CANCER RESEARCH 27 Part 1, 309-313, February 1967]
Restoration of Immunologie Reactivity of Thymectomized Mice by
Calf Thymus Extracts1
NATHAN
TRAININ AND MARIANA
Department of Experimental
LINKER-ISRAELI
Biology, Isaac Wolfson Building, The Weizmann Institute of Science, Rehovoth, Israel
Summary
Various calf organ extracts were injected into newborn thymectomized mice, and into adult thymectomized and irradiated
mice, of C57BL/6 and C3H/eb strains. Whereas mice receiving
either calf muscle or calf kidney extracts manifested an impaired
immunologie response, of the same magnitude as equivalent con
trols, animals injected with calf thymus extracts resembled intact
normal mice in their immunologie behavior. The immunologie
repair, produced by thymus extracts, appears therefore to be
specific to this organ, and to involve a hormone-like mechanism.
Evidence for the contribution of this calf thymus factor to the
restoration of immune competence in spleen cells of thymecto
mized mice is presented. The precise mechanism of this apparent
hormone-cell interaction is still unknown.
Introduction
Surgical removal of the thymus in neonatal mice has been
shown to elicit a triple syndrome: (a) a decline in lymphocyte
number in peripheral blood, with atrophy of lymphoid tissues;
(6) a failure of normal bodily growth and early mortality; and
(c) an impaired immunologie competence (20, 25). Partial preven
tion of all 3 syndromes by implantation of mouse thymic tissue
within cell-tight Millipore diffusion chambers suggested the
possible existence of a diffusible humoral factor secreted by the
thymus which is able to endow lymphoid cells with immunologie
competence (16, 17, 28). It has also been reported that injection
of calf thymus extract into thymectomized mice protects such
animals from the fatal effect of a viral infection, prevents the
appearance of a wasting syndrome, and increases the number of
lymphocytes in peripheral blood (4).
In normal mice the thymus has been shown to produce a
lymphocytosis-stimulating factor (LSF) which enhances lympho
cyte production, and also appears to play a role in the regulation
of normal lymphocyte homeostasis (3, 21). It has been suggested
that LSF activity is associated with the epithelial-reticular cell
complex of the medulla and not with the cells of the lymphoid
cortex of the thymus (10, 22).
Thymus extracts fiom various sources have also been demon
strated to increase glycine-2-14C or thymidine-3H incorporation
into total protein or DNA of peripheral lymph nodes of normal
mice, thus suggesting the presence of a thymic lymphocytopoietic factor in these extracts (12).
1This investigation was supported in part by USPHS Research
Grant No. AM-08460.
Received July 18, 1966; accepted September 1, I960.
It was considered desirable to evaluate the efficiency of
thymus extracts in the repair of other aspects of thymus depriva
tion. The present investigation was specifically directed to
examine calf thymus extracts vis-Ã -vissome immunologie def
icits induced in newborn mice by thymeetomy and in adult
mice by the combination of thymeetomy and irradiation.
Materials
and
Methods
MICE. Mice originally obtained from the Roscoe B. Jackson
Memorial Laboratory, Bar Harbor, were bred at the Weizmann
Institute of Science by sibling mating. C57HL/6 and C3H/eb
inbred mice, as well as (C57BL/6 XC3H/eb) FI, were used in the
present experiments. The animals were housed in metal cages in
air-conditioned rooms kept at 21°-25°C,
and after weaning, at
5 weeks of age, were fed Purina laboratory chow pellets, sup
plemented by barley and sunflower seeds, and tap water ad
libitum.
THYMECTOMY.
Neonatal mice were thymectomized either
within 24 hr after birth, or at 3 days of age, as indicated, by an
adaptation of Miller's technic (24). Adult mice were thymecto
mized at 8 weeks of age by a technic similar to that described by
Kaplan (11). At the end of the experiment, or after death, the
mice were autopsied, and the absence of all mediastinal thymic
tissue was ascertained by gross inspection and microscopic
sectioning when indicated. Any operated animal found to con
tain a thymic remnant was discarded from the experiment.
SKINGRAFTING.Full-thickness skin grafts were performed by
the method of Billingham and Medawar (2). Mice were grafted
at 12 weeks of age and the grafts were examined periodically
from the 7th day, survival of the grafts being estimated from
their gross appearance.
IRRADIATION.Irradiation was performed with a G. E. Maximar 250-III machine. The physical properties were: 200 kv,
15 ma, with 1-mm Al and 0.5-mm Cu filter and a dose rate of 55
Il/min FSD, 50 cm. During the irradiation, the mice were
placed in a Lucite container, 10 per group in a field of 20 x 20 cm.
