CELLULAR IMMUNOLOGY 17, 215-227 (1975)

Mechanisms in Immune Tolerance

Ill. Immunosuppression and the Induction of Tolerance--Synergism and Antagonism 1

ISRAEL Z A N - B A R , DAVID NACtlTIGAL, AND MICHAEL FELDIVIAN

Department of Cell Biology, The Weizmann Institute of Science, Rehovot, Israel

Received April 26, 1974

Several immunosuppressive procedures were examined for their facilitation of

inducing immune tolerance to protein antigens in rabbits. Thus, gamma irradiation,
6-mercaptopurine, and cyclophosphamide treatments were found to be mutually poten-
tiating in their effect of predisposing to the induction of tolerance, l-Iydrocortisone
acetate, on the other hand, was found to interfere in the induction of tolerance follow-
ing irradiation. An attempt is made to interpret this observation on a cellular basis.

INTRODUCTION
Immune tolerance to protein antigens can be conveniently studied in adult rabbits
which are made tolerant by appropriate stimulation with normally immunizing
doses of the tolerogen following sublethal total body gamma irradiation. It has
been demonstrated that tolerance so induced manifests marked stability and
durability and lends itself, therefore, to long-range experimentation (1). Similarly,
certain immunosuppressive agents, like 6-mercaptopurine (2, 3) or cyclophos-
phamide (4) will also render animals susceptible to tolerance induction with
massive antigen doses. We report in the following study, however, that nonspecific
immunosuppression per se will not invariably predispose to specific unresponsive-
ness since in all probability immunosuppressants do not affect the same target cells
of the immune apparatus in each case. This investigation was planned to examine
synergistic or antagonistic interactions of various immunosuppressive procedures
in respect to the induction of specific unresponsiveness, the ultimate goal being to
gain thereby a better understanding of the cellular mechanisms involved in the
establishment of immune tolerance. Since tolerance facilitated by irradiation is
more stable than the other models mentioned, we employed it as a reference
routine singly and in combination with the immunosuppressive agents tested.

MATERIALS AND METHODS

Animals

Locally bred New Zealand white rabbits of 2-3 kg wt were obtained from the
Animal Breeding Center of the Weizmann Institute of Science.

x This work was supported by a Grant from the Thyssen Foundation.

215

Copyright O 1975 by Academic Press, Inc.

All rights of reproduction in any form reserved.
216 ZAN-BAR, NACHTIGAL AND I~ELDMAN

Antigens
Crystalline human serum albumin (lISA) and bovine serum albumin (BSA)
were purchased from Calbiochem Inc., Los Angeles, California and were employed
for the induction of tolerance, immunization and antibody assays.

Immunosuppressive drugs
6-Mercaptopurine (6-MP) was supplied by the California Corporation for Bio-
chemical Research, Los Angeles, and cyclophosphamide by the Koch-Light Lab-
oratories, Colnbrook-Bucks, England. Hydrocortisone acetate came from the
Fredriksberg Chemical Laboratories, Copenhagen, and was supplied as a suspen-
sion of 25 mg/ml in saline. 6-Mercaptopurine had to be first dissolved in N/1
NaOH (10 g/100 ml alkali) and this solution was diluted in saline to give a
12 mg/ml concentration,

Irradiation
The animals were irradiated in perspex drums revolving at 1 rpm along their
axis and placed at a distance of 75 cm from the irradiation source, as measured
from the center of each drum. The source was ~°Co device (Gamma Beam 150 A,
Atomic Energy of Canada, Ltd.) and total body irradiation was administered at
the rate of 53 R/min.

