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/ . Embryol. exp. Morph. Vol. 29, 1, pp. 73-85, 1973
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73
Cytodynamics and ontogeny of the immune reponse
of Xenopus laevis against sheep erythrocytes
By GERALD M. KIDDER, 1 LAURENS N. RUBEN,1-2
AND JEAN M. STEVENS 1
From the Department of Biology, Reed College
SUMMARY
The heterologous red cell response of Xenopus laevis larvae and post-metamorphic toadlets
was investigated by means of the immuno-cytoadherence (ICA) technique. Sheep erythrocytes
(SRBC) were employed as immunogen.
Toadlets responded to a single injection of immunogen within 4 days, and exhibited a peak
level of rosette-forming cells (RFC) in their spleens at 8 days post-injection. Toadlets
immunized against sheep erythrocytes gave only a very slight response when tested against
rat erythrocytes. A secondary response, much greater in magnitude than the primary response, wasevidentwithin 2days when previously immunized toadlets were reinjected with the
same immunogen. It was concluded that the ICA technique provides a quantitative measure
of an acute immune response in these animals.
Larvae which had passed through stage 50 of Nieuwkoop & Faber exhibited substantial
increases in RFC in the spleens when tested 6-10 days after injection with sheep erythrocytes.
Significantly increased frequencies of RFC in thymi were also noted in these larvae, but the
numbers involved were very low and varied considerably. Histological observations of these
larvae revealed lymphoid maturation of the spleens and thymi to be essentially complete.
Larvae which had not reached stage 50 according to external morphological criteria, but
whose lymphoid organs had matured to a degree equivalent to stage 50, also exhibited strong
anti-SRBC response in the spleens. Response in the thymi was low and not statistically significant. Larvae injected at a stage when lymphocytic differentiation was complete in the thymi
but had not begun in the spleens did not exhibit an elevated splenic RFC frequency when
tested after the spleens had matured. These data suggest that the heterologous red cell response
in the larval spleen is dependent upon antigenic challenge to spleens which have reached the
stage 50 equivalent in their histogenesis.
INTRODUCTION
The immunological responses of Amphibia have been the subject of experimentation for more than a decade, and it has become clear that the members of
this vertebrate class exhibit a spectrum of immunological capability. Members
of the order of Apoda (Cooper & Garcia-Herrera, 1968) and Urodela (Cohen,
1969), for example, effect sub-acute or chronic allograft rejection responses,
whereas some species of Anura, namely the Ranidae, show the same type of
acute allograft rejection observed in mammals (Hildemann & Haas, 1959;
Bovbjerg, 1966). Even within the Anura, however, there is variation with respect
1
2
Authors'' address: Department of Biology, Reed College, Portland, Oregon 97202, U.S.A.
Author to whom reprint requests should be sent.
74
G. M. KIDDER, L. N. RUBEN AND J. M. STEVENS
to graft rejection: larval and juvenile Xenopus laevis, the South African clawed
toad (Horton, 1969; Bernardini, Chardonnens & Simon, 1969), and Alytes
obstetrleans, the midwife toad (Delson & Flatin, 1967, cited by Cohen &
Borysenko, 1970), both of which are considered to be relatively primitive, appear
to reject allografts sub-acutely. These facts led Horton (1970) to suggest that the
graft rejection system of Xenopus illustrates an evolutionary intermediate
between that of the more primitive Apoda and Urodela and the more advanced
members of the Anura.
Our interest in this subject arose out of similar experiments in this laboratory
(Ruben, 1970), which involved implantation of lymphoreticular tumor foci along
with normal tissues into the tails of larval Xenopus. These experiments had
indicated that lymphocytic destruction of the normal tissue allografts proceeded
as a progessive, chronic phenomenon. Among the possible interpretations of
this finding, it was suggested that a chronic rejection response in the larvae could
represent a stage in the maturation of the more acute response of adult Xenopus
(Simnett, 1965). As Cohen & Borysenko (1970) have pointed out, however, an
animal may fail to respond acutely to an antigenic challenge such as an allograft
either because its immune response is of limited capacity (due to its phylogenetic
or developmental status) or because the antigens involved represent only weak
histocompatibility differentials. In light of these uncertainties, we deemed it of
interest to investigate the maturation of immune competence in Xenopus by
means of a response system which gave promise of behaving in acute fashion
even in the larval stages.
