Proc. Nat. Acad. Sci. USA Vol. 72, No. 5, pp. 1873-1877, May 1975
Somatic Hybrid of Thymus Leukemia (+) and (-) Cells Forms Thymus Leukemia Antigens but Fails to Undergo Modulation (antigenic modulation/membrane antigens/tumor "escape")
WEITZE LIANG AND EDWARD P. COHEN La RabidaUniversity of Chicago Institute and the Department of Microbiology, University of Chicago, E. 65th St. at Lake Michigan, Chicago, Illinois 60649
Communicated by Hewson Swift, February 18, 1975 A somatic hybrid of ASL-1 leukemia cells ABSTRACT [H-2a, thymus leukemia (TL)1,2,3, Thy-lb] and LM(TK)cells [H-2k, TL(-), Thy-i (-), thymidine kinase deficient] was formed with the aid of inactivated Sendai virus. The selection of hybrid cell clones was facilitated by the inability of ASL-1 cells to grow in vitro and of LM(TK)cells to survive in hypoxanthine/amethopterin/thymidine medium. The H-2 antigens of both parental cells were formed in approximately equivalent amounts by the hybrid cells, and they possessed a hybrid karyotype. As determined by five independent experimental procedures (antibody and complement-mediated cytotoxicity tests, the reduction of specific antibody activity of antiserum of known titer, immunofluorescent tests, mixed hemagglutination tests, and their direct isolation), TL antigens but not Thy-i antigens were formed by the hybrid cells. TL antigens of the hybrids failed to undergo modulation under conditions leading to the modulation of TL antigens of parental ASL-1 cells. Modulation was attempted with TL 1,3, TL 2, or TL 1,2,3 antisera of high titer. Hybrid cells were incubated for up to 30 hr in medium with TL antisera. Both direct and indirect immune methods were attempted. These results indicate that cellular mechanisms controlling the expression of TL antigens may be distinguished from the capacity of the cells to undergo modulation.
The capacity of cells to modify the expression of certain membrane-associated antigens in response to specific antisera is exemplified by the thymus-leukemia (TL) system. Thymus cells of several mouse strains and murine leukemias form TL antigens; in the presence of TL antiserum, the TL antigens disappear from the cells (antigenic modulation). Further cellular metabolism in the absence of TL antiserum leads to a reappearance of these antigens (1). The modulation of TL antigens stimulated by TL antiserum allows leukemic cells to "escape" humoral immune defenses. TL(+) neoplastic cells grow without apparent inhibition in specifically preimmunized histocompatible recipients. The capacity of the cells to modulate their TL antigens exceeds the host's capacity to destroy them (1). This phenomenon is not restricted to the TL system
Previously, we determined that modulation of the TL antigens of ASL-1 cells resulted from a faster rate of degradation of TL antigens, than synthesis, resulting in a gradual loss of the antigens from the external membranes of the cells. In this publication, we report the successful fusion of ASL-1 cells, a TL(+) leukemia of strain A mice that possesses the capacity to undergo modulation in the presence of TL antiserum, and LM(TK) - cells, a thymidine kinase deficient, TL(-) mouse fibroblast cell line. The hybrid cells formed a hybrid karyotype, shared H-2 antigens of both parents, and were Thy-lb (-) and TL(+). However, they lost the capacity to undergo the modulation of TL antigens. TL antigens persisted after prolonged exposure to specific antiserum, indicating that the capacity for modulation was under control mechanisms separate from the capacity to form such antigens. MATERIALS AND METHODS
Animals. Inbred mouse strains of A/J, C3H/HeJ, C57BL/ 6J, BALB/CJ, 129/J, and AKR/J were obtained from Jackson Laboratories (Bar Harbor, Maine). F1 crosses of (BALB/C X C3H/He) mice were bred in our animal colony. A/J TL(-) congenic mice were a gift of Dr. E. A. Boyse. Parental Cells. Mouse leukemia ASL-l (TL 1,2,3, H-28, Thy-lb) is a spontaneously occurring neoplasm of strain A mice maintained by serial passage in histocompatible recipients.
LM(TK) - cells, a subline of LM cells, are maintained by growth in vitro in Dulbecco modified Eagle's medium (GIBCO, Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal calf serum (GIBCO), 0.5% lactalbumin hydrolysate (Nutritional Biochemicals Corp., Cleveland, Ohio), 30,gg/ml of 5-bromodeoxyuridine (BrdU) (Sigma, St. Louis, Mo.), 5 ,ug/ml of uridine (Calbiochem., LaJolla, Calif.), and 100 units/ ml of penicillin and 100 jug/ml of streptomycin (GIBCO). This cell line does not form thymidine kinase and is resistant to high concentrations of BrdU (3, 4). Sendai Virus. Sendai virus seed (parainfluenza type 1) (Microbiological Assocs., Bethesda, Md.) was propagated in embryonated eggs and titered by the method of Giles and Ruddle (5). It was inactivated by treatment with 0-propriolactone (Sigma, St. Louis, Mo.). Cell Fusion. ASL-l and LM-(TK) - cells were fused with the aid of ,3-propriolactone-inactivated Sendai virus at high pH (6). Approximately 1.4 X 107 cells from the spleens of A/J mice terminally ill with ASL-1 leukemia were mixed with 2 X
(2).
As yet, little is known of the genetic controls involved in the expression and capacity for modification of membraneassociated antigens. One means of approaching this problem is by fusing parental cells expressing clearly defined and distinguishable membrane antigens. Antigenic expression in the hybrid cells and their capacity for modulation may be investigated and correlated with the presence of other membrane antigens and their known linkage relationships. Abbreviations: TL, thymus leukemia; i.p., intraperitoneally. 1873
1874
Immunology: Liang and Cohen
Proc. Nat. Acad. Sci. USA 72
9
I p
0
FIG. 1. Metaphase chromosome spread of hybrid cells with 85 chromosomes. Both parental cell marker chromosomes are present (arrows).
106 LM(TK)- cells in a total volume of 2.0 ml of Eagle's minimal essential medium at pH 8.0. One thousand hemagglutination units of ,B-propriolactone-inactivated Sendai virus in 0.5 ml was added to the cell mixture, which was cooled to 40 for 20 min and then warmed to 370 for an additional 30 min. After the second incubation, the cells were transferred to 30 ml plastic tissue culture flasks (Falcon) and cultured at 370 in a high humidity incubator gassed with 5% CO2 and 95% air. Growth medium was added and incubation was continued for 24 hr, after which the culture fluid was aspirated and replaced by hypoxanthine/amethopterin/thymidine-selective medium (7). Isolation of Clones of Hybrid Cells. By the end of the second week of culture in hypoxanthine/amethopterin/thymidineselective medium, well-isolated colonies of cells were detected. Each colony was picked carefully and subcultured at low density in fresh hypoxanthine/amethopterin/thymidine-selective medium. Subclones were isolated, replated, and propagated indefinitely in the medium. For analysis of the karyotypes of parental and hybrid cells, we used an air-dry technique (8), with some modifications. The modal number of chromosomes of the parental or hybrid cells was determined by counting at least 30 well-delineated metaphase spreads in each group. Unique chromosomes characteristic of the parental cells were identified (Fig. 1).
