Clin. exp. Immunol. (1971) 9, 79-98.

PROLONGED ADMINISTRATION OFANTITHYMOCYTE SERUM IN MICE

II. HISTOPATHOLOGICAL INVESTIGATION

ELIZABETH SIMPSON AND SANDRA L. NEHLSEN

National Institute for Medical Research, Mill Hill, London

(Received 18 December 1970)

SUMMARY

Prolonged administration ofATS to mice resulted in depletion ofsmall lymphocytesin the thymus-dependent (paracortical) areas of lymph nodes in all mice. Smalllymphocyte depletion of the thymus-dependent periarteriolar region of the spleenwas present in most mice, although this feature was masked by plasmacytosis inthis region in some. Depletion of small lymphocytes in the thymus-dependentareas of Peyer's patches was evident in some of the younger mice. None of thesechanges in lymphoid organs were seen in control mice, untreated or given NRS.The thymus was unaffected except in some ATS- or NRS-treated mice whichwere sick and/or old, in which the narrowing of the thymic cortex was attributedto non-specific stress. Plasmacytosis was seen in the medullae of lymph nodes ofboth ATS- and NRS-treated mice, although it was more intense in the latter.In non-lymphoid organs the most striking changes were seen in the kidneys ofmice treated both with ATS and NRS. Complex-type nephritis followed byamyloidosis was seen in a large proportion of mice over 6 months old in boththese groups and in these mice amyloid was seen frequently in other organs,including spleen and liver. Tumours occurred in fifty-four ATS-treated mice, butin no other group. Fifty-two of these tumours were attributable to polyoma virus;two others were lymphoblastomas. Reticulum cell hyperplasia was seen in twofurther mice.

INTRODUCTION

Although the effects of heterologous antilymphocyte sera (ALS) on various functions havebeen widely investigated during the past few years (Levey & Medawar, 1966a, b, 1967;Gray et al., 1966; Monaco et al., 1966a, b; Starzl et al., 1967), relatively little attention hasbeen paid to the morbid anatomy and histopathology of animals receiving ALS. The effectsof ALS on the lymphoid organs have been variously described by several authors. GrayCorrespondence: Dr S. L. Nehlsen, National Institute for Medical Research, Mill Hill, London NW7

1AA.

F 79

80 Elizabeth Simpson and Sandra L. NehIsenet al. (1966) reported lymphoid depletion affecting thymus, cortex and paracortex oflymph nodes, follicles of spleen and Peyer's patches in mice. Denman & Frenkel (1967)described in rats lymphoid depletion of thymus, the cortex and paracortex of lymph nodesand the follicles of spleen, with hyperplasia of plasma cells in the medulla of lymph nodesand in the spleen. Parrott (1967) and Turk & Willoughby (1967) reported that lymphoiddepletion in the lymph nodes was limited to the paracortex or thymus-dependent areas inthe lymph nodes of both mice and guinea-pigs respectively. This finding was confirmed byTaub & Lance (1968) who also found in mice lymphoid depletion in the periarteriolarregions of the splenic follicles and described hyperplastic changes in the follicles of lymphnodes and spleen, and a marked increase in the number of plasma cells in the medullarycords of lymph nodes. They reported that thymuses and Peyer's patches were normal.The description of macroscopic and microscopic lesions in non-lymphoid organs also

varies. 'Wasting disease' was reported by Gray et al. (1966) in mice and by Denman &Frenkel (1967) in rats. Infections of various organs (lung, peritoneum, liver, spleen, joints)were described by Denman & Frenkel (1967). Lance (1967) and Taub & Lance (1968)both described glomerular nephritis in mice on long term ALS treatment and Guttmanet al. (1967) described similar lesions in the kidneys of rats on ALS. Gaugas et al. (1969)reported a very high incidence of tumours in thymectomized CBA mice receiving prolongedALS treatment and an absence of tumours was noted by Lance (1967) in mice of the samestrain given ALS alone.Many of the differences reported are probably explicable in terms of differing schedules

of ALS preparation and administration, strain and species differences between the animalsunder investigation, and perhaps on whether concurrent endemic or epidemic disease waspresent or not.