Induction of unresponsiveness
a. After Gamma Irradiation. Adult rabbits were given varying doses of total
body irradiation on day zero, as specified. One day (or sometimes 4 wk) later
intravenous injections of 5 mg H S A each, were given in saline solution at weekly
intervals and continued till the end of the tenth week.
b. In Conjunction with 6-MP. Each rabbit was given a daily intramuscular
injection of 6-MP, 6 mg/kg of body wt for 13 day in all. On the first day and at
subsequent weekly intervals saline solution of H S A was administered intra-
venously, a total of 10 antigen injections of 5 mg each.
c. With the Aid of cyelophosphamide. A solution of cyclophosphamide containing
50 mg of the drug/ml of saline was injected subcutaneously to rabbits, 150 mg
drug/each kg of body wt. The next day 5 mg of H S A in saline were administered
intravenously to each animal and the antigen treatment was repeated weekly for a
total of ten injections.
d. By Means of Hydrocortisone Acetate. Rabbits were injected subcutaneously
with a suspension of the drug, 50 mg/kg of body wt. Simultaneously 5 mg of H S A
in saline were given intravenously and the antigen stimulation was repeated nine
times more at weeny intervals.

Immunization
To test for antibody production the animals were challenged with 20 mg H S A
and 20 mg BSA/animal, administered subcutaneously in complete Freund's
adjuvant (Difco Laboratories, Detroit, Michigan).
 IMMUNOSUPPRESSION AND INDUCTION OF TOLERANCE 217

Antibody Assay
Antibody was assayed by its antigen binding capacity (ABC) employing 125I
labeled HSA or BSA (5) and measuring the radioactivity of the ammgnium
sulphate precipitate of the incubated mixture of reactants (6).

Statistical Evaluation
Where required, differences between mean ABC of animal groups were examined
be means of the t test and were considered as significant with P values of 0.02 or
smaller.

RESULTS
The standard procedure of tolerizing rabbits to a soluble protein antigen involves
a total body irradiation in the range of 550 R, following which eight to ten weekly
doses of 5-10 mg antigen are administered intravenously, thus making practically
all the animals tolerant (1). However, when the irradiation dose was decreased to
300 R while an unchanged antigen stimulation with H S A was maintained, only
about half the animals became unresponsive (Table 1). A similar antigen treat-
ment given following 6-mercaptopurine administration resulted in a short-lived
immunosuppression from which the great majority of the animals recovered by the
seventh week. When, however, both low dose irradiation and mercaptopurine were
combined, they interacted synergistically and antigen stimulation resulted in
unresponsiveness in all the animals so treated, as tested on day 45 (Table 1). The
stability of the unresponsive state was checked in these three groups by challenging
the unresponsive rabbits with a mixture of H S A and B S A in Freund's complete

TABLE 1
UNRESPONSIVENESS TO HUMAN SERUM ALBUMIN (HSA) INDUCED IN ADULT RABBITS WHICH
WERE EITHER IRRADIATED SUBLETHALLY OR TREATED WITH 6-MERCAPTOPURINE (6-MP)
OR AFTER BOTH TREATMENTS COMBINED, ALL IN CONJUNCTION WITH HSA STIMULATION

Treatment given Day of experiment

20 32 45

Irradiation 300 R Ratio of responders

to total ~ 3/12 3/9 4/9
Mean ABCof
responders 3.5 -4- 0.4 4.8 -4- 0.6 37.7 ± 5.7

6-MP Ratio of responders ~/9 ~ 5/9 8/9

Mean ABC 9.8 13.8 -4- 1.7 50.0 4- 16.2

Irradiation combined Ratio of responders 0/9 0/9 0/9

with 6-MP Mean ABC

Untreated normal Ratio of responders 6/6 6/6 6/6

controls Mean ABC 30.0 4- 8.1 46.3 4- 1.2 64.9 4- 2.9

a Responsiveness was defined arbitrarily as the antigen binding capacity (ABC) above 10% of
the arithmetical mean response calculated for the respective untreated normal control groups. This
arbitrary division separated the rabbits into two nonoverlapping levels of response.
218 ZAN-BAR, NACI-ITIGAL AND FELDMAN

TABLE 2
T I l E STABILITY OF TOLERANCE IN RABBITS WHICH REMAINED [MMUNOSUPPRESSED
AFTER THE TREATMENTS REPRESENTED IN T A B L E 1

