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 re-sponse, wasevident within 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.

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 to graft rejection: larval and juvenile Xenopus laevis, the South African clawed toad (Horton, 1969; Bernardini, Chardonnens & Simon, 1969), and Alytes obstetricans, 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 erythro-cytes in Alytes tadpoles as early as 2 days after injection. After several un-successful 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 Rana pipiens (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 allo-graft response, and is mediated by cells of other lymphoid organs, such as the spleen.

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 Alsever’s 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 Alsever’s 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 μ m 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, depend-ing 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 suspen-sion 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 × 100 magnification for each assay. In some cases, such as with early larval stages, in which the tissue cell concentration was less than 2 x 106 cells/ml, the entire assay suspension was scanned. An ICA-positive cell (rosette-forming cell, or RFC) was defined as a ‘rosette’, consisting of a spleen or thymus cell bearing four or more adherent sheep erythrocytes.

Cytodynamics of the anti-SRBC response in post-metamorphic toadlets

The timing and magnitude of the cellular anti-SRBC response in immuno-logically 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 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.

Fig. 1

Cytodynamics of the anti-SRBC response in post-metamorphic Xenopus toad-lets, as measured by immuno-cytoadherence. Toadlets of a single breeding were in-jected with 0’0·25 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.

Fig. 1

Cytodynamics of the anti-SRBC response in post-metamorphic Xenopus toad-lets, as measured by immuno-cytoadherence. Toadlets of a single breeding were in-jected with 0’0·25 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.

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 erythro-cytes. 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.

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, 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 well-formed boundary layers and which were populated by numerous lymphoid cells, including many small lymphocytes (Fig. 2A). 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).

Table 1

Summary of immuno-cytoadherence assays with Xenopus larvae

Summary of immuno-cytoadherence assays with Xenopus larvae
Summary of immuno-cytoadherence assays with Xenopus larvae
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 photo-graph D. The spleens at this point had reached the stage 50 equivalent in their histogenesis. Note the well-formed white-pulp boundary.

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 photo-graph D. The spleens at this point had reached the stage 50 equivalent in their histogenesis. Note the well-formed white-pulp boundary.

With respect to RFC frequencies among thymic cells of the series 1 larvae, the data leave some uncertainty. Significant increases in frequencies of RFC were noted in response to injection (P < 0·01 for group A, P < 0·05 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 Aly tes.

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 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 equi-valent 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 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.

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’ and ‘S’ refer to thymi and spleen, respectively; (+) indicates the presence of small lymphocytes, (−) indicates their absence. One experiment from each series is represented.

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’ and ‘S’ refer to thymi and spleen, respectively; (+) indicates the presence of small lymphocytes, (−) indicates their absence. One experiment from each series is represented.

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 intra-peritoneally 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 con-formity 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 post-injection. 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 stage 49, at which time the larva becomes capable of mounting a weak lympho-cytic 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 × 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 × 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 con-tribution 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 neo-natal 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 thymectomy in Xenopus at stage 49 has any effect on the anti-SRBC response of the spleen at stage 50.

The immunocy 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, McIntire & Boyse (1971) that in mouse spleens immunized against sheep erythrocytes, over 90% of RFC are susceptible to anti-Ig 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.

