- Email: [email protected]
Cellular aspects of tolerance
CELLULAR
12, i?ld--a9
IMMUNOLOGY
(1974)
Cellular VII.
Inheritance MICHIO
Institute
of Immunology, Biochemistry,
Aspects
of Tolerance
of the Resistance FUJIWARA
Departments
3 AND
to Tolerance BERNHARD
Induction
CINADER
of Medical Genetics, Medico1 Biophysics, Sciences Building, University of Toronto, Ontario, Canada M5’S lA8
Medical Toronto,
Received
September
1
and
Clinical
4,1973
The half-lives of elimination (Tt) of ?I-RGG from the body of normal A or Balb/c animals was much longer than the Tt of SJL mice. At all ages, the T+ of normal hybrids (A X SJL, SJL X A, Balb/c X S JL) was similar to or longer than that of the A or Balb/c parents. Thus, in terms of the Ta of normal animals, the SJL responsiveness to ‘“I-RGG appeared to be a recessive trait. Tolerance could be induced in newborn animals and, in terms of Ti, the degree of unresponsiveness at the age of 6 weeks, was the same in A, Balb/c, A X SJL, and Balb/c X SJL animals but was much shorter in SJL mice. Thus, in neonatally induced tolerance, the duration of tolerance was recessive for the SJL type. The average Tt after tolerance induction in 3-week-old hybrids (AX SJL, SJL X A, Balb/c X SJL) was similar to that of the A or Balb/c parent, but by the 8th and 12th week it approached the average Tt of the SJL parent. Comparing 8-week-old hybrids, the average Ts was longest in A X SJL hybrids and identical in SJL X A and Balb/c X SJL mice. An examination of T+ distribution in various 8- and 12-week-old crosses and backcrosses revealed a fairly large proportion of individuals with a Ti which was intermediate between SJL and the other parent. There was a tendency for this number to decrease in 12 weeks as compared to 8-week-old mice. In 8-week-old mice, the number of animals with intermediate T+ was smallest when SJL was the maternal animal [(SJL X A) ; SJL X (A X SJL); SJL X (SJL X A)]. There was no link between Ti of tolerant animals and either the immunoglobulin allotype (MuAl/MuA2) or the C.5 eniotype (MuBl positive/MuBl negative).
INTRODUCTION Age-dependent changes in half-lives of 1311-RGGelimination have been observed in normal and in tolerant mice. The extent of these changes is different in normal mice of different strains and is particularly marked in the tolerance induceability of SJL mice ( 1, 2). In the following series of experiments we have examined these changes in hybrids between A and SJL mice and in hybrids between Balb/c and SJL mice. The A parent was chosen because its tolerance induceability is charac1 Supported by the Medical Research Council, the National Cancer Institute and the Ontario Heart Foundation. saline; 2 Abbreviations : IFA : Incomplete Freund’s adjuvant ; PBS : Phosphate-buffered RGG: Rabbit -y-globulin; T+ : Half-life of antigen elimination. 3 Present address : Department of Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan. 214 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
INHERITANCE
OF
TOLERANCE
INDUCEABILITY
215
teristic of the majority of inbred strains so that it may be regarded as a “normal” animal (3) ; Balb/ c was chosen because it seems to belong, like SJL, to a group of mice (SJL, NZB, Balb/c) which have abnormally high immunological responsiveness, resistance to tolerance, and a tendency to develop lymphoid malignancies (2, 4-6). We used half-lives of elimination, Ig allotypes MuA2, MuAl (7, S), and the possession of the 5th component of complement ( MuBl ; Ref. 9) as markers for genetic studies which involved crosses and various types of back-crosses. MATERIALS
AND
METHODS
Inbred mice were purchased from Jackson Laboratory (Bar Harbor, ME). Hybrids and various back-crosses were bred in our own colony from stock mice obtained from Jackson Laboratory. The strain designation J was common to all animals and was, therefore, omitted from designation in the text. Hybrids were identified by the name of the maternal animal, followed by the name of the paternal animal; back-crosses and double back-crosses were designated on the same basis. Experimental mice were caged in groups of five and allowed free access to food and water. Antibodies directed against MuAl, MuA2, and MuBl (C5) were raised as previously described (7-9). Composition of Freund’s incomplete adjuvant, procedures for purification of rabbit y-globulin (RGG ; lo), iodination of RGG (11) , removal of aggregates from RGG and from its lightly iodinated derivative, tolerance induction, and the elimination test are given in preceding papers of this series (1, 3, 12-14). RESULTS Fl Hybrids:
Antigen
Elimination
from Nomal
Animals
The T, of 1311-RGG eliminations were determined in %week-old normal hybrids, normal back-cross animals, and normal mice of the paternal strains A, Balb/c, and SJL. Data are given for A-SJL derived animals only (Fig. 1). The meanT+ of the hybrids (cross-hatched areas in Fig. 1) resembled that of the parental A and Balb/c animals rather than that of the parental SJL mice. The mean T+ of back-cross animals (A x S JL) x (A x S JL) was shorter than that of hybrids. The frequency curves for T+ of hybrids were closer to those of the A and Balb/c strains than to those of SJL mice. There were some hybrids which eliminated with a Tt that was much longer than any observed in a parental animal. Since the T, distribution of each of the sets of parental animals overlapped, it was impossible to classify individuals from crosses or back-crosses as being one or the other parental type (Fig. 1). The mean values shown by cross-hatched areas in Fig. 1 are compatible with either a monogenic pattern of inheritance, the mean T+ phenotype of SJL being recessive to that of A or Balb/c, or with a multifactorial basis. The age-dependent changes in T+ for hybrids from matings between SJL and either Balb/c or A are shown in the upper section of Table 1. At all ages tested, the T+ in the hybrids were longer than (particularly in young SJL X A mice) or as long as the T+ in A or Balb/c mice and distinct from the much shorter Tt in SJL mice.
216
FU JIWARA
AND
CINADER
FIG. 1. Half-lives of ‘311-RGG elimination from the body of ll-week-old parental strains A and SJL, from crosses between animals of the parental back-crosses. Mice were injected with aggregate free RGG when they were of a given duration are plotted as function of the number of animals which with this half-life. Hatched areas indicate mean T$ il standard deviation.
normal mice of strain and from 8 weeks old. T: eliminate antigen
Fl Hybrids: Antigen El&&nation from Animals Rendered Tolerant at Birth Newborn A, Balb/c, SJL, A x SJL, and Balb/c X SJL mice were given 3 SC injections of a total of 0.2 mg aggregate free RGG per gram body weight. At the age of 5 weeks they were immunized SCwith 0.25 mg RGG-IFC and, 1 week later, they were injected with 10 pg 1311-RGG. The Tt were identical in hybrids, A/J and Balb/c mice, but much lower (p
INHERITANCE
Tolerance Induced at Various
OF
TOLERANCE
217
INDUCEABILITY
Times after Birth.
