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Induction of specific anti-guinea pig T cell sera in rabbits
Journal of Immunological Methods, 9 (1976) 211--223
211
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
INDUCTION OF SPECIFIC ANTI-GUINEA PIG T CELL SERA
IN R A B B I T S
H.P. GODFREY Department of Pathology, Health Sciences Center, S. U.N. Y., Sunnybrook, N. Y. 11794, U.S.A. A.F. GECZY
Institute/'or Clinical Immunology, University of Berne, lnselspital, CH-3010 Berne, Switzerland P.G.H. GELL
Department of Experimental Pathology, University of Birmingham Medical School, Birmingham, England B. RUBIN
lmmunobiology Laboratory, Bloodbank/Blood Typing Department, Statens Seruminstitut, DK-2300 Copenhagen S, Denmark (Received 27 May 1975, accepted 18 June 1975)
In an attempt to increase the specificity of antisera raised in rabbits against strain 2 guinea pig thymocytes and brain, the rabbits were screened for titres of natural antibodies to thymocytes and other lymphocytes. Although unimmunized rabbits commonly had moderate titres of cytotoxic antibodies to guinea pig thymocytes, occasional animals had low titres to thymocytes and moderate titres to bone marrow cells. Intravenous immunization of this latter group of rabbits with thymocytes led to the production of high titred anti-thymocyte sera which were easily made specific for thymus-derived lymphocytes (T cells) by absorption with L2 C lymphoma, a bone marrow-derived lymphoma of strain 2 guinea pigs. Sera raised against guinea pig brain in complete Freund's adjuvant which had high titres of antibodies to both thymocytes and bone marrow cells could be made specific for T cells only with great difficulty. The cytotoxic activity of the anti-T cell serum could be absorbed by strain 2 thymocytes and brain homogenates, while high dilutions of this serum inhibited the formation of spontaneous rosettes between guinea pig lymphoid cells and normal rabbit erythrocytes.
INTRODUCTION The small l y m p h o c y t e s of b o t h animals and m a n can be divided into two s u b p o p u l a t i o n s , t h y m u s - d e r i v e d (T cells) a n d b o n e m a r r o w - d e r i v e d (B ceils) b y m e a n s o f s u i t a b l e c y t o t o x i c , i m m u n o f l u o r e s c e n t a n d r o s e t t i n g assays ( S h e v a c h et al., 1 9 7 3 ) . T h e s e t e c h n i q u e s h a v e p e r m i t t e d d i s s e c t i o n o f t h e immune response and clarification of the interaction between various lym-
212
phocyte subpopulations in the induction and elicitation of the immuno response. It was anticipated that anti-guinea pig T cell serum would be useful in dissecting the etiological role of T and B lymphocytes in the delayed-onset hypersensitivities so easily demonstrated in the guinea pig both in vivo and in vitro (for example, delayed hypersensitivity and cutaneous basophil hypersensitivity). Shevach and his coworkers (Shevaeh et al., 1972) raised anti-T cell sera in rabbits by injecting strain 2 guinea pig t h y m o c y t e s in complete Freund's adjuvant (CFA) and rendering the resulting sera T cell specific (cytotoxic assay) by extensive absorption with L~ C l y m p h o m a cells, a guinea pig B cell lymphoma. Despite their specificity, these sera were not completely suitable for some studies because of residual non-T cell antibodies (Shevach, personal communication). We have raised anti-guinea pig T cell sera in rabbits employing a slightly different approach than that of Shevach et al. (1972) by attempting to increase the specificity of sera raised before absorbing them with the L: C l y m p h o m a to remove B cell antibodies. To this end, we tested a large number of rabbits for natural cytotoxic antibodies against guinea pig lymphoid cells before immunizing them with either t h y m o c y t e s (intravenously) or brain homogenates in CFA (intramuscularly). MATERIALS AND METHODS Guinea pigs
Inbred guinea pigs, strain 2, were maintained as closed colonies by brother sister matings at the Statens Seruminstitut (Copenhagen, Denmm'k), MRC Skin Unit, University of Birmingham (Birmingham, England), or at the Institute for Clinical Immunology, Inselspital (Berne, Switzerland). Hartley guinea pigs, 350 ? 50 g, were obtained from Statens Seruminstitut's random-bred colony. Rabbits
Adult albino rabbits were obtained from the colonies maintained at the Department of Experimental Pathology, University of Birmingham or at the Statens Seruminstitut. Tissue cullure medium
Medium RPMI-1640 containing 15 mM N-2-hydroxyethylpiperazine-N'-2ethane sulphonic acid (ttEPES), pH 7.2 and 0.85% NaHCO3 (Biocult, Glascow, Scotland) was supplemented with 10% heat-inactivated foetal calf serum (Biocult), penicillin, streptomycin and 2 mM glutamine. Except as
213
noted, tissue culture medium was used for all cell suspensions and serum dilutions.
Preparation of purified lymphocytes A normal guinea pig was exsanguinated, its blood defibrinated and diluted I : 2 with medium. T hym us , lymph node and spleen were aseptically removed, cell suspensions prepared by a standard m e t h o d (Oppenheim et al., 1967) and washed once, while bone marrow cells were flushed from the isolated femur. L y m p h o c y t e s from blood, bone marrow and l ym phoi d organs were purified by layering cell suspensions over an equal volume of autoclaved F i c o l l - I s o p a q u e 440, density 1.084 (Ficoll: 26.4 g; Isopaque 440, 40.8 ml; distilled water to 359 g) (Rigshospitalet, Copenhagen) in sterile disposeable plastic culture tubes (NUNC A/S, Roskilde, Denmark) and centrifuging for 20 min at r o o m t em pe rat ure as described by SCrensen (1972). The cells at the interface were then carefully removed, washed twice, counted and their viability determined by trypan blue exclusion. Purified cells consisted o f more than 98% l y m p h o c y t e s by m o r p h o l o g y and were over 95% viable.
Microcytotoxicity assay A m e t h o d based on that of Hawker et al. (1972) was used. A tissue typing plate (G.D. Searle and Co. Ltd., High Wycombe, Bucks, England) was filled to overflowing with 8 ml heavy liquid paraffin, 0.87--0.89 g/ml (BDH, Poole, England) and 2/~1 of medium or serum dilutions to be assayed placed in the wells of the plate. The plate was usually prepared a day in advance of the assay and was frozen at --20°C until used. If frozen, the plate was left at r o o m temp er atur e for 10 min before use to thaw. 2 t~l of cells (2 × 10 ('/ml) and 2 t~l o f normal guinea pig serum were added to each serum dilution. After incubation of the plate at 37°C for 1 hr, 2 ~1 4% eosin Y was added to each serum- cell mixture. Three minutes later 2 t~l CaCO3 neutralized 40% formalin was added to fix the cells. Both eosin Y and formalin were centrifuged at high speed before use. The assays were covered with a cleaned coverglass and non-viable cells/200 cells evaluated under phase contrast microscopy. The assays were stable for up to 14 days at r o o m temperature. The results are shown as c y t o t o x i c indices. % cells non-viable~ -- % cells non-viablem C y t o t o x i c index % . . . . 100 -- % cells nonwiablem - - - X 100 where the subscripts s and m refer to assays performed in serum dilutions and in medium alone. For reproducible results, the use of BDH liquid paraffin and o f CaCO3 neutralised formalin was required.