This report concerns the macroscopic findings and histopathology of mice treatedcontinuously from birth with antithymocyte serum (ATS) prepared according to themethod of Levey & Medawar (1966a). Special attention has been paid to changes in thelymphoid organs and to the occurrence and character of any tumours-an investigationmade specially necessary by the reports of an unexpectedly high incidence of neoplasia inhuman patients given immunosuppressive drugs (Penn et al., 1969; McKhann, 1969) andthe increased incidence of virus and chemically induced tumours in mice treated with ALS(Gaugas et al., 1969; Balner & Dersjant, 1969).

MATERIALS AND METHODS

The general experimental plan is described in the accompanying paper (Nehlsen, 1971).Mice. These were inbred CBA/He mice born and raised in isolation from other experi-

mental mice. They were divided into three sub-groups, normal untreated mice, mice givennormal rabbit serm (NRS) continuously from birth and mice given ATS continuously frombirth according to the same schedule.

Methods. Mice from each of the groups described above were killed by ether and cardiacexsanguination at monthly intervals from 1 month of age onwards. Immediately afterdeath the cranial cavity, thorax and abdomen were opened, and the brain, the respiratory,circulatory, gastrointestinal, urinary, reproductive and lymphoid organs examined macro-

scopically. The skeletal system and the skin were also examined, with special attention to thestate ofany skin graft present. It was necessary to remove some organs, particularly lymphoid

ATS administration in mice. IL Histopathology 81

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82 Elizabeth Simpson and Sandra L. NehIsen

organs, from some mice for other experiments at this stage, but whenever possible the follow-ing organs were placed in fixative for histopathologial examination: brain, pituitary, sub-mandibular salivary gland, thyroid, thymus, heart, lungs, liver, adrenals, kidneys, spleen,pancreas, ileum, Peyer's patch, colon, ovary or testis, mesenteric lymph node, axillary,brachial and inguinal lymph nodes, sternum and, when present, skin grafts. The fixativeused in most cases was formal-acetic-alcohol (F.A.A.), consisting of a freshly preparedmixture of 90 ml 90% ethanol, 10 ml 40% formalin and 4 ml glacial acetic acid. Tissueswere left in this fixative for 3 hr, and then transferred to 70% ethyl alcohol, in which theywere left until trimmed prior to embedding in paraffin wax and sectioning. Sternal tissuewas decalcified in 15 % formic acid overnight prior to processing. 4 Pu sections were stainedroutinely with haematoxylin and eosin and methyl green-pyronin. Special stains, vonKossa, alkaline Congo Red for amyloid, Giemsa and periodic acid Schiff were used inaddition when required.

RESULTS

Tables 1 and 2 summarize the main features of the histologial findings in lymphoid and non-lymphoid organs for all three groups, and Table 3 classifies the tumours that have arisen inthe ATS-treated group.Twenty normal untreated mice, sixty-one NRS-treated and 130 ATS-treated mice have

been examined. Their ages ranged from 1 month to 171 months.

TABLE 2. Histological features of non lymphoid organs examined from mice of each group

Liver Kidney

Group No. in Tumours Inflammatory Glomerular Perivasculargroup lesions Amyloid changes Amyloid 'cuffing'

Normal untreated 20 0/20 0/15 0/15 0/15 0/15 0/15NRS-treated 61 0/61 11/59 18/59 40/60 29/60 20/60ATS-treated 130 54/130 22/126 40/126 73/127 48/127 23/127

NRS = Normal rabbit serum. ATS = Antithymocyte serum.

Tumours. Tumours appeared first in ATS-treated mice of various ages during the 9thmonth of the study, and continued to appear only in the ATS-treated mice in various age-groups until the study was terminated. Antibodies to polyoma virus, absent in the serum ofmice at the beginning of the study, were first detected in sera from several mice in the 4thmonth. The sudden appearance of polyoma antibodies suggests that polyoma virus gainedentry to the mouse colony a short time before: the rather long 5 month latent periodbetween the detection of polyoma antibodies and the appearance of the first tumoursmorphologically similar to those caused by polyoma virus (Stewart, Eddy & Borgese, 1958)is possibly a dose effect (Nehlsen, 1971).From when the first tumour appeared, 175 ATS-treated mice (ninety-six males and seventy-

nine females) were at risk, and fifty-four (31 %) of them (twenty-six males and twenty-eightfemales) developed tumours. Their ages ranged from 4j to 14 months. Histologically the

83ATS administration in mice. II. HistopathologyTABLE 3. Types of tumours found in ATS-treated mice of various ages

