c r o p h o t o g r a p h s o f a parental hybrid and a CS v a r i a n t d e r i v e d f r o m it a r e r e p r e s e n t a t i v e for t h e
CONZELMANN ET AL.
1345
morphological appearance of all the T2 hybrids (Fig. 4). Surface gp Patterns. C T L lines express T145 but not T130, whereas T lymphomas express T130 but not T145 (17). To compare surface gp of CS + hybrids and CSvariants with those of the parental cell lines, the oligosaccharides at the surface of intact cells were radiolabeled by treating the cells with galactose oxidase and neuraminidase followed by exposure to tritiated borohydride. Parental hybrids were labeled at the beginning of the variant selection (day 100 after fusion) and again 45 d later (day 145) together with CS- variants. Separation of the labeled gp by gel electrophoresis yielded the autoradiographs of Fig. 5. Glycoprotein patterns of different CS + hybrids resemble each other, although band c of 9.4.M3 has the mobility of the T145 of the B6.1.SF. 1 line, whereas in the other CS + hybrids we find this band at a position that is intermediate between T145 and T130. Apart from this difference and from differences in the relative intensity of various bands, the parental hybrids have comparable gp patterns. Because VV binds a majority of surface gp of C T L lines (17), and no other specific reagent for the T145 gp is available, we have no means to unambiguously identify T145 in the hybrids, where its mobility is not identical with the T145 of the parental lines. The CS- variants show none of the parental bands a - f of the parental CS + hybrids and express no band with the mobility of the T130 of BW5147. We do not know how the bands of the variants are related to those of the parental hybrids. The difference in surface gp between parental hybrids and variants might reflect either the expression of different gp or different processing (e.g., glycosylation) of the same peptides. A similar change between CS + hybrids and CS- variants is observed when the surface gps are labeled via the sialic acid of their oligosaccharide moieties using periodate (data not shown). DNA Content of Hybrids and Variants. CS- variants might arise as a result of massive chromosome loss from the parental clones. Therefore, we decided to compare DNA contents of CS + and CS- hybrids by staining cells with propidium iodide. The results (Fig. 6) are given on a scale, on which the loss of an average size chromosome measures 0.08 U. It appears that all hybrids have DNA contents that are close to the sum of the DNA content of the two parental lines. The DNA content of CS- variants is either lower, equal to, or, in one case, higher than the one of the corresponding parental hybrids. Where CS- variants and CS + subclones were derived in parallel, there was no significant difference between them. The biggest difference among parental CS + clones and CS- variants are in the order of 1.2 U, i.e., ~ 14 chromosomes. Deviations of this size are statistically significant. Coefficients of variance show that the heterogeneity of the cloned CS + hybrids and CS- variants is greater than in the thymocyte standard population but is similar to the one of the parental cell lines. Discussion Among C T L × T lymphoma hybrids, the correlation between the continued expression of cytolytic activity and the addition of CS to the medium during hybrid selection has been observed in each of six independent crosses involving two different CTL-B lines (CSP.2.4 [32] and B6.1.SF.I[21]) and three different T lymphomas (BW5147, AKR.A, and MbC12, all derived from A K R mice), as well as in crosses between BW5147 and cytolytic T cells generated in mixed lymphocyte culture or between BW5147 and CTL-A lines (12). We have tested over a hundred hybrids selected in the absence of CS and all of them had no detectable or very low cytolytic
1346
T CELL GROWTH
FACTOR
DEPENDENCE
IN T CELL
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activity and were, as far as tested, VV resistant (33). We retained the term "CS dependence," as we have not tested whether pure T C G F is able to maintain the growth of CS ÷ hybrids. The purification of T C G F has been based on its capacity to stimulate proliferation of CTL-B lines and the parental C T L lines used for the crosses described here can be grown in partially purified human TCGF (1,000-fold purification from leukocyte culture supernatants containing 0.5% serum) (O. Acuto, unpublished observation). Various preparations of TCGF, partially purified from different sources and assayed on different T cell lines have comparable biochemical properties, and there is no evidence to suggest that more than one type of TCGF exists (6, 8, 34-36). It is unlikely that the CS + hybrids are dependent on another factor in CS that is different from TCGF and not required by either parental cell line. The fact that CS contains comparable amounts of growth-promoting activity for CS ÷ hybrids and the B6.1.SF. 1 parental line is circumstantial evidence against this possibility (Fig. 7). We find that cloned, cytolytic, CS ÷, VV-sensitive hybrids give rise to variants that are CS- and VV-resistant and that have drastically reduced cytolytic activity. Phenotypic changes of this type have also been observed in a cross (MN12) between BW5147 and a CTL-B line called CSP.2.4. (33), in which we analyzed CS- variants from five CS ÷ hybrid clones derived from two independent hybrids. With regard to VV resistance, the CS- variants resemble hybrids selected originally in the absence of CS. For most CS- variants this is also true with regard to cytolytic activity. Reinduction of cytolytic activity by culture in CS has not been observed in CS- hybrids originally selected in the absence of CS and has been the exception
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CONZELMANN ET AL.
