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Mixed agglutination in microtiter U-trays
Journal oflmmunological Methods, 31 (1979) 83--91
83
© Elsevier/North-Holland Biomedical Press
M I X E D A G G L U T I N A T I O N IN M I C R O T I T E R U - T R A Y S i
MARIA CHAN, KYOICHI KANO and FELIX MILGROM 2 Department o f Microbiology, State University o f New York at Buffalo, School o f Medicine, Buffalo, N Y 14214, U.S.A.
(Received 19 December 1978, accepted 27 June 1979)
The principle of mixed agglutination with cell cultures was applied to those cells which could not be obtained as monolayer cell cultures. Artificial monolayers of freshly isolated cells from various tissues were prepared in poly-L-lysine-coated wells of a microtiter U-tray. By means of this technique, murine alloantigens, H-2 private specificities and Thy-1 antigens were demonstrated on cells from thymus, brain, spleen, kidney and liver. Ia.2 antigen was detected on splenocytes but not on thymocytes or fibroblast L cells. Evidence was also presented that HLA A and B locus antigens could be demonstrated on human thymocytes. Sensitivity of this test was shown to be at least 100 times higher than that of the standard lymphocytotoxicity test for detection of HLA antigens.
INTRODUCTION T h e m i x e d a g g l u t i n a t i o n (MA) p r o c e d u r e has been used quite extensively as a research, and m o r e r e c e n t l y diagnostic, tool. A n extensive review o f this t o p i c was published b y C o o m b s and F r a n k s ( 1 9 6 9 ) . H S g m a n ( 1 9 5 9 ) was the first to p e r f o r m M A tests with m o n o l a y e r cell cultures, a n d Fagraeus and E s p m a r k ( 1 9 6 1 ) e m p l o y e d i n d i c a t o r s y s t e m s with a n t i - g a m m a - g l o b u l i n r e a g e n t t o t h e tests with m o n o l a y e r cultures. T h e latter p r o c e d u r e was also used in o u r l a b o r a t o r y (Milgrom et al., 1 9 6 4 ; A b e y o u n i s et al., 1 9 6 4 ; Milgrom et al., 1 9 6 5 ; K a n o , et al., 1 9 6 6 ; K a n o et al., 1 9 6 9 ; K a n o et al., 1 9 7 2 ; K a n o and Milgrom, 1 9 6 4 ) . T h e o b v i o u s l i m i t a t i o n o f this very c o n v e n i e n t a n d sensitive p r o c e d u r e has been the fact t h a t it can be applied o n l y to t h o s e cells t h a t can g r o w in tissue cultures a n d f o r m a m o n o l a y e r . In t h e p r e s e n t s t u d y , we p r e p a r e d 'artificial m o n o l a y e r s ' o f freshly isolated cells in poly-L-lysine ( P L L ) - c o a t e d wells o f a m i c r o t i t e r U - t r a y (Kenn e d y and Axelrad, 1 9 7 1 ; K a n o et al., 1977). This m o d i f i e d p r o c e d u r e allowed us to d e m o n s t r a t e m u r i n e alloantigens as well as H L A antigens on cells t h a t c o u l d n o t be o b t a i n e d as m o n o l a y e r cell cultures. 1 Supported by a Research Grant No. AM-17317 from the National Institute of Arthritis, Metabolism and Digestive Diseases. Please address all correspondence to Dr. F. Milgrom, Department of Microbiology, 203 Sherman Hall, State University of New York at Buffalo, Buffalo, NY 14214, U.S.A.
84 MATERIALS AND METHODS Animals
Inbred mice, 6--8-week-old females, were used in this study. AKR/J, C57BL/6, BALB/c and R F / J mice were purchased from The Jackson Laboratory, Bar Harbor, Me., and C3H/St mice were purchased from Health Research, Inc., West Seneca, N.Y. Female rats of four inbred strains, Fischer, Wistar/Furth, Lewis and Sprague-Dawley, were obtained from Microbiological Associates, Inc., Walkersville, Md. Human thymus
Human thymuses were kindly supplied by Dr. S. Subramanian of the Children's Hospital, Buffalo, N.Y. The specimens were delivered to our laboratory immediately after collection at cardiac surgery. Cell p r e p a r a t i o n s
T h y m o c y t e suspensions from mice, rats or man were prepared by pressing freshly excised, minced t h y m u s tissue through a 50-mesh stainless steel screen in order to remove connective tissue debris. The resulting suspension was then filtered through a second screen of 200 mesh to obtain a single-cell suspension. The cells were washed 3 times with medium 199, resuspended and adjusted to a proper concentration. Suspensions of murine spleen, liver, kidney or brain cells were prepared in a similar manner. Erythrocytes in these preparations were eliminated by hypotonic shock with distilled water. A n rise ra
Anti-Thy-l.2 sera were prepared by immunization of A K R / J mice with C3H/St t h y m o c y t e suspensions (107 cells/mouse). Weekly intraperitoneal injections were given for 9 weeks. The animals were exsanguinated and the antisera were pooled. Anti-Thy-l.1 sera were prepared in a similar manner by injecting C3H/St mice with A K R / J thymocytes. (B10.HTT × A.SW)F1 anti-A.TL (anti-Ia.2) serum was kindly provided by Dr. J. Klein of the University of Texas, Dallas, Texas. Anti-H-2 sera: D-23 for the detection of the private antigen H-2.23 controlled by the H-2 k haplotype, D-2 for H-2.2 of H-2 b, and D-4 for H-2.4 of H-2 d, were supplied by the Transplantation Immunology Branch, NIH, Bethesda, Md. HLA typing sera were supplied by the Serum Bank, Transplantation Immunology Branch, NIH. Normal human sera were obtained from healthy staff members of this department. MA technique
Fifty microliters of a PLL (Sigma Chemical Company, St. Louis, Mo.) at a
85 concentration of 100 ttg/ml were added to each well of microtiter U-trays (Cooke Laboratories, Alexandria, Va.). The trays were left for 30 min at 22°C. Then the wells were rinsed once with Hanks' balanced salt solution (HBSS), and 50 pl of a cell suspension were added to each well. Thymocytes, spleen cells, liver cells or kidney cells were used at a concentration of 5 × 106 cells/ml. Brain tissue was prepared as a 0.5--1.0% (v/v) suspension. After incubation at 22°C for 30 min, the cells settled and adhered to the PLLcoated b o t t o m of the wells and thus formed an 'artificial monolayer'. These monolayers were washed once with HBSS, and 50 pl of an appropriate dilution of antiserum were added to each well. The trays were incubated for 1 h at 22c'C and the monolayers were washed 3 times with HBSS. Thereafter, 50 pl of indicator erythrocytes were added. The trays were incubated for 1 h at 22°C. After incubation, 200 pl of HBSS were added to each well. The U-trays were then covered with transparent sealing tape (Fisher Scientific Co., Rochester, N.Y.), inverted, and rotated gently for a few minutes to remove nonspecifically attached indicator erythrocytes. Adherence of indicator erythrocytes to the monolayers was evaluated microscopically at low magnification. The reactions were graded according to previously described criteria (Milgrom et al., 1964). To demonstrate binding of murine antibodies, the indicator system consisted of human erythrocytes sensitized by murine antiserum to human RBC and agglutinated by rabbit antiserum to murine 3,-globulins (Abeyounis et al., 1964; Kano et al., 1969; Kano et al., 1972). Human antibodies were detected by human group O Rh-positive erythrocytes sensitized by incomplete anti-Rh antibodies and agglutinated by rabbit antiserum to human 3,-globulins (Kano, 1966; Kano et al., 1969; Kano et al., 1972).