Animals of different ages were rendered tolerant, were immunized, and were tested at a constant time after tolerance induction (lower section of Table 1). Among young animals, the mean T+ in hybrids was similar to that of the paternal A or Balb/c mice; in older animals it was similar to that of the parental SJL animals. Since the normal T, of animals varied with strain and age, the T+ of elimination from tolerant animals were normalized to the Tt in corresponding normal animals (Figs. 3 and 4). It remained apparent that 3-week-old hybrids eliminated 1311-RGG like their A or Balb/c parents, while the S- and 12-week-old hybrids resembled their SJL parent. It seemed that the resistance to tolerance induction was an agedependent phenomenon not only in the S JL animal but also in its hybrid off spring, and attention was now given to animals tolerized at the age of 8 weeks. Fl Hybrids: The T+ in Ani%& Rendered Tolerant at the Age of 8 Weeks, Iwwaunizedat 10 Weeks, aud Tested at 11 Weeks of Age The distribution of Tt in tolerized-immunized crosses and in various types of back-crosses for A and SJL are shown in Fig. 5 and for Balb/c and SJL in Fig. 6. It is apparent that the distribution of T, of tolerized immunized A and Balb/c, on the one hand, and SJL, on the other, were quite different. It will be seen from Tables 2 and 3 of T* in A-SJL and Balb/c-SJL derived matings that there were some hybrid and back-cross animals which had intermediate T+ and thus could not be classified by half-life as resembling either of the two parents.
x
SJL
SJL
Balb/cxSJL
-
Mb/c
tolerance. A, SJL, Balb/c, A X SJL, and Balb/c X SJL mice were injected neonatally (SC) with 0.2 mg aggregate free RGG, were immunized (SC) with 0.25 mg RGG-IFA, and injected with 10 pg ‘“I-RGG at the age of 5 and 6 weeks, respectively. Half lives are shown for each type of animal. The heights of the vertical bars indicate the average half-life and the vertical lines indicate TA *l standard deviation from the mean. FIG.
2. Neonatally induced
:1
12
8
15
11
8
6
1.5
11
8
6
(weeks) 0.6
(8/W
0.9
8.4 f
T+
(4
W/30)
12.7 f 1.0 (5/g) 10.3 f 0.7 (5/g) 9.1 f 1.5
10.9 f 1.3 (7/14) 11.2 f 1.6 (28/36) 10.1 f 0.7 w/w
Gm
14.1 f
A
(4
T4
(4
T,
(4
Tt
b)
T+
(4
Tt
(4
W
Ti
Tbb
Age at testing
1.1
3.1
1.6
2.5
2.5
W/22)
11.0 f 1.5 (15/26) 3.7 f 2.4 (11/24) 1.3 f 1.0
P/5)
11.9 f
14.2 f (214) 13.8 f (10/17) 13.2 f (7/13) 13.3 f (8/14)
A X SJL
0.6
X A
2.2
RGG they
0.90 f 0.30 (3/-i)
W/16)
2.3 f
P/12)
11.7 f 2.1 (3/7) 13.2 f 1.7
18.0 f 2.3 (3/7) 14.0 f 3.7 (g/17) 12.9 f 0.4 (3/5)
G/5)
14.7 f
SJL
* Animals designated as tolerant were injected ip with 0.2 mg/g body weight of aggregate free mice were immunized subcutaneously with 0.25 mg RGG-IFA. One week after this immunization, ‘“II-RGG, at the same age. b T+ designates half-life in days. 0 (n) designates the number of females over the total number of animals in the group.
RGG-IFA, 1311-RGG
5
1 3
Aggregate RGG,
free
-
1 -
-
-
Age at injection of aggregate free RGG (weeks)
1311-RGG
Injection
1
1.1
2.5
1.7 f 0.7 (15/15) 0.40 f 0.30 W/10)
3.0 f
-
X SJL
1.4
1.9
1.8
1.5
2.1
0.8
(10/13) these 10 Mg
8.2 f 2.2 (21/27) 7.2 f 1.6
GW)
9.2 f
-
(17/W
9.7 *
W/30)
9.6 f
W/W
9.9 f (l/3) 9.5 f
Balb/c
Two weeks later animals received
1.4 f 0.8 (11/23)
(lo/W
10.0 f 1.6 (11/17) 2.5 f 2.0
-
@/lo)
12.0 f
(6/W
11.8 f 1.7 (4/g) 12.8 f 1.1
Balb/c
(9/l@
0.8
P/6)
6.7 f
W3)
6.5 f
W/20)
8.7 f 1.2 (3/W 8.5 f 1.6 (9/W 7.8 f 1.0
SJL
at the age indicatd in the table. Normal received 10 pg lJ’I-RGG.
Strain
THIS ELIMIN.\TION OF la11 FMIM A, SJL, BALB/C, .IND FROM HYHRIDP
TABLE
B
a z
2 E 5 g
INHERITANCE
OF
TOLERANCE
219
INDUCEABILITY
c 02
LL
1-p-l 1
2
3
4
5
6
7
8
9
10
11
12
Age in weeks FIG. 3. The relative half-life of “‘I-RGG in tolerized and immunized A, SJL mice, and hybrids between them. Inbred mice and hybrids A (U), A X SJL (A), S JL X A (0 ), and SJL (0) were treated as indicated in Table 1. The average Tt of animals tolerized and immunized are shown as a fraction of the average T+ of normal animals of the same age.
A majority of hybrids resembled the SJL parent, but this was only characteristic of all offspring when SJL was the maternal animal. Tolerant State, C atid Ig Markers The distribution of genetic Ig- and complement-markers among Balb/c, SJL, and A-SJL derived animals is shown in Table 4; half-lives are shown in terms of MuAl and MuA in Tables 5 and 6 and in terms of C5(MuBl) in Table 7. It is apparent that there is no genetic link between either the T+ and the immunoglobulin or the complement markers.
z
0.4 ’
2 2 02 I t
I 1
I 2
I 3
4
5
6
7
8
9
10
11
12
Age in weeks FIG. 4. in hybrids SJL (A) nized are
The relative half-life of 9-RGG in tolerized and immunized Balb/c, SJL mice, and between them. Inbred mice and hybrids BALB/c (Cl), S JL (0)) and BALB/c X were treated as indicated in Table 1. The average Tt of animals tolerized and immushown as a fraction of the average T+ of normal animals of the same age.
220 The Relative Shift with
FUJIWARA
AND
Age of Half-Life
CINADER
D&rib&ion
frow
the Body of Hybrids
It has become apparent from preceding data that some hybrids, tolerized at the age of 8 weeks, eliminated antigen with a Tt that could not be classified as being that of either the maternal or the paternal animal. It seemed possible that this
o
4
8
Half lifeiin
12
16
davsl
FIG. 5. Half-lives of ?-RGG elimination from the body of 11-week-old tolerized mice of parental strains A, and S JL, from crosses between animals of the parental ‘strain and from backcrosses. Mice were injected with aggregate free RGG when they were 8 weeks old. Tt of a given duration are plotted as function of the number of animals which eliminate antigen with this half-life. Hatched areas indicate the mean Tt *I standard deviation.
INHERITANCE
OF
TOLERANCE
TABLE
221
INDCCEABILITY
2
CLASSIFICATION OF ANIMALS FROM VARIOUS MaT1NGs IN TERMS LIFE OF mI-RGG ELIMINATION FROM TOLERIZED-IMMUNIZED A, SJL DERIVED MICE~ Types
of cross female
X male
(A
A SJL A X SJL SJL X A A X (A x SJL) (A X SJL) X A (A X SJL) X SJL SJL X (A x SJL) SJL X (SJL X A) X SJL) x (A X SJL)
o Eight-week-old weeks later with
Animals with halflives (in days)
Number Total
Female
30 15 24 16 15 12 14 8 5 12
animals were 0.25 mg RGG-IFA
OF HALF-
23 15 11 10 8 6 8 5 3 6
injected ip with 0.2 mg/g (SC), and after a further
O-4.3
4.4-5.9
6.0-14.0
0 15 11 15 7 5 14 8 5 2
0 0 4 0 0 1 0 0 0 1
30 0 9 1 8 6 0 0 0 9
body weight aggregate free week with 10 rg i3iI-RGG.
RGG,
2
reflected the rate at which tolerant hybrids shifted to the responsiveness of its SJL parent. We, therefore, examined T+ distribution in various types of hybrids and back-cross offspring as a function of age. Even among hybrids tolerized at the age of 12 weeks there were always some individuals with a T, that was considerably longer than that of the S JL parent. This was more marked in A x SJL than in S JL X A hybrids. It was most striking in hybrid offspring of Balb/c x SJL matings (Figs. 7-9).
