AGE-RELATED
CHANGES
PRELEUKEMIC
IN CELL SURFACE AKR
ANTIGENS
OF
THYMOCYTES*
By KOHEI KAWASHIMA,$ HISAMI IKEDA,§ ELISABETH STOCKERT, TOSHITADA TAKAHASHI, AND LLOYD J. OLD (From the Memorial Sloan-Kettering Cancer Center, New York 10021)
Much of t h e v a s t a m o u n t of r e s e a r c h t h a t h a s b e e n done on l e u k e m o g e n e s i s in the m o u s e h a s centered on the A K R strain. In this inbred strain, which Furth developed by selecting for high incidence of leukemia, the majority of mice die of leukemia within 1 yr (1). These leukemias are virtually all of the T-lymphocyte type and originate in the thymus or from cells derived from the thymus. The incidence of leukemia is greatly reduced by thymectomy in early life (2). It is from this strain that Gross (3) first isolated murine leukemia virus (MuLV), 1and AKR mice are now known to carry at least two chromosomal loci, demonstrated by Rowe and his colleagues to be integrated MuLV genomes which independently entail the spontaneous production of MuLV in high titer from around the time of birth (4). Other loci that predispose to leukemia are (a) Fv-1, which governs infective dissemination of MuLV (5) and (b) H-2-1inked genes of which one may control proliferation of MuLV and MuLVinfected cells via immune responsiveness (6, 7), and another which may concern the production of virions (8). L e u k e m i a in A K R mice is obviously r e l a t e d to age, the incidence rising s h a r p l y b e t w e e n 6 a n d 12 mo a f t e r birth. This h a s not b e e n accounted for in t e r m s of M u L V production, which reaches a p l a t e a u in v e r y e a r l y life a n d does not g r e a t l y increase until t h e onset of o v e r t l e u k e m i a (9). M u c h the s a m e h a s b e e n r e p o r t e d of q u a n t i t a t i v e m e a s u r e m e n t s of v i r a l gs a n t i g e n s (10). N o n e t h e less, t h e r e is a m p l e evidence of morphological t h y m i c c h a n g e s , such as cortical a t r o p h y , which precede t h e d e v e l o p m e n t of l e u k e m i a (11). In our w o r k w i t h M u L V - a s s o c i a t e d a n t i g e n s (12), we h a v e b e e n i m p r e s s e d b y t h e i r a p p a r e n t l y far g r e a t e r expression on l e u k e m i a cells as c o m p a r e d w i t h MuLV-infected b u t n o n t r a n s f o r m e d l y m p h o i d cells. We a s s u m e d to b e g i n w i t h t h a t this 'amplification' w a s s y n c h r o n o u s w i t h the l e u k e m o g e n i c t r a n s f o r m a t i o n itself. B u t this we find is not t h e case, as is shown in t h e following r e p o r t on M u L V - a s s o c i a t e d a n d o t h e r surface a n t i g e n s e x p r e s s e d on t h y m o c y t e s of A K R mice a t v a r i o u s ages.
* This work was supported by National Cancer Institute grants CA-08748 and CA-16599. * Present address: Nagoya University School of Medicine, Nagoya, Japan. § Special Fellow of Leukemia Society of America, Inc. Present address: Asahikawa Medical College, Asahikawa, Japan. 1Abbreviations used in this paper: anti-NTD, (W/Fu × BN)FI anti-W/Fu(C58NT)D; B6, C57BL/ 6; GCSA, Gross cell surface antigen; MuLV, murine leukemia virus. THE
JOURNAL
OF
EXPERIMENTAL
MEDICINE
• VOLUME
144, 1976
193
194
CELL S U R F A C E ANTIGENS OF PRELEUKEMIC AKR THYMOCYTES
Materials and Methods Mice. From our colonies or from The Jackson Laboratory, Bar Harbor, Maine. For description of congenic strains see Table I.