T r e a t m e n t before Day after challenge with H S A a n d BSA

challenge in adjuvant=

14 28 75 95

Irradiation 300 R Ratio of responders

to total 7/12 6/11 4/10 5/8
Mean ABC of
responders 18.9 4- 17.0 26.2 4- 10.0 32.4 4- 5.2 36.6 4- 4.2

6-MP Ratio of responders 5/9 5/6 6/6

Mean ABC 25.3 4- 2.5 25.8 4- 2.0 33.6 4- 3.6 --

Irradiation and Ratio of responders 0/5 1/5 1/5 1/4

6-MP combined Mean ABC -- 4.7 9.9 10.7

Untreated normal Ratio of responders 5/5 5/5 5/5 5/5

controls Mean ABC 30.3 4- 5.4 40.7 4- 4.2 50.7 4- 5.1 50.7 4- 8.9

" To test the stability and specificity of tolerance the unresponsive rabbits were challenged
after 4 wk rest with a mixture of HSA and BSA 20 mg each in complete Freund's a d j u v a n t sub-
cutaneously. The specificity of the tolerant state to HSA was confirmed by the observation t h a t
the rabbits, without exception, responded to BSA, the lowest mean ABC in any group being not
less then 54% of the mean BSA response in the corresponding normal controls.

adjuvant after a period of 4 wk rest from the first antigen stimulation, where BSA
was utilized as a control antigen for the specificity of the tolerant state. Where
rabbits had to be tested for the stability of tolerance afer treatments which gave a

Tolerance induced in rabbits after irradiation ond 6-mercaptopurme

treatment
I00

80

5
,~ 60 6- mercoptoporim
m

40
"E 6-MP and
irradiation
EL
2O
Irradiation
(3OOr)
r
20 52 4t5 ¢'' 0 14 2~ / 75 95 days
1
Induction ~ Chr~lienge

FIG. 1. Percentage of rabbits made unresponsive to H S A after either irradiation or 6-

mercaptopurine treatments, or after both treatments combined, as well as the percentage of
those among the unresponsive which remained tolerant after challenge (see Tables 1 and 2).
 I1VflVIUNOSUPPRESSION A N D I N D U C T I O N OF T O L E R A N C E 219

]'ABLE 3
UNRESPONSIVENESS TO H S A INDUCED IN RABBITS WITH THE AID OF IRRADIATION
OR CYCLOPIIOSPHAMIDE OR BOTH COMBINED

Treatment given Day of experiment

14 36 50

Irradiation 300 R Ratio of responders~

to total 3/12 4/9 4/9
Mean ABC of HSA
in responders 3.5 4- 0.4 4.0 4- 0.7 36.7 4- 5.7

Cyclophosphamide Ratio of responders 2/4 3/4 4/4

Mean ABC of responders 5.0 4- 1.5 15.4 4- 2.8 33.8 4- 5.1

Irradiation and Ratio of responders 0/6 0/6 0/6

cyclophosphamide Mean ABC of responders
combined

Untreated normal Ratio of responders 6/6 6/6 6/6

controls Mean ABC 4.9 4- 1.2 26.4 4- 2.8 44.6 4- 4.2

- For definition of responders see Table 1.

low yield of unresponsive animals, animals from several experiments were pooled
(Table 2, Fig. 1). Thus unresponsive 6-mercaptopurine treated and H S A stim-
ulated rabbits all recovered responsiveness within 75 days of the double challenge.
On the other hand, from the animals made tolerant by irradiation and antigen
treatment some (less than one-third) remained specifically unresponsive, while
when mercaptopurine and gamma rays were combined together the result was a
state of tolerance lasting at least up to 3 mo after challenge in all but one of the
rabbits. It was thus demonstrated that the combination of both was more effective
in facilitating tolerance than each marginally immunosuppressive procedure alone.
Similar results were obtained when cyclophosphamide treatment was substituted
for 6-mercaptopurine in conjunction with normally immunizing H S A treatment
(Table 3). In this case cyclophosphamide and antigen treatment brought about a
transient state of immunosuppression which disappeared before day 50, although
in occasional experiments some rabbits remained unresponsive. 300 R Irradiation
combined with H S A stimulation suppressed, as before, about half of the treated
rabbits while combination of cyclophosphamide, irradiation, and antigen resulted in
all the animals remaining unresponsive when tested on day 50. It was found, on
examining the stability and specificity of unresponsiveness in these groups of
animals, that the cyclophosphamide group responded promptly to challenge, so
that all rabbits were found responsive 19 days later (Table 4, Fig. 2). From among
the irradiated rabbits still about one-half remained tolerant on day 16, while at the
same time in the cyclophosphamide plus irradiation group all but one remained
specifically unresponsive. Consequently, both 6-mercaptopurine and cyclophos-
phamide demonstrate a synergistic interaction with irradiation in predisposing rab-
bits to the induction of tolerance, a finding which would indicate a similar inter-
ference with the immune mechanism. Interaction of a different nature was observed,
however, when hydrocortisone acetate was employed in an analogous experimental
 220 ZAN-BAR, NACI-ITIGAL AND FELDMAN