Preparation of Specific Antisera. Antisera specific for membrane-associated antigens of the parental cells, were prepared as follows: (a) TL 1,3 antiserum was raised in 6- 10-week-old female (BALB/c X C3H)F1 mice (TL 2, H-2a) (9) injected intraperitoneally (i.p.) over a 10-week period according to a described schedule (10). (b) TL 1,2,3 antiserum was raised in 6- 10-week-old female A/J congenic TL(-) mice injected i.p. once each week over a 10-week period with 4.5 X 107 thymocytes from A/J mice (TL 1,2,3) (11). (c) TL 2 antiserum was raised in C57BL/6J female mice (TL(-), H-2b] injected i.p. once each week over a 12-week period with about 4.5 X 107 thymocytes from 129/J mice (TL 2, H-2b) (9).
(1975)
(d) H-2a antiserum was raised in adult C3H female mice (H-2k) injected with spleen cells from A/J mice (H-2a). H-2k antiserum was obtained from adult A/J mice injected with spleen cells from C3H animals. H-2b antiserum was raised in adult C3H mice injected with spleen cells from C57BL/6 mice In each instance, recipient animals received i.p., about 4.5 X 107 viable cells weekly over a 10-week period. (e) Thy-lb antiserum was prepared in 8- 12-week-old AKR mice of both sexes injected i.p. with thymocytes from C3H animals; Thy-la antiserum was raised in C3H mice injected i.p. with thymocytes from AKR donors by the protocol described in d. (f) Mouse antiserum against sheep erythrocytes was raised in AKR mice injected weekly i.p. with 0.1-0.5 ml of a 2.5% suspension of washed sheep erythrocytes (Robert Galvin) over a 6-week period. In each instance, the mice used for immunization were bled periodically under ether anesthesia from the retro-orbital plexus. The serum from clotted blood was heated to 560 for 30 min before it was stored in small aliquots at -70°. (g) Rabbit anti-mouse IgG was raised in New Zealand white rabbits injected with mouse IgG in complete Freund's adjuvant. Mouse IgG for immunization was prepared from normal mouse serum (12). Detection of the Sensitivity of Target Cells to Specific Antisera. The immunologic specificity and sensitivity of the cells to specific antisera was detected by several methods: (a)- antibody and complement-mediated microcytotoxicity tests were performed with certain modifications as described (10); (b) viable cells were stained indirectly with appropriate antisera
and fluorescein-conjugated rabbit anti-mouse Ig (10); (c) membrane-associated antigens associated with the cells were detected by mixed hemagglutination reactions (13) and (d) the capacity of hybrid or parental cells to reduce known titers of specific antisera was used for the detection of specific membrane-associated antigens and as an estimation of their relative density with analogous membrane antigens shared by different cell types. Susceptibility of Hybrid and Parental Cells to TL Antiserum and Complement after Incubation in TL Antiserum Without Complement (Antigenic Modulation). The TL antigens of the ASL-l cells used in these experiments possess the capacity to undergo modulation in the presence of specific antiserum (14). Attempts to induce the modulation of the TL antigens of [ASL-1 X LM(TK)-] hybrid cells were performed in the same way, using aliquots of the same TL antisera used for the modulation of ASb-l cells. About 2 X 106 hybrid cells were incubated at 370 in 4 ml of complete medium containing a 0.4 ml aliquot of TL antisera as indicated. At various times, the cells were tested for the presence of TL antigens by complement-mediated microcytotoxicity methods, the reduction of TL antiserum, and indirect fluorescent antibody techniques. Further attempts to induce the modulation of the TL antigens of the hybrid cells were made as follows: 2 X 106 hybrid cells were incubated at 370 for 5 hr in 4 ml of complete medium containing aO.4-ml aliquot of TL 1,3 antiserum. After this initial incubation period, the cells were chilled, centrifuged, and resuspended in fresh complete medium containing rabbit anti-mouse Ig (final concentration 1:10) and incubated at for an additional 20 hr. At the end of the second incubation, the proportion of hybrid cells killed by TL 1,3, TL 2, or
370
Proc. Nat. Acad. Sci. USA 72
(1975)
Failure of TL Antigens to Undergo Modulation 1010
TL 123
0 8410-
Anti H-20
-J -J
U,
._
= 0
-Y
6
I
Co0-
0-
2100
2
4
8
16
32
64
128 256 512 1024
ANTIBODY DILUTIONS (RECIPROCAL) FIG. 2. Cytotoxic effects of guinea pig complement and TL 1,3, TL 1,2,3, or TL 2 antisera on ASL-l cells. The proportion of cells killed was determined by trypan blue dye exclusion. NMS, normal mouse serum.
TL 1,2,3 antiserum and guinea pig complement mined.
was
deter-
Isolatioh and Electrophoresis of TL Antigens from Hybrid Cells. TL and other membrane-associated antigens are released in soluble form from murine cells after treatment with the detergent Nonidet40. They may be recovered by immunoprecipitation with specific antisera (15). TL antigens of Nonidet-40 lysates of parental ASL-1 and hybrid cells were immunoprecipitated (10). The immunoprecipitates were solubilized in sodium dodecyl sulfate and 2-mercaptoethanol, and electrophoresis was performed in 5.6% polyacrylamide gels (10,14). RESULTS Membrane-associated antigens of the parental and hybrid cells
ASL-1. (a) TL. ASL-1 cells express TL 1,2,3 antigenic specificities (9). Incubation of the cells in medium containing TL 1,3, TL 1,2,3, or TL 2 antiserum and fresh guinea pig complement induced their lysis (Fig. 2). The TL antisera used was cytotoxic toward cells of several mouse strains expressing TL antigens. Thymus cells from A/J (TL 1,2,3), C58 (TL 1,2,3), and 129/J mice (TL 2) (10) lysed in the presence of TL antisera and complement. ASL-1 cells reduce known titers of TL 1,3 antiserum and stained positively when incubated with TL 1,3 antiserum followed by fluorescent anti-mouse Ig. The cells exhibited a ring of fluorescent concentrated over the cytoplasm. Control cells incubated with normal mouse serum, followed by fluorescein-conjugated anti-mouse Ig, did not stain and were clearly negative (10). (b) Thy-lb. ASL-1 cells lysed in the presence of Thy-lb antiserum and guinea pig complement. They remained viable under similar circumstances if normal mouse serum or Thy-la antiserum was substituted for Thy-lb antiserum. They reduced, during incubation, a known titer of Thy-lb antiserum, but had no effect upon the titer of Thy-la antiserum. The cells were stained by Thy-lb antiserum and fluorescein-conjugated anti-mouse Ig, but failed to stain under similar conditions in the presence of Thy-la antiserum or normal mouse serum followed by fluorescein-conjugated anti-mouse Ig. (c) H-2a. ASL-1 cells originating in A/J mice are H-2a.
