AMER. ZOOL., 15:73-84 (1975).
Development of Transplantation Immunity and Restoration Experiments in the
Thymectomized Amphibian
JOHN D. HORTON AND TRUDY L. HORTON
Department of Zoology, The University of Durham, Durham DH1 3LE, England
SYNOPSIS. This paper examines the thymic dependence of alloimmunity in amphibians. In
Xenopus, the presence of a thymus during the first 2 weeks of life is essential for the
development of normal first-set skin allograft immunity. Thymectomy during this early
period always impairs the alloimmune response of young adult toads. However, most of
these thymectomized animals are able to completely destroy skin allografts, albeit with
prolonged rejection times. Chronic graft rejection, rather than tolerance, still occurs following thymectomy as early as 5 days, when the thymus contains no small lymphocytes. In
contrast to the considerable differences in first-set allograft survival times in control and
early-thymectomizedXenojIms, second-set grafts, applied subsequent to first-set destruction,
are rejected in acute fashion (< 3 weeks) in both groups. That the defect in first-set
alloimmunity is specifically related to absence of thymus has been confirmed by implanting
allogeneic thymus 2 weeks post-thymectomy. The donor thymus remains healthy and
restores the allograft response to normal. In contrast, allogeneic spleen does not reconstitute
and itself often undergoes destruction. Preliminary autoradiographic experiments on lymphoid tissue involvement in first-set allograft rejection are also described.
Much of our current understanding of
the central role played by the thymus in
immunological maturation comes from
thymectomy experiments on birds and
mammals (Hess, 1968). Experiments of nature, in which animals develop "without" a
thymus due to congenital defects (e.g., the
nude mouse; Words, 1971) have also proved
invaluable in this respect. Such studies have
revealed that, in the absence of an intact
thymus during early development, most
homoiotherms do not develop normally
and, moreover, possess extremely crippled
immune systems. Thus, cell-mediated immunity (e.g., allograft rejection) and
humoral antibody responses to many antigens are usually severely impaired (Miller,
1971). In contrast, recent experiments
(Cole and Morris, 1971) have shown that
early (in utero) thymectomy in the lamb
does not result in abnormal growth and
development. Moreover, since these
This work was supported by grants from the Medical Research Council of Great Britain. Travel grants
from The Royal Society and The University of
Durham are gratefully acknowledged.
Several of the experiments described in this paper
were performed in collaboration with Dr. M. J. Manning. Thanks are also due to J. J. Rimmer for reading
the manuscript.
thymectomized lambs display normal allograft rejection, the universality of thymic
dependence of alloimmunity has been
questioned (Morris, 1973).
We have turned our attention to analyzing the role of the thymus in the immunological development of amphibians,
since these poikilotherms offer certain advantages for thymic extirpation experiments. Thus, in Xenopus laevis successful
thymic ablation is possible as early as 8 days
post-fertilization (Horton and Manning,
1972). At this age all the lymphoid organs
are at a rudimentary state of differentiation, possessing small numbers of
cells (< 2,000 cells in the thymus and
< 1,000 in the spleen) with few, if any, small
lymphocytes. We would like to stress that
following this early thymectomy, Xenopus
develop normally, despite the absence of
any regenerating thymic tissue. This amphibian system, therefore, enables us to
examine the ontogeny of immunity in healthy animals that have experienced, at most,
only minimal thymic influence. To our
knowledge such early thymectomy has not
been achieved in any homoiothermic
species. Even the nude mouse develops a
thymic primordium which later atrophies
(Pantelouris and Hair, 1970); moreover, a
73
74
JOHN D. HORTON AND TRUDY L. HORTON
small population of thymic-dependent cells ments reported in this paper were carried
may exist in these animals (Raff, 1973). out at 221 ± 1 C and skin allografts were
Another advantage of the Xenopus system is from non-siblings.
that, unlike mammals, these anurans are
free from any direct maternal influence FIRST-SET ALLOGRAFT RESPONSES FOLLOWING
which might have important immunologiTHYMECTOMY
cal consequences (Osoba, 1973).