TABLE 4
THE STABILITY a OF TOLERANCE IN RABBITS WHICH REMAINED ~'NRESPONSIVE
AFTER IMMUNOSUPPRESSION AS DESCRIBED IN T A B L E 3

Treatment before Day after challenge with HSA and BSA

challenge
19 32 46

Irradiation 300 R Ratio of responders b

to total 7/12 6/11 6/11
Mean ABC of HSA
in responders 18.5 ~ 2.2 26.2 4- 4.2 22.4 4- 5.2

Cyclophosphamide Ratio of responders 4/4 4/4 4/4

Mean ABC 35.5 4- 2.1 87.1 ± 3.5 72.8 4- 3.6

Irradiation Ratio of responders I/6 I/6 I/6

combined Mth Mean ABC 5.3 13.7 34.5
cyclophosphamide

Untreated normal Ratio of responders 4/4 4/4 4/4

controls Mean ABC 38.7 4- 3.4 33.6 4- 4.2 53 4- 3.5

For details see Table 2. 100% of the animals responded significantly to BSA, thus confirming
the specificity of the unresponsive state.
b See Table i for explanation.

set up. Thus, a combination of hydrocortisone and antigen treatment resulted in a

state of immunosuppression which lasted over 36 days (Table 5) although on day
50 all but one rabbit of this group produced antibody and eventually all animals be-
came responsive. In this experiment a higher dose of irradiation was employed,
namely 400 R, and when it was followed with antigen stimulation unresponsiveness
was induced in all animals as tested on day 50. This was true as well when irradi-
ation, cortisone, and antigen were combined together. When, however, the stability
of unresponsiveness in the last two groups of rabbits was tested by challenge with

[~ oTolerance
lera induction in rabbits after irradiation cnd cyclophesphamide
treatment
100~
I
7
80;

Cyc'opho!i phom~
= 40 irradmtion
(30Or)

C.P.÷irradiation

+
52 46 days
f
Induction ~ Chatlenge

FIG. 2. Percentage of rabbits made unresponsive to H S A after either irradiation or cyclo-

phosphamide treatments of after both treatments combined, as well as the percentage of those
among the unresponsive which remained tolerant after challenge (see Tables 3 and 4).
 IIVfMUNOSUPPRESSION A N D I N D U C T I O N OF T O L E R A N C E 221

TABLE 5
UNRESPONSIVENESS TO H S A INDUCED WITH THE AID OF IRRADIATION~ HYDROCORTISONE,
OR BOTH COMBINED. H S A ADMINISTRATION STARTED ON D A Y 7

Treatment given Day of experiment

14 36 50

Irradiation 400 R Ratio of responders"

on day zero to total 0/5 0/5 0/5
Mean ABC of responders -- -- --

Hydrocortisone Ratio of responders 0/6 0/6 5/6

on day 7 Mean ABC of responders -- -- 5.3 4- 0.8

Hydrocortisone Ratio of responders 0/9 0/9 0/9

combined with Mean ABC of responders -- -- --
irradiation as above

Untreated normal Ratio of responders 5/5 5/5 5/5

controls Mean ABC of responders 4.9 4- 1.5 26.4 4- 4.2 44.6 4- 5.6

a See Table 1.