214 Percentages of T cells in the total l y m p h o c y t e s from various organs were calculated by averaging plateau values of c y t o t o x i c indices for these cells (Raft, 1971) and are presented as means ~ standard deviation. A n t i - t h y m o c y t e sera
Rabbits were immunized with 2 × 10 ~ strain 2 guinea pig t h y m o c y t e s in phosphate-buffered saline (PBS), pH 7.2 by intravenous injection on days 0 and 7. Immunized animals were exsanguinated on day 14 (Levey and Medawar, 1966). A n t i - b r a i n sera
A normal strain 2 guinea pig was exsanguinated, the brain (including cerebellum and medulla) removed and 4 g brain homogenized in 1/2 volume PBS or Eagle's MEM (Biocult). An equal volume of CFA containing I mg/ml mixed strains of human M y c o b a c t e r i u m t u b e r c u l o s i s (Weybridge) was added and the mixture emulsified. Rabbits received 0.5 ml of the emulsion intramuscularly in the hind leg and were bled weekly beginning 2 weeks after immunization. All animals developed paralysis of varying severity during the subsequent weeks, and were exsanguinated when their clinical condition deteriorated 4 to 8 weeks after immunization.
Absorption of
sera
L2 C l y m p h o m a was obtained from Dr. E. Shevach and maintained by serial passage o f leukemia cells in strain 2 guinea pigs at the Institute for Clinical I m m u n o l o g y , Berne. Leukemia cells were purified from the peripheral blood of mo ri bund animals with white cell counts in excess of 200,OO0/pl by F i c o l l Isopaque sedimentation. Minced livers from guinea pigs inoculated intraperitoneally with 1.5 to 2.0 × 10 ~' leukemia cells in 1 ml Hank's solution 14 days previously were used for absorption. These livers were massively infiltrated with l y m p h o m a cells and were several times normal size. Absorptions were d o n e by mixing equal volumes of l y m p h o m a t o u s liver and undiluted serum at 4~'C for 2 hr, followed by centrifugation to remove particles. Rabbit sera were heat-inactivated (56°C for 30 rain) only after the entire series of absorption were completed. Some sera were also absorbed with t h y m o c y t e s or brain to delineate the specificity of the induced antibodies. Equal volumes of strain 2 purified t h y m o c y t e s or brain homogenate were mixed with 50 pl serum at 4'~C for 30 rain, centrifuged to remove antigen and the process repeated on the supernate. The absorbed sera, and a comparably diluted unabsorbed sample were titrated for c y t o t o x i c i t y against strain 2 t h y m o c y t e s . A sample of rabbit anti-guinea pig ~ chain antiserum (a gift from Dr. A. Kelus) was absorbed twice with equal volumes of strain 2 liver mince at 4 ° C
215 for 2 hr followed by absorption twice with equal volumes of packed strain 2 guinea pig red cells (4°C for 1 hr).
Rosette formation with rabbit red cells 25 pl o f a purified guinea pig l y m p h o c y t e suspension (107 cells/ml) was mixed with 100 pl of a 0.4% suspension of fresh, washed rabbit red cells and the mix tu r e incubated at 4°C for 1 hr before centrifugation at low speed. All assays were p e r f o r m e d in duplicate or triplicate. The cell mixture was allowed to remain at 4°C for at least a further 18 hr before gentle resuspension and counting of rosettes ( l y m p h o c y t e s with 4 or more adherent red cells per 200 l y m p h o c y t e s ) by the m e t h o d of Wilson and Coombs (1973). The prolonged incubation at 4°C was f ound to increase the n u m b e r o f rosettes present as compared with counting immediately after eentrifugation. To test whether these spontaneous rosettes could be inhibited by various antisera, 25 pl o f a purified l y m p h o c y t e suspension (107 cells/nil) was mixed with 1 pl PBS, pH 7.2, or with 1 ~1 of undiluted normal rabbit serum (NRS), undiluted rabbit anti-guinea pig K chain undiluted or 1 : 2 diluted specific anti-T cell antisera and incubated for 30 min at 4°C before addition of the rabbit red cells. These assays were performed in triplicate. The percentage of rosette forming cells was evaluated in the standard manner. None of the sera was c y t o t o x i c to bone marrow cells at the dilutions used. RESULTS
Natural anti-guinea pig lymphoid cell antibodies in rabbits Unimmunized rabbits f r equent l y had c y t o t o x i c antibodies to guinea pig t h y m o c y t e s (table 1). 26 of 30 normal rabbit sera tested were e y t o t o x i c to some degree for guinea pig t h y m o c y t e s at a 1 : 6 dilution ( e y t o t o x i c index greater than 5%) and many normal sera showed appreciable c y t o t o x i e i t y even at a dilution of 1 : 24. C y t o t o x i c antibodies to bone marrow cells were markedly less c o m m o n in these sera. Their titres varied independently of a n t i - t h y m o c y t e titres and t he y could be specifically absorbed by L~, C cells. Seven of 30 sera had low titres of anti-lymph node cell c y t o t o x i c i t y only at a 1 : 6 dilution, 2 of 30 sera were c y t o t o x i c for spleen cells. There were no sera with natural antibodies to peripheral blood l y m p h o c y t e s at the 1 : 6 dilution.
Anti-guinea pig lymphoid cell antibodies after immunization Rabbits with different titres of natural antibodies to t h y m o c y t e s and bone marrow cells were immunized with either strain 2 brain or t h y m o c y t e s to evaluate the effect of these natural antibodies on subsequent ant i body production. I mmu n i z a t i on with t h y m o c y t e s induced high titres of c y t o t o x i c
216
TABLE
l
Cytotoxieity
Rabbit
of normal
Marrow
rabbit
sera against guinea pig thymocytes
anti bone.
cells
No.
Thymocyte cytotoxicity (final serum dilution) 1/6 1/24 9326 9627 9691 a 9699 9825 9827 9829 9839 9843 9888 a 9898 a 2a 3 6a 15 19 24 a 25
++ +++ ++ +4+++ +++ ++ ++ ++
+ + + ~ , + +
+
+
++ +++ +++ ++ +++
+ ++ + + ++
++
+ +
4716 4717 4718 4719 b
+ ++ +
+ +
4720 4721 a 4722 a ,1723
+
4724
+++
4726 b
"t
+
2
,:
++
b
± ±
+
4715 b
4725
Bone marrow cell cytotoxicity (final serum dilution) 1/6 1/24
++
+
+
+
++ + +
+
+
+
= cytotoxieity index, 0--5%; ~ = cytotoxicity index, 5--10%; + = cytotoxicity lO--25%; ++ = eytotoxicity index, 25--50%; +++ = cylotoxicity index, 5()--] 00%. aImmunized with strain 2 guinea pig brain. bImmunized with strain 2 guinea pig thymocytes.
index,
anti-thymocyte antibodies independently of the initial titre of natural thymocytotoxic antibodies (compare rabbits 4726 and 4724, fig. 1), but the specificity of the response was greater in those animals with low levels of natural cytotoxic antibodies to thymocytes. Immunization with brain induced high cytotoxic titres to thymocytes only in those animals with moder-
217 9888.
4726,
9888.
100-
& B o n e Marrow Cells
8O"
\
O Thymus
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--
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Poe. Immunization 2 3 4 8 Weeks
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A fte r Immunization
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After Immuntzalton
,\
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,
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24.
4724.
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24,
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3
20"
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|
,
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,
F in al
'~. ........ .,... ......... ;
~12
1/ 48
Serum
DHution
1192
1~7 6 8
~2
!~4e
~192
~'Tes
Fig. 1. C y t o t o x i c antibodies to Hartley guinea pig t h y m o c y t e s and b o n e marrow cells in rabbit sera before and after i m m u n i z a t i o n with strain 2 guinea pig t h y m o c y t e s (rabbits 4726 and 4724) or brain (rabbits 9 8 8 8 and 24). See Materials and m e t h o d s for details of assays and calculation o f c y t o t o x i c indices.
ate titres o f natural antibodies to t h y m o c y t e s (compare rabbits 9 8 8 8 and 24, fig. 1). In all rabbits immunized with brain, t h y m o c y t o t o x i c titres were constant between 2 and 6 weeks after immunization, but usually lower by 8 weeks. C y t o t o x i c antibodies to bone marrow cells showed an increasing titre over the entire 8 week period. Anti-brain antisera were less specifically thym o c y t o t o x i c than anti-thymocyte sera raised in rabbits with low levels of natural t h y m o c y t o t o x i c antibodies. Although strain 2 t h y m o c y t e s and brain were used for immunization, c y t o t o x i c titres for outbred Hartley and strain 2 t h y m o c y t e s and bone marrow cells were the same, and Hartley cells were used routinely to assay the sera.