In 1972 Horton and Manning reported
This paper will concentrate on the effects
of thymectomy on the development of al- on the histological response of larvae to
loimmune responses in Xenopus, while skin allografts and the fate of alloManning (1975) looks at the diversity of transplants applied to post-metamorphic
thymic dependent responses in this species. Xenopus (toadlets) following thymectomy at
Thymectomy experiments in a number of different stages of lymphoid organ maturaanuran species other than Xenopus have tion. Microcautery of the thymus was peralready shown that absence of thymus dur- formed during the third week of life, when
ing early larval life results in severe im- all the lymphoid organs have completed
pairment of alloimmune reactivity (Du their lymphoid histogenesis (Manning and
Pasquier, 1973). Nevertheless, allografts on Horton, 1969), in the second week, when
these thymectomized anurans are generally the peripheral lymphoid organs are still relrejected at later post-transplantation inter- atively undifferentiated, and also at 8 days
vals (Rana catesbeiana: Cooper and Hil- post-fertilization when, as already mendemann, 1965; Cooper, 1967\Alytes obstetri- tioned, all the lymphoid tissues, including
cans: Du Pasquier, 1965; Ranapipiens: Cur- the thymus, are extremely rudimentary.
tis and Volpe, 1971). Difficulties with The experiments in which grafts were
thymic regeneration were often encoun- applied to larvae 16 to 21 days posttered in these experiments (see Curtis and thymectomy were performed simply to deVolpe, 1971, for discussion). Such incom- termine whether the normal lymphocytic
plete thymectomy, or thymectomy after the invasion of larval skin allografts occurred in
thymus has already had a considerable in- thymectomized animals in the second week
fluence, might have been important con- post-transplantation. The ability of the
tributory factors in enabling these anurans thymectomized larva to destroy skin alloto reject transplants. In contrast to this grafts was not assessed, since graft rejection
chronic rejection following thymectomy, end-points are difficult to determine prior
early thymic removal in a number of to metamorphosis. Many thymectomized
urodeles results in definitive tolerance to animals were therefore allowed to
skin allografts (Tournefier, 1973). Tour- metamorphose before allografts were
nefier's experiments on Triturus alpestris are applied 76 to 118 days post-thymectomy: It
of particular interest here since these ani- was on these animals that the outcome of
mals, like Xenopus, do not runt following thymectomy on the ability of Xenopus to rethymectomy, in contrast to the ill-health of ject transplants was assessed.
many anurans {Rana catesbeiana: Hildemann and Cooper, 1963; Alytes obstetri- These experiments showed that the
cans: Du Pasquier, 1968) and other thymus is required for only a relatively
urodeles {Pleurodeles waltlii: Charlemagne short period of larval life to effect normal
and Houillon, 1968) following thymic ex- development of alloimmune reactivity.
tirpation. We were, therefore, particularly Thus, removal of this organ during the
interested to determine the immunological third week had neither effect on the larva's
outcome of applying first-set skin allografts lymphocytic response to allografts nor alto Xenopus that had experienced minimal tered the speed of graft rejection in toadthymic influence, and to examine the rela- lets. inXenopus the thymus would therefore
tionship of alloimmune impairment with appear to have had a significant effect on
stage of lymphoid organ development at the developing immune system in the first 2
the time of thymectomy. All the experi- weeks prior to its removal. Thymic ablation
during this early period—i.e., when the
75
ROLE OF AMPHIBIAN THYMUS IN ALLOIMMUNITY
lymphoid tissues have not completed, or
only just begun their lymphoid
histogenesis—had pronounced effects on
the development of alloimmune responsiveness. Thus, the normal lymphocytic invasion of skin allografts failed to occur in
thymectomized larvae, and graft rejection
was always prolonged (> 4 weeks) in the
adult. However, even when thymectomy
had been performed at 8 days of age, the
majority of such toadlets (5/8) did eventually completely destroy their skin allografts.