The mice were 12 weeks old when submitted to a single total
body exposure of 550 R.
PREPARATION
AND INJECTION
OF EXTRACTS. Calf
thymus
6X-
tracts were derived from organs removed aseptieally from ani
mals killed at a local slaughterhouse and transported to the
laboratory on ice. The capsules and main blood vessels were
removed, the residual pulp mixed with 2 parts of phosphate-buff
ered saline (PBS) solution and then disintegrated in a Vir-Tis
homogenizer. These steps of preparation were carried out on ice
under aseptic conditions. This crude homogenate was centrifugea
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A"«//¡an
Trainin
and Mariana
Linker-Israeli
at 3500 rpm in a Servall-RC-2-cooled centrifuge for 20 min. The
precipitate was discarded and the .supernatant submitted to a
further centrifugaron at 105,000 X g for l hr in a Bookman model
L preparative ultracentrifugo. The precipitate was again dis
carded and the supernatant sterilized by passing it through Millipore filters (0.8 n, pore size). Effectiveness of the filtration was
determined in some cases, by testing the passage of Eschericliia
coli through the filter.
Protein concentration in each sample was determined by the
biuret reaction and the extract was then diluted by adding PBS
to reach a final concentration of 10 nig of total protein/ml
volume.
Calf muscle and calf kidney extracts, used as controls in
different experiments, were prepared under similar conditions
and adjusted to the same concentration of protein. PBS solution
was injected to controls in some of the experiments. All prepara
tions were kept frozen at —
20°Cuntil used, and made up fresh
from 2 to 4 animals were pooled and injected into 2-4 mice; thus,
each recipient litter received cells from donors of 2 different types.
Eight days after cell administration, the animals were killed,
their body and spleen weights recorded, and the relative spleen
weight (mg spleen/10 gm body weight) determined. Finally, the
"spleen index" was obtained by dividing the mean relative
spleen weights of the animals receiving parental strain cells by
the mean relative spleen weights of the mice injected with cells
from syngeneic donors of similar age.
Results
RESTORATION
OF
SKIN
AND
TUMOR
HOMOGRAFT
IMMUNITY
FOLLOWING
NEONATAL
THYMKCTOMY.
Previous work has demon
strated that mice thymectomized during the neonatal period,
failed to reject normal skin homografte from the same or even
different H-2 strong histocompatibility locus (27); and from
subsequent experiments carried out to study the effect of thymec
tomy at birth on the capacity to accept tumor homografts, this
procedure is known to permit successful transplantation of
tumor across the H-2 histocompatibility barrier (19). The present
experiments were performed to test whether calf thymus extracts
may modify the response of neonatally thymectomized mice to
allogeneic skin and tumor grafts.
The results of skin transplantation in which C3H/eb mice
thymectomized at 3 days were grafted with (C57BL/6 X C3H/
eb) F! skin grafts at 12 weeks of age, after repeated injections of
calf thymus or muscle extracts, are summarized in Table 1. In
contrast to intact mice, which rejected the grafts within 15 days,
13/16 mice of the thymectomized controls (injected with calf
muscle extracts—to eliminate any unspccific immunologie
response which may be attributed to parenteral administration
of a foreign protein) failed to reject skin homografts even after 25
days. In 8/16, the skin graft survived up to 50 days, and in the
remaining 5 animals up to 90 days, to the end of the observation
period. The majority of thymectomized mice receiving thymus
every 2nd or 3rd week. In neonatally thymectomized mice,
injection of various extracts was made i.p. twice weekly, from
the 2nd week of age until the end of the experiment. The total
number of injections ranged from 10 to 30 in the different ex
periments, the volume per injection being 0.1 ml before the
animals were 4 weeks old, and 0.2 ml after that age.
In adult mice, beginning 1 week after thymectomy, extracts
were injected i.p. twice weekly throughout the experiment.
These mice received an average of 30 injections of 0.25 ml each,
excepting those which died before this schedule was completed.
TUMORGRAFTING.A solid fibrosarcoma, induced originally
by 3,4-benzpyrene s.c. injection in a C3H/Ja.\ mouse, and since
then serially transferred within recipients of the same strain for
several generations, was used. The tumor was cut into small
pieces, inoculated by trocar i.m. in the right leg of mice, and the
animals observed thereafter for 90 days.
CELL SUSPKNSIONS.