TABLE CLMSIFICSTION
OF ANIMALS ELIMINATION
Types
of cross
Female
Balb/c SJL Balb/c (Balb/c (Balb/c (Balb/c
X SJL) X SJL) X SJL)
3
FROM Vanrous MATINGS IN TKRMS OF H.\LFLIFE FROM TOLERIZ~D (AT 8 WEEKS) AND IMMUNIZHD B.ILB,/-SJL DERIVED MICE~ Number Rlale
X
X X
Balb/c SJL
x”
SJL Balb/c SJL (Balb/c
X X
X SJL)
OF
‘“I-RGG
Number with (in days) Total
Tt
Female
27 15 16 10 11 23
a Eight-week-old animals were injected ip with 0.2 mg aggregate weeks later with 0.25 mg RGG-IFA (SC), and after a further week * Individual !!‘$ were 5.6, 6.2, and 6.3 (for comparison : the average
21 13 9 7 1: free with
o-4.3
5-14
0 15 13 3 11 10
27 0 3* 7 0 13
RGG/g body weight, 2 10 pg r3rI--RGG. T+ of Balb/c was 8.2 f 2.2).
17 15 18 8 8 22
23 12
A X (A X SJL) (AXSJL) XA (A X SJL) x SJL SJL X (A x SJL) SJL X (SJL x A) (A X SJL) X (A X SJL)
A X SJL SJL X A
n
15 12 14 8 5 12
Breeding
A X (A X SJL) (A X SJL) X A (AXSJL)xSJL SJL X (A X SJL) SJL X (SJL x A) (A X SJL) X (A X SJL)
A, SJL :ALLOTYPE
n
Breeding
RELATIVE
0 0
3 2 0 0 0 0
T1(SJL)
MuAl
AND HdLF-LIFE
MuBl+
MuAl
DISTRIBUTION
4
0 0
3 2 0 0 0 0
T+(A)
IN S-WEEK
TABLE
MuBl-
5
OF MuAl-MuA
TABLE
MuAl
4 3 6 8 3 1
1
1
Tt@JL)
TOLERIZED-IMMUNIZED
23 12
5 7 9 8 5 15
MuBl+
MuAl
AND OF MuBl
MuA
0 0
5 4 0 0 0 8
T+(A)
ANIMALS
MuBl-
MuA
0 0 8 0 2 1
WSJL)
0 0
0 0 9 0 3 1
MuBl+
MuA
MuA
0 0
0 0 0 0 0 1
0 0 0 0 0 1
TdA)
MuBl-
% B
2 ‘1
2 $ Lb
+z
6
AND HALF-Lrms
11 23
10
n
7
0 2
3
T;(BaIb/c)
4 4 14 8 5 2
Tt @JL)
MuBl
2
1
positive
5 2 0 0 0 5
T+ (4
OFGWEEKTOLERIZED-IMMUNIZED
TABLE
0 3
0
Tt(SJL)
Muhl
ANIMALS
3 4
4
T:(SJL)
MuAl
0 6
3
T+(Balb/c)
MuA
ALLOTYPE AND H:~LF-LIFE IN ~-WEEK TOLERI~ICD-IMMUNIZED ANIMALS
C5
15 12 14 8 5 12
X SJL)
A X (A X SJL) (A X SJL) X A (A X SJL) x SJL SJL X (A x SJL) SJL X (SJL X A) (A X SJL) X (A X SJL)
X SJL X (Balb/c
n
X SJL) X SJL)
(Balb/c (Balb/c
X Balb/c
Breeding
X SJL)
(Balb/c
Breeding
BALB/C, SJL:
TABLE
3 1 0 0 0 0
T+(SJL)
MuBl
8 4
0
negative
Tt(SJL)
MuA
3 4 0 0 0 3
WA)
0 4
0
T;(Balb/c)
3 4 $ z t: 2
m
2
B
E
ii E
L-4 E
224 DISCUSS
ION
In earlier papers of this series (l-3) we have reported that SJI, mice h;~~c ;I defect in their immune apparatus as manifested by hyper-responsiveness, relatively rapid maturation of the responsi\:eness to RGG aggregates, and resistance to tolerance induction after the age of 8 \vccks. The manifestation of this lesion depended on the accessory cells, but :~ppearcd to result from an abnormality in the thymusderived CCII population. The genetic experiments, described in this paper, were designed to probe further into the nature of the abnormality. The SJI, phenotype was recessive in terms of neonntaiiy induced tolerance (Fig. 2) and in terms of the half-life (Ti) with which aggregate freed (centrifugation at 123,OOOg) lslIRGG was eliminated from the body of otherwise untreated animals (Table 1). A peculiarity, in this latter cast, was the finding that some hybrids eliminated with half-lives which ncre significantly longer than those of parental individuals (Fig. 1) possibly because of a metaholicaiiy manifcstcci interaction between the genes of the two parental animals. ‘IlYe shall now turn to ;I consideration of tolcrizedimmunized animals. in this contest we shall rcfcr to auimnls by the age at which they were toicrized; they were tested 3 weeks later. Broadly speaking, the resistance to toicrance of the adult animals was dominant for SjL characteristics. J-iowever, there were some findings, revealed by detailed esnmination of this dominance (Tables 2-G), ivhich defied a simple monogenic model. The average 7.4 of toierized-immunized S-~ck-old h!.britis approached that of similarly treated SJL parental animals, though it was still somewhat longer. It was longest in A x SJL hybrids and much closer to that of the SJL parent in SJI. X A mice. In the animals, tolerized at the age of 12 weeks, this difference could no longer be observed (Figs. 7 and S). It thus appears as if the identity of the maternal animal affected the rate at which the SJI, phenotype was being reached. ‘i‘here was another, probably related, manifestation of this maternal influence. This was seen when the T! distribution of various 1 l- and 1S-week-old toierizcd-immunized offspring from crosses and back-crosses was examined. The half-life distribution of the parental animals was suci~ that one would have expected that hybrids could be classified as having the 7’4 characteristic either for .\ or for SJI. c4inlination. 111fact. however, a fairly large number of individuals could not be classified since their T+ was intermediate between those of parental animals. Even mot-c striking was the 1‘1 distribution among tolerized-immunized Mb/c x SJ 1. hybrids of which 3/13 showed the T, of Balb/c type while the other 10 had the 7‘, of SJL type (Fig. 6). The general distribution of half-lives in tolerizcd-immunized A-SJT, descended animals might be compatible with a dominant, monogcnic inheritance of the SJL trait. If one assumed that the half-life trait of all A x SJI, mice is of the SJI, type, one would expect that 50 ‘;b of the offspring from :\ X (.A X SJl,) and (X X SJL) X A matings would eliminate 1311-RGG with the same half-life as :2 mice and that none of the offspring from SJL X (:1 x SJL) and SJI, X (SJL X A) matings would have half-lives of the same length as 11 mice. This predicted distribution was in reasonable accord with observation (Table 2). On the other hand, more than the expected 2576 of offspring from (:I x SJL) X (A X SJL) matings had the T, characteristic for the X parent. The finding may be attributable to chance, since the group deviated to the same cstcnt, though in the opposite direction, from the predicted distribution of hIu.41 and Mu.1 2 aliot\;l)e (O’i;, rather than 25’&
INHERITANCE
OF
11
/
TOLERANCE
1
I
I
INDUCEABILITY
I
,
22.5
I
8
4
12
a
4
0
4
8
12
16
Half lifelm days)
FIG. 6. Half-lives of =‘I-RGG elimination from ll-week-old tolerized-immunized inbred mice of strains Balb/c and SJL, crosses between them and back-crosses. Mice were 8 weeks old at the time of injection with aggregate free RGG and were 11 weeks when mI-RGG was injected. Tt of a given duration are plotted as a function of the number of animals which eliminate antigen with this half-life. The mean Tt 2-1 standard deviation for each group is shown as a hatched area. were homozygous for the allotype of A mice ; Table S), while the frequency of MuBl was identical with the predicted incidence of 75% (Table 7). The offspring of matings (Balb/c x SJL) X Balb/c showed much more than 50% Balb/c type T+, though all (Balb/c X SJL) X SJL offspring did have the predicted T+ of SJL (Table 3). In these cases,the distribution of MuAl and MuA was very close to expectation (Table 6). We have already mentioned that the number of hybrids with T+ intermediate between the two parents was smahest when the maternal animal was SJL, i.e., among SJL X A hybrids. This fact must
226
FU JIWARA
AND
CINADER
be taken into account in considering the previously mentioned absence of individual with T, characteristic for A among SJL X (SJL x A) mice. We have previously invoked this as evidence for dominant monogenic inheritance. However, in reality, it might be attributable to a maternally transmitted control over the manifestation of the SJL phenotype. Age:
(in weeks) 1
0g 8 I
RGG in hybrids Half life of “‘I Half life of ‘3’1- RGG in A mice Half life of “‘I - RGG in SJL mice
Half life (in days.) FIG. 7. A X S JL mice : Changes in the half-life distribution of tolerized and then immunized hybrids and parental mice as a function of age at which animals were tolerized. Elimination tests were always carried out 3 weeks after tolerization. The height of the vertical bars indicates the number of animals of a T+ within the range shown on the horizontal axis; the hatched areas indicate the mean C.1 standard deviation for each group.