They lysed in the presence of H-2a antiserum and complement but remained viable in medium containing H-2b, H-2k antiserum, or normal mouse serum. More than 99% of the cells
Or/ Anti H-2K
.cD 410-
TL 1,3
cn
1875
-
.6
I
ir
It
Anti
H-2b
!!.,
--
a
--
4 8 16 32 64 128 256 512 1024 Antibody dilutions (Reciprocal) FIG. 3. Detection of H-2& and H-2k antigens on hybrid cells. Cytotoxic effects of H-2a, H-2k or H-2b antiserum, and complement on hybrid cells. 2
obtained from the spleens of A/J mice terminally ill with ASL-1 leukemia stained positively after incubation with H-2a antiserum and fluorescein-conjugated anti-mouse 1g. Cells incubated with H-2b or H-2k antiserum or normal mouse serum, followed by fluorescein-conjugated anti-mouse Ig, were not stained. (d) Mlodulation of the TL antigens of ASL-1 cells. The TL antigens of ASL-1 cells undergo modulation during incubation in medium containing TL 1,3 or TL 1,2,3 antiserum (see Fig. 5). LJI (TK) - Cells express H-2k antigens. They (1o not reduce known titers of H-2a antiserum or TL 1,3 antiserum, and resist lysis by guinea pig complement and TL 1,3 and H-2a antiserum. They are resistant to high titers of Thy-la, Thylb, and H-2b antisera. They stain positively after incubation with mouse H-2k antiserum and fluorescein-conjugated antimouse Ig, but failed to stain under similar conditions with TL 1,3, H-2a, H-2b, Thy-la, or Thy-lb antisera, followed by fluorescein-conjugated anti-mouse 1g. LMI(TK) - cells reduced the titer of H-2k antiserum during incubation, but did not change the titers of Thy-la or Thy-lb antiserum under similar conditions. [ASL-1 X LuI (TK) -] Hybrid Cells. (a) Expression of H-2a and H-2k antigens. Hybrid cells formed histocomnpatibility antigens characteristic of both parental cell lines. They lysed during incubation in medium containing either H-2a or H-2k antiserum and guinea pig complement (Fig. 3) and stained after incubation with H-2a or H-2k antiserum and fluorescent anti-mouse Ig, but failed to stain if incubated with H-2b antiserum or normal mouse serum, followed by fluorescent anti-mouse 1g. The entire cell population was affected; no evidence of a heterogeneous pol)ulatioll of cells was found. To avoid the possibility that the apparent sharing of H-2a and H-2k determinants by the hybrid cells resulted from antigens in common between H-2a and H-2k complexes, the "H2k" antiserum used was raised in A/J mice (H-2a) immunized with spleen cells from C3H animals (H-2k) and "H-2a" antiserum was raised in C3H mice immunized with spleen cells from A/J mice. The possibility that not all of the determinants unique to each H-2 complex were present on the cells could not be determined with the reagents we used. Similar results have been reported in analogous systems (16, 17). The capacity of the hybrid cells to reduce H-2a or H-2k antiserum was equivalent. During incubation, about 6 X 105 hybrid cells reduce by 50% equal titers of H-2a or H-2k antiserum.
1876
Immunology: Liang and Cohen
Proc. Nat. Acad. Sci. USA 72
100
a LUJ
ANTI TL 123
go
0 -J
-J-180 y 70
TL 1,3
By U)
V) 60
w
i 50 IIJ 0 40 a 30 m 20
0
I 10
Z.
O-
(1975)
(-)
I H
0
ANTIBODY DILUTIONS (RECIPROCAL)
0.5
1.0
2.5
5.0
75
10.0 12.5
15.0 20.0 25.0
NO. OF CELLS (X 106) FIG. 4. Detection of TL antigens of hybrid cells. (Left) Cytotoxic effects of TL 1,3, TL 1,2,3, or TL 2 antisera and guinea pig complement on hybrid cells. (Right) The effect of hybrid cells upon known titers of TL 1,2,3 antiserum. Changes in the titer of TL 1,2,3 antiserum after incubation with cell hybrids, ASL-1 cells, RADA-1 cells, thymocytes from A/J, (A/J X C3H)Fj or (A/J X C57BL/6)Fl mice. NMS, normal mouse serum.
The sharing of H-2a and H-2k antigens by individual hybrid cells was also indicated by the results of mixed hemagglutination studies. Hybrid cells incubated with H-2a antiserum, sheep erythrocytes sensitized previously with sheep cell hemolysin, and anti-mouse Ig formed clearly visible clusters. Similar results were obtained if H-2k antiserum was substituted for H-2a antiserum. Clusters were not found if normal mouse serum was used instead of H-2 antiserum. (b) TL. Sensitivity of the hybrid cells to TL antisera and their capacity to reduce known titers of such antisera were determined. More than 99% of the cells lysed in the presence of TL 1,3, TL 2, or TL 1,2,3 antiserum and complement (Fig. 4, left). They reduced TL antiserum of known titer. Thymus cells from F1 hybrids of TL(+) and TL(-) parents were used for comparison, as well as ASL-1 cells (TL 1,2,3) A/J thymocytes (TL 1,2,3), and RADA-1 cells (TL 1,2,3), a radiationinduced murine leukemia. Three different antisera of known titers were used in this study-TL 1,3, TL 1,2,3, and TL 2 antisera. The results indicated that about 5 X 105 ASL-1 cells and similar numbers of thymocytes from A/J mice reduced a known titer of TL 1,2,3 antiserum by 50%. However, about 10 times as many somatic hybrid cells or cells from F1 100O I
90 -J
a w
80 y
-i
70 V -J
60
-J
C) -J
W
0
50_
'i
LLJ
40 -J e)
30< __
m
IR
e.
0avv;
v-
*c
0c cur% Ab
1vc
ANTIBODY DILUTIONS (RECIPROCAL) FIG. 5. Failure of the TL antigens of hybrid cells to undergo modulation in the presence of TL antiserum. Hybrid cells were incubated for 22 hr at 370 in the presence of TL 1,3 or TL 1,2,3 antiserum. After incubation, the presence of TL antigens was determined by the cytotoxic effects of fresh TL 1,2,3, TL 1,3, or TL 2 antiserum and guinea pig complement on the hybrid cells. Parental ASL-1 cells were treated in the same way.