Lyrnphocytotoxicity test The microcytotoxicity test for HLA typing of human t h y m o c y t e s was conducted according to NIH procedure outlined in the NIAID Manual (1976--1977). Absorption tests For absorption of anti-Thy-1 sera with thymus, spleen, liver, kidney or brain cells, 0.1 ml of properly diluted antiserum was mixed with an equal volume of washed and packed cells. The mixture was incubated at 22°C for 30 min with intermittent agitation. After centrifugation at 4000 rpm for 3 min in a Fisher Centrifuge (Model No. 59, Fisher Scientific Company, Blawnox, Pa.), the supernatant was recovered. Two additional absorptions were carried out using fresh cells. Absorption of selected HLA typing sera with human t h y m o c y t e s or peripheral blood lymphocytes was performed in a similar manner. RESULTS The MA procedure was at first used to demonstrate murine H-2 and Thy-1 alloantigens. Monolayers of t h y m o c y t e s from 5 different inbred strains were
86 prepared at the b o t t o m of PLL-coated wells of microtiter U-trays and tested against anti-H-2 sera and anti-Thy-1 sera. As shown in Table 1, 3 different anti-H-2 sera were e m p l o y e d ; the first contained antibodies to the private H-2 antigen 23 controlled by the H - 2 k haplotype, the second had antibodies to antigen 2 o f the H - 2 b h a p l o t y p e and the third had antibodies to antigen 4 of the H - 2 a haplotype. As expected, the anti-H-2 k serum gave positive MA reactions with t h y m o c y t e s of C3H/ST, A K R / J and R F / J strains but n o t of BALB/c and C 5 7 B L / 6 J strains. The anti-H-2 b serum reacted only with C 5 7 B L / 6 J and anti-H-2 d serum only with BALB/c t h y m o c y t e s . The anti-Thy-l.1 serum reacted with t h y m o c y t e s of A K R / J and R F / J strains b u t it did n o t react with t h y m o c y t e s of any of the remaining 3 strains. In contrast, the a n t i - T h y - l . 2 serum gave positive results with cells of C3H/St, BALB/c and C 5 7 B L / 6 J strains and negative with those of A K R / J and R F /J . Absorption experiments were p e r f o r m e d to ascertain that the described MA reactions with the alloantisera were indeed due to the interaction of H-2 or Thy-1 antibodies with their corresponding antigens on the target cells. As seen in Table 2, absorption of the anti-H-2 k serum with t h y m o c y t e s of C3H/St, A K R / J or R F / J abolished its activity against C3H/St t h y m o c y t e s , whereas similar absorption with BALB/c or C 5 7 B L / 6 J t h y m o c y t e s had no effect on the reactions. The reactivities of the anti-Thy-l.2 serum with C3H/St t h y m o c y t e s were abolished by absorption with t h y m o c y t e s of C3H/St, BALB/c or C 5 7 B L / 6 J but n o t with cells of A K R / J or RF/ J. To study the distribution of the H-2 and Thy-1 antigens on various murine cells, monolayers were prepared f r om cells of t hym us, brain, spleen, kidney and liver of C3H/St mice and tested simultaneously against two selected alloantisera, anti-H-2 k and T h y - l . 2 sera. As exemplified by the results shown in Table 3, monolayers of cells f r om all tissues tested gave strong positive reactions with the anti-H-2 serum even though titers with spleen, kidney and liver cells were higher than with t h y m u s and brain cells. On the other hand, a n t i - T h y - l . 2 gave the highest MA titer of 2560 with cells of t h y m u s and TABLE 1 MIXED AGGLUTINATION WITH ANTI-H-2 SERA AND ANTI-THY-1 SERA Antiserum titers with thymocytes o f C3H/St
BALB/c
C57BL/6J
AKR/J
RF/J
1280 <10 <10 <10 2560
<10 <10 640 <10 2560
<10 1280 <10 <10 2560
1280 <10 <10 1280 <10
1280 <10 <10 1280 <10
A n t i s e r u m to:
H-2k(H-2.23) H-2b (H-2.2) H-2d(H-2.4) Thy-l.1 Thy-l.2
87 TABLE 2 MIXED AGGLUTINATION WITH THYMOCYTES OF C3H/St MICE AND ALLOANTISERA: EFFECTS OF ABSORPTION Antibody titers with
Unabsorbed serum Serum absorbed with thymocytes of: C3H/St BALB/c C57BL/6J AKR/J RF/J
anti-H-2 k serum (H-2.23)
anti-Thy-l.2 serum
1280
2560
<10 1280 1280 <10 <10
<10 <10 <10 2560 2560