Absorption of anti-thyrnocyte and
a n t i - b r a i n sera
A single absorption of sera 4 7 2 6 and 9 8 8 8 caused some reduction of c y t o t o x i c titres to bone marrow cells without affecting titres to thymocytes. After two absorptions with L~ C l y m p h o m a cells, the a n t i t h y m o c y t e serum
218 4726. 100"
k - - T h y m u s Cells 80" 0-- --- Bone Marrow Cells
\
2-3-4 Absorptions 60"
\
\
\
40"
30
• I
2
.dl I
=l. 1
. . . . . . . . . . . . . . . . . . . . . . . .
l
lo 9888.
9888.
o % 8O"
\ \2
60"
~3
\
40"
4, 20' •.3 O--.
_
2 .... O--
I
I
~
~8
.
~,9~ Final
~768
1~12
~48
;,92
T
;~88
Serum Dilution
Fig. 2. Effect of absorption of rabbit anti-strain 2 guinea pig thymoeyte (rabbit ,1726) or pooled rabbit antistrain 2 guinea pig brain (rabbit 9888) with L~,C cells on the titres or cytotoxic antibodies to Hartley guinea pig thymoeytes and bone marrow cells. See Materials and methods for details of absorptions.
raised in r a b b i t 4 7 2 6 was t h y m o c y t e specific (fig. 2). The anti-brain serum raised in r a b b i t 9 8 8 8 (like rabbit 4 7 2 6 , a rabbit with low levels of natural antibodies to t h y m o c y t e s ) was n o t t h y m o c y t e specific after t w o a b s o r p t i o n s and f u r t h e r a b s o r p t i o n s o n l y served to r e d u c e a n t i - t h y m o c y t e activity witho u t increasing the specificity o f the serum (fig. 2). No a t t e m p t was made to absorb the a n t i - t h y m o e y t e sera raised in rabbits with high titres of natural t h y m o e y t o t o x i e a n t i b o d i e s because o f the relatively low specificity o f these sera in c o m p a r i s o n to those raised in rabbits
219
similar to 4726. Despite the relatively low specificity for t h y m o c y t e s of the anti-brain sera raised in rabbits with high titres of natural t h y m o c y t o t o x i c antibodies, an a t t e m p t was made to render them specific by absorption with L2 C cells because of their very high titres of anti-thymocyte antibodies (fig. 3). Anti-brain sera from one of four rabbits, rabbit 2, was rendered specific for guinea pig t h y m o c y t e s after 9 absorptions with L2 C cells. More commonly, as with sera from rabbit 6, extensive absorption with L2 C cells to remove anti-bone marrow cells antibodies led to a loss of anti-thymocyte antibodies as well.
Specificity of anti-thymocyte serum The cytotoxic antibodies of the specific anti-thymocyte serum 4726 could be absorbed by strain 2 t h y m o c y t e s or brain homogenates (table 2). A single absorption with t h y m o c y t e s or two with brain homogenate removed significant t h y m o c y t o t o x i c activity from the serum. Absorption with brain homogenate after exhaustive absorption with t h y m o c y t e s led to a slight further TABLE 2 Specificity of anti-guinea pig T cell serum (4726).
T r e a t m e n t of serum
Cytoxic index, %
None
8O
A.
B.
A b s o r b e d once with equal volume of strain 2 guinea pig t h y m o c y t e s
18
A b s o r b e d twice with equal volume of strain 2 guinea pig t h y m o c y t e s
8
Absorbed thrice with equal volume of strain 2 guinea pig t h y m o c y t e s
8
Absorbed thrice with equal volume of strain 2 guinea pig t h y m o c y t e s and t h e n absorbed with equal volume of strain 2 guinea pig brain h o m o g e n a t e
3
Absorbed once with equal volume of strain 2 guinea pig brain h o m o g e n a t e
43
Absorbed twice with equal volume of strain 2 guinea pig brain h o m o g e n a t e
5
Absorbed thrice with equal volume of strain 2 guinea pig brain h o m o g e n a t e
9
Serum was absorbed as described under Materials and m e t h o d s and tested for c y t o t o x i c activity against strain 2 guinea pig thymocytes.
220 TABLE3 C o m p a r i s o n o f T cell p e r c e n t a g e s in guinea pig l y m p h o i d organs d e t e r m i n e d by specific antisera and s p o n t a n e o u s r o s e t t e f o r m a t i o n with rabbit e r y t h r o e y t e s .
Cell source
C y t o t o x i c index (%) w i t h anti-T cell sera (4726)
R o s e t t e s / 1 0 0 guinea pig l y m p h o e y t e s
Thymus Peripheral blood L y m p h node Spleen Bone m a r r o w
84
86 ~ 5
3a
59 54 22 7
16 10 7 2
19, 6 i9 ~ 3 29 ~ 3 3~2
aMean ~ s t a n d a r d deviation o f tests p e r f o r m e d in parallel o n purified Hartley guinea pig cell s u s p e n s i o n s (5 individuals tested} as described in Materials and m e t h o d s . All assays perf o r m e d b e t w e e n March and O c t o b e r . 2
_~.
X2 ~ \
80
x
60 Q
\,
V,
,.~2
40"
"11,
4
-,
20
" . "'O
Z • z ...........~ . . . ~ . ~ : . : ~ . i
I
....... ,_.ii """
....
l
I
i
I
I
I
6
6
~0(~ o-
Bone Marrow Ceils
\ A , - ~ Thymus Cells 80"
\
x After Irnr~unlzatlOn Z I~e
Immun~zahon
eO 2 3 - 4 5 6 7 8 9
Abso{phorrs 40
1
'J[3
1
',
20"
~-~-~ 'i2
.... ~,,! . ~'.8
.
'~
" ",• .4 I
%2
.
. . 1"768
-_.
1
Final
1'12 Serum
!.8
Y192
1'768
~• i ~2
; ~8
i
!
~'~92
b68
Diluhon
Fig. 3. Efrect of a b s o r p t i o n of rabbit anti-strain 2 guinea pig brain sera with L, C cells on the titres of c y t o t o x i c a n t i b o d i e s w. Hartley guinea pi~4 t h y m o e y t e s and h o n e marrow cells. See Materials and m e t h o d s for deta;le o f a b s o r p t i o n .
221 TABLE 4 Inhibition of rosette formation between normal guinea pig lymphocytes and rabbit erythrocytes by specific anti-T cell serum. Cell source
Thymus Exp. 1 Exp. 2 Peripheral Blood Exp. 1 Exp. 2 Lymph node Exp. 1 Exp. 2
Rosettes per 100 guinea pig lymphocytes after incubation with PBS
NRS, 1:26
Rabbit antiguinea pig chain, 1:26
Rabbit antiguinea pig T cell (4726) 1:26 1:52
79 a 84
76 83
79 82
16 NT
NT 36
43 54
45 50
40 57
6 NT
NT 15
41 48
31 47
33 53
3 NT
NT 9
aMean of triplicates. See Materials and methods for details. Purified lymphocytes were from two individuals Hartley guinea pigs. NT = Not tested.