Age range No. of(months) Sex mice Tumour type(s) Metastases

4-6 FemaleMale

Male

6-9 Female

Female

FemaleFemaleFemaleMaleMaleMale

9-12 Female

Female

Female

Female

Female

Female

Male

Male

Male

Male12-15 Female

MaleMale

Male

Male13 Male

6

1

12

231

Mammary carcinomaMammary carcinomaOsteosarcomamixed salivary tumourMammary carcinomaMammary carcinomamixed salivary tumourMixed salivary tumourOsteosarcomaOsteosarcomaMammary carcinomaOsteosarcomaOsteosarcoma

6 Mammary carcinomaI fMammary carcinoma

mixed salivary tumour1 JMammary carcinoma

osteosarcoma1 XMammary carcinoma

Mosteosarcoma4 Osteosarcoma4 Osteosarcoma

mixed salivary tumour4 Osteosarcoma4 Osteosarcoma

carcinoma in situ, kidney

If

Osteosarcomamixed salivary tumour

1 Mixed salivary tumour2 Osteosarcoma6 Osteosarcoma1 Osteosarcoma1 fOsteosarcoma1 adenocarcinoma, kidney

1 Mammary carcinoma2 Lymphoblastic lymphomas

Lung, liver, peritoneum

Lung, mediastinum

Lung, liver

Lung, liver

Adrenal

Liver

Liver

s Many organs

tumours from fifty-two mice were osteosarcomas (thirty-one), mammary carcinomas (twenty-one) and/or mixed salivary gland tumours (seven) (see Figs 1, 2 and 3). Many were ofmulticentric origin, and nine mice, four males and five females, had tumours of two histo-logical types. Seven tumours, four osteosarcomas, two mammary carcinomas and onemixed salivary gland tumour had metastasized to liver, lung, mediastinum, peritoneumand/or adrenal. These types of tumour have been consistently identified with tumourscaused by polyoma virus in mice although the incidence of metastases appears to be higher

Elizabeth Simpson and Sandra L. Nehisen

FIG. 1. Osteosarcorna from a 91-month-old male ATS-treated mouse (H and E, x 100).

2o ...Mam yclat 10).FIG. 2. Mammary carcinoma from a 7-month-old female ATS-treated mouse (H and E, x 100).

than is reported (Stanton et al., 1959; Dawe, Law & Dunn, 1959). The circumstantialevidence of the apparent introduction of polyoma virus into the mouse colony, and thehistological types of tumours found, suggest that polyoma virus was the cause of the fifty-nine tumours in fifty-two ATS-treated mice in this study. Two kidney tumours were seen, onewas an adencoarcinoma (Fig. 4) which, although lying in the renal pelvis and apparentlygrowing from the epithelium lining the renal pelvis, probably arose from tubular epithelium.

84

ATS administration in mice. IL. Histopathology 85

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FIG. 3. Mixed salivary tumour from a 9-month-old male ATS-treated mouse (H and E, x 100).

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FIG. 4. Renal adenocarcinoma in a 13-month-old female ATS-treated mouse (H and E, x 100).

It was similar to a tumour apparently caused by polyoma virus, described by Stewart et a!.(1958). The other kidney tumour was a small in situ carcinoma, arising from the epitheliumlining the renal pelvis (Fig. 5). It is possible that this tumour was caused by polyoma virus.Both mice with kidney tumours also had osteosarcomas.

Multicentric lymphoblastic lymphomas appeared in two 13-month-old ATS-treated malemice (Fig. 6) and it seems unlikely that these were caused by polyoma virus, as this type of

86 Elizabeth Simpson and Sandra L. Nehisen

FIG. 5. Carcinoma in situ of pelvic epithelium of kidney of a lOi-month-old male ATS-treated mouse (H and E, x 100).

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male ATS-treated mouse (H and E, x 250).

tumour has not been reported following experimental injection of polyoma virus into mice(Stanton et al., 1959; Dawe et al., 1959; Stewart, Eddy & Borgese, 1958).

In addition to the fifty-four mice whose tumours are described above, two mice, one 10-month-old female and one 172-month-old male, showed a hyperplasia of recticulum cells

ATS administration in mice. II. Histopathology 87(with many cells in mitosis) in the paracortical areas which had been depleted of smalllymphocytes. This hyperplasia might have been an early stage of neoplasia but there was noloss of architecture or invasion by these mitotically active reticulum cells.