brain; with s p l e n o c y t e s a titer o f 6 4 0 was observed and the r e a c t i o n s with cells o f k i d n e y a n d liver were very w e a k or negative. T h e MA p r o c e d u r e was also applied to d e m o n s t r a t e Ia antigens. M o n o layers o f C 3 H / S t s p l e n o c y t e s , t h y m o c y t e s a n d c u l t u r e d L cells were tested against an anti-Ia.2 serum. S p l e n o c y t e s gave an M A titer o f 1 2 8 0 while the titers with t h y m o c y t e s or L cells were less t h a n 40 (Table 4). It was o f s o m e interest t o s t u d y t h e d i s t r i b u t i o n o f T h y - 1 antigens a m o n g d i f f e r e n t rat strains. T o this end, t h y m o c y t e s o f 4 inbred rat strains were e x a m i n e d for the presence o f t h e T h y - l . 1 antigen. The a n t i - T h y - l . 1 serum gave M A titers o f 1 6 0 0 with Wistar-Furth a n d Lewis t h y m o c y t e s , 8 0 0 with S p r a g u e - D a w l e y t h y m o c y t e s and 4 0 0 with Fischer t h y m o c y t e s . On the o t h e r h a n d , the a n t i - T h y - l . 2 serum gave negative results with t h y m o c y t e s o f all these strains. T h e M A p r o c e d u r e was applied to d e m o n s t r a t e H L A A and B locus antigens o n h u m a n t h y m o c y t e s . H L A p h e n o t y p e s o f t h y m o c y t e s f r o m 6 individuals were at first d e t e r m i n e d b y t h e s t a n d a r d c y t o t o x i c i t y tests. On the
TABLE 3 MIXED AGGLUTINATION WITH ANTI-H-2k SERUM AND ANTI-THY-1.2 SERUM AND VARIOUS CELLS OF C3H/St MICE Reactions with cells of Thymus
Brain
Spleen
Kidney
Liver
1280 2560
1280 2560
5120 640
5120 10
5120 40
Ant~erato:
H-2 k (H-2.23) Thy-l.2
88 TABLE 4 MIXED AGGLUTINATION WITH ANTI-Ia.2 SERUM AND VARIOUS CELLS OF C3H/St ORIGIN Serum dilution
Reactions with Splenocytes
1 : 1
:
1 1 1 1 1
: : : : :
40
80 160 320 640 1280 2560
Thymocytes
L cells
+++
-+
-+
+++ +++ +++ ++ ++ -+
--
-+ ------
------
basis o f t h e r e s u l t s o b t a i n e d b y t h e c y t o t o x i c i t y t e s t s , 3 4 H L A t y p i n g sera w h i c h gave p o s i t i v e r e a c t i o n s w i t h o n e o r m o r e t h y m o c y t e s p e c i m e n s w e r e selected. Results obtained in preliminary titration experiments showed that r e p r e s e n t a t i v e sera f o r e a c h o f t h e H L A s p e c i f i c i t i e s gave t i t e r s o f 5 0 0 o r m o r e a g a i n s t t h y m o c y t e s c a r r y i n g c o r r e s p o n d i n g a n t i g e n s a n d less t h a n 5 0 a g a i n s t t h y m o c y t e s w i t h o u t c o r r e s p o n d i n g a n t i g e n s . S u b s e q u e n t l y , all t h e 3 4 sera w e r e d i l u t e d 1 : 5 0 0 a n d t e s t e d a g a i n s t t h e t h y m o c y t e s p e c i m e n s b y t h e MA p r o c e d u r e . Results of reactions with 3 t h y m o c y t e specimens given b y means of MA and cytotoxicity tests were almost identical. With one thymoc y t e s p e c i m e n , h o w e v e r , a n t i - B 1 2 sera gave p o s i t i v e M A b u t n e g a t i v e c y t o t o x i c i t y t e s t s a n d w i t h a n o t h e r s p e c i m e n a n t i - B W 1 7 sera gave p o s i t i v e c y t o t o x i c i t y b u t negative M A tests. T o c o r r e l a t e r e s u l t s o f t h e M A t e s t s w i t h t h o s e o f c y t o t o x i c i t y tests, 5 0 h u m a n H L A a n t i s e r a w e r e t e s t e d a g a i n s t t h y m o c y t e s f r o m 12 i n d i v i d u a l s . For the MA tests, 1 : 100 and 1 : 500 diluted serum samples were used, w h e r e a s u n d i l u t e d sera w e r e u s e d f o r t h e c y t o t o x i c i t y tests. A s s u m m a r i z e d i n T a b l e 5, o f 6 0 0 p o s s i b l e r e a c t i o n s , 1 3 8 p o s i t i v e M A t e s t s a n d 1 2 2 p o s i t i v e
TABLE 5 REACTIONS OF 50 HLA ANTISERA IN MIXED AGGLUTINATION AND CYTOTOXICITY TESTS Coefficient of correlation = +-~/(x2 / n ) = O.85. MA with thymocytes
Cytotoxicity with thymocytes
+ -Total
+
--
Total
114 24 138
8 454 462
122 478 600
89 TABLE 6 MIXED AGGLUTINATION WITH ANTI-HLA-A2 SERUM 1243: EFFECT OF ABSORPTION Serum dilution
Reactions of anti-HLA-A2 serum with thymocytes No. 51 Unabsorbed serum
Serum absorbed with leukocytes of HLA-A2 positive donors MN
1:
RC
+++
+++
.
.
.
.
++
+++
1:1250
+
.
.
.
.
.
+
1:6250
.
.
.
KH
.
.
.
CP
.
.
.
MD
+++
250
.
KK
+++
1 :
50
MG
HLA-A2 negative donors
.
.
.
+ .