IKHERITANCE
OF
TOLERANCE
INDUCEARILITY
227
ge : (in Iueeks)
1
1!
7
0 1-
Half life 01 ‘“IRGG in hybrids Holf life of ‘3’1- RGG in * mice tall tite of “’ I - RGG in SJL mke
8 -I
1I
12 8 4 12 4 2 0
4
8
12
16 20
Half life (in days) FIG. 8. SJL X A mice : Changes in the half-life distribution of tolerized and then immunized hybrids and parental mice as a function of age at which animals were tolerized. The height of the vertical bars indicates the number of animals which have a Te within the range shown on
the horizontal axis ; the hatched areas indicate the mean Cl standard deviation for each group. There was a tendency for the number of animals with intermediate Ts to decrease with advancing age, but even at 12 weeks there were some hybrids with T+ which were significantly in excess of those characteristic for the SJL animals of the same age. This was most marked among offspring from Balb/c X SJL matings (Figs. 7-9). Nevertheless, genetic analysis of even older animals might lead to a more definitive analysis. The design of the present study does not permit formal
228
FUJIWARA
AND
CINADER
Hoif
life
of
‘3’1-
Half
lhfe
of
-‘I
tiotf
l,fe
of ‘=I
RGG - RGG - RGG
8 4 I 0
2
4
6 Half
8
10
12
14
lkfe (m days)
9. BALB/c X SJL mice : Changes in the half-life distribution of tolerized and then immunized hybrids and parental mice as a function of age at which animals were tolerized. The height of the vertical bars indicated the number of animals which have a T+ within the range shown on the horizontal axis; the hatched area indicates the mean kl standard deviation for each group. FIG.
it is reasonable to expect that analysis for multifactorial inheritance. However, more than one locus might be involved in the determination of resistance to tolerance induction. We must now come back to the T, of untreated animals. Among the hybrids we noted individuals with T+ in excess of those found in any A or Balb/c animals, i.e., a shift of the Tt distribution to higher values (Fig. 1). It is this interaction which might also affect the T+ of tolerized-immunized animals and thus interfere with the full manifestation of the SJL genotype for responsiveness of tolerized and immunized hybrids (see Figs. 3 and 4). The maternal influence does not reduce the T* of untreated animals (cf. S JL X A and A X SJL in Fig. 1), and thus appears to be a separate factor. In summary, the SJL trait for the resistance to adult tolerance induction appears to be dominant and might be inherited monogenitally. However, the expression of this dominance depended on the identity of the maternal animal and might be affected by a process which results in slow elimination of antigen. Clearly, the question arises whether the effects, so far de-
INHERITANCE
OF
TOLERANCE
INDUCEABILITY
229
scribed, can be explained solely by the transmission of genetic factors or whether other, possibly viral factors, are implicated. There is remarkable age-dependent change in the Tg of tolerance-inducing antigen from the body of hybrids between SJL and either A or Balb/c mice. The young hybrids behave like their Balb/c or the A parents, whereas the old hybrids approach very closely the half-life of the SJL parents (Figs. 3 and 4). It is, therefore, clear that hybrids acquire resistance to tolerance induceability with advancing age. However, hybrid responsiveness is far from uniform and there are always some individuals whose responsiveness is intermediate between that of the two parents (Figs. 5 and 6). The progression with time is different for individuals of identical genotypes. This and the lack of uniformity leads us to think that there is some external event which may stimulate the induction of SJL-type responsiveness. The proneness of SJL mice to a variety of immunoglobulin abnormalities and to neoplastic disease may be connected with the resistance of old SJL and of SJL hybrids to tolerance induction. At any rate, a factor seems to be involved which is responsible for variability. There is some indication that the transmission of this factor occurs more regularly if SJL is the maternal rather than the paternal parent of the hybrids (Figs. 7 and 8) ; this is particularly noticeable in the relative distribution of half-lives in 11-week-old tolerized-immunized S JL X A and A X S JL hybrids. There appear to be, at least, two factors involved in the transmission of the resistance to tolerance induceability; one of these seems to determine the age at which a hybrid or back-cross animal acquires the characteristic of the SJL parent. The lack of uniformity of the hybrid leads to the assumption that an external stimulus or a probabilistic event, such as the activation of a virus, may also enter into the expression of the S JL parent’s resistance to tolerance induction (15, 16). There was no major difference between the age variation of the T3 distribution of A X S JL and Balb/c X S JL mice (Figs. 7 and 9) and the abnormal responsivenesss of Balb/c may not reinforce the SJL trait (Table 1). There was no linkage between the half-life of tolerant-immunized animals and the immunoglobulin allotype (MuAl/MuAZ) or the C5 eniotype (Tables 5-7). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11. 12. 13. 14. 15. 16.
Fujiwara, M., and Cinader, B., Cell. Immunol. 11, 194, 1974. Fujiwara, M., and Cinader, B., Cell. Zmmunol. 11,205, 1974. Fujiwara, M., and Cinader, B., Cell. Immunol. 12, 11, 1974. Evans, M. M., Williamson, W. G., and Irvine, W. J., Cl& Exg. Zmmunol. 3, 375, 1968. Staples, P. J., Steinberg, A. D., and Talal, N., J. Exp. Med. 131, 1223, 1970. Cerottini, J. C., Lambert, P. H., and Dixon, F. J., J. Exp. Med. 130, 1093, 1969. Dubiski, S., and Cinader, B., Na&ure (Lo&on) 197, 705, 1963. Cinader, B., and Dubiski, S., J. Zmmunol. 101, 1236, 1968. Cinader, B., Dubiski, S., and Wardlaw, A. C., J. Expt. Med. 120, 897, 1964. Fujiwara, M., Jap. J. Exp. Med. 41, 59, 1971. Sonoda, S., and Schlamowitz, M., Immunochemistry 7, 885, 1970. Kaplan, A. M., and Cinader, B., Cell. Zmmzmol. 6, 442, 1973. Fujiwara, M., and Cinader, B., Cell. Zmmzmol. 12, 1, 1974. Kaplan, A. M., and Cinader, B., Cell. fmmunol. 6, 429, 1973. Mellors, R. C., and Huang, C. Y., J. Exfi. Med. 126, 53, 1967. Thivolet, J., Morrier, J. C., Ruel, J. P., and Richard, M. H., Natrive (London) 214, 1134, 1967.