Recent reports had indicated that both Xenopus (Auerbach & Ruben, 1970) and
Alytes (Du Pasquier, 1970) are capable of mounting readily demonstrable
cellular anti-SRBC responses. The immuno-cytoadherence (ICA) assay employed
by Du Pasquier allowed the detection of an immune response to sheep erythrocytes in Alytes tadpoles as early as 2 days after injection. After several unsuccessful attempts to apply the in vitro haemagglutinin technique of Auerbach
& Ruben to Xenopus tadpoles and post-metamorphic juveniles, we turned to the
ICA technique, which proved to be well suited for our purposes.
Previous studies of the ontogeny of immune competence in anuran amphibia
have utilized primarily the allograft rejection response. In both Xenopus (Horton,
1969) and Ranapipiens (Horton, 1971) the capacity of a larval host to generate
a lymphocytic invasion of an allograft is correlated with the lymphoid maturation
of the thymus. Our results with the immuno-cytoadherence assay suggest that
this type of response matures slightly later in development than does the allograft response, and is mediated by cells of other lymphoid organs, such as the
spleen.
The immune response 0/Xenopus
75
MATERIALS AND METHODS
Recently metamorphosed Xenopus laevis toadlets were employed for an
investigation of the cytodynamics of the anti-SRBC response. Animals were
anaesthetized in MS 222 (Sandoz; diluted 1:500 in distilled water) and then
injected intraperitoneally with 0-25 ml 20% sheep erythrocytes (SRBC) in
Alsevefs solution. Larval Xenopus, raised from laboratory breedings, were
staged according to the external morphological criteria set out in the Normal
Table of Nieuwkoop & Faber (1956). Larvae were anaesthetized in MS 222
(1:2000) and injected intraperitoneally (i.p.) with 0-003-0-005 ml 75% SRBC
in Alsevefs solution. All injected animals were held at 23 °C until use. In the
case of the larvae, individuals representative of each injected group were collected
on the day of injection and fixed in Bouin's fixative, after which they were
embedded in paraffin, sectioned at 6 jum and then stained with haematoxylin
and eosin. These larvae were examined microscopically to ascertain the degree
of maturation of the thymus and spleen.
Immuno-cytoadherence assays were peformed in a manner similar to that
applied to Alytes by Du Pasquier (1970). Spleens or thymi were removed from
anaesthetized animals which had been bled by aortic puncture. The intact organs
were transferred to 5:4:1 medium (5 parts Leibovitz L-15 medium : 4 parts glass
distilled water: 1 part heat-inactivated [30 min at 56 °C] fetal bovine serum) and
teased apart. In the case of larvae, spleens or thymi of 4-10 individuals, depending on size, were pooled to make a single cell suspension. The resulting cell
suspensions were transferred to 1 ml test-tubes and held for a few minutes to
allow debris to settle out. The supernatant suspensions were then transferred to
calibrated 1 ml tubes and the volumes were adjusted to accurate levels (usually
0-1-0-2 ml).
Viable cell counts were carried out by combining a small aliquot of each cell
suspension with 0-2 volume of 0-4 % trypan blue. After 5 min the dyed suspension was transferred to a haemacytometer for a count of total dye-excluding
cells, excluding erythrocytes. The undyed remainder of each cell suspension was
then combined with a volume of sheep erythrocytes, suspended in L-15 medium,
sufficient to give a 30-fold excess of SRBC over tissue cells. (SRBC were stored
in Alsever's, then washed and suspended to 2 % in L-15 medium prior to use.)
The volumes were adjusted to yield tissue cell concentrations in the range
2-0-3-5 x 106 cells/ml. The tissue cell-SRBC suspensions were mixed thoroughly
and then held at 3 °C for 10-20 h, although preliminary experiments revealed
little or no increase in the number of ICA positive cells ('rosettes') after 5 h.
After the incubation period the tubes were rotated gently by hand to resuspend
the cells. Samples of 0-05 ml were removed and pipetted into counting chambers,
which were then sealed with vacuum grease. Normally, a single 0-05 ml sample
was scanned in its entirety at x 100 magnification for each assay. In some cases,
such as with early larval stages, in which the tissue cell concentration was less
76
G. M. KIDDER, L. N. RUBEN AND J. M. STEVENS
3000
2500 -
2000 -
a- 1500 -
1000 -
10
Days after injection
15
20
Fig. 1. Cytodynamics of the anti-SRBC response in post-metamorphic Xenopus toadlets, as measured by immuno-cytoadherence. Toadlets of a single breeding were injected with 0-025 ml 20% SRBC, and four were killed for each set of spleen cell
assays. Points represent mean RFC frequency for four spleens ± standard deviation,
indicating variation among individual spleens of each set.
than 2 x 106 cells/ml, the entire assay suspension was scanned. An ICA-positive
cell (rosette-forming cell, or RFC) was denned as a 'rosette', consisting of
a spleen or thymus cell bearing four or more adherent sheep erythrocytes.