c y t o t o x i c i t y tests w e r e o b t a i n e d . T h e 138 positive MA r e a c t i o n s i n c l u d e d 24 negative c y t o t o x i c i t y tests. In 8 instances, h o w e v e r , positive c y t o t o x i c i t y tests in t h e face o f negative MA r e a c t i o n s were e n c o u n t e r e d . T h e c o r r e l a t i o n c o e f f i c i e n t was 0.85 w h i c h i n d i c a t e d a significant positive c o r r e l a t i o n b e t w e e n t h e MA tests a n d t h e c y t o t o x i c i t y tests. A t t e m p t s w e r e t h e n m a d e to s t u d y t h e specificity o f t h e MA r e a c t i o n s w i t h a n t i - H L A sera. F o r this p u r p o s e , 3 d i f f e r e n t a n t i - H L A - A 2 sera w e r e selected a n d t e s t e d against 6 individual t h y m u s s p e c i m e n s . T h e MA titers o f r e a c t i o n s with A 2 - p o s i t i v e t h y m u s e s varied f r o m 250 to 1 2 5 0 a n d titers o f r e a c t i o n s w i t h A2-negative s p e c i m e n s were 50 or less. A b s o r p t i o n s o f an antiH L A - A 2 s e r u m w i t h l e u k o c y t e s o f 5 ' A 2 - p o s i t i v e ' a n d 2 ' A 2 - n e g a t i v e ' individuals were p e r f o r m e d . A b s o r b e d s e r u m s p e c i m e n s a l o n g w i t h u n a b s o r b e d s p e c i m e n w e r e t e s t e d against t h y m o c y t e s o f an A 2 - p o s i t i v e individual. As seen in T a b l e 6, u n a b s o r b e d s e r u m gave s t r o n g MA r e a c t i o n s . A b s o r p t i o n o f the s e r u m w i t h a n y of t h e 5 l e u k o c y t e s p e c i m e n s f r o m A2-positive individuals a b o l i s h e d this activity. In c o n t r a s t , similar a b s o r p t i o n s w i t h A2-negative l e u k o c y t e s did n o t a f f e c t its activity. DISCUSSION
T h e results o b t a i n e d in t h e p r e s e n t s t u d y s h o w e d t h a t t h e m o d i f i e d MA p r o c e d u r e c o u l d be a p p l i e d f o r d e m o n s t r a t i o n o f m u r i n e a n d h u m a n alloantigens o n various n u c l e a t e d cells w i t h o u t c u l t u r i n g these cells. T h e antib o d y titers o f b o t h m u r i n e a n d h u m a n a l l o a n t i s e r a in this p r o c e d u r e w e r e quite c o m p a r a b l e to t h o s e o b t a i n e d b y MA w i t h m o n o l a y e r cell c u l t u r e s (Milgrom et al., 1 9 6 5 ; K a n o , 1 9 6 6 ; K a n o et al., 1969). This w o u l d m e a n t h a t t h e sensitivity o f this MA p r o c e d u r e was at least 50 t i m e s higher t h a n t h a t o f o t h e r p r o c e d u r e s such as t h e l y m p h o c y t o t o x i c i t y t e s t f o r H L A t y p i n g .
90 The real advantage of this technical development is the fact that freshly isolated cells from various organs can be readily tested; in particular, cells from liver and brain which are very difficult to test for cell-surface antigens by other procedures. Experiments performed on t h y m o c y t e s of five inbred strains of mice provided evidence that the MA test is capable of detecting private antigens of the H-2 complex and the antigens of the Thy-1 system. The results obtained with the anti-H-2 and anti-Thy-1 sera were in agreement with the established distribution of the H-2 antigens and Thy-1 antigens among the different inbred strains (Reif and Allen, 1966; Snell and Cherry, 1972). Furthermore, Thy-l.1 antigen but not T h y - l . 2 antigen was demonstrated on t h y m o c y t e s of 4 rat strains -- Fischer, Lewis, Wistar-Furth and Sprague-Dawley. These results confirmed those reported by other investigators (Douglas, 1972; Micheel et al., 1973). In taking advantage of this procedure, we were able to confirm the well established unique distribution of the H-2 and T h y - l . 2 antigens on cells from various murine organs (Reif and Allen, 1966; Snell and Cherry, 1972). Whereas the anti-H-2 serum to a private antigen 23 gave high MA titers against cells from all organs, the anti-Thy-l.2 sera gave positive MA reactions with cells from thymus, brain and spleen and virtually negative results with cells of kidney and liver. In a similar way, the Ia.2 antigen was demonstrated on C3H splenocytes but not on t h y m o c y t e s or a fibroblast line, L cells (Klein, 1975). In comparing results of the MA procedure and the l y m p h o c y t o t o x i c i t y tests with human t h y m o c y t e s and 50 HLA typing sera, a highly significant value of correlation coefficient (0.85) was obtained. Although the MA titers of the sera were at least 100 times higher than those given by the lymphocytotoxicity tests, major specificities demonstrated by the former appear to be HLA A and B locus antigens. The MA reactions with 2 anti-B12 sera in the face of negative c y t o t o x i c i t y tests could be due to weak cytotoxic antibodies in the serum or additional antibodies of non-HLA variety. The latter possibility could n o t be dismissed at present since MA procedure with monolayer cell cultures was shown to be able to detect such non-HLA antibodies (Kano et al., 1975). Discrepancy given by a few anti-BWl7 sera, i.e. positive l y m p h o c y t o t o x i c i t y tests in the face of negative MA tests, might have been due to the IgM nature of the antibodies in the particular sera since the MA procedure as employed in this study detected only IgG antibodies (Kano et al., 1975). REFERENCES Abeyounis, C.J., F. Milgrom and E. Witebsky, 1964, Nature 203,313. Coombs, R.R.A. and D. Franks, 1969, Progr. Allergy 13, 272. Douglas, T.C., 1972, J. Exp. Med. 136, 1054. Fagraeus, A. and A. Espmark, 1961, Nature 190, 370.
91 HSgman, C.F., 1959, Vox Sang. 4, 12. Kano, K., 1966, Int. Arch. Allergy 30, 281. Kano, K. and F. Milgrom, 1964, Proc. Soc. Exp. Biol. Med. 115, 172. Kano, K., W. Baranska, B. Knowles, H. Koprowski and F. Milgrom, 1969, J. Immunol. 103, 1050. Kano, K., B. Knowles, H. Koprowski and F. Milgrom, 1972, Eur. J. Immunol. 2, 198. Kano, K., C.A. Kussmaul and F. Milgrom, 1975, Transplant. Proc. 7, 201. Kano, K., A. Fjelde and F. Milgrom, 1977, J. Immunol. 119, 945. Kennedy, J.C. and M.A. Axelrad, 1971, Immunology 20, 253. Klein, J., 1975, in: Biology of the Mouse Histocompatibility-2 Complex, ed. J. Klein (Springer-Verlag, New York) p. 450. Micheel, B., G. Pasternak and J. Steuden, 1973, Nature New Biol. 241,221. Milgrom, F., K. Kano, A.L. Barron and E. Witebsky, 1964, J. Immunol. 92, 8. Milgrom, F., K. Kano and E. Witebsky, 1965, J. Am. Med. Assoc. 192, 845. NIAID Manual of Tissue Typing Techniques 1976--1977, eds. J.G. Ray, D.B. Hare, P.D. Pedersen and D.T. Mullally (NIH, Bethesda, Md.). Reif, A.E. and J.M. Allen, 1966, Nature 2 0 9 , 5 2 1 . Snell, G.D. and M. Cherry, 1972, in: RNA Viruses and Host Genome in Oncogenesis, eds. P. Emmelot and P. Bentvelzen (North-Holland, Amsterdam) p. 221.