12, i?ld--a9
IMMUNOLOGY
(1974)
Cellular VII.
Inheritance MICHIO
Institute
of Immunology, Biochemistry,
Aspects
of Tolerance
of the Resistance FUJIWARA
Departments
3 AND
to Tolerance BERNHARD
Induction
CINADER
of Medical Genetics, Medico1 Biophysics, Sciences Building, University of Toronto, Ontario, Canada M5’S lA8
Medical Toronto,
Received
September
1
and
Clinical
4,1973
The half-lives of elimination (Tt) of ?I-RGG from the body of normal A or Balb/c animals was much longer than the Tt of SJL mice. At all ages, the T+ of normal hybrids (A X SJL, SJL X A, Balb/c X S JL) was similar to or longer than that of the A or Balb/c parents. Thus, in terms of the Ta of normal animals, the SJL responsiveness to ‘“I-RGG appeared to be a recessive trait. Tolerance could be induced in newborn animals and, in terms of Ti, the degree of unresponsiveness at the age of 6 weeks, was the same in A, Balb/c, A X SJL, and Balb/c X SJL animals but was much shorter in SJL mice. Thus, in neonatally induced tolerance, the duration of tolerance was recessive for the SJL type. The average Tt after tolerance induction in 3-week-old hybrids (AX SJL, SJL X A, Balb/c X SJL) was similar to that of the A or Balb/c parent, but by the 8th and 12th week it approached the average Tt of the SJL parent. Comparing 8-week-old hybrids, the average Ts was longest in A X SJL hybrids and identical in SJL X A and Balb/c X SJL mice. An examination of T+ distribution in various 8- and 12-week-old crosses and backcrosses revealed a fairly large proportion of individuals with a Ti which was intermediate between SJL and the other parent. There was a tendency for this number to decrease in 12 weeks as compared to 8-week-old mice. In 8-week-old mice, the number of animals with intermediate T+ was smallest when SJL was the maternal animal [(SJL X A) ; SJL X (A X SJL); SJL X (SJL X A)]. There was no link between Ti of tolerant animals and either the immunoglobulin allotype (MuAl/MuA2) or the C.5 eniotype (MuBl positive/MuBl negative).
INTRODUCTION Age-dependent changes in half-lives of 1311-RGGelimination have been observed in normal and in tolerant mice. The extent of these changes is different in normal mice of different strains and is particularly marked in the tolerance induceability of SJL mice ( 1, 2). In the following series of experiments we have examined these changes in hybrids between A and SJL mice and in hybrids between Balb/c and SJL mice. The A parent was chosen because its tolerance induceability is charac1 Supported by the Medical Research Council, the National Cancer Institute and the Ontario Heart Foundation. saline; 2 Abbreviations : IFA : Incomplete Freund’s adjuvant ; PBS : Phosphate-buffered RGG: Rabbit -y-globulin; T+ : Half-life of antigen elimination. 3 Present address : Department of Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan. 214 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
INHERITANCE
OF
TOLERANCE
INDUCEABILITY
215
teristic of the majority of inbred strains so that it may be regarded as a “normal” animal (3) ; Balb/ c was chosen because it seems to belong, like SJL, to a group of mice (SJL, NZB, Balb/c) which have abnormally high immunological responsiveness, resistance to tolerance, and a tendency to develop lymphoid malignancies (2, 4-6). We used half-lives of elimination, Ig allotypes MuA2, MuAl (7, S), and the possession of the 5th component of complement ( MuBl ; Ref. 9) as markers for genetic studies which involved crosses and various types of back-crosses. MATERIALS
AND
METHODS
Inbred mice were purchased from Jackson Laboratory (Bar Harbor, ME). Hybrids and various back-crosses were bred in our own colony from stock mice obtained from Jackson Laboratory. The strain designation J was common to all animals and was, therefore, omitted from designation in the text. Hybrids were identified by the name of the maternal animal, followed by the name of the paternal animal; back-crosses and double back-crosses were designated on the same basis. Experimental mice were caged in groups of five and allowed free access to food and water. Antibodies directed against MuAl, MuA2, and MuBl (C5) were raised as previously described (7-9). Composition of Freund’s incomplete adjuvant, procedures for purification of rabbit y-globulin (RGG ; lo), iodination of RGG (11) , removal of aggregates from RGG and from its lightly iodinated derivative, tolerance induction, and the elimination test are given in preceding papers of this series (1, 3, 12-14). RESULTS Fl Hybrids:
Antigen
Elimination
from Nomal
Animals
The T, of 1311-RGG eliminations were determined in %week-old normal hybrids, normal back-cross animals, and normal mice of the paternal strains A, Balb/c, and SJL. Data are given for A-SJL derived animals only (Fig. 1). The meanT+ of the hybrids (cross-hatched areas in Fig. 1) resembled that of the parental A and Balb/c animals rather than that of the parental SJL mice. The mean T+ of back-cross animals (A x S JL) x (A x S JL) was shorter than that of hybrids. The frequency curves for T+ of hybrids were closer to those of the A and Balb/c strains than to those of SJL mice. There were some hybrids which eliminated with a Tt that was much longer than any observed in a parental animal. Since the T, distribution of each of the sets of parental animals overlapped, it was impossible to classify individuals from crosses or back-crosses as being one or the other parental type (Fig. 1). The mean values shown by cross-hatched areas in Fig. 1 are compatible with either a monogenic pattern of inheritance, the mean T+ phenotype of SJL being recessive to that of A or Balb/c, or with a multifactorial basis. The age-dependent changes in T+ for hybrids from matings between SJL and either Balb/c or A are shown in the upper section of Table 1. At all ages tested, the T+ in the hybrids were longer than (particularly in young SJL X A mice) or as long as the T+ in A or Balb/c mice and distinct from the much shorter Tt in SJL mice.
216
FU JIWARA
AND
CINADER
FIG. 1. Half-lives of ‘311-RGG elimination from the body of ll-week-old parental strains A and SJL, from crosses between animals of the parental back-crosses. Mice were injected with aggregate free RGG when they were of a given duration are plotted as function of the number of animals which with this half-life. Hatched areas indicate mean T$ il standard deviation.
normal mice of strain and from 8 weeks old. T: eliminate antigen
Fl Hybrids: Antigen El&&nation from Animals Rendered Tolerant at Birth Newborn A, Balb/c, SJL, A x SJL, and Balb/c X SJL mice were given 3 SC injections of a total of 0.2 mg aggregate free RGG per gram body weight. At the age of 5 weeks they were immunized SCwith 0.25 mg RGG-IFC and, 1 week later, they were injected with 10 pg 1311-RGG. The Tt were identical in hybrids, A/J and Balb/c mice, but much lower (p
INHERITANCE
Tolerance Induced at Various
OF
TOLERANCE
217
INDUCEABILITY
Times after Birth.