Spleens were removed aseptically from
intact and newborn thymectomized mice, injected previously
with different calf organ extracts (see below). The spleens were
diced and gently pushed through a fine stainless steel screen
(50 x 50 mesh), and the cells obtained were suspended in a
TABLE 1
cooled sterile saline and washed twice employing low speed
THE EFFECT OF CALF THYMUSEXTRACTINJECTIONSINTO
centrifugation. Cell counts were made and adjusted to a concen
NEONATALLYTHYMECTOMIZED
C3H/eb MICE ON THE
tration of 10-15 million nucleated cells in approximately 0.05
RESTORATION
OF IMMUNOLOGIC
REACTIVITYTO
ml volume.
ALLOGENEIC
SKIN GRAFTS"
ASSAYOF GRAFT-versus-HosTACTIVITY.The graft-1'ersîis-host
SHOWINGSKIN
OF UICE
assay of Simonsen et al. (29) was used for evaluation of immuno
FOR:<15days210141S-25days3625-50days8>50days5
GRAFT SURVIVAL
TREATMENTOF
OFHICE211620No.
STRAIN*(C57BL/6
logie capacity of spleen cells from newborn thymectomized mice
RECIPIENTSNormal
submitted to a previous short treatment (10 injections in 5 weeks)
or to prolonged treatment (20 injections in 10 weeks) of calf
thymus or kidney extract. This assay measures the spleen enlarge
controlsThymectomy XC3H/eb)F,(C57BL/6
ment induced in young FI hybrids receiving lymphoid or spleen
cells from one of the parental strains.
andcalf
XC3H/eb)F,(C57BL/6
Litters of 4-8 (C57BL/6 X C3H/eb) Ft hybrid mice were
muscle ex
injected i.p., at 8 days of age, with 10-15 million dissociated
tractThymectomy'
andcalf
spleen cells from (a) intact syngeneic (C57BL/6 X C3H/eb) FI
XC3H/eb)F,No.
thymus ex
mice; (6) intact parental C57BL/6 mice; (e) C57BL/6 mice
tractDONOR
thymectomized within 24 hr after birth and injected twice weekly
with calf thymus extract during 5 and 10 weeks, respectively; or
" Calf thymus and calf muscle extracts injected twice weekly,
(d) C57BL/6 mice thymectomized within 24 hr after birth and beginning at 3 wk. of age.
injected twice weekly with calf kidney extract during 5 and 10
6Skin grafts performed at 12 wk. of age.
' Thymectomy performed at 3 days old.
weeks, respectively. To attain randomization, the spleen cells
310
CANCER RESEARCH VOL. 27
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Restoralion of Immunologie Reactivity of Thymectomized Mice
TABLE 2
THE EFFECT OF CALF THYMUS EXTRACT INJECTIONS INTO NEONATALLVTHYMECTOMIZED
C57BL/6 MICE MICE ON THE RESTO
RATION OF IMMUNOLOGICREACTIVITY TO ALLOGENIC C3H
TUMOR GRAFTS"
recipientsNormal
Treatment
takes/No,
of lethal
of mice
grafted0/157/150/14% takes0470
of
TABLE 3
THE EFFECT OF CALF THYMUS EXTRACT INJECTIONS INTO THY
MECTOMIZED
IRRADIATEDADULTC57BL/G ON THE RESTORATION
OF IMMUNOLOGICREACTIVITY TO ALLOGENEIC C3H TUMOR
GRAFTS"
takes/
time
GroupiiiinIVVVITreatmentof
recipientsX-rays»Thymectomy'Thymectomy
No. of mice
lethal
death(days)484550,
of
grafted0/140/1211/1412/154/200/17%of
takes007880200Av.
controlsThymectomy6
PBSCThymectomy
and
thymusextract0Lethal
and calf
andX-raysThymectomy,
" C3H fibrosarcoma grafted i.m. at 12 wk. of age.
b Thymectomy performed within 24 hr after birth.
c Calf thymus and phosphate-buffered
saline (PBS) injected
twice weekly beginning at 3 wk. of age.
X-rays,and
calf kidney ex
*Thymectomy,
tract
X-rays,and
65,72"
53,
calf thymus ex
dIntact
tract
extracts rejected the grafts within 15 days (14/20), as did the
normal controls, and the rest showed slightly prolonged rejection
time, up to 25 days.
Experiments
in which C57BL/6 mice were thymectomized
within 24 hr after birth and challenged at 12 weeks of age with
homograft C'3H/Jax tumor are summarized in Table 2.