83
© Elsevier/North-Holland Biomedical Press
M I X E D A G G L U T I N A T I O N IN M I C R O T I T E R U - T R A Y S i
MARIA CHAN, KYOICHI KANO and FELIX MILGROM 2 Department o f Microbiology, State University o f New York at Buffalo, School o f Medicine, Buffalo, N Y 14214, U.S.A.
(Received 19 December 1978, accepted 27 June 1979)
The principle of mixed agglutination with cell cultures was applied to those cells which could not be obtained as monolayer cell cultures. Artificial monolayers of freshly isolated cells from various tissues were prepared in poly-L-lysine-coated wells of a microtiter U-tray. By means of this technique, murine alloantigens, H-2 private specificities and Thy-1 antigens were demonstrated on cells from thymus, brain, spleen, kidney and liver. Ia.2 antigen was detected on splenocytes but not on thymocytes or fibroblast L cells. Evidence was also presented that HLA A and B locus antigens could be demonstrated on human thymocytes. Sensitivity of this test was shown to be at least 100 times higher than that of the standard lymphocytotoxicity test for detection of HLA antigens.
INTRODUCTION T h e m i x e d a g g l u t i n a t i o n (MA) p r o c e d u r e has been used quite extensively as a research, and m o r e r e c e n t l y diagnostic, tool. A n extensive review o f this t o p i c was published b y C o o m b s and F r a n k s ( 1 9 6 9 ) . H S g m a n ( 1 9 5 9 ) was the first to p e r f o r m M A tests with m o n o l a y e r cell cultures, a n d Fagraeus and E s p m a r k ( 1 9 6 1 ) e m p l o y e d i n d i c a t o r s y s t e m s with a n t i - g a m m a - g l o b u l i n r e a g e n t t o t h e tests with m o n o l a y e r cultures. T h e latter p r o c e d u r e was also used in o u r l a b o r a t o r y (Milgrom et al., 1 9 6 4 ; A b e y o u n i s et al., 1 9 6 4 ; Milgrom et al., 1 9 6 5 ; K a n o , et al., 1 9 6 6 ; K a n o et al., 1 9 6 9 ; K a n o et al., 1 9 7 2 ; K a n o and Milgrom, 1 9 6 4 ) . T h e o b v i o u s l i m i t a t i o n o f this very c o n v e n i e n t a n d sensitive p r o c e d u r e has been the fact t h a t it can be applied o n l y to t h o s e cells t h a t can g r o w in tissue cultures a n d f o r m a m o n o l a y e r . In t h e p r e s e n t s t u d y , we p r e p a r e d 'artificial m o n o l a y e r s ' o f freshly isolated cells in poly-L-lysine ( P L L ) - c o a t e d wells o f a m i c r o t i t e r U - t r a y (Kenn e d y and Axelrad, 1 9 7 1 ; K a n o et al., 1977). This m o d i f i e d p r o c e d u r e allowed us to d e m o n s t r a t e m u r i n e alloantigens as well as H L A antigens on cells t h a t c o u l d n o t be o b t a i n e d as m o n o l a y e r cell cultures. 1 Supported by a Research Grant No. AM-17317 from the National Institute of Arthritis, Metabolism and Digestive Diseases. Please address all correspondence to Dr. F. Milgrom, Department of Microbiology, 203 Sherman Hall, State University of New York at Buffalo, Buffalo, NY 14214, U.S.A.
84 MATERIALS AND METHODS Animals
Inbred mice, 6--8-week-old females, were used in this study. AKR/J, C57BL/6, BALB/c and R F / J mice were purchased from The Jackson Laboratory, Bar Harbor, Me., and C3H/St mice were purchased from Health Research, Inc., West Seneca, N.Y. Female rats of four inbred strains, Fischer, Wistar/Furth, Lewis and Sprague-Dawley, were obtained from Microbiological Associates, Inc., Walkersville, Md. Human thymus
Human thymuses were kindly supplied by Dr. S. Subramanian of the Children's Hospital, Buffalo, N.Y. The specimens were delivered to our laboratory immediately after collection at cardiac surgery. Cell p r e p a r a t i o n s
T h y m o c y t e suspensions from mice, rats or man were prepared by pressing freshly excised, minced t h y m u s tissue through a 50-mesh stainless steel screen in order to remove connective tissue debris. The resulting suspension was then filtered through a second screen of 200 mesh to obtain a single-cell suspension. The cells were washed 3 times with medium 199, resuspended and adjusted to a proper concentration. Suspensions of murine spleen, liver, kidney or brain cells were prepared in a similar manner. Erythrocytes in these preparations were eliminated by hypotonic shock with distilled water. A n rise ra
Anti-Thy-l.2 sera were prepared by immunization of A K R / J mice with C3H/St t h y m o c y t e suspensions (107 cells/mouse). Weekly intraperitoneal injections were given for 9 weeks. The animals were exsanguinated and the antisera were pooled. Anti-Thy-l.1 sera were prepared in a similar manner by injecting C3H/St mice with A K R / J thymocytes. (B10.HTT × A.SW)F1 anti-A.TL (anti-Ia.2) serum was kindly provided by Dr. J. Klein of the University of Texas, Dallas, Texas. Anti-H-2 sera: D-23 for the detection of the private antigen H-2.23 controlled by the H-2 k haplotype, D-2 for H-2.2 of H-2 b, and D-4 for H-2.4 of H-2 d, were supplied by the Transplantation Immunology Branch, NIH, Bethesda, Md. HLA typing sera were supplied by the Serum Bank, Transplantation Immunology Branch, NIH. Normal human sera were obtained from healthy staff members of this department. MA technique
Fifty microliters of a PLL (Sigma Chemical Company, St. Louis, Mo.) at a
85 concentration of 100 ttg/ml were added to each well of microtiter U-trays (Cooke Laboratories, Alexandria, Va.). The trays were left for 30 min at 22°C. Then the wells were rinsed once with Hanks' balanced salt solution (HBSS), and 50 pl of a cell suspension were added to each well. Thymocytes, spleen cells, liver cells or kidney cells were used at a concentration of 5 × 106 cells/ml. Brain tissue was prepared as a 0.5--1.0% (v/v) suspension. After incubation at 22°C for 30 min, the cells settled and adhered to the PLLcoated b o t t o m of the wells and thus formed an 'artificial monolayer'. These monolayers were washed once with HBSS, and 50 pl of an appropriate dilution of antiserum were added to each well. The trays were incubated for 1 h at 22c'C and the monolayers were washed 3 times with HBSS. Thereafter, 50 pl of indicator erythrocytes were added. The trays were incubated for 1 h at 22°C. After incubation, 200 pl of HBSS were added to each well. The U-trays were then covered with transparent sealing tape (Fisher Scientific Co., Rochester, N.Y.), inverted, and rotated gently for a few minutes to remove nonspecifically attached indicator erythrocytes. Adherence of indicator erythrocytes to the monolayers was evaluated microscopically at low magnification. The reactions were graded according to previously described criteria (Milgrom et al., 1964). To demonstrate binding of murine antibodies, the indicator system consisted of human erythrocytes sensitized by murine antiserum to human RBC and agglutinated by rabbit antiserum to murine 3,-globulins (Abeyounis et al., 1964; Kano et al., 1969; Kano et al., 1972). Human antibodies were detected by human group O Rh-positive erythrocytes sensitized by incomplete anti-Rh antibodies and agglutinated by rabbit antiserum to human 3,-globulins (Kano, 1966; Kano et al., 1969; Kano et al., 1972).