Auerbach
,
R.
&
Ruben
,
L. N.
(
1970
).
Studies of antibody formation in Xenopus laevis
.
J. Immun
.
104
,
1242
1246
.
Bernardini
,
N.
,
Chardonnens
,
X.
&
Simon
,
D.
(
1969
).
Développement après métamorphose de compétences immunologiques envers les homogreffes cutanées chez Xenopus laevis Daudin
.
C.r. hebd. Séanc. Acad. Sci., Paris
269
,
1011
1014
.
Bovbjerg
,
A. M.
(
1966
).
Rejection of skin homografts in larvae of Ranapipiens
.
J. exp. Zool
.
162
,
69
80
.
Cohen
,
N.
(
1969
).
Immunogenetic and developmental aspects of tissue transplantation immunity in urodele amphibians
.
In Recent Results in Cancer Research, Biology of Amphibian Tumors
(ed.
M.
Mizell
), pp.
153
168
.
New York
:
Springer-Verlag
.
Cohen
,
N.
(
1971
).
Reptiles as models for the study of immunity and its phylogenesis
.
J. Am. vet. med. Ass
.
159
,
1662
1671
.
Cohen
,
N.
&
Borysenko
,
M.
(
1970
).
Acute and chronic graft rejections. Possible phylogeny of transplantation antigens
.
Transplantation Proc
.
2
,
333
336
.
Cooper
,
E. L.
&
Garcia-Herrera
,
F.
(
1968
).
Chronic skin allograft rejection in the apodan, Typhlonectes compressicauda
.
Copeia
2
,
224
229
.
Delson
,
M.
&
Flatin
,
J.
(
1967
).
Premières observations d’ensemble sur des homogreffes réalisées chez le têtard ú’Alytes obstetricans Laur
.
C.r. Ass. Anat
.
138
,
398
.
Diener
,
E.
&
Marchalonis
,
J.
(
1970
).
Cellular and humoral aspects of the primary immune response of the toad, Bufo mar inus
.
Immunology
18
,
279
293
.
Duffus
,
W. P. H.
&
Allan
,
D.
(
1971
).
The kinetics and morphology of the rosette-forming cell response in the popliteal lymph nodes of rats
.
Immunology
20
,
345
361
.
Du Pasquier
,
L.
(
1970
).
Ontogeny of the immune response in animals having less than one million lymphocytes: The larvae of the toad Aly tes obstetricans
.
Immunology
19
,
353
362
.
Greaves
,
M. F.
(
1970
).
Biological effects of anti-immunoglobulins: Evidence for immunoglobulin receptors on ‘T’ and ‘B’ lymphocytes
.
Transplantation Rev
.
5
,
45
75
.
Hildemann
,
W. H.
&
Haas
,
R.
(
1959
).
Homotransplantation immunity and tolerance in the bullfrog
.
J. Immun
.
83
,
478
485
.
Horton
,
J. D.
(
1969
).
Ontogeny of the immune response to skin allografts in relation to lymphoid organ development in the amphibian Xenopus laevis Daudin
.
J. exp. Zool
.
170
,
449
466
.
Horton
,
J. D.
(
1970
).
Phylogenetic status of immune system in Xenopus
.
Transplantation Proc
.
2
,
282
284
.
Horton
,
J. D.
(
1971
).
Ontogeny of the immune system in amphibians
.
Am. Zool
.
11
,
219
228
.
Manning
,
M. J.
(
1971
).
The effect of early thymectomy on histogenesis of the lymphoid organs in Xenopus laevis
.
J. Embryol. exp. Morph
.
26
,
219
229
.
Manning
,
M. J.
&
Horton
,
J. D.
(
1969
).
Histogenesis of lymphoid organs in larvae of the South African clawed toad, Xenopus laevis (Daudin)
.
J. Embryol. exp. Morph
.
22
,
265
277
.
Miller
,
J. F. A. P.
(
1964
).
The thymus and the development of immunologic responsiveness
.
Science, N.Y
.
144
,
1544
1551
.
Nieuwkoop
,
P. D.
&
Faber
,
J.
(
1956
).
Normal Table of Xenopus laevis (Daudin)
.
Amsterdam
:
North-Holland Publishing Co
.
Ruben
,
L. N.
(
1970
).
Immunological maturation and lymphoreticular cancer transformation in larval Xenopus laevis, the South African clawed toad
.
Devi Biol
.
22
,
43
58
.
Ruben
,
L. N.
,
Stevens
,
J. M.
&
Kidder
,
G. M.
(
1972
).
Suppression of the allograft response by implants of mature lymphoid tissues in larval Xenopus laevis
.
J. Morph, (in the Press)
.
Simnett
,
J. D.
(
1965
).
The prolongation of homograft survival time in the platanna, Xenopus laevis laevis (Daudin), by exposure to low environmental temperature
.
J. cell comp. Physiol
.
65
,
293
298
.
Takahashi
,
T.
,
Old
,
L. J.
,
McIntire
,
K. R.
&
Boyse
,
E. A.
(
1971
).
Immunoglobulin and other surface antigens of cells of the immune system
.
J. exp. Med
.
134
,
815
832
.