an antigen in ad j u v a n t , a m a r k e d difference was observed ( T a b l e 6, Fig. 3 ) . T h u s

the irradiated g r o u p of animals r e m a i n e d specifically u n r e s p o n s i v e for over 2 m o
after challenge while in the g r o u p treated with a combination of g a m m a irradiation
and h y d r o c o r t i s o n e over half the animals responded as soon as day 14. T h e s e
e x p e r i m e n t s demonstrate, therefore, that hydrocortisone interfered with the induc-
tion of stable tolerance after irradiation. Essentially similar findings w e r e observed

TABLE 6
T H E STABILITY a OF TOLERANCE IN IMMUNOSUPPRESSED RABBITS AS DESCRIBED IN TABLE 5

Treatment before Day after challenge with HSA and BSA

challenge
14 30 48 62

Irradiation 400 R Ratio of responders b

on day zero to total 0/7 0/6 0/6 0/6
Mean ABC of HSA
in responders

Hydrocortisone Ratio of responders All animals responded before challenge

on day 7 Mean ABC Not tested

Hydrocortisone Ratio of responders 8/15 7/14 8/13 7/12

combined with Mean ABC 20.4 4- 8.5 19.6 4- 4.2 22.0 4- 5.6 20.8 4- 2.1
irradiation as above

Untreated normal Ratio of responders 4/4 4/4 4/4 4/4

controls Mean ABC 33.8 4- 6.3 43.0 4- 0.8 43.6 4- 7.4 34.9 4- 6.4

a See Table 2. All the anirnals responded significantly to BSA.

b See Table I.
222 ZAN-BAR, NACHTIGAL AND FELDMAN

t , , -7--4/r-i L J
Tolerance induced in rabbits after irradiation and hydrocorfisone treatmen!
I00

8q-

o 60 Hydrocorfisone
+irradiation(4OOr

40 U
Hydrocorfisone -q

Irradiation
Op (40Or)

14 36 2'0 ~~ 36-- 4s 6e d0>

Induction -
TChfl[lenge
FIG. 3. Percentage of rabbits made unresponsive to HSA after either irradition of 400 R of
after hydrocortisone treatment or after both treatments combined, as well as the percentage of
those among the unresponsive which remained tolerant after challenge (see Tables 5 and 6).

also in rabbits made tolerant after irradiation of 550 R with and without hydro-
cortisone (Table 7).
I n order to define the temporal correlation of the interference by hydrocortisone,
an experiment was designed which was based on previous observations on the
mechanism of tolerance induction after irradiation (1). According to these earlier
findings irradiated rabbits can be made tolerant, even when antigen stimulation is
postponed for 1 mo after irradiation, so that the tolerizing antigen treatment is
maintained solely over the second month of recovery from radiation injury. Anti-
gen administration over the first month only does not, however, result in lasting
specific unresponsiveness. This model of delayed antigen stimulation was utilized
in two variant experiments. I n the first variation (Table 8) the irradiated rabbits
were given hydrocortisone 7 days after irradiation, then rested 3 wk whereupon

TABLE 7
THE STABILITY OF TOLERANCE INDUCEDWITHTHE AID OF IRRADIATION OF 550 R
OR IRRADIATION COMBINED WITH HYDROCORTISONE a

Treatment before Day after challenge with HSA and BSA

challenge
14 24 30 48

Irradiation 550 R Ratio of responders

on day zero to total o/5 0/5 o/5 0/4
Mean ABC of HSA
in responders

Irradiation combined Ratio of responders 3/9 3/9 4/8 4/8

with hydrocortisone Mean ABC 5.0=t=3.7 3.14-1.2 6.84-3.2 22.5 4-3.2

Untreated normal Ratio of responders 2/2 2/2 2/2 2/2

controls Mean ABC 8.5 4-1.5 9.84-0.9 10.54-1.5 84.94-3.2

a As in Table 6. All animals responded to BSA.