hybrid mice were required to reduce the titer to the same extent (Fig. 4, right). Experiments performed with TL 1,3 and TL 2 antisera yielded essentially the same results-the capacity of the somatic hybrid cells to reduce antiserum titers paralleled that of the F1 hybrids and was significantly less than parental ASL-1 cells or A/J thymocytes. The capacity of somatic hybrid cells sensitive to lysis by complement and TL antisera to reduce titers of TL antisera paralleled that of RADA-1 leukemia cells (TL 1,2,3). (c) Thy-i. Hybrid cells did not lyse in the presence of Thylb or Thy-la antiserum nor did they reduce known titers of Thy-1 antisera of either specificity (data not shown). Thy-lb and Thy-la antisera used were cytotoxic for thymocytes from A/J mice (Thy-lb) and for thymocytes from AKR mice (Thy-la), respectively. Hybrid cells incubated with Thy-lb or Thy-la antiserum and fluorescent anti-mouse Ig failed to stain and were clearly negative. The possibility that Thy-1 antigens were formed by the cells but were not detectable by immunologic methods detecting only "surface" antigens was not excluded by these experiments. Modulation of TL antigens on hybrid cells
The TL antigens of ASL-1 cells undergo modulation in the presence of TL antiserum; however, all attempts to induce modulation in hybrid cells were unsuccessful. Two different antiserum preparations, specific for TL 1,2,3 and TL 1,3 determinants, were used in attempts to induce modulation. [TL 2 antiserum does not induce modulation (11).] Each preparation was found to be capable of inducing complete modulation of the TL antigens of ASL-1 parental cells within 10 hr. However, incubation of hybrid cells for up to 30 hr in medium containing excessive concentrations of TL antiserum failed to induce modulation (Fig. 5). Further attempts to stimulate the modulation of hybrid cells were made with TL 1,3 antiserum and anti-mouse Ig. Cells were exposed to TL 1,3 antiserum for up to 5 hr, followed by an additional 20 hr in medium with anti-mouse Ig. At the end of the second incubation, their susceptibility to fresh TL 1,3, TL 1,2,3, and TL 2 antisera and complement was determined. Control ASL-1 cells, treated in the same way, were resistant, whereas hybrid cells lysed. TL, along with other membrane-associated antigens of mammalian cells, is released in soluble form during extraction
Nat. Acad. Sci. USA 72 (1975) Proc. PFailure of TL Antigens to Undergo Modulation with the nonionic detergent, Nonidet-40 (10, 14, 15). It may be iinmunoprecipitated with specific antisera. TL antigens, as well as both H-2a and H-2k antigens, were recoverable from Nonidet-40 extracts of the hybrid cells both before and after (Fig. 6) modulation was attempted. The antigens recovered with TL 1,3, TL 2, or TL 1,2,3 antiserum migrated in polyacrylamide gel electrophoresis as a single peak (Fig. 6). Similar results were obtained previously (10, 14) with Nonidet-40 extracts of ASIA cells. Immunoprecipitations of Nonidet-40 extracts of hybrid cells with TL 1,3 antiserum revealed the presence of a second slower migrating peak. It was identified previously as a tumor-associated antigen of ASL-1 cells, not found in extracts of thymocytes of nontumorbearing A/J mice (10). DISCUSSION
These data indicate that genetic mechanisms controlling the expression of TL antigens are separable from their capacity to undergo modulation. Previously, it was determined that only one of two antigenically distinct membrane structures of parental ASL-1 cells underwent modulation in the presence of antisera specific for both. ASIA cells form TL 1,2,3 antigenic determinants as well as a tumor-associated membrane antigen not associated with thymus cells of normal A/J mice. In the presence of both TL antiserum and antiserum specific for the tumor-associated antigen found on the membranes of the cells, the TL antigens underwent modulation but the tumorassociated antigen did not (14). This finding suggested that modulation was not "simply" the result of interaction of membrane antigens with specific antibody, i.e., the formation of an antigen-antibody complex on the surface of the cell, but that more complicated cellular control mechanisms might be involved. The finding that the TL antigens of the cellular hybrid of LM(TK)- and ASL-1 cells failed to undergo modulation under conditions that led to the modulation of the ASL-1 parental cells strongly supports this concept. The relative capacity of the hybrid cells to reduce titers of H-2a or Hj2k antisera was equivalent, likely indicating that the concentrations of H-2 antigens of each parent expressed by the cells was approximately the same. This is taken as an indication that the formation of H-2 antigens was regulated in a codominant manner. Modulation of the TL antigens of the hybrid was attempted by both direct (TL antiserum) and indirect (TL antiserum and anti-mouse Ig) methods. The overall time of exposure of the cells to antisera was up to 5-fold longer, using 16 times more Concentrated TL antiserum than required to completely modulate the TL antigens of the ASL-1 parents. "Capping" without endocytosis was observed when fluorescein-conjugated anti-mouse Ig was added, indicating that antigen-anti-
H chain
600 ,
1877
L chain
4'
49+
TL1 ,2,3
500
U
TL1, 3
* 4000 CL
TL2
E 300 U
W_
2001
C/)
^-~ 1oUl 0
4
8
12
20 !6 24 No of gel slice
28
32
FIG. 6. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the immunoprecipitates formed with TL 1,3, TL 1,2,3, or TL 2 antiserum from Nonidet-40 extracts of "5I-labeled hybrid cells. Hybrid cells were labeled with 126I by lactoperoxidase and incubated at 370 for 20 hr in medium containing TL antiserum. After washing, the cells were extracted with Nonidet-40.
body complexes remained on the surfaces of the cells and that complement activation might occur upon the addition of fresh guinea pig complement. This work was supported by grants from the National Science Foundation no. GB-40659 and the Leukemia Research Foundation. 1. Boyse, E. A., Stockert, E. & Old, L. J. (1970) Proc. Nat. Acad. Sci. USA 65, 933-938. 2. Aoki, J. & Johnson, P. A. (1972) J. Nat. Cancer Inst. 49, 183-192. 3. Kit, S., Dubbs, D. R., Piekarski, L. J. & Hsu, T. C. (1963) Exp. Cell Res. 31, 297-312. 4. Dubbs, D. R. & Kit, S. (1964) Exp. Cell Res. 33, 19-28. 5. Giles, R. E. & Ruddle, F. H. (1973) In Vitro 9, 103-107. 6. Croce, C. M., Koprowski, H. & Eagle, H. (1972) Proc. Nat. Acad. Sci. USA 69, 1953-1956. 7. Littlefield, J. W. (1964) Science 145, 709-710. 8. Rothfels, K. H. & Simmovitch, L. (1958) Stain Technol. 33, 73-77. 9. Old, L. J., Stockert, E., Boyse, E. A. & Kim, J. H. (1968) J. Exp. Med. 127, 523-539. 10. Yu, A. & Cohen, E. P. (1974) J. Immunol. 112, 1285-1295. 11. Boyse, E. A., Stockert, E. & Old, L. J. (1968) Int. Convoc. on Immunol. (Buffalo, N.Y.), pp. 353-357. 12. Campbell, D. H., Garvey, F. S., Cremer, N. E. & Sussdorf, D. H. (1970) Methods in Immunology (W. A. Benjamin, Inc., New York), 2nd ed., pp. 189-202. 13. Milgrom, E., Kano, K., Barron, S. L. & Witebsky, E. (1964) J. Immunol. 92, 8-16. 14. Yu, A. & Cohen, E. P. (1974) J. Immunol. 112, 1296-1307. 15. Schwartz, B. D. & Nathenson, S. G. (1971) J. Immunol. 107, 1363-1367. 16. Frye, L. D. & Edidin, M. (1970) J. Cell Sci. 7, 319-333. 17. Spenser, R. A., Hauschka, T. S., Amos, D. B. & Ephrussi, B. (1964) J. Nat. Cancer Inst. 33, 892-903.