1347
among CS- variants derived from CS + hybrids, as it occurred only in one of four CSvariants of the T2 fusion and was not observed in variants from the MN12 cross (A. Conzelmann, unpublished observation). The CS- variants also show a characteristic surface gp pattern different from the one of CS + hybrids which, in the case of the MN12 cross, is identical with the one of hybrids originally selected in the absence of CS (A. Conzelmann, unpublished observation). Because, with regard to their phenotype, the CS- variants derived from CS + hybrids and the CS- hybrids originally selected without CS are indistinguishable, we consider the latter as early arising CS- variants of CS + hybrids. This implies that the primary fusion product of a C T L with a T lymphoma line is CS dependent. This hypothesis is also supported by the fact that we consistently found significantly more hybrid colonies when selecting in the presence of CS than without (12). If the primary fusion product is CS +, but can give rise to CS- variants, we can postulate a genetic element present in the C T L line that represses the TCGFindependent mode of proliferation of the T lymphoma parent. In the simplest case, this may be a gene, switched on in C T L but inactive or lost in A K R - T lymphoma cells, the product of which prevents cell division unless it is inactivated or blocked in its action as a result of the exposure of the cell to TCGF. Loss or inactivation of such a gene in the hybrids would render the cell TCGF-independent. Because three out of four CS- variants from fusions MN12 and T2 (Fig. 4) (M.C. and A.C., unpublished) still express TCGF-receptors, it appears that the gene(s) controlling their expression has (have) to be different from this hypothetical "repressor gene." The mechanisms of growth control in normal and transformed cells have been studied by means of somatic cell fusion between transformed and nontransformed cells (for review see 37) and both "dominance" as well as "recessiveness" of the nontransformed phenotype have been reported. The contradictory findings might in part be caused by the fact that different parameters such as saturation density, serum requirement, senescence, malignancy, etc., were used to distinguish the normal from the transformed phenotype and that most fusions involved cells differing with regard to species origin, histiotype, and mode of transformation. Our hybrids belong to the class in which the nontransformed growth control regulation is "dominant." We are not aware of another report where a well-defined growth factor dependence has been analyzed in somatic cell hybrids. A better interpretation of our results may be possible when we understand the way by which the BW5147 thymoma parent is transformed. There is compelling evidence to imply endogenous murine leukemia virus in A K R leukemogenesis. However, the mechanism of transformation in this system is not yet understood (38). Although it is conceivable that some T lymphocyte leukemias respond to T C G F that they produce themselves (39), there is no indication that this is the case for BW5147, as this line does not produce T C G F either constitutively or after stimulation with mitogens (34, 40) and has no or only low amounts of T C G F receptors (Fig. 4). Nevertheless, we have to consider the possibility that TCGF-independent hybrids produce and respond to TCGF. We could not detect T C G F in the spent medium of several independent CS- variants even after stimulation with Con A and phorbol myristate acetate, but we cannot exclude the possibility that T C G F is produced and used at rates that preclude the accumulation of detectable amounts in the medium. If CS- variants are CS independent because they make TCGF, then they have to be able to utilize it