In contrast to normal mice, where the percentage of takes was
nil, 7/15 of the thymectomized
controls injected with PBS
accepted the tumor homograft and died as a consequence of its
progressive growth. Neonatally thymectomized
mice receiving
thymus extracts, on the other hand, behaved completely as
normal, all of them rejecting the tumor challenge.
controlsLethal
" C3H fibrosarcoma
equivalent age.
grafted
i.m. 40 days after irradiation
6X-rays: single total body exposure to 550 R at 12 wk. of age.
' Thymectomy
performed at 8 wk. of age.
d Calf thymus and calf kidney extracts injected
beginning 1 week after thymectomy.
* Day of individual deaths.
Groft-versus-host
REPAIR
OF FAILURE
OF SPLEEN
CELLS
FROM
THYMECTOMIZKI)
twice weekly
assay in (C57 BL> C3H ) F\ recipients
D
Inloct
RESTORATION
OF TUMOR HOMOGRAFT IMMUNITY IN ADULT MICE
FOLLOWING
THYMECTOMY
AND
X-RAY
IRRADIATION.
Recent
investigations suggest that the role of the thymus in immunogenesis is not necessarily restricted to early life. Although thymectomy
of the adult mouse does not seem to impair its immunologie
competence, some decrease in the lymphocyte population does
take place (23). Furthermore, it appears that thymectomy delays
or suppresses the spontaneous restoration of immune response
following sublethal doses of X-rays. This impairment in immune
reactivation has been made evident by the challenge of adult,
irradiated, thymectomized mice with allogeneie skin grafts (26),
and allogeneie tumor tissue (5, 6).
Experiments were therefore set up to assay the effect of calf
thymus extracts on the restoration of immunologie reactivity of
thymectomized,
irradiated, adult C57BL/6 mice to allogeneie
grafts of a C3H/Jax
transplantable
fibrosarcoma. Results of
these experiments are summarized in Table 3.
Like intact controls (Group VI), total-body-irradiated
(Group
I) and thymectomized
animals (Group II) rejected the tumors
in all cases. Those which were only thymectomized and irradiated
(Group III) or which received, in addition, calf kidney extract
(Group IV) exhibited a very high percentage of lethal takes (78
and 8/0%, respectively), as an expression of the profound damage
rendered to the immune response mechanism of these animals
by combined treatment
of thymectomy
and X-irradiation.
However, from those which were thymectomized and irradiated,
and received, in addition, calf thymus extract (Group V), only
4/20 developed tumors, and these died later than the respective
controls.
or
C578L
j Thymectomized
Spleen ceti
j plus
calf
C57
thymus
BL
extract
donors
derived
from
j Thymectomized
j plus
calf
kidney
jThymectomized
J uninjected
CHART 1. The effect of calf thymus
weekly for 10 weeks) into neonatally
mice, on the restoration of immunologie
cells against (C57BL/G X C3H/eb)Fi
represents the average spleen index
standard deviation is indicated by the
each column.
C 57 BL
extract
C57BL
extract injections (twice
thymectomized
C57BL/G
reactivity of their spleen
recipients. Each column
of 12-15 recipients. The
vertical line at the top of
MICE TO INDUCEA GRAFT-t*mfS-HOSTREACTION.Spleen cells
from mice of parental strains thymectomized at birth do not
produce the graft-i'ersws-host response in appropriate FI hybrid
recipients—a reaction which is characteristic of normal parental
cells. This is generally accepted as evidence that immunologie
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311
Xathan Trainin and Mariana Linker-Israeli
TABLE 4
recipient mice (16, 17, 28). Restorative effects, have also been
elicited in thymectomized rats and rabbits by inserting thymus
tissue enclosed within Millipore chambers (1, 30) and in thymec
tomized mice bearing xenogeneic thymic grafts (14). These data
would suggest, then, the existence of a diffusible product from
index2.601.201.41
thymus tissue in different strains and species, which, without
being species specific, enables thymectomized animals to produce
new
and to develop immunologie competence (1416). lymphocytes
"
GRAFT-i>er*us-HosT ASSAY IN (C57BL/6 X C3H/eb)F!
MICE
USING SPLEEN CELLS FROMTHYMECTOMIZEDC57BL/6 MICE
PRETREATED WITH CALF THYMUS EXTRACTS (5 wk.)a
groupsIntact
Experimental
C57BL/6Newborn
thymec-tomized
C57BL/6Newborn
of
donorNonePBS»Calfindices3.02,
2.60,2.75,
2.44,
2.211.13,
1.07,1.06,
1.36,
1.28,1.081.43,
1.42,
thymec-tomized
thymusextractSpleen
1.46,1.36,
1.21,
1.36,1.43Mean
1.70,
C57BL/6Treatment
" Calf thymus extract was injected twice weekly.