Animals of different ages were rendered tolerant, were immunized, and were tested at a constant time after tolerance induction (lower section of Table 1). Among young animals, the mean T+ in hybrids was similar to that of the paternal A or Balb/c mice; in older animals it was similar to that of the parental SJL animals. Since the normal T, of animals varied with strain and age, the T+ of elimination from tolerant animals were normalized to the Tt in corresponding normal animals (Figs. 3 and 4). It remained apparent that 3-week-old hybrids eliminated 1311-RGG like their A or Balb/c parents, while the S- and 12-week-old hybrids resembled their SJL parent. It seemed that the resistance to tolerance induction was an agedependent phenomenon not only in the S JL animal but also in its hybrid off spring, and attention was now given to animals tolerized at the age of 8 weeks. Fl Hybrids: The T+ in Ani%& Rendered Tolerant at the Age of 8 Weeks, Iwwaunizedat 10 Weeks, aud Tested at 11 Weeks of Age The distribution of Tt in tolerized-immunized crosses and in various types of back-crosses for A and SJL are shown in Fig. 5 and for Balb/c and SJL in Fig. 6. It is apparent that the distribution of T, of tolerized immunized A and Balb/c, on the one hand, and SJL, on the other, were quite different. It will be seen from Tables 2 and 3 of T* in A-SJL and Balb/c-SJL derived matings that there were some hybrid and back-cross animals which had intermediate T+ and thus could not be classified by half-life as resembling either of the two parents.
x
SJL
SJL
Balb/cxSJL
-
Mb/c
tolerance. A, SJL, Balb/c, A X SJL, and Balb/c X SJL mice were injected neonatally (SC) with 0.2 mg aggregate free RGG, were immunized (SC) with 0.25 mg RGG-IFA, and injected with 10 pg ‘“I-RGG at the age of 5 and 6 weeks, respectively. Half lives are shown for each type of animal. The heights of the vertical bars indicate the average half-life and the vertical lines indicate TA *l standard deviation from the mean. FIG.
2. Neonatally induced
:1
12
8
15
11
8
6
1.5
11
8
6
(weeks) 0.6
(8/W
0.9
8.4 f
T+
(4
W/30)
12.7 f 1.0 (5/g) 10.3 f 0.7 (5/g) 9.1 f 1.5
10.9 f 1.3 (7/14) 11.2 f 1.6 (28/36) 10.1 f 0.7 w/w
Gm
14.1 f
A
(4
T4
(4
T,
(4
Tt
b)
T+
(4
Tt
(4
W
Ti
Tbb
Age at testing
1.1
3.1
1.6
2.5
2.5
W/22)
11.0 f 1.5 (15/26) 3.7 f 2.4 (11/24) 1.3 f 1.0
P/5)
11.9 f
14.2 f (214) 13.8 f (10/17) 13.2 f (7/13) 13.3 f (8/14)
A X SJL
0.6
X A
2.2
RGG they
0.90 f 0.30 (3/-i)
W/16)
2.3 f
P/12)
11.7 f 2.1 (3/7) 13.2 f 1.7
18.0 f 2.3 (3/7) 14.0 f 3.7 (g/17) 12.9 f 0.4 (3/5)
G/5)
14.7 f
SJL
* Animals designated as tolerant were injected ip with 0.2 mg/g body weight of aggregate free mice were immunized subcutaneously with 0.25 mg RGG-IFA. One week after this immunization, ‘“II-RGG, at the same age. b T+ designates half-life in days. 0 (n) designates the number of females over the total number of animals in the group.
RGG-IFA, 1311-RGG
5
1 3
Aggregate RGG,
free
-
1 -
-
-
Age at injection of aggregate free RGG (weeks)
1311-RGG
Injection
1
1.1
2.5
1.7 f 0.7 (15/15) 0.40 f 0.30 W/10)
3.0 f
-
X SJL
1.4
1.9
1.8
1.5
2.1
0.8
(10/13) these 10 Mg
8.2 f 2.2 (21/27) 7.2 f 1.6
GW)
9.2 f
-
(17/W
9.7 *
W/30)
9.6 f
W/W
9.9 f (l/3) 9.5 f
Balb/c
Two weeks later animals received
1.4 f 0.8 (11/23)
(lo/W
10.0 f 1.6 (11/17) 2.5 f 2.0
-
@/lo)
12.0 f
(6/W
11.8 f 1.7 (4/g) 12.8 f 1.1
Balb/c
(9/l@
0.8
P/6)
6.7 f
W3)
6.5 f
W/20)
8.7 f 1.2 (3/W 8.5 f 1.6 (9/W 7.8 f 1.0
SJL
at the age indicatd in the table. Normal received 10 pg lJ’I-RGG.
Strain
THIS ELIMIN.\TION OF la11 FMIM A, SJL, BALB/C, .IND FROM HYHRIDP
TABLE
B
a z
2 E 5 g
INHERITANCE
OF
TOLERANCE
219
INDUCEABILITY
c 02
LL
1-p-l 1
2
3
4
5
6
7
8
9
10
11
12
Age in weeks FIG. 3. The relative half-life of “‘I-RGG in tolerized and immunized A, SJL mice, and hybrids between them. Inbred mice and hybrids A (U), A X SJL (A), S JL X A (0 ), and SJL (0) were treated as indicated in Table 1. The average Tt of animals tolerized and immunized are shown as a fraction of the average T+ of normal animals of the same age.
A majority of hybrids resembled the SJL parent, but this was only characteristic of all offspring when SJL was the maternal animal. Tolerant State, C atid Ig Markers The distribution of genetic Ig- and complement-markers among Balb/c, SJL, and A-SJL derived animals is shown in Table 4; half-lives are shown in terms of MuAl and MuA in Tables 5 and 6 and in terms of C5(MuBl) in Table 7. It is apparent that there is no genetic link between either the T+ and the immunoglobulin or the complement markers.
z
0.4 ’
2 2 02 I t
I 1
I 2
I 3
4
5
6
7
8
9
10
11
12
Age in weeks FIG. 4. in hybrids SJL (A) nized are
The relative half-life of 9-RGG in tolerized and immunized Balb/c, SJL mice, and between them. Inbred mice and hybrids BALB/c (Cl), S JL (0)) and BALB/c X were treated as indicated in Table 1. The average Tt of animals tolerized and immushown as a fraction of the average T+ of normal animals of the same age.
220 The Relative Shift with
FUJIWARA
AND
Age of Half-Life
CINADER
D&rib&ion
frow
the Body of Hybrids
It has become apparent from preceding data that some hybrids, tolerized at the age of 8 weeks, eliminated antigen with a Tt that could not be classified as being that of either the maternal or the paternal animal. It seemed possible that this
o
4
8
Half lifeiin
12
16
davsl
FIG. 5. Half-lives of ?-RGG elimination from the body of 11-week-old tolerized mice of parental strains A, and S JL, from crosses between animals of the parental ‘strain and from backcrosses. Mice were injected with aggregate free RGG when they were 8 weeks old. Tt of a given duration are plotted as function of the number of animals which eliminate antigen with this half-life. Hatched areas indicate the mean Tt *I standard deviation.
INHERITANCE
OF
TOLERANCE
TABLE
221
INDCCEABILITY
2
CLASSIFICATION OF ANIMALS FROM VARIOUS MaT1NGs IN TERMS LIFE OF mI-RGG ELIMINATION FROM TOLERIZED-IMMUNIZED A, SJL DERIVED MICE~ Types
of cross female
X male
(A
A SJL A X SJL SJL X A A X (A x SJL) (A X SJL) X A (A X SJL) X SJL SJL X (A x SJL) SJL X (SJL X A) X SJL) x (A X SJL)
o Eight-week-old weeks later with
Animals with halflives (in days)
Number Total
Female
30 15 24 16 15 12 14 8 5 12
animals were 0.25 mg RGG-IFA
OF HALF-
23 15 11 10 8 6 8 5 3 6
injected ip with 0.2 mg/g (SC), and after a further
O-4.3
4.4-5.9
6.0-14.0
0 15 11 15 7 5 14 8 5 2
0 0 4 0 0 1 0 0 0 1
30 0 9 1 8 6 0 0 0 9
body weight aggregate free week with 10 rg i3iI-RGG.
RGG,
2
reflected the rate at which tolerant hybrids shifted to the responsiveness of its SJL parent. We, therefore, examined T+ distribution in various types of hybrids and back-cross offspring as a function of age. Even among hybrids tolerized at the age of 12 weeks there were always some individuals with a T, that was considerably longer than that of the S JL parent. This was more marked in A x SJL than in S JL X A hybrids. It was most striking in hybrid offspring of Balb/c x SJL matings (Figs. 7-9).