 IMMUNOSUPPRESSION AND INDUCTION OF TOLERANCE 223

TABLE 8
INTERFERENCE OF HYDROCORTISONE IN TOLERANCE INDUCTION TO HSA
AND ITS TEMPORAL RELATION TO ANTIGEN STIMULATION a

Treatment given Day of experiment

14 23 45 69

550 R irradiation Ratio of responders

and postponed to total 0/6 0/6 0/6 0/6
antigen Mean ABC of
treatment ~ responders . . . .

Irradiation, Ratio of responders 0/8 1/8 1/7 1/7

hydrocortisone Mean ABC -- 7.6 16.3 13.8
and postponed
antigen treatment

Irradiation and Ratio of responders t/10 3/8 6/9 5/9

postponed both Mean ABC 3.1 3.5 4- 2.9 22.6 4- 12.0 27.3 4- 10.5
antigen and
hydrocortisone
treatments

Untreated normal Ratio of responders 4/4 4/4 4/4 4/4

controls Mean ABC 8.3 4- 0.4 9.1 4- 0.7 81.9 4- 2.4 81.3 4- 9.1

a All but two rabbits responded significantly to BSA.

b Irradiation given on day zero. Postponed treatment started at the end of 4 wk. Cortisone
treatment, when not postponed, was given on day 7.

antigen stimulation was started and repeated four times at weekly intervals. The
animals were given a second rest of 4 wk after which they were challenged with
antigen in adjuvant. When tested 2 mo later, six out of seven rabbits were found
tolerant. In the second variation (Table 8) hydrocortisone administration was also
deferred and combined with the first antigen dose 4 wk after irradiation. In this
group in which hydrocortisone and antigen were administered concurrently the
ratio of tolerant animals was reduced to four out of nine, suggesting thus that
the observed antagonistic action of hydrocortisone depends on a concomitant antigen
stimulation.
Besides interfering with the establishment of tolerance during the induction
period, hydrocortisone will also facilitate the abrogation of the normally stable
specific state of unresponsiveness in rabbits made tolerant following irradiation
(Table 9). This, however, will take place only when the animals are stimulated
concomitantly with hydrocortisone and antigen in adjuvant. When, instead, the
antigen is administered intravenously the established state of tolerance will not
be affected by the drug (Table 9, Fig. 4).

DISCUSSION
The common denominator to which the immunosuppressive effect of gamma
irradiation, cyclophosphamide, and 6-mercaptopurine treatments may be traced
is the antimitotic activity common to these procedures ( 7 - 9 ) . Moreover, according
to recent evidence irradiation and cyclophosphamide, at least, show preferential anti
224 ZAN-BAR~ NACItTIGAL AND FELDMAN

TABLE 9
ABROGATION OF ESTABLISHED TOLERANCE TO HSA ix RABBITS WITH THE AID
OF HYDROCORTISONE a

T r e a t m e n t given to Day of experiment

tolerant and
normal rabbits 7 21 33 49

Tolerant, Ratio of responders

hydrocortisone, to total 0/7 0/7 3/6 4/6
and HSA Mean ABC -- -- 4.0 4- 2.3 5.7 4- 2.6
in a d j u v a n t

Tolerant, Ratio of responders 0/5 0/5 0/5 0/5

hydrocortisone, Mean ABC . . . .
and intravenously
HSA

Tolerant, Ratio of responders 0/4 0/4 0/3 0/3

HSA in a d j u v a n t Mean ABC . . . .

Tolerant, Ratio of responders 0/4 /04 0/4 0/4

intravenous HSA Mean ABC . . . .

Normal, Ratio of responders 2/2 2/2 2/2 2/2

hydrocortisone, Mean ABC 14.0 4- 1.1 20.1 4- 1.5 46.6 4- 2.8 45.6 =t= 10.1
and intravenous
HSA

a Tolerant or normal rabbits were injected with 5 mg HSA either in a d j u v a n t subcutaneously or

in solution intravenously and some were given a t the same time hydrocortisone acetate sub-
cutaneously, as specified.