r e d u c t i o n in c y t o t o x i c titre. F u r t h e r evidence t h a t the specific anti-thym o c y t e serum was an anti-T cell serum can be seen in table 3 where the m e a n plateau c y t o t o x i c i t y o f the serum f o r l y m p h o i d cells f r o m various organs is c o m p a r e d with the percentages o f cells able to f o r m s p o n t a n e o u s rosettes with r a b b i t red cells (T cell rosettes). The data are derived f r o m parallel assays o f cells o f 5 individual H a r t l e y guinea pigs. All tests were d o n e b e t w e e n March and O c t o b e r ( G o d f r e y , 1975). The p e r c e n t a g e o f cells killed b y specific a n t i - t h y m o c y t e s e r u m are in reasonable a g r e e m e n t with the n u m ber o f T cells as m e a s u r e d b y rosette f o r m a t i o n . Table 4 shows t h a t the specific a n t i - t h y m o c y t e serum was able to inhibit f o r m a t i o n o f s p o n t a n e o u s rosettes with rabbit red cells, c o n s i s t e n t with its T cell specificity, while n o r m a l r a b b i t serum or r a b b i t anti-guinea pig K chain sera lacked this ability. DISCUSSION T h e presence o f serum a n t i b o d i e s to guinea pig l y m p h o i d cells in u n i m m u n i z e d rabbits m i g h t be e x p e c t e d to interfere with the raising o f anti-T cell a n t i b o d i e s either b y c o m p e t i n g with l y m p h o c y t e r e c e p t o r s for antigen or b y a rapid clearance o f antigen before a n t i b o d y i n d u c t i o n c o u l d occur. The sensitive and c o n v e n i e n t m i c r o c y t o t o x i c plate assay enabled us to e x a m i n e
222 the sera o f a large n u m b e r of rabbits for titres of natural antibodies to guinea pig ly mp h o id cells prior to immunization. Such screening d e m o n s t r a t e d that many rabbits had m oder a t e to high levels of a n t i - t h y m o c y t e antibodies and low to moderate levels of anti-bone marrow cell antibodies, as noted in passing by Shevach er al. (1972). It proved easier to raise specific anti-T cell sera in rabbits by intravenous injection o f t h y m o e y t e s than by intramuscular injection of brain. Intravenous immunization was most sucessful in those animals with low titres of natural antibodies to t h y m o c y t e s and m od erat e titres to bone marrow cells. The sera raised in these selected rabbits had high titres of a n t i - t h y m o c y t e antibodies and could easily be made specific by absorption with L2 C cells. Perhaps these observations are related to those of Pincus et al. (1971), who showed that immunization with mixtures of a complex antigen and ant i body directed against one of the antigenic determinants of this complex antigen led to suppression of ant i body f or m at i on against this det erm i nant and increased a n t i b o d y f o r m a t i o n against non-suppressed determinants. If our observations are another example of this general p h e n o m e n o n , it might be possible to raise high titred specific antisera for guinea pig T cells routinely by immunizing rabbits with low titres of natural antibodies to t h y m o c y t e s and mo d er ate titres to bone marrow cells with mixtures of autologous norreal serum and t h y m o c y t e s . A somewhat similar approach has been used to raise specific rabbit antisera to mouse leukemia cells (Weiner et al., 1972). All rabbits immunized with guinea pig brain produced c y t o t o x i c antibodies for guinea pig t h y m o c y t e s and bone marrow cells (Golub, 1971). Regardless of the levels of natural bone marrow cell antibodies, all rabbits had elevated titres of c y t o t o x i c antibodies to bone marrow cells 2 weeks after immunization, which rose steadily over the next 6 weeks. Repeated absorption with L2 C cells could not always specifically remove these antibodies, perhaps indicating that this c y t o t o x i c activity was associated with anti-stem cell antibodies (Golub, 1972). In contrast to results from work with rabbit anti-mouse brain sera (Golub, 1971), we found antisera raised against guinea pig t h y m o c y t e s to be considerably more specific for thym ocy tes than sera raised against guinea pig brain. The most t h y m o c y t e specific anti-brain sera were raised in rabbits with m oderat e (rather than very high or very low) titres of natural antibodies to guinea pig t h y m o c y t e s . Since anti-brain sera required considerably more absorptions with L: C cells to be rendered t h y m o c y t e specific than did a n t i - t h y m o c y t e sera, and since the titres of the two types of specific sera were similar after absorption, immunization of rabbits with guinea pig brain to raise specific anti-guinea pig T cell antibodies has few if any advantages over immunization with t h y m o cytes directly. It is not clear why our specific a n t i - t h y m o c y t e and anti-brain antisera killed no more than 85 90% (rather than 100%) of t h y m o c y t e s at the lowest dilutions tested (Raff, 1971). However, the results we obtained for percent T cells in t h y m u s and other organs are in reasonable agreement with
223 t h o s e of S h e v a c h et al. ( 1 9 7 2 ) a n d S t a d e c k e r et al. ( 1 9 7 3 ) w h o used specific r a b b i t o r b u r r o anti-T cell antisera and a s t Cr release m e t h o d to m e a s u r e c y t o t o x i c i t y . This c o n c o r d a n c e o f o b s e r v a t i o n s w o u l d s e e m to indicate t h a t the c y t o t o x i c i t y plate m e t h o d we e m p l o y (with its relatively high c o n c e n t r a tions o f r e a g e n t s and long i n c u b a t i o n times) is n o t m u c h less sensitive t h a n r a d i o a c t i v e release m e t h o d s and t h a t t h e l o w e r c y t o t o x i c p l a t e a u is n o t d u e to t h e t e c h n i c a l s h o r t c o m i n g s o f t h e assay e m p l o y e d . T h e p e r c e n t a g e o f T cells in various l y m p h o i d organs was f o u n d to be similar w h e n m e a s u r e d b y s p o n t a n e o u s r o s e t t e f o r m a t i o n or b y c y t o t o x i c i t y ; diluted anti-T cell s e r u m was able to p r e v e n t s p o n t a n e o u s r o s e t t e f o r m a t i o n in t h e a b s e n c e of c o m p l e m e n t . B o t h of t h e findings c o n f i r m t h e r e p o r t o f S t a d e c k e r et al. ( 1 9 7 3 ) . In view o f the f r e q u e n c y o f n a t u r a l anti-guinea pig t h y m o c y t e a n t i b o d i e s in rabbits, s p o n t a n e o u s r o s e t t e f o r m a t i o n b e t w e e n guinea pig T cell and r a b b i t red cells m i g h t d u e to t h e p r e s e n c e o f n a t u r a l anti-guinea pig t h y m o c y t e a n t i b o d i e s a d s o r b e d to the surface o f the red cells, a s u p p o s i t i o n c o n f i r m e d b y t h e ability o f a n t i - a l l o t y p e sera to b l o c k r o s e t t e f o r m a t i o n (H. G o d f r e y , u n p u b l i s h e d results). ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d b y grants f r o m Statens L a e g e v i d e n s k a b e l i g e F o r s k n i n g s r ~ d (to P r o f e s s o r M. S i m o n s e n and B. R u b i n ) , Arvid Nilssons F o n d , M a r y Rosenkjaers Legat, a n d t h e Swiss N a t i o n a l F o u n d a t i o n for Scientific R e s e a r c h ( G r a n t No. 3 . 7 6 0 0 . 7 2 ) . We wish to t h a n k J . F . L o w e and H a n n e SCrensen for valuable technical assistance.
REFERENCES Godfrey, H.P., 1975, Int. Arch. Allergy Appl. Immunol., in press. Golub, E.S., 1971, Cellular Immunol. 2, 353. Golub, E.S., 1972, J. Exp. Med. 136, 369. Hawker, R.J., A.D. Barnes and L.A. King, 1972, Tissue Antigens 2,293. Levey, R.H. and P.B. Medawar, 1966, Ann. N.Y. Aead. Sei. 129, 164. Oppenheim, J.J., R.A. Wolsteneroft and P.G.H. Gell, 1967, Immunology 12, 89. Pineus, C.S., M.E. Lamm and V. Nussenzweig, 1971, J. Exp. Med. 133,987. Raft, M.C., 1971, Transplant. Rev. 6, 52. Shevaeh, E., I. Green, L. Ellman and J.M. Maillard, 1972, Nature New Biol. 235, 19. Shevaeh, E.I., E.S. Jaffe and I. Green, 1973, Transplant. Rev. 16, 3. S~brensen, S.F., 1974, Aeta Path. Microbiol. Seand. Sect. B. suppl. 230. Stadeeker, M.J., G. Bishop and H.H. Wortis, 1973, J. Immunol. 111, 1834. Weiner, R.S., J.D. Hubbard and M.R. Mardiney, Jr., 1972, J. Nat. Cancer Inst. 49, 1063. Wilson, A.B. and R.R.A. Coomhs, 1973, Int. Arch. Allergy Appl. Immunol, 44, 544.