It is interesting that this impairment of allograft immunity is not concomitant with
dramatic changes in lymphoid organ morphology following thymic ablation at 8
days. In fact, both larvae and toadlets develop relatively normal lymphoid tissues
following early thymectomy, although
spleens often become reduced in size and
display varying degrees of lymphoid depletion (Manning, 1971; Horton and Manning, 1974).
The finding that toadlets thymectomized
early in larval life can effect chronic allograft rejection has now been confirmed with
experiments on larger numbers of animals
(see Table 1). The data in this Table reveal
that, of the 26 toadlets receiving skin allografts at varying ages following thymectomy
at 7 or 8 days, 12 rejected their grafts 26 to
50 days post-transplantation. Six of the remaining 14 animals rejected allografts at
later post-transplantation intervals (rang-
ing from 53 to 72 days), the other 8 still
bearing intact grafts when killed (57 to 105
days). Half the latter were displaying mild
rejection phenomena, while the other four
grafts still looked healthy. Regular
stereomicroscopic observations during
first-set allograft rejection in these thymectomized animals revealed that chronic rejection reactions were always heralded by
prolonged post-transplantation intervals
when grafts remained in excellent condition. Thus, at a time (18 days) when grafts
on most intact animals were in the terminal
stages of rejection, allotransplants on all
thymectomized toadlets appeared extremely healthy with, at the most, only
slight vascular disturbances. Alongside this
delay in onset of immune reactivity,
another externally observable feature,
sometimes related to chronicity of graft rejection, was a decrease in intensity of incompatibility phenomena (enlarged and
hemorrhaged blood vessels, pigmentary
defects). However, in many thymectomized
animals the intensity of the alloimmune response, once begun, was essentially no different from that seen in intact toadlets. Indeed, extremely chronic graft rejection
(> 50 days) was nearly always characterized
by a prolonged interval in which the graft
remained fully viable. This was followed by
a vigorous destructive phase. For example,
two thymectomized toadlets that had (by
external criteria) perfect grafts at 50 days
TABLE 1. Summary of first-set skin allograft rejection times in thymectomized and control toadlets.
Control
Toadlets
(sham-thymectomized or
non-thymectomized)
Thymectomized
Toadlets
1
Complete rejection within
Larval age
(days) at
thymectomy or
sham-thymectomy
Age (days)
when firstset grafts
applied
Number
of
toadlets
grafted
7-8
7-8
5
70 - 280
70 - 124
280 - 370
187
22
10
2
3
20
10
2
3
2
0
0
0
0
0
0
0
37
35
2
0
19
7
6
0
0
0
9
3
4
10
4
2
32
0
16
16"
7-8
7-8
5
70- 124
280 - 370
187
18-25
days
26-50
days > 50 days
7 of these thymectomized animals had rejected their grafts when killed (53 to 107 days). The remaining 9 still
had grafts intact when killed (57 to 153 days).
76
JOHN D. HORTON AND TRUDY L. HORTON
had completely rejected these within mals thymectomized at 7 or 8 days of age.
another 22 days.
Thymectomy as early as 5 days postIncluded in Table 1 are data from an fertilization has now been achieved and
experiment performed on seven toadlets, these animals still develop normally, dethymectomized at 7 or 8 days post- spite absence of thymic tissue. At 5 days
fertilization, but left for longer intervals (stage 46/47 of Nieuwkoop and Faber,
(up to 1 year) before allografts were 1967) the thymus is extremely undifferenapplied. This experiment was to determine tiated (see Fig. 1), the thymic cells being
whether or not an increased time interval large and few in number (< 500 per
post-thymectomy would result in im- thymus). The thymus is, in fact, only first
provement of alloimmune responsiveness. recognized as a bud from the pharyngeal
Such an improvement could occur if the epithelium at about 3 days of age (Manning
defect following thymectomy is a numerical and Horton, 1969). The results of this exone, i.e., due to a paucity of lymphocytes. periment are included in Table 1. Four of
Such a quantitatively reduced cellular the six toadlets rejected allografts between
population might be gradually built up 26 and 50 days. Of the other two, one refrom extra-thymic sources. Rejection times jected at 107 days, the other bore a fully
afforded by the seven animals do not, how- viable graft when killed 153 days postever, indicate improvement of alloimmune transplantation. Clearly, 5-day thymeccompetence associated with increasing in- tomy has no greater effect on toadlet alterval post-thymectomy. Thus 1-year old loimmune reactivity than the 7 to 8 day
toadlets, thymectomized early in life, be- operation.