1348
T CELL GROWTH FACTOR DEPENDENCE IN T CELl. HYBRIDS
without expressing TCGF-receptors at the surface, as we have found one C S - variant (15.4.CS146) that expresses no detectable absorbing capacity for T C G F (data not shown). We have proposed chromosome loss as one possible mechanism by which C S variants arise from CS ÷ hybrids. T h e hypothesis predicts that cloned C S - variants cannot give rise to CS ÷ hybrids. Although we have not observed this type of transition in the limited n u m b e r of clones derived from 9.4.M3CS69 and 15.4.CS146, we cannot exclude that it occurs at low frequency since we have no powerful selection system for CS ÷ variants. T h e comparison of the D N A content of CS ÷ and their C S - variants allows us to exclude massive chromosome loss as a cause of the appearance of the C S phenotype. T o obtain conclusive evidence for or against models implying different chromosome constitutions of CS + and C S - hybrids, it will be necessary to reproduce our findings in crosses where parental chromosomes can be karyotypically distinguished. Alternative explanations for the transition from the CS + to the C S - state imply regulatory events involving a change in the state of the genetic element of C T L lines, which, in the hybrids, represses the CS independent replication of A K R - t h y m o m a s . It is difficult to build experimentally testable models of this kind, but it is worth noting that we could not obtain C S - variants from parental C T L lines (M. Nabholz, unpublished observations). T h e maturation of most CTL-precursors into mature, cytolytically active cells involves the acquirement of TCGF-dependence. 2 We do not know whether the coordinate expression of cytolytic activity, CS dependence, and V V resistance that was observed in several independent crosses between C T L lines and T l y m p h o m a s is related to a regulation mechanism that is governing normal T lymphocyte maturation. C S - hybrids might fail to lyse target cells for fortuitous reasons. T h e y might not, e.g., properly interact with Con A, which is needed in the C T L assay as the T2-hybrids, unlike the parental B6.1.SF. 1 C T L line, have no activity in its absence. This possibility is however unlikely, as C S - variants from other crosses were noncytolytic in spite of the fact that the parental hybrids were killing specifically. O n the other hand, a genetic element that represses a whole set of liver-specific functions has been found in dedifferentiated rat h e p a t o m a lines (41, 42). An analogous genetic element might for instance be present in BW5147, become inactive upon fusion with a C T L line and become activated again in the C S - variants. Summary Somatic cell fusion between cytolytically active, T cell growth factor- (TCGF) dependent murine T cell lines (CTL lines) and noncytolytic, T C G F - i n d e p e n d e n t murine T l y m p h o m a lines has yielded two types of somatic cell hybrids (5): cytolytic hybrids, growth of which is dependent on T C G F , and hybrids with very weak or undetectable cytolytic activity which grow at the same rate with or without T C G F . Here we report that the former can produce stable variants that resemble the latter type. Some of these T C G F - i n d e p e n d e n t variants still have T C G F receptors. High susceptibility to the cytotoxic effects of Vicia villosa lectin, a marker distinguishing the parental C T L lines from T lymphomas, is expressed by the T C G F - d e p e n d e n t hybrids 2 A. Glasebrook. Cytolytic T lymphocyte clones which proliferate aulonomously in response to antigenic stimulation. Manuscript submitted for publication.
CONZELMANN ET AL.