Somatic Hybrid of Thymus Leukemia (+) and (-) Cells Forms Thymus Leukemia Antigens but Fails to Undergo Modulation (antigenic modulation/membrane antigens/tumor "escape")
WEITZE LIANG AND EDWARD P. COHEN La RabidaUniversity of Chicago Institute and the Department of Microbiology, University of Chicago, E. 65th St. at Lake Michigan, Chicago, Illinois 60649
Communicated by Hewson Swift, February 18, 1975 A somatic hybrid of ASL-1 leukemia cells ABSTRACT [H-2a, thymus leukemia (TL)1,2,3, Thy-lb] and LM(TK)cells [H-2k, TL(-), Thy-i (-), thymidine kinase deficient] was formed with the aid of inactivated Sendai virus. The selection of hybrid cell clones was facilitated by the inability of ASL-1 cells to grow in vitro and of LM(TK)cells to survive in hypoxanthine/amethopterin/thymidine medium. The H-2 antigens of both parental cells were formed in approximately equivalent amounts by the hybrid cells, and they possessed a hybrid karyotype. As determined by five independent experimental procedures (antibody and complement-mediated cytotoxicity tests, the reduction of specific antibody activity of antiserum of known titer, immunofluorescent tests, mixed hemagglutination tests, and their direct isolation), TL antigens but not Thy-i antigens were formed by the hybrid cells. TL antigens of the hybrids failed to undergo modulation under conditions leading to the modulation of TL antigens of parental ASL-1 cells. Modulation was attempted with TL 1,3, TL 2, or TL 1,2,3 antisera of high titer. Hybrid cells were incubated for up to 30 hr in medium with TL antisera. Both direct and indirect immune methods were attempted. These results indicate that cellular mechanisms controlling the expression of TL antigens may be distinguished from the capacity of the cells to undergo modulation.
The capacity of cells to modify the expression of certain membrane-associated antigens in response to specific antisera is exemplified by the thymus-leukemia (TL) system. Thymus cells of several mouse strains and murine leukemias form TL antigens; in the presence of TL antiserum, the TL antigens disappear from the cells (antigenic modulation). Further cellular metabolism in the absence of TL antiserum leads to a reappearance of these antigens (1). The modulation of TL antigens stimulated by TL antiserum allows leukemic cells to "escape" humoral immune defenses. TL(+) neoplastic cells grow without apparent inhibition in specifically preimmunized histocompatible recipients. The capacity of the cells to modulate their TL antigens exceeds the host's capacity to destroy them (1). This phenomenon is not restricted to the TL system
Previously, we determined that modulation of the TL antigens of ASL-1 cells resulted from a faster rate of degradation of TL antigens, than synthesis, resulting in a gradual loss of the antigens from the external membranes of the cells. In this publication, we report the successful fusion of ASL-1 cells, a TL(+) leukemia of strain A mice that possesses the capacity to undergo modulation in the presence of TL antiserum, and LM(TK) - cells, a thymidine kinase deficient, TL(-) mouse fibroblast cell line. The hybrid cells formed a hybrid karyotype, shared H-2 antigens of both parents, and were Thy-lb (-) and TL(+). However, they lost the capacity to undergo the modulation of TL antigens. TL antigens persisted after prolonged exposure to specific antiserum, indicating that the capacity for modulation was under control mechanisms separate from the capacity to form such antigens. MATERIALS AND METHODS
Animals. Inbred mouse strains of A/J, C3H/HeJ, C57BL/ 6J, BALB/CJ, 129/J, and AKR/J were obtained from Jackson Laboratories (Bar Harbor, Maine). F1 crosses of (BALB/C X C3H/He) mice were bred in our animal colony. A/J TL(-) congenic mice were a gift of Dr. E. A. Boyse. Parental Cells. Mouse leukemia ASL-l (TL 1,2,3, H-28, Thy-lb) is a spontaneously occurring neoplasm of strain A mice maintained by serial passage in histocompatible recipients.
LM(TK) - cells, a subline of LM cells, are maintained by growth in vitro in Dulbecco modified Eagle's medium (GIBCO, Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal calf serum (GIBCO), 0.5% lactalbumin hydrolysate (Nutritional Biochemicals Corp., Cleveland, Ohio), 30,gg/ml of 5-bromodeoxyuridine (BrdU) (Sigma, St. Louis, Mo.), 5 ,ug/ml of uridine (Calbiochem., LaJolla, Calif.), and 100 units/ ml of penicillin and 100 jug/ml of streptomycin (GIBCO). This cell line does not form thymidine kinase and is resistant to high concentrations of BrdU (3, 4). Sendai Virus. Sendai virus seed (parainfluenza type 1) (Microbiological Assocs., Bethesda, Md.) was propagated in embryonated eggs and titered by the method of Giles and Ruddle (5). It was inactivated by treatment with 0-propriolactone (Sigma, St. Louis, Mo.). Cell Fusion. ASL-l and LM-(TK) - cells were fused with the aid of ,3-propriolactone-inactivated Sendai virus at high pH (6). Approximately 1.4 X 107 cells from the spleens of A/J mice terminally ill with ASL-1 leukemia were mixed with 2 X
(2).
As yet, little is known of the genetic controls involved in the expression and capacity for modification of membraneassociated antigens. One means of approaching this problem is by fusing parental cells expressing clearly defined and distinguishable membrane antigens. Antigenic expression in the hybrid cells and their capacity for modulation may be investigated and correlated with the presence of other membrane antigens and their known linkage relationships. Abbreviations: TL, thymus leukemia; i.p., intraperitoneally. 1873
1874
Immunology: Liang and Cohen
Proc. Nat. Acad. Sci. USA 72
9
I p
0
FIG. 1. Metaphase chromosome spread of hybrid cells with 85 chromosomes. Both parental cell marker chromosomes are present (arrows).
106 LM(TK)- cells in a total volume of 2.0 ml of Eagle's minimal essential medium at pH 8.0. One thousand hemagglutination units of ,B-propriolactone-inactivated Sendai virus in 0.5 ml was added to the cell mixture, which was cooled to 40 for 20 min and then warmed to 370 for an additional 30 min. After the second incubation, the cells were transferred to 30 ml plastic tissue culture flasks (Falcon) and cultured at 370 in a high humidity incubator gassed with 5% CO2 and 95% air. Growth medium was added and incubation was continued for 24 hr, after which the culture fluid was aspirated and replaced by hypoxanthine/amethopterin/thymidine-selective medium (7). Isolation of Clones of Hybrid Cells. By the end of the second week of culture in hypoxanthine/amethopterin/thymidineselective medium, well-isolated colonies of cells were detected. Each colony was picked carefully and subcultured at low density in fresh hypoxanthine/amethopterin/thymidine-selective medium. Subclones were isolated, replated, and propagated indefinitely in the medium. For analysis of the karyotypes of parental and hybrid cells, we used an air-dry technique (8), with some modifications. The modal number of chromosomes of the parental or hybrid cells was determined by counting at least 30 well-delineated metaphase spreads in each group. Unique chromosomes characteristic of the parental cells were identified (Fig. 1).