haved in a similar manner to those 19 toadWe may conclude that in anurans the
lets allografted at a much shorter interval capacity for chronic allograft rejection will
post-thymectomy, i.e., they rejected trans- exist however early one removes the
plants in chronic fashion. It is pertinent to thymus. Tolerance induction to skin alloremember at this juncture that we do not grafts in the "absence" of thymic influence
know the eventual immunological outcome rarely occurs in this group, which therefore
in the larva following thymectomy. displays a differing degree of thymic deWhether or not a real difference between pendency of allograft rejection when comlarva and adult exists in the thymic depen- pared with urodeles.
dency of allograft reactivity requires
further experimentation (see Du Pasquier,
AUTORADIOGRAPHIC STUDIES DURING ALLO1973, for discussion of this point).
One possible explanation emerging from
the foregoing experiments is that allograft
rejection in early thymectomized Xenopus is
effected by thymic independent lymphocytes. Alternatively such responsiveness
may be dependent upon a small residual
population of thymic-schooled cells. Until
specific markers for thymic dependent
lymphocytes are available in Xenopus we
cannot, for the moment, conclude that such
cells are absent in the early thymectomized
animal. However, we feel it unlikely that
any significant thymic influence could have
occurred prior to thymectomy at 7 days.
Nevertheless an attempt to perform
thymectomy even earlier in life appeared
warranted in order to determine if such
animals display a more pronounced impairment of allograft reactivity than ani-
GRAFT REJECTION
It is difficult to learn more about the level
at which the thymus controls the developing alloimmune system in amphibians
without first having a much greater understanding of allograft destruction in the intact animal. The involvement of lymphocytes in allograft rejection in amphibians
has been described by many authors, but a
more detailed analysis of how these and
other cells effect graft destruction is clearly
warranted (see also Cohen, 1971; Baculi
and Cooper, 1970). Moreover, studies on
the role of lymphoid organs, other than the
thymus, in this process would prove invaluable to understanding the mechanism of
thymic control of allograft rejection in amphibians. Some of these issues are being
examined in Xenopus.
ROLE OF AMPHIBIAN THYMUS IN ALLOIMMUNITY
FIG. 1. Section through the center of the thymus of a
5-day-old Xenopus larva, the earliest age at which
thymectomy was performed. The anlage is mainly
comprised of immature lymphoid cells and epithelial
cells with pale nuclei and prominent nucleoli. Several
mitoses are apparent. No small lymphocytes are found
at this stage of development. Stain: haematoxylin and
Lymphoid tissues and skin transplants
were removed from 12 allografted and 8
autografted intact toadlets at regular intervals from 5 to 19 days post-transplantation.
Lymphoid organs from 4 non-grafted siblings were also included in this preliminary
experiment. Four hours before killing,
most of the 24 toadlets were injected with
tritiated thymidine (1 /^Ci/g body wt) via the
dorsal lymph sac. Serial sections were prepared from Carnoy-fixed material and
mounted on gelatine-coated slides. They
were then dipped in Ilford K5 nuclear
emulsion, exposed for 4 weeks prior to development, following the autoradiographic
procedure used by Turner and Manning
(1973) in their study on Xenopus spleen. Sections were subsequently stained in methyl
green-pyronin.
Analysis of coded sections revealed intense
lymphoid pyroninophilia in many (6/12) of
the spleens taken from allografted animals.
In contrast, the levels of pyroninophilia in
all autografted and non-grafted toadlets
were much lower. It would appear that in-
crease in numbers of pyroninophilic cells in
the spleen occurs following allografting.