Antisera, Reference Cells, and Complement Source. See Table II. Cytotoxic Test (16). Equal volumes of cells (5 x 10e/ml), a serial dilution of a n t i s e r u m and an appropriate dilution of complement were mixed and incubated a t 37°C for 45 rain. After addition of 0.16% freshly prepared trypan blue, a count of stained and unstained cells was made. Quantitative Absorption Test (22, and Table II). After absorbing aliquots of diluted a n t i s e r u m with a range of counted numbers of cells, the residual activity of the serum was tested in the standard cytotoxic test. Absorption Index. (a) Glx and Gross cell surface antigen (GCSA): the n u m b e r of cells (B6-GI× + thymocytes from 2-too-old donors for Gjx assays and pooled spleen cells from 2 to 3-too-old AKR mice for GCSA assays) required to reduce the cytotoxicity of reference a n t i s e r u m to 40% lysis of the standard test cell divided by the n u m b e r of AKR thymocytes or leukemia cells required to reduce the cytotoxicity of the a n t i s e r u m to the same level. (b) H-2, Thy-1, and Ly-2: the n u m b e r of cells from 2-too-old donors (10 x 10e B6-H-2 k thymocytes for H-2% 3 x 108 A-Thy-l.1 thymecytes for Thy-1.1 assays, and 3 × 106 B6-Ly-2.1 thymocytes for Ly-2.1 assays) divided by the n u m b e r of AKR thymocytes or leukemia cells which give the same percent lysis of the standard test cell. Membrane Immunofluorescence Test for gp70 and p30 Antigens (23). A mixture of 1:40 diluted a n t i s e r u m (50 ftl) and cell suspension (25 ~l, 2 x 107/ml) was incubated at 4°C for 30 min. The cells were washed twice and resuspended in 50/~l fluoresceinated antigoat or rabbit immunoglobulin depending on the source of the antiserum. After incubation for 30 rain at 4°C, the cells were rewashed twice and examined with a Leitz Orthoplan microscope equipped with an Osram HBO 200 mercury lamp, and BC 38 exciter, KP 490 interference, and K 530 harrier filters. The two a n t i s e r a used in these tests were: goat anti-gp69/71 MuLV-Rauscher (24), provided by Doctors M. S t r a n d and J. T. August, Albert Einstein College of Medicine, and rabbit anti-p30 MuLV-Rauscher (25), provided by Doctors E. Fleissner and W. D. Hardy, Jr., of this Institute. Bovine Serum Albumin (BSA) Gradient Separation (26). Approximately l0 s AKR thymocytes were suspended in 1 ml of 35% BSA (Path-o-cyte 5, Pentex, Miles Laboratories Inc., Kankakee, Ill.) in a 5-ml Beckman cellulose-nitrate centrifuge tube. On this was layered 1 ml each of 26, 23, 20, and 10% BSA (diluted in medium 199), and t h e tube was t h e n centrifuged at 4°C at 13,000 rpm for 30 min in a Beckman SW 50.1 rotor. The four layers obtained were designated A (at the 10-20% interface), B(20-23%), C(23-26%), and D(26-35%). Cytograf Analysis. AKR thymocyte suspensions were adjusted to 2 x 105 cells per ml and examined with a Cytograf Model 6300 (Bio/Physics Systems Inc., New York), coupled with a pulse height analyzer (NS Econ Series, N o r t h e r n Scientific Inc., Wisconsin).
Results
Cytotoxic Tests with Broadly Reactive MuLV Antisera.