RESULTS
Cytodynamics of the anti-SRBC response in post-metamorphic toadlets
The timing and magnitude of the cellular anti-SRBC response in immunologically mature Xenopus were investigated as a background to the study of the
ontogeny of this response. Thirty-two toadlets were given single i.p. injections
of SRBC, and four at a time were killed at intervals up to 20 days after injection.
ICA assays were performed on individual spleens. Assays were also performed
on eight spleens from non-immunized animals of the same breeding. Standard
The immune response of Xenopus
77
Table 1. Summary of immuno-cytoadherence assays with Xenopus larvae
RFC/106 cells*
Series
I
II
m
Group
A
Stage at
injection
Late-50
B
51
C
53
D
49/50
E
48/49
F
48
G
48
Days after
injection
(Background)
6-lOf
(Background)
6-10t
(Background)
6-8 %
(Background)
6-10§
(Background)
6-8
(Background)
13-22J
(Background)
10-11
No.
larvae
34
12
34
11
4
10
20
28
20
16
(
A
Spleens
Thymi
168 ±21
308 ± 20
168 + 21
725 ±77
10±l
30±4
10±l
46 ±13
32
7
130 ±170
94 ±5
1240 ±400
94±5
.1840 ±370
58 ±10
8±1
45 ±22
8±1
13±1
15
257
11
17
281 ±48
214 ±24
171 ±21
9±1
22
16
30±4
10 ±1
* Except for background determinations in groups C and F, numbers indicate means of
multiple assays of each group of larvae carried out during the designated time intervals.
Variation is expressed in terms of standard error of the mean.
t Since larvae of groups A and B were siblings of approximately the same chronological
age, a single set of background determinations was taken for both groups.
X A single background determination was carried out for this group of larvae.
§ A single set of background determinations was taken for groups D and E as explained in
footnote f.
deviations were computed as a measure of the variation among the individual
spleens of each set.
The results, expressed as mean RFC per 106 viable spleen cells, are given in
Fig. 1. It was found that non-immunized animals of that breeding harbored
a background level of 28 ± 8 RFC per 106 viable spleen cells. No change in this
level was noted in the injected animals until 4 days, when a twofold increase
above background was evident. The response increased thereafter until it
reached a peak at 8 days, after which it declined to a near-background level by
16 days. At 20 days there had been no further decrease. In a repeat of this
experiment it was determined that a response was still not evident 3 days after
injection; with this second group of toadlets the background RFC level was
re-established 22 days after injection.
In order to test the specificity of this response, a set of four spleens collected
10 days after sheep SRBC injection was assayed with both sheep and rat erythrocytes. Whereas the spleens contributed a mean of 1300 ± 700 RFC per 106 cells
against sheep erythrocytes, the mean response against rat erythrocytes was
17-fold lower, 77 + 50. Although this latter value may indicate a small degree
of cross-reactivity (the background level for rat RBC was found to be 33 ± 19),
the degree of specificity of the response is nonetheless great.
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G. M. KIDDER, L. N. RUBEN AND J. M. STEVENS
The immune response 0/Xenopus
79
Preliminary data on the secondary response were obtained by re-injecting
toadlets which had been immunized 20-30 days earlier. A secondary response in
the spleen was evident within 2 days after re-injection, with a level as high as
220 ± 110 in one experiment, as compared with a background of 32 ±20. One
set of toadlets exhibited a secondary response at 11 days which involved, on the
average, more than 2 % of the cells in the spleens.
It was concluded on the basis of these experiments that the Xenopus anti-SRBC
response measured by the ICA assay afforded a rapid and pronounced indication
of immunological competence with which to survey the larval stages.
Cellular anti-SRBC response in Xenopus larvae
Manning & Horton (1969) have described the histogenesis of the lymphoid
organs in Xenopus, and they reported that the larval thymus acquires its mature
lymphoid histology beginning in stage 49, when many small lymphocytes can be
seen populating the thymic cortex. In the spleen, on the other hand, lymphocytic
differentiation was observed to be completed slightly later, in stage 50. We
designed our experiments to test the immunological responsiveness of thymus
and spleen cells during this crucial period of lymphoid maturation.