High levels of tritiated thymidine uptake
were recorded in the splenic white pulp
regions from most (6/8) of the allografted,
thymidine-injected toadlets. Many of the
pyroninophilic cells in this splenic region
were labeled, indicating that they were a
proliferating population. In this initial experiment, autografted and non-grafted
animals invariably displayed low levels of
label throughout their spleens. However, subsequent studies revealed that spleens from
non-allografted animals of a different
batch frequently displayed intense white
pulp activity. Experiments to date are,
therefore, inconclusive in implicating a
splenic involvement in the alloimmune response of intact Xenopus. Moreover, our
preliminary studies did not reveal any apparent histological changes in lymphoid tissues other than the spleen following allografting: Thus, levels of label and
pyroninophilia in the thymus, liver, kidney
and intestine were similar in allografted,
78
JOHN D. HORTON AND TRUDY L. HORTON
autografted and non-grafted toadlets. Of
particular relevance here is the work of
Ruben et al. (1972). These authors suggest,
from histological studies on the early immune response to diverse tissue implants in
Xenopus larvae, that allograft reactivity may
not center in the host's lymphoid organs. In
contrast, increases in small lymphocytes in
lymph glands, spleen, liver, kidney, and intestine were reported in larval bullfrogs following skin allografting (Baculi and
Cooper, 1970).
It has been suggested elsewhere (Simnett, 1965) that the lymphocytic mass which
accumulates at the graft site in Xenopus may
itself be an important center of immunological reactivity. Our autoradiographic
studies on allografts certainly strengthen
this suggestion. Uptake of tritiated
thymidine by the lymphoid accumulation
in allografts was extremely heavy in contrast to the low level of label in autografts
(see Fig. 2a,b), indicating intense lymphocytic proliferation at the site of antigen application. Studies are now in progress to
analyze in more detail the cellular
mechanism of graft rejection in the thymectomized animal. In particular, we wish to
determine if the lymphoid cells which eventually accumulate under skin allografts on
thymectomized toadlets (Horton and Manning, 1972) are a rapidly proliferating
population. Deficiencies in proliferative
ability of lymphocytes following contact
with antigen might be expected in the
thymectomized animal if one considers that
an important thymic function is to stimulate mitosis of lymphoid cells (see discussion
in Weiss et al., 1973).
SECOND-SET ALLOGRAFT REACTIONS FOLLOWING
EARLY THYMECTOMY
We next turned our attention to assessing
the immunological outcome of first-set allograft destruction on the subsequent alloimmune capabilities of the thymectomized animal. Would a thymectomized
toadlet reject a second skin transplant from
the same donor in accelerated fashion? To
answer this question we initially transplanted first-set skin allografts to three
toadlets (70 days old) that had been thymectomized on the seventh day of life. These
animals took from 42 to 65 days to reject
their first-set transplants in contrast to two
sibling sham-thymectomized toadlets and
three non-thymectomized siblings, which
rejected first-set grafts in 18 to 21 days.
Following first-set allografting all toadlets
were left until the last thymectomized animal had rejected its transplant (65 days).
Second-set grafts (of approximately the
same size as the first-set transplants) from
the original donors were then applied when
all animals were 140 days old. Thus, the
interval between rejection of first-set allograft and application of second-set transplant ranged from 5 to 28 days in the
thymectomized group, but was considerably longer in the controls (116 to 119 days).