Thymocytes from AKR mice of various ages were examined in direct cytotoxic tests with (W/Fu x BN)F1 anti-W/Fu(C58NT)D serum. This antiserum (which is referred to as antiNTD) was produced by immunization with a MuLV-induced transplantable lymphoma of W/Fu origin and contains antibodies to a spectrum of MuLVrelated antigens (15). Fig. 1 illustrates cytotoxic tests with thymocytes from individual 2 wk to 6-mo-old AKR mice and with thymic leukemia cells. Thymocytes from young AKR mice show a low level of cytotoxicity with anti-NTD serum, whereas thymic leukemia cells are strongly reactive. Cytotoxic tests with thymocytes from individual 6-mo-old mice reveal that anti-NTD reactivity falls into two categories, low sensitivity comparable to young mice and high sensitivity approaching that seen with AKR leukemia cells. Characteristics of Thymocytes from Preleukemic Mice. Before detailed analysis of the amplified expression of MuLV antigens in the thymus of 6-mo-old AKR mice, it was essential to exclude the possibility of microscopic leukemia
KAWASHIMA, IKEDA, STOCKERT, TAKAHASHI, AND OLD
195
TABLE I
Description of Congenic Stocks Derived from C57BL/6(B6) and A Strains Used in this Study Name
Different locus
Donor strain of allele
Reference
B6-H-2 k B6-Ly-2. I
H-2 Ly-2
AKR C3H/An
13 13
B6-GIx+ A-Thy-1.1
Gv-1, Gv-2 Thy-1 CO")
129 AKR
14 13
TABLE II
Description of Serological Typing Systems for Quantitative Absorption Tests Antigenic system
Antiserum
Standard test cell
Complement source (serum dilution)
G~x
Rat (W/Fu x BN)F, anti-W/ Fu leukemia (C58NT)D (anti-NTD) (1:600")
B6-G~x +thymocytes
Rabbit
GCSA
B6 antitransplanted spontaneous AKR leukemia K36 (1:30")
EdG2 leukemia cells
Guinea pig (1:2)
17
H-2k
(B6 × BALB/c)F~ anti-C3H ascites sarcoma BP8 (1:900")
B6-H-2k lymphocytes
Guinea pig (1:4)
18
Thy-l.1
(B6 x A)F~ anti-A-Thy-l.1 thymocytes (1:120")
A-Thy-l.1 thymocytes
Rabbit
(1:15)
19, 20
Thy-l.2
(A-Thy-l.1 × AKR-H-2b)F~ anti-A strain spontaneous leukemia ASL1 (1:500")
B6 thymocytes
Rabbit
(1:15)
19, 20
Ly-2.1
B6-H-2k anti-CE thymocytes {1:100")
B6-Ly-2.1 thymocytes
Rabbit
(1:15)
20, 21
(i:15)
Reference 15, 16
* Dilution of antiserum used in the quantitative absorption tests as determined by preliminary assays. cell infiltration a n d r e p l a c e m e n t . As a rule, t h y m i c l e u k e m i a cells a r e clearly l a r g e r t h a n n o r m a l t h y m o c y t e s , a n d the experienced microscopist h a s little difficulty in d i s t i n g u i s h i n g t h e m . Such inspection r e v e a l e d no significant difference in size b e t w e e n t h y m o c y t e s from 2-mo a n d 6-mo-old A K R mice. E x a m i n a tion of cell size distribution b y C y t o g r a f a n a l y s i s s u b s t a n t i a t e d this. In addition, s e p a r a t i o n of cells by BSA g r a d i e n t c e n t r i f u g a t i o n showed e s s e n t i a l l y the s a m e distribution p a t t e r n for t h y m o c y t e s from 2-mo a n d 6-mo-old mice. T h e predomin a n t cell type of the t h y m u s , the s m a l l cortical t h y m o c y t e , is found in t h e D layer; t h y m o c y t e s from 6-mo-old A K R mice showing increased a n t i - N T D sensitivity were also found in this layer. The critical test for the p r e s e n c e of l e u k e m i a cells is t r a n s p l a n t a b i l i t y to syngeneic hosts. 5-10 × 106 l e u k e m i a cells from A K R mice with t h y m o m a produces evident disease in y o u n g A K R recipients w i t h i n 3 wk; l e u k e m i a did not r e s u l t (observation period 100 days) f r o m the inoculation of 50-100 × 106 t h y m o -
196
CELL SURFACE ANTIGENS OF PRELEUKEMIC AKR THYMOCYTES 1 mo
>90
70
or
2-4
less
mo
J
50
5O ~J
90
6
L
mo
>I..-(.,.)