Amyloidosis. Amyloidosis was a common finding in both ATS- and NRS-treated miceover 6 months of age. It affected more than 50% of the mice. It did not occur in normaluntreated mice. Amyloid first appeared in the perifollicular areas in the spleen, but laterliver, spleen, kidney, adrenal and many other organs were infiltrated, and kidney and liverfailure were a common sequel. Three mice killed when they were 171-19 months old, andwho had been off ATS for 2-3 months, did not show amyloidosis, and this suggeststhat amyloid had been resorbed because no ATS- or NRS-treated mice over 1 year ofage were free of amyloid.

Lymphoid organsThe lymphoid tissue in normal untreated mice showed lymph nodes with small relatively

inactive follicles in the cortex, a paracortex populated with predominantly small lympho-cytes and a small medulla in which cords and sinuses were identified. Just over half thespleens in this group had active follicles, containing lymphoblasts and cells in mitosis.NRS-treated mice had more active follicles in lymph nodes and spleen, and more than two-thirds of the mice in this group had an increased number of plasma cells in the medulla: incontrast to this, the ATS-treated mice showed paracortices depleted of small lymphocytes,and in over 900% of nodes examined the medullary cords were packed with plasma cells(Fig. 7). There was activity in many of the follicles of lymph nodes and spleen. Periarteriolardepletion in the spleen as seen in most ATS-treated mice (Fig. 8), but in some an increasednumber of plasma cells in this area masked the depletion by small lymphocytes (Fig. 9).Perifollicular amyloid was seen in the spleen of fifty-three out of eighty-six ATS-treatedmice over 6 months of age, and in the spleens of thirty-three out of forty-six NRS-treatedmice in the same age range (Fig. 10). Amyloidosis in the spleen varied from mild, when asmall collar could be seen around some follicles, to very severe, when all splenic architecturewas obliterated and replaced by amyloid. Peyer's patches appeared to be similar in mice ofall three groups, with germinal centre formation, and no depletion of small lymphocytesexcept in some ofthe younger ATS-treated mice, in which interfollicular areas were depletedof small lymphocytes (Fig. 11), but this was not a consistent finding in ATS-treated mice.The thymuses of mice of the three groups were essentially similar, and there was no consistentdepletion of small lymphocytes in the ATS-treated group (Fig. 12); twenty-six mice over6 months old in this group had thymic cortices which were narrower than normal, but allthese mice appeared ill before they were killed, and either had severe kidney lesions or werecarrying large tumours. The depletion of their thymuses was therefore attributed to non-specific stress. Six NRS-treated mice with severe amyloid also had narrow thymic cortices.

Oxazalone treatment of ATS-treated mice produced no lymphoid changes, but in normaluntreated mice and in NRS-treated mice the paracortical areas of lymph nodes were en-larged and contained many lymphoblasts and cells in mitosis.

Non-lymphoid organsThe kidneys of mice under 6 months old showed no consistent lesions in any group, but

thereafter many mice in both the NRS- and ATS-treated groups showed glomerular changes:73/86 ATS-treated mice and 40/46 NRS-treated mice over 6 months old showed some degree

88 Elizabeth Simpson and Sandra L. Nehisen

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FI.7 xlay .yp..efo.a4-ot-l fml T -raed mue hwn~tyiacage of acieflils aao xdpeedo ml ypoye n eulrcod pake wit plsacls( n ,x7

FIG. 8. Spleen showing periarteriolar depletion of small lymphocytes in a 10-month-old maleATS-treated mouse (H and E, x 120).

of change. These changes appeared to start as increased thickening ofthe mesangium witheosinophilic material, and the glomerular capillaries had a 'wire loop' appearance (Fig. 13).Later changes consisted of the laying down of amyloid in the mesangium (Fig. 14a) and

ATS administration in mice. II. Histopathology 89

FIG. 9. Spleen from a 10I-month-old male ATS-treated mouse showing accumulation of plasmacells in the periarteriolar region (H and E, x 250).