The results of this second-set experiment
were striking. Thus, despite the dissimilarity in first-set rejection times, second-set rejection end-points were comparable in
thymectomized and control toadlets, these
being 13 to 17 days in the former and 16 to
17 days in the latter. The experiments were
therefore repeated on three other toadlets
which had been thymectomized at 5 days of
age. These animals rejected first-set allografts (applied at 187 days) at 27, 36, and 45
days post-transplantation. They were then
regrafted with second-set transplants at 13,
4, and 30 days respectively after rejection of
the first-set grafts. The controls in this second experiment (three sham-thymectomized and one non-operated) rejected first-set grafts in 18 to 22 days, and
were also regrafted shortly (8 to 12 days)
after first-set rejection had occurred. Despite the earlier thymic ablation in this second experiment, second-set grafts were
again rejected rapidly by the thymectomized toadlets (9 to 17 days), these times
comparing favorably with rejection endpoints in the controls (9 to 14 days). The
results of the two experiments are grouped
together in Figure 3. This similarity in
second-set rejection capacity of control and
thymectomized animals has since been confirmed in thymectomized toadlets that took
over 100 days to reject first-set allografts.
The experiments indicate that following
chronic rejection of a first-set skin allograft,
ROLE OF AMPHIBIAN THYMUS IN ALLOIMMUNITY
2a
100 pm
2b
+i:::
. SG
*
•
•
sc
<
. * • '
FIG. 2. Autoradiographs of an allograft (a) and autograft (b) taken from intact toadlets 12 days posttransplantation. Both animals had been injected with
tritiated thymidine 4 hr prior to removal of grafts. The
allograft appears necrotic with loss of normal structure and has been infiltrated by a large mass of cells,
most of which are lymphocytes. Many of the invading
lymphocytes display heavy uptake of tritiated
thymidine. In contrast, the autograft retains its normal
morphology with very low level of label. (EP) epidermis; (M) melanin; (SC) stratum compactum; (SG) skin
glands. Stain: methyl green-pyronin.
80
JOHN D. HORTON AND TRUDY L. HORTON
skin transplants to non-grafted, thymectomized toadlets. The donors and hosts of
the transferred lymphoid tissues are mutually tolerant, following reciprocal embryonic transplantation of flank tissue
(Clark and Newth, 1972).
THYMECTOMPZED
60
FIRST-SET
SHAMTHYMECTOMIZED
REJECTION TIMES
NONTHYMECTOMIZED
40
RESTORATION OF THE THYMECTOMIZED AMPHIBIAN
20
»
17
20
17
17
»
17
SECOND-SET
REJECTION TIMES
FIG. 3. Comparison of first- and second-set skin allograft rejection times in individual thymectomized
and control toadlets.
a toadlet which has experienced little, if
any, thymic influence, can subsequently destroy allogeneic tissue from the same donor
in acute fashion. This confirms the immunological nature of transplant rejection
in the thymectomized Xenopus. Moreover,
the range of second-set allograft survival
times (9 to 17 days) is identical in thymectomized and control groups, which
suggests that following first-set rejection a
thymectomized animal is now as immunologically competent (with respect to a
particular donor) as the control toadlet.
The specificity of this dramatic change in
alloimmune reactivity following first-set
grafting has not yet been critically assessed.
The nature of the changes occurring in
thymectomized animals during first-set rejection which account for their competency
in attacking second-set allografts is currently being examined. Histological studies
reveal no readily observable differences in
lymphoid organ morphology between
thymectomized, non-grafted toadlets and
those thymectomized animals that have destroyed first-set allografts. Experiments are
now in progress to assess the ability of lymphoid tissues, lymphoid cells, and serum
factors to transfer allograft immunity from
thymectomized animals that have rejected
Experiments have been performed to
check the specificity of the immunological
defect caused by early thymectomy in
Xenopus. Following thymic ablation,
thymuses were implanted to determine if
first-set skin allograft rejection returned to
normal. The restorative capacity of spleen
implants has also been examined. All the
implants used in this work were from allogeneic (non-tolerant) sibling donors since
mutually tolerant pairs of larvae were not
available in the laboratory when this work
was performed.
These experiments involved 27 toadlets
thymectomized at 7 days of age and 8 control animals. Most of these toadlets were
siblings. Eleven of the thymectomized animals and all 8 controls received no lymphoid organ implants in larval life. In contrast, 8 thymectomized larvae were given a
thymus, 6 a spleen implant, and 2 animals
were implanted with small pieces of tail
muscle that acted as non-lymphoid allogeneic stimulation. All these implants
were performed 2 weeks post-thymectomy
when larvae were at stage 52/53 of development. Both alloimplanted and nonimplanted animals were then allografted at
70 days of age with non-sibling skin transplants. Two of the thymus-implanted hosts
were simultaneously grafted with skin from
their original thymic donors.