I- J ~--F-/ AF , - - q

I00~

I
_ !I
The liming of hydrocorfisone and
antigen-administration for inlerference
'l
6C
wilh tolerance induction

- 4C--
o ~ Irradialic~ alone i

~_ (550r)
2C-

28 515
.... i
~3 46 58 doys
t ~ I f
Trredlation HSA HSA HSA HSA
I t /
II ~ Challenge
H'/drocorfisone Hydrocor tisone

FIG. 4. Interference of cortisone in tolerance induction after irradiation (day zero) and
delayed antigen treatment (day 28) when cortisone is administered either on day 7 or with
the antigen on day 28. Expressed as percentage of tolerant rabbits (see Table 8).
 IMMUNOSUPPRESSION AND I N D U C T I O N O F T O L E R A N C E 225

B lymphocyte activity (10, 11). When combined with antigen stimulation, this
type of immunosuppression seems to facilitate the induction of specific immune
tolerance as demonstrated in sublethally irradiated adult rabbits treated with
moderate doses of protein antigen. 6-Mercaptopurine or cyclophosphamide treat-
ment, when combined with a massive antigen stimulus will induce likewise a pro-
nounced degree of unresponsiveness (2). If, however, the protocols of the experi-
mental treatments are modified, either by reducing the irradiation dose prior to
antigen administration or by decreasing the amount of antigen given in conjunction
with the tolerance inducing drugs, the result is a borderline specific immuno-
suppression. In consequence only part of the animals become tolerant after
irradiation, while 6-mercaptopurine and cyclophosphamide facilitate merely a short-
lived state of unresponsiveness. However, on combining two such modified pro-
cedures together a synergistic interaction in facilitating tolerance is observed. We
could not study the synergism of 6-mercaptopurine combined with cyclophosphamide
since this combination proved to be lethal for all the animals. On the other hand,
irradiation when combined with either one of the two drugs under conditions where
each treatment alone gave only marginal results, brought about a solid state of
tolerance in the great majority of rabbits. That the combined action of both these
drugs even in minimal immunosuppressive doses is invariably lethal for rabbits
which can, on the other hand, survive sublethal irradiation in combination with
each drug alone, would point to important differences in the mechanism of action
of the drugs as compared with gamma irradiation and would additionally justify
the abandonment of the term "radiomimetic drugs." However, as far as facilitation
of tolerance is concerned, both mercaptopurine and cyclophosphamide seem to be
more than just "radiomimetic" since their synergistic interaction with gamma rays
may suggest that they all affect closely associated or perhaps identical targets of
the immune mechanism. Contrary to this, hydrocortisone demonstrated a definite
antagonistic function in this respect. Thus hydrocortisone treatment alone brought
about a pronounced immunosuppression of long duration. Unexpected results were
obtained, however, when this immunosuppressive treatment was combined with a
regime of irradiation and antigen stimulation, which renders normally all the rabbits
tolerant. The outcome of such a combination of two treatments, each one immuno-
suppressive for itself, was a reduction in the percentage of tolerant rabbits from
100% to around 40%. Similar interference of corticosteroids with tolerance induc-
tion has been observed also by Dukor ond Dietrich (12) in mice made tolerant to
sheep red blood cells (SRBC) with the aid of eyclophosphamide. These findings
demonstrate therefore clearly that nonspecific immunosuppression will not
invariably facilitate the establishment of immune tolerance as is sometimes claimed
(13) and in fact, as in the case of corticosteroids, it may even antagonize tolerance.
Moreover, the finding that the establishment of tolerance can be interfered with
actively would seem to have significant theoretical repercussions. This is par-
ticularly so since corticosteroids are known to affect the immune mechanism mainly
through the elimination of the immature compartment of the thymus derived
lymphoid cell population (14, 15). It could be argued, of course, that interference
with the elicitation of tolerance may not necessarily depend on the cytotoxic
activity of hydrocortisone since adrenal steroids are known also to act as inducers
of adaptive enzymes (16) and it could be speculated that it is a stimulative rather
than a cytotoxic mechanism which counteracts the establishment of unresponsive-
226 ZAN-BAR, NACHTIGAL AND FELDMAN