Preparation of Specific Antisera. Antisera specific for membrane-associated antigens of the parental cells, were prepared as follows: (a) TL 1,3 antiserum was raised in 6- 10-week-old female (BALB/c X C3H)F1 mice (TL 2, H-2a) (9) injected intraperitoneally (i.p.) over a 10-week period according to a described schedule (10). (b) TL 1,2,3 antiserum was raised in 6- 10-week-old female A/J congenic TL(-) mice injected i.p. once each week over a 10-week period with 4.5 X 107 thymocytes from A/J mice (TL 1,2,3) (11). (c) TL 2 antiserum was raised in C57BL/6J female mice (TL(-), H-2b] injected i.p. once each week over a 12-week period with about 4.5 X 107 thymocytes from 129/J mice (TL 2, H-2b) (9).
(1975)
(d) H-2a antiserum was raised in adult C3H female mice (H-2k) injected with spleen cells from A/J mice (H-2a). H-2k antiserum was obtained from adult A/J mice injected with spleen cells from C3H animals. H-2b antiserum was raised in adult C3H mice injected with spleen cells from C57BL/6 mice In each instance, recipient animals received i.p., about 4.5 X 107 viable cells weekly over a 10-week period. (e) Thy-lb antiserum was prepared in 8- 12-week-old AKR mice of both sexes injected i.p. with thymocytes from C3H animals; Thy-la antiserum was raised in C3H mice injected i.p. with thymocytes from AKR donors by the protocol described in d. (f) Mouse antiserum against sheep erythrocytes was raised in AKR mice injected weekly i.p. with 0.1-0.5 ml of a 2.5% suspension of washed sheep erythrocytes (Robert Galvin) over a 6-week period. In each instance, the mice used for immunization were bled periodically under ether anesthesia from the retro-orbital plexus. The serum from clotted blood was heated to 560 for 30 min before it was stored in small aliquots at -70°. (g) Rabbit anti-mouse IgG was raised in New Zealand white rabbits injected with mouse IgG in complete Freund's adjuvant. Mouse IgG for immunization was prepared from normal mouse serum (12). Detection of the Sensitivity of Target Cells to Specific Antisera. The immunologic specificity and sensitivity of the cells to specific antisera was detected by several methods: (a)- antibody and complement-mediated microcytotoxicity tests were performed with certain modifications as described (10); (b) viable cells were stained indirectly with appropriate antisera
and fluorescein-conjugated rabbit anti-mouse Ig (10); (c) membrane-associated antigens associated with the cells were detected by mixed hemagglutination reactions (13) and (d) the capacity of hybrid or parental cells to reduce known titers of specific antisera was used for the detection of specific membrane-associated antigens and as an estimation of their relative density with analogous membrane antigens shared by different cell types. Susceptibility of Hybrid and Parental Cells to TL Antiserum and Complement after Incubation in TL Antiserum Without Complement (Antigenic Modulation). The TL antigens of the ASL-l cells used in these experiments possess the capacity to undergo modulation in the presence of specific antiserum (14). Attempts to induce the modulation of the TL antigens of [ASL-1 X LM(TK)-] hybrid cells were performed in the same way, using aliquots of the same TL antisera used for the modulation of ASb-l cells. About 2 X 106 hybrid cells were incubated at 370 in 4 ml of complete medium containing a 0.4 ml aliquot of TL antisera as indicated. At various times, the cells were tested for the presence of TL antigens by complement-mediated microcytotoxicity methods, the reduction of TL antiserum, and indirect fluorescent antibody techniques. Further attempts to induce the modulation of the TL antigens of the hybrid cells were made as follows: 2 X 106 hybrid cells were incubated at 370 for 5 hr in 4 ml of complete medium containing aO.4-ml aliquot of TL 1,3 antiserum. After this initial incubation period, the cells were chilled, centrifuged, and resuspended in fresh complete medium containing rabbit anti-mouse Ig (final concentration 1:10) and incubated at for an additional 20 hr. At the end of the second incubation, the proportion of hybrid cells killed by TL 1,3, TL 2, or
370
Proc. Nat. Acad. Sci. USA 72
(1975)
Failure of TL Antigens to Undergo Modulation 1010
TL 123
0 8410-
Anti H-20
-J -J
U,
._
= 0
-Y
6
I
Co0-
0-
2100
2
4
8
16
32
64
128 256 512 1024
ANTIBODY DILUTIONS (RECIPROCAL) FIG. 2. Cytotoxic effects of guinea pig complement and TL 1,3, TL 1,2,3, or TL 2 antisera on ASL-l cells. The proportion of cells killed was determined by trypan blue dye exclusion. NMS, normal mouse serum.
TL 1,2,3 antiserum and guinea pig complement mined.
was
deter-
Isolatioh and Electrophoresis of TL Antigens from Hybrid Cells. TL and other membrane-associated antigens are released in soluble form from murine cells after treatment with the detergent Nonidet40. They may be recovered by immunoprecipitation with specific antisera (15). TL antigens of Nonidet-40 lysates of parental ASL-1 and hybrid cells were immunoprecipitated (10). The immunoprecipitates were solubilized in sodium dodecyl sulfate and 2-mercaptoethanol, and electrophoresis was performed in 5.6% polyacrylamide gels (10,14). RESULTS Membrane-associated antigens of the parental and hybrid cells
ASL-1. (a) TL. ASL-1 cells express TL 1,2,3 antigenic specificities (9). Incubation of the cells in medium containing TL 1,3, TL 1,2,3, or TL 2 antiserum and fresh guinea pig complement induced their lysis (Fig. 2). The TL antisera used was cytotoxic toward cells of several mouse strains expressing TL antigens. Thymus cells from A/J (TL 1,2,3), C58 (TL 1,2,3), and 129/J mice (TL 2) (10) lysed in the presence of TL antisera and complement. ASL-1 cells reduce known titers of TL 1,3 antiserum and stained positively when incubated with TL 1,3 antiserum followed by fluorescent anti-mouse Ig. The cells exhibited a ring of fluorescent concentrated over the cytoplasm. Control cells incubated with normal mouse serum, followed by fluorescein-conjugated anti-mouse Ig, did not stain and were clearly negative (10). (b) Thy-lb. ASL-1 cells lysed in the presence of Thy-lb antiserum and guinea pig complement. They remained viable under similar circumstances if normal mouse serum or Thy-la antiserum was substituted for Thy-lb antiserum. They reduced, during incubation, a known titer of Thy-lb antiserum, but had no effect upon the titer of Thy-la antiserum. The cells were stained by Thy-lb antiserum and fluorescein-conjugated anti-mouse Ig, but failed to stain under similar conditions in the presence of Thy-la antiserum or normal mouse serum followed by fluorescein-conjugated anti-mouse Ig. (c) H-2a. ASL-1 cells originating in A/J mice are H-2a.