Implantation of the lymphoid tissues was
performed in the following manner: a
whole thymus or spleen was carefully removed from the donor and immediately
transferred to the thymectomized host,
where it was gently pushed into a small
cavity prepared under the dorsal skin medial and just posterior to one eye. This region was chosen for implantation because,
being anterior to the host's own thymic region the implant did not interfere with
ROLE OF AMPHIBIAN THYMUS IN ALLOIMMUNITY
checking for absence of host thymic tissue
during larval life and again at autopsy. The
transplanted organs become vascularised
and grow; the red-coloured spleens are
particularly striking in appearance in vivo.
Following metamorphosis the implants
come to lie close to the skin and adjacent to
the eye (see Fig. 4a,b).
81
The results of these restoration experiments are recorded in Table 2. Implantation of allogeneic thymus clearly reconstitutes, since all the thymectomized, thymusimplanted toadlets rejected third-party
skin allografts in under 25 days (see Fig.
4a), in contrast to the chronic rejection displayed by their non-implanted, thymec4b
FIG. 4. a and b, Two 14-week-old toadlets that were
thymectomized on the seventh day of life. The toadlet
in a subsequently received an allogeneic thymus implant in larval life which now appears as a distinct
nodule lying medial to the left eye. The toadlet in b, on
the other hand, had been implanted with allogeneic
spleen 2 weeks post-thymectomy; this implant is just
distinguishable as a dark region medial to the right
eye. Both animals had received skin allografts from
third-party donors at 10 weeks of age. The thymusimplanted, thymectomized animal rejected its skin allograft 18 days post-transplantation and only scar tissue remains. In contrast, the spleen-implanted animal
still retains a healthy skin allograft 4 weeks posttransplantation, c and d, Sections through the lymphoid organ transplants taken at autopsy from toadlets depicted in a and b. The thymus alloimplant c
appears extremely healthy, with normal cortical and
medullary differentiation. The spleen alloimplant d
also contains extensive lymphoid tissue and both white
and red pulp regions are evident. However, spleen
implants in many other toadlets had either disappeared or looked abnormal at autopsy. Skin (SK) covers these implants in vivo. (E) eye; (H) healthy graft;
(R) rejected graft; (S) spleen implant; (T) thymus implant. Stain: haematoxylin and eosin.
82
JOHN D. HORTON AND TRUDY L. HORTON
TABLE 2. The effect of implantation ofallogeneic thymus or spleen during larval life on thefirst-set skin allograft reactions of
thymectomized toadlets.
Complete rejection within
Thymectomized
Toadlets
Control
Toadlets
Lymphoid tissue
implanted at
21 days
Number of
toadlets
grafted
18 -25
days
26 - 50
days
> 50 days
None
Spleen
Thymus
11
6
8
0
0
8
4
4
0
7
2
0
None
8
8
0
0
tomized siblings. Spleen-implanted,
thymectomized animals, on the other hand,
still rejected skin allografts in chronic fashion (Fig. 4b) as did the two muscleimplanted toadlets. The two thymectomized, thymus-implanted animals which
also received skin allografts from their original thymus donors, displayed tolerance to
these grafts, while simultaneously rejecting
adjacent third-party allografts in acute
fashion. These tolerated skin grafts were
observed for 200 days when they were still
in perfect condition. It is important to stress
here that host thymic remnants were never
found at histological autopsy in any of the
thymectomized animals.
The fact that defective first-set alloimmune reactivity following thymectomy can
be specifically abrogated by allogeneic
thymus implantation pin-points the absence of thymus as the causative factor. To
our knowledge this is the first demonstration that a thymus transplant can restore to
normal the alloimmune response of the
thymectomized amphibian. However, similar findings have been well documented in
mammals. For example, the alloimmune
response of the neonatally thymectomized
mouse can be restored by allogeneic
thymus implantation (Miller, 1965).