(b
70-
_
leukemia
~
50
30
CHANGES
PRELEUKEMIC
IN CELL SURFACE AKR
ANTIGENS
OF
THYMOCYTES*
By KOHEI KAWASHIMA,$ HISAMI IKEDA,§ ELISABETH STOCKERT, TOSHITADA TAKAHASHI, AND LLOYD J. OLD (From the Memorial Sloan-Kettering Cancer Center, New York 10021)
Much of t h e v a s t a m o u n t of r e s e a r c h t h a t h a s b e e n done on l e u k e m o g e n e s i s in the m o u s e h a s centered on the A K R strain. In this inbred strain, which Furth developed by selecting for high incidence of leukemia, the majority of mice die of leukemia within 1 yr (1). These leukemias are virtually all of the T-lymphocyte type and originate in the thymus or from cells derived from the thymus. The incidence of leukemia is greatly reduced by thymectomy in early life (2). It is from this strain that Gross (3) first isolated murine leukemia virus (MuLV), 1and AKR mice are now known to carry at least two chromosomal loci, demonstrated by Rowe and his colleagues to be integrated MuLV genomes which independently entail the spontaneous production of MuLV in high titer from around the time of birth (4). Other loci that predispose to leukemia are (a) Fv-1, which governs infective dissemination of MuLV (5) and (b) H-2-1inked genes of which one may control proliferation of MuLV and MuLVinfected cells via immune responsiveness (6, 7), and another which may concern the production of virions (8). L e u k e m i a in A K R mice is obviously r e l a t e d to age, the incidence rising s h a r p l y b e t w e e n 6 a n d 12 mo a f t e r birth. This h a s not b e e n accounted for in t e r m s of M u L V production, which reaches a p l a t e a u in v e r y e a r l y life a n d does not g r e a t l y increase until t h e onset of o v e r t l e u k e m i a (9). M u c h the s a m e h a s b e e n r e p o r t e d of q u a n t i t a t i v e m e a s u r e m e n t s of v i r a l gs a n t i g e n s (10). N o n e t h e less, t h e r e is a m p l e evidence of morphological t h y m i c c h a n g e s , such as cortical a t r o p h y , which precede t h e d e v e l o p m e n t of l e u k e m i a (11). In our w o r k w i t h M u L V - a s s o c i a t e d a n t i g e n s (12), we h a v e b e e n i m p r e s s e d b y t h e i r a p p a r e n t l y far g r e a t e r expression on l e u k e m i a cells as c o m p a r e d w i t h MuLV-infected b u t n o n t r a n s f o r m e d l y m p h o i d cells. We a s s u m e d to b e g i n w i t h t h a t this 'amplification' w a s s y n c h r o n o u s w i t h the l e u k e m o g e n i c t r a n s f o r m a t i o n itself. B u t this we find is not t h e case, as is shown in t h e following r e p o r t on M u L V - a s s o c i a t e d a n d o t h e r surface a n t i g e n s e x p r e s s e d on t h y m o c y t e s of A K R mice a t v a r i o u s ages.
* This work was supported by National Cancer Institute grants CA-08748 and CA-16599. * Present address: Nagoya University School of Medicine, Nagoya, Japan. § Special Fellow of Leukemia Society of America, Inc. Present address: Asahikawa Medical College, Asahikawa, Japan. 1Abbreviations used in this paper: anti-NTD, (W/Fu × BN)FI anti-W/Fu(C58NT)D; B6, C57BL/ 6; GCSA, Gross cell surface antigen; MuLV, murine leukemia virus. THE
JOURNAL
OF
EXPERIMENTAL
MEDICINE
• VOLUME
144, 1976
193
194
CELL S U R F A C E ANTIGENS OF PRELEUKEMIC AKR THYMOCYTES
Materials and Methods Mice. From our colonies or from The Jackson Laboratory, Bar Harbor, Maine. For description of congenic strains see Table I.