They lysed in the presence of H-2a antiserum and complement but remained viable in medium containing H-2b, H-2k antiserum, or normal mouse serum. More than 99% of the cells
Or/ Anti H-2K
.cD 410-
TL 1,3
cn
1875
-
.6
I
ir
It
Anti
H-2b
!!.,
--
a
--
4 8 16 32 64 128 256 512 1024 Antibody dilutions (Reciprocal) FIG. 3. Detection of H-2& and H-2k antigens on hybrid cells. Cytotoxic effects of H-2a, H-2k or H-2b antiserum, and complement on hybrid cells. 2
obtained from the spleens of A/J mice terminally ill with ASL-1 leukemia stained positively after incubation with H-2a antiserum and fluorescein-conjugated anti-mouse 1g. Cells incubated with H-2b or H-2k antiserum or normal mouse serum, followed by fluorescein-conjugated anti-mouse Ig, were not stained. (d) Mlodulation of the TL antigens of ASL-1 cells. The TL antigens of ASL-1 cells undergo modulation during incubation in medium containing TL 1,3 or TL 1,2,3 antiserum (see Fig. 5). LJI (TK) - Cells express H-2k antigens. They (1o not reduce known titers of H-2a antiserum or TL 1,3 antiserum, and resist lysis by guinea pig complement and TL 1,3 and H-2a antiserum. They are resistant to high titers of Thy-la, Thylb, and H-2b antisera. They stain positively after incubation with mouse H-2k antiserum and fluorescein-conjugated antimouse Ig, but failed to stain under similar conditions with TL 1,3, H-2a, H-2b, Thy-la, or Thy-lb antisera, followed by fluorescein-conjugated anti-mouse 1g. LMI(TK) - cells reduced the titer of H-2k antiserum during incubation, but did not change the titers of Thy-la or Thy-lb antiserum under similar conditions. [ASL-1 X LuI (TK) -] Hybrid Cells. (a) Expression of H-2a and H-2k antigens. Hybrid cells formed histocomnpatibility antigens characteristic of both parental cell lines. They lysed during incubation in medium containing either H-2a or H-2k antiserum and guinea pig complement (Fig. 3) and stained after incubation with H-2a or H-2k antiserum and fluorescent anti-mouse Ig, but failed to stain if incubated with H-2b antiserum or normal mouse serum, followed by fluorescent anti-mouse 1g. The entire cell population was affected; no evidence of a heterogeneous pol)ulatioll of cells was found. To avoid the possibility that the apparent sharing of H-2a and H-2k determinants by the hybrid cells resulted from antigens in common between H-2a and H-2k complexes, the "H2k" antiserum used was raised in A/J mice (H-2a) immunized with spleen cells from C3H animals (H-2k) and "H-2a" antiserum was raised in C3H mice immunized with spleen cells from A/J mice. The possibility that not all of the determinants unique to each H-2 complex were present on the cells could not be determined with the reagents we used. Similar results have been reported in analogous systems (16, 17). The capacity of the hybrid cells to reduce H-2a or H-2k antiserum was equivalent. During incubation, about 6 X 105 hybrid cells reduce by 50% equal titers of H-2a or H-2k antiserum.
1876
Immunology: Liang and Cohen
Proc. Nat. Acad. Sci. USA 72
100
a LUJ
ANTI TL 123
go
0 -J
-J-180 y 70
TL 1,3
By U)
V) 60
w
i 50 IIJ 0 40 a 30 m 20
0
I 10
Z.
O-
(1975)
(-)
I H
0
ANTIBODY DILUTIONS (RECIPROCAL)
0.5
1.0
2.5
5.0
75
10.0 12.5
15.0 20.0 25.0
NO. OF CELLS (X 106) FIG. 4. Detection of TL antigens of hybrid cells. (Left) Cytotoxic effects of TL 1,3, TL 1,2,3, or TL 2 antisera and guinea pig complement on hybrid cells. (Right) The effect of hybrid cells upon known titers of TL 1,2,3 antiserum. Changes in the titer of TL 1,2,3 antiserum after incubation with cell hybrids, ASL-1 cells, RADA-1 cells, thymocytes from A/J, (A/J X C3H)Fj or (A/J X C57BL/6)Fl mice. NMS, normal mouse serum.
The sharing of H-2a and H-2k antigens by individual hybrid cells was also indicated by the results of mixed hemagglutination studies. Hybrid cells incubated with H-2a antiserum, sheep erythrocytes sensitized previously with sheep cell hemolysin, and anti-mouse Ig formed clearly visible clusters. Similar results were obtained if H-2k antiserum was substituted for H-2a antiserum. Clusters were not found if normal mouse serum was used instead of H-2 antiserum. (b) TL. Sensitivity of the hybrid cells to TL antisera and their capacity to reduce known titers of such antisera were determined. More than 99% of the cells lysed in the presence of TL 1,3, TL 2, or TL 1,2,3 antiserum and complement (Fig. 4, left). They reduced TL antiserum of known titer. Thymus cells from F1 hybrids of TL(+) and TL(-) parents were used for comparison, as well as ASL-1 cells (TL 1,2,3) A/J thymocytes (TL 1,2,3), and RADA-1 cells (TL 1,2,3), a radiationinduced murine leukemia. Three different antisera of known titers were used in this study-TL 1,3, TL 1,2,3, and TL 2 antisera. The results indicated that about 5 X 105 ASL-1 cells and similar numbers of thymocytes from A/J mice reduced a known titer of TL 1,2,3 antiserum by 50%. However, about 10 times as many somatic hybrid cells or cells from F1 100O I
90 -J
a w
80 y
-i
70 V -J
60
-J
C) -J
W
0
50_
'i
LLJ
40 -J e)
30< __
m
IR
e.
0avv;
v-
*c
0c cur% Ab
1vc
ANTIBODY DILUTIONS (RECIPROCAL) FIG. 5. Failure of the TL antigens of hybrid cells to undergo modulation in the presence of TL antiserum. Hybrid cells were incubated for 22 hr at 370 in the presence of TL 1,3 or TL 1,2,3 antiserum. After incubation, the presence of TL antigens was determined by the cytotoxic effects of fresh TL 1,2,3, TL 1,3, or TL 2 antiserum and guinea pig complement on the hybrid cells. Parental ASL-1 cells were treated in the same way.