ness. Evidence is available, however, which may support the first alternative. Thus
Pevnitsky and Andreson who made mice tolerant to SRBC by means of cyclophos-
phamide, observed that concomitant administration of anti lymphocytic serum
antagonized the induction of tolerance (17). Taking these findings together it
may be assumed that agents which are cytotoxic for T lymphocytes, particularly
for cortisone sensitive thymocytes of the cortical origin in the thymus, can interfere
with the induction of at least certain types of immune tolerance. Our recent find-
ings in mice (18) support this contention. Thus, when lethally irradiated mice are
reconstituted with syngeneic thymus and bone marrow cells from normal donors,
they can be made tolerant to H S A by a single injection of aggregate-free antigen.
When, however, the donors of the thymus are pretreated with hydrocortisone,
the recipients cannot be rendered tolerant. On the other hand, pretreatment with
hydrocortisone of the bone marrow donors does not affect the results. This would
imply that immunologically immature T lymphocytes (or their equivalent in
rabbits) play a positive role in the mechanism leading to this type of specific
unresponsiveness and the question arises how could such an assumption be inte-
grated into the present concepts of cellular immunology. The explanation which
we would suggest is based on recently emerging evidence showing that unrespon-
siveness can be passed to normal lymphocytes by means of lymphoid cells from
tolerant donors (19-21). We have demonstrated that the relevant cells of tolerant
mice which suppress the immune response of normal spleen in adoptive transfers
are antigen-specific suppressor T cells which in themselves are hydrocortisone
resistant (18). Thus, although suppressor T cells would not be eliminated by
hydrocortisone, it could be assumed that the drug will delete their immature pre-
cursors (8) and might interfere in this way with the induction of those types of
unresponsiveness which depend on specific suppression by T cells. This hypothesis
may find support in the observations of Phillips-Quagliata et al. (22), who found
that tolerance to BSA could not be induced in a proportion of rats after neonatal
thymectomy and that this failure to become tolerant could not be repaired by
reconstitution with mature T lymphocytes. On the strength of these findings it
could perhaps be speculated, that the induction of " T cell" tolerance may result
from the interaction of antigen with an immature cortisone sensitive T cell pre-
cursor, which will thus be converted into a specific T suppressor cell. Our finding
(Table 8) that the interference of hydrocortisone in tolerance induction is most
effective when the drug is administered concomitantly with the tolerizing antigen
might be relevant in this context. It could suggest, perhaps, that the cortisone-
sensitive precursors are antigen-specific cells that might arise as a stage in the
differentiation process which is triggered by antigen. A phenomenon depending
perhaps on a similar mechanism was observed by Stavy, Cohen, and Feldman (23)
in a system of immune cytolysis in vitro. They found that rat lymph node cells
sensitized on a monolayer of mouse fibroblasts will demonstrate an increase in
replication and immune lysis, when treated with hydrocortisone and transferred
after washing onto a fresh syngeneic monolayer target. It could be speculated that
the increase is replication and lysis in this experimental model occurs when sup-
pression by T cells is eliminated through the killing of their cortisone-sensitive
precursors. And indeed, immune sensitization concurrent with the cortisone treat-
ment was essential, in this case as well, for the immunostimulative effect of the
drug to manifest itself.
 IMMUNOSUPPRESSION A N D I N D U C T I O N OF T O L E R A N C E 227

Besides its obvious theoretical interest, the study of corticosteroids as blocking

agents in tolerance-induction m a y have also practical repercussions. I t would be
important to ascertain whether corticosteroids will also interfere with the induc-
tion of transplantation tolerance, since in such an eventuality the use of these agents
as immunosuppressants in the m a n a g e m e n t of transplantation cases m a y defeat
a priori chances to establish lasting tolerance to the grafted tissue.

ACKNOWLEDGMENTS
The competent technical assistance of Mrs. V. Segal, Mrs. R. Eschel-Zussman, and Mr.
E. Shasha is gratefully acknowledged.

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