The results of these experiments are summarized in Table 1. In general,
a pronounced increase over the background level of RFC in the spleens was
observed upon immunization of larvae which were judged to be stage 50 or later
according to external morphological criteria (series 1). The elevated frequency
of spleen RFC was evident in all samplings of each immunized population of
larvae of this series, from 6 to 10 days after injection. The differences between
experimental and control means in group A and B were tested for significance,
FIGURE 2
Fig. 2. (A) Spleen of a late stage 50 Xenopus larva of group A (Table 1), killed on day
that group A larvae were injected. Arrows indicate boundary-layer cells partially
delimiting a white pulp follicle.
(B) Thymus of same larva; cortex densely packed with lymphocytes. s.L, Small
lymphocytes.
(C) Thymus of a group E larva (stage 48/49) killed on day of injection, showing
distinct cortico-medullary differentiation and lymphocyte accumulation in cortex
equivalent to stage 50.
(D) Spleen of same larva; note distinct white-pulp follicle outlined by boundary
cells (arrows). s.L, Small lymphocytes.
(E) Thymus of a group G larva (stage 48) killed on day of injection, showing
distinct cortico-medullary differentiation with numerous small lymphocytes in the
cortex.
(F) Spleen of same larva: small lymphocytes have yet to appear at this stage.
(G) Spleen of a group G larva killed 10 days after injection; compare with photograph D. The spleens at this point had reached the stage 50 equivalent in their
histogenesis. Note the well-formed white-pulp boundary.
80
G. M. KIDDER, L. N. RUBEN AND J. M. STEVENS
and found to be highly significant (P < 0-01, Student's t test). Histological
examination of siblings from these groups, killed on the day of injection and
then sectioned and stained, verified the assumption that the spleens of these
stage 50-51 larvae were well on the way to completing lymphoid maturation:
white pulp areas were evident which in many cases were delineated by wellformed boundary layers and which were populated by numerous lymphoid
cells, including many small lymphocytes (Fig. 2 A). The magnitude of the response
varied considerably from one group of tadpoles to another, even within the same
breeding; groups A and B, for example, did not differ in any obvious way with
regard to spleen size or degree of maturation, despite the fact that group B
larvae were slightly more advanced morphologically and gave a much greater
response. The thymi of all larvae examined from series I exhibited mature
lymphoid histology (Fig. 2B) as described by Manning & Horton (1969).
With respect to RFC frequencies among thymic cells of the series I larvae, the
data leave some uncertainty. Significant increases in frequencies of RFC were
noted in response to injection (P < 001 for group A, P < 005 for group B),
but the actual numbers of rosettes involved were very low. In most cases, the
frequency of RFC in the thymi was 10 % or less of that in the spleens. Whereas
the background frequency of RFC in the thymi was very low and did not change
during development, the levels in the spleens of stage 48-51 larvae were
considerably greater than the level in stage 53 larvae and post-metamorphic
toadlets. Such a decline in the non-immunized level of RFC in spleens as
development progresses was observed also by Du Pasquier (1970) in Alytes.
The rate of development of Xenopus larvae is greatly influenced by factors
such as temperature, crowding, nutrient supply, etc., and under sub-optimal
conditions individuals of a population may vary considerably with respect to
the progress of morphogenesis. Thus, from among larvae in a population from
a single breeding, it is often possible to pick out subpopulations which represent
different morphological stages. The experiments reported in series II of Table 1
were carried out with such a heterogeneous population: these stage 49-50 and
48-49 larvae were retarded in their external morphological development since
they were the same age as stage 50 larvae of the same population. These larvae
gave strong but variable anti-SRBC responses in the spleens within 6 days of
injection (P = 0-06 for group D, P < 0-05 for group E). RFC frequencies in the
thymi, however, were not significantly different from background (P = 0-2 for
group D, P = 0 1 for group E). Histological examination of sibling larvae of
the same stages, killed on the day of injection, revealed both groups D and E to
have reached a level of thymic and splenic lymphoid maturation equivalent to
stage 50 (Fig. 2C and D show thymus and spleen from one larva of group E).
It therefore appears that lymphoid differentiation of the thymus and spleen can
proceed at the maximal rate regardless of the fact that morphological maturation,
as judged by the appearance and growth of limb-buds, distribution of pigment
cells, and elongation of the intestine, has been considerably retarded.