TABLE CLMSIFICSTION
OF ANIMALS ELIMINATION
Types
of cross
Female
Balb/c SJL Balb/c (Balb/c (Balb/c (Balb/c
X SJL) X SJL) X SJL)
3
FROM Vanrous MATINGS IN TKRMS OF H.\LFLIFE FROM TOLERIZ~D (AT 8 WEEKS) AND IMMUNIZHD B.ILB,/-SJL DERIVED MICE~ Number Rlale
X
X X
Balb/c SJL
x”
SJL Balb/c SJL (Balb/c
X X
X SJL)
OF
‘“I-RGG
Number with (in days) Total
Tt
Female
27 15 16 10 11 23
a Eight-week-old animals were injected ip with 0.2 mg aggregate weeks later with 0.25 mg RGG-IFA (SC), and after a further week * Individual !!‘$ were 5.6, 6.2, and 6.3 (for comparison : the average
21 13 9 7 1: free with
o-4.3
5-14
0 15 13 3 11 10
27 0 3* 7 0 13
RGG/g body weight, 2 10 pg r3rI--RGG. T+ of Balb/c was 8.2 f 2.2).
17 15 18 8 8 22
23 12
A X (A X SJL) (AXSJL) XA (A X SJL) x SJL SJL X (A x SJL) SJL X (SJL x A) (A X SJL) X (A X SJL)
A X SJL SJL X A
n
15 12 14 8 5 12
Breeding
A X (A X SJL) (A X SJL) X A (AXSJL)xSJL SJL X (A X SJL) SJL X (SJL x A) (A X SJL) X (A X SJL)
A, SJL :ALLOTYPE
n
Breeding
RELATIVE
0 0
3 2 0 0 0 0
T1(SJL)
MuAl
AND HdLF-LIFE
MuBl+
MuAl
DISTRIBUTION
4
0 0
3 2 0 0 0 0
T+(A)
IN S-WEEK
TABLE
MuBl-
5
OF MuAl-MuA
TABLE
MuAl
4 3 6 8 3 1
1
1
Tt@JL)
TOLERIZED-IMMUNIZED
23 12
5 7 9 8 5 15
MuBl+
MuAl
AND OF MuBl
MuA
0 0
5 4 0 0 0 8
T+(A)
ANIMALS
MuBl-
MuA
0 0 8 0 2 1
WSJL)
0 0
0 0 9 0 3 1
MuBl+
MuA
MuA
0 0
0 0 0 0 0 1
0 0 0 0 0 1
TdA)
MuBl-
% B
2 ‘1
2 $ Lb
+z
6
AND HALF-Lrms
11 23
10
n
7
0 2
3
T;(BaIb/c)
4 4 14 8 5 2
Tt @JL)
MuBl
2
1
positive
5 2 0 0 0 5
T+ (4
OFGWEEKTOLERIZED-IMMUNIZED
TABLE
0 3
0
Tt(SJL)
Muhl
ANIMALS
3 4
4
T:(SJL)
MuAl
0 6
3
T+(Balb/c)
MuA
ALLOTYPE AND H:~LF-LIFE IN ~-WEEK TOLERI~ICD-IMMUNIZED ANIMALS
C5
15 12 14 8 5 12
X SJL)
A X (A X SJL) (A X SJL) X A (A X SJL) x SJL SJL X (A x SJL) SJL X (SJL X A) (A X SJL) X (A X SJL)
X SJL X (Balb/c
n
X SJL) X SJL)
(Balb/c (Balb/c
X Balb/c
Breeding
X SJL)
(Balb/c
Breeding
BALB/C, SJL:
TABLE
3 1 0 0 0 0
T+(SJL)
MuBl
8 4
0
negative
Tt(SJL)
MuA
3 4 0 0 0 3
WA)
0 4
0
T;(Balb/c)
3 4 $ z t: 2
m
2
B
E
ii E
L-4 E
224 DISCUSS
ION
In earlier papers of this series (l-3) we have reported that SJI, mice h;~~c ;I defect in their immune apparatus as manifested by hyper-responsiveness, relatively rapid maturation of the responsi\:eness to RGG aggregates, and resistance to tolerance induction after the age of 8 \vccks. The manifestation of this lesion depended on the accessory cells, but :~ppearcd to result from an abnormality in the thymusderived CCII population. The genetic experiments, described in this paper, were designed to probe further into the nature of the abnormality. The SJI, phenotype was recessive in terms of neonntaiiy induced tolerance (Fig. 2) and in terms of the half-life (Ti) with which aggregate freed (centrifugation at 123,OOOg) lslIRGG was eliminated from the body of otherwise untreated animals (Table 1). A peculiarity, in this latter cast, was the finding that some hybrids eliminated with half-lives which ncre significantly longer than those of parental individuals (Fig. 1) possibly because of a metaholicaiiy manifcstcci interaction between the genes of the two parental animals. ‘IlYe shall now turn to ;I consideration of tolcrizedimmunized animals. in this contest we shall rcfcr to auimnls by the age at which they were toicrized; they were tested 3 weeks later. Broadly speaking, the resistance to toicrance of the adult animals was dominant for SjL characteristics. J-iowever, there were some findings, revealed by detailed esnmination of this dominance (Tables 2-G), ivhich defied a simple monogenic model. The average 7.4 of toierized-immunized S-~ck-old h!.britis approached that of similarly treated SJL parental animals, though it was still somewhat longer. It was longest in A x SJL hybrids and much closer to that of the SJL parent in SJI. X A mice. In the animals, tolerized at the age of 12 weeks, this difference could no longer be observed (Figs. 7 and S). It thus appears as if the identity of the maternal animal affected the rate at which the SJI, phenotype was being reached. ‘i‘here was another, probably related, manifestation of this maternal influence. This was seen when the T! distribution of various 1 l- and 1S-week-old toierizcd-immunized offspring from crosses and back-crosses was examined. The half-life distribution of the parental animals was suci~ that one would have expected that hybrids could be classified as having the 7’4 characteristic either for .\ or for SJI. c4inlination. 111fact. however, a fairly large number of individuals could not be classified since their T+ was intermediate between those of parental animals. Even mot-c striking was the 1‘1 distribution among tolerized-immunized Mb/c x SJ 1. hybrids of which 3/13 showed the T, of Balb/c type while the other 10 had the 7‘, of SJL type (Fig. 6). The general distribution of half-lives in tolerizcd-immunized A-SJT, descended animals might be compatible with a dominant, monogcnic inheritance of the SJL trait. If one assumed that the half-life trait of all A x SJI, mice is of the SJI, type, one would expect that 50 ‘;b of the offspring from :\ X (.A X SJl,) and (X X SJL) X A matings would eliminate 1311-RGG with the same half-life as :2 mice and that none of the offspring from SJL X (:1 x SJL) and SJI, X (SJL X A) matings would have half-lives of the same length as 11 mice. This predicted distribution was in reasonable accord with observation (Table 2). On the other hand, more than the expected 2576 of offspring from (:I x SJL) X (A X SJL) matings had the T, characteristic for the X parent. The finding may be attributable to chance, since the group deviated to the same cstcnt, though in the opposite direction, from the predicted distribution of hIu.41 and Mu.1 2 aliot\;l)e (O’i;, rather than 25’&
INHERITANCE
OF
11
/
TOLERANCE
1
I
I
INDUCEABILITY
I
,
22.5
I
8
4
12
a
4
0
4
8
12
16
Half lifelm days)
FIG. 6. Half-lives of =‘I-RGG elimination from ll-week-old tolerized-immunized inbred mice of strains Balb/c and SJL, crosses between them and back-crosses. Mice were 8 weeks old at the time of injection with aggregate free RGG and were 11 weeks when mI-RGG was injected. Tt of a given duration are plotted as a function of the number of animals which eliminate antigen with this half-life. The mean Tt 2-1 standard deviation for each group is shown as a hatched area. were homozygous for the allotype of A mice ; Table S), while the frequency of MuBl was identical with the predicted incidence of 75% (Table 7). The offspring of matings (Balb/c x SJL) X Balb/c showed much more than 50% Balb/c type T+, though all (Balb/c X SJL) X SJL offspring did have the predicted T+ of SJL (Table 3). In these cases,the distribution of MuAl and MuA was very close to expectation (Table 6). We have already mentioned that the number of hybrids with T+ intermediate between the two parents was smahest when the maternal animal was SJL, i.e., among SJL X A hybrids. This fact must
226
FU JIWARA
AND
CINADER
be taken into account in considering the previously mentioned absence of individual with T, characteristic for A among SJL X (SJL x A) mice. We have previously invoked this as evidence for dominant monogenic inheritance. However, in reality, it might be attributable to a maternally transmitted control over the manifestation of the SJL phenotype. Age:
(in weeks) 1
0g 8 I
RGG in hybrids Half life of “‘I Half life of ‘3’1- RGG in A mice Half life of “‘I - RGG in SJL mice
Half life (in days.) FIG. 7. A X S JL mice : Changes in the half-life distribution of tolerized and then immunized hybrids and parental mice as a function of age at which animals were tolerized. Elimination tests were always carried out 3 weeks after tolerization. The height of the vertical bars indicates the number of animals of a T+ within the range shown on the horizontal axis; the hatched areas indicate the mean C.1 standard deviation for each group.