FIG. 10. Spleen from the same mouse as Fig. 9, showing perifollicular amyloid (H and E, x 100).

between tubules of the medulla (Fig. 14b). At this stage many tubules would be dilated, andcontain casts, and in some kidneys wedge shaped areas of tubular collapse were apparent.Perivascular accumulations of lymphocytes and/or plasma cells (Fig. 15) were also a featureof kidneys of ATS- and NRS-treated mice, and were found in twenty-three ATS-treated andtwenty NRS-treated mice. This feature was not seen in untreated mice. Microfoci of calcium

90 Elizabeth Simpson and Sandra L. Nehisen

~~~~~~~~~~~~7~~~~~~~~~7

FIG. 1 1. Peyer's patch from a 44-month-old female ATS-treated mouse showing small lympho-cyte depletion in the interfollicular areas (H and E, x 70).

FIG. 12. Thymus from a 13i-month-old male ATS-treated mouse (H and E, x 100).

with no surrounding cellular reaction were present in the cortices ofsome mice over 6 monthsold: there were fourteen cases in the ATS-treated, twelve in the NRS-treated, but none inthe untreated group.

The livers of mice under 6 months of age were normal in mice of all groups, but of eighty-six ATS-treated mice over 6 months old the livers of forty showed some degree of amyloi-

ATS administration in mice. II. Histopathology

FIG. 13. Kidney from a 64-month-old female ATS-treated mouse, showing glomerularcapillaries with a 'wire loop' appearance (H and E, x 250).

k3.r .1 eY s D '

FIG. 14. (a) Kidney from a 10-month-old male ATS-treated mouse, showing amyloid in themesangium of two glomerulae. (b) Kidney from the same mouse as (a), showing amyloid inbetween tubules (H and E, x 175).

dosis (Fig. 16), and twenty-two had focal accumulations of inflammatory cells, mainlypolymorphs, although in three livers, giant cells were present and the lesions were suggestiveof MHV infection. Tumour metastases appeared in the liver of five ATS-treated mice.Of forty-six NRS-treated mice over 6 months, eighteen showed amyloid and eleven hadfocal accumulations of inflammatory cells, mononuclear and/or polymorphonuclear.The heart. The only lesions seen were microfoci of calcium in the myocardium of fourteen

ATS-treated mice, ranging in age from 1 month to 12 months.

91

92 Elizabeth Simpson and Sandra L. NehIsen

FIG. 15. Kidney from a 10i-month-old female ATS-treated mouse showing perivascularaccumulations of lymphocytes (H and E, x 100).

t ~~~~~~~~~~-X--AFARv.6zP.;,.W.

FIG. 16. Liver from a 10k-month-old female ATS-treated mouse showing moderate amyloido-sis (H and E, x 125).

Skin grafts from forty mice of the ATS-treated group were examined histologically;five were rat xenografts and the remaining thirty-five were mouse allografts. They had beengrafted for varying times before examination: twenty-five did not show any signs of rejectionor cellular infiltration (Fig. 17), and this included one of the rat xenografts. The other rat

ATS administration in mice. II. Histopathology

FIG. 17. A-strain skin graft from a 14-month-old male ATS-treated mouse showing practicallyno cellular infiltration (H and E, x 100).

FIG. 18. C57 skin graft from a 9-month-old female ATS-treated mouse showing mild to moderateperivascular accumulations of lymphocytes and plasma cells (H and E, x 100).

xenografts showed moderate infiltration by polymorphonuclear and mononuclear cells andone was haemorrhagic. Of the remaining ten mouse allografts, seven showed a minimalamount ofdiffuse infiltration by lymphocytes and plasma cells, mainlyperivascularly (Fig. 18).Three showed heavy lymphocytic infiltration but all three recipient mice had been off ATS

93

94 Elizabeth Simpson and Sandra L. NehIsen

for over 3 weeks. Six of the mouse allografts had been taken from nude congenitally athymicmice (Nu/Nu) (Pantelouris, 1968) which frequently carry fungal hyphae and spores in thehair follicles and epidermis (Simpson, unpublished observation). Morphologically similar

fungi were seen growing in the adjacent ATS-treated recipient's epidermis on two occasions,

suggesting that depression of cell-mediated immunity enabled this unusual microorganismto establish itself.

Other non-lymphoid organs routinely examined in mice from all three groups were lung,

brain, pituitary, bone marrow, thyroid, submandibular salivary gland, ovaries, testis,

pancreas, adrenals, small and large intestine. No abnormalities were detected apart from

amyloid infiltration of practically all organs, especially in the adrenals of some ATS- and

NRS-treated mice and the metastatic deposit of mammary carcinoma in an adrenal of one