More recent experiments indicate that
the immunological outcome of transplanting allogeneic thymus tissue into the
thymectomized mammal depends to a certain extent upon the genetic relatedness of
donor and host. Thus, thymuses from
donors with only minor, non-H2 histocompatibility differences are invariably successful in restoring, in contrast to the poor success achieved when allogeneic neonatal
thymic grafts are made across major H2
histocompatibility barriers (Biggar et al.,
1972). In the latter situation, neonatally
thymectomized mice may, following reconstitution, reject the thymus transplant
(Stutman et al., 1972). Such animals not
only reject skin allografts from third-party
donors but also skin from the same origin as
the thymus grafts (Stutman et al., 1969). In
contrast, these latter authors have shown
that with weak histocompatibility differences between thymus donor and thymectomized host, thymus alloimplants are not
rejected but remain fully viable with typical
cortical and medullary differentiation.
This latter situation is, therefore, comparable with the present findings on Xenopus,
where a very healthy thymic graft was always seen (see Fig. 4c). Another similarity
of the present experiments to those performed by Stutman et al. (1969) on H2
compatible mice is that animals remain tolerant to skin from the thymic donor while
effecting normal allograft rejection of
third-party transplants. It is known from
cytological studies on thymectomized mice
that virtually all the lymphocytes eventually
found in thymus allografts are of host origin (Dukor et al., 1965), the donor tissue
possibly acting as a reticular framework
within which the host cells differentiate.
The origin of lymphocytes seen in allogeneic thymus transplants in Xenopus is
not known, but a small population of donor
cells might account for tolerance induction
to subsequent skin allografts from the same
animal.
In contrast to allogeneic thymus implants, spleen alloimplants did not restore
skin allograft immunity to normal. How-
ROLE OF AMPHIBIAN THYMUS IN ALLOIMMUNITY
ever, the disability of spleen in this respect
might be related to our observations that
these alloimplants, unlike the thymus
grafts, did not generally remain healthy.
Thus, in the six spleen-implanted, thymectomized toadlets, four of the spleens enlarged considerably during chronic skin allograft rejection and subsequently two
completely disappeared. Of the four spleen
implants still present following skin allograft destruction, only two possessed relatively normal histology; one of these is seen
in Figure 4d. The other two spleens, although containing many lymphocytes,
were disorganized, possessing large necrotic regions and greatly enlarged sinuses.
More recent observations on several
spleen-alloimplanted, thymectomized
Xenopus indicate that disappearance of
these implants also occurs in non-skin allografted animals. Graft-versus-host reactivity might account for the loss of spleens
since Clark and Newth (1972) have noted
that transplanted Xenopus spleens involved
in GVH reactions may themselves undergo
destruction. Because of the loss of spleen
implants the need arises to perform experiments in which the lymphoid organ donor
is mutually tolerant with the thymectomized host. Only then can the restorative
ability of spleen and other lymphoid tissues
be critically assessed. The capacity of bone
marrow to restore allograft immunity was
shown by Cooper and Schaefer's study
(1970) on irradiated adult Rana pipiens.
Implantation of allogeneic thymus has
recently been used in our laboratory to reconstitute the response of thymectomized
Xenopus to circulating, xenogeneic antigens. Thus, following early thymectomy,
the number of rosette-forming cells in the
spleen does not increase above background
level following sheep erythrocyte immunization (unpublished). However, thymusreconstituted, thymectomized Xenopus respond to sheep erythrocytes with levels of
rosette-forming cells directly comparable
to those reported for intact Xenopus by Kidder et al. (1973). Our success with thymus
reconstitution should allow experiments to
be devised that can examine whether the
amphibian thymus, like its mammalian
counterpart (Davies, 1969; Goldstein and
83
White, 1973; Trainin and Small, 1973),
exerts a humoral influence in its control of
immunological maturation.
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