Antisera, Reference Cells, and Complement Source. See Table II. Cytotoxic Test (16). Equal volumes of cells (5 x 10e/ml), a serial dilution of a n t i s e r u m and an appropriate dilution of complement were mixed and incubated a t 37°C for 45 rain. After addition of 0.16% freshly prepared trypan blue, a count of stained and unstained cells was made. Quantitative Absorption Test (22, and Table II). After absorbing aliquots of diluted a n t i s e r u m with a range of counted numbers of cells, the residual activity of the serum was tested in the standard cytotoxic test. Absorption Index. (a) Glx and Gross cell surface antigen (GCSA): the n u m b e r of cells (B6-GI× + thymocytes from 2-too-old donors for Gjx assays and pooled spleen cells from 2 to 3-too-old AKR mice for GCSA assays) required to reduce the cytotoxicity of reference a n t i s e r u m to 40% lysis of the standard test cell divided by the n u m b e r of AKR thymocytes or leukemia cells required to reduce the cytotoxicity of the a n t i s e r u m to the same level. (b) H-2, Thy-1, and Ly-2: the n u m b e r of cells from 2-too-old donors (10 x 10e B6-H-2 k thymocytes for H-2% 3 x 108 A-Thy-l.1 thymecytes for Thy-1.1 assays, and 3 × 106 B6-Ly-2.1 thymocytes for Ly-2.1 assays) divided by the n u m b e r of AKR thymocytes or leukemia cells which give the same percent lysis of the standard test cell. Membrane Immunofluorescence Test for gp70 and p30 Antigens (23). A mixture of 1:40 diluted a n t i s e r u m (50 ftl) and cell suspension (25 ~l, 2 x 107/ml) was incubated at 4°C for 30 min. The cells were washed twice and resuspended in 50/~l fluoresceinated antigoat or rabbit immunoglobulin depending on the source of the antiserum. After incubation for 30 rain at 4°C, the cells were rewashed twice and examined with a Leitz Orthoplan microscope equipped with an Osram HBO 200 mercury lamp, and BC 38 exciter, KP 490 interference, and K 530 harrier filters. The two a n t i s e r a used in these tests were: goat anti-gp69/71 MuLV-Rauscher (24), provided by Doctors M. S t r a n d and J. T. August, Albert Einstein College of Medicine, and rabbit anti-p30 MuLV-Rauscher (25), provided by Doctors E. Fleissner and W. D. Hardy, Jr., of this Institute. Bovine Serum Albumin (BSA) Gradient Separation (26). Approximately l0 s AKR thymocytes were suspended in 1 ml of 35% BSA (Path-o-cyte 5, Pentex, Miles Laboratories Inc., Kankakee, Ill.) in a 5-ml Beckman cellulose-nitrate centrifuge tube. On this was layered 1 ml each of 26, 23, 20, and 10% BSA (diluted in medium 199), and t h e tube was t h e n centrifuged at 4°C at 13,000 rpm for 30 min in a Beckman SW 50.1 rotor. The four layers obtained were designated A (at the 10-20% interface), B(20-23%), C(23-26%), and D(26-35%). Cytograf Analysis. AKR thymocyte suspensions were adjusted to 2 x 105 cells per ml and examined with a Cytograf Model 6300 (Bio/Physics Systems Inc., New York), coupled with a pulse height analyzer (NS Econ Series, N o r t h e r n Scientific Inc., Wisconsin).
Results
Cytotoxic Tests with Broadly Reactive MuLV Antisera.