Lyrnphocytotoxicity test The microcytotoxicity test for HLA typing of human t h y m o c y t e s was conducted according to NIH procedure outlined in the NIAID Manual (1976--1977). Absorption tests For absorption of anti-Thy-1 sera with thymus, spleen, liver, kidney or brain cells, 0.1 ml of properly diluted antiserum was mixed with an equal volume of washed and packed cells. The mixture was incubated at 22°C for 30 min with intermittent agitation. After centrifugation at 4000 rpm for 3 min in a Fisher Centrifuge (Model No. 59, Fisher Scientific Company, Blawnox, Pa.), the supernatant was recovered. Two additional absorptions were carried out using fresh cells. Absorption of selected HLA typing sera with human t h y m o c y t e s or peripheral blood lymphocytes was performed in a similar manner. RESULTS The MA procedure was at first used to demonstrate murine H-2 and Thy-1 alloantigens. Monolayers of t h y m o c y t e s from 5 different inbred strains were
86 prepared at the b o t t o m of PLL-coated wells of microtiter U-trays and tested against anti-H-2 sera and anti-Thy-1 sera. As shown in Table 1, 3 different anti-H-2 sera were e m p l o y e d ; the first contained antibodies to the private H-2 antigen 23 controlled by the H - 2 k haplotype, the second had antibodies to antigen 2 o f the H - 2 b h a p l o t y p e and the third had antibodies to antigen 4 of the H - 2 a haplotype. As expected, the anti-H-2 k serum gave positive MA reactions with t h y m o c y t e s of C3H/ST, A K R / J and R F / J strains but n o t of BALB/c and C 5 7 B L / 6 J strains. The anti-H-2 b serum reacted only with C 5 7 B L / 6 J and anti-H-2 d serum only with BALB/c t h y m o c y t e s . The anti-Thy-l.1 serum reacted with t h y m o c y t e s of A K R / J and R F / J strains b u t it did n o t react with t h y m o c y t e s of any of the remaining 3 strains. In contrast, the a n t i - T h y - l . 2 serum gave positive results with cells of C3H/St, BALB/c and C 5 7 B L / 6 J strains and negative with those of A K R / J and R F /J . Absorption experiments were p e r f o r m e d to ascertain that the described MA reactions with the alloantisera were indeed due to the interaction of H-2 or Thy-1 antibodies with their corresponding antigens on the target cells. As seen in Table 2, absorption of the anti-H-2 k serum with t h y m o c y t e s of C3H/St, A K R / J or R F / J abolished its activity against C3H/St t h y m o c y t e s , whereas similar absorption with BALB/c or C 5 7 B L / 6 J t h y m o c y t e s had no effect on the reactions. The reactivities of the anti-Thy-l.2 serum with C3H/St t h y m o c y t e s were abolished by absorption with t h y m o c y t e s of C3H/St, BALB/c or C 5 7 B L / 6 J but n o t with cells of A K R / J or RF/ J. To study the distribution of the H-2 and Thy-1 antigens on various murine cells, monolayers were prepared f r om cells of t hym us, brain, spleen, kidney and liver of C3H/St mice and tested simultaneously against two selected alloantisera, anti-H-2 k and T h y - l . 2 sera. As exemplified by the results shown in Table 3, monolayers of cells f r om all tissues tested gave strong positive reactions with the anti-H-2 serum even though titers with spleen, kidney and liver cells were higher than with t h y m u s and brain cells. On the other hand, a n t i - T h y - l . 2 gave the highest MA titer of 2560 with cells of t h y m u s and TABLE 1 MIXED AGGLUTINATION WITH ANTI-H-2 SERA AND ANTI-THY-1 SERA Antiserum titers with thymocytes o f C3H/St
BALB/c
C57BL/6J
AKR/J
RF/J
1280 <10 <10 <10 2560
<10 <10 640 <10 2560
<10 1280 <10 <10 2560
1280 <10 <10 1280 <10
1280 <10 <10 1280 <10
A n t i s e r u m to:
H-2k(H-2.23) H-2b (H-2.2) H-2d(H-2.4) Thy-l.1 Thy-l.2
87 TABLE 2 MIXED AGGLUTINATION WITH THYMOCYTES OF C3H/St MICE AND ALLOANTISERA: EFFECTS OF ABSORPTION Antibody titers with
Unabsorbed serum Serum absorbed with thymocytes of: C3H/St BALB/c C57BL/6J AKR/J RF/J
anti-H-2 k serum (H-2.23)
anti-Thy-l.2 serum
1280
2560
<10 1280 1280 <10 <10
<10 <10 <10 2560 2560