1349
b u t not b y the T C G F - i n d e p e n d e n t variants. T h e two types o f h y b r i d s also differ in the expression o f surface glycoproteins. W e propose t h a t there exists a genetic e l e m e n t in the C T L line t h a t represses the T C G F - i n d e p e n d e n t r e p l i c a t i o n m e c h a n i s m o f the T l y m p h o m a p a r e n t in the T C G F - d e p e n d e n t h y b r i d s a n d t h a t this genetic element is lost or switched off in the T C G F - i n d e p e n d e n t variants. We thank Dr. R. Hyman for BW5147 TG r OU r, Dr. J. P. Mach for the gift o f V V lectin, Dr. F. Fitch for monoclonal antibody against Thy-l.2, P. Zaech for FACS analysis, and R. Lees for helping to improve the style of the manuscript.
Receivedfor publication 1June 1982. References 1. Morgan, D. A., F. W. Ruscetti, and R. C. Gallo. 1976. Selective in vitro growth of Tlymphocytes from normal human bone marrows. Science (Wash. D. C.). 193:1007. 2. Gillis, S., and K. A. Smith. 1977. Long term culture of tumour-specific cytotoxic T cells. Nature ( Lond.). 268:154. 3. Nabholz, M., H. D. Engers, D. Collavo, and M. North. 1978. Cloned T-cell lines with specific cytolytic activity. Curr. Top. Microbiol. Immunol. 81:176. 4. Baker, P. E., S. Gillis, and K. A. Smith. 1979. Monoclonal cytolytic T cell lines. J. Exp. Med. 149:273. 5. Nabholz, M., M. Cianfriglia, O. Acuto, A. Conzelmann, W. Haas, H v. Boehmer, H. R. MacDonald, H. Pohlit, and J. P. Johnson. 1980. Cytolytically active murine T-cell hybrids. Nature (Lond.). 287:437. 6. Robb, R. J., A. Monck, and K. A. Smith. 1981. T cell growth factor receptors. Quantitation, specificity, and biological relevance.J. Exp. Med. 154:1455. 7. Gillis, S., K. A. Smith, and J. Watson. 1980a. Biochemical characterization of lymphocyte regulatory molecules. II. Purification of a class of rat and human lymphokines.J. Immunol. 124:1954. 8. Watson, J., S. Gillis, J. Marbrook, D. Mochizuki, and K. A. Smith. 1979. Biochemical and biological characterization of lymphocyte regulatory molecules. I. Purification of a class of murine lymphokines.J. Exp. Med. 150:849. 9. Coutinho, A., E.-L. Larsson, K. O. Gr6nvik, and J. Andersson. 1979. Studies on T lymphocyte activation. II. The target cells for concanavalin A-induced growth factors. Eur. J. Immunol. 9:587. 10. Larsson, E.-L., and A. Coutinho. 1979. The role of mitogenic lectins in T-cell triggering. Nature (Lond.). 280:239. 11. Larsson, E.-L. 1981. Mechanism of T cell activation. II. Antigen- and lectin-dependent acquisition of responsiveness to TCGF is a nonmitogenic, active response of resting T cells. Nature (Lond.). 126:1323. 12. Nabholz, M., M. Cianfriglia, O. Acuto, A. Conzelmann, A. Weiss, W. Haas, and H. von Boehmer. 1981. The production of murine cytolytic T cell hybrids. In Monoclonal Antibodies and T Cell Hybridomas. G. H. H~mmerling, U. H~.mmerling, and J. F. Kearney, editors. Elsevier/North-Holland, New York. 539-548. 13. Johnson, J.-P., M. Cianfriglia, and M. Nabholz. 1982. Karyotype evolution of cytolytic Tcell lines. In Isolation, Characterization and Utilization of T Lymphocyte Clones. C. G. Fathman and F. W. Fitch, editors. Academic Press, Inc., New York. 183-191. 14. Sekaly, R. P., H. R. MacDonald, and M. Nabholz. 1982. Growth regulation ofcytolytic Tcell lines by IL-2. In Isolation, Characterization and Utilization of T Lymphocyte Clones. C. G. Fathman, and F. W. Fitch, editors. Academic Press, Inc., New York. 193-203.
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T CELL GROWTH FACTOR DEPENDENCE IN T CELL ttYBRIDS
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