211
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
INDUCTION OF SPECIFIC ANTI-GUINEA PIG T CELL SERA
IN R A B B I T S
H.P. GODFREY Department of Pathology, Health Sciences Center, S. U.N. Y., Sunnybrook, N. Y. 11794, U.S.A. A.F. GECZY
Institute/'or Clinical Immunology, University of Berne, lnselspital, CH-3010 Berne, Switzerland P.G.H. GELL
Department of Experimental Pathology, University of Birmingham Medical School, Birmingham, England B. RUBIN
lmmunobiology Laboratory, Bloodbank/Blood Typing Department, Statens Seruminstitut, DK-2300 Copenhagen S, Denmark (Received 27 May 1975, accepted 18 June 1975)
In an attempt to increase the specificity of antisera raised in rabbits against strain 2 guinea pig thymocytes and brain, the rabbits were screened for titres of natural antibodies to thymocytes and other lymphocytes. Although unimmunized rabbits commonly had moderate titres of cytotoxic antibodies to guinea pig thymocytes, occasional animals had low titres to thymocytes and moderate titres to bone marrow cells. Intravenous immunization of this latter group of rabbits with thymocytes led to the production of high titred anti-thymocyte sera which were easily made specific for thymus-derived lymphocytes (T cells) by absorption with L2 C lymphoma, a bone marrow-derived lymphoma of strain 2 guinea pigs. Sera raised against guinea pig brain in complete Freund's adjuvant which had high titres of antibodies to both thymocytes and bone marrow cells could be made specific for T cells only with great difficulty. The cytotoxic activity of the anti-T cell serum could be absorbed by strain 2 thymocytes and brain homogenates, while high dilutions of this serum inhibited the formation of spontaneous rosettes between guinea pig lymphoid cells and normal rabbit erythrocytes.
INTRODUCTION The small l y m p h o c y t e s of b o t h animals and m a n can be divided into two s u b p o p u l a t i o n s , t h y m u s - d e r i v e d (T cells) a n d b o n e m a r r o w - d e r i v e d (B ceils) b y m e a n s o f s u i t a b l e c y t o t o x i c , i m m u n o f l u o r e s c e n t a n d r o s e t t i n g assays ( S h e v a c h et al., 1 9 7 3 ) . T h e s e t e c h n i q u e s h a v e p e r m i t t e d d i s s e c t i o n o f t h e immune response and clarification of the interaction between various lym-
212
phocyte subpopulations in the induction and elicitation of the immuno response. It was anticipated that anti-guinea pig T cell serum would be useful in dissecting the etiological role of T and B lymphocytes in the delayed-onset hypersensitivities so easily demonstrated in the guinea pig both in vivo and in vitro (for example, delayed hypersensitivity and cutaneous basophil hypersensitivity). Shevach and his coworkers (Shevaeh et al., 1972) raised anti-T cell sera in rabbits by injecting strain 2 guinea pig t h y m o c y t e s in complete Freund's adjuvant (CFA) and rendering the resulting sera T cell specific (cytotoxic assay) by extensive absorption with L~ C l y m p h o m a cells, a guinea pig B cell lymphoma. Despite their specificity, these sera were not completely suitable for some studies because of residual non-T cell antibodies (Shevach, personal communication). We have raised anti-guinea pig T cell sera in rabbits employing a slightly different approach than that of Shevach et al. (1972) by attempting to increase the specificity of sera raised before absorbing them with the L: C l y m p h o m a to remove B cell antibodies. To this end, we tested a large number of rabbits for natural cytotoxic antibodies against guinea pig lymphoid cells before immunizing them with either t h y m o c y t e s (intravenously) or brain homogenates in CFA (intramuscularly). MATERIALS AND METHODS Guinea pigs
Inbred guinea pigs, strain 2, were maintained as closed colonies by brother sister matings at the Statens Seruminstitut (Copenhagen, Denmm'k), MRC Skin Unit, University of Birmingham (Birmingham, England), or at the Institute for Clinical Immunology, Inselspital (Berne, Switzerland). Hartley guinea pigs, 350 ? 50 g, were obtained from Statens Seruminstitut's random-bred colony. Rabbits
Adult albino rabbits were obtained from the colonies maintained at the Department of Experimental Pathology, University of Birmingham or at the Statens Seruminstitut. Tissue cullure medium
Medium RPMI-1640 containing 15 mM N-2-hydroxyethylpiperazine-N'-2ethane sulphonic acid (ttEPES), pH 7.2 and 0.85% NaHCO3 (Biocult, Glascow, Scotland) was supplemented with 10% heat-inactivated foetal calf serum (Biocult), penicillin, streptomycin and 2 mM glutamine. Except as
213
noted, tissue culture medium was used for all cell suspensions and serum dilutions.
Preparation of purified lymphocytes A normal guinea pig was exsanguinated, its blood defibrinated and diluted I : 2 with medium. T hym us , lymph node and spleen were aseptically removed, cell suspensions prepared by a standard m e t h o d (Oppenheim et al., 1967) and washed once, while bone marrow cells were flushed from the isolated femur. L y m p h o c y t e s from blood, bone marrow and l ym phoi d organs were purified by layering cell suspensions over an equal volume of autoclaved F i c o l l - I s o p a q u e 440, density 1.084 (Ficoll: 26.4 g; Isopaque 440, 40.8 ml; distilled water to 359 g) (Rigshospitalet, Copenhagen) in sterile disposeable plastic culture tubes (NUNC A/S, Roskilde, Denmark) and centrifuging for 20 min at r o o m t em pe rat ure as described by SCrensen (1972). The cells at the interface were then carefully removed, washed twice, counted and their viability determined by trypan blue exclusion. Purified cells consisted o f more than 98% l y m p h o c y t e s by m o r p h o l o g y and were over 95% viable.
Microcytotoxicity assay A m e t h o d based on that of Hawker et al. (1972) was used. A tissue typing plate (G.D. Searle and Co. Ltd., High Wycombe, Bucks, England) was filled to overflowing with 8 ml heavy liquid paraffin, 0.87--0.89 g/ml (BDH, Poole, England) and 2/~1 of medium or serum dilutions to be assayed placed in the wells of the plate. The plate was usually prepared a day in advance of the assay and was frozen at --20°C until used. If frozen, the plate was left at r o o m temp er atur e for 10 min before use to thaw. 2 t~l of cells (2 × 10 ('/ml) and 2 t~l o f normal guinea pig serum were added to each serum dilution. After incubation of the plate at 37°C for 1 hr, 2 ~1 4% eosin Y was added to each serum- cell mixture. Three minutes later 2 t~l CaCO3 neutralized 40% formalin was added to fix the cells. Both eosin Y and formalin were centrifuged at high speed before use. The assays were covered with a cleaned coverglass and non-viable cells/200 cells evaluated under phase contrast microscopy. The assays were stable for up to 14 days at r o o m temperature. The results are shown as c y t o t o x i c indices. % cells non-viable~ -- % cells non-viablem C y t o t o x i c index % . . . . 100 -- % cells nonwiablem - - - X 100 where the subscripts s and m refer to assays performed in serum dilutions and in medium alone. For reproducible results, the use of BDH liquid paraffin and o f CaCO3 neutralised formalin was required.