hybrid mice were required to reduce the titer to the same extent (Fig. 4, right). Experiments performed with TL 1,3 and TL 2 antisera yielded essentially the same results-the capacity of the somatic hybrid cells to reduce antiserum titers paralleled that of the F1 hybrids and was significantly less than parental ASL-1 cells or A/J thymocytes. The capacity of somatic hybrid cells sensitive to lysis by complement and TL antisera to reduce titers of TL antisera paralleled that of RADA-1 leukemia cells (TL 1,2,3). (c) Thy-i. Hybrid cells did not lyse in the presence of Thylb or Thy-la antiserum nor did they reduce known titers of Thy-1 antisera of either specificity (data not shown). Thy-lb and Thy-la antisera used were cytotoxic for thymocytes from A/J mice (Thy-lb) and for thymocytes from AKR mice (Thy-la), respectively. Hybrid cells incubated with Thy-lb or Thy-la antiserum and fluorescent anti-mouse Ig failed to stain and were clearly negative. The possibility that Thy-1 antigens were formed by the cells but were not detectable by immunologic methods detecting only "surface" antigens was not excluded by these experiments. Modulation of TL antigens on hybrid cells
The TL antigens of ASL-1 cells undergo modulation in the presence of TL antiserum; however, all attempts to induce modulation in hybrid cells were unsuccessful. Two different antiserum preparations, specific for TL 1,2,3 and TL 1,3 determinants, were used in attempts to induce modulation. [TL 2 antiserum does not induce modulation (11).] Each preparation was found to be capable of inducing complete modulation of the TL antigens of ASL-1 parental cells within 10 hr. However, incubation of hybrid cells for up to 30 hr in medium containing excessive concentrations of TL antiserum failed to induce modulation (Fig. 5). Further attempts to stimulate the modulation of hybrid cells were made with TL 1,3 antiserum and anti-mouse Ig. Cells were exposed to TL 1,3 antiserum for up to 5 hr, followed by an additional 20 hr in medium with anti-mouse Ig. At the end of the second incubation, their susceptibility to fresh TL 1,3, TL 1,2,3, and TL 2 antisera and complement was determined. Control ASL-1 cells, treated in the same way, were resistant, whereas hybrid cells lysed. TL, along with other membrane-associated antigens of mammalian cells, is released in soluble form during extraction
Nat. Acad. Sci. USA 72 (1975) Proc. PFailure of TL Antigens to Undergo Modulation with the nonionic detergent, Nonidet-40 (10, 14, 15). It may be iinmunoprecipitated with specific antisera. TL antigens, as well as both H-2a and H-2k antigens, were recoverable from Nonidet-40 extracts of the hybrid cells both before and after (Fig. 6) modulation was attempted. The antigens recovered with TL 1,3, TL 2, or TL 1,2,3 antiserum migrated in polyacrylamide gel electrophoresis as a single peak (Fig. 6). Similar results were obtained previously (10, 14) with Nonidet-40 extracts of ASIA cells. Immunoprecipitations of Nonidet-40 extracts of hybrid cells with TL 1,3 antiserum revealed the presence of a second slower migrating peak. It was identified previously as a tumor-associated antigen of ASL-1 cells, not found in extracts of thymocytes of nontumorbearing A/J mice (10). DISCUSSION
These data indicate that genetic mechanisms controlling the expression of TL antigens are separable from their capacity to undergo modulation. Previously, it was determined that only one of two antigenically distinct membrane structures of parental ASL-1 cells underwent modulation in the presence of antisera specific for both. ASIA cells form TL 1,2,3 antigenic determinants as well as a tumor-associated membrane antigen not associated with thymus cells of normal A/J mice. In the presence of both TL antiserum and antiserum specific for the tumor-associated antigen found on the membranes of the cells, the TL antigens underwent modulation but the tumorassociated antigen did not (14). This finding suggested that modulation was not "simply" the result of interaction of membrane antigens with specific antibody, i.e., the formation of an antigen-antibody complex on the surface of the cell, but that more complicated cellular control mechanisms might be involved. The finding that the TL antigens of the cellular hybrid of LM(TK)- and ASL-1 cells failed to undergo modulation under conditions that led to the modulation of the ASL-1 parental cells strongly supports this concept. The relative capacity of the hybrid cells to reduce titers of H-2a or Hj2k antisera was equivalent, likely indicating that the concentrations of H-2 antigens of each parent expressed by the cells was approximately the same. This is taken as an indication that the formation of H-2 antigens was regulated in a codominant manner. Modulation of the TL antigens of the hybrid was attempted by both direct (TL antiserum) and indirect (TL antiserum and anti-mouse Ig) methods. The overall time of exposure of the cells to antisera was up to 5-fold longer, using 16 times more Concentrated TL antiserum than required to completely modulate the TL antigens of the ASL-1 parents. "Capping" without endocytosis was observed when fluorescein-conjugated anti-mouse Ig was added, indicating that antigen-anti-
H chain
600 ,
1877
L chain
4'
49+
TL1 ,2,3
500
U
TL1, 3
* 4000 CL
TL2
E 300 U
W_
2001
C/)
^-~ 1oUl 0
4
8
12
20 !6 24 No of gel slice
28
32
FIG. 6. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the immunoprecipitates formed with TL 1,3, TL 1,2,3, or TL 2 antiserum from Nonidet-40 extracts of "5I-labeled hybrid cells. Hybrid cells were labeled with 126I by lactoperoxidase and incubated at 370 for 20 hr in medium containing TL antiserum. After washing, the cells were extracted with Nonidet-40.
body complexes remained on the surfaces of the cells and that complement activation might occur upon the addition of fresh guinea pig complement. This work was supported by grants from the National Science Foundation no. GB-40659 and the Leukemia Research Foundation. 1. Boyse, E. A., Stockert, E. & Old, L. J. (1970) Proc. Nat. Acad. Sci. USA 65, 933-938. 2. Aoki, J. & Johnson, P. A. (1972) J. Nat. Cancer Inst. 49, 183-192. 3. Kit, S., Dubbs, D. R., Piekarski, L. J. & Hsu, T. C. (1963) Exp. Cell Res. 31, 297-312. 4. Dubbs, D. R. & Kit, S. (1964) Exp. Cell Res. 33, 19-28. 5. Giles, R. E. & Ruddle, F. H. (1973) In Vitro 9, 103-107. 6. Croce, C. M., Koprowski, H. & Eagle, H. (1972) Proc. Nat. Acad. Sci. USA 69, 1953-1956. 7. Littlefield, J. W. (1964) Science 145, 709-710. 8. Rothfels, K. H. & Simmovitch, L. (1958) Stain Technol. 33, 73-77. 9. Old, L. J., Stockert, E., Boyse, E. A. & Kim, J. H. (1968) J. Exp. Med. 127, 523-539. 10. Yu, A. & Cohen, E. P. (1974) J. Immunol. 112, 1285-1295. 11. Boyse, E. A., Stockert, E. & Old, L. J. (1968) Int. Convoc. on Immunol. (Buffalo, N.Y.), pp. 353-357. 12. Campbell, D. H., Garvey, F. S., Cremer, N. E. & Sussdorf, D. H. (1970) Methods in Immunology (W. A. Benjamin, Inc., New York), 2nd ed., pp. 189-202. 13. Milgrom, E., Kano, K., Barron, S. L. & Witebsky, E. (1964) J. Immunol. 92, 8-16. 14. Yu, A. & Cohen, E. P. (1974) J. Immunol. 112, 1296-1307. 15. Schwartz, B. D. & Nathenson, S. G. (1971) J. Immunol. 107, 1363-1367. 16. Frye, L. D. & Edidin, M. (1970) J. Cell Sci. 7, 319-333. 17. Spenser, R. A., Hauschka, T. S., Amos, D. B. & Ephrussi, B. (1964) J. Nat. Cancer Inst. 33, 892-903.