mouse and metastases of tumours of various types in the lungs of four mice (see Table 3).A few mice in both NRS and ATS groups had received BSA/KLH (Nehlsen, 1971) withina few weeks of being killed. Some of these mice had evidence of mild granulomatousperitonitis.

DISCUSSION

The most consistent findings in the lymphoid tissue of ATS-treated mice in this study

have been depletion of small lymphocytes from the paracortical or thymus-dependentareas of lymph nodes and spleen, hyperplasia of medullary plasma cells in the lymph nodes

and a hyperactivity of follicles in lymph nodes and spleen. The thymuses and Peyer's patches

with a few exceptions have appeared to be quite normal. In these findings we concur with

Parrott (1967), Lance (1967) and Taub & Lance (1968). The similarity of these findings is

probably due to our use of the same method of preparation of the ATS. Sera raised in this

manner are relatively non-toxic, and this is reflected in the tolerance shown to continuousadministration (this paper, Lance, 1967; Taub & Lance, 1968) and the limited nature of the

lymphoid lesion. In contrast to this, many authors have described severe lymphoid depletion,

involving the thymic cortex in mice (Gray et al., 1966), and in rats (Denman & Frenkel, 1967),

the follicles in the cortex of lymph nodes in mice (Gray et al., 1966), rats (Denman &

Frenkel, 1967), dogs (Monaco et al., 1966c) and man (Penn et al., 1969); and the folliclesof the spleen in mice (Gray et al., 1966) and rats (Denman & Frenkel, 1967, 1968; Guttman

et al., 1967; Nagaya & Sieker, 1965). In all reports of such severe, non-selective lymphoid

depletion, the antilymphocyte sera have been raised with the use of adjuvants and have

often followed a long course of hyperimmunization with cells from a variety of sources.

Many such sera are poorly tolerated when given over long periods (Gray et al., 1966;

Denman & Frenkel, 1967; Balner & Dersjant, 1969).In our experience hyperplasia of the plasma cells in the medulla of lymph nodes has been

a consistent finding in both ATS- and NRS-treated mice. Similar results were obtained by

Lance (1967) and Taub & Lance (1968). However, this hyperplasia appears to be more

intense in the ATS group. This may be related to the fact that ATS/ALS is more immuno-

genic than NRS (Lance & Dresser, 1967), or it could be caused by an exaggerated humoral

response to other antigens in ALS-treated mice (Hirsch, Murphy & Hicklin, 1968).The kidney lesions in our NRS- and ATS-treated mice over 6 months of age may be the

result of many factors, of which one could be loss of tolerance to rabbit serum proteins,with the resultant 'complex type' nephritis as described by Lance (1967) and Taub & Lance

ATS administration in mice. II. Histopathology

(1968) under similar circumstances; but an antibody response to other antigens and theresultant formation of antibody/antigen complexes may also have been causal. Polyomavirus, present in our colony after the 4th month of the study, is known to be capable ofeliciting an antibody response which can lead to complex nephritis (Dixon, Oldstone &Tonietti, 1969). The exaggerated humoral response of ALS-treated mice (Hirsch et al.,1968) may have exacerbated their kidney lesions.The reports of increased tumour incidence in human patients given immunosuppressive

drugs have placed ALS under suspicion, as it has been used, in conjunction with other drugs,in some of the patients who developed tumours (McKhann, 1969). The experimental evidenceon the enhanced incidence of growth rate of tumours in animals given ALS covers a widefield ranging from transplantable tumours to those induced by viruses and chemicalcarcinogens.

In the case of syngeneic transplantable tumours Fisher, Soliman & Fisher (1969a, b)have shown that ALS treatment of recipients increases 'take', growth rate and metastaticrate. Deohdar, Crile & Schofield (1968) have been able to abolish active immunity to sarcoma180 in mice treated with ALS. There have been several reports of human tumours xeno-grafted into ALS-treated mice (Phillips & Gazet, 1967; Stanbridge & Perkins, 1969) andhamsters (Sommers, Reeves & Reeves, 1966; Davis & Lewis, 1968). Certain oncogenicviruses have been shown to produce a higher incidence of tumours in ALS-treated animalsthan in untreated controls (Gaugas et al., 1969; Law, Ting & Allison, 1968; Allison,Berman & Levey, 1967). Balner & Dersjant (1969) have shown that in mice given methyl-cholanthrene, and a 5 or 9 week course of ALS, tumours arise earlier and grow faster thanin controls, although the final incidence in all groups is the same.That ALS should establish and maintain the growth of transplantable tumours is hardly