brain; with s p l e n o c y t e s a titer o f 6 4 0 was observed and the r e a c t i o n s with cells o f k i d n e y a n d liver were very w e a k or negative. T h e MA p r o c e d u r e was also applied to d e m o n s t r a t e Ia antigens. M o n o layers o f C 3 H / S t s p l e n o c y t e s , t h y m o c y t e s a n d c u l t u r e d L cells were tested against an anti-Ia.2 serum. S p l e n o c y t e s gave an M A titer o f 1 2 8 0 while the titers with t h y m o c y t e s or L cells were less t h a n 40 (Table 4). It was o f s o m e interest t o s t u d y t h e d i s t r i b u t i o n o f T h y - 1 antigens a m o n g d i f f e r e n t rat strains. T o this end, t h y m o c y t e s o f 4 inbred rat strains were e x a m i n e d for the presence o f t h e T h y - l . 1 antigen. The a n t i - T h y - l . 1 serum gave M A titers o f 1 6 0 0 with Wistar-Furth a n d Lewis t h y m o c y t e s , 8 0 0 with S p r a g u e - D a w l e y t h y m o c y t e s and 4 0 0 with Fischer t h y m o c y t e s . On the o t h e r h a n d , the a n t i - T h y - l . 2 serum gave negative results with t h y m o c y t e s o f all these strains. T h e M A p r o c e d u r e was applied to d e m o n s t r a t e H L A A and B locus antigens o n h u m a n t h y m o c y t e s . H L A p h e n o t y p e s o f t h y m o c y t e s f r o m 6 individuals were at first d e t e r m i n e d b y t h e s t a n d a r d c y t o t o x i c i t y tests. On the
TABLE 3 MIXED AGGLUTINATION WITH ANTI-H-2k SERUM AND ANTI-THY-1.2 SERUM AND VARIOUS CELLS OF C3H/St MICE Reactions with cells of Thymus
Brain
Spleen
Kidney
Liver
1280 2560
1280 2560
5120 640
5120 10
5120 40
Ant~erato:
H-2 k (H-2.23) Thy-l.2
88 TABLE 4 MIXED AGGLUTINATION WITH ANTI-Ia.2 SERUM AND VARIOUS CELLS OF C3H/St ORIGIN Serum dilution
Reactions with Splenocytes
1 : 1
:
1 1 1 1 1
: : : : :
40
80 160 320 640 1280 2560
Thymocytes
L cells
+++
-+
-+
+++ +++ +++ ++ ++ -+
--
-+ ------
------
basis o f t h e r e s u l t s o b t a i n e d b y t h e c y t o t o x i c i t y t e s t s , 3 4 H L A t y p i n g sera w h i c h gave p o s i t i v e r e a c t i o n s w i t h o n e o r m o r e t h y m o c y t e s p e c i m e n s w e r e selected. Results obtained in preliminary titration experiments showed that r e p r e s e n t a t i v e sera f o r e a c h o f t h e H L A s p e c i f i c i t i e s gave t i t e r s o f 5 0 0 o r m o r e a g a i n s t t h y m o c y t e s c a r r y i n g c o r r e s p o n d i n g a n t i g e n s a n d less t h a n 5 0 a g a i n s t t h y m o c y t e s w i t h o u t c o r r e s p o n d i n g a n t i g e n s . S u b s e q u e n t l y , all t h e 3 4 sera w e r e d i l u t e d 1 : 5 0 0 a n d t e s t e d a g a i n s t t h e t h y m o c y t e s p e c i m e n s b y t h e MA p r o c e d u r e . Results of reactions with 3 t h y m o c y t e specimens given b y means of MA and cytotoxicity tests were almost identical. With one thymoc y t e s p e c i m e n , h o w e v e r , a n t i - B 1 2 sera gave p o s i t i v e M A b u t n e g a t i v e c y t o t o x i c i t y t e s t s a n d w i t h a n o t h e r s p e c i m e n a n t i - B W 1 7 sera gave p o s i t i v e c y t o t o x i c i t y b u t negative M A tests. T o c o r r e l a t e r e s u l t s o f t h e M A t e s t s w i t h t h o s e o f c y t o t o x i c i t y tests, 5 0 h u m a n H L A a n t i s e r a w e r e t e s t e d a g a i n s t t h y m o c y t e s f r o m 12 i n d i v i d u a l s . For the MA tests, 1 : 100 and 1 : 500 diluted serum samples were used, w h e r e a s u n d i l u t e d sera w e r e u s e d f o r t h e c y t o t o x i c i t y tests. A s s u m m a r i z e d i n T a b l e 5, o f 6 0 0 p o s s i b l e r e a c t i o n s , 1 3 8 p o s i t i v e M A t e s t s a n d 1 2 2 p o s i t i v e
TABLE 5 REACTIONS OF 50 HLA ANTISERA IN MIXED AGGLUTINATION AND CYTOTOXICITY TESTS Coefficient of correlation = +-~/(x2 / n ) = O.85. MA with thymocytes
Cytotoxicity with thymocytes
+ -Total
+
--
Total
114 24 138
8 454 462
122 478 600
89 TABLE 6 MIXED AGGLUTINATION WITH ANTI-HLA-A2 SERUM 1243: EFFECT OF ABSORPTION Serum dilution
Reactions of anti-HLA-A2 serum with thymocytes No. 51 Unabsorbed serum
Serum absorbed with leukocytes of HLA-A2 positive donors MN
1:
RC
+++
+++
.
.
.
.
++
+++
1:1250
+
.
.
.
.
.
+
1:6250
.
.
.
KH
.
.
.
CP
.
.
.
MD
+++
250
.
KK
+++
1 :
50
MG
HLA-A2 negative donors
.
.
.
+ .