214 Percentages of T cells in the total l y m p h o c y t e s from various organs were calculated by averaging plateau values of c y t o t o x i c indices for these cells (Raft, 1971) and are presented as means ~ standard deviation. A n t i - t h y m o c y t e sera
Rabbits were immunized with 2 × 10 ~ strain 2 guinea pig t h y m o c y t e s in phosphate-buffered saline (PBS), pH 7.2 by intravenous injection on days 0 and 7. Immunized animals were exsanguinated on day 14 (Levey and Medawar, 1966). A n t i - b r a i n sera
A normal strain 2 guinea pig was exsanguinated, the brain (including cerebellum and medulla) removed and 4 g brain homogenized in 1/2 volume PBS or Eagle's MEM (Biocult). An equal volume of CFA containing I mg/ml mixed strains of human M y c o b a c t e r i u m t u b e r c u l o s i s (Weybridge) was added and the mixture emulsified. Rabbits received 0.5 ml of the emulsion intramuscularly in the hind leg and were bled weekly beginning 2 weeks after immunization. All animals developed paralysis of varying severity during the subsequent weeks, and were exsanguinated when their clinical condition deteriorated 4 to 8 weeks after immunization.
Absorption of
sera
L2 C l y m p h o m a was obtained from Dr. E. Shevach and maintained by serial passage o f leukemia cells in strain 2 guinea pigs at the Institute for Clinical I m m u n o l o g y , Berne. Leukemia cells were purified from the peripheral blood of mo ri bund animals with white cell counts in excess of 200,OO0/pl by F i c o l l Isopaque sedimentation. Minced livers from guinea pigs inoculated intraperitoneally with 1.5 to 2.0 × 10 ~' leukemia cells in 1 ml Hank's solution 14 days previously were used for absorption. These livers were massively infiltrated with l y m p h o m a cells and were several times normal size. Absorptions were d o n e by mixing equal volumes of l y m p h o m a t o u s liver and undiluted serum at 4~'C for 2 hr, followed by centrifugation to remove particles. Rabbit sera were heat-inactivated (56°C for 30 rain) only after the entire series of absorption were completed. Some sera were also absorbed with t h y m o c y t e s or brain to delineate the specificity of the induced antibodies. Equal volumes of strain 2 purified t h y m o c y t e s or brain homogenate were mixed with 50 pl serum at 4'~C for 30 rain, centrifuged to remove antigen and the process repeated on the supernate. The absorbed sera, and a comparably diluted unabsorbed sample were titrated for c y t o t o x i c i t y against strain 2 t h y m o c y t e s . A sample of rabbit anti-guinea pig ~ chain antiserum (a gift from Dr. A. Kelus) was absorbed twice with equal volumes of strain 2 liver mince at 4 ° C
215 for 2 hr followed by absorption twice with equal volumes of packed strain 2 guinea pig red cells (4°C for 1 hr).
Rosette formation with rabbit red cells 25 pl o f a purified guinea pig l y m p h o c y t e suspension (107 cells/ml) was mixed with 100 pl of a 0.4% suspension of fresh, washed rabbit red cells and the mix tu r e incubated at 4°C for 1 hr before centrifugation at low speed. All assays were p e r f o r m e d in duplicate or triplicate. The cell mixture was allowed to remain at 4°C for at least a further 18 hr before gentle resuspension and counting of rosettes ( l y m p h o c y t e s with 4 or more adherent red cells per 200 l y m p h o c y t e s ) by the m e t h o d of Wilson and Coombs (1973). The prolonged incubation at 4°C was f ound to increase the n u m b e r o f rosettes present as compared with counting immediately after eentrifugation. To test whether these spontaneous rosettes could be inhibited by various antisera, 25 pl o f a purified l y m p h o c y t e suspension (107 cells/nil) was mixed with 1 pl PBS, pH 7.2, or with 1 ~1 of undiluted normal rabbit serum (NRS), undiluted rabbit anti-guinea pig K chain undiluted or 1 : 2 diluted specific anti-T cell antisera and incubated for 30 min at 4°C before addition of the rabbit red cells. These assays were performed in triplicate. The percentage of rosette forming cells was evaluated in the standard manner. None of the sera was c y t o t o x i c to bone marrow cells at the dilutions used. RESULTS
Natural anti-guinea pig lymphoid cell antibodies in rabbits Unimmunized rabbits f r equent l y had c y t o t o x i c antibodies to guinea pig t h y m o c y t e s (table 1). 26 of 30 normal rabbit sera tested were e y t o t o x i c to some degree for guinea pig t h y m o c y t e s at a 1 : 6 dilution ( e y t o t o x i c index greater than 5%) and many normal sera showed appreciable c y t o t o x i e i t y even at a dilution of 1 : 24. C y t o t o x i c antibodies to bone marrow cells were markedly less c o m m o n in these sera. Their titres varied independently of a n t i - t h y m o c y t e titres and t he y could be specifically absorbed by L~, C cells. Seven of 30 sera had low titres of anti-lymph node cell c y t o t o x i c i t y only at a 1 : 6 dilution, 2 of 30 sera were c y t o t o x i c for spleen cells. There were no sera with natural antibodies to peripheral blood l y m p h o c y t e s at the 1 : 6 dilution.
Anti-guinea pig lymphoid cell antibodies after immunization Rabbits with different titres of natural antibodies to t h y m o c y t e s and bone marrow cells were immunized with either strain 2 brain or t h y m o c y t e s to evaluate the effect of these natural antibodies on subsequent ant i body production. I mmu n i z a t i on with t h y m o c y t e s induced high titres of c y t o t o x i c
216
TABLE
l
Cytotoxieity
Rabbit
of normal
Marrow
rabbit
sera against guinea pig thymocytes
anti bone.
cells
No.
Thymocyte cytotoxicity (final serum dilution) 1/6 1/24 9326 9627 9691 a 9699 9825 9827 9829 9839 9843 9888 a 9898 a 2a 3 6a 15 19 24 a 25
++ +++ ++ +4+++ +++ ++ ++ ++
+ + + ~ , + +
+
+
++ +++ +++ ++ +++
+ ++ + + ++
++
+ +
4716 4717 4718 4719 b
+ ++ +
+ +
4720 4721 a 4722 a ,1723
+
4724
+++
4726 b
"t
+
2
,:
++
b
± ±
+
4715 b
4725
Bone marrow cell cytotoxicity (final serum dilution) 1/6 1/24
++
+
+
+
++ + +
+
+
+
= cytotoxieity index, 0--5%; ~ = cytotoxicity index, 5--10%; + = cytotoxicity lO--25%; ++ = eytotoxicity index, 25--50%; +++ = cylotoxicity index, 5()--] 00%. aImmunized with strain 2 guinea pig brain. bImmunized with strain 2 guinea pig thymocytes.
index,
anti-thymocyte antibodies independently of the initial titre of natural thymocytotoxic antibodies (compare rabbits 4726 and 4724, fig. 1), but the specificity of the response was greater in those animals with low levels of natural cytotoxic antibodies to thymocytes. Immunization with brain induced high cytotoxic titres to thymocytes only in those animals with moder-
217 9888.
4726,
9888.
100-
& B o n e Marrow Cells
8O"
\
O Thymus
'4
--
6O"
....
\ 2O
Poe. Immunization 2 3 4 8 Weeks
~
4@
Cells
A fte r Immunization
\',
After Immuntzalton
,\
•
,
t
,
!
24.
4724.
!