Thymocytes from AKR mice of various ages were examined in direct cytotoxic tests with (W/Fu x BN)F1 anti-W/Fu(C58NT)D serum. This antiserum (which is referred to as antiNTD) was produced by immunization with a MuLV-induced transplantable lymphoma of W/Fu origin and contains antibodies to a spectrum of MuLVrelated antigens (15). Fig. 1 illustrates cytotoxic tests with thymocytes from individual 2 wk to 6-mo-old AKR mice and with thymic leukemia cells. Thymocytes from young AKR mice show a low level of cytotoxicity with anti-NTD serum, whereas thymic leukemia cells are strongly reactive. Cytotoxic tests with thymocytes from individual 6-mo-old mice reveal that anti-NTD reactivity falls into two categories, low sensitivity comparable to young mice and high sensitivity approaching that seen with AKR leukemia cells. Characteristics of Thymocytes from Preleukemic Mice. Before detailed analysis of the amplified expression of MuLV antigens in the thymus of 6-mo-old AKR mice, it was essential to exclude the possibility of microscopic leukemia
KAWASHIMA, IKEDA, STOCKERT, TAKAHASHI, AND OLD
195
TABLE I
Description of Congenic Stocks Derived from C57BL/6(B6) and A Strains Used in this Study Name
Different locus
Donor strain of allele
Reference
B6-H-2 k B6-Ly-2. I
H-2 Ly-2
AKR C3H/An
13 13
B6-GIx+ A-Thy-1.1
Gv-1, Gv-2 Thy-1 CO")
129 AKR
14 13
TABLE II
Description of Serological Typing Systems for Quantitative Absorption Tests Antigenic system
Antiserum
Standard test cell
Complement source (serum dilution)
G~x
Rat (W/Fu x BN)F, anti-W/ Fu leukemia (C58NT)D (anti-NTD) (1:600")
B6-G~x +thymocytes
Rabbit
GCSA
B6 antitransplanted spontaneous AKR leukemia K36 (1:30")
EdG2 leukemia cells
Guinea pig (1:2)
17
H-2k
(B6 × BALB/c)F~ anti-C3H ascites sarcoma BP8 (1:900")
B6-H-2k lymphocytes
Guinea pig (1:4)
18
Thy-l.1
(B6 x A)F~ anti-A-Thy-l.1 thymocytes (1:120")
A-Thy-l.1 thymocytes
Rabbit
(1:15)
19, 20
Thy-l.2
(A-Thy-l.1 × AKR-H-2b)F~ anti-A strain spontaneous leukemia ASL1 (1:500")
B6 thymocytes
Rabbit
(1:15)
19, 20
Ly-2.1
B6-H-2k anti-CE thymocytes {1:100")
B6-Ly-2.1 thymocytes
Rabbit
(1:15)
20, 21
(i:15)
Reference 15, 16
* Dilution of antiserum used in the quantitative absorption tests as determined by preliminary assays. cell infiltration a n d r e p l a c e m e n t . As a rule, t h y m i c l e u k e m i a cells a r e clearly l a r g e r t h a n n o r m a l t h y m o c y t e s , a n d the experienced microscopist h a s little difficulty in d i s t i n g u i s h i n g t h e m . Such inspection r e v e a l e d no significant difference in size b e t w e e n t h y m o c y t e s from 2-mo a n d 6-mo-old A K R mice. E x a m i n a tion of cell size distribution b y C y t o g r a f a n a l y s i s s u b s t a n t i a t e d this. In addition, s e p a r a t i o n of cells by BSA g r a d i e n t c e n t r i f u g a t i o n showed e s s e n t i a l l y the s a m e distribution p a t t e r n for t h y m o c y t e s from 2-mo a n d 6-mo-old mice. T h e predomin a n t cell type of the t h y m u s , the s m a l l cortical t h y m o c y t e , is found in t h e D layer; t h y m o c y t e s from 6-mo-old A K R mice showing increased a n t i - N T D sensitivity were also found in this layer. The critical test for the p r e s e n c e of l e u k e m i a cells is t r a n s p l a n t a b i l i t y to syngeneic hosts. 5-10 × 106 l e u k e m i a cells from A K R mice with t h y m o m a produces evident disease in y o u n g A K R recipients w i t h i n 3 wk; l e u k e m i a did not r e s u l t (observation period 100 days) f r o m the inoculation of 50-100 × 106 t h y m o -
196
CELL SURFACE ANTIGENS OF PRELEUKEMIC AKR THYMOCYTES 1 mo
>90
70
or
2-4
less
mo
J
50
5O ~J
90
6
L
mo
>I..-(.,.)
(b
70-
_
leukemia
~
50
30