c y t o t o x i c i t y tests w e r e o b t a i n e d . T h e 138 positive MA r e a c t i o n s i n c l u d e d 24 negative c y t o t o x i c i t y tests. In 8 instances, h o w e v e r , positive c y t o t o x i c i t y tests in t h e face o f negative MA r e a c t i o n s were e n c o u n t e r e d . T h e c o r r e l a t i o n c o e f f i c i e n t was 0.85 w h i c h i n d i c a t e d a significant positive c o r r e l a t i o n b e t w e e n t h e MA tests a n d t h e c y t o t o x i c i t y tests. A t t e m p t s w e r e t h e n m a d e to s t u d y t h e specificity o f t h e MA r e a c t i o n s w i t h a n t i - H L A sera. F o r this p u r p o s e , 3 d i f f e r e n t a n t i - H L A - A 2 sera w e r e selected a n d t e s t e d against 6 individual t h y m u s s p e c i m e n s . T h e MA titers o f r e a c t i o n s with A 2 - p o s i t i v e t h y m u s e s varied f r o m 250 to 1 2 5 0 a n d titers o f r e a c t i o n s w i t h A2-negative s p e c i m e n s were 50 or less. A b s o r p t i o n s o f an antiH L A - A 2 s e r u m w i t h l e u k o c y t e s o f 5 ' A 2 - p o s i t i v e ' a n d 2 ' A 2 - n e g a t i v e ' individuals were p e r f o r m e d . A b s o r b e d s e r u m s p e c i m e n s a l o n g w i t h u n a b s o r b e d s p e c i m e n w e r e t e s t e d against t h y m o c y t e s o f an A 2 - p o s i t i v e individual. As seen in T a b l e 6, u n a b s o r b e d s e r u m gave s t r o n g MA r e a c t i o n s . A b s o r p t i o n o f the s e r u m w i t h a n y of t h e 5 l e u k o c y t e s p e c i m e n s f r o m A2-positive individuals a b o l i s h e d this activity. In c o n t r a s t , similar a b s o r p t i o n s w i t h A2-negative l e u k o c y t e s did n o t a f f e c t its activity. DISCUSSION
T h e results o b t a i n e d in t h e p r e s e n t s t u d y s h o w e d t h a t t h e m o d i f i e d MA p r o c e d u r e c o u l d be a p p l i e d f o r d e m o n s t r a t i o n o f m u r i n e a n d h u m a n alloantigens o n various n u c l e a t e d cells w i t h o u t c u l t u r i n g these cells. T h e antib o d y titers o f b o t h m u r i n e a n d h u m a n a l l o a n t i s e r a in this p r o c e d u r e w e r e quite c o m p a r a b l e to t h o s e o b t a i n e d b y MA w i t h m o n o l a y e r cell c u l t u r e s (Milgrom et al., 1 9 6 5 ; K a n o , 1 9 6 6 ; K a n o et al., 1969). This w o u l d m e a n t h a t t h e sensitivity o f this MA p r o c e d u r e was at least 50 t i m e s higher t h a n t h a t o f o t h e r p r o c e d u r e s such as t h e l y m p h o c y t o t o x i c i t y t e s t f o r H L A t y p i n g .
90 The real advantage of this technical development is the fact that freshly isolated cells from various organs can be readily tested; in particular, cells from liver and brain which are very difficult to test for cell-surface antigens by other procedures. Experiments performed on t h y m o c y t e s of five inbred strains of mice provided evidence that the MA test is capable of detecting private antigens of the H-2 complex and the antigens of the Thy-1 system. The results obtained with the anti-H-2 and anti-Thy-1 sera were in agreement with the established distribution of the H-2 antigens and Thy-1 antigens among the different inbred strains (Reif and Allen, 1966; Snell and Cherry, 1972). Furthermore, Thy-l.1 antigen but not T h y - l . 2 antigen was demonstrated on t h y m o c y t e s of 4 rat strains -- Fischer, Lewis, Wistar-Furth and Sprague-Dawley. These results confirmed those reported by other investigators (Douglas, 1972; Micheel et al., 1973). In taking advantage of this procedure, we were able to confirm the well established unique distribution of the H-2 and T h y - l . 2 antigens on cells from various murine organs (Reif and Allen, 1966; Snell and Cherry, 1972). Whereas the anti-H-2 serum to a private antigen 23 gave high MA titers against cells from all organs, the anti-Thy-l.2 sera gave positive MA reactions with cells from thymus, brain and spleen and virtually negative results with cells of kidney and liver. In a similar way, the Ia.2 antigen was demonstrated on C3H splenocytes but not on t h y m o c y t e s or a fibroblast line, L cells (Klein, 1975). In comparing results of the MA procedure and the l y m p h o c y t o t o x i c i t y tests with human t h y m o c y t e s and 50 HLA typing sera, a highly significant value of correlation coefficient (0.85) was obtained. Although the MA titers of the sera were at least 100 times higher than those given by the lymphocytotoxicity tests, major specificities demonstrated by the former appear to be HLA A and B locus antigens. The MA reactions with 2 anti-B12 sera in the face of negative c y t o t o x i c i t y tests could be due to weak cytotoxic antibodies in the serum or additional antibodies of non-HLA variety. The latter possibility could n o t be dismissed at present since MA procedure with monolayer cell cultures was shown to be able to detect such non-HLA antibodies (Kano et al., 1975). Discrepancy given by a few anti-BWl7 sera, i.e. positive l y m p h o c y t o t o x i c i t y tests in the face of negative MA tests, might have been due to the IgM nature of the antibodies in the particular sera since the MA procedure as employed in this study detected only IgG antibodies (Kano et al., 1975). REFERENCES Abeyounis, C.J., F. Milgrom and E. Witebsky, 1964, Nature 203,313. Coombs, R.R.A. and D. Franks, 1969, Progr. Allergy 13, 272. Douglas, T.C., 1972, J. Exp. Med. 136, 1054. Fagraeus, A. and A. Espmark, 1961, Nature 190, 370.
91 HSgman, C.F., 1959, Vox Sang. 4, 12. Kano, K., 1966, Int. Arch. Allergy 30, 281. Kano, K. and F. Milgrom, 1964, Proc. Soc. Exp. Biol. Med. 115, 172. Kano, K., W. Baranska, B. Knowles, H. Koprowski and F. Milgrom, 1969, J. Immunol. 103, 1050. Kano, K., B. Knowles, H. Koprowski and F. Milgrom, 1972, Eur. J. Immunol. 2, 198. Kano, K., C.A. Kussmaul and F. Milgrom, 1975, Transplant. Proc. 7, 201. Kano, K., A. Fjelde and F. Milgrom, 1977, J. Immunol. 119, 945. Kennedy, J.C. and M.A. Axelrad, 1971, Immunology 20, 253. Klein, J., 1975, in: Biology of the Mouse Histocompatibility-2 Complex, ed. J. Klein (Springer-Verlag, New York) p. 450. Micheel, B., G. Pasternak and J. Steuden, 1973, Nature New Biol. 241,221. Milgrom, F., K. Kano, A.L. Barron and E. Witebsky, 1964, J. Immunol. 92, 8. Milgrom, F., K. Kano and E. Witebsky, 1965, J. Am. Med. Assoc. 192, 845. NIAID Manual of Tissue Typing Techniques 1976--1977, eds. J.G. Ray, D.B. Hare, P.D. Pedersen and D.T. Mullally (NIH, Bethesda, Md.). Reif, A.E. and J.M. Allen, 1966, Nature 2 0 9 , 5 2 1 . Snell, G.D. and M. Cherry, 1972, in: RNA Viruses and Host Genome in Oncogenesis, eds. P. Emmelot and P. Bentvelzen (North-Holland, Amsterdam) p. 221.