* PBS, phosphate-buff e red saline.
competence of such cells is thymus dependent (7). It was decided
to analyze the influence of thymus extract on immunologie
activity of spleen cells of thymectomized mice, using as a criterion
the graft-t«rsus-host assay of histocompatibility of Simonsen.
The results of these experiments are summarized in Chart 1.
Spleen indices of different groups of recipients show that spleen
cells of newborn thymectomized C57BL/6 controls, injected with
calf kidney extracts, failed to induce a spleen enlargement in
appropriate FI hybrids, as compared with cells of intact donors.
Spleen cells from similar parental newborn thymectomized
donors, injected twice weekly, for 10 weeks, with calf thymus
extract were, however, able to repair this failure, this restoration
being manifested by a spleen index of recipients significantly
increased (P —0.001). Spleen indices were, nevertheless, lower
than comparable intact controls, indicating that immunologie
repair was not completely achieved.
Table 4 summarizes a further experiment, the critical difference
being that the respective parental donors were given only half
the number of calf thymus and calf kidney extract injections
before testing (10 injections in 5 weeks). Under these conditions,
calf thymus extract improved only to a slight extent the im
munologie performance of donor cells—as compared with the
previous experiment—suggesting the possible existence of a
critical length of time before this humoral factor fulfills its reparative action at the cellular level.
Discussion
The results presented here indicate that the damage to the
immune response, induced in newborn mice by thymectomy, or
in adult mice by thymectomy followed by total body irradiation,
can be prevented by repeated injections of calf thymus extract,
while extracts (of the same protein concentration) derived from
other calf organs (kidney or muscle), are ineffective. It would
seem, therefore, that the observed reparative effect is due to
something specific in thymus extracts, and not a result of a non
specific protein effect.
In early studies, in which syngeneic or allogeneic thymus tissue
enclosed in cell-tight, fluid-permeable, Millipore diffusion cham
bers, were implanted i.p. into neonatally thymectomized mice,
body wasting and lymphoid depletion were prevented, and
restoration of immune response was achieved in many of the
312
The alternative explanation of an inductive mechanism across
cell membranes, involving cell-to-cell contact between thymic
cells and circulating peripheral lymphoid cells (5), can be dis
carded in the light of the present results, since the technic used
in these experiments for the preparation of organ extracts re
moved any possibility of survival of intact thymic cells.
It has been suggested that the thymic humoral factor con
tributes to the establishment of immunologically competent cells,
either within the environment of thymic tissue itself, or through
its elaboration, circulation, and systemic (hormone-like) effect
upon seeded cells within lymphoid organs (13, 16). The restora
tion of competence to spleen cells of neonatally thymectomized
mice, when calf thymus extract is given, as shown by the graftt'ersi«-hostassay of histocompatibility reported here, argues for a
systemic hormone-like mechanism, originating in the thymus
and affecting the cells of the peripheral lymphoid organs. The
kind of information furnished by this humoral factor of thymus
seems to be essential for the establishment of immune competence
of lymphoid cells.
The existence of a lag period required by thymic extracts to
bring back to normal the impaired immunologie reactivity of
spleen cells derived from thymectomized animals calls for further
comment. Graft-versus-host reactions are thought to be initiated
by small lymphocytes, and the sequence small lymphocyte —>
large pyroninophilie cell —»
small lymphocyte has been proposed
(8, 9). Thus there may be 2 categories of small lymphocytes:
those immunologically "committed," which have already met an
antigen, and lymphocytes which are immunologically "uncom
mitted," i.e., free from previous antigenic stimulus, and com
petent to produce a new clone of antibody-forming cells. Accord
ingly, the fact that spleen cells of thymectomized mice are able
to induce a graft-i'ersws-host reaction only after a prolonged treat
ment of the donor with thymus extract, may be related to the
time required for the establishment of a new clone of cells which
are both uncommitted and competent (8, 9, 18). In any case,
more information on the mechanism of function of the thymic
humoral factor is required in order to understand the intimacy
of this hormone-cell interaction. The chemical nature of this
factor is still unknown and further research in this direction is
needed.
Acknowledgments
The authors wish to express their appreciation
to Prof. M.
Feldman for providing the transplantable
tumor used in one of
these experiments, and to Miss T. Eckhaus and Mr. I. Serussi
for valuable technical assistance.
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Restoration of Immunologic Reactivity of Thymectomized Mice
by Calf Thymus Extracts
Nathan Trainin and Mariana Linker-Israeli
Cancer Res 1967;27:309-313.
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