IKHERITANCE
OF
TOLERANCE
INDUCEARILITY
227
ge : (in Iueeks)
1
1!
7
0 1-
Half life 01 ‘“IRGG in hybrids Holf life of ‘3’1- RGG in * mice tall tite of “’ I - RGG in SJL mke
8 -I
1I
12 8 4 12 4 2 0
4
8
12
16 20
Half life (in days) FIG. 8. SJL X A mice : Changes in the half-life distribution of tolerized and then immunized hybrids and parental mice as a function of age at which animals were tolerized. The height of the vertical bars indicates the number of animals which have a Te within the range shown on
the horizontal axis ; the hatched areas indicate the mean Cl standard deviation for each group. There was a tendency for the number of animals with intermediate Ts to decrease with advancing age, but even at 12 weeks there were some hybrids with T+ which were significantly in excess of those characteristic for the SJL animals of the same age. This was most marked among offspring from Balb/c X SJL matings (Figs. 7-9). Nevertheless, genetic analysis of even older animals might lead to a more definitive analysis. The design of the present study does not permit formal
228
FUJIWARA
AND
CINADER
Hoif
life
of
‘3’1-
Half
lhfe
of
-‘I
tiotf
l,fe
of ‘=I
RGG - RGG - RGG
8 4 I 0
2
4
6 Half
8
10
12
14
lkfe (m days)
9. BALB/c X SJL mice : Changes in the half-life distribution of tolerized and then immunized hybrids and parental mice as a function of age at which animals were tolerized. The height of the vertical bars indicated the number of animals which have a T+ within the range shown on the horizontal axis; the hatched area indicates the mean kl standard deviation for each group. FIG.
it is reasonable to expect that analysis for multifactorial inheritance. However, more than one locus might be involved in the determination of resistance to tolerance induction. We must now come back to the T, of untreated animals. Among the hybrids we noted individuals with T+ in excess of those found in any A or Balb/c animals, i.e., a shift of the Tt distribution to higher values (Fig. 1). It is this interaction which might also affect the T+ of tolerized-immunized animals and thus interfere with the full manifestation of the SJL genotype for responsiveness of tolerized and immunized hybrids (see Figs. 3 and 4). The maternal influence does not reduce the T* of untreated animals (cf. S JL X A and A X SJL in Fig. 1), and thus appears to be a separate factor. In summary, the SJL trait for the resistance to adult tolerance induction appears to be dominant and might be inherited monogenitally. However, the expression of this dominance depended on the identity of the maternal animal and might be affected by a process which results in slow elimination of antigen. Clearly, the question arises whether the effects, so far de-
INHERITANCE
OF
TOLERANCE
INDUCEABILITY
229
scribed, can be explained solely by the transmission of genetic factors or whether other, possibly viral factors, are implicated. There is remarkable age-dependent change in the Tg of tolerance-inducing antigen from the body of hybrids between SJL and either A or Balb/c mice. The young hybrids behave like their Balb/c or the A parents, whereas the old hybrids approach very closely the half-life of the SJL parents (Figs. 3 and 4). It is, therefore, clear that hybrids acquire resistance to tolerance induceability with advancing age. However, hybrid responsiveness is far from uniform and there are always some individuals whose responsiveness is intermediate between that of the two parents (Figs. 5 and 6). The progression with time is different for individuals of identical genotypes. This and the lack of uniformity leads us to think that there is some external event which may stimulate the induction of SJL-type responsiveness. The proneness of SJL mice to a variety of immunoglobulin abnormalities and to neoplastic disease may be connected with the resistance of old SJL and of SJL hybrids to tolerance induction. At any rate, a factor seems to be involved which is responsible for variability. There is some indication that the transmission of this factor occurs more regularly if SJL is the maternal rather than the paternal parent of the hybrids (Figs. 7 and 8) ; this is particularly noticeable in the relative distribution of half-lives in 11-week-old tolerized-immunized S JL X A and A X S JL hybrids. There appear to be, at least, two factors involved in the transmission of the resistance to tolerance induceability; one of these seems to determine the age at which a hybrid or back-cross animal acquires the characteristic of the SJL parent. The lack of uniformity of the hybrid leads to the assumption that an external stimulus or a probabilistic event, such as the activation of a virus, may also enter into the expression of the S JL parent’s resistance to tolerance induction (15, 16). There was no major difference between the age variation of the T3 distribution of A X S JL and Balb/c X S JL mice (Figs. 7 and 9) and the abnormal responsivenesss of Balb/c may not reinforce the SJL trait (Table 1). There was no linkage between the half-life of tolerant-immunized animals and the immunoglobulin allotype (MuAl/MuAZ) or the C5 eniotype (Tables 5-7). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11. 12. 13. 14. 15. 16.
Fujiwara, M., and Cinader, B., Cell. Immunol. 11, 194, 1974. Fujiwara, M., and Cinader, B., Cell. Zmmunol. 11,205, 1974. Fujiwara, M., and Cinader, B., Cell. Immunol. 12, 11, 1974. Evans, M. M., Williamson, W. G., and Irvine, W. J., Cl& Exg. Zmmunol. 3, 375, 1968. Staples, P. J., Steinberg, A. D., and Talal, N., J. Exp. Med. 131, 1223, 1970. Cerottini, J. C., Lambert, P. H., and Dixon, F. J., J. Exp. Med. 130, 1093, 1969. Dubiski, S., and Cinader, B., Na&ure (Lo&on) 197, 705, 1963. Cinader, B., and Dubiski, S., J. Zmmunol. 101, 1236, 1968. Cinader, B., Dubiski, S., and Wardlaw, A. C., J. Expt. Med. 120, 897, 1964. Fujiwara, M., Jap. J. Exp. Med. 41, 59, 1971. Sonoda, S., and Schlamowitz, M., Immunochemistry 7, 885, 1970. Kaplan, A. M., and Cinader, B., Cell. Zmmzmol. 6, 442, 1973. Fujiwara, M., and Cinader, B., Cell. Zmmzmol. 12, 1, 1974. Kaplan, A. M., and Cinader, B., Cell. fmmunol. 6, 429, 1973. Mellors, R. C., and Huang, C. Y., J. Exfi. Med. 126, 53, 1967. Thivolet, J., Morrier, J. C., Ruel, J. P., and Richard, M. H., Natrive (London) 214, 1134, 1967.