24,
o
8O-
\
\ 6O-
•,
\ '",
3
20"
!
|
,
,
,
F in al
'~. ........ .,... ......... ;
~12
1/ 48
Serum
DHution
1192
1~7 6 8
~2
!~4e
~192
~'Tes
Fig. 1. C y t o t o x i c antibodies to Hartley guinea pig t h y m o c y t e s and b o n e marrow cells in rabbit sera before and after i m m u n i z a t i o n with strain 2 guinea pig t h y m o c y t e s (rabbits 4726 and 4724) or brain (rabbits 9 8 8 8 and 24). See Materials and m e t h o d s for details of assays and calculation o f c y t o t o x i c indices.
ate titres o f natural antibodies to t h y m o c y t e s (compare rabbits 9 8 8 8 and 24, fig. 1). In all rabbits immunized with brain, t h y m o c y t o t o x i c titres were constant between 2 and 6 weeks after immunization, but usually lower by 8 weeks. C y t o t o x i c antibodies to bone marrow cells showed an increasing titre over the entire 8 week period. Anti-brain antisera were less specifically thym o c y t o t o x i c than anti-thymocyte sera raised in rabbits with low levels of natural t h y m o c y t o t o x i c antibodies. Although strain 2 t h y m o c y t e s and brain were used for immunization, c y t o t o x i c titres for outbred Hartley and strain 2 t h y m o c y t e s and bone marrow cells were the same, and Hartley cells were used routinely to assay the sera.
Absorption of anti-thyrnocyte and
a n t i - b r a i n sera
A single absorption of sera 4 7 2 6 and 9 8 8 8 caused some reduction of c y t o t o x i c titres to bone marrow cells without affecting titres to thymocytes. After two absorptions with L~ C l y m p h o m a cells, the a n t i t h y m o c y t e serum
218 4726. 100"
k - - T h y m u s Cells 80" 0-- --- Bone Marrow Cells
\
2-3-4 Absorptions 60"
\
\
\
40"
30
• I
2
.dl I
=l. 1
. . . . . . . . . . . . . . . . . . . . . . . .
l
lo 9888.
9888.
o % 8O"
\ \2
60"
~3
\
40"
4, 20' •.3 O--.
_
2 .... O--
I
I
~
~8
.
~,9~ Final
~768
1~12
~48
;,92
T
;~88
Serum Dilution
Fig. 2. Effect of absorption of rabbit anti-strain 2 guinea pig thymoeyte (rabbit ,1726) or pooled rabbit antistrain 2 guinea pig brain (rabbit 9888) with L~,C cells on the titres or cytotoxic antibodies to Hartley guinea pig thymoeytes and bone marrow cells. See Materials and methods for details of absorptions.
raised in r a b b i t 4 7 2 6 was t h y m o c y t e specific (fig. 2). The anti-brain serum raised in r a b b i t 9 8 8 8 (like rabbit 4 7 2 6 , a rabbit with low levels of natural antibodies to t h y m o c y t e s ) was n o t t h y m o c y t e specific after t w o a b s o r p t i o n s and f u r t h e r a b s o r p t i o n s o n l y served to r e d u c e a n t i - t h y m o c y t e activity witho u t increasing the specificity o f the serum (fig. 2). No a t t e m p t was made to absorb the a n t i - t h y m o e y t e sera raised in rabbits with high titres of natural t h y m o e y t o t o x i e a n t i b o d i e s because o f the relatively low specificity o f these sera in c o m p a r i s o n to those raised in rabbits
219
similar to 4726. Despite the relatively low specificity for t h y m o c y t e s of the anti-brain sera raised in rabbits with high titres of natural t h y m o c y t o t o x i c antibodies, an a t t e m p t was made to render them specific by absorption with L2 C cells because of their very high titres of anti-thymocyte antibodies (fig. 3). Anti-brain sera from one of four rabbits, rabbit 2, was rendered specific for guinea pig t h y m o c y t e s after 9 absorptions with L2 C cells. More commonly, as with sera from rabbit 6, extensive absorption with L2 C cells to remove anti-bone marrow cells antibodies led to a loss of anti-thymocyte antibodies as well.
Specificity of anti-thymocyte serum The cytotoxic antibodies of the specific anti-thymocyte serum 4726 could be absorbed by strain 2 t h y m o c y t e s or brain homogenates (table 2). A single absorption with t h y m o c y t e s or two with brain homogenate removed significant t h y m o c y t o t o x i c activity from the serum. Absorption with brain homogenate after exhaustive absorption with t h y m o c y t e s led to a slight further TABLE 2 Specificity of anti-guinea pig T cell serum (4726).
T r e a t m e n t of serum
Cytoxic index, %
None
8O
A.
B.
A b s o r b e d once with equal volume of strain 2 guinea pig t h y m o c y t e s
18
A b s o r b e d twice with equal volume of strain 2 guinea pig t h y m o c y t e s
8
Absorbed thrice with equal volume of strain 2 guinea pig t h y m o c y t e s
8
Absorbed thrice with equal volume of strain 2 guinea pig t h y m o c y t e s and t h e n absorbed with equal volume of strain 2 guinea pig brain h o m o g e n a t e
3
Absorbed once with equal volume of strain 2 guinea pig brain h o m o g e n a t e
43
Absorbed twice with equal volume of strain 2 guinea pig brain h o m o g e n a t e
5
Absorbed thrice with equal volume of strain 2 guinea pig brain h o m o g e n a t e
9
Serum was absorbed as described under Materials and m e t h o d s and tested for c y t o t o x i c activity against strain 2 guinea pig thymocytes.
220 TABLE3 C o m p a r i s o n o f T cell p e r c e n t a g e s in guinea pig l y m p h o i d organs d e t e r m i n e d by specific antisera and s p o n t a n e o u s r o s e t t e f o r m a t i o n with rabbit e r y t h r o e y t e s .
Cell source
C y t o t o x i c index (%) w i t h anti-T cell sera (4726)
R o s e t t e s / 1 0 0 guinea pig l y m p h o e y t e s
Thymus Peripheral blood L y m p h node Spleen Bone m a r r o w
84
86 ~ 5
3a
59 54 22 7
16 10 7 2
19, 6 i9 ~ 3 29 ~ 3 3~2
aMean ~ s t a n d a r d deviation o f tests p e r f o r m e d in parallel o n purified Hartley guinea pig cell s u s p e n s i o n s (5 individuals tested} as described in Materials and m e t h o d s . All assays perf o r m e d b e t w e e n March and O c t o b e r . 2
_~.
X2 ~ \
80
x
60 Q
\,
V,
,.~2
40"
"11,
4
-,
20
" . "'O
Z • z ...........~ . . . ~ . ~ : . : ~ . i
I
....... ,_.ii """
....
l
I
i
I
I
I
6
6
~0(~ o-
Bone Marrow Ceils
\ A , - ~ Thymus Cells 80"
\
x After Irnr~unlzatlOn Z I~e
Immun~zahon
eO 2 3 - 4 5 6 7 8 9
Abso{phorrs 40
1
'J[3
1
',
20"
~-~-~ 'i2
.... ~,,! . ~'.8
.
'~
" ",• .4 I
%2
.
. . 1"768
-_.
1
Final
1'12 Serum
!.8
Y192
1'768
~• i ~2
; ~8
i
!
~'~92
b68
Diluhon
Fig. 3. Efrect of a b s o r p t i o n of rabbit anti-strain 2 guinea pig brain sera with L, C cells on the titres of c y t o t o x i c a n t i b o d i e s w. Hartley guinea pi~4 t h y m o e y t e s and h o n e marrow cells. See Materials and m e t h o d s for deta;le o f a b s o r p t i o n .