The immune response o/Xenopus
Stage
Age (days)
Histological
condition
48
10
1
T(-)
s(-)
49
15
T(+)
S(-)
Inject
group A
50 1 51
20
\
Response
group A
52
S( + )
f
1 25
i
T(+)
S(+)
81
|
T( + )
S( + )
B
Stage
Age (days)
47
10
Inject
group E
48
I 49
15 \
1
i
Histological
condition
T(-)
s(-)
S(-)
1
Inject
group G
S(+)
Response
group E
50 f
20
1 1
1
T( + )
51
25
1
-»
S(+)
No response
group G
Fig. 3. Diagrammatic summary of experiments with larval spleens, illustrating injection and assay schedules and the relation between larval age, morphological stage,
and histological condition of the thymi and spleen. Part A refers to experiments with
larvae developing at a near-optimal rate; part B refers to experiments with retarded
or slowly developing larvae. ' T ' a n d ' S ' refer to thymi and spleen, respectively; ( + )
indicates the presence of small lymphocytes, ( —) indicates their absence. One experiment from each series is represented.
The data of series 111 represent attempts to immunize larvae whose thymi
were in the final stage of lymphocytic differentiation but whose spleens had not
yet begun this process. Group F and G larvae were morphologically stage 48
when injected but contained thymi which displayed distinct cortico-medullary
differentiation involving the presence of numerous small lymphocytes in the
cortex (Fig. 2E). Larvae whose thymi had developed to this stage were shown by
Horton (1969) to be capable of mounting a weak lymphocytic response against
skin allografts. The spleens of these larvae at the time of injection were very
small and contained very few, if any, small lymphocytes, though cells in the
medium-to-large lymphocyte category were present (Fig. 2F). By the time the
first assays were performed on these larvae 13 and 10 days after injection (for
groups F and G respectively) their spleens had grown considerably and had
undergone lymphocytic differentiation to the extent that they appeared equivalent to those of stage 50 larvae (Fig. 2G), although no enhancement of the
frequency of RFC above the background level was apparent (P = 0-27 for
group G). Group F larvae were assayed as late as 22 days after injection, and
still displayed no response. Neither of these two groups gave evidence of an
increased RFC frequency in the thymi, and in fact group G larvae exhibited
6
E M B 29
82
G. M. KIDDER, L. N. RUBEN AND J. M. STEVENS
a statistically significant decrease (P < 0-01). Further experiments will be
required to ascertain whether a response eventually develops after a longer
period of time in such larvae. These results invite the tentative conclusion that
an immune response of this kind in the spleen is dependent upon an antigenic
challenge to the histologically mature spleen itself, rather than to the thymus as
a primary lymphoid organ. The data relevant to this conclusion are summarized
diagrammatically in Fig. 3.
DISCUSSION
The immuno-cytoadherence technique has been employed in studying the
immune responses of a variety of vertebrate organisms, including at least two
other species of anurans. Diener & Marchalonis (1970) reported that RFC in the
spleens of adult Bufo marinus increased in frequency within 3 days at 37 °C after
intraperitoneal injection with Salmonella adelaide flagella, and reached a peak
level within 7-14 days. At 22 °C a peak response was attained at 14 days. Du
Pasquier (1970), working with Alytes obstetricans tadpoles injected intraperitoneally with sheep erythrocytes, detected an increase in RFC in the spleens
within 4 days and noted a peak response at 9-11 days; intracardiac injection
resulted in an increase by 2 days and a peak at 4-7 days. We have here reported
that post-metamorphic Xenopus laevis exhibit an augmented frequency of RFC
in the spleen 4 days after intraperitoneal injection of SRBC and reach a peak
level of response at 8 days. These results reflect a considerable degree of conformity among the three anuran species, considering the dependence of response
kinetics on a variety of uncontrolled factors such as quantity and source of the
antigen as well as the route of injection (Du Pasquier, 1970). Furthermore, these
results are in good agreement with the kinetics of the response to SRBC in the
rat measured by the ICA technique: Duffus & Allan (1971) reported on increased
RFC count in rat lymph nodes within 3 days and a peak count at 5-6 days postinjection. Thus there appears to be no fundamental dissimilarity between the
kinetics of these responses of primitive and advanced anurans nor between the
anuran amphibia and the mammals. This is in contrast to the observation, noted
in the Introduction, that primitive anurans reject allografts in sub-acute fashion
as compared with the Ranidae or the mammals, which typically exhibit acute
rejection. Perhaps the best explanation for this discrepancy lies in the suggestion
(Cohen & Borysenko, 1970; Cohen, 1971) that the failure of an organism to
reject an allograft in acute fashion reflects a lack of strong antigenic differences
between individuals rather than a phylogenetically primitive immune system.