surprising, as such a tumour is an allograft bearing transplantation antigens, and ALS isknown to enable such grafts to survive (Levey & Medawar, 1966a). Some of the tumoursarising in human patients under immunosuppression have undoutedly been due to accidentaltumour transplantation (Wilson et al., 1968).Many of the tumours caused by oncogenic viruses, particularly the DNA viruses such as

SV 40, polyoma and the adenoviruses, are 'laboratory tumour artifacts', in as much astumours caused by these agents do not occur under natural conditions. They were firstreported to give rise to tumours when injected into neonatal animals which are known to beimmunologically incompetent but can be induced in adult animals after a very long latentperiod (Allison, Chesterman & Baron, 1967). That ALS reproduces or hastens this effectis therefore not surprising. It would perhaps be unwise to draw too close a parallel betweenexperimental animals given ALS and these viruses, and human patients under ALS treat-ment, as there is very little direct evidence that viruses play a similar role in the pathogenesisof human tumours, with the possible exception of one or two specific tumour entities inwhich the role of virus has still to be defined (leading article, Lancet, 1969). The findings ofBalner & Dersjant (1969) on MCA induced tumours are relevant to chemically inducedtumours, and this type of tumour does occur in man (Hueper, 1957).

There is a dearth ofinformation on the effect ofALS on 'spontaneous' tumours in animals.Such tumours have been shown to be poorly antigenic in comparison with virus or chemicalcarcinogen induced tumours (Prehn & Main, 1957), and if this is also true of 'spontaneous'tumours of man, there are implications relating to the effect of ALS on oncogenesis. ALSwould be expected to enhance the growth of antigenic tumours, as the cellular immune

G

95

96 Elizabeth Simpson and Sandra L. Nehisenreaction of the body to transplantation antigens would be suppressed (see Discussion inBalner & Dersjant, 1969). Conversely, the development of poorly antigenic tumours wouldnot be affected to the same extent.

It is against this background that we chose to look at the effect of ATS on the incidence ofspontaneous tumours in a strain of mouse, CBA, with a low incidence of tumours (Hoag,1963). It is of interest that, after treating a colony of mice continuously for 18 months withATS, the only tumours to arise in any numbers have been attributable to polyoma virus.Two lymphoblastic lymphomas appeared in mice over 1 year old, and two mice showedreticulum cell hyperplasia. Reasoning based on a rather simple form of the surveillancehypothesis would have predicted a higher incidence of 'spontaneous' tumours.

ACKNOWLEDGMENTS

We wish to thank Sir Peter Medawar for help and encouragement, and for criticizing themanuscript. We are indebted to Mr F. P. Wharton and his assistants for histological prepara-tion of the specimens and to Miss Janet Ashwell for typing this manuscript. One of us(S.L.N.) holds a Medical Research Council Research Fellowship.

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ALLISON, A.C., CHESTERMAN, F.C. & BARON, S. (1967b) Induction of tumours in adult hamsters withsimian virus 40. J. nat. Cancer Inst. 38, 567.

BALNER, H. & DERSJANT, H. (1969) Increased oncogenic effect of methylcholanthrene after treatment withanti-lymphocyte serum. Nature (Lond.), 224, 376.

DAVIES, R.C. & LEWIS, J.L., JR (1968) The effect of adult thymectomy on the immunosuppression obtainedby treatment with antilymphocyte serum. Transplantation, 6, 879.

DAWE, C.J., LAW, L.W. & DUNN, T.B. (1959) Studies on parotid-tumor agent in cultures of leukemictissues of mice. J. nat. Cancer Inst. 23, 717.

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DENMAN, A.M. & FRENKEL, E.P. (1968) Mode of action of anti-lymphocyte globulin. II. Changes in thelymphoid cell population in rats treated with anti-lymphocyte globulin. Immunology, 14, 115.

DEODHAR, S.D., CRILE, G., JR & SCHOFIELD, P.F. (1968) Immunosuppression in allogeneic murine tumour

system. A model for the study of anti-lymphocyte serum. Lancet, i, 168.DIXON, F.J., OLDSTONE, M.B.A. & ToNIETTI, G. (1969) Virus-induced immune-complex-type glomerulo-

nephritis. Transpl. Proc. 1, 945.FISHER, E.R., SOLIMAN, 0. & FISHER, B. (1969a) Effect of antilymphocyte serum on parameters of growth

of MCA-induced tumours. Nature (Lond.), 221, 287.FISHER, E.R., SOLIMAN, 0. & FISHER, B. (1969b) Effect of antilymphocyte serum on parameters of tumor

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