221 TABLE 4 Inhibition of rosette formation between normal guinea pig lymphocytes and rabbit erythrocytes by specific anti-T cell serum. Cell source
Thymus Exp. 1 Exp. 2 Peripheral Blood Exp. 1 Exp. 2 Lymph node Exp. 1 Exp. 2
Rosettes per 100 guinea pig lymphocytes after incubation with PBS
NRS, 1:26
Rabbit antiguinea pig chain, 1:26
Rabbit antiguinea pig T cell (4726) 1:26 1:52
79 a 84
76 83
79 82
16 NT
NT 36
43 54
45 50
40 57
6 NT
NT 15
41 48
31 47
33 53
3 NT
NT 9
aMean of triplicates. See Materials and methods for details. Purified lymphocytes were from two individuals Hartley guinea pigs. NT = Not tested.
r e d u c t i o n in c y t o t o x i c titre. F u r t h e r evidence t h a t the specific anti-thym o c y t e serum was an anti-T cell serum can be seen in table 3 where the m e a n plateau c y t o t o x i c i t y o f the serum f o r l y m p h o i d cells f r o m various organs is c o m p a r e d with the percentages o f cells able to f o r m s p o n t a n e o u s rosettes with r a b b i t red cells (T cell rosettes). The data are derived f r o m parallel assays o f cells o f 5 individual H a r t l e y guinea pigs. All tests were d o n e b e t w e e n March and O c t o b e r ( G o d f r e y , 1975). The p e r c e n t a g e o f cells killed b y specific a n t i - t h y m o c y t e s e r u m are in reasonable a g r e e m e n t with the n u m ber o f T cells as m e a s u r e d b y rosette f o r m a t i o n . Table 4 shows t h a t the specific a n t i - t h y m o c y t e serum was able to inhibit f o r m a t i o n o f s p o n t a n e o u s rosettes with rabbit red cells, c o n s i s t e n t with its T cell specificity, while n o r m a l r a b b i t serum or r a b b i t anti-guinea pig K chain sera lacked this ability. DISCUSSION T h e presence o f serum a n t i b o d i e s to guinea pig l y m p h o i d cells in u n i m m u n i z e d rabbits m i g h t be e x p e c t e d to interfere with the raising o f anti-T cell a n t i b o d i e s either b y c o m p e t i n g with l y m p h o c y t e r e c e p t o r s for antigen or b y a rapid clearance o f antigen before a n t i b o d y i n d u c t i o n c o u l d occur. The sensitive and c o n v e n i e n t m i c r o c y t o t o x i c plate assay enabled us to e x a m i n e
222 the sera o f a large n u m b e r of rabbits for titres of natural antibodies to guinea pig ly mp h o id cells prior to immunization. Such screening d e m o n s t r a t e d that many rabbits had m oder a t e to high levels of a n t i - t h y m o c y t e antibodies and low to moderate levels of anti-bone marrow cell antibodies, as noted in passing by Shevach er al. (1972). It proved easier to raise specific anti-T cell sera in rabbits by intravenous injection o f t h y m o e y t e s than by intramuscular injection of brain. Intravenous immunization was most sucessful in those animals with low titres of natural antibodies to t h y m o c y t e s and m od erat e titres to bone marrow cells. The sera raised in these selected rabbits had high titres of a n t i - t h y m o c y t e antibodies and could easily be made specific by absorption with L2 C cells. Perhaps these observations are related to those of Pincus et al. (1971), who showed that immunization with mixtures of a complex antigen and ant i body directed against one of the antigenic determinants of this complex antigen led to suppression of ant i body f or m at i on against this det erm i nant and increased a n t i b o d y f o r m a t i o n against non-suppressed determinants. If our observations are another example of this general p h e n o m e n o n , it might be possible to raise high titred specific antisera for guinea pig T cells routinely by immunizing rabbits with low titres of natural antibodies to t h y m o c y t e s and mo d er ate titres to bone marrow cells with mixtures of autologous norreal serum and t h y m o c y t e s . A somewhat similar approach has been used to raise specific rabbit antisera to mouse leukemia cells (Weiner et al., 1972). All rabbits immunized with guinea pig brain produced c y t o t o x i c antibodies for guinea pig t h y m o c y t e s and bone marrow cells (Golub, 1971). Regardless of the levels of natural bone marrow cell antibodies, all rabbits had elevated titres of c y t o t o x i c antibodies to bone marrow cells 2 weeks after immunization, which rose steadily over the next 6 weeks. Repeated absorption with L2 C cells could not always specifically remove these antibodies, perhaps indicating that this c y t o t o x i c activity was associated with anti-stem cell antibodies (Golub, 1972). In contrast to results from work with rabbit anti-mouse brain sera (Golub, 1971), we found antisera raised against guinea pig t h y m o c y t e s to be considerably more specific for thym ocy tes than sera raised against guinea pig brain. The most t h y m o c y t e specific anti-brain sera were raised in rabbits with m oderat e (rather than very high or very low) titres of natural antibodies to guinea pig t h y m o c y t e s . Since anti-brain sera required considerably more absorptions with L: C cells to be rendered t h y m o c y t e specific than did a n t i - t h y m o c y t e sera, and since the titres of the two types of specific sera were similar after absorption, immunization of rabbits with guinea pig brain to raise specific anti-guinea pig T cell antibodies has few if any advantages over immunization with t h y m o cytes directly. It is not clear why our specific a n t i - t h y m o c y t e and anti-brain antisera killed no more than 85 90% (rather than 100%) of t h y m o c y t e s at the lowest dilutions tested (Raff, 1971). However, the results we obtained for percent T cells in t h y m u s and other organs are in reasonable agreement with
223 t h o s e of S h e v a c h et al. ( 1 9 7 2 ) a n d S t a d e c k e r et al. ( 1 9 7 3 ) w h o used specific r a b b i t o r b u r r o anti-T cell antisera and a s t Cr release m e t h o d to m e a s u r e c y t o t o x i c i t y . This c o n c o r d a n c e o f o b s e r v a t i o n s w o u l d s e e m to indicate t h a t the c y t o t o x i c i t y plate m e t h o d we e m p l o y (with its relatively high c o n c e n t r a tions o f r e a g e n t s and long i n c u b a t i o n times) is n o t m u c h less sensitive t h a n r a d i o a c t i v e release m e t h o d s and t h a t t h e l o w e r c y t o t o x i c p l a t e a u is n o t d u e to t h e t e c h n i c a l s h o r t c o m i n g s o f t h e assay e m p l o y e d . T h e p e r c e n t a g e o f T cells in various l y m p h o i d organs was f o u n d to be similar w h e n m e a s u r e d b y s p o n t a n e o u s r o s e t t e f o r m a t i o n or b y c y t o t o x i c i t y ; diluted anti-T cell s e r u m was able to p r e v e n t s p o n t a n e o u s r o s e t t e f o r m a t i o n in t h e a b s e n c e of c o m p l e m e n t . B o t h of t h e findings c o n f i r m t h e r e p o r t o f S t a d e c k e r et al. ( 1 9 7 3 ) . In view o f the f r e q u e n c y o f n a t u r a l anti-guinea pig t h y m o c y t e a n t i b o d i e s in rabbits, s p o n t a n e o u s r o s e t t e f o r m a t i o n b e t w e e n guinea pig T cell and r a b b i t red cells m i g h t d u e to t h e p r e s e n c e o f n a t u r a l anti-guinea pig t h y m o c y t e a n t i b o d i e s a d s o r b e d to the surface o f the red cells, a s u p p o s i t i o n c o n f i r m e d b y t h e ability o f a n t i - a l l o t y p e sera to b l o c k r o s e t t e f o r m a t i o n (H. G o d f r e y , u n p u b l i s h e d results). ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d b y grants f r o m Statens L a e g e v i d e n s k a b e l i g e F o r s k n i n g s r ~ d (to P r o f e s s o r M. S i m o n s e n and B. R u b i n ) , Arvid Nilssons F o n d , M a r y Rosenkjaers Legat, a n d t h e Swiss N a t i o n a l F o u n d a t i o n for Scientific R e s e a r c h ( G r a n t No. 3 . 7 6 0 0 . 7 2 ) . We wish to t h a n k J . F . L o w e and H a n n e SCrensen for valuable technical assistance.
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