Further research will be required to verify this hypothesis.
Our data concerning the anti-SRBC response in larval spleens conform well
to expectations based on recent studies of the ontogeny of lymphoid organs in
Xenopus carried out in other laboratories (Manning & Horton, 1969; Horton,
1969) as well as our own (Ruben, Stevens & Kidder, 1972). Under optimal
conditions, the larval thymi acquire their mature lymphoid appearance during
The immune response 0/Xenopus
83
stage 49, at which time the larva becomes capable of mounting a weak lymphocytic response to a skin allograft. Allografts made on stage 48 larvae are rejected
after a delay corresponding to the time necessary for the thymi to complete their
maturation (Ruben et al. 1972). The spleen, on the other hand, completes
its lymphocytic differentiation in stage 50. Our results demonstrate that stage 50
spleens or their histological equivalent in morphologically retarded larvae can
respond to an injection of sheep erythrocytes by an increase in the frequency of
rosette-forming cells. Such an increase may also occur in the thymus, but the
number of cells involved is extremely small. These observations underscore the
unreliability of external criteria of staging when dealing with the histogenesis of
lymphoid and perhaps other organs.
Du Pasquier (1970) reported that, in Alytes, the capacity to form RFC in
larval spleens in response to erythrocyte injection was acquired when the spleens
had grown to contain 6-12 x 103 cells. In our experiments the smallest spleens
giving a demonstrable response were those of group D (series II), which yielded
an average of 11 x 103 cells per spleen at the time of injection. The spleens of
group F and G larvae, which failed to respond to SRBC injection, were too
small at the time of injection to determine cell numbers. It appears that the
ability to mount an immune response in spleens made up of relatively few cells
may well be a general feature of amphibian immune systems (refer to Du
Pasquier, 1970, for a discussion of this phenomenon).
Larvae injected with SRBC at a stage when their thymi were completing
lymphocytic differentiation and their spleens were still immature in this regard
failed to exhibit a response when lymphoid maturation of the spleen had been
completed. Although an independent test of thymic immunological maturity at
the time of injection was not carried out in this instance, there is ample reason to
believe, based on other experiments with larvae of this stage (Horton, 1969;
Ruben et al. 1972), that the thymi of these larvae were capable of generating at
least a weak immunological response. Failure of the response to develop in the
spleen, therefore, could mean either that the maturation of the splenic white pulp
is independent of a cellular contribution from the thymi or that, if such a contribution is essential, the cells involved are unable to respond to the antigen
until they have taken up residence in the spleen. A third possibility must also
be considered, namely that in the present experiments the injection of a massive
dose of antigen into the developing immune system induced a state of tolerance
or otherwise retarded the normal generation of a response. The first possibility
receives support from a recent report by Manning (1971), in which it was
demonstrated that removal of the thymi from Xenopus larvae at stage 49 failed
to prevent histogenesis of the splenic white pulp. In view of the fact that neonatal thymectomy in rodents results in a depletion of lymphoid organs (Miller,
1964), the situation in Xenopus may reflect a fundamental phylogenetic difference
between the immune systems of amphibians and mammals, and as such is worthy
of further investigation. It would be of interest, for example, to determine whether
6-2
84
G. M. KIDDER, L. N. RUBEN AND J. M. STEVENS
thymectomy in Xenopus at stage 49 has any effect on the anti-SRBC response of
the spleen at stage 50.
The immuno-cy toad here nee technique as employed in this study measures
only the frequency of cells bearing specific immunoglobulin on their surfaces,
and does not distinguish between cells actively producing the antibody and
those which are merely antigen binding. It has been demonstrated by Greaves
(1970) and by Takahashi, Old, Mclntire & Boyse (1971) that in mouse spleens
immunized against sheep erythrocytes, over 90% of RFC are susceptible to
anti-lg inhibition and therefore bear surface immunoglobulin. While the present
studies do provide an indication of the immunocompetence of larval spleens,
they do not provide quantitative information regarding antibody synthesis.
This work was supported in part by a grant (CA-08268) from the National Cancer Institute,
National Institutes of Health, U.S.A. G. M. Kidder was supported as a Postdoctoral
Research Fellow in the Biological Sciences on a grant to Reed College from the Albert Sloan
Foundation. The assistance of Ms Sheryl Swink is gratefully acknowledged.
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